EA040921B1 - LONG-LASTING BLOOD COAGULATION FACTOR VII AND METHODS FOR ITS PRODUCTION - Google Patents

Description

The field of technology to which the invention belongs

Disclosed are polypeptides containing at least one carboxyl-terminal peptide (CTP) of human chorionic gonadotropin attached to the carboxyl end of a blood coagulation factor, and polynucleotides encoding them. Also disclosed are pharmaceutical compositions and pharmaceutical compositions containing polypeptides and polynucleotides according to the present invention, and methods for their use and preparation.

Background of the invention

The development of clotting factor replacement therapy has transformed the lives of many individuals with hemophilia. Hemophilia is a group of inherited genetic disorders that impair the body's ability to regulate blood clotting or coagulation. Patients with hemophilia do not produce enough factor VIII or factor IX proteins, which are essential for effective blood clotting. In subjects with severe hemophilia, even minor damage can result in blood loss that lasts for days or weeks, and complete healing may not occur, leading to the possibility of debilitating, irreversible damage to joints and other organs and premature death.

Hemophilia is an X-linked inherited hemorrhagic disorder caused by defects or absence of critical factors in the coagulation cascade. In patients with hemophilia, thrombin formation and fibrin clot formation are significantly reduced, resulting in episodes of spontaneous bleeding, most often in the joints and internal organs, and profuse bleeding during and after surgery or trauma. In patients with hemophilia, frequent bleeding can also lead to joint swelling, joint damage, severe deformities, frequent infections, and reduced mobility (Mayo Clinic). Hemophilia A is caused by defects or deficiencies in the expression of coagulation factor VIII, while hemophilia B is caused by defects or deficiencies in the expression of coagulation factor IX.

Hemophilia B results in a deficiency in the procoagulant activity of FIX. Patients with hemophilia B have spontaneous soft tissue hemorrhages and recurrent hemarthroses, which often lead to disabling arthropathy. Existing treatments for these patients include intravenous administration of recombinant FIX. However, the problems of cost and the relatively rapid clearance of FIX from the bloodstream make the development of sustained release FIX a difficult task. The commercial availability of FVIII and FIX has led to improved control of life-threatening bleeding episodes. Many patients receive prophylactic therapy that reduces the risk of bleeding and associated complications. However, a significant proportion of patients (10-30%) develop inhibitory antibodies to exogenously administered FVIII and FIX. Administration of FVIIa, which is a bypass drug, can induce hemostasis and provide effective treatment in patients with Ab inhibition.

Recombinant FVIIa (NovoSeven®) is commercially available and was approved in 1996 for the treatment of bleeding episodes in hemophiliac patients treated with inhibitors. However, rFVIIa is rapidly cleared with a terminal half-life of 2.5 hours. As a result, patients typically require multiple frequent infusions (2-3 doses given at 2-3 hour intervals) to achieve adequate hemostasis after mild to moderate bleeding. . In this regard, there is considerable interest in the development of a form of FVIIa sustained release, which would provide an increase in the duration of hemostatic activity after a single dose and would allow you to enter doses much less frequently. Long-acting FVIIa will also increase the feasibility of long-term prophylactic therapy.

Various technologies are being developed to extend the half-life of FVIIa. However, there remains a need to achieve a long half-life of this protein while maintaining its biological activity and ensuring that modifications will not induce significant immunogenicity. In the present invention, this need is resolved by attaching carboxyl-terminal peptides (CTP) of gonadotropin to FVIIa, thus modifying it to prolong its half-life and biological activity.

Brief description of the invention

In one aspect, a method is disclosed for producing a polypeptide that is a human blood coagulation factor VII (FVII) modified with a carboxy-terminal peptide (CTP) of human chorionic gonadotropin, where the specified polypeptide contains three CTP molecules attached sequentially one after the other to the C-terminus of FVII, while the method includes the steps of stable transfection of a predetermined number of cells with an expression vector containing a coding portion encoding said CTP-modified FVII, where said transfected cells express and secrete said CTP-modified FVII; obtaining cell clones overexpressing said CTP-modified FVII; propagating said clones in solution to a predetermined scale; collecting said solution containing said clones; filtering said solution containing said clones to obtain a clarified

- 1 040921 collected solution; and purifying said polypeptide from said clarified pooled solution to obtain a purified protein solution having the desired concentration of CTP-modified FVII; thereby obtaining a CTP-modified FVII, wherein the amino acid sequence of the resulting CTP-modified FVII is given under SEQ ID NO: 7.

In another aspect, a method is disclosed for producing a polypeptide that is an active human blood coagulation factor VII (FVIIa) modified with a carboxy-terminal peptide (CTP) of human chorionic gonadotropin, where the specified polypeptide contains three CTP molecules attached sequentially one after the other to the C-terminus of FVII, while the method includes the steps of: stable transfection of a predetermined number of cells with an expression vector containing a coding portion encoding a CTP-modified FVII, wherein said transfected cells express and secrete said CTP-modified FVII; obtaining cell clones overexpressing said CTP-modified FVII; propagating said clones in solution to a predetermined scale; collecting said solution containing said clones; filtering said solution containing said clones to obtain a clarified collected solution; and purifying and activating said polypeptide from said clarified pooled solution to obtain a purified protein solution having the desired concentration of CTP-modified FVIIa; thereby obtaining a CTP-modified FVIIa, wherein the amino acid sequence of the resulting CTP-modified FVIIa is given under SEQ ID NO: 7.

In another aspect, a polypeptide is disclosed, which is human blood coagulation factor VII (FVII), a modified carboxy-terminal peptide (CTP) of human chorionic gonadotropin, containing three CTP molecules attached sequentially one after the other to the C-terminus of FVII, where the specified CTP-modified FVII is obtained with using a method comprising the steps of: stable transfection of a predetermined number of cells with an expression vector containing a coding portion encoding said CTP-modified FVII, where said transfected cells express and secrete said CTP-modified FVII; obtaining cell clones overexpressing said CTP-modified FVII; propagating said clones in solution to a predetermined scale; collecting said solution containing said clones; filtering said solution containing said clones to obtain a clarified collected solution; and purifying said polypeptide from said clarified pooled solution to obtain a purified protein solution having the desired concentration of CTP-modified FVII; wherein said resulting CTP-modified FVII contains the amino acid sequence shown under SEQ ID NO: 7.

In another aspect, a polypeptide is disclosed that is an activated human coagulation factor VII (FVIIa), a modified carboxy-terminal peptide (CTP) of human chorionic gonadotropin, containing three CTP molecules attached sequentially one after the other to the C-terminus of FVII, where the specified CTP-modified FVIIa is obtained using a method comprising the steps of: stable transfection of a predetermined number of cells with an expression vector containing a coding portion encoding said CTP-modified FVII, where said transfected cells express and secrete said CTP-modified FVII; obtaining clones of cells overexpressing said CTP-modified FVII; propagating said clones in solution to a predetermined scale; collecting said solution containing said clones; filtering said solution containing said clones to obtain a clarified collected solution; and purifying and activating said polypeptide from said clarified pooled solution to obtain a purified protein solution having the desired concentration of CTP-modified FVIIa; wherein said resulting CTP-modified FVIIa contains the amino acid sequence shown under SEQ ID NO: 7.

In a related aspect, the resulting human clotting factor VII (FVII) polypeptide, modified with human chorionic gonadotropin carboxy-terminal peptide (CTP), is highly glycosylated. In another related aspect, the glycosylation profile of the resulting CTP-modified FVII is glycosylated at at least 4 O-linked glycosylation sites on the CTP. In another related aspect, said CTP-modified FVII has a high percentage of charged N-glycans. In another related aspect, the resulting human human chorionic gonadotropin carboxy-terminal peptide (CTP) modified carboxy-terminal peptide (CTP) is highly sialylated.

In a related aspect, the resultant human coagulation factor VII (FVII) polypeptide, a modified carboxy-terminal peptide (CTP) of human chorionic gonadotropin, has a high percentage of carboxylated glutamic acid residues.

In a related aspect, propagating clones includes propagating clones derived from a working cell bank (WCB) or from a master cell bank (MCB). In another related aspect, said clones

- 2 040921 express and secrete CTP-modified FVII at a level of at least 600 mg/l. In another related aspect, these clones are propagated in solution through a series of subculturing steps to a level characteristic of a production bioreactor. In another related aspect, the bioreactor provides for a disposable or stainless steel bioreactor. In another related aspect, said bioreactor functions as a fed-batch bioreactor.

In a related aspect, at least 60% of the purified human clotting factor VII (FVII) human chorionic gonadotropin carboxy-terminal peptide (CTP) polypeptide from said clarified harvest is the highly glycosylated form of the CTP-modified FVII. In a related aspect, at least 60% of the purified human chorionic gonadotropin carboxy-terminal peptide (CTP) modified human carboxy-terminal peptide (CTP) polypeptide of said clarified harvest contains a high percentage of carboxylated glutamic acid residues.

In a related aspect, purification comprises the steps of passing said clarified collected material through an affinity chromatography column, a multimodal matrix or mixed media chromatography column, a hydrophobic interaction chromatography column, and an anion exchange chromatography column; inactivation of viruses present in the clarified collected material or in the eluate collected after column chromatography, or in any combination thereof, where the inactivation of viruses includes incubation in a solution toxic to these viruses, or nanofiltration, or any combination thereof; and where the eluate after anion exchange chromatography passes the stage of ultrafiltration/diafiltration.

In a related aspect, the resulting polypeptide, which is a (CTP)-modified human blood coagulation factor VII (FVII), includes a polypeptide that is active CTP-modified FVII (a polypeptide that is a CTP-modified FVIIa).

In a related aspect, at least 20% recovery of highly glycosylated CTP-modified FVII is achieved by the preparation method.

In another aspect, the composition comprises the resulting CTP-modified FVII and a pharmaceutically acceptable carrier.

In another aspect, this document discloses a polypeptide that is an active human blood coagulation factor VII (FVIIa), a modified carboxy-terminal peptide (CTP) of human chorionic gonadotropin, containing three CTP molecules attached sequentially one after the other to the C-terminus of FVIIa, where the specified polypeptide, which is a CTP-modified FVIIa, is in an essentially pure and active form, while the specified polypeptide, which is a CTP-modified FVIIa, provides: (a) a high content of sialic acid; (b) low content of oxidized form; (c) a highly glycosylated form; (d) a high percentage of carboxylated glutamic acid residues; (e) a high percentage of charged N-glycans; and (f) a high specific activity, or any combination thereof, wherein said CTP-modified FVIIa comprises the amino acid sequence shown under SEQ ID NO: 7.

In a related aspect, the high content of sialic acid is at least 15 mol/mol. In another related aspect, the highly glycosylated form provides for an O-glycan content of at least 10 mol/mol. In another related aspect, the substantially pure and active form contains at least 60% of the highly glycosylated form of said active CTP-modified FVIIa. In another related aspect, at least 60% of the substantially pure and CTP-modified form of FVIIa contains a high percentage of carboxylated glutamic acid (Gla) residues. In another related aspect, a high percentage of carboxylated glutamic acid (Gla) residues is at least 90% of Gla residues. In another related aspect, the low percentage of oxidized form is less than 5%. In another related aspect, said substantially pure and active polypeptide, which is a CTP-modified FVII, is at least 90% pure. In a further related aspect, the purity percentage is selected from the group consisting of 97.3%, 97.6%, 97.4%, and 97.0%. In another related aspect, the specific activity of said substantially pure and active CTP-modified FVII polypeptide is at least 10,500 U/mg. In a further related aspect, the specific activity is selected from the group consisting of 15563, 16720, 22478 and 23608 U/mg.

In another aspect, this document discloses a composition containing CTP-modified FVIIa, which provides: (a) high content of sialic acid; (b) low content of oxidized form; (c) a highly glycosylated form; (d) a high percentage of carboxylated glutamic acid residues; (e) a high percentage of charged N-glycans; and (f) a high specific activity, or any combination thereof, wherein said CTP-modified FVIIa comprises the amino acid sequence shown under SEQ ID NO: 7 and a pharmaceutically acceptable carrier.

- 3 040921

Other features and advantages will become apparent from the following detailed description, examples and figures. It should, however, be understood that the detailed description and specific examples, while indicative of preferred embodiments of the present invention, are provided for purposes of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed descriptions.

Brief description of graphic materials

The patent or application file contains at least one graphic material in color. Copies of this patent or patent application publication with color(s) graphic(s) will be made available by the Office upon request and upon payment of the required fee.

Fig. 1. The pCI-dhfr-MOD-5014 plasmid map is shown.

Fig. 2A. A schematic representation of the FVII-CTP3 purification process is shown. Lot 31 was received for the PK/PD study.

Fig. 2b. A schematic representation of the FVII-CTP3 purification process is shown. Lot 38 was received for a survival study.

Fig. 3A. SDS-PAGE and Western blotting patterns of final FVII and FVIIa are shown. Each lane of Coomassie-stained SDS-PAGE gel was loaded with 10 μg (batch 31) or 5 μg (batch 38). 1. FVII-CTP3 polypeptide. 2. Heavy chain including 3x CTP. 3. Light chain. All three antibodies detected FVII.

Fig. 3B. SDS-PAGE and Western blotting patterns of final FVII and FVIIa are shown. Each lane of Coomassie-stained SDS-PAGE gel was loaded with 10 μg (batch 31) or 5 μg (batch 38). 1. FVII-CTP3 polypeptide. 2. Heavy chain including 3x CTP. 3. Light chain.

Fig. 3C. SDS-PAGE and Western blotting patterns of final FVII and FVIIa are shown. Each lane of Coomassie-stained SDS-PAGE gel was loaded with 10 μg (batch 31) or 5 μg (batch 38). 1. FVII-CTP3 polypeptide. 2. Heavy chain including 3x CTP. 3. Light chain.

Fig. 3D. SDS-PAGE and Western blotting patterns of final FVII and FVIIa are shown. Each lane of Coomassie-stained SDS-PAGE gel was loaded with 10 μg (batch 31) or 5 μg (batch 38). 1. FVII-CTP3 polypeptide. 2. Heavy chain including 3x CTP. 3. Light chain.

Fig. 3E. SDS-PAGE and Western blotting patterns of final FVII and FVIIa are shown. Each lane of Coomassie-stained SDS-PAGE gel was loaded with 10 μg (batch 31) or 5 μg (batch 38). 1. FVII-CTP3 polypeptide. 2. Heavy chain including 3x CTP. 3. Light chain.

Fig. 3F. SDS-PAGE and Western blotting patterns of final FVII and FVIIa are shown. Each lane of the western blotting gel was loaded with 1 μg of protein. 1. FVII-CTP3 polypeptide. 2. Heavy chain including 3x CTP. 3. Light chain. All three antibodies detected FVII. The light chain of FVIIa was detected using both α-FVII.

Fig. 3g. SDS-PAGE and Western blotting patterns of final FVII and FVIIa are shown. Each lane of the western blotting gel was loaded with 1 μg of protein. 1. FVII-CTP3 polypeptide. 2. Heavy chain including 3x CTP. 3. Light chain. All three antibodies detected FVII. The heavy chain of FVIIa was detected using α-CTP.

Fig. 3H. SDS-PAGE and Western blotting patterns of final FVII and FVIIa are shown. Each lane of the western blotting gel was loaded with 1 μg of protein. 1. FVII-CTP ₃ polypeptide. 2. Heavy chain including 3x CTP. 3. Light chain. All three antibodies detected FVII. The heavy chain of FVIIa was detected using α-Gla.

Fig. 4. It has been shown that purification on a ceramic hydroxyapatite (HA) column increases the chromogenic activity of FVII-CTP3. Comparative in vitro specific activity assessment of harvested FVII-CTP ₃ containing material, process fractions and purified FVII-CTP3 versus a pool of normal human plasma was performed using the commercially available BIOPHEN Chromogenic Activity Test Kit (Hyphen BioMed 221304). The harvested material containing FVII-CTP3 and the protein itself were serially diluted, and the specific activity was evaluated by comparing the dose-response curve with that of a normal human plasma reference preparation.

Fig. 5. Shows the PK profile of FVIIa-CTP ₃ versus NovoSeven® in FVIII deficient mice. FVIIa-CTP3 was obtained after selection of FVII, purification process for HA and activation. FVIIa-CTP3 or NovoSeven® was administered as a single intravenous injection of FVIII -/- mice with hemophilia. Blood samples were taken from the retroorbital sinus at 0.083, 0.5, 2, 8, 24, 48 and 72 hours post-dose. Citrated blood plasma (0.38%) was obtained immediately after sampling and stored at -20°C until analysis, and the PK profile was established based on the clotting activity of FVIIa using the commercial STACLOT kit.

Fig. 6A. It is shown that FVIIa-CTP ₃ was obtained after selection of FVII, purification process for HA and activation. FVIIa-CTP ₃ or NovoSeven® was administered as a single intravenous injection of FVIII-/- mice with

- 4 040921 hemophilia. Blood samples were taken from the retroorbital sinus at 0.083, 0.5, 2, 8, 24, 48 and 72 hours post-dose. Citrated blood plasma (0.38%) was obtained immediately after sampling and stored at -20°C until analysis. Thrombin formation parameters were evaluated during the PK experiment, and parameters including the ratio of the maximum amount to the time to reach the peak were evaluated.

Fig. 6b. It is shown that FVIIa-CTP ₃ was obtained after selection of FVII, purification process for HA and activation. FVIIa-CTP ₃ or NovoSeven® was administered as a single intravenous injection of FVIII -/- mice with hemophilia. Blood samples were taken from the retroorbital sinus at 0.083, 0.5, 2, 8, 24, 48 and 72 hours post-dose. Citrated blood plasma (0.38%) was obtained immediately after sampling and stored at -20°C until analysis. Thrombin formation parameters were evaluated during the PK experiment, and parameters including the amount of thrombin at each time point were evaluated.

Fig. 6C. It is shown that FVIIa-CTP ₃ was obtained after selection of FVII, purification process for HA and activation. FVIIa-CTP ₃ or NovoSeven® was administered as a single intravenous injection of FVIII -/- mice with hemophilia. Blood samples were taken from the retroorbital sinus at 0.083, 0.5, 2, 8, 24, 48 and 72 hours post-dose. Citrated blood plasma (0.38%) was obtained immediately after sampling and stored at -20°C until analysis. Thrombin generation parameters were evaluated during the PK experiment, and parameters including the thrombin generation rate were evaluated.

Fig. 7A. Survival curves of mice with hemophilia after tail vein dissection (TVT) are shown. TVT was performed 15 min after administration. Mice survival was monitored for 24 h after TVT and recorded every hour for the first 12 h and 24 h later. Data for the control group (inert medium) are total of 3 experiments with 5 mice in each experiment.

Fig. 7B. Survival curves of mice with hemophilia after tail vein dissection (TVT) are shown. TVT was performed 24 hours after administration. Mice survival was monitored for 24 h after TVT and recorded every hour for the first 12 h and 24 h later. Data for the control group (inert medium) are total of 3 experiments with 5 mice in each experiment.

Fig. 7C. Survival curves of mice with hemophilia after tail vein dissection (TVT) are shown. TVT was performed 48 hours after administration. Mice survival was monitored for 24 h after TVT and recorded every hour for the first 12 h and 24 h later. Data for the control group (inert medium) are total of 3 experiments with 5 mice in each experiment.

Fig. 7D. Summarized is the survival of mice recorded 24 hours after TVT.

Fig. 8. Comparison of substrate cleavage activity (Pefachrome FVIIa) between FVIIa (NovoSeven) and CTP-modified coagulation factor VIIa (MOD-5014) is shown.

Fig. 9. Comparison of substrate activity (Pefachrome FVIIa) between FVIIa (NovoSeven) and tissue factor-associated CTP-modified coagulation factor VIIa (MOD-5014) is shown.

Fig. 10. Comparison of the production of activated coagulation factor X by FVIIa (NovoSeven) or CTP-modified FVIIa (MOD-5014) taking into account the concentration of coagulation factor VIIa is shown.

Fig. 11. Comparison of production of activated coagulation factor X by FVIIa (NovoSeven) or CTP-modified FVIIa (MOD-5014) taking into account the concentration of coagulation factor X is shown.

Fig. 12A and B. Comparison of the rate of formation of activated coagulation factor X by FVIIa (NovoSeven) or CTP-modified FVIIa (MOD-5014) in the absence of tissue factor and lipid concentration is shown (Fig. 12A). Comparison of activated coagulation factor X production by FVIIa (NovoSeven) or CTP-modified FVIIa (MOD-5014) in the absence of tissue factor and lipid concentration is shown (Fig. 12B).

Fig. 13. Comparison of activated coagulation factor X production between FVIIa (NovoSeven) and MOD-5014 in the absence of tissue factor and factor X concentration is shown.

Fig. 14. Comparison of inhibition of substrate cleavage (Pefachrome FVIIa) by FVIIa (NovoSeven) and CTP-modified FVIIa (MOD-5014) is shown, taking into account the influence of polybrene.

Fig. 15A-C. The inhibition of substrate cleavage (Pefachrome FXa) by FVIIa (NovoSeven) and CTP-modified FVIIa (MOD-5014) was compared with respect to TFPI concentration (Fig. 15A) and duration of TFPI exposure for FVIIa (Fig. 15B) and MOD- 5014 (FIG. 15C).

Fig. 16. Shown is a flow chart of the production process for the accumulation of biomass containing CTP-modified FVII-CTP3.

Fig. 17. A flowchart of the purification process for CTP-modified FVII-CTP3 is shown.

Fig. 18. Reducing SDS-PAGE results for purified CTP-modified FVII-CTP3 are shown.

Fig. 19. It was shown that the percentage of charged N-glycans in total N-glycans was constant during the purification process, with the initial percentage of charged N-glycans being determined by the accumulation of cell culture biomass.

Fig. 20. The content of oxidized forms and other related forms is presented, which decreases throughout the purification process. The purification steps on multimodal array chromatography and HIC columns have the most significant effect on the reduction of oxidized species and related species.

Fig. 21. Removal of non-gamma carboxylated proteins on a multimodal array chromatography column is shown. The CHT column enriches the fraction of gamma-carboxylated proteins by removing proteins that are not gamma-carboxylated.

Fig. 22. The content of sialic acid throughout the purification process is presented. The content of sialic acid was constant during the purification process, while the initial content of sialic acid is determined by the accumulation of cell culture biomass.

Detailed description

In one embodiment, a method is disclosed for preparing a human chorionic gonadotropin carboxy-terminal peptide (CTP) modified carboxy-terminal peptide (CTP), wherein said FVII contains three CTP molecules attached sequentially one after the other to its C-terminus, wherein the method includes the steps of stable transfection of a predetermined number of cells with an expression vector containing a coding portion encoding said CTP-modified FVII, wherein said transfected cells express and secrete said CTP-modified FVII; obtaining cell clones overexpressing said CTP-modified FVII; propagating said clones in solution to a predetermined scale; collecting said solution containing said clones; filtering said solution containing said clones to obtain a clarified collected solution; and purifying said clarified pooled solution to obtain a purified protein solution having the desired concentration of CTP-modified FVII; thereby obtaining a CTP-modified FVII, wherein the amino acid sequence of the resulting CTP-modified FVII is given under SEQ ID NO: 7.

In one embodiment, a human human chorionic gonadotropin carboxy-terminal peptide (CTP) modified carboxy-terminal peptide (CTP) is disclosed, comprising three CTP molecules attached sequentially one after the other to its C-terminus, wherein said CTP-modified FVII is obtained by the method comprising the steps of stable transfection of a predetermined number of cells with an expression vector containing a coding portion encoding said CTP-modified FVII, where said transfected cells express and secrete said CTP-modified FVII; obtaining cell clones overexpressing said CTP-modified FVII; propagating said clones in solution to a predetermined scale; collecting said solution containing said clones; filtering said solution containing said clones to obtain a clarified collected solution; and purifying said clarified pooled solution to obtain a purified protein solution having the desired concentration of CTP-modified FVII; wherein said resulting CTP-modified FVII contains the amino acid sequence shown under SEQ ID NO: 7.

In one embodiment, a composition is disclosed comprising human blood coagulation factor VII (FVII) modified with a carboxy-terminal peptide (CTP) of human chorionic gonadotropin, comprising three CTP molecules attached sequentially one after the other to its C-terminus. In another embodiment, the CTP-modified FVII includes activated CTP-modified FVII (CTP-modified FVIIa).

Coagulation factor VII modified carboxy-terminal peptide (CTP) human chorionic gonadotropin polypeptide

Blood coagulation factor VII (FVII) is a 444 amino acid (50 kDa) glycoprotein secreted into the bloodstream by hepatocytes as an inactive proenzyme (zymogen). Upon tissue damage and contact with circulating blood, FVII forms a complex with tissue factor (TF), which is the true receptor protein for FVII and is expressed by various cells localized in the deeper layers of the vessel wall. The formation of this FVII-TF complex leads to the activation of FVII. Activated FVII (FVIIa) triggers the extrinsic coagulation pathway through activation of coagulation factor IX and factor X.

FVII belongs to a group of vitamin K-dependent glycoproteins associated with the coagulation system. FVII is synthesized as a precursor with an N-terminal propeptide followed by the mature amino acid sequence. The propeptide contains a docking site for gamma-carboxylase, which converts glutamic acid residues (Glu) into gamma-carboxylated glutamic acid residues (Gla). Carboxyglutamic acid (Gla) is a non-standard amino acid introduced into proteins via post-translational carboxylation of glutamic acid residues. This modification introduces an affinity for calcium ions, while vitamin K is required to introduce gamma-carboxylation of blood coagulation factors, including the blood coagulation factor FVII. The Gla domain is responsible for binding calcium ions with high affinity, which plays an important role in coagulation . Behind this domain are two epidermal growth factor (EGF)-like domains, a junction region (CR), and a C-terminal serine protease domain. Prior to secretion, the FVII propeptide is cleaved and the signal peptide is removed to form the single chain FVII glycoprotein as a 406 amino acid zymogen. After secretion, the protein can be activated to form a disulfide-stabilized double-stranded FVIIa heterodimer by cleavage at CR. The plasma concentration of FVII is 10 nM, and in healthy individuals approximately 1% circulates in active form.

In one embodiment, provided herein is a method for extending the biological half-life or a method for improving the area under the curve (AUC) of FVII or FVIIa, comprising the step of attaching three CTPs to the carboxyl terminus of FVII or FVIIa, thereby prolonging the biological half-life or improving the AUC of FVII or FVIIa.

In another embodiment, this document discloses a method for reducing the frequency of dosing of a blood coagulation factor VIIa (FVIIa) polypeptide, comprising the step of attaching three carboxy-terminal peptides (CTPs) of human chorionic gonadotropin to the carboxyl terminus of said FVIIa polypeptide, thereby reducing the dosing frequency of said FVIIa polypeptide.

In another embodiment, this document discloses a method for reducing the rate of clearance from a coagulation factor VIIa (FVIIa) polypeptide, comprising the step of attaching three carboxy-terminal peptides (CTP) of chorionic gonadotropin to the carboxyl terminus of said FVIIa polypeptide, thereby reducing the rate of clearance from said FVIIa polypeptide.

In one embodiment, this document discloses a method for producing a polypeptide that is an activated CTP-modified coagulation factor VII (FVIIa), comprising the step of attaching three carboxy-terminal peptides (CTP) of human chorionic gonadotropin to the carboxyl terminus of said FVIIa polypeptide, thereby obtaining a polypeptide that is a CTP -modified FVIIa.

In another embodiment, the blood coagulation factor of the present invention is a protein. In another embodiment, the blood coagulation factor of the present invention is a peptide. In another embodiment, the blood coagulation factor of the present invention is a polypeptide. In another embodiment, the blood clotting factor is an enzyme. In another embodiment, the blood clotting factor is a serine protease. In another embodiment, the blood clotting factor is a glycoprotein. In another embodiment, the blood clotting factor is a vitamin K dependent glycoprotein. In another embodiment, the blood clotting factor is a vitamin-K-independent glycoprotein. In another embodiment, the blood clotting factor is a transglutaminase. In another embodiment, the blood clotting factor is an inactive zymogen. In another embodiment, the blood clotting factor is any blood clotting factor known to the person skilled in the art. In another embodiment, the blood clotting factor is blood clotting factor VIIa (FVIIa).

In another embodiment, the blood clotting factor is a recombinant protein. In another embodiment, the blood clotting factor is a recombinant glycoprotein. In another embodiment, the blood clotting factor is a recombinant FVII. In another embodiment, the blood clotting factor is a recombinant FVIIa. In another embodiment, the blood clotting factor contains a signal peptide. In another embodiment, the recombinant blood coagulation factor does not contain a signal peptide. In another embodiment, the activated coagulation factor does not contain a signal peptide.

In another embodiment, the coagulation factor contains 3 CTPs attached to the C-terminus and no CTPs attached to the N-terminus.

In another embodiment, disclosed herein is a CTP-modified coagulation factor VIIa (FVIIa) polypeptide consisting of a FVIIa polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) fused to the carboxyl terminus of said FVIIa.

In another embodiment, the blood clotting factor is a blood clotting factor having a domain organization similar to or identical to that of FVII. In another embodiment, the blood coagulation factor is synthesized as a precursor with an N-terminal propeptide (signal sequence). In another embodiment, the coagulation factor described herein is in the form of an inactive proenzyme. In another embodiment, the coagulation factor described herein is an inactive zymogen that has been secreted by the cell and lacks an N-terminal signal.

- 7 040921 sequence. In another embodiment, the blood clotting factor described herein is an activated blood clotting factor. In another embodiment, the CTP-modified FVII described herein is in the form of an inactive proenzyme until activated. In another embodiment, the CTP-modified FVII described herein is an inactive zymogen that has been secreted by the cell and lacks an N-terminal signal sequence. In another embodiment, the CTP-modified FVII described herein is an activated coagulation factor. In another embodiment, the blood clotting factor is produced by hepatocytes. In another embodiment, the blood coagulation factor contains a docking site for a gamma carboxylase that converts glutamic acid (Glu) residues to gamma-carboxylated glutamic acid (Gla) residues. In another embodiment, the blood clotting factor used herein is a commercially available blood clotting factor.

In one embodiment, this document discloses a polypeptide that is an active human blood coagulation factor VII (FVIIa), a modified carboxy-terminal peptide (CTP) of human chorionic gonadotropin, containing three CTP molecules attached sequentially one after the other to the C-terminus of FVIIa, where the specified polypeptide , which is a CTP-modified FVIIa, is in an essentially pure and active form, while the specified polypeptide, which is a CTP-modified FVIIa, provides: (a) a high content of sialic acid; (b) low content of oxidized form; (c) a highly glycosylated form; (d) a high percentage of carboxylated glutamic acid residues; (e) a high percentage of charged N-glycans; and (f) a high specific activity, or any combination thereof, wherein said CTP-modified FVIIa comprises the amino acid sequence shown under SEQ ID NO: 7.

In one embodiment, this document discloses a polypeptide that is an active human blood coagulation factor VII (FVIIa), a modified carboxy-terminal peptide (CTP) of human chorionic gonadotropin, containing three CTP molecules attached sequentially one after the other to the C-terminus of FVIIa, where the specified polypeptide , which is a CTP-modified FVIIa, is in an essentially pure and active form, while the specified polypeptide, which is a CTP-modified FVIIa, provides: (a) a high content of sialic acid; (b) low content of oxidized form; (c) a highly glycosylated form; (d) a high percentage of carboxylated glutamic acid residues; (e) a high percentage of charged N-glycans and (f) a specific activity of at least 10 U/mg; or any combination thereof, wherein said CTP-modified FVIIa contains the amino acid sequence shown under SEQ ID NO: 7.

In a related embodiment, the high content of sialic acid is at least 15 mol/mol. In another related aspect, the highly glycosylated form provides for an O-glycan content of at least 10 mol/mol. In another related aspect, the substantially pure and active form contains at least 60% of the highly glycosylated form of said active CTP-modified FVIIa. In another related aspect, at least 60% of the substantially pure and CTP-modified form of FVIIa contains a high percentage of carboxylated glutamic acid (Gla) residues. In another related aspect, a high percentage of carboxylated glutamic acid (Gla) residues is at least 90% of Gla residues. In another related aspect, the low percentage of oxidized form is less than 5%. In another related aspect, said substantially pure and active polypeptide, which is a CTP-modified FVII, is at least 90% pure. In a further related aspect, the purity percentage is selected from the group consisting of 97.3%, 97.6%, 97.4%, and 97.0%. In another related aspect, the specific activity of said substantially pure and active CTP-modified FVII polypeptide is at least 10,500 U/mg. In a further related aspect, the specific activity is selected from the group consisting of 15563, 16720, 22478 and 23608 U/mg.

In another embodiment, this document discloses a composition containing a CTP-modified FVIIa, which provides: (a) a high content of sialic acid; (b) low content of oxidized form; (c) a highly glycosylated form; (d) a high percentage of carboxylated glutamic acid residues; (e) a high percentage of charged N-glycans; and (f) a high specific activity, or any combination thereof, wherein said CTP-modified FVIIa comprises the amino acid sequence shown under SEQ ID NO: 7 and a pharmaceutically acceptable carrier.

In another embodiment, this document discloses a composition containing a CTP-modified FVIIa, which provides: (a) a high content of sialic acid; (b) low content of oxidized form; (c) a highly glycosylated form; (d) a high percentage of carboxylated glutamic acid residues; (e) a high percentage of charged N-glycans and (f) a specific activity of at least 10,500 units/mg;

or any combination thereof, wherein said CTP-modified FVIIa contains the amino acid sequence shown under SEQ ID NO: 7 and a pharmaceutically acceptable carrier.

In another embodiment, this document discloses a composition comprising a CTP-modified FVIIa, wherein said CTP-modified FVIIa polypeptide is in substantially pure and active form, wherein said CTP-modified FVIIa polypeptide provides

a) high content of sialic acid;

b) a highly glycosylated form; where said CTP-modified FVIIa further provides at least one of the following:

c) low content of oxidized form;

d) a high percentage of carboxylated glutamic acid residues;

e) at least 60% charged N-glycans, or

f) a specific activity of at least 10500 units/mg;

or any combination thereof; and wherein said CTP-modified FVIIa contains the amino acid sequence shown under SEQ ID NO: 7.

In another embodiment, this document discloses a composition comprising a CTP-modified FVIIa, wherein said CTP-modified FVIIa polypeptide is in substantially pure and active form, wherein said CTP-modified FVIIa polypeptide provides

a) high content of sialic acid;

b) a highly glycosylated form; where said CTP-modified FVIIa further provides at least one of the following:

c) low content of oxidized form;

d) a high percentage of carboxylated glutamic acid residues;

e) at least 60% charged N-glycans, or

f) a specific activity of at least 10500 units/mg;

or any combination thereof; and wherein said CTP-modified FVIIa comprises the amino acid sequence shown under SEQ ID NO: 7, wherein the amino acid sequence of said CTP-modified FVIIa is structurally a disulfide-stabilized double-stranded heterodimer containing a disulfide (S-S) bridge between the cysteine residue 135 and the cysteine residue 262 in SEQ ID NO: 7, and wherein said two chains include a light chain containing amino acids 1-152 and a heavy chain containing amino acids 153-490 of SEQ ID NO: 7.

In one embodiment, the nucleic acid sequence encoding blood coagulation factor VII includes the following nucleic acid sequence:

ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttgggcttcagggctgcc tggctgcagtcttcgtaacccaggaggaagcccacggcgtcctgcaccggcgccggcgcgccaa cgcgttcctggaggagctgcggccgggctccctggagagggagtgcaaggaggagcagtgctcc ttcgaggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctggatttcttaca

- 9 040921 gtgatggggaccagtgtgcctcaagtccatgccagaatgggggctcctgcaaggaccagctcca gtcctatatctgcttctgcctccctgccttcgagggccggaactgtgagacgcacaaggatgac cagctgatctgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgggcacca agcgctcctgtcggtgccacgaggggtactctctgctggcagacggggtgtcctgcacacccac agttgaatatccatgtggaaaaatacctattctagaaaaaagaaatgccagcaaaccccaaggc cgaattgtggggggcaaggtgtgccccaaaggggagtgtccatggcaggtcctgttgttggtga atggagctcagttgtgtggggggaccctgatcaacaccatctgggtggtctccgcggcccactg tttcgacaaaatcaagaactggaggaacctgatcgcggtgctgggcgagcacgacctcagcgag cacgacggggatgagcagagccggcgggtggcgcaggtcatcatccccagcacgtacgtcccgg gcaccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtcctcactgaccatgt ggtgcccctctgcctgcccgaacggacgttctctgagaggacgctggccttcgtgcgcttctca ttggtcagcggctggggccagctgctggaccgtggcgccacggccctggagctcatggtcctca acgtgccccggctgatgacccaggactgcctgcagcagtcacggaaggtgggagactccccaaa tatcacggagtacatgttctgtgccggctactcggatggcagcaaggactcctgcaagggggac agtggaggcccacatgccacccactaccggggcacgtggtacctgacgggcatcgtcagctggg gccagggctgcgca accgtgggccactttggggtgtacaccagggtctcccagtacatcgagtg gctgcaaaagctcatgcgctcagagccacgcccaggagtcctcctgcgagccccatttccctga ggatgcggccgc (SEQ ID NO: 1).

In another embodiment, the amino acid sequence of blood coagulation factor VII includes the following amino acid sequence:

MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEE AREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLI CVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIV GGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWWSAAHCFDKIKNWRNLIAVLGEHDLSEHDG DEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPWLTDHVVPLCLPERTFSERTLAFVRFSLVS GWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGG PHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP (SEQ ID NO: 2).

In another embodiment, the amino acid sequence of blood coagulation factor VII includes the following amino acid sequence:

MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEE AREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLI CVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIV GGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDG DEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPWLTDHWPLCLPERTFSERTLAFVRFSLVS GWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGG PHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPGCGR (SEQ ID NO: 3).

In another embodiment, the nucleic acid sequence encoding the coagulation factor VII-CTP-CTP-CTP polypeptide (attached to the carboxyl terminus) comprises the following nucleic acid sequence:

- 10 040921 ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttgggcttcagggctgcc tggctgcagtcttcgtaacccaggaggaagcccacggcgtcctgcaccggcgccggcgcgccaa cgcgttcctggaggagctgcggccgggctccctggagagggagtgcaaggaggagcagtgctcc ttcgaggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctggatttcttaca gtgatggggaccagtgtgcctcaagtccatgccagaatgggggctcctgcaaggaccagctcca gtcctatatctgcttctgcctccctgccttcgagggccggaactgtgagacgcacaaggatgac cagctgatctgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgggcacca agcgctcctgtcggtgccacgaggggtactctctgctggcagacggggtgtcctgcacacccac agttgaatatccatgtggaaaaatacctattctagaaaaaagaaatgccagcaaaccccaaggc cgaattgtggggggcaaggtgtgccccaaaggggagtgtccatggcaggtcctgttgttggtga atggagctcagttgtgtggggggaccctgatcaacaccatctgggtggtctccgcggcccactg tttcgacaaaatcaagaactggaggaacctgatcgcggtgctgggcgagcacgacctcagcgag cacgacggggatgagcagagccggcgggtggcgcaggtcatcatccccagcacgtacgtcccgg gcaccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtcctcactgaccatgt ggtgcccctctgcctgcccgaacggacgttctctgagaggacgctggccttcgtgcgcttctca ttggtcagcggct ggggccagctgctggaccgtggcgccacggccctggagctcatggtcctca acgtgccccggctgatgacccaggactgcctgcagcagtcacggaaggtgggagactccccaaa tatcacggagtacatgttctgtgccggctactcggatggcagcaaggactcctgcaagggggac agtggaggcccacatgccacccactaccggggcacgtggtacctgaccggcatcgtgagctggg gccagggctgcgccaccgtgggccacttcggcgtgtacaccagggtgtcccagtacatcgagtg gctgcagaaactgatgagaagcgagcccagacccggcgtgctgctgagagcccccttccccage agcagctccaaggcccctccccctagcctgcccagccctagcagactgcctgggcccagtgaca cccctatcctgcctcagtccagctccagcaaggccccaccccctagcctgccttctccttctcg gctgcctggccccagcgatactccaattctgccccagtcctccagcagtaaggctccccctcca tctctgccatcccccagcagactgccaggcccttctgatacacccatcctcccacagtgatgag gatccgcggccgcttaattaa (SEQ ID NO: 4).

In another embodiment, the amino acid sequence of the coagulation factor VII-CTP-CTP-CTP polypeptide (attached to the carboxyl terminus) includes the following amino acid sequence:

MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEE AREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLI CVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIV GGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDG DEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVS GWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGG PHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSS KAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLP SPSRLPGPSDTPILPQ (SEQ ID NO: 5) .

In another embodiment, amino acids 1-38 of SEQ ID NO: 5 form a signal sequence. In another embodiment, the signal sequence has the amino acid sequence

MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRR (SEQ ID NO: 6).

In another embodiment, the amino acid sequence of a coagulation factor VII-CTP-CTP-CTP (carboxyl-terminated) polypeptide having no signal peptide comprises the following amino acid sequence:

- 11 040921

ANAFLEELRP GSLERECKEE QCSFEEAREI FKDAERTKLF

WISYSDGDQC ASSPCQNGGS CKDQLQSYIC FCLPAFEGRN CETHKDDQLI CVNENGGCEQ YCSDHTGTKR SCRCHEGYSL LADGVSCTPT VEYPCGKIPI LEKRNASKPQ GRIVGGKVCP KGECPWQVLL LVNGAQLCGG TLINTIWWS AAHCFDKIKN WRNLIAVLGE HDLSEHDGDE QSRRVAQVII PSTYVPGTTN HDIALRLHQ PWLTDHWP LCLPERTFSE RTLAFVRFSL VSGWGQLLDR GATALELMVL NVPRLMTQDC LQQSRKVGDS PNITEYMFCA GYSDGSKDSC KGDSGGPHAT HYRGTWYLTG IVSWGQGCAT VGHFGVYTRV SQYIEWLQKL MRSEPRPGVL LRAPFPSSSS KAPPPSLPSP SRLPGPSDTP ILPQSSSSKA

PPPSLPSPSR LPGPSDTPIL PQSSSSKAPP PSLPSPSRLP GPSDTPILPQ (SEQ ID

NO: 7).

In another embodiment, the amino acid sequence of the activated coagulation factor VII-CTP-CTP-CTP (carboxyl-terminated) polypeptide (FVIIaCTP3) does not have a signal peptide and includes the amino acid sequence shown under SEQ ID NO: 7. In another embodiment, FVIIa-CTP3 lacks a signal peptide and contains a homologue of SEQ ID NO: 7. In another embodiment, FVIIa-CTP3 does not have a signal peptide and contains a variant of SEQ ID NO: 7. In another embodiment, the amino acid sequence of FVIIa-CTP3 is cleaved between an arginine residue (R) at position 152 and an isoleucine residue (I) at position 153. In another embodiment, the amino acid sequence of FVIIa-CTP ₃ is structurally represented as a disulfide-stabilized double-stranded heterodimer containing an SS disulfide bridge between the cysteine residues present in each from chains. In another embodiment, the amino acid sequence of FVIIa-CTP ₃ is structurally represented as a heterodimer comprising a light chain and a heavy chain connected by a -SS- disulfide bond between a cysteine residue present in the light chain and a cysteine residue present in the heavy chain. In another embodiment, the light chain contains the N-terminal fragment of the FVIIa-CTP3 amino acid sequence and the heavy chain contains the C-terminal fragment of the FVIIa-CTP3 amino acid sequence. In another embodiment, the cysteine residues may be any cysteine residues in any chain. In another embodiment, the amino acid sequence of FVIIa-CTP ₃ is structurally represented as a disulfide-stabilized double-stranded heterodimer containing an SS bridge between cysteine residue 135 and cysteine residue 262 in SEQ ID NO: 7, wherein said two chains include a light chain , containing amino acids 1-152, and a heavy chain containing amino acids 153-490 of SEQ ID NO: 7.

In another embodiment, the light chain migrates at about 25 kDa into the SDSPAGE under denaturing conditions. In another embodiment, the heavy chain migrates at about 50 kDa into SDS-PAGE under denaturing conditions. In another embodiment, the heavy chain migrates at about 60 kDa in SDS-PAGE under denaturing conditions.

In another embodiment, the light chain of activated FVII modified by adding 3 CTP to its C-terminus (FVIIa-CTP-CTP-CTP) contains SEQ ID NO: 8:

ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQ

LQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCT

PTVEYPCGKIPILEKRNASKPQGR (SEQ ID NO: 8).

In another embodiment, the heavy chain of activated FVII modified by adding 3 CTP to its C-terminus (FVIIa-CTP-CTP-CTP) contains SEQ ID NO: 9:

IVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEH

DGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPWLTDHWPLCLPERTFSERTLAFVRFSL VSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDS GGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSS SSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPS LPSPSRLPGPSDTPILPQ (SEQ ID NO: 9).

In another embodiment, furin is added to a cell expressing a coagulation factor-CTP polypeptide of the present invention. In another embodiment, furin increases the production efficiency of the coagulation factor-CTP polypeptide of the present invention in a cell. In another embodiment, furin is administered by co-transfection

- 12 040921 with a vector containing a sequence encoding a coagulation factor-CTP polypeptide according to the present invention. In another embodiment, furin is encoded by a separate vector. In another embodiment, the furin and the coagulation factor-CTP polypeptide are encoded by the same vector. In another embodiment, a furin coding sequence is inserted into pCI-DHFR. In another embodiment, the furin coding sequence is inserted into pCI-dhfr/SmaI+NotI, Furin/AsisI F.I.+NotI.

In another embodiment, the nucleic acid sequence encoding furin includes the following nucleic acid sequence:

tctagagtcgaccccgccatggagctgaggccctggttgctatgggtggtagcagcaacaggaa ccttggtcctgctagcagctgatgctcagggccagaaggtcttcaccaacacgtgggctgtgcg catccctggaggcccagcggtggccaacagtgtggcacggaagcatgggttcctcaacctgggc cagatcttcggggactattaccacttctggcatcgaggagtgacgaagcggtccctgtcgcctc accgcccgcggcacagccggctgcagagggagcctcaagtacagtggctggaacagcaggtggc aaagcgacggactaaacgggacgtgtaccaggagcccacagaccccaagtttcctcagcagtgg tacctgtctggtgtcactcagcgggacctgaatgtgaaggcggcctgggcgcagggctacacag ggcacggcattgtggtctccattctggacgatggcatcgagaagaaccacccggacttggcagg caattatgatcctggggccagttttgatgtcaatgaccaggaccctgacccccagcctcggtac acacagatgaatgacaacaggcacggcacacggtgtgcgggggaagtggctgcggtggccaaca acggtgtctgtggtgtaggtgtggcctacaacgcccgcattggaggggtgcgcatgctggatgg cgaggtgacagatgcagtggaggcacgctcgctgggcctgaaccccaaccacatccacatctac agtgccagctggggccccgaggatgacggcaagacagtggatgggccagcccgcctcgccgagg aggccttcttccgtggggttagccagggccgaggggggctgggctccatctttgtctgggcctc ggggaacgggggccgggaacatgacagctgcaactgcgacggctacaccaacagtatctacacg ctgtccatcagcagcgccacgcagt ttggcaacgtgccgtggtacagcgaggcctgctcgtcca cactggccacgacctacagcagtggcaaccagaatgagaagcagatcgtgacgactgacttgcg gcagaagtgcacggagtctcacacgggcacctcagcctctgcccccttagcagccggcatcatt gctctcaccctggaggccaataagaacctcacatggcgggacatgcaacacctggtggtacaga cctcgaagccagcccacctcaatgccaacgactgggccaccaatggtgtgggccggaaagtgag ccactcatatggctacgggcttttggacgcaggcgccatggtggccctggcccagaattggacc acagtggccccccagcggaagtgcatcatcgacatcctcaccgagcccaaagacatcgggaaac ggctcgaggtgcggaagaccgtgaccgcgtgcctgggcgagcccaaccacatcactcggctgga gcacgctcaggcgcggctcaccctgtcctataatcgccgtggcgacctggccatccacctggtc agccccatgggcacccgctccaccctgctggcagccaggccacatgactactccgcagatgggt ttaatgactgggccttcatgacaactcattcctgggatgaggatccctctggcgagtgggtcct agagattgaaaacaccagcgaagccaacaactatgggacgctgaccaagttcaccctcgtactc tatggcaccgcccctgaggggctgcccgtacctccagaaagcagtggctgcaagaccctcacgt ccagtcaggcctgtgtggtgtgcgaggaaggcttctccctgcaccagaagagctgtgtccagca ctgccctccaggcttcgccccccaagtcctcgatacgcactatagcaccgagaatgacgtggag accatccgggccagcgtctgcgccccctgccacgcctcatgtgccacatg ccaggggccggccc tgacagactgcctcagctgccccagccacgcctccttggaccctgtggagcagacttgctcccg gcaaagccagagcagccgagagtccccgccacagcagcagccacctcggctgcccccggaggtg gaggcggggcaacggctgcgggcagggctgctgccctcacacctgcctgaggtggtggccggcc tcagctgcgccttcatcgtgctggtcttcgtcactgtcttcctggtcctgcagctgcgctctgg

- 13 040921 ctttagttttcggggggtgaaggtgtaaccatggaccgtggcctcatctcctacaaggggctg ccccctgaagcctggcaggagaggagtgcccgtctgactcagaagaggacgagggccggggcgaga ggaccgcctttatcaaagaccagagcgccctctgaacgcggccgc (SEQ ID NO: 10).

In another embodiment, the amino acid sequence of furin includes the following amino acid sequence:

MELRPWLLWVVAATGTLVLLAADAQGQKVFTNTWAVRIPGGPAVANSVARKHGFLNLGQIFGDY

YHFWHRGVTKRSLSPHRPRHSRLQREPQVQWLEQQVAKRRTKRDVYQEPTDPKFPQQWYLSGVT QRDLNVKAAWAQGYTGHGIVVSILDDGIEKNHPDLAGNYDPGASFDVNDQDPDPQPRYTQMNDN RHGTRCAGEVAAVANNGVCGVGVAYNARIGGVRMLDGEVTDAVEARSLGLNPNHIHIYSASWGP EDDGKTVDGPARLAEEAFFRGVSQGRGGLGSIFVWASGNGGREHDSCNCDGYTNSIYTLSISSA TQFGNVPWYSEACSSTLATTYSSGNQNEKQIVTTDLRQKCTESHTGTSASAPLAAGIIALTLEA NKNLTWRDMQHLWQTSKPAHLNANDWATNGVGRKVSHSYGYGLLDAGAMVALAQNWTTVAPQR KCIIDILTEPKDIGKRLEVRKTVTACLGEPNHITRLEHAQARLTLSYNRRGDLAIHLVSPMGTR STLLAARPHDYSADGFNDWAFMTTHSWDEDPSGEWVLEIENTSEANNYGTLTKFTLVLYGTAPE GLPVPPESSGCKTLTSSQACWCEEGFSLHQKSCVQHCPPGFAPQVLDTHYSTENDVETIRASV CAPCHASCATCQGPALTDCLSCPSHASLDPVEQTCSRQSQSSRESPPQQQPPRLPPEVEAGQRL RAGLLPSHLPEVVAGLSCAFIVLVFVTVFLVLQLRSGFSFRGVKVYTMDRGLISYKGLPPEAWQ EECPSDSEEDEGRGERTAFIKDQSAL (SEQ ID NO: 11).

In one embodiment, the term blood clotting factor further includes a homologue of a known blood clotting factor. In one embodiment, the homologue has coagulative activity. In some embodiments, the implementation of the homology in accordance with the present invention also covers the corresponding options with divisions, insertions or substitutions, including amino acid substitutions, and the corresponding biologically active fragments of the polypeptides. In one embodiment, the variant contains conservative substitutions or deletions, insertions or substitutions that do not significantly alter the three-dimensional structure of the coagulation factor. In another embodiment, the deletion, insertion, or substitution does not alter the function of the coagulation factor of interest, which in one embodiment is binding to a particular binding partner.

In another embodiment, the present invention encompasses a coagulation factor homologue. In another embodiment, the present invention encompasses a blood clotting factor homologue having coagulative activity. In another embodiment, the present invention encompasses a coagulation factor homologue characterized by functional binding. In another embodiment, the present invention encompasses coagulation factor homologues as described herein. In another embodiment, the present invention encompasses homologues of the blood coagulation factor described herein, characterized by functional binding. In another embodiment, the homologues, e.g., polypeptides, are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% homologous to a coagulation factor, as determined by the National Center for Biotechnology Information (NCBI) BlastP software using default parameters.

In another embodiment, the ((CTP)n>1 coagulation factor) described herein comprises a full-length coagulation factor or active fragment thereof linked at its carboxyl terminus by a peptide bond to at least one CTP unit and containing no CTP. at its amino end. In another embodiment, the ((CTP)n>1 coagulation factor) described herein comprises a coagulation factor or active fragment thereof linked by a peptide bond to at least one CTP unit, which is linked by a peptide bond to an additional CTP unit , and does not contain CTP at its amino terminus. In another embodiment, one nucleic acid molecule encodes an engineered coagulation factor containing at least one CTP attached to its C-terminus and not containing a CTP at its amino terminus.

In another embodiment, the CTP is attached to a coagulation factor via a linker. In another embodiment, a linker connecting the CTP sequence to the factor

- 14 040921 blood coagulation, is a covalent bond. In another embodiment, the linker connecting the CTP sequence to a blood coagulation factor is a peptide bond. In another embodiment, the linker connecting the CTP sequence to a blood coagulation factor is a substituted peptide bond. In another embodiment, the CTP sequence contains

DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID NO: 12).

In another embodiment, the CTP sequence contains

SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 13).

In another embodiment, the CTP sequence includes an amino acid sequence selected from the sequences listed under SEQ ID NO: 12 and SEQ ID NO: 13.

In another embodiment, the carboxy-terminal peptide (CTP) disclosed herein comprises the amino acid sequence of amino acids at positions 112-145 of human chorionic gonadotropin as set forth under SEQ ID NO: 12. In another embodiment, the CTP sequence disclosed herein comprises the amino acid sequence of amino acids at positions 118-145 of human chorionic gonadotropin as set forth under SEQ ID NO: 13. In another embodiment, the CTP sequence also starts at any position between positions 112 and 118 and ends at position 145 of human chorionic gonadotropin. In some embodiments, the CTP peptide sequence is 28, 29, 30, 31, 32, 33, or 34 amino acids long and starts at amino acid position 112, 113, 114, 115, 116, 117, or 118 of the CTP sequence.

In another embodiment, the CTP peptide is a human chorionic gonadotropin CTP variant that differs from native CTP 1-5 by conservative amino acid substitutions as described in US Pat. No. 5,712,122, which is incorporated herein by reference. In another embodiment, the CTP peptide is a variant of human chorionic gonadotropin CTP that differs from native CTP 1 by a conservative amino acid substitution. In another embodiment, the CTP peptide is a human chorionic gonadotropin CTP variant that differs from native CTP 2 by conservative amino acid substitutions. In another embodiment, the CTP peptide is a variant of human chorionic gonadotropin CTP that differs from native CTP 3 by conservative amino acid substitutions. In another embodiment, the CTP peptide is a human chorionic gonadotropin CTP variant that differs from native CTP 4 by conservative amino acid substitutions. In another embodiment, the CTP peptide is a human chorionic gonadotropin CTP variant that differs from native CTP 5 by conservative amino acid substitutions.

In another embodiment, the amino acid sequence of the CTP peptide disclosed herein is at least 70% homologous to the amino acid sequence of native CTP or the corresponding peptide. In another embodiment, the amino acid sequence of the CTP peptide disclosed herein is at least 80% homologous to the amino acid sequence of native CTP or the corresponding peptide. In another embodiment, the amino acid sequence of the CTP peptide disclosed herein is at least 90% homologous to the amino acid sequence of native CTP or the corresponding peptide. In another embodiment, the amino acid sequence of the CTP peptide disclosed herein is at least 95% homologous to the amino acid sequence of native CTP or the corresponding peptide. In another embodiment, the amino acid sequence of the CTP peptide disclosed herein is at least 98% homologous to the amino acid sequence of native CTP or the corresponding peptide.

In another embodiment, the polynucleotide encoding the CTP peptide disclosed herein is at least 70% homologous to the DNA sequence encoding native human CTP or the corresponding peptide. In another embodiment, a polynucleotide encoding a CTP peptide disclosed herein is at least 80% homologous to a DNA sequence encoding native human CTP or the corresponding peptide. In another embodiment, the polynucleotide encoding the CTP peptide disclosed herein is at least 90% homologous to the DNA sequence encoding the native CTP or the corresponding peptide. In another embodiment, the polynucleotide encoding the CTP peptide disclosed herein is at least 95% homologous to the DNA sequence encoding the native CTP or the corresponding peptide. In another embodiment, a polynucleotide encoding a CTP peptide disclosed herein is at least 98% homologous to a DNA sequence encoding a native CTP or corresponding peptide.

In one embodiment, at least one of the human chorionic gonadotropin CTP amino acid sequences is truncated. In another embodiment, both human chorionic gonadotropin CTP amino acid sequences are truncated. In a different

- 15 040921 embodiment 2 of the human chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, 3 of the human chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, 4 of the human chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, 5 of the human chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, 2 or more of the human chorionic gonadotropin CTP amino acid sequences are truncated. In another embodiment, all of the human chorionic gonadotropin CTP amino acid sequences are truncated. In one embodiment, the truncated CTP contains the first 10 amino acids of SEQ ID NO: 14. In another embodiment, SEQ ID NO: 14 contains the following amino acid (AA) sequence: SSSSKAPPPSLP. In another embodiment, the first 10 amino acids of SEQ ID NO: 14 are listed under SEQ ID NO: 15: SSSSKAPPPS.

In one embodiment, the truncated CTP contains the first 10 amino acids of SEQ ID NO: 13.

In one embodiment, the truncated CTP contains the first 11 amino acids of SEQ ID NO: 13. In one embodiment, the truncated CTP contains the first 12 amino acids of SEQ ID NO: 13. In one embodiment, the truncated CTP contains the first 8 amino acids of SEQ ID NO: 13 or SEQ ID NO: 14. In one embodiment, the truncated CTP contains the first 13 amino acids of SEQ ID NO: 13. In one embodiment, the truncated CTP contains the first 14 amino acids of SEQ ID NO: 13. In one embodiment, the truncated CTP contains the first 6 amino acids of SEQ ID NO: 13 or SEQ ID NO: 14. In one embodiment, the truncated CTP contains the first 5 amino acids of SEQ ID NO: 13 or SEQ ID NO: 14.

In one embodiment, at least one of the human chorionic gonadotropin CTP amino acid sequences is glycosylated. In another embodiment, 2 of the human chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, 3 of the human chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, 4 of the human chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, 5 of the human chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, 2 or more of the human chorionic gonadotropin CTP amino acid sequences are glycosylated. In another embodiment, all of the human chorionic gonadotropin CTP amino acid sequences are glycosylated.

In one embodiment, the CTP sequence disclosed herein contains at least one glycosylation site. In one embodiment, the CTP sequence disclosed herein contains 2 glycosylation sites. In one embodiment, the CTP sequence disclosed herein contains 3 glycosylation sites. In one embodiment, the CTP sequence disclosed herein contains 4 glycosylation sites. In one embodiment, one or more of the human chorionic gonadotropin CTP amino acid sequences are fully glycosylated. In another embodiment, one or more of the human chorionic gonadotropin CTP amino acid sequences are partially glycosylated. In one embodiment, "partially glycosylated" means that glycosylation occurs at one of the CTP glycosylation sites. In another embodiment, glycosylation occurs at two of the CTP glycosylation sites. In another embodiment, glycosylation occurs at three of the CTP glycosylation sites.

In some embodiments, the modification of the CTP sequence is advantageous in that it allows lower doses to be used. In some embodiments, modification of the CTP sequences is advantageous in that fewer doses can be used. In some embodiments, modification of the CTP sequences is advantageous in that it allows a safe, sustained effect to be achieved.

In some embodiments, a polypeptide, engineered coagulation factor, or protein as used herein includes native polypeptides (degradants, synthetically synthesized polypeptides, or recombinant polypeptides) and peptidomimetics (typically synthetically synthesized polypeptides) as well as peptoids. and semi-peptoids, which are analogs of polypeptides having, in some embodiments, modifications that make the coagulation factor-containing polypeptides even more stable in the body or more capable of penetrating cells.

In some embodiments, modifications include, but are not limited to, C-terminal modification, polypeptide bond modification, including but not limited to CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S= C-NH, CH=CH or CF=CH, backbone modifications and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are defined, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press

- 16 040921 (1992), incorporated by reference as if it were set forth in its entirety in this document. Additional details in this regard are provided later in this document.

In some embodiments, the polypeptide bonds (-CO-NH-) in the polypeptide are substituted. In some embodiments, polypeptide bonds are substituted with N-methylated bonds (-N(CH3)-CO-). In some embodiments, the polypeptide bonds are substituted with ester bonds (-C(R)H-C-O-O-C(R)-N-). In some embodiments, polypeptide bonds are substituted with ketomethylene bonds (-CO-CH2-). In some embodiments, the polypeptide bonds are substituted with -aza bonds (-NH-N(R)-CO-), where R is any alkyl, eg methyl, carbas bonds (-CH2-NH-). In some embodiments, the polypeptide bonds are replaced with hydroxyethylene bonds (-CH(OH)-CH2-). In some embodiments, polypeptide bonds are substituted with thioamide bonds (-CS-NH-). In some embodiments, the polypeptide bonds are substituted with olefinic double bonds (-CH=CH-). In some embodiments, the polypeptide bonds are substituted with retroamide bonds (-NH-CO-). In some embodiments, the polypeptide bonds are substituted with polypeptide derivatives (-N(R)-CH2-CO-), where R is the normal side chain naturally present at carbon. In some embodiments, these modifications take place in any of the bonds along the polypeptide chain, and in one embodiment, in multiple bonds (2-3 bonds) simultaneously.

In some embodiments, the natural aromatic amino acids of the polypeptide, such as Trp, Tyr, and Phe, are replaced with synthetic non-natural acids, such as phenylglycine, TIC, naphthyl-L-alanine (Nol), methylated ring Phe derivatives, halogenated Phe derivatives, or O-methyl -tyr. In some embodiments, the polypeptides disclosed herein contain one or more modified amino acids or one or more non-amino acid monomers (eg, fatty acids, complex carbohydrates, etc.).

In some embodiments, the naturally occurring amino acid glutamic acid (Glu) undergoes post-translational carboxylation such that carboxyglutamic acid (Gla) is present in the CTP-modified FVII or CTP-modified FVIIa described herein.

In one embodiment, an amino acid or amino acid sequence is understood to include 20 naturally occurring amino acids; those amino acids often resulting from in vivo post-translational modifications, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; as well as other unusual amino acids, including, without limitation, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine. In one embodiment, the amino acid includes both D- and L-amino acids.

In some embodiments, the polypeptides disclosed herein are used in therapeutic agents that require the coagulation factor-containing polypeptides to be in soluble form. In some embodiments, the polypeptides disclosed herein contain one or more non-natural or naturally occurring polar amino acids, including, but not limited to, serine and threonine, which are capable of increasing the solubility of the polypeptides due to their hydroxyl-containing side chain.

In some embodiments, the engineered coagulation factor disclosed herein is used in a linear form, although one skilled in the art will appreciate that where cyclization does not significantly interfere with the performance of the engineered coagulation factors, cyclic forms of the engineered coagulation factors can also be used. coagulation factors.

In some embodiments, the engineered coagulation factors disclosed herein are biochemically synthesized, such as by using standard solid phase techniques. In some embodiments, these biochemical methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis.

In some embodiments, recombinant protein techniques are used to produce the engineered coagulation factors disclosed herein. In some embodiments, recombinant protein techniques are used to produce relatively long polypeptides (eg, longer than 18-25 amino acids). In some embodiments, recombinant protein techniques are used to produce large amounts of the engineered coagulation factors disclosed herein. In some embodiments, recombinant techniques are described in Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol. cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463, which are incorporated herein by reference in their entirety.

- 17 040921

In another embodiment, the present invention provides a polynucleotide molecule comprising the coding portion of a gene encoding a polypeptide containing a blood coagulation factor and carboxy-terminal gonadotropin peptides fused to the carboxyl terminus of a blood coagulation factor, as described herein above. In another embodiment, the present invention provides a polynucleotide molecule consisting of the coding portion of a gene encoding a polypeptide containing a blood coagulation factor and carboxy-terminal gonadotropin peptides attached to the carboxyl terminus of a blood coagulation factor, as described herein above. In another embodiment, the present invention provides a polynucleotide molecule consisting essentially of the coding portion of a gene encoding a polypeptide containing a blood coagulation factor and carboxy-terminal gonadotropin peptides attached to the carboxyl terminus of a blood coagulation factor, as described herein above.

In another embodiment, the present invention provides a polynucleotide encoding a polypeptide comprising a blood coagulation factor and three carboxyl-terminal gonadotropin peptides attached to the carboxyl terminus of the blood coagulation factor, as described herein above. In another embodiment, the present invention provides a polynucleotide encoding a polypeptide consisting of a blood coagulation factor and three carboxy-terminal gonadotropin peptides attached to the carboxyl terminus of the blood coagulation factor, as described herein above. In another embodiment, the present invention provides a polynucleotide encoding a polypeptide consisting essentially of a blood coagulation factor and three carboxy-terminal gonadotropin peptides attached to the carboxyl terminus of the blood coagulation factor, as described herein above. In one embodiment, the polynucleotide is a polynucleotide sequence. In one embodiment, the polynucleotide is a polynucleotide molecule.

In another embodiment, the present invention provides an expression vector containing the polynucleotide molecule described herein. In another embodiment, this document discloses an expression vector containing a polynucleotide encoding a CTP-modified polypeptide consisting of a coagulation factor VII polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of said FVII polypeptide. In another embodiment, a CTP-modified FVII expressed from an expression vector described herein can be activated at some point in time after expression, resulting in a CTP-modified FVIIa.

In another embodiment, the present invention provides a cell containing the expression vector described in this document. In another embodiment, this document discloses a cell containing an expression vector containing a polynucleotide encoding a CTP-modified polypeptide consisting of a blood coagulation factor VII (FVII) polypeptide and three carboxyl-terminal peptides (CTP) of gonadotropin attached to the carboxyl terminus of said FVIIa polypeptide. In another embodiment, CTP-FVII expressed from an expression vector described herein and contained in a cell can be activated upon secretion from the cell.

In another embodiment, the present invention provides a composition containing the expression vector described in this document. In another embodiment, this document discloses a composition containing an expression vector containing a polynucleotide encoding a CTP-modified polypeptide consisting of a blood coagulation factor VII (FVII) polypeptide and three carboxyl-terminal peptides (CTP) of gonadotropin attached to the carboxyl terminus of said FVIIa polypeptide.

In another embodiment, the present invention provides a composition containing the cell described in this document. In another embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is a prokaryotic cell.

In one embodiment, this document discloses a method for producing a CTP-modified FVII, comprising the step of attaching three carboxy-terminal peptides (CTP) of human chorionic gonadotropin to the carboxyl terminus of said FVII, thereby obtaining a CTP-modified FVII.

In another embodiment, the engineered coagulation factors disclosed herein are synthesized using a polynucleotide molecule encoding a polypeptide disclosed herein. In some embodiments, a polynucleotide molecule encoding the engineered coagulation factors is ligated into an expression vector containing a cis-acting transcription control regulatory sequence (eg, a promoter sequence). In some embodiments, the cis-acting regulatory sequence is suitable for directing constitutive expression of the engineered coagulation factor disclosed herein. In some embodiments, the cis-acting regulatory sequence is suitable for directing tissue-specific expression of the engineered coagulation factors. In some embodiments, a cis-acting regulatory sequence is suitable for directing inducible expression of the engineered coagulation factors.

In some embodiments, tissue-specific promoters suitable for use in the present invention include sequences that function in one or more specific cell populations. Examples include, without limitation, promoters such as the albumin gene promoter, which is liver specific (Pinkert et al., (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame et al., (1988) Adv. Immunol 43:235-275); in particular promoters of T-cell receptor genes (Winoto et al., (1989) EMBO J. 8:729-733) and immunoglobulins; (Banerji et al. (1983) Cell 33729-740), neuron-specific promoters such as the neurofilament protein gene promoter (Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), promoters pancreas-specific (Edlunch et al. (1985) Science 230:912-916), or breast-specific promoters such as the whey protein gene promoter (US Patent No. 4,873,316 and European Patent Application Publication No. 264,166 ). Suitable inducible promoters for use in the present invention include, for example, the tetracycline-inducible promoter (Srour, M.A., et al., 2003. Thromb. Haemost. 90: 398-405).

In one embodiment, the phrase polynucleotide molecule refers to a single-stranded or double-stranded nucleic acid sequence, isolated and presented in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence, and/or multiple polynucleotide sequences (e.g., combinations of the above).

In one embodiment, the complementary polynucleotide sequence refers to a sequence resulting from the reverse transcription of messenger RNA using reverse transcriptase or any other RNA-dependent DNA polymerase. In one embodiment, the sequence can be subsequently amplified in vivo or in vitro using DNA polymerase.

In one embodiment, the genomic polynucleotide sequence refers to a sequence derived (isolated) from a chromosome, and thus is a contiguous part of the chromosome.

In one embodiment, a composite polynucleotide sequence refers to a sequence that is at least partially complementary and at least partially genomic. In one embodiment, the composite sequence may contain some of the exon sequences required to encode the polypeptide disclosed herein, as well as some of the intron sequences located between them. In one embodiment, the intron sequences may be derived from any source, including other genes, and will typically contain conserved splicing signal sequences. In one embodiment, the intron sequences contain cis-acting expression control elements.

In one embodiment, after expression and before secretion, the signal peptides are cleaved from engineered coagulation factor precursors, resulting in mature engineered coagulation factors lacking the signal peptide. In another embodiment, after secretion, said mature engineered coagulation factor is activated.

In some embodiments, the polynucleotides disclosed herein are obtained using PCR techniques or any other method or procedure known to a person skilled in the art. In some embodiments, the procedure involves the ligation of two different DNA sequences (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons, 1992).

In one embodiment, the polynucleotides disclosed herein that encode engineered clotting factors are inserted into expression vectors (ie, a nucleic acid construct) to allow expression of the recombinant polypeptide. In one embodiment, the expression vector disclosed herein contains additional sequences that make the vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector disclosed herein contains additional sequences that make the vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector disclosed herein includes a shuttle vector, which makes this vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, cloning vectors contain transcription and translation initiation sequences (eg, promoters, enhancers) and transcription and translation terminators (eg, polyadenylation signals).

In one embodiment, for the expression of blood clotting factors disclosed in

- 19 040921 of this document, you can use a variety of prokaryotic or eukaryotic cells as host cells in expression systems. In some embodiments, they include, but are not limited to, microorganisms such as bacteria transformed with a recombinant expression vector based on bacteriophage DNA, plasmid DNA, or cosmid DNA containing a polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing a sequence encoding a polypeptide; plant cell systems infected with recombinant viral expression vectors (eg, cauliflower mosaic virus CaMV; tobacco mosaic virus TMV) or transformed with recombinant plasmid expression vectors, such as the Ti plasmid, containing the sequence encoding the polypeptide.

In some embodiments, non-bacterial expression systems (eg, mammalian expression systems such as CHO cells) are used to express the clotting factors disclosed herein. In one embodiment, the expression vector used to express the polynucleotides disclosed herein in mammalian cells is a pCI-DHFR vector containing the CMV promoter and a neomycin resistance gene. The construction of the pCI-dhfr vector is described according to one embodiment in International Application No. PCT/IL 2016/050645, which is incorporated herein in its entirety.

In some embodiments, a range of expression vectors in the bacterial systems disclosed herein can advantageously be selected depending on the intended use of the expressed polypeptide. In one embodiment, large amounts of the polypeptide are desirable. In one embodiment, vectors driving high levels of expression of a protein product are desirable, possibly as a fusion product with a hydrophobic signal sequence that directs the expressed product to the bacterial periplasm or culture medium where the protein product is readily purified. In one embodiment, certain fusion proteins are designed with a specific cleavage site to facilitate recovery of the polypeptide. In one embodiment, vectors amenable to such manipulation include, without limitation, E. coli expression vectors of the pET series (Studier et al., Methods in Enzymol. 185:60-89 (1990)).

In one embodiment, yeast expression systems are used. In one embodiment, yeast can use a number of vectors containing constitutive or inducible promoters, as disclosed in US patent application No. 5932447, incorporated herein by reference in its entirety. In another embodiment, vectors are used to facilitate the integration of foreign DNA sequences into yeast chromosomes.

In one embodiment, the expression vector disclosed herein may further comprise additional polynucleotide sequences that, for example, allow translation of multiple proteins from a single mRNA, such as an internal ribosome entry site (IRES), and sequences for integration into the promoter genome and region encoding the chimeric polypeptide.

In some embodiments, recombinant viral vectors are useful for in vivo expression of the coagulation factors disclosed herein because they have advantages such as lateral infection and targeting specificity. In one embodiment, lateral infection is inherent in the life cycle of, for example, a retrovirus and is a process by which many progeny virions are produced in one infected cell, which bud off and infect neighboring cells. In one embodiment, this quickly infects a large area, most of which was not originally infected with the original virus particles. In one embodiment, viral vectors incapable of lateral spread are obtained. In one embodiment, this feature may be applicable if the desired goal is to introduce a particular gene into only a limited number of target cells.

In one embodiment, various methods can be used to introduce the expression vector disclosed herein into cells. Such methods are outlined in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore , Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. (Biotechniques 4 (6): 504-512, 1986) and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. Also, see US Pat. Nos. 5,464,764 and 5,487,992 incorporated herein by reference for positive-negative selection methods.

In some embodiments, the introduction of a nucleic acid by infection with a virus has some advantages over other methods such as lipofection and electroporation because, due to the infectious nature of viruses,

- 20 040921 higher transfection efficiency.

In one embodiment, it will be understood that the engineered clotting factors disclosed herein can also be expressed from a nucleic acid construct administered to an individual using any suitable method of administration described herein above (eg, in vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into a suitable cell using an appropriate gene delivery agent/method (transfection, transduction, homologous recombination, etc.) and expression system as needed, and then the modified cells are expanded in culture and returned to an individual (ie, via ex vivo gene therapy).

In one embodiment, plant expression vectors are used. In one embodiment, expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters are used, such as the CaMV 35S RNA and 19S RNA gene promoters (Brisson et al., Nature 310:511-514 (1984)) or the TMV coat protein gene promoter (Takamatsu et al., EMBO J. 6 :307-311 (1987)). In another embodiment, plant promoters are used, such as, for example, promoters of the RUBISCO small subunit gene (Coruzzi et al., EMBO J. 3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984 )) or genes for heat shock proteins, for example soybean hsp17.5-E or hsp17.3-B (Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)). In one embodiment, the constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant expression viral vectors, DNA direct transformation, microinjection, electroporation, and other techniques well known to one of skill in the art. See, for example, Weissbach & Weissbach (Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)). Other expression systems can also be used in the present invention, such as those based on insect and mammalian host cells well known in the art.

It will be understood that in addition to the fact that the expression construct disclosed herein contains the necessary elements for transcription and translation of the inserted coding sequence (coding for the polypeptide), it may also contain sequences designed to optimize the stability, production, purification, yield or activity of the expressed polypeptide.

In some embodiments, the transformed cells are cultured under efficient conditions that allow high levels of the recombinant engineered coagulation factors to be expressed. In some embodiments, effective culture conditions include, without limitation, effective environmental, bioreactor, temperature, pH, and oxygen conditions that allow for protein production. In one embodiment, an effective medium refers to any medium in which cells are cultured to produce the recombinant polypeptide disclosed herein. In some embodiments, the medium typically includes an aqueous solution having sources of usable carbon, nitrogen, and phosphate, as well as essential salts, minerals, metals, and other nutrients such as vitamins. In some embodiments, cells disclosed herein can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and Petri dishes. In some embodiments, the implementation of the cultivation is carried out at a temperature, pH and oxygen content suitable for the recombinant cell. In some embodiments, determining the culture conditions is within the skill of the average person skilled in the art.

In some embodiments, depending on the vector and host cells in the expression system used to produce, the resulting engineered clotting factors disclosed herein remain in the recombinant cell, are secreted into the fermentation medium, are secreted into the space between two cell membranes , such as the periplasmic space in E. coli; or are retained on the outer surface of the cell or viral membrane.

In one embodiment, after a predetermined period of time has elapsed, the recombinant engineered coagulation factor is extracted from the culture.

In one embodiment, the phrase "retrieval of the recombinant engineered blood clotting factor" as used herein refers to the collection of the entire fermentation medium containing the polypeptide and does not necessarily imply additional separation or purification steps.

In one embodiment, the engineered coagulation factors disclosed herein are purified using a variety of standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography (HIC), size exclusion chromatography. , reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.

- 21 040921

In one embodiment, the type of hydrophobic interaction chromatography column is a Capto Phenyl ImpRes (CIP) column.

In one embodiment, to facilitate extraction, the expressed coding sequence can be designed to encode the engineered coagulation factor disclosed herein and the fusion cleavage component. In one embodiment, the fusion protein can be designed such that the polypeptide can be easily isolated by affinity chromatography; for example, by immobilization on a column with a sorbent specific for the component to be cleaved off. In one embodiment, a cleavage site is inserted between the engineered coagulation factor and the cleavage component, and release of the polypeptide from the chromatographic column can be accomplished by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at that site (e.g., see Booth et al., Immunol Lett 19:65-70 (1988) and Gardella et al., J Biol Chem 265:15854-15859 (1990)).

In one embodiment, the engineered coagulation factor disclosed herein is recovered in a substantially pure form. In another embodiment, the substantially pure engineered coagulation factor may further include an active form of the coagulation factor. In another embodiment, the substantially pure form is at least 90% pure. In another embodiment, the substantially pure form is at least 95-99% pure.

In one embodiment, the engineered coagulation factor disclosed herein can also be synthesized using in vitro expression systems. In one embodiment, in vitro synthesis methods are well known in the art and the components of the system are commercially available.

In some embodiments, recombinant engineered coagulation factors are synthesized and purified; their therapeutic efficacy can be evaluated in vivo or in vitro. In one embodiment, the binding activity values of the recombinant engineered coagulation factors disclosed herein can be determined using various assays known to one of skill in the art.

It should be understood that the polypeptides, compositions, compositions, and methods disclosed herein, including the elements or steps described herein, may in another embodiment consist of these elements or steps, or, in another embodiment, consist essentially of these elements or stages. In some embodiments, the term contain refers to the inclusion of the specified active substance, such as CTP-modified blood clotting factor, as well as the inclusion of other active substances and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc. known in the pharmaceutical industry. . In some embodiments, the term consisting essentially of refers to a composition whose said active ingredient is the only active ingredient, however, other compounds intended to stabilize, preserve, etc. may be included. composition, but not directly involved in providing a therapeutic effect of the specified active ingredient. In some embodiments, the term consisting essentially of may refer to components that facilitate the release of the active ingredient. In some embodiments, the term consisting refers to a composition containing the active ingredient and a pharmaceutically acceptable carrier or excipient.

In another embodiment, disclosed herein is a polynucleotide encoding a CTP-modified polypeptide consisting of a blood coagulation factor VII (FVII) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) fused to the carboxyl terminus of said FVII polypeptide. In another embodiment, this document discloses a composition comprising an expression vector comprising a polynucleotide encoding a blood coagulation factor VII (FVII) polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) fused to the carboxyl terminus of said FVIIa polypeptide. In one embodiment, the CTP-modified FVII contains a signal peptide. In another embodiment, the CTP-modified FVII does not contain a signal peptide.

In one embodiment, this document discloses the recombinant blood clotting factor described herein above. In one embodiment, this document discloses the engineered coagulation factor described herein above. In one embodiment, the engineered coagulation factor described herein above is referred to as a CTP-modified coagulation factor.

In one embodiment, the CTPs attached to the carboxyl end of a coagulation factor are attached sequentially one after the other to the carboxyl end.

In one embodiment, the engineered clotting factor described herein has equivalent or improved biological activity compared to a non-CTP modified clotting factor. In another embodiment, the engineered coagulation factor described herein has equivalent or improved pharmacological profiles compared to non-CTP modified coagulation factor. In another embodiment, the engineered coagulation factor described herein has equivalent or improved pharmacokinetic parameters compared to a non-CTP modified coagulation factor. In another embodiment, the engineered coagulation factor described herein has equivalent or improved pharmacodynamic characteristics compared to a non-CTP modified coagulation factor.

In another embodiment, the terms CTP peptide, carboxy-terminal peptide, and CTP sequence are used interchangeably herein. In another embodiment, the carboxy-terminal peptide is a full length CTP.

In other embodiments, the term engineered coagulation factor refers to the amino acid sequence of a mature coagulation factor. In other embodiments, the term engineered coagulation factor refers to the amino acid sequence of a coagulation factor containing its signal sequence or signal peptide.

In another embodiment, the signal sequence and the signal peptide are used interchangeably herein in all cases with the same properties and meanings. In another embodiment, the sequence in relation to the polynucleotide molecule may refer to the coding portion. In another embodiment, an engineered coagulation factor containing at least one CTP described herein has increased in vivo biological activity compared to the same coagulation factor lacking at least one CTP. In one embodiment, the increased biological activity is due to a longer half-life of the engineered coagulation factor while maintaining at least some biological activity. In another embodiment, the increased biological activity is due to the increased biological activity as a result of modification with CTP. In another embodiment, the increased biological activity is due to both a longer half-life and increased functionality of the CTP-modified coagulation factor.

In some embodiments, at least one CTP sequence at the carboxyl terminus of a coagulation factor provides increased protection against degradation of the coagulation factor. In some embodiments, at least one CTP sequence at the carboxyl terminus of a coagulation factor provides increased protection against clearance. In some embodiments, at least one CTP sequence at the carboxyl terminus of a coagulation factor provides for an extended clearance time. In some embodiments, at least one CTP sequence at the carboxyl terminus of a coagulation factor provides for an increase in its Cmax. In some embodiments, at least one CTP sequence at the carboxyl terminus of a coagulation factor provides for an increase in its Tmax. In some embodiments, at least one CTP sequence at the carboxyl terminus of a coagulation factor provides for the extension of its tV ₂ .

In another embodiment, a conjugated coagulation factor of the present invention is used in the same manner as an unconjugated coagulation factor. In another embodiment, the conjugated coagulation factor of the present invention is characterized by increased circulating half-life and plasma retention time, reduced clearance rate, and increased in vivo clinical activity. In another embodiment, due to the improved properties of the conjugated coagulation factor described herein, the conjugate is administered at a lower frequency than the unmodified form of the same coagulation factor.

In another embodiment, reducing the frequency of administration results in an improved treatment strategy, which in one embodiment results in improved patient adherence to therapy, resulting in improved treatment outcomes as well as an improvement in the patient's quality of life. In another embodiment, compared to conventional coagulation factor conjugates, conjugates having the molecular weight and structure of the conjugate linkers of the present invention have been found to have improved specific activity, improved stability, increased AUC levels, and increased circulating half-life.

Compositions and uses

In another embodiment, the engineered coagulation factor disclosed herein may be provided to an individual per se. In one embodiment, the engineered clotting factor disclosed herein may be provided

- 23 040921 to an individual as part of a pharmaceutical composition in which it is mixed with a pharmaceutically acceptable carrier.

In one embodiment, a pharmaceutical composition or pharmaceutical formulation refers to a formulation of one or more of the active ingredients described herein and other chemical components such as physiologically acceptable carriers and excipients. The purpose of using a pharmaceutical composition or pharmaceutical composition is to facilitate the introduction of the compound into the body. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation is a pharmaceutical dosage form of a drug. Pharmaceutical compositions or pharmaceutical formulations in certain embodiments include sustained release technologies, transdermal patches, or any dosage form known in the art.

In another embodiment, the active ingredient refers to the polypeptide sequence of interest that is responsible for the biological effect.

In another embodiment, any of the compositions disclosed herein will contain at least one CTP sequence linked only to the carboxyl terminus of the engineered clotting factor of interest, in any form. In one embodiment, combination preparations are disclosed herein. In one embodiment, the combination preparation is specifically defined as a set of parts in the sense that the combination partners defined above can be dosed independently or by using different fixed combinations with distinguishable numbers of combination partners, i. simultaneously, in parallel, separately or in series. In some embodiments, parts of a set of parts may thus, for example, be administered simultaneously or chronologically apart, that is, at different points in time and at equal or different time intervals for each part of a set of parts. The ratio of the total number of partners in combination in some embodiments, the implementation can be entered in the form of a combined preparation. In one embodiment, the components of the combination preparation may be varied, for example, to meet the needs of a subgroup of patients to be treated, or the needs of a single patient, whose different needs may be due to a particular disease, disease severity, age, sex, or body weight, which can easily be done specialist in this field.

In another embodiment, this document discloses a pharmaceutical composition or pharmaceutical formulation comprising a CTP-modified coagulation factor VIIa (FVIIa) polypeptide consisting of a FVIIa polypeptide and three gonadotropin carboxy-terminal peptides (CTPs) attached to the carboxyl terminus of said FVIIa.

In another embodiment, provided herein is a composition comprising a conjugated coagulation factor as described herein. In another embodiment, provided herein is a pharmaceutical composition comprising a conjugated coagulation factor as described herein. In another embodiment, provided herein is a pharmaceutical composition containing a therapeutically effective amount of a conjugated blood coagulation factor described herein. In one embodiment, a therapeutically effective amount of a conjugated blood clotting factor is determined in accordance with such factors as the particular condition being treated, the condition of the patient being treated, and other ingredients in the composition.

In one embodiment, the present invention provides a pharmaceutical composition for use in compositions, formulations and methods according to the present invention. In another embodiment, the present invention provides a pharmaceutical composition comprising a polypeptide consisting of a blood coagulation factor and three carboxy-terminal human chorionic gonadotropin peptides attached to the carboxyl terminus of the blood coagulation factor. In another embodiment, the pharmaceutical formulation further comprises a buffer and a tonicity agent. In another embodiment, the buffer is 20 mM citrate and 13.3 mM glycine and the tonicity agent is 150 mM NaCl. In another embodiment, the formulation has a pH of about 6.4. In another embodiment, the buffer is 20 mM citrate and 13.3 mM glycine and the tonicity agent is 150 mM NaCl and the pH is 6.4. In another embodiment, the buffer is 20 mM citrate, 100 mM arginine and 2% trehalose and the pH is 6.2. In another embodiment, the formulation is a liquid formulation. In another embodiment, the formulation is a lyophilized formulation. In another embodiment, a liquid formulation can be prepared using a lyophilized CTP-modified coagulation factor. In another embodiment, the CTP-modified coagulation factor is FVII-CTP3. In another embodiment, the CTP-modified coagulation factor is FVIIa-CTP3.

- 24 040921

In another embodiment, provided herein is a dosage form for administration once a week containing the pharmaceutical composition provided herein. In another embodiment, provided herein is a dosage form for administration once a day containing the pharmaceutical composition provided herein. In another embodiment, provided herein is a dosage form for administration once every two days, containing the pharmaceutical composition provided herein. In another embodiment, provided herein is a dosage form for administration once every three days, containing the pharmaceutical composition provided herein. In another embodiment, provided herein is a dosage form for administration twice a week containing the pharmaceutical composition provided herein. In another embodiment, provided herein is a dosage form for administration twice a week containing the pharmaceutical composition provided herein. In another embodiment, provided herein is a weekly dosage form containing the pharmaceutical composition provided herein. In another embodiment, provided herein is a biweekly (every two weeks) dosage form containing the pharmaceutical composition provided herein.

In another embodiment, this document discloses a composition comprising a polypeptide consisting of a blood coagulation factor and three human chorionic gonadotropin CTPs attached to the carboxyl terminus of said blood coagulation factor, and wherein said polypeptide optionally consists of a signal peptide, wherein said composition has increased stability. In one embodiment, the formulation is stable for at least one year. In another embodiment, the composition is stable for at least two years.

In one embodiment, the CTP-modified coagulation factor is formulated as a liquid formulation. In another embodiment, CTP-modified coagulation factor VII is formulated as a liquid formulation. In another embodiment, CTP-modified coagulation factor VIIa is formulated as a liquid formulation. In another embodiment, the CTP-modified coagulation factor is formulated for intranasal administration. In another embodiment, the CTP-modified coagulation factor is formulated into an injectable dosage form.

In another embodiment, the methods of the present invention comprise increasing adherence to clotting factor therapy, comprising providing a subject in need thereof with a CTP-modified clotting factor, thereby increasing adherence to clotting factor therapy.

In another embodiment, the CTP-modified clotting factor is administered to the subject once a day. In another embodiment, a polypeptide containing a coagulation factor modified with CTP is administered to a subject once every two days. In another embodiment, the CTP-modified clotting factor is administered to the subject once every three days. In another embodiment, the CTP-modified clotting factor is administered to the subject once every four days. In another embodiment, a CTP-modified clotting factor is administered to a subject once every five days. In another embodiment, the CTP-modified clotting factor is administered to the subject once every six days. In another embodiment, the CTP-modified clotting factor is administered to the subject once a week. In another embodiment, a CTP-modified clotting factor is administered to a subject once every 7-14 days. In another embodiment, a CTP-modified clotting factor is administered to a subject once every 10-20 days. In another embodiment, a CTP-modified clotting factor is administered to a subject once every 5-15 days. In another embodiment, a CTP-modified clotting factor is administered to a subject once every 15-30 days.

In one embodiment, the preparation disclosed herein is formulated as liquid formulations for injection using a syringe or pen.

In one embodiment, the formulations provided herein also contain preservatives such as benzalkonium chloride and thimerosal, and the like; chelating agents such as sodium edetate and others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerol, mannitol, and others; antioxidants such as ascorbic acid, acetylcysteine, sodium metabisulphite and others; flavoring agents; viscosity regulators such as polymers, including cellulose and its derivatives and polyvinyl alcohol; as well as acids and bases for adjusting the pH of these aqueous compositions as needed. The compositions also contain local anesthetics or other active substances. The compositions can be applied in the form of sprays, mist aerosols, drops, and the like.

In one embodiment, the blood clotting factor described herein is a human blood clotting factor.

- 25 040921

In another embodiment, the conjugated coagulation factor described herein is useful for the treatment of subjects suffering from a coagulation or blood clotting disorder. In another embodiment, the coagulation or clotting disorder is hemophilia. In another embodiment, the conjugated coagulation factor described herein is useful for the prophylactic treatment of hemophilia, thereby reducing the risk of bleeding and associated complications. In another embodiment, while reducing the risk of bleeding and associated complications, the risk of spontaneous bleeding is reduced. In another embodiment, while reducing the risk of bleeding and associated complications, the risk of developing heavy bleeding is reduced. In another embodiment, the conjugated coagulation factor described herein is useful for the treatment of subjects suffering from hemophilia, while providing a reduction in the risk of developing inhibitory antibodies to exogenously administered blood coagulation factors. In another embodiment, the conjugated coagulation factor described herein is useful in the treatment of subjects suffering from hemophilia, thereby inducing hemostasis.

In one embodiment, the CTP-modified coagulation factor disclosed herein has therapeutic uses. In another embodiment, the CTP-modified coagulation factor disclosed herein has prophylactic uses.

In another embodiment, the conjugated coagulation factor described herein is useful in the treatment of subjects who experience profuse bleeding or bruising or who have a long prothrombin time (PT) or partial thromboplastin time (PTT). In another embodiment, the conjugated coagulation factor described herein is useful for the treatment of subjects having an acquired bleeding condition such as vitamin K deficiency or liver disease. In another embodiment, the conjugated coagulation factor described herein is useful for the treatment of subjects having coagulation factor deficiencies that are acquired (due to other diseases) or hereditary, mild or severe, permanent or temporary. In another embodiment, the conjugated coagulation factor described herein is applicable to the treatment of subjects suffering from hemophilia A. In another embodiment, the conjugated coagulation factor described herein is applicable to the treatment of subjects suffering from hemophilia B. In another embodiment, an embodiment, the conjugated coagulation factor described herein is useful for the treatment of subjects having acquired deficiencies due to chronic diseases such as liver disease or cancer; in connection with an acute condition such as disseminated intravascular coagulation (DIC), in which clotting factors are consumed at a high rate; or due to vitamin K deficiency or treatment with a vitamin K antagonist such as warfarin (vitamin K is required for the production of clotting factors II, VII, IX, and X). In another embodiment, the conjugated coagulation factor described herein is useful in the treatment of subjects suffering from a disease that causes blood clotting imbalances, such as, but not limited to, liver disease, uremia, cancer, bone marrow disorder. , exposure to snake venom, vitamin K deficiency, anticoagulation therapy, accidental ingestion of the anticoagulant warfarin, multiple blood transfusions (stored blood units lose some of their clotting factors), or a combination of these. In another embodiment, disclosed herein is a method for treating deep vein thrombosis in a subject, comprising administering a CTP-modified coagulation factor disclosed herein. In another embodiment, disclosed herein is a method for preventing uncontrolled bleeding in a subject with hemophilia, comprising administering a CTP-modified coagulation factor disclosed herein. In another embodiment, disclosed herein is a method for preventing bleeding episodes in a subject with hemophilia, comprising administering a CTP-modified coagulation factor disclosed herein. In another embodiment, this document discloses a method for controlling bleeding episodes in a subject with hemophilia B (congenital deficiency of blood clotting factor IX).

In one embodiment, the composition according to the present invention contains the composition described in this document. In another embodiment, the method according to the present invention includes the introduction of the composition described in this document. In another embodiment, the method according to the present invention includes the introduction of a composition containing the composition described in this document.

In one embodiment, this document discloses a method for preventing or treating a bleeding or coagulation disorder. In another embodiment, in this

- 26 040921 document discloses a method for the prevention or treatment of hemophilia in a subject, including the introduction of CTP-modified clotting factor disclosed in this document. In another embodiment, disclosed herein is a method for preventing and treating hemophilia in a subject, comprising administering a CTP-modified coagulation factor disclosed herein. In another embodiment, disclosed herein is a method for treating hemophilia in a subject, comprising administering a CTP-modified coagulation factor VIIa disclosed herein.

In one embodiment, the hemophilia is hemophilia A. In another embodiment, the hemophilia is hemophilia B. In another embodiment, the methods of the present invention for preventing or treating a bleeding or coagulation disorder prevent or treat hemophilia in patients having hemophilia A or B with FVIII or FIX inhibitors, respectively. In another embodiment, the methods of the present invention are for the prevention or treatment of patients with acquired hemophilia (hemophilia without inhibitors). In another embodiment, the methods of the present invention for preventing or treating a clotting or coagulation disorder prevent or treat hemophilia A or B without inhibitors. In another embodiment, the hemophilia is severe hemophilia. In another embodiment, the hemophilia is moderate hemophilia. In another embodiment, the hemophilia is moderate to severe hemophilia with or without inhibitors. One skilled in the art will appreciate that the term moderate to severe hemophilia refers to a subject having an FVIII or FIX content of less than or equal to 3%. In another embodiment, severe hemophilia may involve clotting factor levels of approximately 0-1%. In another embodiment, the hemophilia is moderate hemophilia, which in another embodiment describes hemophilia in which clotting factor levels are 1-5%. In another embodiment, the hemophilia is mild hemophilia, which in another embodiment describes hemophilia in which clotting factor levels are 5-50%.

In another embodiment, disclosed herein is a method for preventing or treating a bleeding or coagulation disorder in a subject, comprising administering a CTP-modified coagulation factor VII (FVII) polypeptide comprising a FVIIa polypeptide and three carboxy-terminal chorionic gonadotropin (CTP) peptides fused to the carboxyl terminus of said FVIIa polypeptide in said subject, thereby preventing or treating a clotting or coagulation disorder in said subject.

In another embodiment, disclosed herein is a method for preventing or treating hemophilia in a subject, comprising administering a CTP-modified coagulation factor VIIa (FVIIa) polypeptide comprising a FVIIa polypeptide and three carboxyl-terminal chorionic gonadotropin (CTP) peptides fused to the carboxyl terminus of said FVIIa polypeptide. to said subject, thereby preventing or treating hemophilia in said subject.

In another embodiment, disclosed herein is a method for treating hemophilia in a subject, comprising administering one or more of the CTP-modified coagulation factors described herein to said subject. Thus, in one embodiment, disclosed herein is a method of treating hemophilia in a subject comprising administering a CTP-modified coagulation factor VIIa (FVIIa) polypeptide comprising a FVIIa polypeptide and three carboxyl-terminal chorionic gonadotropin (CTP) peptides attached to the carboxyl terminus said FVIIa polypeptide to said subject, thereby providing treatment for hemophilia in said subject. In one embodiment, the CTP-modified FVIIa is administered in the same formulation at the same time. In another embodiment, the CTP-modified FVIIa is administered in different formulations at the same time. In another embodiment, the CTP-modified FVIIa is administered in different formulations at different times.

In another embodiment, disclosed herein is a method for preventing or treating hemophilia in a subject, comprising administering a FVIIa polypeptide and three carboxy-terminal chorionic gonadotropin (CTP) peptides fused to the carboxyl terminus of said FVIIa polypeptide to said subject, thereby preventing or treating hemophilia in specified subject.

In another embodiment, disclosed herein is a method for preventing or treating hemophilia in a subject, comprising subcutaneously or intravenously administering a CTP-modified coagulation factor VIIa polypeptide comprising an FVIIa polypeptide and three carboxyl-terminal chorionic gonadotropin (CTP) peptides attached to the carboxyl terminus of said a FVIIa polypeptide to said subject, thereby preventing or treating hemophilia in said subject. In some embodiments, in this

A method for preventing or treating hemophilia in a subject is provided, the method comprising the step of administering to the subject a CTP-modified coagulation factor comprising three to five carboxy-terminal peptides (CTP) of human chorionic gonadotropin attached to the carboxyl terminus of said coagulation factor polypeptide blood, where the sequence of the specified CTP-modified coagulation factor is selected from the group consisting of SEQ ID NO: 5 or 7, thereby ensuring the prevention of hemophilia in the specified subject. In another embodiment, said CTP-modified coagulation factor is selected from the group consisting of SEQ ID NO: 5 or 7. In another embodiment, said CTP-modified coagulation factor consists of SEQ ID NO: 7. In another embodiment, said CTP -modified coagulation factor, consisting of SEQ ID NO: 7, includes activated FVII (FVIIa).

In another embodiment, this document discloses a method for treating hemophilia in a subject, comprising administering a polypeptide that is an activated CTP-modified coagulation factor VII (FVIIa) containing a FVIIa polypeptide and three carboxy-terminal chorionic gonadotropin (CTP) peptides attached to the carboxyl terminus of said polypeptide FVIIa to said subject, thereby providing treatment for hemophilia in said subject.

In another embodiment, subcutaneous (SC) administration results in higher bioavailability of CTP-modified FVII compared to recombinant FVII. In another embodiment, the half-life is longer and the bioavailability (AUC SC/AUC IV) is higher after SC administration of FVIIa-CTP3 compared to SC administration of NovoSeven®. In another embodiment, subcutaneous injection of MOD-5014 demonstrates improved mouse survival compared to recombinant FVII (NovoSeven®).

In one embodiment, MOD-5014 is FVIIa-CTP ₃ (having three CTP peptides attached to the C-terminus). In one embodiment, MOD-5014 is a long acting clotting factor. In one embodiment, MOD-5014 provides a more robust and prolonged clotting response compared to recombinant human FVIIa. One skilled in the art will appreciate that the terms MOD-5014 or FVIIa-CTP ₃ can be used interchangeably in all instances with the same properties and meanings, and in one embodiment they refer to a disulfide bond-stabilized double-stranded heterodimeric structure comprising the amino acid sequence of SEQ ID NO: 7. In addition, one of skill in the art will appreciate that when describing coagulation factors or CTP-modified coagulation factors in this document, e.g., FVII-CTP3, the term FVII-CTP3 may in some cases refer to the inactive form of FVII. -CTP3. One of skill in the art will definitely understand which form is being referred to based on related details such as activity. Similarly, while the term MOD-5014 is interchangeable with the term FVIIa-CTP ₃ , i. represents the active form of the CTP-modified coagulation factor, in some cases the term MOD-5014 may be used to refer to the active form of FVII or the nucleotide sequence encoding FVII-CTP3, which will then be expressed and secreted from the cell, and cleared and activated in vitro to form the result of the active form of FVIIa, presented as a molecule MOD-5014.

In one embodiment, the inactivation of MOD-5014 by a tissue factor pathway inhibitor (TFPI) is dose dependent. In one embodiment, deactivation of MOD-5014 by TFPI exhibits a dose-dependent deactivation pattern similar to that of recombinant FVIIa (NovoSeven®) by TFPI. In one embodiment, MOD-5014 is inhibited by antithrombin III. In one embodiment, inhibition of MOD-5014 by antithrombin III is enhanced in the presence of heparin. In one embodiment, inhibition of MOD-5014 by antithrombin III exhibits an inhibition pattern similar to that of recombinant FVIIa (NovoSeven®), in the presence or absence of heparin.

In one embodiment, MOD-5014 provides thrombin generation in a dose-dependent manner. In one embodiment, MOD-5014 reduces the duration of the lag phase of thrombin formation. In one embodiment, MOD-5014 reduces blood clotting time. In one embodiment, MOD-5014 increases the efficiency of blood clot formation. In one embodiment, administering MOD-5014 decreases blood clotting time in a subject. In one embodiment, administering MOD-5014 increases the efficiency of blood clot formation in a subject. In one embodiment, thrombin generation by MOD-5014 is similar to that induced by recombinant FVIIa (NovoSeven®). In one embodiment, the decrease in the duration of the lag phase of thrombin formation under the action of MOD-5014 is similar to that caused by recombinant FVIIa (NovoSeven®). In one embodiment, the reduction in clotting time by MOD-5014 is similar to that caused by recombinant FVIIa (NovoSeven®). In one embodiment, the increase in blood clot formation by MOD-5014 is similar to that induced by recombinant FVIIa (NovoSeven®).

As provided herein, when CTP is attached to blood clotting factors, for example, blood clotting factor FVII, the half-life of the blood clotting factor is increased. The examples demonstrate that the attachment of a CTP, for example, the attachment of three CTPs to FVIIA, does not appear to affect the patterns of blood coagulation activity. In one embodiment, the attachment of CTP to FVII does not prevent the formation of blood clots. In one embodiment, the attachment of CTP to FVII does not interfere with an increase in the efficiency of blood clot formation. In one embodiment, the attachment of CTP to FVII does not interfere with the reduction in clotting time. In one embodiment, the binding of the phospholipid to FVII is maintained after CTP has been attached to a blood coagulation factor. In one embodiment, the attachment of CTP to FVIIA does not interfere with the formation of blood clots. In one embodiment, the addition of CTP to FVIIA does not prevent an increase in the efficiency of blood clot formation. In one embodiment, the attachment of CTP to FVIIA does not interfere with the reduction in blood clot formation. In one embodiment, the binding of the phospholipid to FVIIA is maintained after CTP has been attached to a blood coagulation factor.

In another embodiment, disclosed herein is a method for treating hemophilia in a subject, comprising administering the CTP-modified coagulation factors described herein to said subject.

In other embodiments, the engineered coagulation factor is for the treatment of patients with hemophilia B. The results of administering MOD-5014 to large mammals (dogs) are presented. Administration of MOD-5014 provided an effective and safe sustained release FVIIa for blood coagulation (see International Application No. PCT/IL 2016/050645, which is incorporated herein in its entirety). Treatment with MOD-5014 can be prophylactic or on demand. In one embodiment, disclosed herein is a method for treating hemophilia in a subject, comprising administering MOD-5014 to said subject, thereby providing treatment for hemophilia in said subject. In one embodiment, disclosed herein is a method for preventing profuse bleeding in a subject, comprising administering MOD-5014 to said subject, thereby preventing profuse bleeding in said subject. In one embodiment, disclosed herein is a method for the prophylactic treatment of hemophilia in a subject, comprising administering MOD-5014 to said subject, thereby providing prophylactic treatment for hemophilia in said subject.

In one embodiment, the treatment of hemophilia in a subject with MOD-5014 provides for a reduced frequency of administration of MOD-5014 compared to recombinant FVIIa (NovoSeven®). In one embodiment, prophylactic treatment of hemophilia in a subject with MOD-5014 provides for a reduced frequency of administration of MOD-5014 compared to recombinant FVIIa (NovoSeven®). In one embodiment, prevention of excessive bleeding in a subject with MOD-5014 involves a reduced frequency of administration of MOD-5014 compared to recombinant FVIIa (NovoSeven®).

In one embodiment, coagulation factor VII containing 3 CTPs located one after the other at its carboxyl terminus exhibits an improved PK profile compared to NovoSeven® while maintaining its coagulative activity.

In another embodiment, the compositions, formulations, and methods disclosed herein are for the treatment of bleeding episodes in patients with hemophilia A or B having FVIII or FIX inhibitors, and in patients with acquired hemophilia; prevention of bleeding during surgery or invasive procedures in patients with hemophilia A or B who have FVIII or FIX inhibitors, and in patients with acquired hemophilia; treatment of bleeding episodes in patients with congenital FVII deficiency; and prevention of bleeding during surgery or invasive procedures in patients with congenital FVII deficiency. Acquired hemophilia is a spontaneous autoimmune disorder in which patients with previously normal hemostasis develop autoantibodies to clotting factors, most commonly FVIII. The production of autoantibodies to FVIII leads to deficiency of FVIII, which causes insufficient formation of thrombin under the action of the complex of blood coagulation factor IXa and blood coagulation factor VIIIa through the internal pathway of the coagulation cascade. The following conditions may be associated with acquired hemophilia A: idiopathic disorders, pregnancy, autoimmune disorders, inflammatory bowel disease, ulcerative colitis, dermatological disorders (eg, psoriasis, pemphigus), respiratory disorders (eg, asthma, chronic obstructive pulmonary disease), allergic reactions drugs, diabetes mellitus, acute infectious hepatitis B, acute infectious hepatitis C, solid malignant tumors (tumors of the prostate, lung, colon, pancreas, stomach, biliary tract, head and neck, cervix, breast, melanoma, kidney tumors), hematological malignant tumors. One of skill in the art will appreciate that autoimmune disorders may include rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, temporal arteritis, Sjögren's syndrome, autoimmune hemolytic anemia, Goodpasture's syndrome, myasthenia gravis, Graves' disease, autoimmune hypothyroidism. The person skilled in the art will understand that allergic reactions can occur in a subject who is administered penicillin and its derivatives, sulfonamides, phenytoin, chloramphenicol, methyldopa, thioxanthene depot, interferon-alpha, fludarabine, Bacillus Calmette-Guerin vaccine ( BCG), desvenlafaxine. One skilled in the art will appreciate that hematologic malignancies may include chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, Waldenström's macroglobulinemia, myelodysplastic syndrome, myelofibrosis, and erythroleukemia. Accordingly, in one embodiment, provided herein is a method of treating acquired hemophilia in a subject, comprising administering to the subject any of the compositions provided herein.

In another embodiment, the compositions, formulations, and methods disclosed herein are for the treatment or prevention of muscle bleeding. In another embodiment, the compositions, formulations, and methods disclosed herein are for the treatment or prevention of articular bleeding. In another embodiment, the compositions, formulations, and methods disclosed herein provide therapeutic or prophylactic treatment for epistaxis and gingival bleeding, mucosal bleeding, and central nervous system bleeding. In another embodiment, the compositions, formulations, and methods disclosed herein provide therapeutic or prophylactic treatment for gastrointestinal or cerebral bleeding. In another embodiment, the compositions, formulations, and methods disclosed herein provide therapeutic or prophylactic treatment for infrequent minor bleeding. In another embodiment, the compositions, formulations, and methods disclosed herein provide therapeutic or prophylactic treatment for infrequent moderate bleeding. In another embodiment, the compositions, formulations, and methods disclosed herein provide therapeutic or prophylactic treatment for common light bleeding. In another embodiment, the compositions, formulations, and methods disclosed herein provide therapeutic or prophylactic treatment for moderate to moderate bleeding.

In one embodiment, the compositions, formulations, and methods disclosed herein provide therapeutic or prophylactic treatment for asymptomatic hemophilia. In another embodiment, the compositions, formulations, and methods disclosed herein provide therapeutic or prophylactic treatment for mild to moderate hemophilia. In another embodiment, the compositions, formulations, and methods disclosed herein provide therapeutic or prophylactic treatment for severe hemophilia.

In one embodiment, the compositions, formulations, and methods disclosed herein provide therapeutic or prophylactic treatment for a hemorrhage, which in one embodiment is an uncontrolled hemorrhage, and in another embodiment, an intracerebral hemorrhage. In another embodiment, the compositions, formulations, and methods disclosed herein provide therapeutic or prophylactic treatment for forms of neonatal coagulopathy; severe liver disease; surgical procedures with a high level of risk; traumatic blood loss; bone marrow transplants; forms of thrombocytopenia and platelet dysfunction; emergency withdrawal of oral anticoagulants; congenital deficiencies of coagulation factors V, VII, X, and XI, or von Willebrand disease, in one embodiment, von Willebrand disease with von Willebrand factor inhibitors.

In one embodiment, the CTP-modified coagulation factor disclosed herein is for the treatment of hemophilia or a concomitant disease described herein in a subject. In one embodiment, the subject is a human. In another embodiment, the subject is a human child. In another embodiment, the subject is a domesticated animal. In another embodiment, the subject is a mammal. In another embodiment, the subject is a farm animal. In another embodiment, the subject is a monkey. In another embodiment, the subject is a horse. In another embodiment, the subject is a cow. In another embodiment, the subject is a mouse. In another embodiment, the subject is a rat. In another embodiment, the subject is a canine. In another embodiment, the subject is a feline. In another embodiment, the subject is a bovine, ram, porcine, equine, murine, or deer animal. In one embodiment, the subject is male. In another embodiment, the subject is female. In one embodiment, the subject is a child, in another embodiment, a teenager, in another embodiment, an adult, or, in another embodiment, an elderly subject. In another embodiment, the subject is a pediatric

- 30 040921 sky subject, in another embodiment, a geriatric subject.

In another embodiment, the phrases physiologically acceptable carrier and pharmaceutically acceptable carrier are used interchangeably with respect to a carrier or diluent that does not cause significant irritation to the body and does not inhibit the biological activity and properties of the administered compound. An aid is included within the scope of these phrases. In one embodiment, one of the ingredients included in the pharmaceutically acceptable carrier may be, for example, polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media.

In another embodiment, excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the active ingredient. In one embodiment, excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

Methods for formulating and administering drugs are in the latest edition of Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, which is incorporated herein by reference.

The present invention contemplates various embodiments of dosage ranges. The dose of the engineered coagulation factor disclosed herein, in one embodiment, is in the range of 0.005-100 mg/day. In another embodiment, the dose is in the range of 0.005-5 mg/day. In another embodiment, the dose is in the range of 0.01-50 mg/day. In another embodiment, the dose is in the range of 0.1-20 mg/day. In another embodiment, the dose is in the range of 0.1-10 mg/day. In another embodiment, the dose is in the range of 0.01-5 mg/day. In another embodiment, the dose is in the range of 0.001-0.01 mg/day. In another embodiment, the dose is in the range of 0.001-0.1 mg/day. In another embodiment, the dose is in the range of 0.1-5 mg/day. In another embodiment, the dose is in the range of 0.5-50 mg/day. In another embodiment, the dose is in the range of 0.2-15 mg/day. In another embodiment, the dose is in the range of 0.8-65 mg/day. In another embodiment, the dose is in the range of 1-50 mg/day. In another embodiment, the dose is in the range of 5-10 mg/day. In another embodiment, the dose is in the range of 8-15 mg/day. In another embodiment, the dose is in the range of 10-20 mg/day. In another embodiment, the dose is in the range of 20-40 mg/day. In another embodiment, the dose is in the range of 60-120 mg/day. In another embodiment, the dose is in the range of 12-40 mg/day. In another embodiment, the dose is in the range of 40-60 mg/day. In another embodiment, the dose is in the range of 50-100 mg/day. In another embodiment, the dose is in the range of 1-60 mg/day. In another embodiment, the dose is in the range of 15-25 mg/day. In another embodiment, the dose is in the range of 5-10 mg/day. In another embodiment, the dose is in the range of 55-65 mg/day.

In another embodiment, the dose is in the range of 50-500 mg/day. In another embodiment, the dose is in the range of 50-150 mg/day. In another embodiment, the dose is in the range of 100-200 mg/day. In another embodiment, the dose is in the range of 150-250 mg/day. In another embodiment, the dose is in the range of 200-300 mg/day. In another embodiment, the dose is in the range of 250-400 mg/day. In another embodiment, the dose is in the range of 300-500 mg/day. In another embodiment, the dose is in the range of 350-500 mg/day.

In one embodiment, the dose is 20 mg/day. In one embodiment, the dose is 30 mg/day. In one embodiment, the dose is 40 mg/day. In one embodiment, the dose is 50 mg/day. In one embodiment, the dose is 0.01 mg/day. In another embodiment, the dose is 0.1 mg/day. In another embodiment, the dose is 1 mg/day. In another embodiment, the dose is 0.530 mg/day. In another embodiment, the dose is 0.05 mg/day. In another embodiment, the dose is 50 mg/day. In another embodiment, the dose is 10 mg/day. In another embodiment, the dose is 20-70 mg/day. In another embodiment, the dose is 5 mg/day.

In one embodiment, the dose of CTP-modified coagulation factor is 1-5 mg/day. In one embodiment, the dose of CTP-modified coagulation factor is 1-3 mg/day. In another embodiment, the dose of CTP-modified coagulation factor is 2 mg/day.

In another embodiment, the dose is 1-90 mg/day. In another embodiment, the dose is 1-90 mg/2 days. In another embodiment, the dose is 1-90 mg/3 days. In another embodiment, the dose is 1-90 mg/4 days. In another embodiment, the dose is 1-90 mg/5 days. In another embodiment, the dose is 1-90 mg/6 days. In another embodiment, the dose is 1-90 mg/week. In another embodiment, the dose is

- 31 040921

1-90 mg/9 days. In another embodiment, the dose is 1-90 mg/11 days. In another embodiment, the dose is 1-90 mg/14 days.

In another embodiment, the dose of coagulation factor is 10-50 mg/day. In another embodiment, the dose is 10-50 mg/2 days. In another embodiment, the dose is 10-50 mg/3 days. In another embodiment, the dose is 10-50 mg/4 days. In another embodiment, the dose is 10-50 mg/5 days. In another embodiment, the dose is 10-50 mg/6 days. In another embodiment, the dose is 10-50 mg/week. In another embodiment, the dose is 10-50 mg/9 days. In another embodiment, the dose is 10-50 mg/11 days. In another embodiment, the dose is 10-50 mg/14 days.

In another embodiment, a polypeptide comprising a blood coagulation factor and at least one CTP unit is formulated for intranasal administration. In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is formulated into an injectable dosage form. In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 0.0001 mg to 0.6 mg. In another embodiment, a polypeptide comprising a blood coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 0.001 mg to 0.005 mg. In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 0.005 mg to 0.01 mg. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 0.01 mg to 0.3 mg. In another embodiment, a polypeptide comprising a blood coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 0.2 mg to 0.6 mg. In another embodiment, the blood coagulation factor does not contain CTP at its amino terminus.

In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 1-100 μg. In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 10-80 μg. In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 20-60 μg. In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 10-50 μg. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 40-80 μg. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 10-30 μg. In another embodiment, a polypeptide comprising a coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 30-60 μg.

In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 0.2 mg to 2 mg. In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 2 mg to 6 mg. In another embodiment, a polypeptide comprising a blood coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 4 mg to 10 mg. In another embodiment, a polypeptide comprising a blood coagulation factor and at least one CTP unit is administered to a subject at a dose in the range of 5 mg to 15 mg.

In one embodiment, a CTP-modified clotting factor is administered to a subject at a dose in the range of 10-1000 μg/kg. In another embodiment, a CTP-modified coagulation factor is administered to a subject at a dose in the range of 25-600 μg/kg. In another embodiment, a CTP-modified coagulation factor is administered to a subject at a dose in the range of 50-400 μg/kg. In another embodiment, a CTP-modified clotting factor is administered to a subject at a dose of about 25 μg/kg. In another embodiment, a CTP-modified clotting factor is administered to a subject at a dose of about 50 μg/kg. In another embodiment, a CTP-modified coagulation factor is administered to a subject at a dose of about 100 μg/kg. In another embodiment, a CTP-modified coagulation factor is administered to a subject at a dose of about 200 μg/kg. In another embodiment, a CTP-modified clotting factor is administered to a subject at a dose of about 300 μg/kg. In another embodiment, a CTP-modified coagulation factor is administered to a subject at a dose of approximately 400 μg/kg. In another embodiment, a CTP-modified coagulation factor is administered to a subject at a dose of approximately 500 μg/kg. In another embodiment, a CTP-modified coagulation factor is administered to a subject at a dose of about 600 μg/kg.

In one embodiment, the dose of CTP-modified FVIIa contains 50% of the amount of FVIIa administered as part of the recommended dose of recombinant FVIIa (eg, NovoSeven®) to patients over the same time period. In one embodiment, the dose of CTP-modified FVII contains 50% of the amount of FVII administered as part of the recommended dose of recombinant FVII to patients for the same period of time. For example, if NovoSeven® is given at a dose of 90 mcg/kg once every 2 hours to a patient before or after surgery (i.e. 7.65 mg once every 2 hours or 45.9 mg in six doses for 12- hour period for an 85 kg patient), then the CTP-modified coagulation factor disclosed herein can be given at a dose of 50% of the 12-hour dose of recombinant FVIIa for a patient (i.e., at a dose of 23 mg given once over a 12 hour period).

In another embodiment, the dose of CTP-modified coagulation factor is such that it contains 45% of the amount of coagulation factor administered using non-CTP-modified coagulation factor. In another embodiment, the dose of CTP-modified coagulation factor is such that it contains 10% of the amount of coagulation factor administered using non-CTP-modified coagulation factor. In another embodiment, the dose of CTP-modified coagulation factor is such that it contains 25% of the amount of coagulation factor administered using non-CTP-modified coagulation factor. In another embodiment, the dose of CTP-modified coagulation factor is such that it contains 35% of the amount of coagulation factor administered using non-CTP-modified coagulation factor. In another embodiment, the dose of CTP-modified clotting factor is such that it contains 75% of the amount of clotting factor administered when using a non-CTP-modified clotting factor. In another embodiment, the dose of CTP-modified clotting factor is such that it contains 100% of the amount of clotting factor administered when using a non-CTP-modified clotting factor. However, even if a dose contains the same amount of a coagulation factor (eg, FIX) as a non-CTP modified coagulation factor, it is still advantageous to subjects in that it will be administered with less frequency due to its increased half-life compared to recombinant blood coagulation factors.

In another embodiment, the therapeutically effective amount of conjugated coagulation factor is 10-500μg/kg for FVIIa. In another embodiment, the therapeutically effective amount of conjugated coagulation factor is 150250 IU per kg of body weight when administered once a day. In another embodiment, the pharmaceutical composition containing the conjugated coagulation factor is formulated at a strength effective for administration to a human patient by various routes.

In one embodiment, the CTP-modified coagulation factor is administered to the subject on a weekly basis. In another embodiment, the CTP-modified coagulation factor is administered to the subject twice a week. In another embodiment, the CTP-modified coagulation factor is administered to the subject once every two weeks (every two weeks). In another embodiment, the CTP-modified coagulation factor is administered to the subject twice a month. In another embodiment, the CTP-modified coagulation factor is administered to the subject once a month. In another embodiment, the CTP-modified coagulation factor is administered to the subject daily. In another embodiment, the CTP-modified coagulation factor is administered to the subject once every two days.

In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is administered to a subject once every three days. In another embodiment, a polypeptide containing a coagulation factor and at least one CTP unit is administered to a subject once every four days. In another embodiment, a polypeptide containing a blood clotting factor and at least one CTP unit is administered to a subject once every five days. In another embodiment, a polypeptide containing a blood clotting factor and at least one CTP unit is administered to a subject once every six days. In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is administered to a subject once every 7-14 days. In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is administered to a subject once every 10-20 days. In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is administered to a subject once every 5-15 days. In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is administered to a subject once every 15-30 days.

In another embodiment, the methods according to the present invention include increasing adherence to the use of therapy with blood clotting factors, including providing a subject in need thereof, a polypeptide containing a blood clotting factor and at least one carboxy-terminal peptide (CTP) of human chorionic gonadotropin attached to the carboxyl terminus of the factor blood coagulation, thereby increasing adherence to the use

- 33 040921 coagulation factor therapy.

In another embodiment, the methods according to the present invention include increasing adherence to therapy in patients suffering from chronic diseases who are in need of therapy with blood clotting factors. In another embodiment, the methods of the present invention provide for reducing the frequency of dosing of a coagulation factor by modifying the coagulation factor with CTP, as described herein above.

In another embodiment, the term adherence to therapy includes adherence to therapy. In another embodiment, the methods of the present invention include increasing adherence to therapy in patients in need of clotting factor therapy by reducing the frequency of clotting factor administration. In another embodiment, the reduction in the frequency of administration of the clotting factor is achieved through modifications with CTP, which make the CTP-modified clotting factor more stable. In another embodiment, the reduction in the frequency of administration of the coagulation factor is achieved by increasing the T1/ ₂ of the coagulation factor. In another embodiment, the reduction in the frequency of administration of the clotting factor is achieved by increasing the clearance time or reducing the rate of clearance from the clotting factor.

In another embodiment, the reduction in the frequency of administration of the clotting factor is achieved by increasing the AUC of the clotting factor.

In another embodiment, provided herein is a method for reducing the dosing frequency of a coagulation factor, comprising the step of attaching one to ten CTPs to the carboxyl terminus of the coagulation factor, thereby reducing the dosing frequency of the coagulation factor. In another embodiment, provided herein is a method for reducing the frequency of dosing of a blood coagulation factor, comprising the step of attaching one to five CTPs to the carboxyl terminus of the blood coagulation factor, thereby reducing the frequency of dosing of the blood coagulation factor. In another embodiment, provided herein is a method for reducing the dosing frequency of a coagulation factor, comprising the step of attaching three CTPs to the carboxyl terminus of the coagulation factor, thereby reducing the dosing frequency of the coagulation factor. In another embodiment, provided herein is a method for reducing the frequency of dosing of a coagulation factor, comprising the step of attaching three to five CTPs to the carboxyl terminus of the coagulation factor, thereby reducing the frequency of dosing of the coagulation factor.

In another embodiment, provided herein is a method of increasing adherence to the use of therapy with blood clotting factors, including providing to a subject in need thereof, a polypeptide containing a blood clotting factor and from one to ten carboxy-terminal chorionic gonadotropin peptides attached to the carboxyl terminus of a blood clotting factor, thereby increasing adherence to the use of therapy with blood coagulation factors. In another embodiment, provided herein is a method of increasing adherence to clotting factor therapy, comprising providing to a subject in need thereof a polypeptide comprising a clotting factor and one to five carboxy-terminal chorionic gonadotropin peptides attached to the carboxy terminus of the clotting factor, thereby increasing adherence to the use of therapy with blood coagulation factors. In another embodiment, provided herein is a method of increasing adherence to clotting factor therapy, comprising providing to a subject in need thereof a polypeptide comprising a clotting factor and three carboxy-terminal chorionic gonadotropin peptides attached to the carboxy terminus of the clotting factor, whereby increased adherence to the use of therapy with blood clotting factors. In another embodiment, provided herein is a method of increasing adherence to the use of therapy with blood clotting factors, comprising providing to a subject in need thereof, a polypeptide containing a blood clotting factor and from three to five carboxy-terminal chorionic gonadotropin peptides attached to the carboxyl terminus of a blood clotting factor, thereby increasing adherence to the use of therapy with blood coagulation factors.

In another embodiment, provided herein is a method for preventing or treating a bleeding or coagulation disorder in a subject, comprising providing said subject with a polypeptide comprising a blood coagulation factor and from one to ten carboxy-terminal chorionic gonadotropin peptides attached to the carboxyl terminus of the blood coagulation factor by which provides the treatment of disorders of blood clotting or coagulation in the specified subject. In another embodiment, provided herein is a method for preventing or treating a bleeding or coagulation disorder in a subject, comprising providing to a subject in need thereof a polypeptide comprising a blood coagulation factor and one to five carboxy-terminal chorionic gonadotropin peptides attached to a car

- 34 040921 to the boxy end of a blood coagulation factor, thereby preventing or treating a disorder of blood coagulation or coagulation in the specified subject. In another embodiment, provided herein is a method for preventing or treating a bleeding or coagulation disorder in a subject, comprising providing to a subject in need thereof a polypeptide comprising a blood coagulation factor and three carboxy-terminal chorionic gonadotropin peptides attached to the carboxyl terminus of the blood coagulation factor, for thereby preventing or treating a bleeding or coagulation disorder in said subject.

In another embodiment, provided herein is a method for preventing hemophilia in a subject, comprising providing to a subject in need thereof a polypeptide comprising a blood coagulation factor and three carboxy-terminal chorionic gonadotropin peptides attached to the carboxyl terminus of the blood coagulation factor, thereby preventing hemophilia in specified subject. In another embodiment, provided herein is a method for preventing hemophilia in a subject, comprising providing to a subject in need thereof a polypeptide comprising a blood coagulation factor and three to five carboxy-terminal chorionic gonadotropin peptides attached to the carboxyl terminus of the blood coagulation factor, thereby providing preventing hemophilia in said subject.

In another embodiment, the compositions provided herein are surprisingly more efficiently absorbed into the bloodstream after SC administration (see PCT/IL 2016/050645, incorporated herein by reference in its entirety). The possibility of subcutaneous administration of FVIIa is advantageous as it can be used in prophylactic applications. Subcutaneous injections are also much easier for patients to self-inject and are advantageous in cases where the patients are very young and their veins are small and difficult to locate.

In another embodiment, provided herein is a method for treating hemophilia in a subject, comprising providing said subject with a polypeptide comprising a blood coagulation factor and one to ten carboxy-terminal chorionic gonadotropin peptides attached to the carboxyl terminus of the blood coagulation factor, thereby providing treatment for hemophilia in said subject. In another embodiment, provided herein is a method of treating hemophilia in a subject, comprising providing to a subject in need thereof a polypeptide comprising a blood coagulation factor and one to five carboxy-terminal chorionic gonadotropin peptides attached to the carboxyl terminus of the blood coagulation factor, thereby providing treatment of hemophilia in said subject. In another embodiment, provided herein is a method for treating hemophilia in a subject, comprising providing to a subject in need thereof a polypeptide comprising a blood coagulation factor and three carboxy-terminal chorionic gonadotropin peptides attached to the carboxyl terminus of the blood coagulation factor, thereby providing treatment for hemophilia in specified subject. In another embodiment, provided herein is a method of treating hemophilia in a subject, comprising providing to a subject in need thereof a polypeptide comprising a blood coagulation factor and three to five carboxy-terminal chorionic gonadotropin peptides attached to the carboxyl terminus of the blood coagulation factor, thereby providing treatment of hemophilia in said subject.

Oral administration in one embodiment involves the use of a unit dosage form, including tablets, capsules, lozenges, chewable tablets, suspensions, emulsions, and the like. Such unit dosage forms contain a safe and effective amount of the desired blood clotting factor according to the present invention, which in each case in one embodiment is from about 0.7 or 3.5 mg to about 280 mg/70 kg, or in another embodiment from about 0.5 or 10 mg up to approximately 210 mg/70 kg. Pharmaceutically acceptable carriers suitable for the preparation of oral unit dosage forms are well known in the art. In some embodiments, tablets typically contain conventional pharmaceutically compatible excipients such as inert diluents such as calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose; binders such as starch, gelatin and sucrose; disintegrators such as starch, alginic acid and croscarmellose; lubricants such as magnesium stearate, stearic acid and talc. In one embodiment, glidants such as silica can be used to improve the flow characteristics of the powder mixture. In one embodiment, coloring agents, such as FD&C dyes, may be added to provide an aesthetic appearance. Sweeteners and flavorings such as aspartame, saccharin, menthol flavors, peppermint flavors and fruit flavors are useful chewable tablet auxiliaries. Capsules usually contain one or more of the solid diluents disclosed above. In some embodiments, the choice of media components depends on secondary considerations.

- 35 040921 factors such as taste, cost and storage stability, which are not critical for the purposes of the present invention, and can be easily made by a person skilled in the art.

In one embodiment, the oral dosage form has a predetermined release profile. In one embodiment, the oral dosage form disclosed herein includes sustained release tablets, capsules, lozenges, or chewable tablets. In one embodiment, the oral dosage form disclosed herein includes sustained release tablets, capsules, lozenges, or chewable tablets. In one embodiment, the oral dosage form disclosed herein includes immediate release tablets, capsules, lozenges, or chewable tablets. In one embodiment, the oral dosage form is formulated according to the desired release profile of the pharmaceutical active ingredient known to those skilled in the art.

In some embodiments, oral compositions include liquid solutions, emulsions, suspensions, and the like. In some embodiments, pharmaceutically acceptable carriers suitable for preparing such compositions are well known in the art. In some embodiments, oral liquid compositions contain from about 0.001% to about 0.933% of the desired compound or compounds, or, in another embodiment, from about 0.01% to about 10%.

In some embodiments, compositions for use in the methods of the present invention include solutions or emulsions, which in some embodiments are aqueous solutions or emulsions, containing a safe and effective amount of the compounds disclosed herein, and optionally other compounds intended for local intranasal administration. In some embodiments, the compositions contain from about 0.001% to about 10.0% w/v. of the compound in question, more preferably from about 0.01% to about 2.0, which is used for systemic delivery of compounds via the intranasal route.

In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is injected into a muscle (intramuscular injection). In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is injected under the skin (subcutaneous injection). In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is injected into a muscle. In another embodiment, a polypeptide containing a blood coagulation factor and at least one CTP unit is injected into the skin. In another embodiment, the blood clotting factor described herein is administered via systemic administration. In another embodiment, the blood clotting factor described herein is administered by intravenous injection. In another embodiment, administration may be parenteral, intrapulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, transnasal, intraocular, ophthalmic, epidural, buccal, rectal, transmucosal, intraintestinal, or parenteral delivery, including intramedullary injections, as well as intrathecal or direct intraventricular administration.

In another embodiment, the drug is administered locally and not systemically, for example, by direct injection of the drug into a specific area of the patient's body.

In one embodiment, the route of administration may be enteral. In another embodiment, the pathway may be conjunctival, transdermal, intradermal, intraarterial, vaginal, rectal, intratumoral, parasacral, transmucosal, intramuscular, intravascular, intraventricular, intracranial, intranasal, sublingual, or a combination thereof.

In another embodiment, pharmaceutical compositions and pharmaceutical formulations are administered by intravenous, intra-arterial, or intramuscular injection of a liquid preparation. In some embodiments, liquid formulations include solutions, suspensions, dispersions, emulsions, oils, and the like. In one embodiment, pharmaceutical compositions and pharmaceutical formulations are administered intravenously and are therefore formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical compositions and pharmaceutical formulations are administered intra-arterially and are therefore formulated to be suitable for intra-arterial administration. In another embodiment, the pharmaceutical compositions and pharmaceutical formulations are administered intramuscularly and are therefore formulated to be suitable for intramuscular administration.

In addition, in another embodiment, the pharmaceutical compositions and pharmaceutical compositions are administered topically on the surface of the body, and therefore they are formulated in a form suitable for topical administration. Suitable topical formulations include gels, ointments, creams, lotions, drops, and the like. For topical administration, the compounds disclosed herein are combined with additional appropriate therapeutic agent or agents obtained and

- 36 040921 used as solutions, suspensions or emulsions in a physiologically acceptable diluent with or without a pharmaceutical carrier.

In one embodiment, the pharmaceutical compositions and pharmaceutical formulations disclosed herein are prepared using processes well known in the art, such as conventional mixing, dissolving, granulating, panning, wet triturating, emulsifying, encapsulating, capture or lyophilization.

In one embodiment, pharmaceutical compositions and pharmaceutical formulations for use in accordance with the present invention are formulated in the traditional manner using one or more physiologically acceptable carriers, including excipients and excipients that facilitate the processing of active ingredients into formulations that can be used in pharmaceuticals. . In one embodiment, the formulation depends on the chosen route of administration.

In one embodiment, the injectable formulations of the present invention are formulated as aqueous solutions. In one embodiment, the injectable formulations of the present invention are formulated as physiologically compatible buffers such as Hank's solution, Ringer's solution, or buffered physiological saline. In some embodiments, for transmucosal administration, the formulation uses penetration agents appropriate for the barrier to be penetrated. Such penetration agents are well known in the art.

In one embodiment, the formulations described herein are formulated for parenteral administration, such as by bolus injection or continuous infusion. In some embodiments, formulations for injection are presented in unit dosage form, eg, in ampoules or in multi-dose containers, optionally with an added preservative. In some embodiments, the compositions are suspensions, solutions or emulsions in oily or aqueous inert media and contain formulation aids such as suspending, stabilizing and/or dispersing agents.

In some embodiments, the compositions also contain preservatives such as benzalkonium chloride and thimerosal, and the like; chelating agents such as sodium edetate and others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerin, mannitol, and others; antioxidants such as ascorbic acid, acetylcysteine, sodium metabisulphite and others; flavoring agents; viscosity regulators such as polymers, including cellulose and its derivatives and polyvinyl alcohol; as well as acids and bases for adjusting the pH of these aqueous compositions as needed. In some embodiments, the compositions also contain local anesthetics or other active agents. The compositions can be applied in the form of sprays, mist aerosols, drops, and the like.

In some embodiments, pharmaceutical compositions and pharmaceutical compositions for parenteral administration contain aqueous solutions of the active drug in a water-soluble form. In addition, suspensions of the active ingredients, in some embodiments, are provided as appropriate oily or aqueous injection suspensions.

Suitable lipophilic solvents or inert media in some embodiments include fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions in some embodiments contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. In another embodiment, the suspension also contains suitable stabilizers or solubility enhancers of the active ingredients to enable highly concentrated solutions to be obtained.

In another embodiment, the active compound can be delivered in a vesicle, in particular in a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); LopezBerestein, ibid., pp. 317-327; J. E. Diederichs and al., Pharm./nd. 56 (1994) 267-275 ).

In another embodiment, the pharmaceutical composition delivered in a controlled release system is formulated for intravenous infusion, administration via an implantable osmotic pump, transdermal patch, liposomes, or other routes of administration. In one embodiment, a pump is used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N Engl J Med 321:574 (1989) In another embodiment, polymeric materials can be used In yet another embodiment, the controlled release system can be placed near the therapeutic target, i.e., the brain, thereby requiring is only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlled release systems are discussed in the review article Langer (Science 249: 1527-1533 (1990).

- 37 040921

In some embodiments, the active ingredient is in powder form to be diluted with a suitable inert medium, such as a sterile, pyrogen-free aqueous solution, prior to use. In some embodiments, the compositions are formulated to be sprayed and administered by inhalation. In another embodiment, the compositions are contained in a container with a fine spray device attached thereto.

In one embodiment, the formulation disclosed herein is formulated into rectal compositions such as suppositories or retention enemas using, for example, traditional suppository bases such as cocoa butter or other glycerides.

In some embodiments, pharmaceutical compositions and pharmaceutical compositions suitable for use in the context disclosed herein include compositions in which the active ingredients are contained in an amount effective to achieve the intended purpose. In some embodiments, a therapeutically effective amount means an amount of active ingredients that is effective in preventing, alleviating, or ameliorating the symptoms of a disease or prolonging the survival of a subject being treated.

In one embodiment, determining a therapeutically effective amount is well within the skill of those skilled in the art.

Some examples of substances that can act as pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and methylcellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween™ brand emulsifiers; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring additives; means for tableting, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline and phosphate buffer solutions. The choice of a pharmaceutically acceptable carrier for use in combination with a compound is determined primarily by the route by which the compound is to be administered. If the compound in question is to be injected, then the pharmaceutically acceptable carrier in one embodiment is sterile saline containing a blood-compatible suspending agent that has been adjusted to a pH of about 7.4.

In addition, the compositions additionally contain binders (for example, gum arabic, corn starch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose, povidone), disintegrants (for example, corn starch, potato starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g. Tris-HCI, acetate, phosphate) with different pH and ionic strengths, additives such as albumin or gelatin to prevent adsorption to surfaces, detergents (e.g. Tween 20, Tween 80, Pluronic F68, bile salts), protease inhibitors, surfactants (eg sodium lauryl sulfate), penetration enhancers, solubilizing agents (eg glycerol, polyethylene glycol), antioxidants (eg ascorbic acid, sodium metabisulphite, butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, hydroxypropylmethylcellulose), viscosity enhancers (eg carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (eg aspartame, citric acid), preservatives (eg thimerosal, benzyl alcohol, parabens), lubricants (eg stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g. poloxamers or poloxamines), coating and film-forming agents (eg ethyl cellulose, acrylates, polymethacrylates) and/or auxiliaries.

Typical carrier components in syrups, elixirs, emulsions and suspensions include ethanol, glycerin, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. In suspensions, typical suspending agents include methylcellulose, sodium carboxymethylcellulose, cellulose (eg Avicel™, RC-591), tragacanth, and sodium alginate; typical wetting agents include lecithin and polyoxyethylene sorbitan (eg polysorbate 80). Typical preservatives include methyl paraben and sodium benzoate. In another embodiment, liquid compositions for oral use also contain one or more of the components such as sweeteners, flavors and colors disclosed above.

The compositions also provide for the inclusion of the active material in preparations or compositions.

- 38 040921 preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc., in the form of particles or in liposomes, microemulsions, micelles, monolayer or multilayer vesicles, shadows of erythrocytes or spheroplasts. Such compositions will affect physical condition, solubility, stability, in vivo release rate, and in vivo clearance rate.

The present invention also encompasses particulate compositions coated with polymers (eg, poloxamers or poloxamines) and a compound associated with antibodies directed to tissue-specific receptors, ligands or antigens, or associated with tissue-specific receptors.

In some embodiments, the compounds are modified by covalent attachment of water-soluble polymers such as polyethylene glycol, polyethylene glycol-polypropylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, or polyproline. In another embodiment, the modified compounds exhibit substantially longer blood half-lives following intravenous injection than the corresponding unmodified compounds. In one embodiment, the modifications also provide an increase in the solubility of the compound in aqueous solution, the elimination of aggregation, an increase in the physical and chemical stability of the compound, and a significant decrease in the immunogenicity and reactivity of the compound. In another embodiment, the desired in vivo biological activity is achieved by administering such polymer-compound adducts at a lower frequency or at lower doses than is the case with the unmodified compound.

In some embodiments, obtaining an effective amount or dose can first be calculated in in vitro assays. In one embodiment, the dose may be formulated in animal models and such information may be used to more accurately determine applicable doses in humans.

In one embodiment, the toxicity and therapeutic efficacy of the active ingredients described herein can be determined using standard pharmaceutical procedures in vitro, in cell cultures, or in experimental animals. In one embodiment, the data obtained from these in vitro assays and from cell culture and animal studies can be used to formulate a dosage range for use in humans. In one embodiment, the doses vary depending on the dosage form used and the route of administration used. In one embodiment, the exact composition, route of administration, and dose may be selected by the attending physician based on the condition of the patient. (See, for example, Fingl, et al., (1975) The Pharmacological Basis of Therapeutics, Ch. 1 p. 1).

In one embodiment, depending on the severity and susceptibility of the condition being treated, the administration of doses may consist of one or multiple administrations, with the course of treatment continuing for a period of several days to several weeks, or until a cure or relief of the disease state is achieved.

In one embodiment, the amount of the composition to be administered will, of course, depend on the subject being treated, the severity of the lesion, the route of administration, the judgment of the prescribing physician, and so forth.

In one embodiment, compositions containing the drug disclosed herein, formulated in a compatible pharmaceutical carrier, are also prepared, placed in an appropriate container, and labeled as intended for the treatment of the indicated condition.

In another embodiment, the blood coagulation factor described herein is a lyophilized (i.e., freeze-dried) preparation in combination with complex organic excipients and stabilizers, such as nonionic surfactants (i.e., surfactants), various sugars, organic polyols and/or human serum albumin. In another embodiment, the pharmaceutical composition contains the described lyophilized blood coagulation factor in sterile water for injection. In another embodiment, the pharmaceutical composition contains the disclosed lyophilized coagulation factor in sterile injectable PBS. In another embodiment, the pharmaceutical composition contains the described lyophilized coagulation factor in sterile 0.9% NaCl for injection.

In another embodiment, the pharmaceutical composition contains a blood coagulation factor as described herein and complex carriers such as human serum albumin, polyols, sugars, and anionic surfactant stabilizers. In another embodiment, the pharmaceutical composition contains a blood coagulation factor as described herein, as well as lactobionic acid and an acetate/glycine buffer. In another embodiment, the pharmaceutical composition contains a blood coagulation factor as described herein and amino acids, such as arginine or glutamate, which increase the aqueous solubility of the interferon-based compositions. In another embodiment, the pharmaceutical composition contains a lyophilized coagulation factor as described herein, as well as glycine or human serum albumin (HSA), a buffer (eg, acetate) and an isotonic agent (eg, NaCl). In another embodiment, the pharmaceutical composition contains the lyophilized coagulation factor described herein, as well as phosphate buffer, glycine and HSA.

In another embodiment, a pharmaceutical composition containing a blood clotting factor described herein is stabilized when placed in buffered solutions having a pH of about 4 to 7.2. In another embodiment, the pharmaceutical composition containing the blood clotting factor is in a buffered solution having a pH of about 4 to 8.5. In another embodiment, the pharmaceutical composition containing the blood clotting factor is in a buffered solution having a pH of about 6 to 7. In another embodiment, the pharmaceutical composition containing the blood clotting factor is in a buffered solution having a pH of about 6 ,5. In another embodiment, the pharmaceutical composition containing the blood clotting factor is in a buffered solution having a pH of about 6.4. In another embodiment, a pharmaceutical composition containing a blood coagulation factor described herein is stabilized using an amino acid as a stabilizer and in some cases a salt (if the amino acid does not contain a charged side chain).

In another embodiment, a pharmaceutical composition containing a blood coagulation factor described herein is a liquid composition containing a stabilizer in an amount of from about 0.3% to 5% by weight, which is an amino acid.

In another embodiment, a pharmaceutical composition containing a blood coagulation factor described herein provides dosing accuracy and product safety. In another embodiment, a pharmaceutical composition containing a blood coagulation factor described herein provides a biologically active, stable liquid formulation for use in injection routes of administration. In another embodiment, the pharmaceutical composition contains a non-lyophilized coagulation factor as described herein.

In another embodiment, a pharmaceutical composition containing a blood coagulation factor described herein provides a liquid formulation capable of being stored in a liquid state for an extended period of time, which facilitates storage and transport prior to administration.

In another embodiment, the pharmaceutical composition containing the blood coagulation factor described herein contains solid lipids as a matrix material. In another embodiment, an injectable pharmaceutical composition containing a blood coagulation factor described herein contains solid lipids as a matrix material. In another embodiment, the preparation of lipid microparticles by spray curing as described by Speiser (Speiser and al., Pharm. Res. 8 (1991) 47-54) is presented, followed by the preparation of lipid nanopellets for oral administration (Speiser EP 0167825 (1990)). In another embodiment, the lipids used (eg, fatty acid glycerides that are present in parenteral nutrition emulsions) are well tolerated by the body.

In another embodiment, the pharmaceutical composition containing the blood coagulation factor described herein contains polymeric microparticles. In another embodiment, the pharmaceutical composition containing the blood coagulation factor described herein contains nanoparticles. In another embodiment, the pharmaceutical composition containing the blood coagulation factor described herein contains liposomes. In another embodiment, the pharmaceutical composition containing the blood coagulation factor described herein contains a lipid emulsion. In another embodiment, the pharmaceutical composition containing the blood coagulation factor described herein contains microspheres. In another embodiment, the pharmaceutical composition containing the blood coagulation factor described herein contains lipid nanoparticles. In another embodiment, the pharmaceutical composition containing the blood coagulation factor described herein contains lipid nanoparticles containing amphiphilic lipids. In another embodiment, a pharmaceutical composition containing a blood coagulation factor described herein contains lipid nanoparticles containing a drug, a lipid matrix, and a surfactant. In another embodiment, the lipid matrix has a monoglyceride content of at least 50% w/w.

In one embodiment, the compositions disclosed herein are presented in a package or dispenser device, such as an FDA-approved kit, that contains one or more unit dosage forms containing the active ingredient. In one embodiment, the package, for example, contains a metal or plastic foil, such as a blister pack. In one embodiment, instructions for administration are included with the package or dispenser. In one embodiment, the package or dispenser is provided with a notice, provided with the container, in the form prescribed by a government agency that regulates the manufacture, use or sale of pharmaceuticals, which notice reflects the authority's approval of the form of the composition or its administration in medicine or in veterinary medicine. Such notice, in one embodiment, is a US Food and Drug Administration-approved prescription drug label or approved package leaflet.

In one embodiment, it will be understood that the clotting factors disclosed herein can be provided to an individual along with additional active agents to achieve an improved therapeutic effect compared to treating each of the agents alone. In another embodiment, certain steps are taken (eg, dosing and choice of complementary agent) to avoid unwanted side effects associated with combination therapies.

Receipt

In one embodiment, a method is disclosed for producing a coagulation factor VIIa polypeptide modified with human chorionic gonadotropin CTP peptide, the method comprising the steps of: (a) stably transfecting a predetermined number of cells with an expression vector containing a coding moiety encoding said CTP-modified clotting factor blood VII, where the specified transfected cell expresses and optionally secretes the specified CTP-modified coagulation factor VII; (b) obtaining cell clones overexpressing said CTP-modified coagulation factor VII; (c) propagating said clones in solution to a predetermined scale; (d) collecting said solution containing said clones; (e) filtering said solution containing said clones to obtain a clarified collected solution; and (f) purifying and activating said clarified pooled solution to provide a purified protein solution having a desired concentration of CTP-modified coagulation factor VIIa, thereby obtaining a coagulation factor VIIa polypeptide modified with human chorionic gonadotropin CTPpeptide. In another embodiment, the CTP-modified coagulation factor VII is secreted. In another embodiment, the CTP-modified coagulation factor VII is not secreted. In obtaining CTP-modified coagulation factor VII, transfection is an early step. After selection of the final clone with a high level of expression, each procedure of obtaining includes the thaw of the master cell bank (MBC), propagation, collection of material and purification. (See example 3, steps 1-5, describing the processes from propagation of clones to collection of material; steps 6-14, describing purification and activation). In one embodiment, a method is disclosed for preparing a human human chorionic gonadotropin carboxy-terminal peptide (CTP) modified polypeptide, which is an active human blood coagulation factor VII (FVIIa), wherein said polypeptide comprises three CTP molecules attached sequentially one after the other to the C-terminus of FVII, with this method includes the steps of: stable transfection of a predetermined number of cells with an expression vector containing a coding portion encoding said CTP-modified FVII, where said transfected cells express and secrete said CTP-modified FVII; obtaining cell clones overexpressing said CTP-modified FVII; propagating said clones in solution to a predetermined scale; collecting said solution containing said clones; filtering said solution containing said clones to obtain a clarified pooled solution containing said CTP-modified FVII; and purifying and activating CTP-modified FVII from said clarified pooled solution to obtain a purified protein solution having the desired concentration of CTP-modified FVIIa; where the specified obtained CTP-modified FVIIa provides at least one of the following:

a) low content of oxidized form;

b) a high percentage of carboxylated glutamic acid residues;

c) at least 60% charged N-glycans, or

d) a specific activity of at least 10500 U/mg;

thereby obtaining a CTP-modified FVIIa, and wherein the amino acid sequence of the resulting CTP-modified FVIIa is given under SEQ ID NO: 7.

In one embodiment, the polynucleotides disclosed herein are inserted into expression vectors (ie, a nucleic acid construct) to allow expression of a recombinant polypeptide. In one embodiment, the expression vector disclosed herein contains additional sequences that make the vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector disclosed herein contains additional sequences that make the vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector disclosed herein includes a shuttle vector, making the vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, cloning vectors contain transcription and translation initiation sequences (eg, promoters, enhancers) and transcription and translation terminators (eg, polyadenylation signals).

In one embodiment, a method for producing a CTP-modified FVIIa polypeptide comprises the step of using an expression vector, wherein said expression vector contains a promoter, a sequence encoding a polypeptide that is a CTP-modified FVII, and a polyadenylation sequence. In another embodiment, the polyadenylation sequence is the simian virus (SV) 40 polyadenylation sequence.

In one embodiment, for the expression of the polypeptides disclosed herein, you can use a variety of prokaryotic or eukaryotic cells as host cells in expression systems. In some embodiments, they include, but are not limited to, microorganisms such as bacteria transformed with a recombinant expression vector based on bacteriophage DNA, plasmid DNA, or cosmid DNA containing a polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing a sequence encoding a polypeptide; plant cell systems infected with recombinant viral expression vectors (eg, cauliflower mosaic virus CaMV; tobacco mosaic virus TMV) or transformed with recombinant plasmid expression vectors, such as the Ti plasmid, containing the sequence encoding the polypeptide.

In one embodiment, non-bacterial expression systems (eg, mammalian expression systems such as CHO cells or cells derived from CHO cells) are used to express a polypeptide disclosed herein. In one embodiment, the expression vector used to express the polynucleotides disclosed herein in mammalian cells is a pCI-DHFR vector containing the CMV promoter and a neomycin resistance gene.

In one embodiment, the expression vector disclosed herein may further comprise additional polynucleotide sequences that, for example, allow translation of multiple proteins from a single mRNA, such as an internal ribosome entry site (IRES), and sequences for integration into the promoter genome and region encoding the chimeric polypeptide.

In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1 , pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV available from Stratagene, pTRES available from Clontech, and derivatives thereof.

In some embodiments, the methods disclosed herein use expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses. SV40-based vectors include pSVT7 and pMT2. In some embodiments, bovine papillomavirus-derived vectors include pBV-1MTHA and Epstein-Barr virus-derived vectors include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV00 9/A+, pMTO10/A ⁺ , pMAMneo-5, baculovirus vector, pDSVE, and any other vector capable of expressing proteins under the control of the SV-40 early promoter, SV-40 late promoter, a metallothionein gene, a mouse mammary tumor virus promoter, a Rous sarcoma virus promoter, a polyhedrin gene promoter, or other promoters that have been shown to be effective for expression in eukaryotic cells.

In one embodiment, various methods can be used to introduce an expression vector encoding a CTP-modified coagulation factor VII disclosed herein into cells. Transfection of eukaryotic host cells with a polynucleotide or expression vector resulting in genetically modified cells or transgenic cells can be performed using any method well known in the art and described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. (Biotechniques 4 (6): 504-512, 1986) and includes, for example, stable or transient transfection and electroporation. Also see US Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods. Transfection methods further include, without limitation, liposome-mediated transfection, calcium phosphate coprecipitation, electroporation, polycation-mediated transfection (such as DEAE-dextran), protoplast fusion, viral infection (including infection with recombinant viruses), and microinjection. The transfection is preferably a stable transfection. Advantageous is a transfection method that provides optimal transfection frequency and expression of heterologous genes encoding a peptide of interest disclosed herein in a particular host cell line and type. Suitable methods can be determined using conventional procedures. To obtain stable transfectants, the constructs are integrated into the host cell genome or artificial chromosome/minichromosome, or are positioned episomally so as to be stably maintained in the host cell.

In the practice of the present invention disclosed herein, unless otherwise indicated, conventional techniques of cell biology, molecular biology, cell culture, immunology, and the like will be used, which are within the skill of the person skilled in the art. These techniques are fully disclosed in modern literature. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, ^2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel et al., Current Protocols in Molecular Biology (1987, updated edition); Brown ed., Essential Molecular Biology, IRL Press (1991); Goeddel ed., Gene Expression Technology, Academic Press (1991); Bothwell et al. eds., Methods for Cloning and Analysis of Eukaryotic Genes, Bartlett Publ. (1990); Wu et al., eds., Recombinant DNA Methodology, Academic Press (1989); Kriegler, Gene Transfer and Expression, Stockton Press (1990); McPherson et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); Gait ed., Oligonucleotide Synthesis (1984); Miller & Calos eds., Gene Transfer Vectors for Mammalian Cells (1987); Butler ed., Mammalian Cell Biotechnology (1991); Pollard et al., eds., Animal Cell Culture, Humana Press (1990); Freshney et al., eds., Culture of Animal Cells, Alan R. Liss (1987); Studzinski, ed., Cell Growth and Apoptosis, A Practical Approach, IRL Press at Oxford University Press (1995); Melamed et al., eds., Flow Cytometry and Sorting, Wiley-Liss (1990); Current Protocols in Cytometry, John Wiley & Sons, Inc. (updated edition); Wirth & Hauser, Genetic Engineering of Animals Cells, in: Biotechnology Vol. 2, Puhler ed., VCH, Weinheim 663-744; Methods of Enzymology series (Academic Press, Inc.) and Harlow et al., eds., Antibodies: A Laboratory Manual (1987).

A heterologous gene of interest encoding a CTP-modified coagulation factor VII can be introduced into the cell disclosed herein by various methods, such as viral transformation, transfection, or microinjection. The heterologous gene of interest may be introduced into the cell as linear DNA or as part of an expression vector. A number of expression vectors are known in eukaryotic cells whose multiple cloning sites allow the insertion of one or more heterologous genes and their expression. Commercial suppliers include, among others, Stratagene, La Jolla, CA, USA; Invitrogen, Carlsbad, California, USA; Promega, Madison, WI, USA or BD Biosciences Clontech, Palo Alto, CA, USA. Transfection of cells with DNA or an expression vector encoding one or more genes of interest is carried out using conventional methods described, for example, in Sambrook et al., 1989 or Ausubel et al., 1994. Suitable transfection methods include, for example, liposome mediated transfection, calcium phosphate co-precipitation, electroporation, polycation mediated transfection (eg DEAE-dextran), protoplast fusion, microinjection and virus infection. Preferably, a stable transfection is carried out, in which the DNA molecules are integrated into the genome of the host cell or artificial chromosome/mini-chromosome, or are contained in an episomally stable manner in the host cell. Preferred is a transfection method that provides optimal transfection frequency and expression of one or more heterologous genes of interest in the host cell in question.

In some embodiments, the introduction of the nucleic acid by infection with a virus has some advantages over other methods such as lipofection and electroporation because higher transfection efficiency can be achieved due to the infectious nature of the viruses.

In one embodiment, it will be understood that the polypeptides disclosed herein can also be expressed from a nucleic acid construct administered to an individual using any suitable method of administration described herein above (eg, in vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into a suitable cell using an appropriate gene delivery agent/method (transfection, transduction, homologous recombination, etc.) and expression system as needed, and then the modified cells are expanded in culture and returned to an individual (ie, via ex vivo gene therapy).

A heterologous gene of interest is typically operably linked to a promoter that allows transcription of the gene of interest and to other regulatory elements that allow or increase the efficiency of transcription and translation (expression) of the gene of interest.

One of skill in the art will appreciate that the term promoter may encompass a polynuclear

- 43 040921 leotide sequence, which provides and controls the transcription of genes or sequences functionally associated with it. The promoter contains recognition sequences for RNA polymerase binding and a transcription initiation site (transcriptional initiation site). For purposes of expressing the desired sequence in a particular cell type or host cell, an appropriate functional promoter should be selected. A person skilled in the art will be aware of a variety of promoters from various sources, including constitutive, inducible and repressible promoters. They are deposited in databanks such as, for example, GenBank, and can be obtained as single elements or cloned elements within polynucleotide sequences from commercial or personal sources. In inducible promoters, promoter activity can decrease or increase in response to a signal. One example of an inducible promoter is the tetracycline sensitive (tet) promoter. It contains tetracycline-responsive operator (tetO) sequences that can be induced by the tetracycline-regulated transactivator protein (tTA). In the presence of tetracycline, the binding of tTA to tetO is inhibited. Examples of other inducible promoters are the jun, fos, metallothioneins, and heat shock protein promoters (see also Sambrook, J., Fritsch, E. F. & Maniatis, T., Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. , 1989; Gossen, M. et al., Curr Opi Biotech 1994, 5, 516-520). Promoters particularly suitable for high level expression in eukaryotes include, for example, the hamster ubiquitin/S27a gene promoter (WO 97/15664), SV40 early promoter, adenovirus major late promoter, mouse metallothionein-1 gene promoter, virus long terminal repeat region Rous sarcomas and the human cytomegalovirus early promoter. Examples of other mammalian heterologous promoters are promoters for actin genes, immunoglobulins, or heat shock proteins.

For example, a promoter may be operably linked to enhancer sequences for the purpose of increasing transcriptional activity. This can be done using one or more enhancers and/or multiple copies of an enhancer sequence, such as the CMV or SV40 enhancer. For example, a promoter may be operably linked to enhancer sequences for the purpose of increasing transcriptional activity. This can be done using one or more enhancers and/or multiple copies of an enhancer sequence, such as the CMV or SV40 enhancer.

The person skilled in the art will understand that the term enhancer can encompass a polynucleotide sequence that affects the activity of a promoter in the cis position and thus stimulates the transcription of a gene operably linked to the promoter. Unlike promoters, the effect of enhancers is independent of position and orientation, and as a result, they can be located in front of or behind the transcription unit, in the intron, or even in the coding region. The enhancer can be located both in the immediate vicinity of the transcription unit and at a considerable distance from the promoter. There may also be physical and functional overlap with the promoter. A person skilled in the art will be aware of a number of enhancers from various sources (and deposited in databanks such as GenBank, e.g. SV40 enhancers, CMV enhancers, polyomavirus enhancers, adenovirus enhancers) available as independent elements or cloned elements within polynucleotide sequences ( such as those deposited with the ATCC or available from commercial and individual sources). A number of promoter sequences also contain enhancer sequences, such as the frequently used CMV promoter. The human CMV enhancer is one of the most potent enhancers identified to date. One example of an inducible enhancer is the metallothionein gene enhancer, which can be stimulated by glucocorticoids or heavy metals.

Generally speaking, regulatory elements include promoters, enhancers, termination and polyadenylation signals, and other expression control elements. Both inducible and constitutive regulatory sequences are known for various cell types. Transcriptional regulatory elements typically include a promoter upstream of the gene sequence to be expressed, transcription initiation and termination sites, and a polyadenylation signal.

One skilled in the art will appreciate that the term transcription initiation site may encompass a nucleic acid in a construct that corresponds to the first nucleic acid included in the primary transcript, i.e. mRNA precursor. The transcription initiation site may overlap with promoter sequences.

One of ordinary skill in the art will appreciate that the term transcription termination site may encompass a nucleotide sequence, typically found at the 3' end of a gene of interest or region of a gene to be transcribed, resulting in termination of transcription by RNA polymerase.

One skilled in the art will appreciate that the term polyadenylation signal may encompass a signal sequence that causes cleavage at a particular site at the 3' end of eukaryotic mRNA and post-transcriptional incorporation of a sequence of approximately 100-200 adenine nucleotides (poly(A)-tail ) at the cleaved 3' end. The polyadenylation signal contains the AATAAA sequence approximately 10-30 nucleotides upstream of the cleavage site, as well as a sequence downstream of it. Various polyadenylation units are known, such as poly(A) tk, early and late poly(A) SV40 or poly(A) BGH (described, for example, in US Pat. No. 5,122,458).

Translational regulatory elements include a translation initiation site (AUG), a stop codon, and a poly(A) signal for each polypeptide to be expressed. For optimal expression, it may be desirable to remove, add, or alter the 5' and/or 3' untranslated regions of the nucleic acid sequence to be expressed, in order to eliminate any potentially inappropriate additional translation initiation codons or other sequences that may affect expression at the level transcription or expression. For purposes of promoting expression directly upstream of the start codon, ribosome consensus binding sites may alternatively be inserted. For the purposes of generating a secreted polypeptide, the gene of interest typically contains a signal sequence that encodes a precursor signal peptide for transport of the synthesized polypeptide to and across the ER membrane. The signal sequence is often, but not always, located at the amino terminus of the secreted protein and is cleaved off by signal peptidases after the protein has been filtered through the ER membrane. The gene sequence will usually, but not necessarily, contain its own signal sequence. If the native signal sequence is not present, then a heterologous signal sequence can be introduced in a known manner. Numerous signal sequences of this kind are known to the person skilled in the art and are deposited in sequence data banks such as GenBank and EMBL.

One skilled in the art will appreciate that the terms polypeptides, polypeptide, or their grammatical equivalents can be used interchangeably to cover amino acid sequences or proteins, and can cover polymers of amino acids of any length. The term also includes proteins that have undergone post-translational modifications through reactions such as glycosylation, phosphorylation, acetylation, or protein processing. The structure of a polypeptide can be modified, for example, by substitutions, deletions or insertions of amino acids and fusion with other proteins while maintaining its biological activity.

In order to obtain one or more products of genes of interest in cells, cells can be grown in serum-free culture medium and in suspension culture under conditions that allow expression of the gene of interest. If, for example, the gene of interest is under the control of a constitutive promoter, then there is no need to add special inducers. If the expression of the gene of interest is under the control of an inducible promoter, then, for example, an appropriate inducer should be added to the cell culture medium at a sufficient but non-toxic concentration. Cells can be expanded at will by multiple subpassaging and transferred to suitable cell culture vessels. The product(s) of the genes are, or are produced as such, cellular, membrane-bound or secretory products.

In one embodiment, the step of obtaining a CTP-modified coagulation factor VIIa comprises stably transfecting a predetermined number of cells with an expression vector containing a coding portion encoding a CTP-modified coagulation factor VIIa. In another embodiment, the step of obtaining a CTP-modified coagulation factor VIIa comprises stable transfection of cells with an expression vector containing a coding portion encoding said CTP-modified coagulation factor VIIa. In one embodiment, the cells are CHO cells. In another embodiment, the cells are DG44 cells. In another embodiment, the cells are any cells known in the art suitable for the expression and secretion of CTP-modified coagulation factor VII. In one embodiment, the expressed CTP-modified FVII is a FVII-CTP3 zymogen lacking a signal peptide. In another embodiment, the amino acid sequence of the expressed CTP-modified FVII is given under SEQ ID NO: 7.

One skilled in the art will appreciate that while the CTP-modified FVII may be expressed as a zymogen in an inactive state, the zymogen may be activated during or after the purification process. The term CTP-modified FVII may be used interchangeably with CTP-modified FVII/FVIIa and may encompass inactive and active forms of a polypeptide that is a CTP-modified FVII. Similarly, for individual polypeptides that are CTP-modified coagulation factors VII, for example, FVII/FVIIa-CTP3, the name FVII/FVIIa can also be used, representing the inactive or active forms, or both. In one embodiment, the CTP-modified polypeptide, including activated FVIIa-CTP ₃ , contains a light chain and a heavy chain connected by a disulfide bond.

- 45 040921

In another embodiment, the transfected cells express CTP-modified coagulation factor VII. In another embodiment, the expressed and produced FVII/FVIIa-CTP3 consists of two CTPs attached to the carboxy terminus of said coagulation factor VII/VIIa and one carboxy-terminal chorionic gonadotropin peptide attached to the amino terminus of said coagulation factor VII/VIIa. In another embodiment, the expressed and produced CTP-modified coagulation factor VII/VIIa consists of a single carboxy-terminal chorionic gonadotropin peptide attached to the carboxyl terminus of said coagulation factor VII/FVIIa. In another embodiment, the expression of the CTP-modified coagulation factor VII is at a high level of expression. In another embodiment, said CTP-modified coagulation factor VII/VIIa is highly glycosylated. In another embodiment, said CTP-modified coagulation factor VII/VIIa is highly sialylated. In another embodiment, said CTP-modified coagulation factor VII/VIIa has a high content of O-glycans. In another embodiment, said CTP-modified coagulation factor VII/VIIa has a high N-glycan content. In another embodiment, said CTP-modified coagulation factor VII/VIIa has a high content of charged N-glycans. In another embodiment, said CTP-modified coagulation factor VII/VIIa has a high percentage of carboxyglutamic acid. As described in detail herein, CTP-modified coagulation factor VIIa can have different levels and patterns of glycosylation. The CTP-modified coagulation factor VIIa produced by the methods disclosed herein may include any of the glycosylation profiles and degrees disclosed herein. In general, the method of preparation presented herein provides for the production of a CTP-modified coagulation factor VIIa having a high level of glycosylation and a high percentage of glycosylated glycosylation sites.

In another embodiment, said high content of O-glycans in the CTP-modified coagulation factor VII/VIIa is at least 2 mol/mol. In another embodiment, said high content of O-glycans in the CTP-modified coagulation factor VII/VIIa is at least 4 mol/mol. In another embodiment, said high content of O-glycans in the CTP-modified coagulation factor VII/VIIa is at least 5 mol/mol. In another embodiment, said high content of O-glycans in the CTP-modified coagulation factor VII/VIIa is at least 6 mol/mol. In another embodiment, said high content of Oglycans in the CTP-modified coagulation factor VII/VIIa is at least 8 mol/mol. In another embodiment, said high content of O-glycans in the CTP-modified coagulation factor VII/VIIa is at least 10 mol/mol. In another embodiment, said high content of O-glycans in the CTP-modified coagulation factor VII/VIIa is at least 12 mol/mol. In another embodiment, said high content of O-glycans in the CTP-modified coagulation factor VII/VIIa is at least 14 mol/mol. In another embodiment, said high content of O-glycans in the CTP-modified coagulation factor VII/VIIa is at least 16 mol/mol.

In one embodiment, a high level or content of O-glycans is achieved as a result of the process of accumulation of biomass in the production process disclosed in this document. In another embodiment, a high level or content of O-glycans is achieved by using the clone in the manufacturing process disclosed herein. In another embodiment, high content or levels of O-glycans are maintained until the final drug substance is obtained.

In another embodiment, the CTP-modified coagulation factor VII/VIIa has a high sialic acid content. In another embodiment, said high content of sialic acid in the CTP-modified coagulation factor VII/VIIa is at least 4 mol/mol. In another embodiment, said high content of sialic acid in the CTP-modified coagulation factor VII/VIIa is at least 6 mol/mol. In another embodiment, said high content of sialic acid in the CTP modified coagulation factor VII/VIIa is at least 8 mol/mol. In another embodiment, said high content of sialic acid in the CTP modified coagulation factor VII/VIIa is at least 10 mol/mol. In another embodiment, said high content of sialic acid in the CTP modified coagulation factor VII/VIIa is at least 12 mol/mol. In another embodiment, said high content of sialic acid in the CTP modified coagulation factor VII/VIIa is at least 14 mol/mol. In another embodiment, said high content of sialic acid in CTP-46 040921 modified coagulation factor VII/VIIa is in another embodiment, said high content of modified blood coagulation factor VII/VIIa is in another embodiment, said high content of modified blood coagulation factor VII/ VIIa is in another embodiment said high content of modified blood coagulation factor VII/VIIa is in another embodiment said high content of modified blood coagulation factor VII/VIIa is in another embodiment said high content of modified blood coagulation factor VII/VIIa is in another embodiment implementation of the specified high content of the modified blood coagulation factor VII/VIIa is in another embodiment, the specified high content of at least 15 mol/mol. In sialic acid, the CTP is less than 17 mol/mol. In sialic acid, the CTP is less than 18 mol/mol. In sialic acid, the CTP is less than 20 mol/mol. In sialic acid, the CTP is less than 22 mol/mol. In sialic acid, the CTP is less than 25 mol/mol. In sialic acid, the CTP is less than 27 mol/mol. In sialic acid, the CTP of modified coagulation factor VII/VIIa is at least 30 mol/mol.

In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 40%. In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 50%. In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 60%. In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 70%. In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 80%. In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 85%. In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 90%. In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 91%. In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 92%. In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 93%. In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 94%. In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 95%. In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 96%. In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 97%. In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 98%. In another embodiment, said high percentage of carboxyglutamic acid in the CTP-modified coagulation factor VII/VIIa is at least 99%. The person skilled in the art will appreciate that the percentage of carboxyglutamic acid residues can be expressed in inverse form as the % fraction without domains with carboxyglutamic acid residues (the fraction without Gla domains), where, for example, if the high percentage of Gla is 40%, then the proportion without Gla domains is 60%.

In one embodiment, the step of obtaining CTP-modified coagulation factor VIIa includes obtaining clones of cells overexpressing CTP-modified coagulation factor VII. In another embodiment, expression of the CTP-modified coagulation factor VII is optimal. In another embodiment, the expression level is 30-1500 mg/l. In another embodiment, the expression level is at least 30 mg/L. In another embodiment, the expression level is at least 40 mg/L. In another embodiment, the expression level is at least 50 mg/l. In another embodiment, the expression level is at least 60 mg/l. In another embodiment, the expression level is at least 70 mg/l. In another embodiment, the expression level is 50-70 mg/l. In another embodiment, the expression level is at least 200 mg/l. In another embodiment, the expression level is

- 47 040921 at least 300 mg/l. In another embodiment, the expression level is at least 400 mg/l. In another embodiment, the expression level is at least 500 mg/l. In another embodiment, the expression level is at least 600 mg/l. In another embodiment, the expression level is at least 700 mg/l. In another embodiment, the expression level is at least 800 mg/l. In another embodiment, the expression level is at least 900 mg/l. In another embodiment, the expression level is at least 1000 mg/l. In another embodiment, the expression level is at least 1100 mg/l. In another embodiment, the expression level is at least 1200 mg/l. In another embodiment, the expression level is at least 1300 mg/l. In another embodiment, the expression level is at least 1400 mg/l. In another embodiment, the expression level is at least 1500 mg/l. In another embodiment, clones are propagated in media to form a master cell bank (MCB) and a working cell bank (WCB). In one embodiment, the clones in step (c) are obtained from MCB. In another embodiment, clones are obtained from WCB.

The CTP-modified FVII is obtained from the cell culture medium as a secreted gene product. If a protein or polypeptide is expressed without a secretion signal, the gene product can still be isolated from cell lysates, however. In order to obtain a pure homogeneous product, essentially free of other recombinant and host cell proteins, conventional purification procedures are carried out. First of all, cells and cell debris are repeatedly removed from the culture medium or lysate. The desired gene product can then be freed from contaminating soluble proteins, polypeptides, and nucleic acids, for example, by fractionation on immunoaffinity and ion exchange chromatography columns, affinity chromatography columns, ethanol precipitation, reverse phase HPLC, or Sephadex, hydroxyapatite, silica, or cation exchange chromatography. resins such as DEAE (see examples in this document). General techniques known in the art and leading to the purification of a heterologous protein expressed by recombinant host cells are known to the person skilled in the art and are described in the literature, for example, Harris et al. (Harris et al., Protein Purification: A Practical Approach, Pickwood and Hames, eds., IRL Press, Oxford, 1995) and Scopes (Scopes, R., Protein Purification, Springer Verlag, 1988). These methods can be used in whole or in part in the methods disclosed herein.

In another embodiment, this document discloses a method for producing CTP-modified FVII in mammalian cells under serum-free conditions, characterized in that (i) the mammalian cells contain a gene encoding CTP-modified FVII; (ii) the mammalian cells are grown under serum-free conditions allowing reproduction of the mammalian cells; (iii) in each case, at least one (1) of these mammalian cells is placed in a cell culture vessel under serum-free conditions; (iv) suitably placed mammalian cells are replicated under serum-free conditions; (v) reproducing cells are cultured under serum-free conditions in which the CTP-modified FVII is expressed; and (vi) the CTP-modified FVII product is then isolated from the cells or culture supernatant and purified and activated. In another embodiment of this process, the mammalian cell is a transfected mammalian cell into which the CTP-modified FVII gene has been introduced. Accordingly, the methods disclosed herein also relate to a method for producing recombinant gene products, characterized in that prior to step (i) of the process described above, mammalian cells are transfected with a nucleic acid that at least encodes a CTP-modified FVII. Stable transfection of an appropriate mammalian cell is preferred.

Examples of serum-free, protein-free, or chemically defined media include, for example, the commercially available F12 Ham's Medium (Sigma, Deisenhofen, Germany), RPMI 1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), Minimal Nutrient Medium (MEM ; Sigma), Iskov's Modified Dulbecco's Medium (IMDM; Sigma), CDCHO (Invitrogen, Carlsbad, CA, USA), CHO-S-SFMII (Invitrogen), CHO Serum-Free Cell Culture Medium (Sigma), CD-PowerCHO2 Medium ( Lonza) and protein-free CHO cell culture medium (Sigma). Each of these media can optionally be supplemented with various compounds such as hormones and/or other growth factors (e.g. insulin, transferrin, epidermal growth factor, insulin-like growth factor), salts (e.g. sodium chloride, calcium salts, magnesium salts, phosphates ), buffers (eg HEPES), nucleosides (eg adenosine, thymidine), glutamine, glucose or other equivalent nutrients, antibiotics and/or trace elements, or a commercially available feed such as Power Feed A (Lonza). If the reproducible cells are recombinant cells expressing one or more selectable markers, one or more suitable selection agents, such as antibiotics, may also be added to the medium.

It will be understood that in addition to the fact that the expression construct disclosed herein contains the necessary elements for transcription and translation of the inserted coding sequence (encoding polypeptide), it may also contain sequences designed to optimize stability, production, purification, the yield or activity of the expressed polypeptide. In one embodiment, the nucleotide sequence encoding

CTP-modified FVII is listed under SEQ ID NO: 4.

In some embodiments, the transformed cells are cultured under efficient conditions that allow high levels of the recombinant polypeptide to be expressed. In some embodiments, effective culture conditions include, without limitation, effective environmental, bioreactor, temperature, pH, and oxygen conditions that allow for protein production. The person skilled in the art will understand that the term "effective medium" can cover any medium in which the cell is cultured to obtain the recombinant polypeptide disclosed herein. In some embodiments, the medium typically includes an aqueous solution having sources of usable carbon, nitrogen, and phosphate, as well as essential salts, minerals, metals, and other nutrients such as vitamins. In some embodiments, cells disclosed herein can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and Petri dishes. In some embodiments, the implementation of the cultivation is carried out at a temperature, pH and oxygen content suitable for the recombinant cell. In some embodiments, establishing culture conditions is within the skill of the average person skilled in the art.

In one embodiment, culture conditions include a dissolved oxygen (DO) content of about 20-80%. In another embodiment, the DO content is about 20-30%. In another embodiment, the DO content is about 30-40%. In another embodiment, the DO content is about 40-50%. In another embodiment, the DO content is about 50-60%. In another embodiment, the DO content is about 60-70%. In another embodiment, the DO content is about 70-80%.

In one embodiment, the culture conditions include setting one pH as the initial value and shifting it to another during production. In another embodiment, the pH starts at about 7.3 and shifts to about 6.7 during incubation in the bioreactor. In another embodiment, the pH starts at about 7.3, about 7.2, or about 7.1 and shifts to about 6.7, about 6.8, about 6.9, or about 7.0 during incubation. in a bioreactor.

In some embodiments, depending on the vector and host cells in the expression system used for production, the resulting polypeptides disclosed herein remain in the recombinant cell or are secreted into the environment.

In one embodiment, after a predetermined period of time has elapsed, a recombinant polypeptide is recovered from the culture.

One of skill in the art will appreciate that the phrase recovering a recombinant polypeptide as used herein may encompass the collection of all media containing the polypeptide and may involve additional separation or purification steps.

In one embodiment, the polypeptides disclosed herein are purified using a variety of standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, size exclusion chromatography, reverse phase chromatography, chromatography on concanavalin A, chromatography on hydroxyapatite, chromatography and differential solubilization.

In one embodiment, each column may be operated at controlled or uncontrolled temperature.

In one embodiment, all chromatography steps are carried out in downstream mode. In another embodiment, all chromatography steps are performed in upstream mode.

In one embodiment, to facilitate extraction, the expressed coding sequence can be designed to encode a polypeptide disclosed herein and fused to a cleavable component. In one embodiment, the fusion protein can be designed such that the polypeptide can be easily isolated by affinity chromatography; for example, by immobilization on a column with a sorbent specific for the component to be cleaved off. In one embodiment, a cleavage site is inserted between the polypeptide and the cleavage component, and release of the polypeptide from the chromatographic column can be accomplished by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at that site (e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988) and Gardella et al., J Biol Chem 265:15854-15859 (1990)).

- 49 040921

In one embodiment, the polypeptide disclosed herein is recovered in a substantially pure form. One of skill in the art will appreciate that the phrase substantially pure can encompass a degree of purity that allows the protein to be used effectively in the uses described herein. Such form may also include highly glycosylated (with O-glycans and/or N-glycans) and highly sialylated forms, also disclosed herein. In another embodiment, such a form may include forms with a high percentage of carboxylated glutamic acid residues and/or a low percentage of the oxidized form. In yet another embodiment, a polypeptide that is a CTP-modified FVII disclosed herein may include a substantially pure and active form of the polypeptide.

In another embodiment, the percentage of oxidized CTP-modified FVII/FVIIa described herein is less than 20% of the oxidized form. In another embodiment, the percentage of oxidized CTP-modified FVII/FVIIa described herein is less than 15% of the oxidized form. In another embodiment, the percentage of oxidized CTP-modified FVII/FVIIa described herein is less than 10% of the oxidized form. In another embodiment, the percentage of oxidized CTP-modified FVII/FVIIa described herein is less than 8% of the oxidized form. In another embodiment, the percentage of oxidized CTP-modified FVII/FVIIa described herein is less than 5% of the oxidized form. In another embodiment, the percentage of oxidized CTP-modified FVII/FVIIa described herein is less than 4% of the oxidized form. In another embodiment, the percentage of oxidized CTP-modified FVII/FVIIa described herein is less than 3% of the oxidized form.

In one embodiment, oxidized form reduction and oxidized form control is performed throughout the manufacturing process from biomass accumulation to final drug substance.

In another embodiment, the degree of purity of said essentially pure and active polypeptide, which is a CTP-modified FVII, is at least 40%. In another embodiment, said substantially pure and active polypeptide, which is a CTP-modified FVII, is at least 50% pure. In another embodiment, the degree of purity of said essentially pure and active polypeptide, which is a CTP-modified FVII, is at least 60%. In another embodiment, said substantially pure and active FVII CTP-modified polypeptide is at least 70% pure. In another embodiment, said substantially pure and active FVII CTP-modified polypeptide is at least 75% pure. In another embodiment, said substantially pure and active FVII CTP-modified polypeptide is at least 80% pure. In another embodiment, said substantially pure and active FVII CTP-modified polypeptide is at least 85% pure. In another embodiment, said substantially pure and active FVII CTP-modified polypeptide is at least 90% pure. In another embodiment, said substantially pure and active FVII CTP-modified polypeptide is at least 91% pure. In another embodiment, said substantially pure and active FVII CTP-modified polypeptide is at least 92% pure. In another embodiment, said substantially pure and active FVII CTP-modified polypeptide is at least 93% pure. In another embodiment, said substantially pure and active FVII CTP-modified polypeptide is at least 94% pure. In another embodiment, said substantially pure and active FVII CTP-modified polypeptide is at least 95% pure. In another embodiment, the purity of said substantially pure and active FVII CTP-modified polypeptide is at least 96%. In another embodiment, said substantially pure and active FVII CTP-modified polypeptide is at least 97% pure. In another embodiment, said substantially pure and active FVII CTP-modified polypeptide is at least 98% pure. In another embodiment, said substantially pure and active FVII CTP-modified polypeptide is at least 99% pure. In a further embodiment, the purity percentage is selected from the group consisting of 97.3%, 97.6%, 97.4%, and 97.0%.

- 50 040921

In one embodiment, the subject disclosed herein is a human subject. In another embodiment, the subject is a domesticated animal. In another embodiment, the subject is a pet. In another embodiment, the subject is a mammal. In another embodiment, the subject is a farm animal. In another embodiment, the subject is a monkey. In another embodiment, the subject is a horse. In another embodiment, the subject is a cow. In another embodiment, the subject is a mouse. In another embodiment, the subject is a rat. In another embodiment, the subject is a canine. In another embodiment, the subject is a feline. In another embodiment, the subject is a bovine, ram, porcine, equine, murine, or deer animal. In one embodiment, the subject is male. In another embodiment, the subject is female. In one embodiment, the subject is a child, in another embodiment, a teenager, in another embodiment, an adult, or, in another embodiment, an elderly subject. In another embodiment, the subject is a pediatric subject, in another embodiment, a geriatric subject.

In one embodiment, the CTP-modified FVII polypeptide disclosed herein is synthesized using in vitro expression systems. In one embodiment, in vitro synthesis methods are well known in the art and the components of the system are commercially available.

In one embodiment, the generation of coagulation factor VIIa modified with CTP is performed using recombinant DNA technology.

In some embodiments, recombinant polypeptides are synthesized and purified; their therapeutic efficacy can be evaluated in vivo or in vitro. In one embodiment, the binding activity values of the recombinant coagulation factor VII/VIIa modified with the CTP disclosed herein can be determined using various assays.

In one embodiment, a method for producing a CTP-modified coagulation factor VIIa includes the step of obtaining clones characterized by optimal expression of said CTP-modified coagulation factor VII from said WCB and propagating said clones. In another embodiment, a method for producing CTP-modified coagulation factor VIIa includes the step of obtaining clones characterized by optimal expression of said CTP-modified coagulation factor VII from said MCB and propagating said clones. In another embodiment, cell clones are propagated in solution through a series of subculturing steps to the level of a production bioreactor. In another embodiment, a solution containing said subcultured clones is seeded into a bioreactor. In another embodiment, the bioreactor is a disposable bioreactor. In another embodiment, the bioreactor comprises a stainless steel bioreactor, an oscillating bioreactor such as GE's Wave system, a perfusion bioreactor, or any other bioreactor system known in the art. In one embodiment, the extraction of cells from the bioreactor is carried out by using a disposable filtration system. If large-scale production is performed, then continuous centrifugation can be used before using the filtration system.

In one embodiment, cell clones are further expanded or scaled up by serially culturing said cells in increasing size bioreactors until the desired scale is achieved. In another embodiment, the bioreactor is operated in fed-batch mode. In another embodiment, the bioreactor is operated in a batch mode. In another embodiment, the bioreactor is operated in a repetitive batch mode. In another embodiment, the bioreactor is operated in perfusion culture mode.

Peak viable cell densities vary depending on the type of bioreactor used. In one embodiment, the peak viable cell density in the bioreactor used in the preparation methods disclosed herein is approximately 0.2x106-1.4x106 cells/mL. In another embodiment, the peak viable cell density in the bioreactor used in the production methods disclosed herein is approximately 0.05x106-100x106. In another embodiment, the peak viable cell density in the bioreactor is approximately 0.05x106-0.5x106. In another embodiment, the peak viable cell density in the bioreactor is approximately 0.5x106-5x106. In another embodiment, the peak viable cell density in the bioreactor is approximately 5.0x106-50x106. In another embodiment, the peak viable cell density in the bioreactor is approximately 50x106-100x106.

Feed schemes for using a bioreactor can be different, for example, with repetitive daily feed starting from a certain day, or with a fixed feed after a certain number of days, in addition, the percentage of added feed may be different, ranging from a few percent to even 50% or more.

DMSO can be added to the bioreactor at various concentrations, as is known in the art. In one embodiment, 0.1-3% DMSO is added to the bioreactor during use. In another embodiment, 0.1-0.5% DMSO is added. In another embodiment, 0.5-1.0% DMSO is added. In another embodiment, 1.0-1.5% DMSO is added. In another embodiment, 1.5-2.0% DMSO is added. In another embodiment, 2.0-2.5% DMSO is added. In another embodiment, 2.5-3.0% DMSO is added.

In one embodiment, the method for producing CTP-modified coagulation factor VIIa includes the step of purifying and activating the clarified pooled solution to obtain a purified solution of active CTP-modified FVII. In another embodiment, the purified protein solution obtained using the methods presented herein contains at least 5-95% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution obtained using the methods presented herein contains at least 5% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution obtained using the methods presented herein contains at least 10% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution obtained using the methods presented herein contains at least 20% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution obtained using the methods presented herein contains at least 30% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution obtained using the methods presented herein contains at least 40% CTP-modified clotting factor VII/VIIa. In another embodiment, the purified protein solution contains at least 50% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution contains at least 60% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution contains at least 70% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution contains at least 80% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution contains at least 90% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution obtained using the methods presented herein contains at least 95% CTP-modified clotting factor VH/VIIa.

In one embodiment, the clarified harvest is held for up to 24 hours at 2-25°C. In another embodiment, the clarified harvest is stored at at least 5° C. for up to one month.

In one embodiment, the clarified harvest obtained in this step is tested for bioburden, presence of bacterial endotoxins, specific protein content, presence of residual DNA, viruses, virus-like particles and/or mycoplasmas, or any combination thereof.

In one embodiment, purification of the clarified collected material is carried out by sequentially performing the steps including: (g) concentration, diafiltration and purification of said clarified collected solution, where said concentration, diafiltration and purification are carried out using a hollow fiber membrane cassette or a tangential in-line filtration cassette, sequentially passing said clarified collected solution through an anion exchange chromatography column and a hydrophobic interaction chromatography column; (h) receiving the specified clarified collected material obtained after this stage; and (i) inactivating the viruses present in said clarified harvest material by incubation in a solution toxic to said viruses; (j) obtaining said clarified collected solution after (h) and concentrating, diafiltering and purifying said clarified collected solution, where after said concentration, diafiltration, activation and purification, sequentially passing said clarified collected solution through an affinity chromatography column, a chromatography column on multimodal matrices or mixed media, a hydrophobic interaction chromatography (HIC) column and an anion exchange chromatography column; (j) obtaining said clarified pool solution after step (i) and physically removing viruses from said clarified pool solution by nanofiltration; (k) obtaining said clarified harvest solution after step (j) and concentrating, diafiltering and purifying said clarified harvest solution to obtain a maximally purified clarified harvest containing said highly glycosylated form of CTP-modified FVII/FVIIa.

In one embodiment, the CTP-modified FVII is activated during purification. In an alternative embodiment, the CTP-modified FVII is activated after purification. In another embodiment, the CTP-modified FVII is activated concurrently with any purification step.

In one embodiment, ultrafiltration and diafiltration using a hollow fiber membrane cartridge or an equivalent UF/DF stage based on the TFF method can be performed to concentrate and filter the clarified collected material. The nominal molecular weight cutoff value for the cartridge is 10,000 kDa. In another embodiment, the membrane cartridge may fit PES/PS/RC membranes with a cutoff of 3 to 30 kDa. In another embodiment, the UF/DF step can be performed between chromatography steps. In another embodiment, the UF/DF step may be performed prior to using the anion exchange chromatography column. In another embodiment, the UF/DF step may be performed prior to using hydrophobic interaction chromatography (HIC). In another embodiment, the UF/DF step can be performed after using hydrophobic interaction chromatography (HIC). In yet another embodiment, UF/DF can be performed in multiple steps, for example, UF/DF can be performed after collection of material, between chromatography steps, or as a step after virus removal by nanofiltration, or any combination thereof.

In another embodiment, the anion exchange chromatography column is a DEAE-Sepharose Fast Flow column. In another embodiment, a DEAE column purifies the highly glycosylated form of said

CTP-modified coagulation factor VII/VIIa. In one embodiment, the higher the degree of glycosylation, the better the pharmacodynamic characteristics of the CTP-modified coagulation factor VII/VIIa. In another embodiment, the anion exchange chromatography column may include other anion exchange chromatography columns known in the art, such as an anion exchange chromatography column with Capto DEAE or other resins such as Eshmuno Q.

In one embodiment, the hydrophobic interaction chromatography column is a hydrophobic interaction chromatography (HIC) column with phenyl sorbents. The number of cycles using HIC with phenyl sorbents can be in the range of approximately 1-10. In one embodiment, 1-3 cycles are performed. In another embodiment, 1-5 cycles are performed. In another embodiment, 1-6 cycles are performed. In another embodiment, 1-7 cycles are performed. In another embodiment, 1-8 cycles are performed. In another embodiment, 1-9 cycles are performed. In another embodiment, 1-10 cycles are performed. In another embodiment, for washing and elution using buffers known from the prior art. In one embodiment, the elution buffer contains ammonium sulfate and propylene glycol. In one embodiment, the elution buffer contains ammonium sulfate and ethylene glycol.

In one embodiment, the hydroxyapatite mixed bed chromatography column includes a ceramic hydroxyapatite mixed bed chromatography (CHT) column. The number of CHT usage cycles may be in the range of approximately 1-10. In one embodiment, 1-3 cycles are performed. In another embodiment, 1-5 cycles are performed. In another embodiment, 16 cycles are performed. In another embodiment, 1-7 cycles are performed. In another embodiment, 1-8 cycles are performed. In another embodiment, 1-9 cycles are performed. In another embodiment, 1-10 cycles are performed. Elution from a CHT column can be done with approximately 3-10 column volumes (CV). In one embodiment, elution is performed at approximately 3 CV. In another embodiment, the elution is performed at approximately 4 CV. In another embodiment, elution is performed at about 5 CV. In another embodiment, the elution is performed at approximately 6 CV. In another embodiment, the elution is performed at approximately 7 CV. In another embodiment, the elution is performed at approximately 8 CV. In another embodiment, elution is performed at approximately 9 CV. In another embodiment, elution is performed at about 10 CV.

In one embodiment, viruses that may be present in the clarified harvest due to infection are inactivated in the clarified harvest. In another embodiment, the viruses are inactivated with a 1% solution of Triton X-100. In another embodiment, the viruses are inactivated with a 0.2-2% solution of Triton X-100. In another embodiment, the viruses are inactivated with a 0.5% solution of Triton X-100. In another embodiment, the viruses are inactivated with a 1-4% solution of Triton X-100. In another embodiment, the viruses are inactivated with a 0.2-0.5% solution of Triton X-100. In another embodiment, the viruses are inactivated with a 0.5-1.0% solution of Triton X-100. In another embodiment, the viruses are inactivated with a 2% solution of Triton X-100. In another embodiment, the viruses are inactivated with a 3% solution of Triton X-100. In another embodiment, the viruses are inactivated with a 4% solution of Triton X-100. In another embodiment, the viruses are inactivated with a 5-10% solution of Triton X-100. In another embodiment, virus inactivation with Triton X-100 solution is continued for about 0.5-24 hours. In another embodiment, virus inactivation with Triton-X solution is continued for about 0.5-1 hour. In another embodiment, Virus inactivation with Triton-X solution continues for approximately 1-2 hours. In another embodiment, virus inactivation with Triton-X solution continues for approximately 2-3 hours. In another embodiment, virus inactivation with Triton-X solution X continues for about 3-4 hours. In another embodiment, virus inactivation with Triton-X solution continues for about 4-6 hours. In another embodiment, virus inactivation with Triton-X solution continues for about 6-8 hours. h. In another embodiment, virus inactivation with Triton-X solution continues for approximately about 8-10 hours. In another embodiment, virus inactivation with Triton-X solution continues for about 10-12 hours. In another embodiment, virus inactivation with Triton-X solution continues for about 12-24 hours.

One skilled in the art will appreciate that other concentrations or other solutions available in the art that are toxic to these viruses, including but not limited to sodium cholate and Tween 80, may be used in the methods disclosed herein. In another embodiment, for virus inactivation in step (h), a mixture of tri-n-butyl phosphate (TNBP) and polysorbate 80 (Tween 80) is used.

In one embodiment, viruses are physically removed using nanofiltration. One of skill in the art will appreciate that any filter known in the art can be used to remove viruses in the methods disclosed herein. In another embodiment, nanofiltration is carried out using a Planova or Planova type filter cartridge (1-60 mm ² ). After carrying out such methods, confirmation of the purification of the clarified collected material from viruses is carried out using methods known from the prior art.

In one embodiment, at least 20% recovery of highly glycosylated CTP-modified coagulation factor VII/VIIa is achieved using the methods disclosed herein. In another embodiment, the methods achieve a recovery corresponding to at least 5%, at least 10%, at least 15%, 20%, at least 25%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9% recovery of highly glycosylated CTP-modified coagulation factor VII/VIIa.

In one embodiment, the methods disclosed herein further comprise characterizing said CTP-modified polypeptide after purification of highly glycosylated CTP-modified coagulation factor VII/VIIa. In another embodiment, the purity of the CTP-modified coagulation factor VII is determined. In another embodiment, the level of glycosylation is determined. In another embodiment, the degree of occupancy of glycosylation sites is determined. In one embodiment, the resulting CTP-modified coagulation factor VII/VIIa is determined for purity, glycosylation level, and glycosylation site occupancy.

In another embodiment, the cell clones used in the methods disclosed herein are stored in a frozen cell bank. In another embodiment, the cell clones are stored in a lyophilized cell bank.

In another embodiment, the cell bank used in the methods and compositions disclosed herein is a master cell bank. In another embodiment, the cell bank is a working cell bank. In another embodiment, the cell bank is a Good Manufacturing Practice (GMP) cell bank. In another embodiment, the cell bank is designed to obtain clinically pure material. In another embodiment, the cell bank complies with regulatory practices for use in medicine. In another embodiment, the cell bank is any other type of cell bank known in the art.

In another embodiment, Good Manufacturing Practice is defined by the US Code of Federal Regulations (CFR Title 21 Parts 210-211). In another embodiment, good manufacturing practice is defined by other standards for the production of a clinically pure material or material intended for human consumption; for example, the standards of a state other than the United States. Each possibility represents a separate embodiment disclosed in this document.

In another embodiment, the medium used for cell propagation contains the method

- 54 040921 rexat (MXT). In another embodiment, the medium is a medium that does not contain methotrexate. In another embodiment, the concentration of MXT present in the medium is approximately 0.1-2 μM. In another embodiment, the concentration of MXT present in the medium is approximately 0.1-0.5 μM. In another embodiment, the concentration of MXT present in the medium is approximately 0.5-1.0 μM. In another embodiment, the concentration of MXT present in the medium is approximately 1.0-1.5 μM. In another embodiment, the concentration of MXT present in the medium is approximately 1.5-2.0 μM. It will be well understood that the term medium may encompass a liquid or gel or powder suitable for growing or culturing cells containing the CTP-modified coagulation factor VII disclosed herein. Such media may alternatively be referred to as growth media or culture media, and may include, without limitation, nutrient media, enriched media, minimal media, differential media, transport media, or selective media. In an additional aspect, the selection medium may be suitable for selecting a specific group of cells during the manufacturing process.

In one embodiment, the purified protein solution contains at least 5-95% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution contains at least 5% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution contains at least 10% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution contains at least 15% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution contains at least 20% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution contains at least 30% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution contains at least 40% CTP-modified coagulation factor VIIa. In another embodiment, the purified protein solution contains at least 50% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution contains at least 60% CTP-modified coagulation factor VIIa. In another embodiment, the purified protein solution contains at least 70% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified protein solution contains at least 80% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified solution contains at least 90-95% CTP of modified coagulation factor VII/VIIa. In another embodiment, the purified solution contains 95.1-99.9% CTP-modified coagulation factor VII/VIIa. In another embodiment, the purified solution contains 100% CTP-modified coagulation factor VII/VIIa.

In one embodiment, the resulting CTP-modified coagulation factor has a high % level of gamma carboxylation. One skilled in the art will appreciate that the percentage level (%) of coagulation factor gamma-carboxylation may be directly proportional to the specific activity of the CTP-modified coagulation factor. In one embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes at least 50% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes at least 60% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVIICTP3 or FVIIa-CTP3 includes at least 70% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes at least 80% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes at least 90% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes at least 92% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes at least 93% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes at least 94% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes at least 95% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes at least 96% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes at least 97% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes at least 98% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes at least 99% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes 100% gamma carboxylation.

In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes 40-50% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes 50-60% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes 60-70% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes 70-80% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes 80-85% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes 85-90% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes 90-92% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes 90-95% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes 95-97% gamma carboxylation. In another embodiment, the % gamma carboxylation level of FVII-CTP3 or FVIIa-CTP3 includes 95-100% gamma carboxylation.

In one embodiment, the removal of low gamma carboxylation forms is performed in a multimodal matrix or vehicle chromatography step of the mixed type manufacturing process disclosed herein (see step 9, FIG. 17).

In one embodiment, the resulting CTP-modified coagulation factor VII/VIIa is highly glycosylated. It will be well understood by one of skill in the art that the term highly glycosylated in relation to CTP-modified coagulation factor VII/VIIa may encompass a glycosylation level of approximately 70-80% of the total polypeptide that is CTP-modified coagulation factor VII/VIIa. In another embodiment, the highly glycosylated CTP-modified coagulation factor VII/VIIa has a glycosylation level of at least 70%. In another embodiment, the highly glycosylated CTP-modified coagulation factor VII/VIIa has a glycosylation level of at least 80%. In another embodiment, the term may encompass a glycosylation level of approximately 81-90% of the total CTP-modified coagulation factor VII/VIIa polypeptide. In another embodiment, the highly glycosylated CTP-modified coagulation factor VII/VIIa has a glycosylation level of at least 90%. In another embodiment, the term may encompass a glycosylation level of approximately 91-95% of the total CTP-modified coagulation factor VIIa polypeptide. In another embodiment, the term may encompass a glycosylation level of approximately 95.1-99% of the total CTP-modified coagulation factor VII/VIIa polypeptide. In another embodiment, the term may encompass a glycosylation level of 100% of the total polypeptide that is a CTP-modified coagulation factor VII/VIIa. Highly glycosylated polypeptides that are CTP-modified coagulation factors VII/VIIa may have advantageous properties in long-acting methods of using coagulation factor VII/VIIa to reduce the frequency of administration. High levels of glycosylation promote a significant increase in hydrodynamic volume of CTP-modified coagulation factor VII/VIIa, eg CTP-modified coagulation factor VIIa, compared to recombinant coagulation factor VII/VIIa. This can lead to a prolongation of the circulation time in the bloodstream of the CTP-modified coagulation factor VIIa.

In one embodiment, the number of O-glycans per CTP is at least 4-6. In another embodiment, the number of O-glycans per CTP is 4-6. In another embodiment, the number of O-glycans per CTP is at least 4-8. In another embodiment, the number of O-glycans per CTP is 4-8. In one embodiment, the number of O-glycans per CTP is at least 6-8. In one embodiment, the number of O-glycans per CTP is 6-8. In another embodiment, the number of O-glycans per CTP is at least 4. In another embodiment, the number of O-glycans per CTP is at least 5. In another embodiment, the number of O-glycans per CTP is at least 4. The CTP is at least 6. In another embodiment, the number of O-glycans per CTP is at least 7. In another embodiment, the number of O-glycans per CTP is 8.

In one embodiment, the number of O-glycans per polypeptide, which is a CTP-modified coagulation factor VII/VIIa, having one attached

- 56 040921

CTP is at least 4-6. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having one CTP attached is at least 6-8. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having one CTP attached is at least 4-8. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having two CTP units attached is at least 8-12. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having two CTP units attached is at least 12-16. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having two CTP units attached is at least 8-16. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having three CTP units attached is at least 12-18. In another embodiment, the number of Oglycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having three CTP units attached is at least 18-24. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having three CTP units attached is at least 12-24. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having four CTP units attached is at least 16-24. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having four attached CTP units is at least 24-32. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having four attached CTP units is at least 16-32. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having five CTP units attached is at least 20-30. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having five attached CTP units is at least 30-40. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having five CTP units attached is at least 20-40. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having six attached CTP units is at least 24-36. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having six CTP units attached is at least 36-48. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having six attached CTP units is at least 24-48. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having seven CTP units attached is at least 28-35. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having seven attached CTP units is at least 42-56. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having seven CTP units attached is at least 28-56. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having eight attached CTP units is at least 32-48. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having eight CTP units attached is at least 48-64. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having eight attached CTP units is at least 32-64. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having nine CTP units attached is at least 36-54. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having nine attached CTP units is at least 54-72. In a different

- 57 040921 embodiment, the number of O-glycans per polypeptide, which is a CTP-modified coagulation factor VII/VIIa, having nine attached CTP units, is at least 36-72. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having ten attached CTP units is at least 40-60. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having ten CTP units attached is at least 60-80. In another embodiment, the number of O-glycans per polypeptide that is a CTP-modified coagulation factor VII/VIIa having five attached CTP units is at least 40-80.

In one embodiment, the O-glycan occupancy per CTP is at least 70%. In another embodiment, the O-glycan occupancy per CTP is at least 80%. In another embodiment, the Oglycan occupancy per CTP is at least 90%. In another embodiment, the O-glycan occupancy per CTP is 100%.

In one embodiment, a high percentage of N-glycans per CTP-modified FVII/FVIIa is charged. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is at least 40%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is at least 50%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is at least 60%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is at least 70%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is at least 80%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is at least 85%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is at least 90%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is at least 95%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is 100%.

In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is approximately 40%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is approximately 50%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is approximately 60%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is approximately 70%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is approximately 80%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is approximately 85%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is approximately 90%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is approximately 95%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is approximately 100%.

In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 10% to 30%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 20% to 40%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 30% to 40%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 20% to 50%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 40% to 50%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 30% to

- 58 040921

60%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 50% to 60%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 40% to 70%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 60% to 70%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 50% to 80%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 70% to 80%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 55% to 85%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 80% to 85%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 60% to 90%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 85% to 90%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 65% to 95%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 90% to 95%. In another embodiment, the percentage of N-glycans per CTP-modified FVII/FVIIa that are charged is from about 95% to 100%.

In one embodiment, a high level of charged N-glycans is achieved as a result of the accumulation of biomass in the manufacturing process disclosed herein. In another embodiment, a level in excess of 60% charged N-glycans is reached early in the DSP and this level is maintained until the final drug substance is obtained.

In one embodiment, a high level of charged N-glycans is achieved as a result of the accumulation of biomass in the manufacturing process disclosed herein. In another embodiment, a level in excess of 60% charged N-glycans is reached early in the DSP and this level is maintained until the final drug substance is obtained.

In one embodiment, the CTP-modified coagulation factor VII/VIIa is highly sialylated. One skilled in the art will appreciate that the term highly sialylated in relation to CTP-modified coagulation factor VII/VIIa may encompass a sialylation level of approximately 70-80% of the total polypeptide that is CTP-modified coagulation factor VIIa. In another embodiment, the term may encompass a level of sialylation that is approximately 80-90% of the total CTP-modified coagulation factor VII/VIIa polypeptide. In another embodiment, the term may encompass a sialylation level of approximately 90-95% of the total CTP-modified coagulation factor VII/VIIa polypeptide. In another embodiment, the term may encompass a sialylation level of approximately 95.1-99% of the total CTP-modified coagulation factor VII/VIIa polypeptide. In another embodiment, the term may encompass a sialylation level of 100% of the total CTP-modified coagulation factor VII/VIIa polypeptide. In another embodiment, the O-glycan structure in the CTP-modified coagulation factor VIIa contains a monosialylated core 1.

In one embodiment, the high percentage of sialylation and O-linked glycans in the CTP-modified FVII/FVIIa described herein results in an increase in the specific activity of the CTP-modified FVII/FVIIa. In another embodiment, the high percentage of sialylation and O-linked glycans in the CTP-modified FVII/FVIIa described herein results in an increase in the hydrodynamic volume of the CTP-modified FVII/FVIIa.

In one embodiment, the CTP-modified FVII/FVIIa described herein has specific activity. In another embodiment, the CTP-modified FVII/FVIIa is a substantially pure and active polypeptide that is a CTP-modified FVII. In another embodiment, CTP-modified FVII/FVIIa is produced using the method described herein. In another embodiment, the specific activity is at least 5000 U/mg. In another embodiment, the specific activity is at least 7500 units/mg. In another embodiment, the specific asset

- 59 040921 the value is at least 10000 units/mg. In another embodiment, the specific activity is at least 10500 U/mg. In another embodiment, the specific activity is at least 15,000 units/mg. In another embodiment, the specific activity is at least 20,000 U/mg. In another embodiment, the specific activity is at least 25,000 U/mg. In another embodiment, the specific activity is at least 27500 U/mg. In another embodiment, the specific activity is at least 30,000 U/mg. In another embodiment, the specific activity is at least 35,000 U/mg. In another embodiment, the specific activity is at least 40,000 U/mg. In a further embodiment, the specific activity is selected from the group consisting of 15563 U/mg, 16720 U/mg, 22478 U/mg and 23608 U/mg.

In one embodiment, the CTP-modified coagulation factor VII/VIIa polypeptide consists of two CTPs attached to the carboxyl terminus of said coagulation factor VII/VIIa and one carboxyl terminus of said coagulation factor VII/VIIa and one carboxyl-terminal chorionic gonadotropin peptide attached to the amino terminus of said coagulation factor VII/ VIIa. In another embodiment, the CTP-modified coagulation factor VII/VIIa polypeptide consists of a single carboxy-terminal chorionic gonadotropin peptide attached to the carboxyl terminus of said coagulation factor VII/VIIa.

In one embodiment, an expression vector containing a coding portion encoding said CTP-modified coagulation factor VII/VIIa also contains a promoter, a sequence encoding said CTP-modified polypeptide, and a polyadenylation sequence. In one embodiment, the polyadenylation sequence is the simian virus (SV) 40 polyadenylation sequence.

In one embodiment, the CTP-modified coagulation factor VII is expressed at a level of 30-1500 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 30 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 40 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 50 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 60 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 70 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 50-70 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 80 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 90 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 70-100 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 100 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 200 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 100-200 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 300 mg/L. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 200-300 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 400 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 300-400 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 500 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 500-600 mg/L. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 600 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 600-700 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 700 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 701-800 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 800 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 801-900 mg/l. In another variant

The CTP-modified coagulation factor VII is expressed at a level of at least 900 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 901-1000 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 1000 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 1001-1100 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 1100 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 1101-1200 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 1200 mg/L. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 1201-1300 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 1300 mg/l. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 1301-1400 mg/L. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 1400 mg/L. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 1401-1500 mg/L. In another embodiment, the CTP-modified coagulation factor VII is expressed at a level of at least 1500 mg/L.

One of skill in the art will appreciate that the term expression may encompass the transcription and/or translation of a heterologous nucleic acid sequence in a host cell. The level of expression of the desired product/protein of interest in the host cell can be determined based on the amount of the corresponding mRNA or cDNA present in the cell, or the amount of the desired polypeptide/protein of interest encoded by the selected sequence, as indicated in these examples. For example, mRNA transcribed from a selected sequence can be quantified by hybridization using northern blotting, RNA protection against ribonucleases, in situ hybridization with cellular RNA, or by PCR (see Sambrook et al., 1989; Ausubel et al., 1987, updated edition). Proteins encoded by the selected sequence can be quantified using various methods, for example, using ELISA, using Western blotting, using radioimmunoassays, using immunoprecipitation, using analysis of the biological activity of the protein, using protein immunostaining followed by FACS- by analysis (see Sambrook et al., 1989; Ausubel et al., 1987, updated edition) or by time-resolved homogeneous fluorescence (HTRF) analyses. In one embodiment, quantification of CTP-modified coagulation factor VIIa involves the use of reverse phase high performance liquid chromatography (RP-HPLC). In another embodiment, the system for RP-HPLC contains a C-18 column. In another embodiment, the system for RP-HPLC contains a C-8 column. In another embodiment, the methods disclosed herein use RP-HPLC to quantify CTP-modified coagulation factor VII in a harvest (see Example 3, steps 2-5). In another embodiment, the methods disclosed herein use RP-HPLC to quantify CTP-modified coagulation factor VII during purification.

In another embodiment, the cell bank or stock of frozen cells disclosed herein exhibits a viability after thawing in excess of 90%. In another embodiment, storage continues for an indefinite period of time.

In another embodiment, storage continues for 2 weeks. In another embodiment, storage continues for 3 weeks. In another embodiment, storage continues for 1 month. In another embodiment, storage continues for 2 months. In another embodiment, storage continues for 3 months. In another embodiment, storage continues for 5 months. In another embodiment, storage continues for 6 months. In another embodiment, storage continues for 9 months. In another embodiment, storage continues for 1 year.

In another embodiment, the cell bank or stock of frozen cells disclosed herein is cryopreserved by a method comprising growing a cell culture in a defined medium disclosed herein, freezing the culture in a solution containing glycerol, and storing the cell clones at a temperature below -20°С. In another embodiment, the temperature is approximately -70°C. In another embodiment, the temperature is approximately -70 - -80°C. In another embodiment, any defined media disclosed herein can be used in the method.

- 61 040921

Each environment of a certain composition represents a separate embodiment, disclosed in this document.

In another embodiment of the methods and compositions disclosed herein, the culture is seeded from a cell bank. In another embodiment, the culture is seeded from a stock of frozen cells. In another embodiment, the culture is seeded from a starter culture. In another embodiment, the culture is seeded from a colony. In another embodiment, the culture is sown in the middle of the logarithmic growth phase. In another embodiment, the culture is sown approximately in the middle of the logarithmic growth phase. In another embodiment, the culture is sown in a different growth phase.

In another embodiment of the methods and compositions disclosed herein, the solution used for freezing contains DMSO in an amount of 2-20%. In another embodiment, the amount is 2%. In another embodiment, the amount is 20%. In another embodiment, the amount is 1%. In another embodiment, the amount is 1.5%. In another embodiment, the amount is 3%. In another embodiment, the amount is 4%. In another embodiment, the amount is 5%. In another embodiment, the amount is 2%. In another embodiment, the amount is 2%. In another embodiment, the amount is 7%. In another embodiment, the amount is 7.5%. In another embodiment, the amount is 9%. In another embodiment, the amount is 10%. In another embodiment, the amount is 12%. In another embodiment, the amount is 14%. In another embodiment, the amount is 16%. In another embodiment, the amount is 18%. In another embodiment, the amount is 22%. In another embodiment, the amount is 25%. In another embodiment, the amount is 30%. In another embodiment, the amount is 35%. In another embodiment, the amount is 40%.

In another embodiment, the additive is sucrose. In another embodiment, the additive is any other additive with colligative properties or additive with antifreeze properties known in the art. Each possibility represents a separate embodiment disclosed in this document.

In one embodiment, the freezing solution used in the methods and compositions disclosed herein contains conditioned medium and DMSO. In one embodiment, the freeze solution used in the methods and compositions disclosed herein contains approximately 46.255% conditioned medium and 7.5% DMSO.

In one embodiment, the cell culture is grown using techniques conventional in the art. In another embodiment, during cell culture growth, a constant pH value is maintained. In another embodiment, the pH is maintained at about 7.0. In another embodiment, the pH is about 6. In another embodiment, the pH is about 6.5. In another embodiment, the pH is about 7.5. In another embodiment, the pH is about 8. In another embodiment, the pH is about 6.5-7.5. In another embodiment, the pH is about 6-8. In another embodiment, the pH is about 6-7. In another embodiment, the pH is about 7-8.

In another embodiment, a constant temperature is maintained during the growth of the culture. In another embodiment, the temperature is maintained at approximately 37°C. In another embodiment, the temperature is 37°C. In another embodiment, the temperature is 25°C. In another embodiment, the temperature is 27°C. In another embodiment, the temperature is 28°C. In another embodiment, the temperature is 30°C. In another embodiment, the temperature is 32°C. In another embodiment, the temperature is 34°C. In another embodiment, the temperature is 35°C. In another embodiment, the temperature is 36°C. In another embodiment, the temperature is 38°C. In another embodiment, the temperature is 39°C.

In another embodiment, a constant concentration of dissolved oxygen is maintained during culture growth. In another embodiment, the concentration of dissolved oxygen is maintained at a 20% saturation level. In another embodiment, the concentration corresponds to a 15% saturation level. In another embodiment, the concentration corresponds to a 16% saturation level. In another embodiment, the concentration corresponds to an 18% saturation level. In another embodiment, the concentration corresponds to a 22% saturation level. In another embodiment, the concentration corresponds to a 25% saturation level. In another embodiment, the concentration corresponds to a 30% saturation level. In another embodiment, the concentration corresponds to a 35% saturation level. In another embodiment, the concentration corresponds to a 40% saturation level. In another embodiment, the concentration corresponds to a 45% saturation level. In another embodiment, the concentration corresponds to a 50% saturation level. In another embodiment, the concentration corresponds to a 55% saturation level. In another embodiment, the concentration corresponds to a 60% saturation level. In another embodiment, the concentration corresponds to a 65% saturation level. In another embodiment, the concentration corresponds to the 70% saturation level. In another embodiment, the concentration corresponds to a 75% saturation level. In another embodiment, the concentration corresponds to the 80% saturation level. In another embodiment, the concentration corresponds to the 85% saturation level. In another embodiment, the concentration corresponds to the 90% saturation level. In another embodiment, the concentration corresponds to a 95% saturation level. In another embodiment, the concentration corresponds to a 100% saturation level. In another embodiment, the concentration corresponds to a saturation level close to 100%.

In another embodiment of the methods and compositions disclosed herein, the culture is grown in a medium having a maximum volume of 2 liters per vessel. In another embodiment, the medium has a maximum volume of 200 ml per vessel. In another embodiment, the medium has a maximum volume of 300 ml per vessel. In another embodiment, the medium has a maximum volume of 500 ml per vessel. In another embodiment, the medium has a maximum volume of 750 ml per vessel. In another embodiment, the medium has a maximum volume of 1 liter per vessel. In another embodiment, the medium has a maximum volume of 1.5 liters per vessel. In another embodiment, the medium has a maximum volume of 2.5 liters per vessel. In another embodiment, the medium has a volume of 3 liters per vessel. In another embodiment, the medium has a volume of 5 liters per vessel. In another embodiment, the medium has a volume of at least 5 liters per vessel. In another embodiment, the medium has a volume of at least 10 liters per vessel.

In another embodiment, the medium has a minimum volume of 2 liters per vessel. In another embodiment, the medium has a minimum volume of 500 ml per vessel. In another embodiment, the medium has a minimum volume of 750 ml per vessel. In another embodiment, the medium has a minimum volume of 1 liter per vessel. In another embodiment, the medium has a minimum volume of 1.5 liters per vessel. In another embodiment, the medium has a minimum volume of 2.5 liters per vessel. In another embodiment, the medium has a minimum volume of 3 liters per vessel. In another embodiment, the medium has a minimum volume of 4 liters per vessel. In another embodiment, the medium has a minimum volume of 5 liters per vessel. In another embodiment, the medium has a minimum volume of 6 liters per vessel. In another embodiment, the medium has a minimum volume of 8 liters per vessel. In another embodiment, the medium has a minimum volume of 10 liters per vessel.

In another embodiment, the freeze step is performed when the culture has a density of 1x106 viable cells (VC/ml. In another embodiment, the biomass is 1.5x106 VC/ml. In another embodiment, the biomass is 1.5x106 VC/ml. In another embodiment, the biomass is 2x106 VC/ml In another embodiment the biomass is 3x106 VC/ml In another embodiment the biomass is 4x106 VC/ml In another embodiment the biomass is 5x106 VC/ml In another embodiment the biomass is 7x106 VC/ml In another embodiment, the biomass is 9x106 VC/ml In another embodiment, the biomass is 10x106 VC/ml In another embodiment, the biomass is 12x106 VC/ml In another embodiment, the biomass is 15x106 VC/ml In another embodiment, the biomass is 20x107 VC/ml In another embodiment, the biomass is 25x106 VC/ml In another embodiment those exercise biomass is 30x107 VC/mL. In another embodiment, the biomass is 33x106 VC/ml. In another embodiment, the biomass is 40x106 VC/ml. In another embodiment, the biomass is 50x106 VC/ml. In another embodiment, the biomass is greater than 50x106 VC/ml.

In another embodiment of the methods and compositions disclosed herein, the cell culture is subjected to liquid nitrogen flash freezing followed by storage at the final freezing temperature. In another embodiment, the culture is frozen more smoothly; for example, by placing the culture in a vial with a final storage temperature. In another embodiment, the culture is frozen using any other cell culture freezing method known in the art.

One skilled in the art will appreciate that the terms cell culture and tissue culture can be used interchangeably and refer to the maintenance of cells in vitro in suspension culture in a liquid medium or on a surface such as glass, plastic or agar on which the liquid medium is placed. In general, cell culture requires a buffered medium to maintain a suitable constant pH. The media used for culturing cells are generally formulated to contain a sufficient supply of essential nutrients and their osmotic properties can be tailored to the specific cells supported, while also controlling the temperature and composition of the gas phase within suitable limits. Cell culture techniques are well known in the art. See, for example, Morgan et al. 1993 Animal

- 63 040921

Cell Culture, BIOS Scientific Publishers, Oxford, UK; and Adams, R. L. P. 1990 Cell Culture for Biochemists,

Second edition, Elsevier.

One of skill in the art will appreciate that the term passage may encompass the act of subculturing a population of cells. One of skill in the art will appreciate that the term subculture may encompass a cell culture formed by seeding into a sterile medium, which in one embodiment is a fresh sterile medium, sampled from a previous culture.

One of skill in the art will also appreciate that the term cell line can encompass a population of cells obtained from a primary culture using subculturing techniques. Thus, the primary culture can be subcultured to produce two or more new cultures, and the subculture can be repeated at regular intervals over several months to maintain the cell line. Subculturing can be carried out using well-known cell culture techniques.

In one embodiment, passaged cell lines and immortalized cell lines may be characterized by their expression of specific functional markers such as keratins, hormone and growth factor receptors, and the like.

In some aspects, the cultivation can be carried out in serum-free media of a certain composition with the addition of growth factors. In other aspects, the medium contains blood serum with or without the addition of growth factors. Such modifications can be empirically determined by one of ordinary skill in the art in order to optimize cell proliferation.

One skilled in the art will appreciate that the term cell line may encompass a population of cells derived from a single explant that have the potential to proliferate indefinitely in vitro. A cell line can be isolated from a primary culture based on its ability to survive and continue to grow in culture. Cell lines that were originally derived from tumor tissue can be transformed in vivo, although not all neoplastic cell populations have the ability to grow indefinitely in vitro. In addition, cell lines tend to retain their differentiated nature through many cycles of division.

Suitable cell culture substrates are typically a container that can be sterilized, that does not leach toxic factors, and that does not distort microscope images. Thus, plates made from glass and plastic are suitable substrates in this document. Plastic containers can be further treated to promote cell adhesion using techniques known in the art (Ramsey et al. 1984 In vitro 20:802). Suitable tissue culture media typically consist of an isotonic buffered basic nutrient medium, which provides an energy source, combined with inorganic salts, amino acids, vitamins, and various supplements. Supplements may include blood serum (eg, fetal bovine serum or the like), various antibiotics to prevent infection or to provide selective conditions, adhesion and growth factors, or the like. A number of media formulations are known in the art, such as, without limitation, Minimal Culture Medium (MEM), Roswell Park Memorial Institute (RPMI) 1640 medium, or Dulbecco's Modified Eagle's Medium (DMEM). Suitable tissue culture conditions are also known in the art. See, for example, Morgan et al. 1993 Animal Cell Culture, BIOS Scientific Publishers Ltd., Oxford, UK, and Adams, R.L.P. 1990 Cell Culture for Biochemists, Second Edition, Elsevier. In another embodiment disclosed herein, the method for producing CTP-modified coagulation factor VIIa is a process without the use of blood serum. In another embodiment disclosed herein, the method for producing CTP-modified coagulation factor VIIa is a method that does not use materials of animal origin.

In another embodiment of the methods and compositions disclosed herein, the culture storage temperature is -20 to -80°C. In another embodiment, the temperature is significantly less than -20°C. In another embodiment, the temperature is at most -70°C. In another embodiment, the temperature is -70°C. In another embodiment, the temperature is approximately -70°C. In another embodiment, the temperature is -20°C. In another embodiment, the temperature is approximately -20°C. In another embodiment, the temperature is -30°C. In another embodiment, the temperature is -40°C. In another embodiment, the temperature is -50°C. In another embodiment, the temperature is -60°C. In another embodiment, the temperature is -80°C. In another embodiment, the temperature is -30 - -70°C. In another embodiment, the temperature is -40 - -70°C. In another embodiment, the temperature is -50 - -70°C. In another embodiment, the temperature is -60 - -70°C. In another embodiment, the temperature is -30 - -80°C. In another embodiment, the temperature is -40 - -80°C. In another embodiment, the temperature is -50 - -80°C. In another embodiment, the temperature is -60 - -80°C. In another embodiment, the temperature is -70 - -80°C. In another embodiment, the temperature is below -70°C.

In another embodiment, the temperature is below -80°C.

In another embodiment, for cryopreservation, the cells are slowly frozen until they reach a temperature below -70°C in a medium containing a cryoprotectant, and the vials are then transferred to a liquid nitrogen cryogenic apparatus to maintain them at temperatures below -130°C.

In another embodiment of the methods and compositions disclosed herein, cryopreservation, or frozen storage, is continued for no more than 24 hours. In another embodiment, cryopreservation, or frozen storage, is continued for no more than 2 days, for up to 3 days, lasts for up to 4 days, lasts for up to 1 week, lasts for up to 2 weeks, lasts for up to 3 weeks, lasts for up to 1 month, lasts for 2 months or less, 3 months or less, 5 months or less, 6 months or less, 9 months or less, or 1 year or less. Each possibility listed above is an implementation option disclosed in this document.

In another embodiment, cryopreservation, or frozen storage, lasts for at least 1 week, lasts for at least 2 weeks, lasts for at least 3 weeks, lasts for at least 1 month, lasts for at least 2 months, continues for at least 3 months, continues for at least 5 months, continues for at least 6 months, continues for at least 9 months, continues for at least 1 year, continues for at least 1.5 years, continued for at least 2 years, continued for at least 3 years, continued for at least 5 years, continued for at least 7 years, continued for at least 10 years, or continued for more than 10 years. Each possibility listed above is an implementation option disclosed in this document.

In another embodiment of the methods and compositions disclosed herein, the cells show growth after thawing following an extended period of cryopreservation or frozen storage. In another embodiment, the cells show growth over a period of approximately 15-22 hours after seeding into fresh cell media from a cell bank or starter culture. In another embodiment, the cells show growth over a period of approximately 12-20 hours after seeding into fresh cell media from a cell bank or starter culture. In one embodiment, to ensure viability, genetic stability and phenotypic stability, cell lines must be maintained in an exponential growth phase (by regular subculturing).

The long term cryopreservation, or frozen storage, in another embodiment is 1 month. In another embodiment, the period is 2 months. In another embodiment, the period is 3 months. In another embodiment, the period is 5 months. In another embodiment, the period is 6 months. In another embodiment, the period is 9 months. In another embodiment, the period is 1 year. In another embodiment, the period is 1.5 years. In another embodiment, the period is 2 years. In another embodiment, the period is 2-7 years. In another embodiment, the period is at least 7 years. In another embodiment, the period is at least 10 years.

In another embodiment, cells in the methods and compositions disclosed herein retain greater than 90% viability after thawing following cryopreservation. In another embodiment, viability after thawing following a cryopreservation period is close to 100%. In another embodiment, viability after thawing is close to 90%. In another embodiment, the viability after thawing is at least 90%. In another embodiment, viability after thawing is greater than 80%.

In another embodiment, the cell bank, frozen cell stock, or vaccine dose lot disclosed herein is grown in a defined cell culture medium. Such media are known in the art and may include, without limitation, Dulbecco's Modified Eagle's Medium (DMEM) (ATCC® No. 30-2002), Iskov's Modified Dulbecco's Medium (IMDM) (ATCC® No. 30-2005), Hybri- Care (ATCC® no. 46-X), McCoy 5A and RPMI-1640 media (ATCC® no. 30-2007), Ham's formulas (ATCC® CCL-61™), chemically defined serum-free cell culture media CHO PowerCHO™ (Lonza, Cat # 12-771Q) or any other media known in the art. In another embodiment, these media may be supplemented with antibiotics or animal serum samples as determined empirically by one of skill in the art.

- 65 040921

In one embodiment, this document discloses bioreactors and methods that enable the cultivation of mammalian cells in large scale volumes. In addition, in another embodiment, these bioreactors and methods allow the mammalian cells to be cultured under optimal conditions even when grown in large scale, and thus allow production and product quality to be achieved regardless of the size of the bioreactor. The length of the incubation time in the bioreactor can be varied by simply changing the scale and the bioreactor system, for example, the duration can be 8-9 or it can be 15-16 days. In another embodiment, the duration of incubation in the bioreactor is about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, approximately 18 days, approximately 19 days, approximately 20 days or more. In another embodiment, when using a perfusion bioreactor, the duration of incubation can be up to 7-120 days.

In another embodiment, this document discloses large-scale bioreactors that allow the cultivation of mammalian cells in an environment that is homogeneous in terms of process parameters such as pH, dissolved oxygen partial pressure (DOT) and temperature, while maintaining a well-mixed suspension of cells and mixing the supplied nutrients in the bioreactor. In another embodiment, the bioreactor is a disposable bioreactor.

The methods disclosed herein solve the technical problems underlying the methods disclosed herein by providing bioreactors, bioreactor systems and methods for culturing eukaryotic cells, in particular mammalian cells, in accordance with the claims of the present invention.

In one embodiment, the bioreactor has a volume of at least 250 liters. In another embodiment, the bioreactor has a volume of at least 500 liters. In another embodiment, the volume is at least 1000 liters, at least 2000 liters, at least 5000 liters, at least 10000 liters, at least 12000 liters or at least 15000 liters.

In another embodiment, the cells are subcultured in increasing volume bioreactors (see examples).

As is generally known in the art, the modified peptides and proteins of the present invention can be associated with labels, drugs, targeting agents, carriers, solid supports, and the like. depending on the desired application. Labeled forms of modified biologicals can be used to track their metabolic fate; suitable labels for this purpose include, in particular, radioisotope labels such as iodine-131, technetium-99, indium-111, and the like. Labels can also be used to mediate the detection of modified proteins or peptides in assay systems; in this case, radioactive isotopes can also be used, as well as enzyme labels, fluorescent labels, chromogenic labels, and the like. The use of such labels is particularly useful when the peptide or protein itself is a targeting agent, such as an antibody or receptor ligand.

Similar linkage techniques, among others, can be used to link the modified peptides and proteins of the present invention to solid supports. Once linked, these modified peptides and proteins can then be used as affinity reagents to isolate desired components with which a specific reaction is demonstrated.

Finally, the modified peptides and proteins of the present invention can be used to generate antibodies that are specifically immunoreactive for these novel compounds. These antibodies are useful in a variety of diagnostic and therapeutic applications depending on the nature of the biological activity of the unmodified peptide or protein. It should be understood that the present invention provides antibodies that are immunoreactive against FVII or FVIIa as described herein. In one embodiment, such antibodies can be used to identify introduced CTP-modified clotting factors or distinguish between them and endogenous clotting factors. In another embodiment, antibodies can be used to localize administered CTP-modified clotting factors.

Additional objects, advantages, and novel features disclosed herein will become apparent to those of ordinary skill in the art upon examination of the following examples, which are not intended to be limiting. In addition, each of the various embodiments and aspects disclosed herein, as outlined herein above and claimed in the Claims below, finds experimental confirmation in the following examples.

- 66 040921

Examples

Generally, the naming system used herein and the laboratory procedures used in the present invention include molecular biology, biochemistry, microbiology, and recombinant DNA techniques. Such techniques are comprehensively explained in the literature. See, for example, Molecular Cloning: A laboratory Manual Sambrook et al., (1989); Current Protocols in Molecular Biology Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988); Watson et al., Recombinant DNA, Scientific American Books, New York; Birren et al. (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); the techniques set forth in US patent No. 4666828; 4683202; 4801531; 5192659 and 5272057; Cell Biology: A Laboratory Handbook, Volumes I-III, Cellis, J. E., ed. (1994); Culture of Animal Cells - A Manual of Basic Technique, Freshney, Wiley-Liss, N. Y. (1994), Third Edition; Current Protocols in Immunology Volumes I-III, Coligan J. E., ed. (1994); Stites et al. (eds), Basic and Clinical Immunology (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, W. H. Freeman and Co., New York (1980); available immunological assays are widely described in the patent and scientific literature, see, for example, US patent No. 3791932; 3839153; 3850752; 3850578; 3853987; 3867517; 3879262; 3901654; 3935074; 3984533; 3996345; 4034074; 4098876; 4879219; 5011771 and 5281521; Oligonucleotide Synthesis Gait, M. J., ed. (1984); Nucleic Acid Hybridization Hames, B. D., and Higgins S. J., eds. (1985); Transcription and Translation Hames, B. D., and Higgins S. J., eds. (1984); Animal Cell Culture Freshney, R. I., ed. (1986); Immobilized Cells and Enzymes IRL Press, (1986); A Practical Guide to Molecular Cloning Perbal, B., (1984) and Methods in Enzymology Vol. 1-317, Academic Press; PCR Protocols: A Guide To Methods And Applications, Academic Press, San Diego, CA (1990); Marshak et al., Strategies for Protein Purification and Characterization - A Laboratory Course Manual CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

Example 1

Feasibility studies on the use of FVII-CTP3 in FVIII-deficient hemophiliac mice

Conducted studies that tested the PK profile and coagulation activity of FVIICTP, FVII-CTP ₂ and FVII-CTP3 in the collected material in comparison with commercial FVII. FVII-CTP3 showed an improved PK profile compared to FVII-CTP and FVII-CTP2 in harvest or rhFVII while maintaining its coagulation activity. In order to further characterize the properties of FVII-CTP3 in vitro and in vivo, a stable mini-pool expressing and secreting the protein was created and purification and activation processes were developed.

In the present study, the pharmacokinetic and pharmacodynamic properties of FVIIa-CTP ₃ were tested in FVIII deficient mice. The PK profile of the protein was assessed. The PK profile of FVIIa was established based on specific activity and compared with that of the commercial product NovoSeven®. In addition, the long-term in vivo hemostatic ability of FVIIa-CTP ₃ , which induces coagulation in FVIII-deficient mice after tail vein dissection, was tested (survival study).

Research objectives.

Evaluation of pharmacokinetic and pharmacodynamic parameters of FVIIa-CTP ₃ versus commercial rhFVIIa (NovoSeven®) in FVIII-deficient mice after a single IV injection at doses similar in activity.

Determination of the ability of FVIIa-CTP ₃ to maintain hemostasis in FVIII-deficient mice in vivo by single IV administration of FVIIa-CTP ₃ and NovoSeven® at similar potency doses followed by tail vein transection challenge (survival study).

Obtaining harvested material containing FVII-CTP3.

FVII-CTP3 expression was achieved in-house in Dg44 cells using the pCI-DHFR vector (FIG. 1). Stably transfected pool no. 71 was grown in shake flasks in the presence of 25 ng/l vitamin K3 (Sigma). The cell suspension was cultured and harvested after viability dropped to 60-80%. The collected material was filtered and frozen at -70°C.

Determination of the level of FVII antigen in the collected material.

FVII antigen level was determined using a human FVII ELISA kit (Zymotest HyPhen) (Table 1). The level of antigen in each pooled batch of collected material was calculated.

- 67 040921

Table 1. FVII-CTP3 antigen level

FVII antigen level PK-PD Study Survival Study Collected material 31A Collected material 31B collected material 38 Αν (µg/ml) 16.0 15, 9 16.6 STD 1.5 0, 0 0.8 %CV 9, 1 0, 1 4, 9

FVII-CTP3 purification process (FIGS. 2A-2B).

Brief summary of the process.

After a short purification study, the following purification process was performed using 2 columns. The γ-carboxylated FVII-CTP3 enriched protein was purified on a VII-Select (GE) affinity chromatography column with 40 μm ceramic hydroxyapatite (HA) type 1 (HA) (Bio Rad). Self-activation was induced by incubating purified FVII-CTP3 in the presence of CaCl ₂ overnight at 2-8°C. The purification process is in its final stages of development and is being optimized, so while most of the purification steps are similar, some of the purification steps are not identical between the two batches.

Ultrafiltration/diafiltration (UF/DF) with 10 kDa hollow fiber membrane or Pellicon cassette.

The clarified harvest was thawed at 4° C. over the weekend (2-3 days).

In Lot 31, the clarified harvest (12 L) was concentrated 4-fold (two consecutive runs) using a hollow fiber membrane cartridge (GE Healthcare, Cat # UFP-10-C-4X2MA) with a 10 kDa molecular weight cutoff. The concentrated harvest was diafiltered against 1-2 volumes of TBS (50 mM Tris, 150 mM NaCl, pH 7.4).

In Lot 38, the clarified pool (8.5 liters) was concentrated 4-fold using a Pellicon 2 cassette (Millipore) with a 10 kDa molecular weight cutoff. The concentrated collected material was loaded directly onto the VII-Select column.

Both ultrafiltration procedures were performed on ice using ice-cold buffers. Samples subjected to UF/DF were filtered through a filter with a pore size of 0.22 μm before loading.

Capture column with FVII-Select.

The UF/DF exposed or concentrated harvest was loaded onto a VIISelect column (XK16/20, CV 18 ml) pre-equilibrated with TBS pH 7.4. The column was washed with 50 mM Tris-HCl, 0.5 M NaCl, pH 7.5, and FVII-CTP3 was eluted with 50 mM Tris-HCl, 1 M NaCl, 50% (v/v) propylene glycol, pH 7, 5. The process was performed in two consecutive cycles using the same column.

Separation by gamma-carboxylation on a ceramic hydroxyapatite column.

The eluted product was diluted 1:10 with 10 mM sodium phosphate, pH 6.8, and loaded onto ceramic hydroxyapatite columns (XK16/20, CV 24 ml). The column was washed with 59 mM sodium phosphate, pH 6.8, and the coagulation factor VII fraction rich in γ-carboxylated residues was eluted with 500 mM sodium phosphate, pH 6.8. This process was performed in two consecutive cycles on the same column. In each batch, the eluates from two cycles were pooled and concentrated to 1.7-2 mg/mL and diafiltered using 20 mM Tris-HCl, 100 mM NaCl, pH 8.2 to reduce volume and obtain material for the activation step.

FVII activation.

Purified FVII-CTP3 was diluted to 1 mg/ml and incubated in 20 mM Tris-HCl, 100 mM NaCl and 1 mM CaCl ₂ , pH 8.2, at 2-8°C for 24 h. UF/DF) to preformulation buffer (20 mM citrate buffer, 240 mM NaCl, 13.3 mM glycine, pH 6.9).

Analytical properties of FVII-CTP3 and FVIIa-CTP3.

SDS-PAGE and Western blot procedures.

Purified FVII-CTP ₃ and FVIIa-CTP ₃ were loaded onto a 12% Tris-glycine gel using a Precision Plus Protein two-color marker (Bio-Rad). SDS-PAGE analysis with Coomassie staining was performed by staining the gel with Coomassie Brilliant Blue reagent (5 or 10 μg protein/lane). Western blot analysis was performed (1 μg protein/lane) using a polyclonal anti-human FVII Ab (R&D Systems; AF2338), a monoclonal anti-human gamma carboxylated protein antibody (American Diagnostics, Cat # 499, 3570), and a polyclonal anti-CTP Ab. Under reducing conditions, FVII-CTP3 migrated at 75 kDa and FVIIa-CTP3 migrated as two major bands, the heavy chain at 50 kDa and the light chain at 25 kDa, which are shown in FIG. 3A-3H as lanes 2 and 3, respectively.

As a result of the purification procedure, the part corresponding to FVII-CTP3 was significantly enriched, while reducing the amount of impurities. The yield of the purification process was 25-30% FVII (according to ELISA). Most of the protein that was lost during purification had little or no FVII chromogenic activity. As follows from the SDS-PAGE pattern with Coomassie staining, the recovered FVIIa-CTP ₃ contains more bands than predicted. The band migrating to about ~75 kDa represents non-activated FVII (FIGS. 3A-3H, lane 1). This band consists of two bands slightly different in MW, which may reflect different levels of γ-carboxylation. Additional bands were observed with MW less than 20 kDa. They, as previously reported, correspond to the degradation products of the heavy chain.

Chromogenic activity of FVII-CTP3.

Comparative in vitro specific activity assessment of harvested FVIICTP3 containing material, process fractions, and purified FVII-CTP3 versus pooled normal human plasma was performed using the commercially available BIOPHEN chromogenic activity test kit (Hyphen BioMed 221304). The harvested material containing FVII-CTP3 and the protein itself were serially diluted, and the specific activity was evaluated by comparing the dose-response curve with that of a normal human plasma reference preparation. After purification of FVII-CTP ₃ , chromogenic activity improved significantly and inactive fractions were separated mainly on the HA column (FIG. 4). A strong correlation was observed between FVII chromogenic activity and FVII detection by anti-Gla monoclonal antibodies in Western blotting. The strength of the chromogenic activity of FVII, reflected in the EC ₅₀ value in the collected material, is affected by both carboxylated and non-carboxylated fractions of FVII. After purification and enrichment of the γ-carboxylated fraction of FVII-CTP _3, activity improved, demonstrating the important contribution of γ-carboxylation to FVII activity (FIG. 4). This parameter is critical for proper FVII activity in vivo and will be further explored in the cloning program.

Determination of protein according to A280.

The theoretical extinction coefficient of FVIIa-CTP ₃ and NovoSeven® was calculated using the ProtParam algorithm (http://web.expasy.org/protparam). The calculation was based on amino acid sequence data. The calculated extinction coefficients for FVII-CTP ₃ and NovoSeven® are 1.186 and 1.406, respectively. These values represent absorbance at 1 g/l and 280 nm.

The difference in extinction coefficients between the two proteins is due solely to the increase in molecular weight of FVIIa-CTP ₃ compared to NovoSeven®, since CTP has no aromatic or cysteine residues and thus does not contribute to absorption.

The A280 protein determination is applied to the final FVII and to the purified samples taken during the process, starting from the elution from the VII-Select column.

Determination of the level of FVIIa antigen.

FVIIa antigen level was determined using a human FVIIa ELISA kit (IMUBIND, American Diagnostica). The level of antigen in each batch was calculated. However, this tool was not applicable for determining the dose for injection because it did not represent the amount of active product.

Analysis of FVIIa clotting activity using Staclot® VIIa-rTF.

FVIIa is formed by intrachain cleavage of single chain FVII. Native tissue factor (TF) is a cofactor for FVIIa. When bound to TF, FVII mediates the activation of coagulation factor X with the formation of Xa, and itself is converted into FVIIa. Soluble tissue factor is the extracellular portion of native tissue factor. It can no longer activate FVII by self-activation, however tissue factor bound FVIIa can activate FX to form FXa.

The specificity of the recombinant soluble tissue factor (rsTF) used in this assay for FVIIa is used to develop a test for FVIIa clotting activity. rsTF in the presence of FVIIa, calcium and phospholipids leads to coagulation of blood plasma without activating FVII with the formation of FVIIa.

The observed clotting time in this system is inversely proportional to the FVIIa content in the test sample and is not distorted by the presence of FVII in the sample.

The analysis was performed at Omri Laboratories (Ness Ziona, Israel). FVIIa activity was assessed for both NovoSeven® after dilution and FVIIa-CTP ₃ before each study. The activity of NovoSeven® did not correlate with the expected activity indicated in the vial, and this discrepancy may be due to a different approach to assessing the activity. In table. 39 summarizes FVIIa clotting activity per unit volume without regard to protein concentration.

- 69 040921

Table 2. FVIIa clotting activity in product lots

Study

PK study of survival

Activity (u/ml) FVIIa-3*STR (FVIIa 31) 1,ZE+06 NovoSeven® 2.5E+05 FVIIa-3*STR (FVIIa 38) 1,ZE+06 NovoSeven® 7.4E+05

Specific activity of FVIIa-CTP ₃ .

The specific activity of FVIIa (calculated as activity/mL divided by protein concentration) was calculated from the A280 value and is shown in Table 1. 3. When comparing the specific activity of two molecules that differ in MW, a correction should be introduced in order to normalize the activity (i.e., due to the difference in molecular weight, the number of active sites in 1 mg of NovoSeven® is 1.185 times higher than in FVIIa-CTP ₃ ) . The conversion factor calculation is presented in the following equation:

Normalized SA = SA (FVIa-CTP ₃ ) x MW (native FVII) =

MW. (FVII STRz) = SA (FVIIa STRz) x 45079.1 Yes = SA (FVIIA-STRz) * 1.185

53419.5 Yes

Table 3. Specific activity of FVIIa-CTP ₃ compared to NovoSeven®

Sample Average value A280 STDV (n=9) % CV Extinction coefficient Kontsen protein consumption (mg/ ml) units / ML Specific activity Reduction ratio compared to NovoSeven units / MG protein units/ MG FVIIa Novo Seven® 1.274 0.031 2.3 98 1.406 0.906 8.36 E+05 9.23E +05 9.23E +05 1.0 FVIIa- STRz 4,396 0.092 2.0 94 1.186 3.706 7.23 E+05 1.95E +05 2.31E +05 4.0

PK-PD FVIIa-CTP ₃ study.

Brief summary of the study.

FVIIa-CTP ₃ and rhFVIIa (NovoSeven®, NS) were administered as a single intravenous injection to FVIII-deficient C57B mice at a dose of 6.4E6 U/kg body weight (160,000 U/animal). Blood samples were taken from the retroorbital sinus of 4 mice in turn at 0.166, 0.5, 2, 4, 8, 12, 24, 34, 48, 58, and 72 hours post-dose (Table 4). Citrated blood plasma (0.32%) was obtained immediately after sampling and stored at -20°C until analysis. The level of FVIIa clotting activity was assessed and a detailed PK analysis was performed. The study was performed at Omri Laboratories (Ness Ziona, Israel).

- 70 040921

Table 4. Study summary

Treatment groups Investigational drug N animals / group / point in time Path DOSE administration Quantity your units/animal Injection volume (µl) Time points (hours after dose) A rhFVIIa 4 IV 1, 6е5 200 0 (up to dose), 0.166, 0.5, 2, 4, 8, 12, 24, 34, 48, 58, 72 IN FVIIa-STR3 4 IV 1, 6e5 200 0 (up to dose), 0.166, 0.5, 2, 4, 8, 12, 24, 34, 48, 58, 72

PK profile of FVIIa-CTP3 in FVIII-deficient mice.

FVIIa activity in blood samples was quantified using the Staclot® VIIa-rTF kit (Stago, Parsippany, NJ). A pharmacokinetic profile was calculated for each protein and represents the average of 4 animals at each time point. In FIG. 5 shows the PK profile of FVIIa throughout the experiment. Extract FVIIa presented in table. 6. A summary of the PK parameters is presented in Table. 7.

In table. 5 summarizes the values of clotting activity after administration of NovoSeven® or FVIIa-CTP3. FVIIa-CTP3 and NovoSeven® reached maximum activity half an hour after dosing. The highest value of NovoSeven® activity reached only 43% of the value of the maximum activity of FVIIa-CTP ₃ . The clotting activity of FVIIa-CTP ₃ was maintained for a longer period of time, demonstrating sustained activity. Clotting activity in mice treated with NovoSeven® was undetectable at time points later than 12 hours, while mice treated with FVII-CTP3 continued to have measurable activity 48 hours post-dose ( Table 5 and Fig. 5).

When three copies of CTP were added sequentially one after the other to FVIIa, the recovery increased by 100% (Table 6) as measured by the highest post-dose activity and compared to the predicted activity from the in vitro assay, and the half-life and mean retention time (MRT) increased by 5 times. Exposure time (AUC) increased 3 times (Table 7).

Table 5 FVIIa clotting activity after a single IV injection

Time after administration (hours) Average coagulation activity of FVIIa (U/ml) FVIIa-STR3 NovoSeven® 0.16 6.8E+07 3.2E+07 0.5 9.7E+07 4,WE+07 2 2.1E+07 3.9E+06 4 7.7E+06 7,WE+05 8 2.7E+06 4.2E+04 12 3.7E+05 6.2E+03 24 2.4E+04 BLQ 34 4.6E+03 BLQ 48 1.5E+03 BLQ

- 71 040921

Table 6 FVIIa-CTP3 extraction

TREATMENT GROUPS Investigational drug Number of units / animal Practical administered dose (unit/ml) * Estimated Stach (U/ml of blood) Stax (U/ml) % recovery A rFVIIa 1.60E+05 1.20E+06 1.40E+05 4.25E+04 thirty IN FVIIa- PAGE ₃ 1.60E+05 1.29E+06 1.50E+05 9.74E+04 64.6

the estimated Cmax is obtained by dividing the administered dose by the blood volume _____Table 7. PK parameters of FVIIa-CTP3 versus NovoSeven®_____

PK parameters NovoSeven® FVIIa—PAGE ₃ Half-life _a _ _phase (0.5-12 hours) 0.94 1.57 Elimination half-life _₽ _ _faEa (12-48 hours) ΝΑ 4.62 AUC (milliu*h/ml) 5.80E+07 1.80E+08 Vd/kg (ml/kg) 1408 2375 CL/kg (ml/h/kg) 1034 356 MRT (h) 1.3 6, 7

Thrombin Generation Assay (TGA).

Thrombin formation is a key part of the blood coagulation cascade, and therefore an assessment of how well a particular individual can form thrombin may correlate with the risk of bleeding or thrombosis. Variables often measured in the analysis of thrombin production include lag period, time to peak thrombin production, peak magnitude, endogenous thrombin potential (ETP) (i.e. area under the curve and inactivation time), thrombogram (TG) trend. After a lag period, there is a surge in thrombin formation. However, blood clotting occurs at the end of the lag period, when more than 95% of the total thrombin has not yet formed. Thrombin production assays were performed at Omri Laboratories using reagents from Thrombinoscope supplemented with plasma from a person with hemophilia. TGA reflects the clotting ability in mouse plasma due to injection of NovoSeven® and FVIIa-CTP3. In FIG. 6A6C shows plasma TGA values in mice after administration of FVIIa-CTP ₃ or NovoSeven®. After administration of FVIIa-CTP3, all three parameters (rate of thrombin formation, maximum amount of thrombin produced and KIIa) demonstrate the advantage of treatment with FVII-CTP3 compared with NovoSeven®. This further reinforces the potential superiority of FVII-CTP ₃ over NovoSeven® in terms of sustained release.

Examination of FVIIa-CTP ₃ after tail vein dissection (TVT).

Brief summary of the study.

Data from the PK/PD assay for FVIIa-CTP3 provided insight into the functionality of FVIIa-CTP3 and demonstrated that FVIIa-CTP3 was superior to NovoSeven® in terms of pharmacokinetic parameters. However, the ability of the protein to induce blood clots in vivo after a traumatic event has not yet been demonstrated. In order to evaluate the ability of FVIIa-CTP3 to stop bleeding, the same FVIII-deficient mouse model was used for bleeding provocation.

FVIII deficient mice were given a single intravenous injection of FVIIa-CTP3 or NovoSeven®. Mice were dosed with drugs in amounts providing equivalent FVIIa activity (1.6E05 units, 200 μl) calculated according to the specific activity of each drug as assessed in the FVIIa clotting activity assay (Table 8). The doses administered were 9 mg/kg NovoSeven® and 40 mg/kg FVII-CTP3 due to reduced FVIIaCTP ₃ activity. The control group was injected with 200 μl of inert medium.

Tail vein dissection was performed at a distance of 2.7 cm from the tail tip at 15 min (Injection 1), 24 h (Injection 2), or 48 h (Injection 3) after administration, and mouse survival was recorded for 24 h.

- 72 040921

Table 8. Evaluation of injected samples

NovoSeven® FVIIa-CTP ₃ No. of injection Concentrated protein intake (mg/ml) Activity (unit/ml) Specific activity (units/mg) Con- price- protein consumption (mg/ml) Activity (U/ml) Specific activity (units/mg) Specific activity (normalized) 1 0.91 8.0E+05 8.8E+05 3.63 6.6E+05 1.8E+05 2.2E+05 2 0.92 8,WE+05 9.0Е+05 3.81 7.8E+05 2.0E+05 2.4E+05 3 0.89 8.8E+05 9.9E+05 3.68 7,WE+05 2.0E+05 2,WE+05

The protein concentration was determined by A280.

Results.

Data from control groups injected with inert medium for three injections (5 x3 injection animals) were summarized and presented in FIG. 7A-7D. 24 hours after dissection of the tail vein, a 30% survival rate was observed.

Mice treated with NovoSeven® and FVIIa-CTP ₃ demonstrated adequate hemostatic activity after tail vein dissection performed 15 minutes after FVIIa administration. Animals treated with FVIIa-CTP ₃ and NovoSeven® (FIGS. 7A-7D) experienced a 100% survival rate.

The decrease in FVII-CTP ₃ clearance rate that was demonstrated in the PK/PD study is most clearly perceived after tail vein dissection performed 24 hours after administration. There has been a drop in survival rates with NovoSeven® administration. As with the control group, 50% mortality was observed within a 10 hour period. At the same time, 90% of mice treated with FVIIa-CTP ₃ survived (Fig. 7A-D). This result highlights the long-term efficacy of treatment with FVIIa-CTP ₃ .

At 48 hours after administration, the FVIIa-CTP ₃ or NovoSeven® treated groups show a drop in survival rate (FIG. 7C). There was a slight improvement in mice treated with FVIIa-CTP, but the difference did not reach statistical significance.

Discussion.

As a result of the fusion of STRs with recombinant proteins, the half-life of proteins from the bloodstream is extended, while maintaining comparable activity. While the mechanism underlying the reduction in clearance from a protein larger than the 70 kDa threshold is well understood in relation to renal clearance, additional protection is achieved after fusion with STR. It is believed that as a result of fusion with CTP, a shielding is formed around the protein that protects it from proteolytic cleavage, its molecular weight increases in the radial direction due to its high negative charge, and its affinity for receptors involved in hepatic clearance decreases.

The present study was aimed at providing a concrete understanding of the effect of the fusion of CTP with FVII on the half-life of the protein and the intensity of clearance from it, as well as the study of the principles that determine its specific activity after this modification. FVIII-deficient mice were given a single IV injection of FVIIa-CTP ₃ or recombinant commercial FVIIa (NovoSeven®) at similar doses (by number of units) and performed PK analysis based on activity. FVIIa-CTP ₃ showed a longer lifespan, which was reflected in a 5-fold and 3.5-fold increase in its half-life and AUC, respectively. The specific activity (u/mg) of FVIIa-CTP calculated using the Staclot® activity assay kit divided by the protein concentration measured at A280 was shown to be 4-5 times lower than the specific activity of NovoSeven®.

To enhance understanding of how CTP affects the hemostatic effects of FVIIa in vivo, the ability of FVIIa-CTP ₃ to reduce bleeding was investigated. In a tail vein hemorrhage model in a hemophilia mouse model, administration of rFVIIa may improve the survival rate of challenged animals and avoid death from blood loss. In the study described herein, animals were administered FVIIa-CTP ₃ or NovoSeven®. Both molecules were able to maintain hemostasis if the incision was made 0.25 hours post-dose. The FVIIa-CTP ₃ treated group showed a significantly increased duration of activity when the incision was made 24 hours after injection.

-73 040921 doses. The survival rate in the inert medium-treated group was higher than expected and higher than that achieved in previous studies (50% vs.

20% in previous studies, data not shown). The percentage survival rate of treated animals was additionally assessed at earlier time points, including 36 hours post-dose.

In conclusion, FVIIa-CTP ₃ has been shown to have an extended duration of activity in hemophilic mice resulting in a longer duration of hemostatic effect compared to NovoSeven®. The collected data suggest that the fusion of CTP with FVII is a technology with the potential to significantly improve prophylactic treatment in patients with hemophilia.

Example 2

Biochemical properties of MOD-5014 compared to commercial recombinant hFVIIa - effect of carboxy-terminal peptide (CTP) on coagulation factor VIIa activity Rationale and brief description of the project.

These assays were designed to evaluate the biochemical properties of MOD-5014 compared to commercial recombinant hFVIIa, referred to herein as MOD-5000.

The following studies have been studied.

Cleavage of synthetic substrate by MOD-5014.

Binding of MOD-5014 to tissue factor (TF) as measured by degradation of the synthetic substrate.

Binding of MOD-5014 to TF as measured by clotting factor X (FX) activation.

Kinetic parameters of FX activation by TF-bound MOD-5014.

Lipid binding of MOD-5014 as measured by FX activation.

Kinetic parameters of activation of blood coagulation factor under the influence of MOD-5014 associated with lipids.

Inactivation of MOD-5014 by antithrombin (AT).

Inactivation of MOD-5014 by TFPI.

Overall, the data suggest that compared to MOD-5000, MOD-5014 has a similar mechanism of action with slightly reduced catalytic activity. These results showed a slightly reduced activity of TF-bound MOD-5014 and somewhat more reduced activity in the non-TF-bound state.

These effects were reflected mainly in the rate of reactions rather than in the extent of reactions, and reactions for which full dynamics can be measured proceed to completion.

The slightly reduced rate of inhibition by AT suggests an extended in vivo half-life of MOD-5014 with adequate sensitivity to inhibition.

Experimental materials.

MOD-5014, GMP-1: 2.5 mg/mL (according to A280).

NovoSeven, Lot # CU60430: 0.943 mg/mL (per A280), referred to as MOD-5000.

Cleavage of synthetic substrate by MOD-5014.

Rationale. Cleavage of the synthetic substrate should depend solely on the availability of the functional active site.

Ways. MOD-5000 and MOD-5014 were diluted to equal molar concentrations. At the same concentrations, they were then added to the Pefachrome FVIIa substrate (methylsulfonyl-D-cyclohexylalanyl-2-aminobutyrylarginine p-nitroanilide) at a fixed concentration, and the degradation of the substrate was monitored by the appearance of a yellow color.

Results.

Concentrations: FVIIa - 360 nM; substrate - 500 μM.

Analysis. The absorbance at 405 nm was converted to the concentration of p-nitroaniline using a known extinction coefficient. The concentration of p-nitroaniline was plotted against time to determine the rate of degradation of the substrate. The data were approximated by the equation:

speed=k ₁ [VIIa];

k1=27.5 mol π-NΑ/min/mol VIIa.

Conclusion. MOD-5000 and MOD-5014 have the same substrate degradation rate on a molar basis (FIG. 8). For subsequent studies, the measured substrate degradation rate is used as a control for dilution and pipetting.

Binding of MOD-5014 to TF as measured by degradation of the synthetic substrate.

Rationale. When the blood coagulation factor VIIa binds to TF, a conformational change in the blood coagulation factor VIIa occurs, which leads to an increase in the rate of substrate cleavage. This means that increased substrate cleavage can be used to monitor coagulation factor VIIa TF binding.

- 74 040921

Ways. MOD-5000 and MOD-5014 at various concentrations were added to TF at a fixed concentration and incubated for 5 min. Substrate (Pefachrome FVIIa) was added. Substrate degradation was monitored at 405 nm by the appearance of a yellow color.

Results.

Concentrations: FVIIa - 0-25 nM; TF - 8.7 nM; substrate - 500 μM.

Analysis. Since the concentration of TF is much higher than the expected Kd, then at low concentrations, all FVIIa should bind to TF. The rate of degradation of the substrate will correspond to the rate for complex VIIa/TF. Once the FVIIa concentration exceeds the TF concentration, the substrate degradation rate should drop to that of free FVIIa. Since FVIIa and TF form a complex in a 1:1 molar ratio, the concentration of FVIIa at which a change in the rate of degradation of the substrate occurs is a test for the estimated concentration of FVIIa.

The data were approximated by the equation:

speed=k ₁ [Vila] + k _x [VIIa/TF];

MOD-5000 MOD-501

27.5 27.5 mol π-NΑ/min/mol Vila

365 357 mol π-NΑ/min/mol VIIa/TF

Conclusion. MOD-5000 (NovoSeven) and MOD-5014 show the same curve inflection point at the expected TF concentration (8.7 nM) (FIG. 9). This confirms the accuracy of the molar concentrations of MOD-5000 and MOD-5014 predicted from substrate degradation. When bound to TF, MOD-5014 had a very slightly lower substrate degradation rate (98%) compared to MOD-5000 (FIG. 9).

Binding of MOD-5014 to TF as measured by clotting factor X activation.

Rationale. Cleavage of FX by FVIIa is slow compared to cleavage by the FVIIa/TF complex. Therefore, binding of FVIIa to TF can be assessed by measuring the rate of FX activation.

Ways. MOD-5000 and MOD-5014 at various concentrations were added to TF at a fixed concentration, and the activation rate of FX was measured. The activation of blood coagulation factor X was assessed by the cleavage of the synthetic substrate Pefachrome FXa (methoxycarbonyl-D-cyclohexylalanylglycylarginine paranitroanilide). The degradation rate of the synthetic substrate was converted to FXa concentration using a calibration curve. Neither FVIIa nor FVIIa/TF cleave the FX substrate at a measurable rate.

Results.

Concentrations: FVIIa - 0-2 nM; TF - 10 pM; FX - 135 nM; substrate - 500 μM.

The concentration of blood coagulation factor X in blood plasma is 8 µg/ml (~135 nM).

Analysis. The rate of FX activation should increase as FVIIa binds to TF. After all TF is saturated with FVIIa, the FX activation rate will reach its maximum value (Fig. 10).

The data were approximated by the equation:

_ , | I Pi 1' '+ 77- ] +K . I Pa [H] J+ K ' G—I ' I' P I ']

V-Г„ ^: 2 (77 - - ^:

MOD-5000 MOD-50I4 1.4(1 I.3O

Kd 3.3 PLN

Hill coefficient value

Conclusion. There is very little negative cooperativity (Hill coefficient value < 1) of FVIIa binding to TF. It is the same for MOD-5000 and MOD-5014. Being associated with TF, MOD-5014 has a slightly reduced FX activation rate (93%) compared to MOD-5000. The affinity of MOD-5014 for TF is equivalent to that of MOD-5000 (FIG. 10).

FX activation rate as a function of FX concentration.

Rationale. The slight decrease in activation rate for TF-bound MOD-5014 may be due to a decrease in affinity for FXa or a decrease in FX turnover after it binds to the complex. When measuring the activation rate of FX, depending on the concentration of FX, the kinetic parameters of the complex are set.

Ways. FX at various concentrations was incubated with the FVIIa/TF complex at a fixed concentration.

The activation of blood coagulation factor X was assessed by the cleavage of a synthetic substrate (Pefachrome FXa).

The degradation rate of the synthetic substrate was converted to FXa concentration using a calibration curve.

Results.

Concentrations: FVIIa - 1 nM; TF - 5 pM; FX - 0-1500 nM; substrate - 500 μM.

Analysis. The more FX added, the more FVIIa/TF complexes had to bind to nM FXa/min. pM

- 75 040921

FX up to the point where all FVIIa/TF complexes were associated with FX. At this point, the reaction was rate-limited at which FX activated. Thus, the rate of activation of FX should have increased with increasing concentration of FX, asymptotically reaching a maximum value in accordance with the shape of the curve (Fig. 123).

The data were approximated ^by the _equation

MOD-5000 MOD-5014 ^V '--“ ^{L?s LM} nM FXa/min.

Km 140 120 hM

Conclusion. When linked to TF, MOD-5014 had a slightly reduced FX turnover (92%) compared to MOD-5000. The binding of FX to the MOD-5014/TF complex was the same as its binding to the MOD-5000/TF complex (FIG. 11).

Lipid binding of MOD-5014 as measured by FX activation.

Rationale. It is believed that the activation of factor X on platelets contributes to the hemostatic effect of FVIIa. This platelet activity is believed to take place in a low TF environment or in the absence of TF. Coagulation factor X activation without TF can be examined on lipid vesicles.

Ways. Activation of coagulation factor X by FVIIa on lipids depends on the binding of both the enzyme (FVIIa) and the protein substrate (FX). The lipid ratio was PC:PE:PS 41:44:14 and was designed to mimic the composition of highly activated platelets. Lipids were obtained as large unilamellar vesicles (200 nm). Vesicles at increasing concentrations were added to FVIIa and FX. The activation of blood coagulation factor X was assessed by the cleavage of a synthetic substrate (Pefachrome FXa). The degradation rate of the synthetic substrate was converted to FXa concentration using a calibration curve.

Results.

Concentrations: FVIIa - 20 nM; FX - 500 nM; lipids -0-1000 μM; substrate - 500 μM.

Analysis. The rate of formation of FXa was plotted against the concentration of lipid vesicles (FIG. 12A). As expected, the rate of FXa production increased with increasing lipid concentration as more surface area became available for the reaction. At sufficiently high lipid concentrations, the reaction rate decreased as FVIIa and FX were separated into different lipid vesicles. This stereotyped response is expected for this system. The data was not approximated by an equation and the lines are shown as a visual guide only. Differences in the rate of formation of FXa between MOD-5000 and MOD-5014 were not due to differences in lipid affinity. This is shown in FIG. 12B, where for each of them the rate of formation of FXa relative to the maximum is plotted against lipid concentration.

Conclusion. The rate of FX activation in the absence of TF is lower for MOD-5014 (~60%) compared to MOD-5000. The lipid affinity of MOD-5014 is the same as that of MOD-5000.

Kinetic parameters of FX activation by lipid-bound MOD-5014.

Rationale. The reduced rate of FX activation in the absence of TF for MOD-5014 compared to MOD-5000 may be due to decreased affinity for FXa or reduced turnover of FX after it binds to the enzyme on the lipid surface.

Ways. FX at various concentrations was incubated with FVIIa at a fixed concentration and lipid vesicles. The activation of blood coagulation factor X was assessed by the cleavage of a synthetic substrate (Pefachrome FXa). The degradation rate of the synthetic substrate was converted to FXa concentration using a calibration curve.

Results.

Concentrations: FVIIa - 20 nM; FX - 0-2500 nM; lipids - 100 μM; substrate - 500 μM.

Analysis. The more FX added, the more FVIIa on the lipid surface must bind to FX up to the point where all of the FVIIa is bound to FX. At this point, the reaction is limited to the rate at which FX is activated. Thus, the FX activation rate should increase with increasing FX concentration, asymptotically reaching a maximum value according to the shape of the curve. As expected, the affinity of FVIIa for FX decreases (higher Km) in the absence of TF and the rate of formation of FXa decreases in the absence of TF (FIG. 13).

- 76 040921

The data were approximated by the equation

V(W'

V,

MOD-5000 MOD-5014 ^{11253 P5} nM FXa/min.

S78848.

nM FXa/min.

Conclusion. The rate of FX activation on the lipid surface in the absence of TF is lower for MOD-5014 (45%) compared to MOD-5000. The binding of FX to MOD-5014 on the lipid surface was the same as its binding to MOD-5000 (FIG. 13).

Inactivation of MOD-5014 by AT.

Rationale. I believe that a significant part of the clearance from FVIIa in vivo occurs through the formation of a complex of FVIIa and AT. The rate of this reaction is only measurable in vitro if FVIIa is associated with TF. In order for the reaction to proceed in vitro at a measurable rate, high concentrations of heparin are also required, which is believed to mimic the effects of naturally occurring glycosaminoglycans.

Ways. Coagulation factor VIIa was incubated with TF to allow complex formation. The complex was incubated with AT and heparin. The reaction was stopped at timed intervals by adding polybrene (hexadimethrin bromide) to neutralize the heparin. Residual FVIIa/TF activity was measured by degradation of the synthetic substrate (Pefachrome FVIIa). At the concentrations used in the assay, polybrene did not alter substrate degradation.

Results.

Concentrations: FVIIa - 10 nM; TF - 11 nM; AT - 1 μM; heparin - 5 units/ml; FVIIa/TF - 8.2 nM; polybrene - 100 µg/ml; substrate - 500 μM.

Analysis. The concentration of FVIIa/TF, measured by the rate of degradation of the substrate, was plotted against time in minutes (FIG. 14). As expected, the AT/heparin complex inhibited FVIIa, leading to loss of FVIIa/TF activity.

The data were approximated by the equation y _t=o e~ ^k * ^time

MYEM LLC MOD-5014

V _e 11.89 11.93 to 0354 0Α17 min ¹

Conclusion. Similar activity values at time T=0 indicate that equal amounts of MOD-5000 and MOD-5014 were present in the reaction mixture. Inhibition of MOD-5014 was slightly slower (62%) than MOD-5000 (FIG. 14). Both reactions proceeded until complete inhibition.

Inactivation of MOD-5014 by TFPI.

Rationale. TFPI is a physiological inhibitor of the FVIIa/TF complex. The K2 domain of TFPI initially forms a complex with FXa. This complex binds FVIIa/TPI, where the K1 domain of TFPI interacts with FVIIa. Thus, activation of FX under the action of FVIIa/TF should lead to the inhibition of the complexes and to the cessation of the functioning of FVIIa-TFPI.

Ways. Coagulation factors VIIa and TF were incubated together to form a complex. The complex was added to the substrate for TFPI/FX/FXa. The activation of blood coagulation factor X was assessed by the cleavage of a synthetic substrate (Pefachrome FXa). The degradation rate of the synthetic substrate was converted to FXa concentration using a calibration curve.

Results.

Inhibition concentrations: FVIIa - 1 nM; TF -20 pM; FX - 135 nM; TFPI - 0-5 nM; substrate 500 μM.

Analysis. As expected, FXa initially formed at the same rate in all reactions (FIGS. 15A-C). In the presence of TFPI, the rate of FXa formation decreased as the FVIIa/TFPI complex was inhibited by TFPI/FXa (bottom two panels). Termination of the functioning of the TFPI complexes occurred faster at higher concentrations of TFPI (bottom two panels). The amount of FXa formed before FVIIa/TFPI ceases to function is a measure of the interaction of TFPI with FVIIa/TF. Because MOD-5014 has a slightly reduced rate of FXa formation, which will slow the formation of FXa/TFPI complexes, it takes longer for MOD-5014 to reach a plateau than for MOD-5000.

Conclusion. As shown in the top panel, the concentration dependence of TFPI inhibition exerted on FXa formation by MOD-5014/TF is very similar to that of MOD-5000. MOD-5014 may be slightly more sensitive to inhibition by TFPI (124%); alternatively, it may be an artifact due to a slight decrease in the rate of FXa formation.

Example 3

Preparation of CTP-modified activated coagulation factor VII

Target.

The aim of the production method was to develop a biomass accumulation process by recombinant DNA fed-batch culture using CHO cells in a chemically defined medium, followed by a sustainable and scalable product isolation and purification process that purifies a highly glycosylated and high gamma- carboxylation of MOD-5014. In other words, this is the production and purification of MOD-5014 with the highest level of gamma-carboxylation and the effective removal of process and industrial impurities. It was important to analyze in terms of O-glycans, N-glycans, percentage of sialic acid, oxidized cognates, specific activity (tested by STA-CLOT assay), percentage of Gla domains (or alternatively, percentage of non-carboxylated residues). glutamic acid) and the percentage of non-activated FVII.

Receiving procedure.

Transfection and selection of stable clones.

The cDNA encoding MOD-5014 was transfected into CHO cells (DHFR-negative CD DG44 cells adapted for protein-free growth and suspension growth) and stable clones were obtained through limiting dilution steps. Clones with the highest productivity were propagated, and the final clone was selected for further development.

Throughout the process of obtaining master and working cell banks (MCB; WCB) used media that do not contain components of animal origin. Stable clones were isolated through limiting dilution steps during cell culture. Clones with the highest productivity were propagated with a selective agent at increasing concentrations. Based on the clonal population doubling level (PDL), the productivity of CTP-modified coagulation factor VII production (picograms per cell per day, PCD) and the maximum cell density achieved in the chosen medium, clones with the highest productivity were isolated and used to generate cell banks for R&D. followed by a Certified Master Cell Bank (MCB) and a Working Cell Bank (WCB).

The process of biomass accumulation.

A stable clone of CHO cells expressing MOD-5014 was seeded from a single flask from the master cell bank (MCB) (step 1 in Fig. 16) and expanded stepwise to the level of 1000 L or 2000 L bioreactors in serum-free medium with chemically formulated supplemented with vitamin K using a fed-batch approach (steps 2-4 in FIG. 16).

The production cell culture supernatant was tested for bioburden, bacterial endotoxins, productivity, and introduced viruses. The process was performed using 50 L and 200 L bioreactors to seed the bioreactors (stage 3 in FIG. 16) and 1000 or 2000 liter bioreactors to scale up (stage 4 in FIG. 16). All product contact surfaces were disposable while reusable product contact equipment was product specific. These pieces of equipment were cleaned and sanitized between batches. The culture was propagated to levels representative of 50 L and 200 L bioreactors before seeding into a 1000 L or 2000 L bioreactor. Final scale-up and bioreactor production by fed-batch culture was performed in disposable 2000 L bioreactors. Cell extraction was performed using a disposable filtration system (Millipore depth filter).

Cell propagation was carried out in a production bioreactor with a volume of 1000 or 2000 liters (stage 4, Fig. 16).

The culture was incubated in the bioreactor for approximately 11 days (depending on cell viability) at 37° C., 50% dissolved oxygen (DO) and pH 7.1. During the run, the pH was shifted to 6.9 before material was collected, a feed (Cell Boost 6) was added and vitamin K ₃ was added. In addition, DMSO was added to the bioreactor. A glucose solution feed was added to the culture to maintain the desired concentration, and a bolus dose of 1 M sodium bicarbonate was added to maintain the desired culture concentration. Material collection was performed using predetermined criteria. During the first four days, cell culture samples were taken daily to determine the number and viability of cells and analyze metabolic processes. Starting from day 5, culture samples were taken twice a day to determine the number and viability of cells and analysis of metabolic processes, and from day 9 - to determine the specific productivity using the ELISA method or affinity HPLC.

- 78 040921

The example provided herein uses a fed-batch mode, however, one skilled in the art can design a perfusion mode using generally similar growth and purification schemes. Alternatively, a person skilled in the art can develop a perfusion method in which the duration of the incubation can be up to 7-120 days.

Cell Collection and Storage (Step 5 in FIG. 16) Material collection was performed using a disposable filtration line. To clarify the collected material, depth filtration and filtration through a filter with a pore size of 0.2 μm were performed. Clarification was followed by filtration through a filter with a pore size of 0.45/0.2 μm. The depth filters were flushed with a liquid jet and the residual liquid was blown out of the system with air. The filtration process was performed at a pump speed <15 l/min, and the maximum detectable pressure. After that, the filters were washed with Tris-HCl buffer and purged with compressed air to increase the degree of product recovery.

The clarified harvest was tested for bioburden, presence of bacterial endotoxins, specific protein content by ELISA or affinity HPLC, SDSPAGE, Western blot, HCP ELISA, presence of residual DNA, presence of viruses by in vitro assay, presence of virus-like particles, S+L- plaques and mycoplasmas.

Cleaning and activation process.

The cleaning scheme is described in Fig. 17. The purification process was based on the use of four chromatographic columns. The protein was purified by affinity chromatography, mixed media chromatography, hydrophobic interaction chromatography, and anion exchange chromatography. The protein was activated at the stage of anion exchange chromatography. The purification process also included virus inactivation and nanofiltration steps.

Ultrafiltration and diafiltration 1 - UF/DF-1 (stage 6).

The clarified collected material was concentrated and subjected to diafiltration through the stages of ultrafiltration and diafiltration (UF/DF) based on the method of tangential flow filtration (TFF). The nominal molecular weight cutoff threshold for the cartridge was 30 kDa. The concentrated and diafiltered harvest was tested for specific protein content by ELISA, HPLC affinity methods, and endotoxin and bioburden were assessed by SDS-PAGE, Western blot and/or HCP ELISA.

Virus inactivation by incubation (step 7).

The material was filtered through a 0.22 µm filter into a sterile mixing bag. Then a solution was added to inactivate the viral content, for example, a solution of Tris/10% Triton was added to the final volume of the filtrate, bringing the concentration of Triton to 1% (w/w). After incubation, before loading onto the affinity chromatography column, the product solution was again filtered with a filter element with a pore size of 0.2 μm. The filtered product after virus inactivation was tested for endotoxin and bioburden by SDS-PAGE and Western blotting.

Affinity chromatography (step 8).

At this stage, an affinity chromatography column was used. The column was packed to a predetermined bed height. This step was performed in 2-4 cycles depending on the amount of product. The specific protein in the feed was determined prior to the addition of Triton due to the confounding caused by Triton in the assay. The affinity chromatography column was equilibrated and loaded with pooled material after virus inactivation and then washed. A second wash was performed and the material was eluted and then stored at 2-8° C. for processing the next day. All stages of chromatography were carried out in a downstream mode.

The eluate was tested for specific protein product content, endotoxin content, residual DNA, sialic acid content, gamma carboxylation percentage, charged N-glycan content, presence of residual leached affinity ligand, and bioburden using techniques well known in the art, including absorbance at 280 nm, RP-HPLC, AIEX HPLC, SEC-HPLC, HCP ELISA, SDSPAGE and Western blotting.

Chromatography on multimodal matrices or mixed excipients (step 9).

At this stage, a column packed with resin for chromatography on multimodal matrices or mixed media was used. The column was packed to a predetermined bed height. The step was performed in 1-4 cycles depending on the amount of product. The column was equilibrated and loaded with the diluted affinity eluate, washed, and the eluate collected and stored at 2-8° C. until further processing. The eluate was tested for specific protein product content, endotoxin content, residual DNA, sialic acid content, gamma carboxylation percentage, charged N-glycans content, presence of residual leached affinity ligand, and bioburden using techniques that included absorbance at 280 nm, RP-HPLC, AIEX HPLC, SEC-HPLC, HCP ELISA, SDS-PAGE and Western blotting.

- 79 040921

Hydrophobic Interaction Chromatography (HIC) (Step 10).

At this stage, a HIC resin was used. The column was packed to a predetermined bed height. Chromatography by HIC was performed in 1-4 cycles depending on the amount of product. The HIC feed was prepared by adding ammonium sulfate to the protein eluate from the multimodal column chromatography or mixed vehicles as an additive. The column was equilibrated and the eluate containing the additive and filtered through a filter with a pore size of 0.2 μm was charged with eluate from a column for chromatography on multimodal matrices or mixed type excipients, and then it was washed. The product was eluted and then stored at 2-8° C. until further processing. The eluate was tested for specific protein concentration by absorbance at 280 nm and SEC-HPLC. The eluate was also tested for the presence of endotoxins and bioburden.

Ultrafiltration and diafiltration of the eluate after HIC (stage 11).

The HIC eluate was concentrated and diafiltered to reduce volume and provide material for the anion exchange column pass step. Once the pH and electrical conductivity were determined to be in the proper range, the system was dried and the material was filtered into a sterile bag through filtration steps through a 0.5/0.2 µm filter. The final volume of the concentrated and diafiltered HIC eluate was stored at 2-8° C. until further processing. The eluate was tested for specific protein product content, endotoxin content, residual DNA, sialic acid content, gamma carboxylation percentage, charged N-glycans content, presence of residual leached affinity ligand, and bioburden using techniques that included absorbance at 280 nm, RP-HPLC, AIEX HPLC, SEC-HPLC, HCP ELISA, SDS-PAGE and Western blotting.

Anion exchange chromatography (step 12).

At this stage, a column packed with anion exchange chromatography resin was used. The column was packed to a predetermined bed height. The feed material was a concentrated diafiltered fraction of the HIC eluate. Activation of FVII to form FVIIa occurred on an anion exchange chromatography column. After the activation and washing step, the product was eluted and collected for further processing. If necessary, the pH of the eluate was adjusted. The eluate was then filtered through a filter with a pore size of 0.45/0.2 μm. The material was stored at 2-8°C until further processing. All chromatography steps were performed in downstream mode. The eluate was tested for specific protein concentration, residual DNA and bioburden by absorbance at 280 nm, RP-HPLC, AIEX HPLC, SEC-HPLC, HCP ELISA, SDS-PAGE and Western blotting.

Removal of viruses by nanofiltration (stage 13).

Virus removal was performed with an Asahi Planova 20N virus removal filter. A membrane filter with a pore size of 0.45/0.2 µm or 0.1 µm was used as a prefilter before the nanofilter (Planova 20N filter). The Asahi Planova 20N filter was pre-equilibrated and pretreated with an anion exchange chromatography elution buffer or a final formulation prepared with formulation buffer. The eluate after anion exchange chromatography was passed through a filtration line at constant pressure and collected in a sterile biotechnological bag. The filtration line (Planova filter) was flushed with anion exchange chromatography elution buffer or formulation buffer to maximize product recovery. The filter was tested for integrity before and after use according to the manufacturer's recommended procedures. The post-use test includes a colloidal gold particle retention test, also in accordance with the manufacturer's procedure. The virus-filtered material was tested for specific protein content by absorbance at 280 nm, RP-HPLC, AIEX HPLC, SEC-HPLC, SDS-PAGE. The virus-filtered material was also tested for endotoxins and bioburden.

UF/DF-3 and drug substance (DS) filtration and storage (step 14).

The material after virus filtration was concentrated to achieve the target concentration of DS (which may vary in the range of 2-100 mg/ml) in the preparation for filtration in the flow and filling. The last step included filtration by sterile filtration through a filter with a pore size of 0.2 μm. At this stage, a disposable or reusable cassette with a cutoff of 3-30 kDa was used. The product was concentrated in the first step to reach a concentration of 5-25 mg/ml protein and subjected to diafiltration (DF) with the addition of 20 mM citrate, 150 mM NaCl, 13.3 mM glycine, pH 6.4 or 20 mM citrate, 100 mM arginine, 2% trehalose, pH 6.2 (>7 DF volumes). The pooled material after UF/DF-3 was tested for specific protein content by determining absorbance at 280 nm. The pooled material after UF/DF-3 was also tested for the presence of endotoxins and bioburden.

The final product concentration was adjusted and polysorbate-80 (PS-80) was added until a final concentration of 0.04% was reached. Alternatively, no addition was made. The UF/DF-3 treated product containing the additive was filtered using a Millipak 100 or Millipak 200 filter. The filtered product solutions were aliquoted and frozen at

70±5°C. The formulated pooled material was tested for product concentration by determining A280. The formulation buffer was 20 mM citrate, 100 mM arginine, 2% trehalose, 0.04% PS80 at pH 6.2.

Results.

The purification process utilized the capture and purification of MOD-5014, which has a high level of gamma carboxylation, in a multimodal array chromatography step (FIG. 21) to give the highly glycosylated MOD-5014 product. In addition, the initial percentage of highly glycosylated MOD-5014 is determined by the accumulation of cell culture biomass and remains constant throughout the purification process (Fig. 19). The process showed high efficiency in removing process impurities such as oxidized forms and other related forms during purification steps using multimodal matrix chromatography and HIC (FIG. 20) and resulted in a high quality product.

SDS-PAGE analysis of the purified MOD-5014 product under reducing conditions is shown in FIG. 18. The following isolated products have been identified (see numbering on the right): 75 kDa non-activated form of MOD-5014 (1); 55 kDa heavy chain MOD-5014-CTP-CTP-CTP (2); 25 kDa - light chain MOD-5014 (4); low molecular weight (LMW) forms (3, 5 and 6).

In table. 9 shows the results of manufacturing processes for a design run (ER) and various GMP (Good Manufacturing Practice) runs in two different CMOs. Details include specific activity, percentage (%) of non-activated MOD-5014, percentage (%) of oxidized form; the percentage (%) of non-carboxylated glutamic acid residues (the proportion without Gla domains), the content of sialic acid (mol/mol) and the content of Oglycans (mol/mol).

Table 9. Quality characteristics of purified MOD-5014

Specific activity (units/mg) 15.563 16.720 22.478 23.608 Non-activated FVII 2.7 2.4 2.6 3.0 Oxidized forms (%) 4.0 4.9 2.9 3.9 5.4 5.5 0.6 0.6 Share without Gla domains (%) 17.1 17.1 18.4 17.2 Sialic acid (mol/mol) 12.3 13.2 13.2 12.5 O-glycans (mol/mol)

In addition, the results obtained in CMO-1 showed that the percentage of charged N-glycans was 85.3 (ER) and 84.2 (GMP1).

Conclusion.

In conclusion, a large-scale fed-batch manufacturing process has been developed that is suitable to enable the clinical development and commercial production of MOD-5014. The results confirm that this process is a reproducible fed-batch production process to produce high glycosylated sustained release FVIIa-CTP (MOD-5014). The purified MOD-5014 product had high levels of O-glycans and sialic acid. The purified product showed minimal levels of non-activated FVII and no Gla domains (non-carboxylated Glu residues).

Example 4

Receipt of a medicinal product (DP).

The drug formulation process (DP) begins with the thawing of the drug substance (DS). The drug product is prepared by diluting the drug substance (DS) to the desired concentration using formulation buffer or filling without dilution, aseptic filtration and filling into standard 2R vials or other primary packaging such as cartridges or pre-filled syringes. The person skilled in the art will understand that the term drug substance (DS) may cover the active pharmaceutical ingredient (API) or be equivalent to it. In one embodiment, the CTP-modified coagulation factor VII set forth herein is a drug substance (DS) containing a bulk purified drug. One of skill in the art will also appreciate that the term drug product (DP) may encompass the final formulated drug after being dispensed into the final container, eg, vial, under aseptic conditions. In one embodiment, the CTP-modified coagulation factor VII set forth herein is a drug product (DP) containing the final formulated CTP-modified coagulation factor VII.

Characterization of CTP-modified coagulation factor VII.

The content of the CTP-modified polypeptide in the collected material and the percentage of highly glycosylated forms are determined using a specific RP-HPLC method. The total protein in the harvested material is determined by Bradford analysis. The percentage of specific protein in the collected material produced by the selected clone exceeds 70% relative to the amount of total protein in the collected material. In addition, the biomass storage manufacturing process is designed to provide a high percentage of highly glycosylated CTP-modified protein compared to the low glycosylated form. The highly glycosylated form is the target form as it results in a greater prolongation of the half-life of the CTP-modified polypeptide.

The content of O-glycans.

The study of carbohydrate composition is performed by the release of glycans followed by labeling of glycans with 2-aminobenzamide (2AB), purification and analysis using NP-HPLC. Briefly, an O-glycan analysis is performed to calculate the number of moles of O-glycans per mole of CTP-modified coagulation factor VII. The terminal galactose units of O-glycans undergo enzymatic cleavage from the protein under the action of β-galactosidase. These free galactose units are separated on a CarboPac PA20 column and detected by pulse amperometry. Galactose (Gal) is quantified by external calibration against a galactose reference standard. The content of galactose can be directly proportional to the content of the O-glycan structure Gal-GalNAc. The analysis of the batches of the drug substance and the medicinal product demonstrates the stable uniformity of product characteristics from batch to batch. This surprisingly stable level of glycosylation is significant and demonstrates that the number of Oglycans per CTP is improved over what is known in the art.

Intact samples for molecular weight analysis.

Molecular weight analysis of different batches of DS is performed to provide information on the number of O-linked glycosylation sites. Intact samples as well as samples desiallylated with neuraminidase and samples de-O-glycosylated with O-glycosidase were analyzed by real-time LC/ES-MS. The result showing a high % occupancy of serine residues is unexpected when compared to levels known in the art (only 4 glycosylated serine residues compared to up to 6 in the CTP-modified coagulation factor VII produced herein).

Occupancy of O-linked glycosylation sites in CTP-modified protein samples.

Occupancy of O-linked glycosylation sites in 4 different batches of DS is determined by M-Scan to provide information on the number of O-linked glycosylation sites per molecule. Samples are desiallylated with neuraminidase followed by trypsin digestion of the reduced/carboxymethylated samples. Finally, real-time LC/ES-MS is performed on the processed samples and MS data is interpreted using specialized software. Evaluation of the data obtained in the analysis of mixtures after cleavage with trypsin gives signals that allow mapping of 100% of the protein sequence. O-glycosylation can take place in the region of both the N-terminal and C-terminal CTP. Occupied sites are defined as serine residues after proline, as well as two of the four serine residues in the serine repeat regions. In total, up to 18 serine residues can serve as attachment sites for O-glycans. No significant differences were found between the parties.

Purity.

During RP-HPLC, molecules are separated according to their polarity. To elute highly polar molecules before less polar molecules, a mobile phase gradient from a more polar to a less polar solvent is used. Related forms are separated from the native protein by UV detection at 220 nm. The relative peak areas (area %) for the related species and the main peak can be calculated by integrating the respective peak areas. The main peak of the drug substance and drug product account for more than 97% of the peak area, which indicates a high degree of purification of the product and an effective purification process.

Size exclusion HPLC is a chromatographic technique that separates molecules according to size. Within the selected fractionation range, larger molecules are eluted earlier than smaller molecules. The separation mechanism is non-adsorptive and the molecules are eluted under isocratic conditions. SEC separates monomers from higher molecular weight forms of the target molecule (such as dimers and polymers). The SEC method has been developed to analyze the content of dimers and polymers in a drug substance and drug product.

Content determination method using RP-HPLC.

This method is used to determine the content of intermediate samples and determine the percentage of non-glycosylated CTP-modified polypeptide in the intermediate samples using reverse phase chromatography. During reverse phase HPLC, the molecules are separated due to their polarity. Relatively non-polar molecules bind to the column material, while charged and polar molecules elute without interacting with the column.

Bound molecules are eluted using a gradient from a polar to a less polar solution. Molecules with the strongest polarity are eluted first, followed by less polar molecules. The detection is carried out by determining the absorbance at 214 nm.

Virus cleansing.

The object of the preliminary evaluation was the ability of the manufacturing process to resolve issues related to contamination of the final medicinal product with endogenous and introduced viruses, and reduce its degree. A GLP compliant study was conducted in accordance with the applicable study product guidelines using three model viruses added in known amounts to downscaled manufacturing process segments to quantify the ability of these steps to inactivate or clear a known virus population added. . Given that the amount of virus is expressed as a ^^-corrected titer, the log10 clearance factor is determined simply by subtracting the output value from the input value. The log10 clearance factors are added to give the overall clearance factor for all stages evaluated. A-MuLV is considered a model virus representing the possible presence of retroviruses in CHO cells, and as a result of the measures taken to inactivate and remove the A-MuLV virus, a clearance factor of at least antilog10, such as the logarithmic viral load reduction factor (LRF), was achieved. , which is approximately 22, demonstrating that the process as a whole is characterized by exceptional virus removal efficiency. For PPV, which is a small, resistant, non-enveloped virus, robust removal is achieved by a nanofiltration step.

While certain features of the present invention have been illustrated and described herein, many modifications, substitutions, alterations, and equivalents will come to mind to those of ordinary skill in the art. Therefore, it is to be understood that the appended claims are intended to cover all such modifications and changes falling within the spirit of the present invention.

Claims (16)

CLAIM 1. A polypeptide that is an active human blood coagulation factor VII (FVIIa), a modified carboxy-terminal peptide (CTP) of human chorionic gonadotropin, containing three CTP molecules attached sequentially one after the other to the C-terminus of FVIIa, where the specified polypeptide, which is a CTP-modified FVIIa , is in an essentially pure and active form, while the specified polypeptide, which is a CTP-modified FVIIa, contains: a) high sialic acid content of at least 15 mol/mol; b) a highly glycosylated form comprising an O-glycan content of at least 10 mol/mol; c) a low content of the oxidized form of the specified CTP-modified FVIIa, constituting less than 5% of the oxidized form; d) a high percentage of carboxylated glutamic acid residues; e) at least 60% charged N-glycans; And f) a specific activity of at least 10500 U/mg; and wherein said CTP-modified FVIIa consists of the amino acid sequence shown under SEQ ID NO: 7. 2. The CTP-modified FVIIa of claim 1, wherein the amino acid sequence of said CTP-modified FVIIa is structurally a disulfide-stabilized double-stranded heterodimer containing a disulfide (S-S) bridge between cysteine residue 135 and cysteine residue 262 in SEQ ID NO: 7 , and where the two chains include a light chain containing amino acids 1-152 and a heavy chain containing amino acids 153-490 of SEQ ID NO: 7. 3. CTP-modified FVIIa according to any one of claims 1, 2, where a) a high percentage of carboxylated residues of glutamic acid (Gla) with - 83 040921 contains at least 90% Gla residues; b) the indicated percentage of charged N-glycans is selected from the group consisting of 85.3 and 84.2%; c) said specific activity is selected from the group consisting of 15563, 16720, 22478 and 23608 units/mg; or any combination of them. 4. CTP-modified FVIIa according to any one of claims 1-3, where a) said substantially pure and active form contains at least 60% of the highly glycosylated form of carboxylated glutamic acid residues (Gla) of said active CTP-modified FVIIa; b) the degree of purity of the substantially pure and active FVIIa CTP-modified polypeptide is at least 90% or selected from 97.3%, 97.6%, 97.4% and 97.0%; or any combination of them. 5. A method for producing a polypeptide that is an active human blood coagulation factor VII (FVIIa) modified with a carboxy-terminal peptide (CTP) of human chorionic gonadotropin, where the specified polypeptide contains three CTP molecules attached sequentially one after another to the C-terminus of FVII, the method includes stages: I) stable transfection of a predetermined number of cells with an expression vector containing a coding portion encoding said CTP-modified FVII, ii) wherein said transfected cells express and secrete said CTP-modified FVII; iii) obtaining cell clones overexpressing said CTP-modified FVII; iv) propagating said clones in solution to a predetermined scale; V) collecting said solution containing said clones; vi) filtering said solution containing said clones to obtain a clarified pooled solution containing said CTP-modified FVII; And vii) purifying and activating CTP-modified FVII from said clarified pooled solution to obtain a purified protein solution having the desired concentration of CTP-modified FVIIa; wherein said resulting CTP-modified FVIIa contains at least one of the following: a) a low content of the oxidized form of the specified CTP-modified FVIIa, constituting less than 5% of the oxidized form; b) a high percentage of carboxylated glutamic acid residues of at least 90% Gla residues; c) at least 60% charged N-glycans, or d) a specific activity of at least 10500 units/mg; thereby obtaining a CTP-modified FVIIa, and wherein the amino acid sequence of the resulting CTP-modified FVIIa is given under SEQ ID NO: 7. 6. The method according to claim 5, where the degree of purity of the polypeptide, which is CTP-modified FVIIa, is at least 90% or selected from 97.3, 97.6, 97.4 and 97.0%; or any combination of them. 7. The method according to any one of claims 5, 6, where a) said expansion step comprises the expansion of clones derived from a working cell bank (WCB) that are characterized by optimal expression and secretion of said CTP-modified FVII, or where said expansion step comprises the expansion of clones derived from a master cell bank (MCB) that are characterized by optimal expression and secretion of said CTP-modified FVII; b) said production method is one that does not use materials of animal origin; c) these clones express and secrete CTP-modified FVII at a level of at least 40 mg/l; d) these clones are propagated in solution through a series of subculturing steps to a level characteristic of a production bioreactor; e) virus clearance is characterized by a log viral load reduction factor (LRF) of approximately 22; or any combination of them. 8. The method of claim 7, wherein said bioreactor comprises a disposable or stainless steel bioreactor, or wherein said bioreactor functions as a fed-batch bioreactor. 9. The method according to any one of claims 5 to 8, wherein said purification of said clarified collected material comprises performing the following steps, including sequentially passing said clarified collected solution through an affinity chromatography column, a chromatography column on multimodal matrices or fillers - 84 040921 mixed type, a column for hydrophobic interaction chromatography and a column for anion exchange chromatography, where the eluate after anion exchange chromatography passes the stage of ultrafiltration/diafiltration; inactivation of viruses present in the clarified collected material or in the eluate collected after passing through any of these chromatographic columns, or in any combination thereof, where the inactivation of viruses includes incubation in a solution toxic to these viruses, or nanofiltration, or any combination thereof; thus obtaining purified CTP-modified FVII. 10. The method according to any one of claims 5-9, where a) said resulting CTP-modified FVIIa is highly glycosylated; b) said resulting CTP-modified FVIIa contains a high content of Oglycans; the glycosylation profile of the resulting CTP-modified FVIIa includes glycosylation at at least 4 O-linked glycosylation sites on the CTP; or any combination of them. 11. The method according to any one of claims 5-10, where a) said resulting CTP-modified FVIIa is highly sialylated; b) said CTP-modified FVIIa comprises a sialic acid content of at least 15 mol/mol; c) said high O-glycan content includes an O-glycan content of at least 10 mol/mol; d) said low percentage of the oxidized form of said CTP-modified FVIIa is less than 5% of the oxidized form; e) said high percentage of carboxylated glutamic acid residues (Gla) in said CTP-modified FVIIa is at least 90% Gla; f) the indicated percentage of charged N-glycans is selected from the group consisting of 85.3% and 84.2%; g) said specific activity is selected from the group consisting of 15563, 16720, 22478 and 23608 units/mg; h) using said method, at least 20% recovery of highly glycosylated CTP-modified FVIIa is achieved; i) the degree of extraction of the specified polypeptide, which is CTP-modified FVIIa, is at least 90% or selected from the group consisting of 97.3, 97.6, 97.4 and 97.0%; or any combination of them. 12. The method of any one of claims 5 to 11, wherein at least 60% of the CTP-modified FVIIa is the highly glycosylated form of carboxylated glutamic acid residues (Gla). 13. The method according to any one of claims 5 to 12, wherein the amino acid sequence of said resulting CTP-modified FVIIa is structurally a disulfide-stabilized double-stranded heterodimer containing a disulfide (S-S) bridge between cysteine residue 135 and cysteine residue 262 in SEQ ID NO: 7, and wherein said two chains include a light chain containing amino acids 1-152 and a heavy chain containing amino acids 153-490 of SEQ ID NO: 7. 14. A polypeptide that is an active human blood coagulation factor VII (FVIIa), a modified carboxy-terminal peptide (CTP) of human chorionic gonadotropin, containing three CTP molecules attached sequentially one after another to the C-terminus of FVII, where the specified polypeptide, which is a CTP-modified FVIIa , consists of the amino acid sequence shown under SEQ ID NO: 7, and where the specified polypeptide, which is a CTP-modified FVIIa, obtained using the method according to any one of claims 5-12. 15. A composition containing a CTP-modified FVIIa according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier, for the treatment of subjects suffering from a coagulation or blood clotting disorder. 16. The composition of claim 15 wherein said coagulation or blood clotting disorder is hemophilia.