CN115161308A - Purification method of tenecteplase supernatant for injection - Google Patents
Purification method of tenecteplase supernatant for injection Download PDFInfo
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- CN115161308A CN115161308A CN202210661963.0A CN202210661963A CN115161308A CN 115161308 A CN115161308 A CN 115161308A CN 202210661963 A CN202210661963 A CN 202210661963A CN 115161308 A CN115161308 A CN 115161308A
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- injection
- tenecteplase
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- 238000002347 injection Methods 0.000 title claims abstract description 54
- 239000007924 injection Substances 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 52
- 108010039185 Tenecteplase Proteins 0.000 title claims abstract description 46
- 229960000216 tenecteplase Drugs 0.000 title claims abstract description 43
- 239000006228 supernatant Substances 0.000 title claims abstract description 42
- 238000000746 purification Methods 0.000 title claims abstract description 28
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 36
- 239000004472 Lysine Substances 0.000 claims abstract description 30
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims abstract description 30
- 238000005571 anion exchange chromatography Methods 0.000 claims abstract description 17
- 239000007853 buffer solution Substances 0.000 claims abstract description 15
- 239000012535 impurity Substances 0.000 claims abstract description 14
- 239000003480 eluent Substances 0.000 claims description 52
- 239000000872 buffer Substances 0.000 claims description 39
- 108090000623 proteins and genes Proteins 0.000 claims description 32
- 102000004169 proteins and genes Human genes 0.000 claims description 32
- 239000000243 solution Substances 0.000 claims description 29
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 229960005480 sodium caprylate Drugs 0.000 claims description 18
- BYKRNSHANADUFY-UHFFFAOYSA-M sodium octanoate Chemical compound [Na+].CCCCCCCC([O-])=O BYKRNSHANADUFY-UHFFFAOYSA-M 0.000 claims description 18
- 239000012488 sample solution Substances 0.000 claims description 17
- 108010039627 Aprotinin Proteins 0.000 claims description 15
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 15
- 229960004405 aprotinin Drugs 0.000 claims description 15
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 claims description 15
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 15
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 15
- 229940068977 polysorbate 20 Drugs 0.000 claims description 15
- 238000011068 loading method Methods 0.000 claims description 13
- 238000010828 elution Methods 0.000 claims description 11
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 6
- SLXKOJJOQWFEFD-UHFFFAOYSA-N 6-aminohexanoic acid Chemical compound NCCCCCC(O)=O SLXKOJJOQWFEFD-UHFFFAOYSA-N 0.000 claims description 5
- 239000004475 Arginine Substances 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- 229960002684 aminocaproic acid Drugs 0.000 claims description 5
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- 238000011146 sterile filtration Methods 0.000 claims description 4
- 239000000047 product Substances 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 7
- 239000000945 filler Substances 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 6
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- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 230000009286 beneficial effect Effects 0.000 description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 4
- 238000011067 equilibration Methods 0.000 description 4
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 4
- 235000019799 monosodium phosphate Nutrition 0.000 description 4
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 3
- 239000003527 fibrinolytic agent Substances 0.000 description 3
- 230000004952 protein activity Effects 0.000 description 3
- 208000007536 Thrombosis Diseases 0.000 description 2
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000013628 high molecular weight specie Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 206010008092 Cerebral artery thrombosis Diseases 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 208000010378 Pulmonary Embolism Diseases 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 108010023197 Streptokinase Proteins 0.000 description 1
- 108010051181 TNK-tissue plasminogen activator Proteins 0.000 description 1
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 1
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 1
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 206010000891 acute myocardial infarction Diseases 0.000 description 1
- 238000004115 adherent culture Methods 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 229940019700 blood coagulation factors Drugs 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000012560 cell impurity Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 230000020764 fibrinolysis Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 239000013315 hypercross-linked polymer Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- 210000001672 ovary Anatomy 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000036316 preload Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229960005202 streptokinase Drugs 0.000 description 1
- 230000002537 thrombolytic effect Effects 0.000 description 1
- 229960000187 tissue plasminogen activator Drugs 0.000 description 1
- 229960005356 urokinase Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6456—Plasminogen activators
- C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21068—Tissue plasminogen activator (3.4.21.68), i.e. tPA
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Animal Behavior & Ethology (AREA)
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- Pharmacology & Pharmacy (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention relates to a purification method of a tenecteplase supernatant for injection, which comprises the steps of firstly pretreating the tenecteplase supernatant for injection and then adopting WorkBeads TM Purifying by 40TREN anion exchange chromatography and lysine HyperD affinity chromatography; wherein WorkBeads TM 40TREN anion exchange chromatography has low filler cost, low cost of raw materials required by buffer solution and longer processing time compared with Blue Sepharose6 Fasthe t Flow affinity chromatography process is reduced by more than half, and the processing speed is high; and the effect of removing impurities is more obvious by matching with the lysine HyperD affinity chromatography process, so that the production period of the process is effectively shortened, and the quality of the product is improved.
Description
Technical Field
The invention relates to the technical field of purification of downstream protein of recombinant protein of a biological product, in particular to a purification method of a tenecteplase supernatant for injection.
Background
In recent years, people have attracted more and more attention due to high mortality and serious sequelae of thrombotic diseases, especially acute myocardial infarction, pulmonary embolism, cerebral arterial thrombosis and the like. Therefore, the research and development of thrombolytic drugs with high efficiency, specificity, safety and few side effects has been a hot spot of drug research in the world in recent years.
The tenecteplase (TNK-tPA) for injection is a freeze-dried powder injection prepared by adopting a recombinant DNA technology, using recombinant Chinese Hamster Ovary (CHO) cells containing high-expression human tissue plasminogen activator modified protein gene to perform inoculation amplification culture by using an amplification culture medium, then performing inoculation tank amplification, tank harvest culture medium, performing cell culture, separation and purification to obtain a human tissue plasminogen activator modified stock solution, and then adding a proper amount of auxiliary materials; the thrombolytic drug not only can be specifically combined with fibrin in thrombus to achieve the effect of local thrombolysis, but also has small destructive effect on fibrinogen, blood coagulation factors and the like in blood, and overcomes the potential bleeding risk caused by activating a whole body fibrinolysis system by early thrombolytic drugs such as streptokinase, urokinase and the like.
In the preparation process of the injection tenecteplase, the cost and the quality of the product are determined by an efficient purification process technology, and the preparation process has great influence on the safety and the effectiveness of clinical application. The existing purification process scheme of the injection tenecteplase generally applies two-step affinity chromatography, namely Blue Sepharose6 Fast Flow affinity chromatography for primary purification, removes process impurities such as host protein residues and the like, plays a role in primarily removing impurity concentrated protein, and then performs lysine HyperD affinity chromatography, so that a better protein purification effect can be achieved, and the purity of the purified injection tenecteplase is more than 95%.
But the time of the Blue Sepharose6 Fast Flow affinity chromatography process is longer in the production process, the filler cost is high, and the recovery rate is low; meanwhile, the removal rate of HCP (host protein residue) and endotoxin in the injection tenecteplase cannot be well controlled by two-step affinity chromatography.
Therefore, it is necessary to provide a purification method which can accelerate the production cycle of the process and greatly improve the product quality.
Disclosure of Invention
The invention aims to provide a purification method of injection tenecteplase supernatant, which can effectively solve the technical problems.
In order to achieve the purpose of the invention, the following technical scheme is adopted:
a purification method of a tenecteplase supernatant for injection comprises the following steps:
step 1: pre-treating the teniprase supernatant for injection:
adding a sodium caprylate solution into the tenipran supernatant for injection, uniformly mixing, and then carrying out deep filtration and sterile filtration to obtain a sample solution S1;
step 2: carrying out WorkBeads on the sample solution S1 obtained in the step 1 TM 40TREN anion exchange chromatography, and eluting with eluent A1 to obtain target protein active peak eluent S2;
and step 3: and (3) performing lysine HyperD affinity chromatography on the target protein active peak eluent S2 obtained in the step (2), and eluting by using an eluent A2 to obtain the purified injection tenecteplase solution.
Preferably, in the step 1, the dosage of the sodium caprylate solution is as follows: adding (1 +/-0.1) g of sodium caprylate solution into each liter of the tenecteplase supernatant for injection, wherein the prepared concentration of the sodium caprylate solution is 10%.
Preferably, in the step 2, the components in the eluent A1 comprise 10-80mM Tris-HCl and 0.5-2M NaCl, and the pH value of the eluent A1 is 7.2-7.6, and the dosage of the eluent A1 is 5-10 times of the column volume.
Preferably, in the step 2, workBeads is carried out TM In 40TREN anion exchange chromatography, the sample solution S1 needs to be equilibrated with buffer B1 before loading.
Preferably, the composition in the buffer B1 comprises 10-80mM Tris-HCl, and the pH value of the buffer B1 is 7.2-7.6, and the dosage of the buffer B1 is 5-10 times of the column volume.
Preferably, in the step 3, when performing the lysine HyperD affinity chromatography, the binding capacity of the lysine HyperD affinity chromatography to the injection tenecteplase protein is 5-10mg/ml, the components in the eluent A2 comprise 0.2-1.0M arginine, 0.1-0.5M aminocaproic acid, 0.020% -0.050% polysorbate 20, 6-10mg/L aprotinin, and 10.0-20.0mg/ml phosphoric acid, and the pH value of the eluent A2 is 7.4-7.8, and the dosage of the eluent is 3-5 times of the column volume.
Preferably, in step 3, when performing lysine HyperD affinity chromatography, the target protein active peak eluate S2 needs to be equilibrated with buffer B2 before loading.
Preferably, the components in the buffer B2 comprise 10-50mM phosphate buffer, 0.020% -0.050% polysorbate 20 and 1-4mg/L aprotinin, and the pH value of the buffer B2 is 7.2-8.0, and the dosage of the buffer B2 is 5-15 times of the column volume.
Preferably, in the step 3, when performing lysine HyperD affinity chromatography, the target protein activity peak eluate S2 is subjected to impurity elution treatment on the chromatography column after loading with a buffer B3, wherein the buffer B3 comprises 10-50mM phosphate buffer, 1-3M sodium chloride, 0.020-0.050% polysorbate 20, and 1-4mg/L aprotinin, and the PH of the buffer B3 is 7.2-8.0, and the amount of the buffer is 5-15 times of the column volume.
In addition, the invention also provides the injection tenecteplase solution obtained by the purification method, wherein the purity of the injection tenecteplase solution is more than 98.5 percent, and the yield is more than 90 percent.
Compared with the prior art, the invention has the following beneficial effects:
the purification process adopted by the invention comprises the steps of firstly pretreating the supernatant of the tenecteplase for injection and then adopting WorkBeads TM Purifying by 40TREN anion exchange chromatography and lysine HyperD affinity chromatography; wherein WorkBeads TM The 40TREN anion exchange chromatography process has the advantages of low filler cost, low raw material price required by the buffer solution and process treatmentCompared with a Blue Sepharose6 Fast Flow affinity chromatography process, the time is reduced by more than half, and the processing speed is high; and the effect of removing impurities is more obvious by matching with the lysine HyperD affinity chromatography process, so that the production period of the process is effectively shortened, and the quality of the product is improved.
In addition, the pretreatment process adopted by the invention has low cost, can effectively remove cell impurities, reduces the microbial load in the supernatant, and has good treatment effect on the supernatant, thereby effectively reducing the load of the filler in the later lysine HyperD affinity chromatography process, being beneficial to eluting the hybrid protein, and prolonging the service life of the filler.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments.
The invention provides a purification method of a tenecteplase supernatant for injection, which comprises the following steps:
step 1: pre-treating the teniprase supernatant for injection: adding a sodium caprylate solution into the tenipran supernatant for injection, uniformly mixing, and then carrying out deep filtration and sterile filtration to obtain a sample solution S1; wherein the dosage of the sodium caprylate solution is as follows: adding (1 +/-0.1) g of sodium caprylate solution into each liter of the tenecteplase supernatant for injection, wherein the prepared concentration of the sodium caprylate solution is 10%.
Step 2: carrying out WorkBeads on the sample solution S1 obtained in the step 1 TM 40TREN anion exchange chromatography, and eluting with eluent A1 to obtain target protein active peak eluent S2;
wherein, the WorkBeads is carried out TM In 40TREN anion exchange chromatography, the sample solution S1 needs to be equilibrated with buffer B1 before loading.
Specifically, the components in the eluent A1 comprise 10-80mM Tris-HCl and 0.5-2M NaCl, the pH value of the eluent A1 is 7.2-7.6, and the dosage of the eluent A1 is 5-10 times of the column volume. The buffer B1 comprises 10-80mM Tris-HCl, the pH value of the buffer B1 is 7.2-7.6, and the dosage of the buffer B1 is 5-10 times of the column volume.
And step 3: performing lysine HyperD affinity chromatography on the target protein active peak eluent S2 obtained in the step 2, and eluting by using an eluent A2 to obtain a purified tenecteplase solution for injection;
wherein, when the lysine HyperD affinity chromatography is carried out, the binding capacity of the lysine HyperD affinity chromatography to the tenecteplase protein for injection is 5-10mg/ml; before the target protein active peak eluent S2 is loaded, a buffer B2 is needed to carry out equilibrium treatment on the chromatographic column, and after the target protein active peak eluent S2 is loaded, a buffer B3 is needed to carry out impurity elution treatment on the chromatographic column.
Specifically, the components in the eluent A2 comprise 0.2-1.0M arginine, 0.1-0.5M aminocaproic acid, 0.020-0.050% polysorbate 20, 6-10mg/L aprotinin and 10.0-20.0mg/ml phosphoric acid, and the pH value of the eluent A2 is 7.4-7.8, and the dosage of the eluent A2 is 3-5 times of the column volume.
The buffer B2 comprises 10-50mM phosphate buffer, 0.020% -0.050% polysorbate 20 and 1-4mg/L aprotinin, the pH value of the buffer B2 is 7.2-8.0, and the dosage of the buffer B2 is 5-15 times of the column volume.
The buffer B3 comprises 10-50mM phosphate buffer solution, 1-3M sodium chloride, 0.020% -0.050% polysorbate 20 and 1-4mg/L aprotinin, wherein the pH value of the buffer B3 is 7.2-8.0, and the dosage of the buffer B3 is 5-15 times of the column volume.
After the purification method is used for purifying the injection tenecteplase supernatant, the purity of the obtained product is more than 98.5 percent, and the yield is more than 90 percent.
The purification principle of the invention is as follows: the product is prepared by performing CHO cell adherent culture, secreting target protein into supernatant, collecting cell supernatant, adding 1 +/-0.1 g of sodium caprylate solution into per liter of supernatant, mixing uniformly, and performing deep filtration and aseptic filtration to remove a small amount of fallen cell fragments and impurities.
WorkBeads TM The filler used for 40TREN anion exchange chromatography is a multifunctional anion exchange column, and allows cell supernatant to be directly loaded without dilution; the packing is positively charged in an environment below pH9, has been shown to be effective in removing chromatin complexes, endotoxins and host cell proteins, and in most cases the isoelectric point of the protein molecules such as antibodies is above pH 7.4, and so will flow directly through without binding to the packing; almost all HCD and most HCPs bound to it (more than 95% reduction), which means that most High Molecular Weight Species (HMWS), such as soluble aggregates and oligomers of the target antibody, will also be removed; therefore, the impurity removal level of the whole purification process of the target protein is greatly improved, the endotoxin, HCP and HCD residues have stable control effect, and the product purity is improved.
And the sodium caprylate solution is added in the pretreatment process, so that the elution of impurity protein in the process of the lysine HyperD affinity chromatography is facilitated, and the filler load of the lysine HyperD affinity chromatography is effectively reduced through the pretreatment, so that the lysine HyperD affinity chromatography can quickly and efficiently purify the target protein.
Example 1
The invention provides a purification method of tenecteplase supernatant for injection, which comprises the following steps:
step 1: pretreating the injection tenecteplase supernatant: specifically, a sodium caprylate solution is added into the supernatant of the injection tenecteplase and uniformly mixed to ensure that the concentration of the sodium caprylate in the supernatant is 1g/L, and deep filtration and aseptic filtration are carried out after uniform mixing to obtain a sample solution S1.
And 2, step: carrying out WorkBeads on the sample solution S1 obtained in the step 1 TM 40TREN anion exchange chromatography:
WorkBeads TM 40TREN anion exchange chromatography pre-load equilibration: sample solution S1 was equilibrated for 5 column volumes on the chromatography column using buffer B1 before loading, the components in buffer B1 comprising 20mM Tris-HCl, pH 7.2;
WorkBeads TM 40TREN anion exchange chromatography loading and elution: sample solution S1 is carried out after chromatographic column equilibrationSampling, and eluting by using an eluent A1 with 6 times of column volume to obtain a target protein active peak eluent S2; the fraction in eluent A1 comprised 30mM Tris-HCl and 0.5M NaCl at a pH of 7.2.
And 3, step 3: and (3) performing lysine HyperD affinity chromatography on the target protein active peak eluent S2 obtained in the step 2:
equilibration and loading of lysine HyperD affinity chromatography: before the target protein active peak eluent S2 is loaded, 5 column volumes of the chromatographic column are balanced by using a buffer solution B2, the components in the buffer solution B2 comprise 10mM phosphate buffer solution (which can be disodium hydrogen phosphate and sodium dihydrogen phosphate), 0.043% of polysorbate 20 and 2mg/L of aprotinin, and the pH value is 7.4; then carrying out target protein active peak eluent S2 sampling;
lysine HyperD affinity chromatography impurity elution: after the target protein activity peak eluent S2 is loaded, 3 column volumes of the chromatographic column are balanced by using a buffer solution B2, then the hybrid protein is washed out by using a buffer solution B3, the components in the buffer solution B3 comprise 10mM phosphate buffer solution (which can be disodium hydrogen phosphate and sodium dihydrogen phosphate), 1M sodium chloride, 0.043% polysorbate 20 and 2mg/L aprotinin, and the using amount of the buffer solution D is 10 column volumes.
lysine HyperD affinity chromatography peak elution of interest: and finally eluting by using eluent A2 with 3 times of column volume, wherein the components in the eluent A2 comprise 0.2M arginine, 0.1M aminocaproic acid, 0.043% polysorbate 20, 6mg/L aprotinin and 10mg/L phosphoric acid, the PH is 7.4, and the eluted solution is the tenecteplase purified solution for injection.
The total time consumption of the process adopting the purification scheme is reduced to 1.5 days, and compared with the original purification process, the time of the process is shortened by 1 time by 3 days, thereby being beneficial to the amplification production of the later process and the quality control of products.
The purified tenecteplase purified liquid for injection obtained by the purification has the purity of more than 97.5 percent, HCP of less than 0.015 percent, endotoxin of less than 1.2EU/mg, yield of more than 89 percent, and various impurity indexes are effectively controlled.
Example 2
The invention provides a purification method of a tenecteplase supernatant for injection, which comprises the following steps:
step 1: pre-treating the teniprase supernatant for injection: specifically, a sodium caprylate solution is added into the supernatant of the injection tenecteplase and uniformly mixed to ensure that the concentration of the sodium caprylate in the supernatant is 1g/L, and after uniform mixing, deep filtration and sterile filtration are carried out to obtain a sample solution S1.
Step 2: carrying out WorkBeads on the sample solution S1 obtained in the step 1 TM 40TREN anion exchange chromatography:
WorkBeads TM equilibration prior to loading on 40TREN anion exchange chromatography: sample solution S1 was equilibrated for 8 column volumes on the chromatography column using buffer B1 before loading, the components in buffer B1 comprising 50mM Tris-HCl, pH 7.4;
WorkBeads TM 40TREN anion exchange chromatography loading and elution: after the chromatographic column is balanced, sample solution S1 is loaded, and after the sample is loaded, eluent A1 with 5 times of column volume is used for elution to obtain target protein active peak eluent S2; the fraction in eluent A1 comprised 50mM Tris-HCl and 1M NaCl at a pH of 7.4.
And 3, step 3: and (3) performing lysine HyperD affinity chromatography on the target protein active peak eluent S2 obtained in the step 2:
equilibration and loading of lysine HyperD affinity chromatography: before the target protein active peak eluent S2 is loaded, 3 column volumes of the chromatographic column are balanced by using a buffer solution B2, the components in the buffer solution B2 are 20mM phosphate buffer solution (which can be disodium hydrogen phosphate and sodium dihydrogen phosphate), 0.043% of polysorbate 20 and 2mg/L aprotinin, and the pH value is 7.6; then carrying out target protein active peak eluent S2 sampling;
lysine HyperD affinity chromatography impurity elution: after the target protein activity peak eluent S2 is loaded, 3 column volumes of the chromatographic column are balanced by using a buffer B2, and then the hybrid protein is washed out by using a buffer B3, wherein the components in the buffer B3 comprise 20mM phosphate buffer (which can be disodium hydrogen phosphate and sodium dihydrogen phosphate), 1M sodium chloride, 0.043% polysorbate 20 and 2mg/L aprotinin, and the using amount of the buffer D is 10 column volumes.
lysine HyperD affinity chromatography peak elution of interest: and finally eluting by using eluent A2 with 3 times of column volume, wherein the components in the eluent A2 comprise 0.5M arginine, 0.2M aminocaproic acid, 0.043% polysorbate 20, 10mg/L aprotinin and 15mg/L phosphoric acid, the PH is 7.4, and the eluted solution is the tenecteplase purified solution for injection.
The total time of the process adopting the purification scheme is shortened to 1.5 days, and compared with the original purification process, the time of the process is shortened by 1 time by 3 days, thereby being beneficial to the amplification production of the later process and the quality control of products.
The purity of the purified tenecteplase liquid for injection obtained by the purification is more than 98.5 percent, HCP is less than 0.01 percent, endotoxin is less than 1EU/mg, the yield is more than 90 percent, and all impurity indexes are effectively controlled.
Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention.
Claims (10)
1. A purification method of a tenecteplase supernatant for injection is characterized by comprising the following steps: the method comprises the following steps:
step 1: pre-treating the teniprase supernatant for injection:
adding a sodium caprylate solution into the tenipran supernatant for injection, uniformly mixing, and then carrying out deep filtration and sterile filtration to obtain a sample solution S1;
step 2: carrying out WorkBeads on the sample solution S1 obtained in the step 1 TM 40TREN anion exchange chromatography, and eluting with eluent A1 to obtain target protein active peak eluent S2;
and 3, step 3: and (3) performing lysine HyperD affinity chromatography on the target protein active peak eluent S2 obtained in the step (2), and eluting by using an eluent A2 to obtain the purified injection tenecteplase solution.
2. The method for purifying the tenecteplase supernatant for injection as claimed in claim 1, wherein the method comprises the following steps: in the step 1, the dosage of the sodium caprylate solution is as follows: adding (1 +/-0.1) g of sodium caprylate solution into each liter of the tenecteplase supernatant for injection, wherein the prepared concentration of the sodium caprylate solution is 10%.
3. The method for purifying the tenecteplase supernatant for injection according to claim 1, wherein the method comprises the following steps: in the step 2, the components in the eluent A1 comprise 10-80mM Tris-HCl and 0.5-2M NaCl, the pH value of the eluent A1 is 7.2-7.6, and the dosage of the eluent A1 is 5-10 times of the column volume.
4. The method for purifying the tenecteplase supernatant for injection as claimed in claim 1, wherein the method comprises the following steps: in the step 2, workBeads is carried out TM In 40TREN anion exchange chromatography, the sample solution S1 is equilibrated with buffer B1 before loading.
5. The method for purifying the tenecteplase supernatant for injection as claimed in claim 4, wherein the method comprises the following steps: the buffer B1 comprises 10-80mM Tris-HCl, the pH value of the buffer B1 is 7.2-7.6, and the dosage of the buffer B1 is 5-10 times of the column volume.
6. The method for purifying the tenecteplase supernatant for injection as claimed in claim 1, wherein the method comprises the following steps: in the step 3, when the lysine HyperD affinity chromatography is carried out, the binding capacity of the lysine HyperD affinity chromatography to the injection tenecteplase protein is 5-10mg/ml, the components in the eluent A2 comprise 0.2-1.0M arginine, 0.1-0.5M aminocaproic acid, 0.020-0.050% of polysorbate 20, 6-10mg/L of aprotinin and 10.0-20.0mg/ml of phosphoric acid, and the pH value of the eluent A2 is 7.4-7.8, and the dosage of the eluent is 3-5 times of the column volume.
7. The method for purifying the tenecteplase supernatant for injection according to claim 1, wherein the method comprises the following steps: in the step 3, when lysine HyperD affinity chromatography is performed, the target protein active peak eluent S2 needs to be subjected to equilibrium treatment on the chromatographic column by using the buffer solution B2 before loading.
8. The method for purifying the tenecteplase supernatant for injection according to claim 7, wherein the method comprises the following steps: the buffer B2 comprises 10-50mM phosphate buffer, 0.020% -0.050% polysorbate 20 and 1-4mg/L aprotinin, the pH value of the buffer B2 is 7.2-8.0, and the dosage of the buffer B2 is 5-15 times of the column volume.
9. The method for purifying the tenecteplase supernatant for injection as claimed in claim 7, wherein the method comprises the following steps: in the step 3, when lysine hyperD affinity chromatography is performed, after the target protein active peak eluent S2 is loaded, a buffer solution B3 is used for performing impurity elution treatment on the chromatographic column, wherein the buffer solution B3 comprises 10-50mM phosphate buffer solution, 1-3M sodium chloride, 0.020% -0.050% polysorbate 20 and 1-4mg/L aprotinin, the pH value of the buffer solution B3 is 7.2-8.0, and the dosage of the buffer solution B3 is 5-15 times of the column volume.
10. A tenecteplase solution for injection obtained by the purification method according to any one of claims 1 to 9, wherein: the purity of the injection tenecteplase solution is more than 98.5 percent, and the yield is more than 90 percent.
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| CN115386562A (en) * | 2022-09-01 | 2022-11-25 | 江苏丰华生物制药有限公司 | Method for removing recombinant protein endotoxin |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468862A (en) * | 2002-07-19 | 2004-01-21 | 广州铭康生物工程有限公司 | Technological process of producing recombinant human histiotype plasminogen activator TNK mutant |
| US20130330808A1 (en) * | 2010-11-16 | 2013-12-12 | Cadila Healthcare Limited | Novel process for the purification of tissue plasminogen activator |
| US20180223270A1 (en) * | 2014-10-21 | 2018-08-09 | Gennova Biopharmaceuticals Limited | A novel purification process for isolation and commercial production of recombinant tnk-tpa tenecteplase |
| CN109689675A (en) * | 2016-09-07 | 2019-04-26 | 葛兰素史密斯克莱知识产权发展有限公司 | The method of antibody purification |
| CN113166792A (en) * | 2018-12-14 | 2021-07-23 | 比亚分离公司 | A method for removing or eliminating endotoxin from an endotoxin-containing source or a potential endotoxin-containing source |
-
2022
- 2022-06-13 CN CN202210661963.0A patent/CN115161308A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468862A (en) * | 2002-07-19 | 2004-01-21 | 广州铭康生物工程有限公司 | Technological process of producing recombinant human histiotype plasminogen activator TNK mutant |
| US20130330808A1 (en) * | 2010-11-16 | 2013-12-12 | Cadila Healthcare Limited | Novel process for the purification of tissue plasminogen activator |
| US20180223270A1 (en) * | 2014-10-21 | 2018-08-09 | Gennova Biopharmaceuticals Limited | A novel purification process for isolation and commercial production of recombinant tnk-tpa tenecteplase |
| CN109689675A (en) * | 2016-09-07 | 2019-04-26 | 葛兰素史密斯克莱知识产权发展有限公司 | The method of antibody purification |
| CN113166792A (en) * | 2018-12-14 | 2021-07-23 | 比亚分离公司 | A method for removing or eliminating endotoxin from an endotoxin-containing source or a potential endotoxin-containing source |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115386562A (en) * | 2022-09-01 | 2022-11-25 | 江苏丰华生物制药有限公司 | Method for removing recombinant protein endotoxin |
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