WO1996009839A1

WO1996009839A1 - Immunoglobulin preparation

Info

Publication number: WO1996009839A1
Application number: PCT/JP1995/001979
Authority: WO
Inventors: Tuyoshi Takahashi; Kenmi Miyano; Hideo Nishimaki; Koichi Irie; Toshiyuki Saki; Shinji Tomioka
Original assignee: The Green Cross Corporation
Priority date: 1994-09-28
Filing date: 1995-09-28
Publication date: 1996-04-04

Abstract

A more safe immunoglobulin preparation, especially one used for intramuscular injection, that can be obtained by an efficient and well-operable production process without causing viral contamination. The preparation is HCV genome negative, contains HCV genome in a concentration of at most 1 PCR unit/ml in a 0.1-15 w/v % immunoglobulin solution, and has a HCV genome removal rate of 103 or above.

Description

Description Immunoglobulin preparation technology

The present invention relates to an immunoglobulin preparation excellent in safety, and particularly to an immunoglobulin preparation for intramuscular injection. Background technology

Immunoglobulin is a generic name for antibodies and proteins that have structural and functional relationships with them, and is also called imnoglobulin (abbreviated as Ig). It is present in the body fluids of all vertebrates, from fish to mammals, and is produced by lymphoid cells (plasma cells). Classified into five classes (G, M, A, D, E) based on their physicochemical or immunological properties. The basic structure of the molecule is common to each class, and consists of a heavy chain with a molecular weight of 50,000 to 70,000 and a light chain with 23,000. Since each class is a collection of a wide variety of antibodies, there are antibodies that have the same specificity even if the classes are different. In addition, immunoglobulins belonging to the same class have partially different structures depending on the reaction specificity with antigens. Thus, immunoglobulins are a mixture of very diverse proteins.

Immunoglobulin preparations are a combination of normal immunoglobulin preparations prepared from pooled plasma, immunoglobulin preparations consisting of immunoglobulins with particularly high antibody titers to specific antigens, and immunoglobulin and other substances Immunoglobulin preparations, and immunoglobulin preparations collected from plasma obtained by immunizing animals, and so-called special immunoglobulin preparations. Depending on the route of administration, there are preparations for intramuscular administration (intramuscular injection) and preparations for intravenous administration (intravenous injection). Intravenous preparations include non-chemically modified preparations, chemically modified preparations, and enzyme-treated preparations depending on the manufacturing method. Non-chemically modified preparations include polyethylene glycol (PEG) treated preparations, pH 4 treated preparations, and ion exchanger treated preparations. Chemically modified products include alkylated products and sulfonated products. Enzyme-treated preparations include plasmin-treated preparations, pepsin-treated preparations, and tribsine-treated preparations. These immunoglobulin preparations are strictly regulated according to the “Biological Preparation Standards” supervised by the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare.

Since immunoglobulin contains a large amount of antibodies such as viruses and bacteria, it is widely used as an immunoglobulin preparation for the prevention and treatment of infectious diseases.

Immunoglobulin preparations, which are plasma protein preparations, are generally produced from human or other mammalian plasma. However, it is possible that various contaminating viruses may be present in the plasma of humans or other mammals that are the raw materials for immunoglobulin preparations.

Among the contaminating viruses in plasma, hepatitis C virus (hereinafter also referred to as HCV) has only recently been confirmed to be present, and the method of preventing and treating HCV infection has not yet been fully elucidated. .

An object of the present invention is to provide a safer immunoglobulin preparation, particularly an immunoglobulin preparation for intramuscular injection, which can be obtained efficiently and with good operability against virus infection. It is in. Disclosure of the invention

In this study, the present inventors conducted various studies in consideration of the above circumstances, and found that immunoglobulin preparations, particularly immunoglobulin preparations for intramuscular injection, were more safe against virus infection, especially HCV infection. The present inventors have found that it is possible to develop a formulation having a high level of performance and completed the present invention.

That is, the present invention is an immunoglobulin preparation having a negative HCV genome, particularly an immunoglobulin preparation for intramuscular injection.

The term "HCV genome negative" in the present invention indicates a level of HCV genome amount of 1 PCR unit (or CID ₅₀ ) / ml or less.

One of the preferred examples of "negative HCV genome" is that the amount of HCV genome is less than 1 PCR unit Zm1 in 0.1 to 15 wZv% immunoglobulin solution.

Further, "HCV genome negative J, other, in a one favored correct example, in which the removal rate of the HCV genome indicates 1 0 ³ or more. The method of measuring and calculating the “removal rate of the HCV genome” in the present invention is not particularly limited. However, the PCR unit value of the immunoglobulin-containing solution before the preparation of the immunoglobulin preparation of the present invention and the immune unit of the present invention The method by comparing the PCR unit values of the glopurin preparation is most preferable.

To remove or inactivate viruses that may be contaminated with plasma fractionated products, we have developed raw plasma screening, advanced purification, liquid heating, dry heating, TN BP / Tween treatment, so-called SD treatment, etc. Currently, a variety of formulations are available that incorporate one or more virus removal or inactivation methods. For example, JP

No. 1-7 8 7 3 0, No. 6 1 — 1 9 1 6 2 2 No., Japanese Unexamined Patent Application Publication No. Hei 2-8 3 3 3 2 No. 2 0 9 8 14 No. 3, 3-2 1 8 3

No. 2 Publication, No. 3 — 2 5 8 7 2 8 Publication, USPN o. 4 7 2 1 7 7 7, EPAN o. 1 7 7 8 3 6, EPAN o. 1 9 6 7 2 WP IN o. 9 0-1 3 5 6 7 2/1 8. WP IN o. 9 0— 2 5 7 5 8 0/3 4, WPI o. 9 0-2 9 5 5 9 7/39, USPN o. 5 See 1 5 1 4 9 9, EPAN o. 3 7 8 2 0 8, WP IN o. 9 2 — 0 0 4 4 5 1/0 1, etc.

By the way, regarding immunoglobulin preparations for intramuscular injection, there is a very low possibility of virus contamination due to screening of the source plasma and alcohol fractionation, indicating that the preparations are highly safe even from an epidemiological point of view. It is said. However, immunoglobulin preparations for intramuscular injection do not incorporate PEG fractionation, heat treatment or SD treatment, and the possibility of virus contamination cannot be completely ruled out.

Therefore, membrane-based treatment using porous hollow fibers as a new virus removal method (or inactivation method) is attracting attention [Japanese Journal of Transfusion Medicine Medicine, No. 34, Vol. 6, pp. 615-617. 198 8 (Japananese Journalof Transfusion Medicine, 34 (6) 6 15-6 17 (1 988))]. However, the porous hollow fiber has a very fine pore size and therefore has poor protein permeability, and it is difficult to perform membrane filtration using a porous hollow fiber without using a high-molecular-weight protein or a high-concentration protein solution. It was difficult, and it was necessary to consider various conditions in order to improve the permeation performance. Preferred examples of the method for producing the immunoglobulin preparation of the present invention include: Guropuri down-containing solution Hitahama porous hollow fibers the average pore diameter. 1 to 1 0 0 nm respect, a method of performing 4-5 Te 0, the membrane filtration treatment with逋過pressure 0. 1~1 kgf / cm ² Are mentioned.

① Starting material

The immunoglobulin-containing solution, which is the starting material for this production method, is not particularly limited as long as it basically contains immunoglobulin as a main component.

Therefore, the immunoglobulin contained as an active ingredient in the immunoglobulin preparation obtained by this production method is highly purified to the extent that it is used as a pharmaceutical. Specific starting materials include those derived from human or plasma plasma and derived from the immunoglobulin fraction.

For example, as a starting material, a fraction II obtained from the ethanol fraction of corn from plasma or an equivalent paste containing immunoglobulin and the like can be mentioned.

In this production method, particularly preferably, the II fraction or the II + III fraction of the colon, which is a raw material of the immunoglobulin preparation, is used. In addition, this production method can be applied to an immunoglobulin preparation comprising an immunoglobulin having an especially high antibody titer to a specific antigen, an immunoglobulin preparation in which the immunoglobulin is complexed with another substance, a so-called special immunoglobulin preparation. Is effective. For example, anti-HBs human immunoglobulin containing a high unit of high HBs antibody (trade name “Hespulin”, manufactured by Green Cross), anti-tetanus human immunoglobulin containing a high unit of tetanus antitoxin Anti-D (Rh0) immunoglobulin (trade name "anti-D human immunity") containing high units of anti-D (Rh0) antibody Includes high-titer anti-CMV (cytomegalovirus) antibody, anti-CMV human immunoglobulin, and high-titer anti-human lymphocyte antibody Anti-immunoglobulin (trade name “Albulin”, manufactured by Green Cross) or histamine-added human immunoglobulin (trade name “Histobrin”, manufactured by Green Cross) ) And the like. In the present invention, an intramuscular formulation is exemplified as a particularly preferred embodiment, but the present invention is not limited to this.For example, an intravenous formulation is also an embodiment of the present invention. Is exemplified.

② Porous hollow fiber Various other requirements can be selected for the porous hollow fiber used in the membrane filtration treatment of this production method as long as the above conditions are satisfied.

The material of the porous hollow fiber is not particularly limited, but is preferably a regenerated cellulose.

The porous hollow fiber of the regenerated cellulose is preferably subjected to a microphase separation method [American Chemical Society (Am. Chem. Soc.), 9, 1997—22] from a cellulose ammonia solution. 8 (19985)]. The average pore size of the porous hollow fiber is from 1 to 100 nm, preferably from 10 to 75 nm, more preferably from 10 to 50 nm, particularly preferably 35 ± 2 nm or 15 ± 2 nm, the inner diameter of the hollow fiber is preferably 330 ± 30 m, the film thickness is preferably 35 ± 3.5 / m or 27 ± 3; um, and the film is preferably hired. Structure.

The porous hollow fiber is preferably used in the form of a module. The module is configured by an adhesive to integrate the porous hollow fiber and container for Takashi塡this and these having the film structure of the membrane area 0. 0 l ~ lm ^2.

③ Filtration method

The solution containing the immunoglobulin in (1) is immersed in the membrane consisting of the porous hollow fiber in (2) and filtered.

The immunoglobulin-containing solution is adjusted to a protein concentration of about 0.1 to 15 wZv%. The pH of the solution is particularly adjusted to about 6.4 to 7.2.

Filtration pressure is 0. 1~ 1 1ζ 2 ί ^ πι 2, is favored properly 0. 1~ 0. 5 kgf Z cm ² or so, more preferred properly is 0. 1 ~ 0. 3 kgfcm ^2.

The processing temperature is 4 to 50 ° C, preferably 30 to 40 ° C (:, more preferably 35 to 37 ° C.

As a form of the dependent treatment, there are a cross-floor pass-through method (circulation type) in which a fluid is strained while giving a strain rate, and a dead-end method (a non-circulation type) in which a fluid passes without a strain rate. is there. Preferably, a method using pressurized air is used.

In addition, the film transfer process under the conditions of the above manufacturing method can be performed a plurality of times. Further, as the immunoglobulin-containing solution to be treated by this production method, the immunoglobulin-containing solution is preliminarily prepared before the membrane filtration step under the above conditions. Other than the above, those which have been subjected to filtration treatment with a hollow fiber membrane filtration or a flat membrane filtration membrane or the like can be used.

The immunoglobulin preparation of the present invention is further formulated or its dosage is adjusted according to the intended efficacy.

Examples of the dispensing method include a method in which various antigens and antibodies are made negative, and a method in which amino acids. Sugars, sugar alcohols, inorganic salts, and other additives are added. By preparing a dry preparation, storage for a longer period can be performed.

The immunoglobulin preparation of the present invention is effective in preventing the onset of various infectious and allergic diseases and in reducing the symptoms.

The dose and frequency of administration of the immunoglobulin preparation of the present invention are appropriately adjusted and studied depending on the composition of the preparation, the patient's symptoms, age, body weight, and the like. BEST MODE FOR CARRYING OUT THE INVENTION

Examples and experimental examples are described in order to explain the present invention in more detail, but the present invention is not limited thereto.

Example 1

The study was conducted using an HCV spike test.

(1) As a starting material, a sample prepared by adding HCV positive plasma to a 5 w / v% immunoglobulin solution was used.

(2) BMM purchased from Asahi Kasei Corporation was used as the porous hollow fiber (the same applies to the following examples and experimental examples).

B MM: Porous hollow fiber [Bemberg Microcroporous Membrane] made from cuprammonium regenerated cellulose as raw material 0.001 to 1. O m ² Having.

A BMM module with an average pore size of 35 nm was used.

The BMM module with an average pore diameter of 35 nm (trade name: Planova 35) has an average pore diameter of 35 ± 2 nm, and the membrane layer has a multilayer structure with a multilayer structure of more than 150 expansions. 0 ± 30 im, hollow fiber membrane with a thickness of 35 ± 3.5 m and a polycarbonate capable of high-pressure steam sterilization The autoclave sterilized product is composed of a plastic container made of Bonnet and a polyurethane adhesive that integrates them, and the module is filled with distilled water for injection. The safety of the various materials that make up Branova has been confirmed by the method specified by the Japanese Pharmacopoeia (BMM product description damage).

③ Filtration method

A sample obtained by adding HCV-positive plasma to a 5 wZv% immunoglobulin solution was adjusted to have a pH of 6.4 to 7.2. After removing bacteria (filtration through a membrane filter with a pore size of 0.2 m and membrane filter), the membrane is filtered for 35 to 37 times at a filtration pressure of 0.2 kgf Z cm ² for 1 to 5 hours. (A dead-end method using air pressure). After cooling, sterilization filtration was performed again, and the mixture was dispensed to prepare a human immunoglobulin preparation for intramuscular injection.

The HCV genome in the solution before and after the treatment of this sample was measured by the PCR method.

The PCR method in this test was performed by a nested (double) [Nested (Double)] reverse transcription polymerase chain reaction. This method involves the steps of extracting RNA from a test sample, preparing (HCV) cDNA, analyzing NestedPCR and analyzing the PCR product by dot hybridization or PAGE.

[Extraction of RNA from Sample] As reagents, guanidine, guanidine thiosinate, sarkosyl, phenol, black-mouthed form, and isoamyl alcohol were used. The reaction conditions were room temperature.

[Preparation of (HCV) cDNA by Reverse Transcriptase] Reverse transcriptase and random hexamer were used as reagents. The reaction condition was ⁴² eC for 1 hour.

[Width of (HCV) cDNA by Nested PCR method] HCV primer (5 ′ non-coding region) and Taq polymerase were used as reagents. The reaction conditions were at least 42 times and at least 20 cycles, and the annealing Lingno PCR was repeated twice.

[Analysis of PCR reaction product] By dot hybridization or 6% PAGE (using ethidium bromide).

As桔果, intramuscular for immune Guropuri emissions formulation adjusted before immunization Guropuri emissions containing solvent solution of the present invention is what was 1 0 ³ PCR unit 1, the formulation of the present invention is below the detection limit Nima Dropped.

Example 2

Example 1 was performed using the immunoglobulin G fraction obtained by fractionating healthy human plasma containing high units of tetanus antitoxin immunized with tetanus toxoid by the low temperature ethanol method of corn as a starting material. In the same manner as described above, an anti-tetanus human immunoglobulin preparation for intramuscular injection was prepared.

Example 3

Example 1 was performed using the immunoglobulin G fraction obtained by fractionating healthy human plasma containing a high unit of anti-D (Rh0) antibody by the cryogenic ethanol method of corn as a starting material. In the same manner as described above, an anti-D (R h0) human immunoglobulin preparation for intramuscular injection was prepared.

Example 4

Using the immunoglobulin G fraction obtained by fractionating healthy human plasma containing a high unit of anti-HBs antibody by the corn low-temperature ethanol method as the starting material, treating in the same manner as in Example 1. Then, an anti-HBs human immunoglobulin preparation for intramuscular injection was prepared.

Example 5

Healthy human plasma was subjected to membrane filtration in the same manner as in Example 1 using the immunoglobulin G fraction obtained by fractionating corn by the low-temperature ethanol method as a starting material. Was added to prepare a histamine-added human immunoglobulin preparation for intramuscular injection.

Experimental example 1 (virus removal effect)

The study was conducted by HCV spike test. A sample obtained by adding HCV-positive plasma to a 5 w / v% immunoglobulin solution was subjected to membrane filtration under the conditions of the present invention (Example 1), and the HCV genome in the solution before and after treatment of this sample was measured by PCR. did. As a result, treatment before those were 1 0 ³ PCR units / m 1, after treatment beat low to below the detection limit.

Experimental example 2 (Properties and stability test of the prepared preparation)

① Properties

The recovery rate of the immunoglobulin preparation for intramuscular injection according to the present invention is determined by using the It was 98% or more with respect to the solution, and it was confirmed that there was no change in properties such as molecular weight distribution, IgG subclass distribution, and antibody titer before and after the treatment.

② Stability test

The immunoglobulin preparation for intramuscular injection prepared according to this example was stored at 25 ° C for 6 power months, and a stability test was performed.

As a result, it was confirmed that the thermostability was good and there was no change in properties such as molecular weight distribution, IgG subclass distribution, and antibody titer.

Experimental example 3 (measurement of endotoxin and endotoxin-like substance)

The measurement was carried out by Procedure I (high sensitivity method) using Toxicolor and Endosc Kit (Seikagaku Corporation). The sample used had been subjected to perchloric acid treatment and deproteinized as a pretreatment, and the absorption was measured at 545 nm using diazo force coupling after the main reaction was completed. The endotoxin level is measured by the endotoxin, and the endotoxin-like substance (; 3-1.3-glucan) value is obtained by subtracting the endotoxin measurement from the toxin capacity measurement. And

As a result, the immunoglobulin preparation for intramuscular injection of the present invention remained low before and after the treatment in any case, and the value did not increase after the treatment.

Experimental example 4 (genome denial test)

The total amount of DNA in the immunoglobulin preparation for intramuscular injection of the present invention was measured using a threshold (Threeshord) system [Morekiula Devices (Molecula Devices)]. DNA was extracted using a DNA extraction kit (Wako Pure Chemical Industries, Ltd.) according to the sample attached to the kit.

As a result, the total DNA amount in the immunoglobulin preparation for intramuscular injection was below the detection limit (40 pg / m 1) in each case.

The concentration of immunoglobulin for intramuscular injection was 15 wZv%.

Example 6

The immunoglobulin fraction obtained by fractionating and recovering the plasma of a mouse containing anti-human lymphocyte antibodies obtained by immunizing the mouse with the human lymphocytes was used as a starting material. The membrane was subjected to membrane filtration in the same manner as in Example 1 to prepare an anti-human lymphocyte / antibody immunoglobulin preparation for intravenous injection. Example 7 The immunoglobulin fraction obtained by fractionating and recovering the plasma of a mouse containing anti-human lymphocyte antibodies obtained by immunizing the mouse with the human lymphocytes was used as a starting material. The membrane filtration treatment was carried out in the same manner as in Example 1 to prepare an anti-human lymphocyte / antibody immunoglobulin preparation for intravenous injection. However, as a porous hollow fiber for membrane filtration treatment, a module with an average pore diameter of 15 nm (brand name "Blanova 15", manufactured by Asahi Kasei Corporation, pore diameter of 15 ± 2 nm) The film thickness was 27 ± 3 m, and the other specifications were the same as in Example 1.)

Experimental example 5

It was examined by HCV spike test. A sample obtained by adding HCV-positive plasma to a 5 w / v% solution of the anti-human immunoglobulin immunoglobulin disclosed in Example 6 was subjected to membrane filtration according to Example 6, and before and after the sample was processed. The amount of HCV genome in the solution was measured by the PCR method. As a result, those before the treatment was 1 0 ³ PCR units Z m 1 is after treatment was reduced to below the detection limit. Industrial applicability

Since the immunoglobulin preparation of the present invention, particularly the immunoglobulin preparation for intramuscular injection, has an extremely small HCV genomic amount, it is expected that the risk of HCV infection is low and the safety is further improved.

Claims

Scope of Claim 1. An immunoglobulin preparation with a negative HCV genome.

2. The immunoglobulin preparation according to claim 1, wherein the amount of HCV genome is measured by a PCR method.

3. The immunoglobulin preparation according to claim 1, wherein the amount of the HCV genome is 1 PCR unit Zm 1 or less.

4. The immunoglobulin preparation according to claim 1, which is for intramuscular or intravenous administration.

5. The immunoglobulin preparation according to claim 1, which is derived from human or plasma.

6. Human immunoglobulin, anti-HBs human immunoglobulin, anti-tetanus human immunoglobulin, anti-D (Rh0) human immunoglobulin, anti-CMV human immunoglobulin, anti-human 2. The immunoglobulin preparation according to claim 1, wherein the immunoglobulin preparation is a lymphocyte spheroid immunoglobulin or a histamine-added human immunoglobulin.

7. The immunoglobulin preparation according to claim 1, comprising 0.1 to 15 w / v% of immunoglobulin in a solution state.

8. The immunoglobulin preparation according to claim 1, comprising an amino acid, a sugar, a sugar alcohol or an inorganic salt as an additive.

9. The immunoglobulin preparation according to item 1, wherein the pH in a solution state is 6.4 to 7.2.