JP2011184299A - Method for producing preparation containing monoclonal antibody - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a highly concentrated monoclonal antibody solution. <P>SOLUTION: The method for producing the preparation containing a monoclonal antibody is characterized in that viruses originated on the production of the monoclonal antibody and viruses originated on the production of a stabilizer are removed in a virus-removing process; the virus-removing ability of the virus-removing process is LRV≥4; the concentration of the monoclonal antibody in the virus-removing process is in a range of 3.0 to 10.0 wt.%; both of the average penetration rates of the virus-removing membrane used in the virus-removing process, for the monoclonal antibody contained in the solution at 0 to 10 min after the start of the filtration and at the passage of 0 to 3 hrs are ≥1.0 (kg/m<SP>2</SP>/hour). <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for producing a preparation containing a monoclonal antibody.

  A monoclonal antibody preparation, which is a kind of protein preparation, is an antibody having a single composition that is produced using genetically modified cells. Antibodies obtained by purifying blood are a mixture of various antibodies called polyclonal antibodies, whereas monoclonal antibodies with a single composition can trigger certain pathologies in combination with genetic recombination techniques. It can specifically recognize specific proteins and cancer cells and cause antibody reactions, and is widely used for the treatment of intractable diseases such as rheumatism, cancer, and leukemia.

  The manufacturing process of a monoclonal antibody preparation varies depending on the type of monoclonal antibody itself to be produced, but at least a culture process for producing a monoclonal antibody using cells or animals having a modified gene, a sterilization membrane, an adsorption column, Purification step to purify monoclonal antibody by chromatography, virus removal step to remove or inactivate virus in protein solution containing monoclonal antibody, stabilizer addition step to add stabilizer that suppresses aggregation of monoclonal antibody In general, an encapsulation step for encapsulating the monoclonal antibody from which the virus has been removed is necessary.

  The process of inactivating or removing viruses is a very important process since many cases of virus contamination from raw materials and processes have been reported in all biopharmaceuticals including monoclonal antibody preparations. Virus inactivation methods include heat treatments and chemical treatments, but these alone are not sufficient to inactivate viruses, and in these methods, useful proteins themselves in protein preparations are used. May also be denatured. From such a background, filtration using a virus removal membrane is performed as a means for removing a physical virus without chemical denaturation. Since the virus removal membrane is a removal method based on the size of the virus, it has the advantage that it is less affected by the characteristics of the virus compared to other methods such as heat treatment and chemical treatment, and removes viruses with a pore size of about 20 nm. If a membrane is used, it is possible to remove all currently known viruses to about 1 / 10,000 before filtration.

  When using monoclonal antibody preparations, liquid preparations may be used as they are, or lyophilized preparations may be used by dissolving them in distilled water, etc. Therefore, it is desired to liquefy the preparation and to increase the concentration in order to reduce the amount of the preparation to be administered and reduce the burden on the patient. However, a monoclonal antibody naturally exists in blood in its composition / ratio, and unlike a polyclonal antibody obtained by purification by Cohn fractionation, it can be produced by a method such as culturing genetically modified cells, Due to the inclusion of non-naturally-occurring antibodies such as chimeric antibodies and the fact that they are single components, their physical properties are more diverse than polyclonal antibodies, and some of them are unstable. To do.

  One factor that is particularly important with respect to the stability of monoclonal antibodies is the concentration of the antibody solution. That is, at a high concentration exceeding the final formulation concentration of 2.0% by weight, it is difficult to ensure the stability of the protein itself in the solution, and multimers and aggregates may be mixed. Many. Therefore, in order to stabilize the monoclonal antibody having unstable properties, it is necessary to use an additive such as a stabilizer in the purification step after the antibody concentration (Patent Document). 1). Multimers and aggregates are known to cause serious side effects such as anaphylactic shock, and even if the monoclonal antibody itself can be produced, protein denaturation during the purification process cannot be prevented. The formulation may be abandoned.

  Conventional filtration with a virus removal membrane has been performed at a low concentration of about 0.1 to 1.0% by weight. It was done. For this reason, there is little need to add stabilizers to suppress protein multimer formation in the system, and concentration with a UF membrane is performed after filtration through a virus removal membrane, and a stabilizer is added to form a final formulation. Was common.

  However, many additives / stabilizers that are effective in suppressing aggregation and multimer formation of the monoclonal antibody preparation described above are produced from organisms or fermentation products (non-contained). Patent documents 1, 2, 3, 4, 5). In this case, the integrity of the monoclonal antibody itself as a preparation with respect to virus removal is ensured, but it has been difficult to ensure the above-described completeness of virus removal from the stabilizer. For this reason, it is difficult to use biological or fermentation-derived stabilizers, or when used, the purification process of the stabilizer and the stabilizer itself becomes complicated.

  In addition, it has been difficult in the prior art to perform the virus removal step with an antibody having a concentration corresponding to the final preparation and a solution having a stabilizer concentration added thereto. That is, when a solution having such a composition is used, it is impossible to perform filtration that achieves both virus removal integrity and solution permeability. In particular, a virus removal membrane capable of removing small viruses such as parvovirus is used. When the filtration is performed, it is necessary to dilute the monoclonal antibody concentration to 1.0% by weight or less at the maximum. And as above-mentioned, it was presupposed in the prior art that after that, the final formulation was made through the concentration step using the UF membrane and the stabilizer addition step. In addition, when components such as stabilizers are mixed with this, there are many cases where the permeability is further lowered.

  In addition to the above-mentioned problems related to virus safety, this restriction cannot be made compact until the bulk reaches the downstream, resulting in inevitable complexity and high costs in the management of the manufacturing process, and unstable properties. In the case of the protein possessed, the stability in the subsequent concentration step cannot be ensured, and it has been a cause of many problems such as difficulty in formulation.

Special table 2001-503781 gazette "Latest Microorganism Handbook" (1986) `` Bio Industry '' 4, 554 (1984) “Fermentation Handbook” (Edited by Fermentation and Metabolism Research Association, Bioindustry Association) (ISBN 4-320-05575-6) "Chemical products of 14303" (Chemical Industry Daily) (ISBN 4-87326-404-9) Japan Science and Technology Agency (JST) website http // ww.go.jp / pr / report / report68 / details.html Introduction to virus experiments (National Institute of Preventive Health)

In view of the above problems, an object of the present invention is to provide a stabilizer that realizes both high permeability and virus removability of a high-concentration monoclonal antibody solution, and has a risk of virus contamination derived from a living organism or a fermentation product. The present invention provides a method for producing a monoclonal antibody that can be used in various preparation manufacturing processes.
In view of this problem, it is the virus removal process that is most difficult to solve. Due to the characteristics of the membrane that removes the virus by the mechanism of size removal (see below [Best Mode for Carrying Out the Invention]), the permeability of the virus removal membrane is generally low, and it is suitable for filtration with a high monoclonal antibody concentration. Since this is generally difficult, it is the biggest problem in the manufacturing process. Furthermore, since the permeability of the virus removal process is proportional to the pore size of the dense layer of the virus removal membrane, and the virus removal property is inversely proportional to the pore size of the dense layer, it is possible to achieve both of these contradictory properties with high performance. This is a technical issue.

As a result of intensive studies to solve the above problems, the present inventors have obtained the present invention. That is, the present invention includes the following.
[1] At least a culture process for producing monoclonal antibodies, a purification process for purifying monoclonal antibodies, a stabilizer addition process for adding at least one stabilizer that suppresses aggregation of monoclonal antibodies, and a solution containing the monoclonal antibodies A method for producing a preparation comprising a monoclonal antibody having a virus removal step of removing a virus with a virus removal membrane and an encapsulation step of encapsulating the monoclonal antibody from which the virus has been removed, wherein the virus removal step originates from the time of production of the monoclonal antibody. The virus and the virus derived from the production of the stabilizer are removed, the virus removal ability of the virus removal step is LRV ≧ 4, and the concentration of the monoclonal antibody at the virus removal step is 3.0 to 10.0 weight % Virus removal used in the virus removal step For, a monoclonal antibody contained in the solution, min filtration started after 0-10, the average permeability at the time lapse of 0-3 hours, the both is ^{1.0 (kg / m 2 / hour} ) or more A method for producing a preparation containing a monoclonal antibody, which is characterized.
[2] The method for producing a preparation containing the monoclonal antibody according to [1], wherein the stabilizer is derived from a living organism or a fermentation product.
[3] The method for producing a preparation containing the monoclonal antibody according to [2], wherein the stabilizer is selected from saccharides, amino acids, amino sugars, organic acids, or derivatives thereof.
[4] The method for producing a preparation containing the monoclonal antibody according to any one of [1] to [3], wherein the virus removal membrane is a hollow fiber membrane made of a synthetic polymer.
[5] The method for producing a preparation containing the monoclonal antibody according to [4], wherein the virus removal membrane is a multilayer microporous membrane.
[6] The amount of each stabilizer added in the stabilizer addition step is in the range of 0.1 to 15.0% by weight, and the monoclonal antibody at the end of the stabilizer addition step and at the end of the encapsulation step Purity is 80% or more, The manufacturing method of the formulation containing the monoclonal antibody in any one of [1]-[5] characterized by the above-mentioned.

By using the present invention, it is possible to use a preparation containing a stabilizer that achieves both high permeability and virus removability of a high-concentration monoclonal antibody solution, and there is a risk of virus contamination from biological or fermented products. Therefore, it is possible to provide a method for producing a monoclonal antibody that can be easily used in various pharmaceutical production processes, thereby making it possible to comprehensively achieve production process simplification, compactness, and cost reduction. .

  The method for producing a monoclonal antibody proposed in the present invention includes at least a culturing step for producing a monoclonal antibody by CHO cells, hybridoma, etc., a purification step for purifying the monoclonal antibody by chromatography, ultrafiltration membrane, polishing, etc., monoclonal antibody Stabilizer addition process to add a stabilizer that suppresses aggregation of the virus, virus removal process to remove the virus in the protein solution containing the monoclonal antibody by the virus removal membrane, encapsulation process to enclose the monoclonal antibody from which the virus has been removed It is a manufacturing method. The important evaluation items in the virus removal step are the filtration rate and virus removal ability in the virus removal step of the intermediate product. This step is intended to remove viruses derived from the production of monoclonal antibodies and viruses derived from the production of stabilizers. Therefore, it is essential that the step of adding an additive derived from an organism or fermentation product with a high risk of virus contamination be performed before the virus removal step by the virus removal membrane. There is no particular limitation on the order of steps for adding additives such as inorganic salts, petroleum products, or additives that are not derived from biological or fermentation products, such as those synthesized therefrom. When the step of concentrating with a UF membrane or the like after virus filtration is performed, it is usually not necessary to re-add the stabilizer as the solution composition is prepared. As described above, by filtering the biological or fermentation product-derived stabilizer together with the preparation in the final step of the monoclonal antibody preparation as one of the purposes of this method, against the risk of virus contamination during the production of the stabilizer, A more essential safety measure can be mentioned. Stabilizers derived from biological or fermented products include fungal / bacterial fermentation products such as “dextran obtained by lactic acid bacteria fermentation” or “neuramic acid obtained from egg yolk derived from an enzyme reaction” It refers to a product that corresponds to a product obtained using a product obtained by direct extraction from a living organism as a raw material (Patent Document 1). Specific production methods of these stabilizers by fermentation are already known from many literatures and the like, and production on an industrial scale is performed (Non-patent Documents 1 to 5). The risk of virus contamination with these stabilizers means, for example, when fermentation methods are used, when using biological materials such as bovine serum when cultivating biological or fermentation product-derived stabilizers, Viruses that are derived from raw materials and infect humans in the manufacturing process of stabilizers that use biological materials such as by decomposing biological materials such as (by enzymes) to obtain the desired stabilizer Means that there is a risk of contamination by viruses derived from these raw materials if they are not sufficiently removed or inactivated in the production process of the stabilizer.

  This method is not particularly limited with respect to the type of stabilizer derived from the organism or the fermentation product, but more preferably sugars (typically monosaccharides, disaccharides, trisaccharides, oligosaccharides, sugar alcohols). Etc., and polysaccharides can also be used), amino acids, amino sugars, organic acids, or derivatives thereof. More preferably, the sugars are glucose, mannose, galactose, fructose, sorbose, sucrose, lactose , Trehalose, maltose, raffinose, dextran, mannitol, sorbitol, erythritol, amino acids leucine, isoleucine, alanine, threonine, glutamic acid, aspartic acid, phenylalanine, tyrosine, tryptophan, proline, hydroxyproline, citrulline, amino As glucosamine, galactosamine, sialic acid, as organic acid α-ketoglutaric acid, 2-ketogluconic acid, 5-ketogluconic acid, ascorbic acid, erythorbic acid, alloisocitric acid, isocitric acid, itaconic acid, caproic acid, citric acid, gluconic acid Succinic acid, pyruvic acid, fumaric acid, mevalonic acid, malic acid, kojic acid, tartaric acid, acetic acid, lactic acid, oxalic acid, 2,5-diketogluconic acid, 2-ketogulonic acid, lecithin, lysolecithin. Derivatives and the like referred to here are salts of those having an acidic or basic site, such as an ester of a carboxyl group and a hydroxyl group, an alkyl or acyl substituent of an amino group and a hydroxyl group, or a salt thereof, Means a combination of multiple derivatizations. The type of salt is not particularly limited as long as it is legally recognized as an additive for foods and pharmaceuticals. Of the polysaccharides, dextran (derived from lactic acid bacteria) is mainly used as a surfactant as an additive derived from a fermentation product, and dextran having an average molecular weight of 40 to 70 kDa is used. It is preferable to do.

  Monomers / aggregates of monoclonal antibodies are those in which monoclonal antibodies are chemically bound to form a dimer, or an assembly of trimers or more, or two or more monoclonal antibodies are produced by intermolecular forces. Or, it means that the material is agglomerated randomly for some reason (such as denaturation).

  In this method, if the amount of stabilizer added in the stabilizer addition step is too low, a sufficient stabilizing effect of the monoclonal antibody cannot be obtained, and if it is too high, permeability is lowered in the virus filtration step. Therefore, the amount of each stabilizer added in the stabilizer addition step is preferably 0.1 to 15.0% by weight, more preferably 0.5 to 10.0% by weight. More preferably, it is in the range of 0.5 to 5.0% by weight. Further, if the monoclonal antibody purity at the end of the encapsulation process is 80% or more, it is considered that a sufficient stabilization effect has been obtained, more desirably 85% or more, more desirably 90% or more, and most desirably 95% or more. It is.

  The permeability of a monoclonal antibody solution generally depends on the antibody solution concentration and the inlet pressure. That is, the permeability has a negative correlation with the monoclonal antibody concentration, and when the protein concentration increases, the filtration rate tends to decrease. On the other hand, the inlet pressure when filtering the monoclonal antibody solution depends on the protein concentration. There is a positive correlation. Therefore, it is desirable that the virus removal membrane itself has a pressure resistance of 600 kPa or higher in order to perform filtration without decreasing the filtration rate while maintaining a high concentration of the monoclonal antibody. As the material of the film having a pressure resistance of 600 kPa or more, for example, a film made of a synthetic polymer such as polyvinylidene fluoride (PVDF), polyethersulfone (PES), or polysulfone (PS) and having a hydrophilic property is desirable. . The membrane may have a flat membrane structure or a hollow fiber structure, but preferably has a hollow fiber structure. The structure of the membrane is a microporous membrane containing a thermoplastic resin having a coarse structure layer with a high porosity and a dense structure layer with a low porosity, and the coarse structure layer is formed on at least one membrane surface. A multilayer microporous membrane which is present and has a thickness of 5.0 μm or more, the thickness of the dense structure layer is 50% or more of the entire film thickness, and the coarse structure layer and the dense structure layer are integrated Is desirable. Further, the coarse structure layer is a layer having an opening ratio of an average opening ratio of the entire film thickness + 2.0% or more, and the dense structure layer has an opening ratio of an average opening ratio of the entire film thickness + 2. A layer that is less than 0.0% and that is within the range of [average average aperture ratio of entire film thickness + partial aperture ratio of layers less than 2.0] ± 2.0% (including both ends). Some are preferred. The coarse structure layer is preferably an inclined structure in which the partial pore ratio continuously decreases from the film surface toward the dense structure layer and exists only on one film surface.

As described above, the permeability has a negative correlation with the monoclonal antibody concentration, and the permeability (filtration rate) tends to decrease as the protein concentration increases. The monoclonal antibody concentration of the monoclonal antibody solution in the virus filtration step is in the range of 3.0 to 10.0% by weight, more preferably in the range of 3.0 to 7.5% by weight, and even more preferably 3%. The range is from 0.0 to 5.0% by weight, and most desirably from 3.0 to 4.0% by weight. As long as the pressure and the concentration of the monoclonal antibody are satisfied, the membrane area and the permeability per hour of the monoclonal antibody are preferably 1.0 (kg / m ² / hour) or more, more preferably 1.5 (kg / m ² / hour) or more, more desirably 2.0 (kg / m ² / hour) or more.

The inlet pressure in the virus removal process using the virus removal membrane has the merit that high pressure can be filtered quickly in a short time. However, if the pressure is too high, the risk of damage and leakage due to pressure on the device increases. The pressure is 150 kPa to 600 kPa, preferably 196 kPa to 500 kPa, more preferably 245 kPa to 400 kPa, further preferably 294 kPa to 400 kPa, and most preferably 294 to 300 kPa.
Further, in the present invention, in the virus filtration step, as an indicator for showing that there is sufficient permeability and no rapid decrease in permeability has occurred, 10 minutes after the start of filtration, and filtration It is essential that the average permeability of the monoclonal solution at 3 hours after the start is 1.0 (kg / m ² / hour) or more, preferably 1.25 (kg / m ² / hour) or more More preferably, it is 1.5 (kg / m ² / hour) or more. The calculation of the average permeability at the time when 10 minutes have elapsed since the start of filtration (or when 3 hours have elapsed after the start of filtration) can be obtained by calculating back from the amount of permeation from 0 to 10 minutes (or from 0 to 3 hours). . That is, the amount of permeation at 10 minutes (or 3 hours) was measured as a weight, calculated as the weight per unit area of the membrane, and divided by 10 minutes (or 3 hours). / M ² / h] is calculated. )
In order to achieve such desired permeability, use a virus removal membrane that has high resistance to protein adhesion due to protein clogging, and use a membrane that does not cause a decrease in permeability due to protein clogging. When suppressing the formation of aggregates of monoclonal antibodies, which is the biggest cause of clogging, controlling the permeation pressure to maintain membrane permeability, and increasing the pressure to increase permeability For example, a film having pressure resistance may be used.

  The virus removal capability of the virus removal membrane should be determined based on known requirements such as ICH (Japan-EU EU Pharmaceutical Regulation Harmonization International Conference) guidelines. That is, for the purpose of analyzing how much the manufacturing process generally has with regard to virus removal and inactivation, that is, for the purpose of analyzing the characteristics (robustness) in terms of ensuring that the process exhibits virus clearance ability, It is stipulated that a filtration test is performed on a pharmaceutical solution to which a virus is artificially added. The virus removal rate LRV is a logarithmic value of the rate of decrease in the amount of virus in the solution after filtration relative to the amount of virus in the solution before filtration through the virus removal membrane, and the actual manufacturing process was accurately reduced. It is common to perform the evaluation in a laboratory-scale small-scale filtration operation.

  Commonly used virus quantification methods include the plaque method and the TCID50 method (Non-patent Document 6). On the other hand, the virus removal membrane used in the present method removes the virus by a sieve removal mechanism based on the size difference between the monoclonal antibody and the virus. Thus, the viruses that are currently known and that are most difficult to remove by the sieve removal mechanism are Parvoviridae viruses (18-25 nm in diameter) with the smallest size. The virus removal membranes used in this method include porcine parvovirus (PPV), mouse minimal virus (MVM), canine parvovirus (CPV), etc. as viruses corresponding to the worst case in analyzing the above robustness. Can be used. In the present invention, a virus removal test using a virus by porcine parvovirus (PPV) is shown as an example.

Porcine parvovirus (PPV) can be quantified by the TCID50 method. The following is given as an example of the detailed procedure. D-MEM (Gibco, high-glucose) containing 3% by volume of bovine serum (upstate, used for 30 minutes by heating in a water bath at 56 ° C. and inactivated) of any cell infected with porcine parvovirus The suspension and the monoclonal antibody solution containing porcine parvovirus are mixed in a 1: 1 ratio. Similarly, a 10-fold dilution of a monoclonal antibody solution containing the same porcine parvovirus is mixed with the cell suspension at a ratio of 1: 1. Thereafter, the same procedure is followed for the 10 ² fold, 10 ³ fold, 10 ⁴ fold, 10 ⁵ fold, 10 ⁶ fold, and 10 ⁷ fold dilutions of the monoclonal antibody solution containing the porcine parvovirus. Eight of these mixed solutions are prepared for each virus solution or diluted solution and cultured in an incubator at 37 ° C. in a 5% carbon dioxide atmosphere. Next, TCID50 (50% infectivity titer) is measured by an erythrocyte adsorption method (Non-patent Document 6) for each suspension in which the cells are cultured. That is, chicken erythrocytes are diluted 5-fold with PBS (-) (manufactured by Nissui Pharmaceutical Co., Ltd., prepared by the method attached to the product), centrifuged at 2500 (rpm) for 5 minutes, and the supernatant is removed. The residue of the obtained preserved blood diluted solution is again diluted 200 times with PBS (−), and then added to each culture solution in which the cells are cultured in the same amount as the above cell suspension and left to stand for 1 to 2 hours. . The presence or absence of red blood cells adsorbed on the surface of the cultured cell tissue is visually confirmed, and the one confirmed to be adsorbed is counted as a culture solution in which virus infection has occurred. Regarding the presence or absence of virus infection for each obtained culture solution, confirm the ratio of the culture solution in which infection was confirmed for each monoclonal antibody solution or diluted solution concentration containing each porcine parvovirus initially added to the cell suspension, Log (TCID50 / ml) is calculated as an infectious titer by the Reed-Munch method (Non-patent Document 6). The above operation is performed on both the solution before and after filtration by the virus removal membrane, and LRV is calculated by the following formula.

LRV = log ₁₀ A-log ₁₀ B where
A = Infectivity titer of solution before filtering through virus removal membrane (TCID 50 / ml)
B = Infectivity titer of solution after filtering through virus removal membrane (TCID 50 / ml)
However, this is an example of a virus quantification method, and the present invention is not limited to the virus quantification method by this method.

  The virus removal membrane used in this method has a sufficient amount of virus removal in a test for analyzing the above characteristics (robustness). LRV ≧ 4 is sufficient in the mixed solution of the total amount of the corresponding solution, and LRV ≧ 5 is more desirable.

  The present method can be easily applied to a monoclonal antibody production process of a biopharmaceutical having the above production process, but is preferably applicable to a human monoclonal antibody, more preferably human γ globulin G.

The adjustment of the membrane area of the virus removal membrane is widely known that there is no large disturbance factor at the time of scale-up / scale-down, so it can be easily scaled up / down, and it is easy to obtain scalability. There is the feature that it is. That is, if the membrane area changes, only the filtration volume per membrane area changes, and if it is constant, it may be considered that the filtration performance is maintained, so any membrane area proportional to the actual production scale can be used. .
In this method, the time for filtering the monoclonal antibody solution with the virus removal membrane is not particularly limited. If you want to complete the virus removal process in the manufacturing process in a short time even if the membrane area of the virus removal filter is large, in a short time, if you want to reduce the membrane area of the virus removal filter even if it is a little longer Can be set for a long time. In addition, the conditions for filtering the monoclonal antibody solution with the virus removal membrane include the temperature of the solution at the time of filtration, the room temperature, the presence or absence of additives such as inorganic salts that are not derived from organisms and fermentation products, the type of solvent, and contamination that is not derived from monoclonal antibodies. There are no particular restrictions on the presence or absence of objects.
[Example]

In the following examples, a filtration device in which a hollow fiber membrane made of hydrophilic polyvinylidene fluoride is used as a virus removal membrane, and the hollow fiber membrane is partitioned into an inlet side space and an outlet side space in the inner space of the container, and A SUS304 tank and silicon tube (manufactured by Tigers Polymer) sterilized by steam at 121 ° C. for 15 minutes were used for the purpose of sending the monoclonal antibody solution to the filtration device.
A monoclonal antibody (hereinafter referred to as antibody A) was prepared and used as a model of an intermediate product of a monoclonal antibody formulation according to the method described in WO2004 / 087761.
Porcine parvovirus (PPV) solution used in the following Examples (obtained from the Association of Animal Biological Products, 90HS strain, Lot No. VS0201, infectivity titer: 8.67 log (TCID50 / ml), hereinafter described as PPV ), Or PK-13 cells (obtained from ATCC, Lot No. ATCC CRL-6489) used as cells for preparation of PPV solution and LRV measurement, 75 (cm ² ) tissue culture flask (manufactured by BD Falcon) ) And bovine serum (Upstate, heated for 30 minutes in a 56 ° C. water bath for 30 minutes to use after inactivation) and 10% by weight penicillin / streptomycin (+10000 Units / ml Penicillin, +10000 μg / ml Streptomycin, manufactured by Invitrogen) ) It was used after being repeatedly cultured with 1% by volume D-MEM (manufactured by Invitrogen, high-glucose).

[Production method of virus removal membrane]
Polyvinylidene fluoride resin having a melt flow index (MFI) of 2.5 (g / 10 ml) (manufactured by Kureha Chemical Co., Ltd., T # 1300), 49% by weight, dicyclohexyl phthalate (manufactured by Osaka Organic Chemical Industry Co., Ltd.) Product) A composition consisting of 51% by weight was stirred and mixed at 70 ° C. using a Henschel mixer (Mitsui Mining Co., Ltd., type 20B), and then cooled to a powder form. It injected into the direction screw type extruder (Technobel Co., Ltd. product KZW25TW-50MG-NH (-600)), and it melted and mixed uniformly at 210 degreeC. Subsequently, while flowing dibutyl phthalate (industrial product manufactured by Daihachi Chemical Industry Co., Ltd.) having a temperature of 130 ° C. into the hollow interior, from a nozzle made of an annular orifice having an inner diameter of 0.8 mm and an outer diameter of 1.05 mm, Each homogeneously dissolved product was extruded into a hollow fiber shape, cooled and solidified in a cooling water bath adjusted to 10, 20, 30, 40 ° C., and wound around a metal frame at a speed of 50 m / min. Thereafter, dicyclohexyl phthalate and dibutyl phthalate were extracted and removed with 58% isopropyl alcohol aqueous solution (industrial product manufactured by Daihachi Chemical Industry Co., Ltd.), and the attached 58% isopropyl alcohol aqueous solution was replaced with water and then immersed in water. In this state, heat treatment was performed at 125 ° C. for 4 hours using a high-pressure steam sterilizer (HV-85 manufactured by Hirayama Seisakusho). Thereafter, the attached water was replaced with isopropyl alcohol (industrial product manufactured by Daihachi Chemical Industry Co., Ltd.), and then dried at a temperature of 60 ° C. with a vacuum dryer (manufactured by STEC Co., Ltd.) to form a hollow fiber-like microporous material. A membrane was obtained. In all steps from winding to drying, the hollow fiber was fixed in a fixed length state and processed.
Subsequently, the microporous membrane was subjected to a hydrophilic treatment by a graft method. In the reaction solution, hydroxypropyl acrylate (Industrial product manufactured by Osaka Organic Chemical Co., Ltd.) was dissolved in a 25% by volume aqueous solution of 3-butanol (Industrial product manufactured by Pure Chemical Co., Ltd.) so as to be 8% by volume. What was nitrogen bubbling for 30 minutes was used for the state hold | maintained at (degreeC). First, in a nitrogen atmosphere, the microporous film was cooled to −60 ° C. with dry ice and irradiated with 25 kGy of γ rays using Co60 as a radiation source. After irradiation, the microporous membrane was allowed to stand for 15 minutes under a reduced pressure of 13.4 Pa or less, and then the reaction solution and the microporous membrane were brought into contact at 60 ° C. for 1 hour. Thereafter, the microporous membrane was washed with a 58% by volume isopropyl alcohol aqueous solution and vacuum dried at 60 ° C. for 4 hours to obtain a hydrophilic microporous membrane. It was confirmed that when the microporous membrane was brought into contact with water, water spontaneously penetrated into the pores. Both ends of the bundle of 12 microporous membranes are sealed with polyurethane, and bonded to a cartridge in which a polystyrene hollow fiber membrane is partitioned into an inlet side space and an outlet side space, and a filtration device (effective membrane area 0. 001 m ² ).

[Manufacturing process of monoclonal antibody]
10 (mmol / l) of CHO cell serum-free culture supernatant (expression level: 700 mg / L) 1500 (ml) containing human monoclonal antibody (human IgG1) clarified with depth filter and 0.2 (μm) membrane filter It was added to a Protein A column (Mabselect 20 mm ID × 20 cm, manufactured by Amercham Biosciences) equilibrated with sodium phosphate buffer (pH 6.0) (linear velocity: 500 cm / h). Subsequently, the human monoclonal antibody was eluted with 5 column volumes of 20 (mmol / l) sodium citrate buffer (pH 3.4) (linear velocity: 500 cm / h). The eluate was neutralized with 10 (mmol / l) sodium phosphate buffer (pH 8.2), adjusted to pH 8.0 with 1.5 (mol / l) Tris-HCl, and then 10 (mmol / l). ) An anion exchange column equilibrated with Tris-HCl (Q Sepharose XL 10 mm ID × 15 cm manufactured by Amercham Biosciences) was added (linear velocity: 300 cm / h). After completion of the addition, 3 column volumes of equilibration buffer were passed through the column (linear velocity: 300 cm / h), the non-adsorbed portion of the column was adjusted to pH 5.0 with 1.0 (mol / l) acetic acid, mmol / l) It was added to a cation exchange column (SP Sepharose FF 26 mm ID × 15 cm, manufactured by Amercham Biosciences) equilibrated with sodium acetate buffer (pH 5.0) (linear velocity: 300 cm / h). After completion of the addition, the column was washed with 5 column volumes of equilibration buffer (linear velocity: 300 cm / h), and 5 column volumes of 20 (mmol / l) sodium acetate / 0.30 (mol / l) sodium chloride buffer ( pH 5.0) was passed through and eluted as a human monoclonal antibody solution (linear velocity: 300 cm / h). This eluate was concentrated to an antibody concentration of 10.0% by weight using an ultrafiltration membrane (Biomax 30 50 cm ² manufactured by Millipore).
Using antibody A solution obtained by the above method, stabilizer (see Table 1 below), sodium chloride, and water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), the ratio of antibody A and stabilizer listed in Table 1 below 0.1 (mol / l) sodium chloride aqueous solution was prepared. Then, the antibody A and the stabilizer solution are mixed with a small amount of 1.0 to 0.10 (mol / l) hydrochloric acid (manufactured by Wako Pure Chemical Industries) or 1.0 to 0.10 (mol / l) sodium hydroxide. The pH was adjusted to 5.5 using an aqueous solution (manufactured by Wako Pure Chemical Industries, Ltd.). Subsequently, the stabilizer solution was allowed to stand for 1 hour, and then the monoclonal antibody purity at this stage was determined by HPLC (Prominence, manufactured by Shimadzu Corporation, column: Tosoh GPC column TSK gel G3000SWXL, mobile phase: phosphate buffer (pH 6). .9) /0.3 (mol / l) sodium chloride aqueous solution), the purity of the monoclonal antibody preparation at the end of the preparation encapsulation step was measured from the peak area ratio, and the following [Table 1] was obtained.
After adding 0.5% by volume of the above PPV solution to the monoclonal antibody and stabilizer solution after pH adjustment and stirring well, a hollow fiber filter (PLANOVA35N) having a pore size of 35 (nm) is prepared for each solution. Pre-filtration was performed.
For each antibody A and stabilizer solution (including PPV) after pre-filtration, a filtration membrane (0.001 m ² ) produced by the method of [Example 1] and the above filtration device were prepared, respectively, and LRV For measurement, each antibody A and stabilizer solution (hereinafter referred to as the original solution) was collected in 5 (ml) and sterilized PP screw vials (BD Falcon) and immediately stored frozen at -78 ° C. Dead-end filtration was performed at a pressure of 294 kPa. The filtration amount of the monoclonal antibody solution at each time and the permeation amount of the monoclonal antibody calculated from the antibody concentration were as shown in Table 1 below, and the required monoclonal antibody permeability could be satisfied. Each of the above filtrates was dispensed into 50 ml sterilized screw vials to obtain the desired monoclonal preparations.

[Virus removal test and monoclonal antibody purity measurement]
Cultured PK-13 cells were 3% by volume of bovine serum (Upstate, heated for 30 minutes in a 56 ° C. water bath and inactivated), and penicillin / streptomycin (+10000 Units / ml Penicillin, +10000 μg) / ml Streptomycin (manufactured by Invitrogen) 1% by volume D-MEM (Invitrogen, high-glucose) (this mixture is hereinafter referred to as 3% FBS / D-MEM) and cell concentration 2.0 × 10 ⁵ A diluted suspension of (cells / ml) was prepared. Ten 96-well round bottom cell culture plates (Falcon) were prepared from this cell suspension, and 100 (μl) was dispensed into all wells.
Then, the total amount mixture of the filtrate subjected to the 3 hour filtered, 10x by their 3% FBS / D-MEM, 10 2 -fold, 10 ³ times, 10 ⁴ times, was prepared ^105-fold dilution. Furthermore, about each original solution extract | collected just before filtration, the 10 ² times, 10 ³ times, 10 ⁴ times, 10 ⁵ times, 10 ⁶ times, 10 ⁷ times dilution liquid by those 3% FBS / D-MEM was prepared. . In 96-well cell culture plate was dispensed the cell suspension min, 10 times of the filtrate and the filtrate, 10 ^twice, 10 ^three times, ¹⁰⁴ times, and ^105-fold dilutions, 10 ^double Motoeki, 10 ³ fold, 10 ⁴ fold, 10 ⁵ fold, 10 ⁶ fold, 10 ⁷ fold dilutions were dispensed 100 μl into 8 wells at 37 ° C. in a 5% carbon dioxide atmosphere in an incubator for 10 days. Cultured.
Subsequently, TCID50 (50% infectivity titer) was measured by the erythrocyte adsorption method (Non-patent Document 6) on the cell culture plate cultured for 10 days. Chicken stored blood (manufactured by Nippon Biotest) is diluted 5-fold with PBS (-) (manufactured by Nissui Pharmaceutical Co., Ltd., prepared by the method attached to the product), and centrifuged at 2500 (rpm) and 4 ° C for 5 minutes. After erythrocytes were precipitated, the supernatant was removed by aspiration, and the resulting precipitate containing erythrocytes was again diluted 200-fold with PBS (−).
Next, 100 (μl) of the prepared erythrocyte precipitate in PBS (−) was dispensed into all wells of the cell culture plate and allowed to stand for 2 hours, after which erythrocytes were adsorbed on the surface of the cultured cell tissue. The presence / absence of adsorption was visually confirmed, and those in which adsorption was confirmed were counted as wells in which virus infection occurred, and those in which adsorption was not confirmed were counted as wells without infection. About the presence or absence of the virus infection for every obtained culture solution, a ratio is confirmed for every filtrate, its diluted solution, or every diluted solution of the original solution, and log (TCID50) as an infectious titer by Reed-Munch method (nonpatent literature 6). / Ml) and the virus removal rate LRV was calculated, and the results were as shown in [Table 2] below.
Furthermore, HPLC (Shimadzu Corporation Prominence, column: Tosoh GPC column TSK gel G3000SWXL, mobile phase: phosphate buffer solution (pH 6.9) /0.3 (mol / l) sodium chloride aqueous solution) When the purity of the monoclonal antibody preparation was measured from the peak area ratio, it was as shown in [Table 2] below. As a result, for entries 1 to 10, the required virus removability, the purity of the monoclonal antibody preparation, and the guarantee of stability in the solution of the monoclonal antibody during the production process were all satisfied.

  By using the present invention, when producing a monoclonal antibody preparation, a high concentration monoclonal antibody solution can be filtered through a virus removal membrane while maintaining high permeability and high virus removal properties, and can be derived from organisms or fermentation. Stabilizers that are at risk of product-derived virus contamination can also be used. This makes it possible to comprehensively achieve the simplification, compactness, and cost reduction of the monoclonal antibody production process.

Claims (6)

At least a culture process for producing a monoclonal antibody, a purification process for purifying the monoclonal antibody, a stabilizer addition process for adding at least one stabilizer that suppresses aggregation of the monoclonal antibody, and a virus in the solution containing the monoclonal antibody as a virus A method for producing a preparation comprising a monoclonal antibody having a virus removal step of removing by a removal membrane and an encapsulation step of encapsulating the monoclonal antibody from which the virus has been removed, wherein the virus removal step comprises a virus derived at the time of production of the monoclonal antibody, Virus from the production of the stabilizer is removed, the virus removal ability of the virus removal step is LRV ≧ 4, and the concentration of the monoclonal antibody at the virus removal step is in the range of 3.0 to 10.0% by weight And for the virus removal membrane used in the virus removal step. That, of the monoclonal antibody contained in the solution, min filtration started after 0-10, the average permeability at the time lapse of 0-3 hours, the both is ^{1.0 (kg / m 2 / hour} ) or more A method for producing a preparation containing a monoclonal antibody, which is characterized.   The method for producing a preparation containing a monoclonal antibody according to claim 1, wherein the stabilizer is derived from a living organism or a fermentation product.   The method for producing a preparation containing a monoclonal antibody according to claim 2, wherein the stabilizer is selected from saccharides, amino acids, amino sugars, organic acids, or derivatives thereof.   The method for producing a preparation containing a monoclonal antibody according to any one of claims 1 to 3, wherein the virus removal membrane is a hollow fiber membrane made of a synthetic polymer.   The method for producing a preparation containing a monoclonal antibody according to claim 4, wherein the virus removal membrane is a multilayer microporous membrane.   In the stabilizer addition step, the amount of each stabilizer added is in the range of 0.1 to 15.0% by weight, and the monoclonal antibody purity at the end of the stabilizer addition step and the encapsulation step is 80. % Or more, The manufacturing method of the formulation containing the monoclonal antibody in any one of Claims 1-5 characterized by the above-mentioned.