TW201002821A - Method of producing agent containing single useful material that has polypeptide - Google Patents

Abstract

The present invention provides a method of producing a high-concentration agent solution containing a single useful material that has polypeptides, characterized in that the virus removal step removes the virus originated from the formation of the single useful material having polypeptides, and originated from the formation of the stabilizer, wherein the virus removal capability of the virus removal step is (LRV, logarithmic reduction value) ≥4, the concentration of the single useful material having polypeptides during the virus removal step is between the range of 3.0-10 wt%, and the average permeability of the single useful material having polypeptides against the virus removal membrane used in the virus removal step is 1.0(kg/m.sup.2/hour) or more both in 0-10 minutes and 2-3 hours after the start of filtration.

Description

201002821 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a method for producing a single useful substance having a polypeptide, particularly a monoclonal antibody preparation or a recombinant preparation. [Prior Art] A single antibody preparation or a recombinant preparation which is a species in a protein preparation has a single component produced by using a recombinant cell or the like. By using these

The preparation 'specifically recognizes a specific protein or cancer cell which induces a certain condition and causes an antibody reaction, and is therefore widely used for the treatment of a difficult condition such as rheumatism, cancer, and leukemia. The step of forming the protein preparation represented by the monoclonal antibody preparation or the recombinant preparation or the like depends on the kind of the desired substance f to be produced. (4) Usually, at least the following steps are required: using the recombinant cell or animal of the gene a step of cultivating the above-mentioned useful substance; a purification step of purifying the above-mentioned useful substance by using a sterilization membrane, an adsorption column, a chromatograph, etc.; a virus removal step of removing or inactivating a virus in a solution containing the above-mentioned useful substance; a stabilizer for the stabilizer of agglomerates of a useful substance (IV); a sealing step of the above-mentioned useful substance (IV) with a virus removed. For a preparation comprising a single substance having a polypeptide represented by a monoclonal antibody preparation as a whole, useful Virus...Drawing, Dali reports that there are steps from the raw material, which are derived from the steps of the step, so the process of purifying or removing the virus is very important. It is used as a passivation method for viruses or treatment with chemicals, etc. == The passivation of the poison is not sufficient, but the product is processed and the disease is treated. Useful material 139962.doc 201002821 The quality itself is also denatured. From the above background, as a physical virus removal method without chemical degeneration, the virus removal membrane is used for filtration. The virus removal membrane is based on the size of the virus. The removal method is therefore less susceptible to the influence of virus characteristics than other methods such as heat treatment or treatment using chemicals, and if the money has a pore diameter of about 20 nm

When the virus is removed from the membrane, all the viruses known to the target can be removed to about 1 in 10,000 before filtration. When a protein preparation represented by a single antibody preparation is used, the case where the liquid preparation is used as it is, and the case where the cold-dried preparation is dissolved in distilled water or the like is used, and the risk of medical care and medical errors is reduced. In view of the above, it is desirable to liquefy the preparation and to reduce the amount of the administration agent to reduce the burden on the patient. However, the protein preparation represented by the monoclonal antibody preparation is originally composed of the components and ratios thereof. Naturally, it exists in the blood, and the multi-strain antibody obtained by Cohn isolation and purification is different, and it is produced by a method such as culturing a genetic recombinant cell, and includes a naturally occurring non-existent antibody such as a chelate antibody, and is a single component. The reason is that the physical properties of the multi-strain antibody are various, and there are also unstable traits. One of the most important factors regarding the stability of the protein preparation represented by the monoclonal antibody preparation is the concentration of the preparation solution. That is, when the concentration is higher, for example, more than 2% by weight of the final formulation concentration, it is often difficult to confirm that the protein itself is in solution. Qualitative, resulting in the incorporation of multimers, aggregates, etc. Therefore, it is necessary to use a stabilizer or the like in the stage after concentration of the antibody in the purification step, and the stabilizer is used to stabilize the protein preparation having the unstable state. (Patent Document 1). It is known that multimers, aggregates, and the like may cause serious side effects such as anaphylactic shock, and ή ή ή 便于 便于 便于 便于 便于 便于 便于 便于 便于 便于 单 单 单 单 单 单 单 单 单 单 单 单 单 单 单 单 单 单 单 单 单 单Degradation of the protein in the purification step and abandonment of the final formulation. 2 The transition of the membrane to remove the virus before is lower than the weight of the 〇w by the lower = lower, it is relatively difficult to form a polymer, agglomerate, etc. The step of concentrating until the purification is concentrated. Therefore, the stabilizer for inhibiting the formation of multimers of proteins is added to the system: ν 'usually using the virus removal membrane and using the sage (10)ra F gamma, ultrafiltration, membranes are concentrated, and stabilizers are added for finalization. However, 'coagulation of protein preparations represented by the above-mentioned monoclonal antibody preparations A stabilizer which exhibits an inhibitory effect on the formation of a body, and is produced by a large amount of a manufacturer derived from an acid-derived product (Non-Patent Document 2, 3, 4' 1) 1 It is difficult to ensure the complete removal of the virus derived from the above stabilizers. It is difficult to use the stabilizer derived from the organism, from the acidifier, or the stabilizer itself. The purification step becomes complicated, etc. Further, it is difficult in the prior art to carry out the virus removal step in a solution corresponding to the concentration of the above-mentioned final preparation and the concentration of the stabilizer added thereto, that is, if such a component is used. In the case of the solution, the filtration of the completeness of the virus and the permeability of the solution cannot be performed. Therefore, the virus removal film which can remove the small virus such as the small virus is subjected to i% by weight or less; Above; 1 "Intermediate class is released into the highest order for filtering. Further, as described above, the prior art 139962.doc 201002821 is premised on the final formulation by the concentration and stabilizer addition step of the UF film. In addition, in many cases, the permeability is further lowered if a stabilizer or the like is mixed therein. In addition to the above-mentioned limitations that may cause problems related to the above-mentioned virus safety, many of the following problems may arise: the main body cannot be simplified until the downstream; the result cannot avoid the complexity of the management of the manufacturing steps, and the cost is high or unstable. In the case of the protein of the trait, the stability of the subsequent concentration step cannot be ensured, and formulation or the like is difficult. [Patent Document 1] Japanese Patent Laid-Open Publication No. 2001-503781 [Non-Patent Document 1] "Latest Microbiology Manual" (丨 986) [Non-Patent Document 2] "Bio Industry" 4, 554 (1984) [Non-Patent Document 3] "The Fermentation Handbook" (edited by the Institute of Fermentation and Metabolism of the Bi〇industry Association) (ISBN 4-320-05575-6) [Non-Patent Document 4] "Chemical Products of 14303" (Chemical Industry News) (ISBN 4-87326-404-9) [Non-Patent Document 5] Homepage of the Independent Administrative Corporation Science and Technology Promotion Agency (JST) http//ww.go.jp/pr/report/report68/details.html [Non-Patent Document 6 In general, the problem of the problem is to solve the problem of the invention. In view of the above problems, an object of the present invention is to provide a method for producing a monoclonal antibody, which can be Achieving a high concentration of a single useful substance having a polypeptide, 139962.doc 201002821, a high permeability and virus removal of the preparation solution, and the extract Z contains a source derived from the substance or the source (4) Formulations that incorporate risky stabilizers can be easily used for each The formulation manufacturing processes. In view of this issue, the most difficult to solve is the virus removal step. In terms of the characteristics of the membrane for removing the virus by the mechanism for removing the size (refer to the following [Embodiment]), the permeability of the virus removal membrane is generally low, and it is generally difficult to adapt to the transition of a higher concentration of the preparation. Become the biggest = problem in the manufacturing steps. Furthermore, the permeability of the virus removal step is proportional to the pore size of the dense layer of the virus removal membrane, and the virus-removing system is inversely proportional to the pore size of the dense layer. Therefore, the two technologies having the high performance and the relative properties become technologies. The topic. [Technical means for solving the problem] ... The inventors conducted intensive studies to solve the above problems, and as a result, obtained the present invention. That is, the present invention includes the following. (1) A method for producing a preparation comprising a single useful substance having a polypeptide, characterized in that it comprises at least the following steps: a step of cultivating a useful substance having a polypeptide early, 'a single useful substance having a polypeptide is pure: a purification step of adding at least one stabilizer addition step for inhibiting a cohesive stabilizer having a polymorphic single substance = a virus-removed virus in a solution of a single-useful substance having a polypeptide by a virus removal membrane a step of sealing the single useful substance having the polypeptide with the virus removed, and the above-mentioned virus removal step is derived from the disease of the single-useful substance having the polypeptide Stabilizer production The disease removal ability of the disease removal step is LRvy, 139962.doc 201002821 The concentration of a single useful substance having a polypeptide in the virus removal step is in the range of 3.0 to 10.0% by weight, and is contained in the solution. A single substance having a polypeptide: the useful substance is removed relative to the virus used in the above virus removal step: after the start of the membrane (2) The average permeability of the fraction # and the passage of 2 to 3 hours was 1.0 (kg/m2/hour) or more. [2] A method of producing a preparation comprising a single useful male meringue having a polypeptide, wherein the stabilizer is derived from a living organism or derived from an acid yeast product, as in [1]. Or a method for producing a preparation comprising a single-useful substance f of a polypeptide, wherein the stabilizer is selected from the group consisting of a saccharide, an amino acid, an amino sugar, an organic acid, or a derivative thereof in. [4] The method for producing a preparation comprising a single useful substance having a polypeptide according to any one of (1) to [3], wherein the virus removal membrane is a hollow fiber membrane comprising a synthetic polymer. [5] As included in any of [1] to [4], there are many. In addition, the preparation method of the preparation of the early useful substance of the peptide, the upper layer of the pot, and the base of the forgotten soil. The diseased mites are removed into a multi-layer microporous membrane. [6] A preparation comprising a monoclonal antibody, wherein the method comprises at least the following steps: a step of culturing a single antibody, and a purification step of purifying the individual antibody Adding at least a lambda stabilizer addition step for inhibiting the aggregation of the early antibody, and removing the virus containing the monoclonal antibody solution from the virus removal membrane in the main vortex removal a removal step, and a sealing step of sealing the virus-removed monoclonal antibody. The virus removal step is derived from the production of the monoclonal antibody, and the production of the stabilizer. When the virus is removed, the virus removal ability of the virus removal step τ ν 乡 is LRV = 4, and the concentration of the monoterpenoid oxime is 3.0 to 10% by weight in the virus removal step. 139962.doc The range of the solution in the range of '02102821' The average permeability of the strain antibody was 1.0 (kg/m 2 /h 〇 ur) or more after two minutes and two hours after the start of filtration of the virus removal enthalpy used in the virus removal step. [7] The method for producing a preparation comprising a monoclonal antibody according to [6], wherein the above-mentioned stable Qishen is derived from a living organism or derived from a fermentation product. [8] The method for producing a preparation comprising a monoclonal antibody according to [6] or [7], wherein the above-mentioned stabilizer is selected from the group consisting of a saccharide, an amino acid, an amino sugar, an organic acid, or the like .

[9] The method for producing a preparation comprising a monoclonal antibody according to any one of [6] to [8] wherein the virus removal membrane is a hollow fiber membrane comprising a synthetic polymer. [10] A method for producing a preparation comprising a monoclonal antibody according to [6] to [9], wherein the virus removal membrane is a multilayer microporous membrane. [11] A method for producing a preparation comprising a monoclonal antibody according to any one of [6] to [1G] wherein the amount of each stabilizer added in the stabilizer addition step is 0.1 to 15% by weight. The range of /〇 is, and the purity of the individual antibody at the end of the stabilizer addition step and at the end of the sealing step is 8% or more. [Effects of the Invention] By using the present invention, it is possible to produce a high-quality single-concentration antibody solution preparation or a recombinant preparation of a recombinant preparation which achieves both high permeability and virus-removability. Further, even if a seven-person Ή A L u 1 is used for the preparation of a stabilizer containing a stabilizer derived from a living organism or a virulence-derived product, the above-mentioned quality preparation or intermediate can be produced. Because of the squeezing, it can provide a simple process for the preparation of various preparations, including a single 139962.doc 201002821 peptide represented by 罝矬P touch 4 ± 13 as an early strain antibody - A method for producing a preparation of a useful substance, whereby the steps of the manufacturing step can be simplified, simplified, reduced in cost, and the like. [Embodiment] The method for producing a so-called "substance-containing substance having a polypeptide" as proposed by the present invention refers to a method comprising at least the following steps: by bacterium clothing ra (chlnese hamster 〇vary, Chinese hamster printing) a step of culturing a single useful substance having a polypeptide by using a cell, a fusion tumor, or the like; and purifying the single-purity purification of the polypeptide by using a chromatograph, an ultrafiltration membrane, a polishing, or the like; Stabilizer addition step of a stabilizer for agglomeration of a single useful monoclonal antibody; removal of a virus in a protein solution containing a monoclonal antibody by a virus removal membrane: a removal step; and sealing of the individual antibody from which the virus has been removed Sealing step: The single useful substance having a polypeptide cultured in the above-mentioned culture step refers to a recombinant protein f or a monoclonal antibody produced by applying a genetic recombination technique to a bacterial or ch〇 cell and cultured, or a technique using cell fusion or the like. A single tumor resistant material produced by culturing a cell containing a single-(four) substance in nature, and its application is (4) not limited, and more specifically: & recombinant colony stimulating factor +, recombinant erythropoietin gene recombination Interferon, gene recombinant fusion protein, monoclonal antibody, humanized antibody, chimeric antibody, and the like. An important evaluation item in the above virus removal step is the transition speed and virus removal ability of the intermediate product of the preparation in the virus removal step. The purpose of this step is to remove the virus from the production of the monoclonal antibody and the virus from the generation of the stabilizer. Therefore, in particular, the addition step of the organism-derived organism-derived additive derived from the organism or from the fermented product 139962.doc 201002821 must be performed before the virus removal step of the virus removal membrane in Nantun. There is no particular limitation on the inorganic salt or petroleum product, or the non-source of the D-forming agent which is derived from the organism or derived from the vinegar-derived product. In the case of virus 2, the order of the addition step is as follows: when the virus is filtered and the step of using a UF membrane or the like is performed, it is usually adjusted with the composition of the solution, and therefore it is preferable to have no such step. Further, as described above, as the object of the method, there may be mentioned a stabilizer or a stabilizer derived from an acid yeast product in a final step of a preparation containing a single-useful substance having a polypeptide in a spoon. The preparation has been carried out together, and the risk of mixing the virus in the manufacture of the stabilizer is more essentially safe. The so-called:: a self-biological or stabilizer derived from a fermented product refers to a fungus, a bacterial acid yeast product such as a "glucan obtained by fermentation," or a "reacted from an enzyme by an enzyme reaction." The production of the "neuraminic acid" or the like is obtained by the production of the raw material of the product obtained by the extraction of the raw material (four) (Patent Document 1). The specific production method for the use of the stabilizers by the fermentation method has been known from a large number of documents and the like, and has been produced on an industrial scale (Non-Patent Documents 1 to 5). The risk of mixing human viruses in such tinctures means, for example, when using a fermentation method, when cultivating a raw material derived from living organisms derived from a biological or a stabilizer derived from a fermented product, or A step of decomposing a biologically derived material such as egg yolk (using an enzyme or the like) to obtain a target stabilizer, etc., and using a stabilizer derived from a material derived from a living organism = a virus derived from a raw material and infected by a human, the virus is mixed with In the case where the stabilizer is not sufficiently removed or passivated in the manufacturing step, there is a risk of virus incorporation from the raw materials. 139962.doc -11 - 201002821 No =: 12:,: The type of stabilizer from the product and more '------------------------------------------------------------------------------------------------------------- Amino acid, amino sugar, and organic sugar bismuth ' Further desirable as sugar, there are Portuguese, galactose, fructose, sorbose 1 sugar, lactose, sea sugar / cockroach, raffinose, Portuguese Glycan, mannitol, sorbitol, alcohol; as amino acid, there are leucine, isoleucine, alanine, :::, face acid, aspartic acid, phenylalanine, road Amine acid, tryptamine diterpene: acid, trans-proline glutamic acid, citrulline; as amino sugar, grape glucosamine, sialic acid; as organic acid, α-ketoglutaric acid, 2': Dyrosic acid, 5-ketone gluconic acid, ascorbic acid, isoascorbic acid, iso-citric acid, iso-citric acid, sputum deficiency, prohexitol, hexanoic acid, citric acid, glucose-glucone, amber, C-acid , fumaric acid, methyl divaleric acid, thin fruit acid, citric acid, tartaric acid, acetic acid, lactic acid, oxalic acid, 2,5-dioxalic acid, 2-ketogulonic acid. Here, the term "derivative" or the like refers to a base of a buffer or a base of a buffer, an amine group, and a base group, or a base substituent, etc., and if it has an acidic or basic moiety, it means such a Salt' or a combination of a plurality of such derivatives. There is no particular limitation on the type of the salt as long as it is legally permitted as an additive to the product or the pharmaceutical product. The term "polymer, agglomerate, and the like of a single-useful substance having a polypeptide" means a substance in which a single-use substance having a polypeptide is scientifically bonded to form a dimer or a trimer or more, or two or more Right eve & 〇α,, 'Early peptide, a useful substance, due to intermolecular forces or any cause (denaturation, etc.). 139962.doc 12 201002821 时本=二: When the stabilizer addition step is too low in the stabilizer addition step, = sufficient stability of the single-useful substance with the polypeptide is obtained. Therefore: the second 'sickness filter step may be The decrease in permeability is caused, and the addition amount of each of the tanning agents in the addition step is preferably in the range of 0.1 to 15% by weight, and further preferably. When there is a single useful substance of the polypeptide, @ 'right control sealing step ends A 'mussel purity is 8 ()% or more, it can be regarded as - the stability effect of the V knife, more desirably 85% or more, and further The phase is 9_, the most ideal is the 9 system. The permeability of a single peptide-soluble substance solution usually depends on the antibody to the nighttime and the inlet pressure. That is, the permeability is negatively correlated with the concentration of a single useful substance having a polymorphism. 'If the protein concentration becomes high, there is a tendency for the transition velocity to decrease'. In addition, regardless of the protein concentration, the permeability and the peptide are mono- The inlet pressure when filtering with a useful substance solution is positively correlated. Therefore, in order to maintain the concentration of a single useful substance having a polypeptide at a high concentration without causing filtration and a decrease in speed, it is preferable that the (four) property of the virus removing film itself is _ or more. As a material having a film having a financial property of more than _ café, for example, it is preferable to use poly(vinylidene fluoride) (PVDF, P〇lyvinylidene flu〇ride) or poly sapphire (pEs, test sulfone) to gather hard ( A synthetic polymer such as PS or polysulf_) is used as a raw material and has a hydrophilic film. Regarding the shape of the film, a flat film structure or a hollow fiber structure may be used, and a hollow fiber structure is preferred. Further, the structure of the film is preferably a multilayer microporous film having a large structural layer having a large opening ratio and a dense structural layer having a small opening ratio. 139962.doc 201002821 Microporous containing a thermoplastic resin a film, and the coarse structural layer is present on at least the surface of the film and has a thickness of 5.0 μm or more, the thickness of the dense structural layer being more than 5% by weight of the entire film thickness, and the coarse structural layer and the dense structural layer are - Physicalization. Further preferably, the large-structure layer of the upper filament is an average opening ratio of the film thickness of +2. 〇% or more, and the average opening ratio of the dense structure layer is less than the entire film thickness. +2〇%, and 胄 [the average opening ratio of the entire film thickness + the average of the partial opening ratio of the 2-layer layer] the layer within the range of the pin (including both ends). The above-mentioned coarse structural layer is preferably an inclined structure in which the opening ratio of the crucible portion continuously decreases from the film surface toward the dense structural layer, and exists only on one of the film surfaces. As described above, the permeability is negatively correlated with the concentration of the single-useful substance having the polypeptide, and if the protein concentration becomes high, the permeability (filtration rate) is lowered. The filtering step is based on a single useful substance solution having a polypeptide. The concentration is in the range of 〇10% by weight, more preferably in the range of 3.0 to 7.5 wt/〇, and further preferably in the range of 3 〇~5 〇% by weight. The most ideal 疋3 _0~4_〇 weight The range of %. Within the range of satisfying the above pressure and the concentration of the individual antibody, the permeation of the unit membrane area per unit time and the per unit time is more than 1.0 (kg/m2/hour), more preferably! 5 (kg/m2/h〇ur) or more is preferably 2.0 (kg/m2/hour) or more. For the inlet pressure of the virus removal step using the virus removal membrane, the high pressure has the advantage of being able to rapidly filter in a short time, but if the pressure is excessive, the risk of damage or leakage of the C force to the device increases, so the virus is removed. The system is carried out at an inlet pressure of 1 50 kPa or more and 600 kPa or less, and is more preferably 196 kPa or more and 500 kPa or less, more preferably 245 139962.doc -14 - 201002821 kPa or more and 400 kPa or less. It is ideally 294 kPa or more and 4 〇 0 kPa or less 'the most ideal is 294 to 300 kPa. In the present invention, as an indicator for indicating that the virus has sufficient permeability in the passing step and that there is no rapid decrease in permeability, 10 minutes after the start of filtration and 2 to 3 hours after the start of filtration. The average permeability of the preparation solution of the single useful substance having the oxime peptide must be 1.0 (kg/m 2 /h 〇 ur) or more, and more preferably 丨 25 (kg/m 2 /h 〇 ur) or more. Further, it is preferably 1·5 (1^/Γη2/1ι〇ιΙΓ) or more. The calculation of the average permeability after 1 minute of the above filtration (or 3 hours after the start of filtration) can be calculated based on the total amount of 〇~1〇 minutes (or 2 to 3 hours overall). Out. That is, the weight per unit area of the film was measured at a time point of 1 minute (or 2 to 3 hours), and the weight per unit area of the film was calculated and divided by 10 minutes (or 60 minutes) to calculate the IgG permeation amount. [kg/m2/h]. In order to achieve such a desired permeability, a virus removal membrane having high protein adhesion resistance is used due to clogging of the protein, and a membrane which does not cause a decrease in permeability due to clogging of the protein is used, and clogging is suppressed. The most important factor is the formation of agglomerates of a preparation containing a single useful substance of a polypeptide, controlling the permeation pressure for maintaining the permeability of the membrane, and using a membrane having pressure resistance when the pressure is increased to improve the permeability. Regarding the virus removal ability of the virus removal membrane, it should be based on ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for

The Human Use ‘Japan-EU (European Union) International Medicine Law Association) guidelines are determined by known requirements. That is to say, for the following purposes of 139962.doc •15-201002821, artificially add the filtration test of the preparation solution of the ancient, the owner λ café, and the addition of two preparations: analysis of the manufacturing steps in the removal of the virus or The extent to which purification generally has the ability to analyze the steps to achieve virality in terms of viral clearance. The virus removal rate lrv is a logarithmic value indicating the rate of decrease in the amount of virus in the solution after the transition of the virus removal membrane relative to the amount of virus in the filtered solution, usually in a laboratory that accurately measures the actual manufacturing steps. This evaluation was carried out in a horizontal small-scale filtration operation. The quantitative method of the virus used for the sling is, for example, a speckle method, a DID CID50 method, or the like (Non-Patent Document 6). 5 From one, the virus-removing membrane used in the method removes the virus according to the monoclonal antibody and the virus ruler mechanism. Therefore, it is currently known and most difficult to utilize a sieve: the virus removed by the structure is the virus of the small virus family (the diameter is 1 8 25 nm) having the smallest size. The virus-removing membrane used in the method can be used as a pig virus (PPV, pig parvovirus) or a mouse parvovirus (mvm, ☆us 〇f mice), a canine parvovirus (cpv' café heart-shaving heart (four), etc. A disease equivalent to the worst case in terms of the above-mentioned characteristics is described in the present invention, and as an example, a virus removal test using a virus vaccinated with porcine virus (ppv) is shown. Pig prion (PPV) The TCID50 method can be used for quantification. An example of a detailed sequence is shown below. Any cell that infects porcine virulence virus is added in a volume of 3. The bovine serum (manufactured by Upstate Inc.) is heated in a water bath of 56. After inactivation, iD_MEM (Mig_gluc〇se) suspension is mixed with s. Similarly, for the same cell suspension, the same 10-fold dilution of the preparation solution containing the porcine parvovirus containing a single useful substance having the polypeptide is mixed in the ratio of 丨:i. The procedure is as follows: 102, 103, 104, 105, 1, 6 and 107 times dilutions of the preparation solution containing the same useful substance as the virus, and the virus is mixed. Each of the solution or the diluent is prepared by separately preparing 8 such mixtures, and culturing them in an incubator under a 5% carbon dioxide atmosphere, respectively, and then using the red blood cell adsorption method for each suspension of the cell culture (Non-Patent Document 6) Measure the TCID50 (5〇% of the infection price). That is, the chicken red blood cells are diluted 5 times with pBS (1) (manufactured by Rishui Pharmaceutical Co., Ltd., using the method attached to the product), and 2500 (rpm) is used. The supernatant was removed by centrifugation in a minute, and the residue of the obtained preserved blood dilution was diluted to 200-fold with PBS (-), and then added to each culture medium of the cell culture in an amount equivalent to the above-mentioned cell suspension liquid phase or the like. In the middle, it was allowed to stand for 1 to 2 hours, and it was confirmed by visual observation whether or not the adsorption of the red blood cells on the surface of the cultured cell tissue was observed, and it was confirmed that the adsorbent was counted as a culture solution causing the disease infection. The presence or absence of a virus infection is determined by the ratio of the concentration of the preparation solution or the diluent containing the single useful substance having the polypeptide, which is initially added to the cell suspension, and the concentration of the culture solution in which the infection is observed, using the Reed_Munch method (non- Patent Document 6) Calculates log (TCID5〇/mL) as the infection price. The above operation is performed on both solutions before and after filtration using the virus removal membrane, and LRV is calculated by the following formula:

LRV=l〇g10A-l〇g10B 139962.doc •17· 201002821 The infection price of the solution, where A=use the virus to remove the membrane over the sputum, before (TCID50/mL); the infection price of the solution after the ferry is not Limited to use B = use virus to remove 臈 (TCID50 / mL). However, this is an example of a virus quantification method, and the virus quantification method by the § Hai method. The virus-removing film used in the method exhibits sufficient value of virus removal in the test for analyzing the above-mentioned particularity, and is equivalent to the virus of the steam type, 疋仃ί 3 The total amount of the solution in the filtration solution of the hourly preparation solution is 0 Υ in the total mixture, and the right 乙 〇 / 24 is a cross, more preferably LRVS5. The method can also be easily applied to the manufacturing steps of any biopharmaceutical having the above manufacturing steps, and is preferably applicable to monoclonal antibodies, humanized antibodies, chimeric antibodies, recombinant colony stimulating factors, and recombinant red blood cells. The production steps of the genomicin, the recombinant interferon, and the recombinant fusion protein are more preferably applied to human monoclonal antibodies, and thus can be applied to human gamma globulin G. Regarding the adjustment of the membrane area of the virus-removing membrane, it has been widely known that there is no large disturbance factor in scale-up and scale-down, so it has the following characteristics: scale-up and scale reduction can be easily performed, and (6) can be easily adjusted. ability. That is, if the membrane area is changed, only the filtration volume per unit membrane area is changed, and it is considered that if the filtration performance is maintained, the membrane area can be maintained in any ratio proportional to the production scale. In the present method, the filtration of a single antibody solution is carried out by using a virus removing membrane. 139962.doc • 18- 201002821 Introduction, ..., particularly limited. In order to complete the virus removal step in the manufacturing step in a short time even if the virus removes the membrane area of the device

% ' can be u for a short time'. When it is desired to reduce the membrane area of the virus cutting device even if the time is long, it can be set to a long time. X, as a condition for filtering a single antibody material by removing cockroaches by a virus, the temperature of the material, the temperature of the liquid, the room temperature, the inorganic salt, and the like are not derived from the addition of the organism or the mash, the type of the solvent, or the source The presence or absence of the inclusions from the preparation containing the substance alone with the polypeptide is not particularly limited. [Examples] In the following examples, the following transition chambers, SUS304 tanks, and Shishi tube mills P〇lymer were used, and the above-mentioned ruthenium apparatus was a hollow fiber membrane using hydrophilicized polyvinylidene fluoride as the #质. The membrane is removed as a virus, and the hollow fiber membrane is interposed between the inlet side space and the outlet side space in the interior of the fitting; the SUS304 groove and the manifold are used to transport the single antibody solution to the filtering device. , already at 121. (: The steam sterilization was carried out for 15 minutes. As a model of the intermediate product of the monoclonal antibody preparation, a monoclonal antibody (hereinafter referred to as antibody A) was prepared and used according to the method described in WO2004/087761. A model for the preparation of a useful substance is prepared by using the method described in Japanese Patent Laid-Open No. Hei 7-173 196, and is used as a recombinant gamma interferon (hereinafter referred to as Formulation A). Virus (ppv) solution (obtained from the Association of Biological Agents for Society Animals, 90HS strain, Lot N〇.VS0201, infection price: 8.67 l〇g (TCID 50/mL), 139962.doc 19-201002821 PK), or PK-13 cells used for the preparation of PP V solution and cells for the determination of LRV (obtained from ATCC ' Lot No. ATCC CRL-6489), using 75 (cm 2 ) tissue culture flask (BD Falcon Manufactured) and added 10 volumes of bovine serum (manufactured by Upstate, used in a water bath of 56 for 30 minutes and inactivated) and 1 volume. / patulin/streptomycin (+1 〇〇〇〇 Units/mL) Penicillin, +10000 D-MEM (manufactured by Invitr〇gen) (manufactured by Invitr〇gen, high glucose) was repeatedly cultured and used. [Example 1 Method for Producing Virus Removal Membrane] Using Henschel Mixer (Mitsui Mining Co., Ltd., Model 20B), a melt flow index (MFI, Melt Flow Index) of 2.5 (g/l 〇 mL) of 49 wt% polyvinylidene fluoride resin (Wu Yu Chemical Co., Ltd., T#1300) and A composition comprising 51 wt% of dicyclohexyl phthalate (made by Osaka Organic Chemical Industry Co., Ltd.) was stirred and mixed at 70 ° C, and then cooled to obtain a powder, which was powdered by a funnel. The body is put into a mysterious twin-axis co-rotating extruder (manufactured by Technovel KZW25TW-5〇MG-NH(-600)), melt-mixed at 21 〇 ° C, and uniformly melted. The internal flow temperature of the hollow is 130. (: The product of bis-phthalic acid dibutyl S (Da-8 Chemical Industry Co., Ltd.)) has a ring from the inner diameter of 〇·8 mm and the outer diameter of 1.05 mm. The spinning nozzle formed by the orifices respectively extrudes the uniform melt into a hollow fiber shape, and the temperature is adjusted to 10, 20 The mixture was cooled and solidified in a 3 °, 40 ° C cooling water bath, and wound on a metal frame at a speed of 50 m / min. Thereafter, an industrial product was manufactured using a 58% aqueous solution of isopropyl alcohol (Da Ba Chemical Industry Co., Ltd.). The dicyclohexyl phthalate and dibutyl phthalate are extracted and removed, and the attached 58% aqueous solution of isopropyl alcohol is replaced with water to be immersed in water of 139962.doc -20-201002821. The high-pressure steam sterilization device (Hirayama Manufacturing Co., Ltd. manufactures HV-85) is at 125. The armpit was subjected to heat treatment for 4 days. Thereafter, the adhered water was replaced with isopropyl alcohol (manufactured by Daigo Kasei Co., Ltd.), and then dried by a vacuum dryer (manufactured by Stec Co., Ltd.) at a temperature of 6 ° C. Thereby, a hollow fiber-like microporous film was obtained. Further, in all the steps from the winding up to the drying, the hollow fiber is fixed and treated in a fixed length state. Then, the above microporous membrane was hydrophilized by a grafting method. The reaction liquid was used in the following manner: 5% by volume of 3 -butanol (a pure chemical (manufactured) industrial product) was dissolved in a hydroxypropyl acrylate (manufactured by Osaka Organic Chemical Co., Ltd.) at a volume of 8 vol%. In the aqueous solution, nitrogen gas was bubbled for 30 minutes while being kept at 45 °C. First, the microporous membrane was cooled to _ 6 利用 by dry ice under a nitrogen atmosphere, and the microporous membrane was irradiated with γ-rays of 25 kGy with c 〇 60 as a light source. After the irradiated microporous membrane was allowed to stand under a reduced pressure of 13.4 Pa or less for 15 minutes, the reaction solution was brought into contact with a microporous membrane at 60 ° C, and allowed to stand for 1 hour. Thereafter, the microporous membrane was washed with a μ volume% aqueous solution of isopropyl alcohol at 60. (: vacuum drying for 4 hours to obtain a hydrophilic microporous film. It was confirmed that the microporous film spontaneously permeated into the micropores when it was in contact with water. The tow of 12 microporous membranes was filled with polyurethane. At both ends, a hollow fiber membrane made of polystyrene was interposed between the inlet side space and the outlet side space to form a filtration device (effective membrane area of 0.001 m2). [Example 2] [Single plant Antibody manufacturing procedure] 139962.doc 21 201002821 CHO cell serum-free culture supernatant containing human monoclonal antibody (human IgG1) clarified by a depth filter and a 〇.2 (μπι) membrane filter (expression amount) : 700 mg/L) 1500 (mL) was added to a Protein A column (Mabselect 20 mm ID manufactured by Amercham Biosciences Co., Ltd.) equilibrated with 10 (mmol/L) sodium citrate buffer (pH 6.0). 20 cm) (linear velocity of 500 cm/h). Then, human monoclonal antibody was eluted using a 20-column capacity of 20 (mmol/L) sodium citrate buffer (pH 3.4) (linear velocity was 500 cm/h). The solution was neutralized with 10 (mmol/L) sodium citrate buffer (pH 8_2), and further After 1.5 (mol/L) Tris-HCl was adjusted to pH 8.0, it was added to an anion exchange column equilibrated with 1 〇 (mmol/L) Tris-HCl (Q Sepharose XL 10 mm ID x 15 cm manufactured by Amercham Biosciences) Medium) (linear velocity 300 cm/h). After the addition, the equilibration buffer of the 3-column capacity is passed through the column (linear velocity 300 cm/h), with 1.0 (mol/L) acetic acid. The component which was not adsorbed on the column was adjusted to a pH of 5.0, and then added to a cation exchange column (A Seclose FF 26 manufactured by Amercham Biosciences Co., Ltd.) equilibrated with 20 (mmol/L) sodium acetate buffer (pH 5.0). Mm IDx 1 5 cm) (line speed 300 cm/h). After the addition, clean it with a 5-bar column balance buffer (linear speed 300 cm/h), and then make 5 column capacity 20 (mmol/L) sodium acetate / 0.30 (mol/L) sodium chloride buffer (pH 5.0) was passed as a human monoclonal antibody solution (linear velocity 300 cm / h). UF membrane was used. (Biomax 30 50 cm2 manufactured by Millipore Co., Ltd.) The eluate was concentrated until the antibody concentration was 1% by weight. The antibody A solution obtained by the above method was used. , Stabilizer (refer to Table 139962.doc -22-201002821 Table 1 below), sodium chloride and water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), respectively, to prepare 〇 1 containing antibody A and a stabilizer in the ratios shown in Table 1 below. (mol/L) aqueous sodium chloride solution. Then, a small amount of 1_〇 to 〇.1〇(m〇l/L) hydrochloric acid (made by Wako Pure Chemical Industries) or 1.0 to 10.10 (m〇l/L) aqueous sodium hydroxide solution (made by Wako Pure Chemical Industries, Ltd.) 'The pH of these antibody A and stabilizer solutions was adjusted to 5 · 5. Then, the δ 疋 疋 疋 浴 浴 bath was allowed to stand for 1 hour, and thereafter, the purity of the individual antibody at this stage was measured, and HPLC was used (Prominence manufactured by Shimadzu Corporation, column: Tosoh GPC column TSK gel G3000SWXL, mobile phase: Phosphate buffer (pH 6.9) / 0.3 (mol/L) aqueous sodium chloride solution) The purity of the individual antibody preparation at the end of the preparation sealing step was measured based on the peak area ratio, and the results are shown in the following [Table 1]. For each of the above antibody A and the stabilizer solution (including ppV), a filtration membrane (〇〇〇1 m2) produced by the method of [Example 1] and the above filtration apparatus were prepared, and 5 (mL) was measured for LRV. Each antibody a and a stabilizer solution (hereinafter referred to as a stock solution) were placed in a sterilized PP spiral sample bottle (manufactured by BD Fak〇n), immediately frozen at -78 t:, and then subjected to a pressure of 294 kPa. Dead-end is too late. The amount of permeation of the monoclonal antibody solution at each time and the concentration of 艮 艮 体 计算 《 《 《 《 《 《 《 《 《 《 《 单 单 单 单 单 单 单 单 单 单 单 单 。 。 。 。 。 。 。 。 。 。 Each of the above filtrates was dispensed into 50 mL of the sterilized spiral sample to obtain the target antibody A preparation. 139962.doc •23- 201002821 [Table i] entry (entry) Antibody A concentration (% by weight) Stabilizer ------ Stabilizer concentration (% by weight) - Antibody preparation purity (%) Filter 0~10 minutes Average permeability of antibody kg (kg/m2/hour) Average permeability of antibody A over 2 to 3 hours (kg/m2/hour) Average permeability of antibody A over 0 to 3 hours (kg/m^ Our) 1 3.0 d-甘露ΐϋ 3.0 97.5 2.44 2.15 2.23 — 2 3.0 D-cotton ___3.0 — 93.5 2.38 1.95 2.06 3 3.0 L-Alanine hydrochloride view 3.0 94.8 2.77 2.43 2,53 4 3,0 D - Glucosamine hydrochloride 3.0 -------- 92.0 1.89 1.62 1,69 5 3.0 Disodium citrate 3.0 92.4 3.08 2.60 2.77 ο 5.0 D-glucose - 5.0 96.5 2.92 2.44 Bu 2.58 7 5.0 D-mannose - 5.0 90.6 2.25 1,81 1.90 8 5.0 L-Alanine hydrochloride 5.0 ---- 98.1 2.34 1.95 2.05 9 5.0 D-Glucosamine hydrochloride 5.0 88.9 L95 1.69 1.76 1U 5.0 Disodium citrate 5.0 91.9 2.85 2.39 2.56 [Example 3] [Vacuum removal test and determination of individual antibody purity] 3% by volume of bovine serum (manufactured by Upstate Co., Ltd., added in a water bath of 56 ° C) D-MEM (manufactured by Invitrogen, high-glucose) of 1% by volume of penicillin/streptomycin (+10000 Units/mL Penicillin ' + 10000 pg/mL Streptomycin, manufactured by Invitrogen) (after use) The mixture was recorded as 3% FBS/D_ME]y [) and the cultured PK-13 cells were diluted to prepare a diluted suspension having a cell concentration of 2 〇xl〇5 (cells/mL). Ten 96-well round bottom cell culture plates (manufactured by Falcon Co., Ltd.) were prepared, and 1 〇〇 (μί) of the cell suspension was dispensed into each well. Then, 'Total mixture of the filtrates which were filtered for 3 hours as described above' was prepared by using 3% FBS/D-MEM to prepare 1〇 times, 1〇2 times, 1〇3 times, 104 times, 1〇5 Double dilution. Further, for each stock solution collected immediately before filtration, 102 times, 1〇3 -24-139962.doc 201002821 times, 10 times, l〇5 times, 106 times, etc. were prepared by using 3% FBS/D-MEM. 1〇 7 times dilution. In a 96-well cell culture plate to which the above cell suspension is dispensed, 1×2, 102 times, 1〇3 times, 104 times, 1〇5 times dilution of each filtrate and filtrate, and 1〇2 times of the stock solution, 10 times, 104 times, 1〇5 times, 1〇6 times, and 1〇7 times dilutions were dispensed in each of the 8 wells, and cultured in an incubator for 10 days in a 3rc, 5% carbon dioxide atmosphere.

Then, the TCID50 (50% infection price) was measured by the red blood cell adsorption method (Non-Patent Document 6) on the cell culture plate cultured for 10 days. The chicken preserved blood (manufactured by Nippon Biotest Co., Ltd.) was diluted to 5 times with PBS() (manufactured by Nissui Pharmaceutical Co., Ltd., and prepared by the method attached to the product), and then subjected to 2500 (rpm) at 4C. After centrifugation, the red blood cells were precipitated, and then the supernatant was removed by suction, and the obtained erythrocyte-containing sediment was diluted to 2 times with PBS (1). Then, 100 (μΙ〇 of the prepared pBs(_) dilution of the prepared red blood cell pellet was dispensed into all the wells of the above cell culture plate, and after standing for 2 hours, the surface of the cultured cell tissue was visually confirmed. The presence or absence of red blood cell adsorption will confirm that the adsorber is counted as a hole causing the virus infection, and the adsorbent is not recognized as an uninfected well. Regarding the presence or absence of the virus infection of each culture solution obtained, the filtrate or its dilution The dilution ratio of the liquid or the stock solution was confirmed by the Reed_Muneh method (Non-patent (4), i〇g (TCID 5〇/mL) was calculated as the infection price, and the virus removal rate LRV was obtained. The results are as follows [Table 2] Further, 'Using HPLC (made by Shimadzu Corporation, ρΓ〇ηιίη_β, column, T〇s〇h GPC column TSKgel G3〇〇〇swxl, mobile phase: phosphorus 139962.doc -25· 201002821 acid buffer (pH value) The purity of the individual antibody preparation at the end of the sealing step of the preparation was determined according to the peak area ratio of 6.9)/0.3 (mol/L) of the sodium carbonate aqueous solution. The results are shown in the following [Table 2]. 1〇, what you want The virus removal property, the purity of the monoclonal antibody preparation, and the stability of the monoclonal antibody in the solution in the production step are all satisfied. [Table 2] entry Antibody A concentration (% by weight) Stabilizer stabilizer Concentration (% by weight) stock log (TCID50) travertine log (TCID50) LRV antibody preparation purity (%) 1 3.0 D-mannitol 3.0 6.00 <0.5 > 5.50 97.2 2 3.0 D-raffinose 3.0 5.67 < 0.5 >5.17 92.4 3 3.0 L-Alanine hydrochloride 3.0 5.80 <0.5 > 5.30 95.7 4 3.0 D-Glucosamine hydrochloride 3.0 5.67 <0.5 > 5.17 90.3 5 3.0 Citric acid di-nano 3.0 5.80 <;0.5>5.30 92.5 6 5.0 D-mannose fermentation 5.0 6.12 <0.5 >5.62 95.5 7 5.0 D-raffinose 5.0 6.20 <0.5 > 5.70 89.5 8 5.0 L-alanine hydrochloride 5.0 6.00 <;0.5>5.50 95.6 9 5.0 D-Glucosamine hydrochloride 5.0 6.00 <0.5 > 5.50 88.6 10 5.0 Citric acid di-nano 5.0 5.80 <0.5 > 5.30 91.9 [Example 4] [Genetic recombination gamma interference a manufacturing step] preparing a stock culture in a sterile baffle culture flask containing 150 to 500 mL of sterile medium, Sterile medium having the following composition: tryptone amidine of 10 g / L, yeast extract was 5 g / L, sodium chloride 5~1〇 mg /; L. Next, a primary culture of denier _ W3110/py_CYC5 was inoculated into the medium. < In turn, the inoculated flask was placed on a shaker at 25 to 37. The culture was carried out under the armpit until the absorbance at 5 50 nm reached about i 〇. About 35% (v/v) of dimethyl sulfoxide was added to the culture solution card at about 50% (V/V). Immediately aliquot the i mL 139962.doc •26- 201002821

Assigned to the benefits of _婵+, 丄Q ... 囷衩 in the bottle, close the lid. save. Each 酦觖 γ sample bottle is below -60 ° C. Each fermentation system is started using the inoculation replication reserve % ^ . In the case of Yu Zhen = bottle or cut-slot, the medium (LBb_h) was used to prepare for about 8 hours of incubation, and the inoculum was transferred to a fermentation tank. The inoculum capacity is 2 to 1% of the fermented capacity. The recombinant interferon _ 丫 is produced in a fermentation tank having an action volume of about 10 to 1000 L. The fermentation medium has the following composition per liter:

Glucose (*) : 50-100 g Ammonium sulfate: 4.0-8.0 g Potassium dihydrogen phosphate: 3.0-5.0 g Dipotassium hydrogen phosphate: 5.0-8.0 g Magnesium sulfate heptahydrate: 〇.5-5.1 g sodium citrate Dihydrate: 0.5-2.0 g UCON LB-625 : 0.5-2.0 mL gasified iron hexahydrate: 0.005-0.15 g

Gasified drill hexahydrate: 0.001-0.005 g sodium molybdate dihydrate: 〇.〇〇1_〇.〇〇5 g copper sulfate pentahydrate: 0.001-0.005 g boric acid: 0.001-0.005 g manganese sulfate monohydrate 0.001-0.005 g Hydrochloric acid: 0.0-1.0 mL Thiamine-HC1 : 0.0-0.1 g Tetracycline-HC1 : 0.001-0.01 g 139962.doc •27· 201002821 L-Tryptophanic acid (*) : 0.1-0.5 g Yeast Extract: 2.0-8.0 g 3-β-indole acrylic acid: 0.02-0.1 〇g (*) Glucose and tryptophan are initially supplied in part during the fermentation process. The components which are partially placed in the fermentation tank are left to be sterilized by heat treatment or filtration before being used for fermentation. The acid yeast is carried out under 25~ thief. Other operating conditions are as follows: Stirring (rpm): 100~1〇〇〇 aeration (vvm): 0_5~1.5 (addition of oxygen if necessary) pH: 6.5~7.5 (adjusted by adding ammonium hydroxide) It is contained in a medium containing a salt and a suitable buffer having a pH of 6 to 9, preferably ^9. The cell suspension was homogenized in a high pressure colloid mill such as a Gaulin mill to extract recombinant gamma interferon. A sufficient solution of polyethyleneimine was added to the above solution to prepare a solution. The supernatant contains gamma interferon. The supernatant is adsorbed to an appropriate salt solution having a pH of from $6 to 9 to remove impurities on the dioxosite-based adsorbent. The recombinant gamma interferon is eluted using a solution containing Q 5 to m tetramethylammonium chloride. All operations in this step were performed at a pH of 7 to 9. The above-mentioned eluate is diafiltered, adsorbed to an anion exchange chromatography medium, and then washed to remove impurities. The recombinant gamma interferon is eluted with an increased salt gradient. Typical anion exchange resins which can be used in this step are slow methyl cellulose and decyl ethyl cellulose. All operations were carried out at a pH of 7 to 9. The above-mentioned dissolution was adsorbed to the "medium, and then washed and the silk was mixed with the acid salt. 139962.doc • 28-201002821 The concentration gradient caused the concentration of the salt to increase and the recombinant gamma interferon was eluted. All of the operations in this step were carried out at a pH of 7 to 9. The above-mentioned eluate is dialyzed to be adsorbed to an anion exchange chromatography medium, followed by washing to remove impurities. The recombinant gamma interferon is eluted from the anion exchange medium with a gradient of increasing salt concentration. Typical anion exchange chromatography media are carboxymethyl cellulose and indeed ethyl cellulose. All of the operations in this step were carried out at a pH of 7 to 9. The above-mentioned eluted matter is applied to a gel permeation medium, and is developed in a column with a salt-containing solvent. Appropriate components containing recombinant gamma interferon are pooled, and the Phinenae column is subjected to decoholization; the east is dried, & All operations in this step are performed at values of 7 to 9.

Formulation A, a stabilizer (refer to the following [Table 3]), sodium chloride, and water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) obtained by the above method were separately prepared to contain the preparation in the ratio of § contained in the following [Table 3]. a and stabilizer 〇 ♦ ship) aqueous sodium chloride solution. Then, use a small amount of (4) one called hydrochloric acid (made by Wako Pure Chemical Industries) or 1 · 〇 to 0.! 〇 (m () 1 / L) aqueous sodium hydroxide solution (made by Wako Pure Chemical Industries), and these preparations and The pH of the stabilizer solution was adjusted to 5.5. For each of the above-mentioned preparation A and the stabilizer solution (including PPV), a transition film (G•(9)i m2) manufactured by the method of [Example 1] and the above-mentioned filtration device were prepared for measuring LRV, and 5 (mL) was used. Each of the preparation a and the stabilizer solution (hereinafter referred to as the stock solution) was placed in a sterilized pp spiral sample vial (manufactured by BD Fak〇n), and immediately stored under a cold bundle at C. Then, the pressure was carried out at a pressure of 294 kPa. Dead-end filtering. The filtration amount of the monoclonal antibody solution at each time and the permeation amount of the preparation A calculated according to the concentration of the preparation are as shown in the following [Table 3], 139962.doc •29·201002821 can satisfy the required recombinant gamma interferon transmission. Sex. Distribute each of the above solutions into a 50 mL sterilized spiral sample vial to obtain the target preparation A 分别 [Table 3] entry (investigation) A concentration (% by weight) Stabilizer stabilizer concentration (% by weight)平均~1 minute average permeability of antibody A (kg/m2/hour) Filtered for 2 to 3 hours of average permeability of antibody A (kg/m2/hour) Filtration, 〇~3 hours of average transmission of antibody A Sex (kg/m2/hour, 1 3.0 D-mannitol 3.0 3.07 2.58 2.77 2 3.0 amphetamine hydrochloride 3.0 2.93 2.47 2.65 [Example 5] [Virus removal test] Adding 3 vol% bovine serum (Upstate manufactured by 'heating in a 56 ° C water bath for 30 minutes and inactivated) and 1% by volume of penicillin/streptomycin (+10000 Units/mL Penicillin ' + 10000 pg/mL Streptomycin 'Invitrogen) -MEM (manufactured by Invitrogen 'high-glucose) (hereinafter, the mixture is described as 3% FBS/D-MEM). The cultured PK-13 cells were diluted to prepare a dilution of a cell concentration of 2.0 x 105 (cells/mL). Suspension. Prepare 10 96-well round bottom cell culture plates (manufactured by Falcon) in all wells The cell suspension of 1 was dispensed. Then, the total mixture of the filtrates which were filtered for 3 hours as described above was prepared by using 3% FBS/D-MEM to prepare 10 times, 1〇2 times, 1〇3 times, 1 04 times, 1.00 times the dilution solution. Further, for each stock solution collected immediately before the transition, 1 〇 2 times, 1 〇 3 times, 104 times, 1 〇 5 were prepared using 3% FBS/D-MEM. Multiple, 1 〇 6 times, 1 〇 7-fold dilution. 10 139962.doc • 30- 201002821 times, ίο2 times, ίο3 times of each filtrate and filtrate in a 96-well cell culture plate to which the above cell suspension is dispensed , 104 times, 105 times dilution, and ίο2 times, 103 times, 104 times, 1〇5 times, 〇6 times, 107 times dilution of the stock solution are each dispensed in 8 wells (μΙ〇 at 37 ° C, The cells were cultured in an incubator for 10 days in a 5% carbon dioxide atmosphere. Then, for the above-mentioned cell culture plates cultured for 10 days, TCID50 (50% infection price) was measured by a red blood cell adsorption method (Non-Patent Document 6). (manufactured by Surabaya Pharmaceutical Co., Ltd., prepared by the method attached to the product) to preserve blood from chicken (Japan Biotest Manufacturing) After diluting to 5 times, the red blood cells were pelleted by centrifugation at 2500 (rpm) for 5 minutes at 4 ° C, and then the supernatant was removed by suction, and the obtained pellet containing red blood cells was again obtained with PBS (-). Dilute to 200 times. Then, 1 〇〇 (μί) of the prepared erythrocyte pellet PBS (1) dilution was dispensed in each well of the above cell culture plate for 2 hours, and visually confirmed whether the surface of the cultured cell tissue was adsorbed by red blood cells. It is confirmed that the δ endorses the adsorber as a hole causing the virus infection, and the adsorbent is not confirmed as the uninfected hole. Regarding the presence or absence of the disease f infection of each culture solution obtained, the ratio of the filtrate or the dilution thereof or the dilution of the stock solution was confirmed, and 1 〇g (TCID50/mL) was calculated by the Reed_Munch method (Non-Patent Document 6). The infection price was determined, and the virus removal rate LRv was determined. The results are shown in the following [Table 4]. As a result, as far as _y i~(9) is concerned, the required virus removal property can be satisfied. 139962.doc -31· 201002821 [Table 4] Item (entry) Antibody A concentration (% by weight) Stabilizer stabilizer concentration (% by weight) Stock log (TCID50) Drain log (TCID50) LRV 1 3.0 D-mannitol 3.0 6.20 <0.5 > 5.70 2 3.0 L-Alanine hydrochloride 3.0 6.20 <0.5 > 5.70 [Industrial Applicability] By using the present invention, a preparation comprising a single useful substance having a polypeptide is produced. The virus removal membrane can be used to filter the high concentration preparation solution while maintaining high permeability and high virus removal, and the risk of virus incorporation from the organism or from the fermentation product can also be used. Stabilizer. Thereby, the simplification, reduction, cost reduction, and the like of the manufacturing steps of the above-described preparations can be collectively achieved. 139962.doc -32-

Claims (1)

201002821 VII. Scope of application for patents: 1. A method for the preparation of a preparation comprising a polypeptide having a polypeptide in the form of a π. #, using a substance, the single-use ρ includes the following steps: producing a culture with a polypeptide early useful substance The step-tanning t " word has a single useful substance of the polypeptide, and the purification step of the deuteration is added to the right-handed addition step of the stabilizer of the new species inhibiting the aggregation of a single useful substance of the polypeptide. , Su-extraction, forgetting to remove the membrane will contain the virus in the solution of the useful substance of the main soil team with the polypeptide h胄 fine removed from the disease, and the old-useful substance with the polypeptide removed from the virus. The seal will be derived from a single-utility with a polypeptide. The above-mentioned path removal step is based on the fact that the virus is produced when the main useful substance is produced, and when the virus is derived from the production of the stabilizer (7) (4) The virus of the disease removal step goes to the range of 3.0 to 10.0% by weight of the single useful substance having the polypeptide at the time of the virus removal step, and is contained in the solution. And the polypeptide has a single-(four) (four) (d). The average permeability of the virus-removing membrane used in the above-mentioned virus removal step is 〇10 minutes, and after 2 to 3 hours, the average permeability is 1 _G (kg/m2/ H_) above. 2. A method of preparing a preparation comprising a single-useful substance having a polypeptide, wherein the above stabilizer is derived from a living organism or derived from a fermentation product. 3. The method of claim 2, wherein the stabilizer is selected from the group consisting of a saccharide, an amino acid, an amino sugar, an organic acid, or a derivative thereof. 4. The method for producing a preparation comprising a single-useful substance having a polypeptide according to any one of items 1 to 3, wherein the virus-removing film is a hollow fiber membrane comprising a synthetic high 139962.doc 201002821 molecule. 5. The method of producing a preparation comprising a polypeptide α according to any one of claims 1 to 3, wherein the u-, early-use substance T is a virus-depleted membrane. The film is a multi-layered microporous body. 6. The method of claim 4, comprising the method for producing a polypeptide, wherein the virus removing film is prepared by a preparation of a useful substance. 7. The falling film is a multilayered one, and comprises a preparation of a monoclonal antibody. The system includes at least the following steps: Yan L is characterized by: purification of the individual antibody purification step, Μ. In the step of cultivating, at least one stabilizer for inhibiting the coagulation of the monoclonal antibody is added to the stabilizer (10) < Μ adding step 'by removing the membrane from the virus to remove the sputum in the solution of the early antibody.病毒 f removal of the virus removal step, and the removal of the virus from the individual antibody main limbs in the sealing step; and the above-mentioned path removal step will be derived from the individual antibody, when the sputum is taken The virus removal ability of the virus, the virus removal from the generation of the stabilizer, the removal of the virus is LRV24, and the concentration of the monoclonal antibody in the virus removal step is 3.0~H)% by weight in the solution. The average permeability of the contained monoclonal antibody relative to the virus removal membrane used in the above-mentioned virus removal step is 〇~1〇 minutes and the average permeability after 2 to 3 hours is LCKkg/m'hour) the above. 8. A method of producing a preparation comprising a monoclonal antibody according to the method of claim 7, wherein the above stabilizer is derived from a living organism or a product derived from a vinegar yeast product. A method for producing a preparation comprising a monoclonal antibody according to claim 8, wherein the above-mentioned stabilizer is selected from the group consisting of a saccharide, an amino acid, an amino sugar, an organic acid, or a derivative thereof. 139962.doc 201002821 10. The method of claims 7 to 9, which comprises a h film of a preparation of a monoclonal antibody. *The domain is a hollow fiber containing a synthetic polymer. 11. The preparation of a monoclonal antibody according to any of claims 7 to 9 is as follows: 12. The above-mentioned virus removal membrane is a multilayer microporous membrane. . And the method for producing a monoclonal antibody comprising a monoclonal antibody, wherein the prion removal enthalpy is a multilayer microporous membrane. Wherein the above 13 _ as claimed in items 7 to 9 φ 〆 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲 甲The amount of α is within the range of ”, .54ϊ%, and the stabilizer addition step is completed. At the end of the sealing step, the purity of the individual antibody at the end of the sealing step is 80% or more. ^: The preparation of the monoclonal antibody containing the monoclonal antibody In the production method, the amount of each of the stabilizers added in the step is 〇, 軏 軏, and the purity of the individual antibody at the end of the step of adding the elixirs and at the time of sealing the bundle is 80% or more. The method for producing a preparation comprising a monoclonal antibody according to claim 11, wherein the amount of each stabilizer added in the stabilizer addition step is in the range of ii to wt%, respectively, and the stabilizer addition step is completed. And the purity of the individual antibody at the time of sealing step 2 is 80% or more. . 16. The method for producing a preparation comprising a monoclonal antibody according to claim 12, wherein the stabilizer addition step of each of the stabilizers is in the range of 重量]% by weight and at the end of the stabilizer addition step (four) The purity of the individual antibody at step = bundle is 80% or more. ' ° 139962.doc 201002821 IV. Designated representative map: (1) The representative representative of the case is: (none) (2) The symbolic symbol of the representative figure is simple: 5. If there is a chemical formula in this case, please reveal the best display invention. Characteristic chemical formula: (none) 139962.doc