WO2004092218A1 - Process for producing recombinant antithrombin - Google Patents

Abstract

A process for producing antithrombin (AT) prepared through genetic engineering technique; and a method of purification thereof. In particular, a recombinant antithrombin (rAT) having been improved with respect to production volume and specific activity can be produced by culturing animal cells capable of production of (AT) transformed by genetic engineering technique under conditions of a combination of 6.9 or below pH, 4 mM or more glutamic acid and 35°C or below. Further, increasing of (rAT) specific activity, high-degree purification and turbidity elimination can be accomplished by subjecting a culture supernatant containing rAT or the like to treatment with immobilized heparin, solvent exchange, anion exchanger treatment, polymer removal, filtration through a porous membrane and treatment at 7.5 or higher pH.

Description

 Specification

 Method for producing recombinant antithrombin

 The present invention relates to a method for producing antithrombin prepared by a genetic recombination technique. The present invention also relates to a method for purifying antithrombin prepared by a genetic recombination technique. Background art

 Antithrombin (also called Antithrombin II.

AT) is a single-chain glycoprotein with a molecular weight of about 58,000, is mainly synthesized in the liver, and is present in human normal plasma at about 150 mg / L. AT is a protease inhibitor that plays the most important function in the control mechanism of blood coagulation, and includes thrombin, factor Xa (a indicates activated coagulation factor; the same applies hereinafter), IXa, XI It has activity to inhibit serine protease such as factor a and Xlla. It is known that AT irreversibly inhibits protease activity by forming a stable complex with these serine proteases in one molecule to one molecule. In the presence of heparin, the rate of formation of the complex of AT and thrombin is activated about 100-fold (heparin coffactor activity). AT is composed of 432 amino acid residues, three disulfide bonds, and three or four N-type sugar chains.

Human plasma-derived AT preparations obtained by conventional techniques (Nearart (trade name), Mitsubishi Pharma Corporation, etc.) have a tendency to form thrombus due to congenital AT deficiency and generalized intravascular coagulation with AT reduction (DIC). These AT-derived products derived from human plasma reduce the risk of contamination with infectious agents by various measures, but are unknown because they are derived from human plasma. There is a concern about the risk of contamination factors. Therefore, there is a demand for the development of a safer AT formulation by applying genetic recombination technology.

 AT is a glycoprotein, but it is generally known that the glycan composition and glycan structure of glycoproteins affect immunogenicity, blood half-life, and biological activity. For the production of recombinant AT (rAT), mammalian cells such as CHO cells (Chinesehamsterovarycels), BHK (neonatal hamster kidney cells: ATCCCL10), and COS-11 monkeyeel Is are suitable. ing. Some expressions of rAT using these animal cells have already been reported (Non-patent Documents 1 to 3). However, the production of rAT using conventional techniques has not reached practical use due to low productivity.

 The method of culturing recombinant cells is one of the important factors that determine the productivity of recombinant proteins. As compared with microorganisms such as Escherichia coli and yeast, animal cells have the following drawbacks as hosts for recombinant protein production. That is, the growth rate is low, the culture medium is expensive, and the production per medium and per cell are low. In order to address these drawbacks, in recombinant animal cells, medium composition, basic culture conditions (culture temperature, dissolved oxygen concentration, PH, etc.), densification of cultured cells, and suppression of reduced viability of cultured cells Are being studied. As a culture method for improving productivity by increasing the density of cultured cells or suppressing the decrease in the survival rate of cultured cells, fed-batch culture and perfusion culture are known. These culture methods maintain the growth and viability of the cells for a long time by replenishing the nutrients required by the cells without depletion.

In animal cell culture, glucose and glutamin are major It is commonly used in medium components as a novel carbon source and energy source (Non-Patent Document 4). However, it is known that ammonia produced by the metabolism of glutamin inhibits cell growth and production of a target protein. In fed-patch culture in which glutamin is fed, cell growth may be inhibited due to the accumulation of ammonia, and the productivity may also be reduced. As a method for suppressing the accumulation of ammonia in fed-batch culture, a glutamin fed-batch method for controlling the concentration of glutamin in a medium to a low concentration has been reported (Non-Patent Document 5). Further, it has been reported that the use of a peptide containing glutamin as a substitute for glutamin also suppresses the accumulation of ammonia (Non-Patent Document 6). Since CHO cells naturally have glutamin synthetase activity, a method using glutamate instead of glutamin has been reported in patch culture (Non-Patent Document 7). Even in a cell line that does not originally have glutamine synthetase activity, it can be grown on a glutamine-free medium by introducing the glutamine synthetase gene using gene recombination technology. (Non-Patent Document 8). · Some basal media widely used in mammalian cell culture include Dulbecco's Modified Eag1e Medium (DME), Hams F—12, RPMI 1640, and Iscove's The concentration of glutamate in the medium contained in these basal media is 0.5 mM or less, using a gene recombination technique to transform the glutamin synthetase gene and the monoclonal antibody gene. In the report of the fed-batch culture of the introduced NS0 cells, the concentration of daltamic acid in the medium was 1 mM or less, and glutamin was not added to the medium (Non-Patent Document 9). Thus, the concentration of glutamate in the medium used for culturing animal cells is usually less than or equal to I mM. Although there is a report suggesting the added culture method (Patent Document 1), there is no specific example regarding the high concentration addition of dal pernic acid, and AT is not mentioned as a recombinant protein to be produced. . In addition, examples of patch culture and continuous culture using 7 mM and 6 mM glutamate as an alternative to glutamin have been reported (Non-patent Documents 7 and 10). The relationship between glutamate concentration and recombinant protein productivity was not investigated. Furthermore, there have been no reports to date on the relationship between daltamic acid concentrations of 1 mM or more and recombinant protein productivity in fed-batch culture of transgenic animal cells.

Normal conditions for pH during culture are about 7 to 7.2, but a culture method at pH 6.8 or lower has also been reported (Non-Patent Document 11). However, there is no mention or suggestion regarding improvement of production volume. Normal conditions for the culture temperature are 37 ° C, but a method for producing rAT by low-temperature culture has been reported (Non-Patent Document 12). However, there is no description or suggestion regarding improvement of specific activity. In order to stably supply rAT that is safer than plasma-derived AT, various methods for producing rAT, including a method using animal cells, are being studied. For example, a method using yeast as a host (Non-patent Document 13), a method using animal cells [especially Chinese / Pemster follicle (CHO) cells] (Patent Document 2), non-human mammals (for example, A transgenic non-human animal in which the expression plasmid has been introduced into E. coli, goats, sheep, etc., to produce rAT in vivo, and recover rAT mainly from the milk. Methods (Patent Document 3), etc. have been reported. However, there is little information on the specific activity of these rATs, including pH-dependent variations in specific activity. Almost unknown.

 It has been reported that the production of 1-AT using CHO cells is (almost temporarily) subject to alkaline PH conditions. In some cases, the specific activity of r AT itself has been reported (Non-patent Documents 2, 14, and 15). However, in each case, it is not disclosed at all whether the specific activity of AT fluctuates or the low specific activity is improved by alkaline pH conditions.

 It has been reported that AT derived from plasma is stable at alkaline pH (Patent Documents 4 and 5). However, these are not derived from recombinants, and it is not disclosed at all whether specific activity is improved in a pH-dependent manner.

 Also, little is known about the above-mentioned rAT which is highly purified to such a degree that it can be used as a pharmaceutical. RAT purification methods include immobilized heparin treatment and antibody affinity carrier treatment. Although ammonium sulfate fractions and the like have been reported (Non-Patent Documents 2 and 15), none of them have low purity and cannot be said to have been sufficiently purified. There is also little mention of removing contaminants. On the other hand, immobilized heparin treatment, solvent exchange, anion exchanger treatment, and hydrophobic carrier treatment are already known as means for purifying plasma-derived AT (Patent Documents 6 to 8). However, the contaminants are completely different between the plasma-derived protein and the recombinant protein (other plasma proteins are the main contaminants in the case of plasma-derived proteins, whereas the host-derived substances are in the case of transgenic proteins). Or medium-derived contaminants) .Thus, knowledge of plasma-derived proteins, such as the behavior of each contaminant in each treatment step and the degree of removal, does not directly apply to genetically modified proteins. .

A method of treating plasma-derived AT with a porous membrane has been reported (Non-Patent Document 16), but this does not relate to rAT, Its purpose is also related to virus removal. In addition, a method for suppressing the association of protein molecules by treatment with a porous membrane has been reported (Patent Document 9), but AT is not disclosed here, and the relationship between protein association and turbidity is also clear. Not clear. Furthermore, there has been reported a method of removing a substance which may be a nucleus forming an insoluble foreign substance by performing a porous membrane treatment during preparation of an immunoglobulin preparation (Patent Document 10). However, this is not related to the recombinant protein, nor is it related to AT. (Patent Document 1) Japanese Patent No. 2783392

 (Patent Document 2) Japanese Patent Application Laid-Open No. 63-444489

 (Patent Document 3) U.S. Pat.No. 5,843,705

 (Patent Document 4) International Patent Publication No. 94 / 22247-1 pamphlet

(Patent Document 5) Japanese Patent Application Laid-Open No. H10-14747538

 (Patent Document 6) Japanese Patent Application Laid-Open No. Sho 63-323898

 (Patent Document 7) Japanese Patent Application Laid-Open No. 11-275600

 (Patent Document 8) Japanese Patent Application Laid-Open No. 2-47171

 (Patent Document 9) JP-A-6-27992296

 (Patent Document 10) Japanese Patent Application Laid-Open No. H10-26654

 (Patent Literature 11) Japanese Patent Application Laid-Open No. 58-16692

 (Patent Document 1 2) Japanese Patent Application Laid-Open No. 5-333992

 (Patent Document 13) JP-A-9-1716600

(Non-Patent Document 1) Journal of Biological Chemistry (J. Biol. Chem.), 1987, 1982, Vol. 262, 1 4 7 6 6 — 1 4 7 7 Page 2

 (Non-Patent Document 2) Ibid., 1989, 264, 211 5 3 — 2 1 1 59

(Non-Patent Document 3) Behring Inst.Mitt., 1 9 8 8 years, 8 2 volumes, 2 6 — 3 4 pages

 (Non-Patent Document 4) Ce11Biol.Int.Rep., 1

9 8 2 years, Volume 6, 6 3 5 — 6 4 9

 (Non-Patent Document 5) Biotechnology and Bioengineering (Bi0tech.Bioeng.), 1994, 44, 95-103

 (Non-Patent Document 6) Journal of Biotechnology (J. Biotechnol.), 1994, pp. 37, 27, pp. 27-290

 (Non-Patent Document 7) Biotechnology Progress (Biotechnol. Prog.), 2000, 16th volume, 69-75

 (Non-Patent Document 8) Bio / Technology (Bio / Technoligoy), 1990, 2010, 10 turns, 16 9 — 17 5

 (Non-Patent Document 9) Biotechnology Progress, 1994, Vol. 10, Volume 87-96

 (Non-Patent Document 10) Ibid., 2001, Vol. 17, Vol. 10, page 32 — page 104

 (Non-Patent Document 11) Biotechnology and Bioengineering (Biotech. Bioeng.), 1988, 32, 32, 947 — 965

 (Non-Patent Document 1 2) Enzym 'And' Mycrovial Technoron (EnzymenddMicrobbiaalTechnoylogy), 1996, 18, 423-427

 (Non-Patent Document 13) Protein Exceedment and And'PiuriFication (Prot. Exp. Purif.), 2001, 23rd volume, 55th — 6 page 5

(Non-Patent Document 14) Pio Technology, 1987, Vol. 5, 720 pages

 (Non-Patent Document 15) Bioscience, Biotechnology, and Biochemistry. Biochem. Biochem., 1992, Vol. 56, No. 4, 600- 6 0 4 pages

 (Non-patent document 16) Attached document of Neut (Mitsubishi ELPHARMA)

 (Non-Patent Document 17) FEBSLett., 1993, 3335 Vol. 1, No. 9, pp. 12

 (Non-Patent Document 18) American Chemical 'Chemical' Society (Am. Chem. Soc. 1985, Vol. 9, 1997 — pp. 228)

 (Non-Patent Document 19) Biotechnology And 'Bioengineering, 1997, 56, 577-582 (Non-Patent Document 20) Ibid., 200 0 years, 69 volumes, 5 6 6 — 5 7 6 pages

 (Non-Patent Document 21) BMM Product Description (Asahi Kasei) Disclosure of Invention

 An object of the present invention is to provide a method for producing rAT with an improved production amount and specific activity, that is, a method for culturing animal cells capable of producing AT transformed by using gene recombination technology. It is to provide

An object of the present invention is to provide a method for highly purifying AT prepared by a gene recombination technique. In view of the above circumstances, the present inventors conducted research using animal cells capable of producing AT transformed using genetic recombination technology. As a result, among various culture conditions, p H, Dartami The three conditions of acid concentration and culture temperature were found to be important for the cultivation of the cells and the production of rAT, and by optimizing those conditions, the intended purpose could be achieved. And completed the method for producing rAT of the present invention.

In addition, the present inventors have started research on a method for purifying rAT in consideration of the above circumstances. However, rAT produced by culturing a host prepared by genetic recombination technology has its own specific activity (for example, a purified plasma-derived product, or a commercially available product). (Pharmaceuticals). Therefore, first, as a result of various studies to recover the specific activity, it was found that the specific activity of rAT can be improved by treating the solution containing rAT with a pH of 7.5 or more. found. Further studies were carried out to purify rAT to a high degree based on this technology, and the culture supernatant containing rAT was treated with immobilized heparin, solvent exchange, anion exchanger treatment, and polymer purification. Removal and porous membrane treatment are combined, and at least the steps after the solvent exchange step are performed under conditions of pH 7.5 or more, so that specific activity can be recovered and high purification can be achieved. It has been found that the obtained rAT can be manufactured. Further, since it was confirmed that the present AT-containing solution had turbidity, various treatment methods were examined to remove the turbidity, and it was concluded that the porous membrane treatment was superior in its effect. found. By combining these findings, the purification method of the present invention was completed. <One embodiment of the present invention provides an animal cell capable of producing AT, which has been transformed using a gene recombination technique. The present invention relates to an rAT production method characterized by culturing under a combination of pH 6.9 or less, dal permic acid 4 mM or more and 35 ° C. or less. In one embodiment of the present invention, immobilization is performed using a culture supernatant containing rAT. Treatment, solvent exchange of the treatment solution, anion exchanger treatment, removal of polymer, filtration using a porous membrane having an average pore diameter of 1 to 100 nm, and at least solvent The present invention relates to a method for purifying rAT from a culture supernatant, wherein the steps after the exchange are performed under conditions of pH 7-5 or more.

 The purification method of the present invention has the following inventive features. Improve the specific activity of rAT by treating at pH 7.5 or higher: immobilized heparin treatment, solvent exchange of the treatment solution, anion exchanger treatment, polymer removal treatment To purify the rAT to a high degree: removing turbidity from the rAT-containing solution by filtration using a porous membrane having an average pore size of 1 to 10 O nm: the entire purification process Alternatively, rAT is recovered in a state of high specific activity by performing a part under the condition of PH 7.5 or more. One aspect of the present invention relates to highly purified rAT.

 One aspect of the present invention relates to a pharmaceutical composition comprising highly purified rAT.

 One embodiment of the present invention relates to a novel method for producing rAT by combining a method for producing rAT with improved production amount and specific activity and a method for highly purifying rAT. The present invention is described in detail below. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows the time course of the culture pH. Culture pH 7.0 or more

(Violent), culture pH 6.6 or more (〇).

 FIG. 2 shows the time course of the rAT production concentration (heparin cofactor activity). The description of the culture pH is the same as in FIG.

FIG. 3 shows the time course of the culture pH. Culture Η 6. 6 (+), Culture pH 6.8 (〇), Culture pH 7.0 (▲), Culture pH 7.0

2 (□).

 Figure 4 shows the time course of AT production concentration (heparin cofactor activity). At the end of the cultivation at 7.0, the AT production concentration at the end of the cultivation was shown as a relative value when the concentration was 100%. The description of the culture pH is the same as in FIG.

 Fig. 5 shows the time course of the glutamate concentration in the medium. Glutamate concentration: ImM (〇), 4 mM (△), 8 mM (mouth).

 Figure 6 shows the time course of AT production (heparin factor activator X culture volume). The explanation of the daltamate concentration, expressed as a relative value when the amount of AT produced at the end of the culture in glutamate I mM is 100%, is the same as in FIG.

 Figure 7 shows the time course of AT production (heparin cofactor activity X culture volume). The AT production at the end of the culture at 37 ° C. was shown as a relative value when the AT production was 100%. Incubation temperature: 35 ° C (〇), 37 ° C (ten). BEST MODE FOR CARRYING OUT THE INVENTION

 (Production method)

 The method for producing rAT of the present invention originally has the following claims.

 1) Animal cells capable of producing AT, which have been transformed using genetic recombination techniques, are cultured under conditions combining pH 6.8 or less, glutamate 4 mM or more, and 35 ° C or less. R AT production method characterized by the following.

2) culturing the cells in a step of contacting with a culture solution having a pH of 6.8 or less, culturing in a step of contacting with a culture solution containing 4 mM or more glutamate, and culturing at 35 ° C or less. 1) r AT production method. 3) The culture mode is that the cells in culture are suspended in the culture medium, the cells in culture are in a serum-free medium, and the cells in culture are in a medium that does not contain mammalian proteins. 1) The method for producing rAT according to 1), wherein the culture method is fed-batch culture.

 4) An animal cell capable of producing AT, which has been transformed using a genetic recombination technique, is prepared using at least pH 6.8 or less, glutamate 4 mM or more, and 35 ° C or less. A method for producing rAT, comprising culturing under a combination of two conditions.

 5) Production of rAT by culturing animal cells capable of producing AT, which has been transformed using genetic recombination technology, in a step of contacting a culture solution having a pH of 6.8 or less. A method for producing AT, characterized by increasing the amount.

 6) Fed-batch culturing is performed by contacting animal cells capable of producing AT, which has been transformed by genetic recombination technology, with a culture medium containing 4 mM or more glutamate. A method for producing rAT, characterized by improving the production of rAT.

 7) The method for producing rAT according to 6), wherein the animal cell has a glutamine synthetase activity.

 8) Animal cells capable of producing AT, which have been transformed using genetic recombination technology, are fed-batch cultured at 35 ° C or lower to reduce rAT production and / or specific activity. R AT production method characterized by improvement.

A T gene · host · expression system

(Human) As the AT gene, a known gene can be used (Patent Document 11). The AT gene may be of a mutant type other than the wild type. Wild-type mutants The amino acid sequence is not particularly limited as long as it is a deletion, substitution, or addition of a part of the amino acid sequence and has at least the same physiological activity as the wild type. For example, the amino acid in the reaction site / heparin binding site is substituted with another amino acid (Patent Document 12), and the amino acid / oligopeptide is added to the C-terminal ( Patent Documents 13) and 13-position in which asparagine is substituted with glutamine (Non-Patent Document 17) are exemplified. The mutant may have heparin-independent thrombin inhibitory activity or a high heparin affinity.

 As the host (animal cell), a host known in gene recombination technology can be used. For example, animal cells such as CHO cells (such as CHO-K1 cells), BHK cells, COS-7 cells, and Vero cells are exemplified. These may be auxotrophs, antibiotic-sensitive strains, or certain mutants (strains deficient in certain genes). For example, mutants such as dhfr (dihydrofolate reductase) deficient strains are exemplified. As the animal cells of the present invention, those having glutamin synthetase activity can be used.

As the AT expression system of the present invention, an expression system (promoter, signal sequence, etc.) usually used in animal cells may be used. A gene encoding the above AT is introduced into an expression vector system to construct a host vector system for expression. The vector has a promoter, a signal sequence, a ribosome binding site, and a transcription termination sequence. It may further have a control sequence (enhancer), an RNA splice sequence, and a polyA addition site. It may also have a marker sequence that allows phenotype selection in transformed cells. Further, as a high production system, a gene amplification system utilizing the dhfr gene may be used. Preparation of transformants

 The transformant can be prepared by a known method. That is, a transformant is prepared by introducing the AT gene into an appropriate expression plasmid into a host cell line. Methods for introducing the expression plasmid into the host cell include the calcium phosphate method, the electroporation method, the lipofectin method, the particle cancer method, the DEAE-dextran method, and the virus. Vector method and the like are exemplified. By these methods, the plasmid or its linear fragment can be introduced into the host chromosome. Culture and culture '' r AT production

 The transformant is cultured by a known method. The medium is not particularly limited as long as it is for culturing animal cells. Examples include a basic medium (eg, MEM medium, DMEM medium, RPMI medium, HamF medium, etc.), a serum-containing medium in which basal medium is added to serum, a serum-free medium without serum, and the like. . Serum-free media may be supplemented with mammalian-derived proteins (eg, insulin, serum albumin, transferrin, etc.) or without mammalian-derived proteins (recombinant protein or plant). Derived protein), those without the protein itself (a so-called serum-free protein-free medium, which may contain protein hydrolysates), and those with the addition of only low-molecular-weight synthetic products (sugars, amino acids). For example, it is composed of acid, lipid, vitamin, nucleic acid, mineral, and amines.

The characteristics of the culture conditions of the present invention are that the culture is performed under the conditions of pH 6.9 or less (preferably pH 6.8 or less), glutamate of 4 mM or more, and 35 or less. If each culture condition is always satisfied during the culture period, or temporarily (only within a certain period of time), Both conceptually included.

 As for pH, a method of culturing the transformant in a step of contacting the transformant with a culture solution having a pH of 6.9 or less (preferably pH 6.8 or less) may be mentioned. Specifically, an aspect in which the pH is maintained at 6.8 or less during the culture period, an aspect in which the pH is set to 6.7 to 6.9 (6.8 ± 0.1), A mode in which only the pH is set to pH 6.8 or less is exemplified. As the latter, for example, by initially setting the pH at about 7.2 and then taking a mode in which the pH is not controlled (becoming more and more), the pH is naturally reduced to 6.8 or less. After that, there is a case where the pH is naturally increased to about 7.2. In each case, the pH is preferably 6.6 or more. This form can increase AT production.

 A specific example of glutamate is a method in which feed patch culture is performed in a step of contacting with a culture solution containing 4 mM or more daltamate. Preferably, 7 mM or more is exemplified. Specifically, there are a mode of setting to 3 to 5 mM (4 ± 1 mM), a mode of setting to 4 mM or more during culturing, and a mode of setting to 6 to 8 mM. Is done. Glutamic acid may be in the form of a salt such as a sodium salt and a calcium salt. At this time, it is preferable not to add free glutamin as a culture solution. This format can increase AT production.

 Regarding the cultivation temperature, specifically, there is a method of cultivating fed batch at 35 ° C or less. The temperature is 35 ° C or less, specifically, about 30 to 35 * C, and preferably about 33 to 35 ° C. According to this method, the production amount and specific activity of AT can be improved.

The three conditions (three steps: pH, glutamate concentration, and temperature) were used in combination with at least three or at least one of them. Can be implemented in combination of the two. In this case, the three conditions (three steps) may be performed simultaneously, may be performed partially overlapping (duplicate) in time, or may be performed independently in time (in a separate manner). ) May be implemented.

 The culturing time is about 10 to 500 hours. Ventilation and agitation can be added as needed. As the culture mode, batch culture, fed-batch culture, continuous culture, perfusion culture, and the like can be used. Preferably, a fed-batch culture is used. Depending on the form of cells in culture, adherent culture (cells in culture adhere to the carrier) and suspension culture (cells in culture are suspended in culture medium) are available. Either method may be used. Preferably, suspension culture is used.

 Other culture conditions (cell density, medium composition, dissolved oxygen concentration, dissolved carbon dioxide concentration, agitation speed, medium flow acceleration, etc.) may be appropriately selected according to the type of animal cell. Isolation and purification

 After rAT is produced by culturing, rAT can be obtained from the transformant or its culture (culture solution, culture supernatant).

 r AT can be purified by a known method. Examples include ultrafiltration, gel filtration, ion exchanger treatment, affinity carrier treatment, and the like. (Purification method)

 The method for purifying rAT of the present invention essentially has the following claims.

1) Using the culture supernatant containing rAT, immobilized heparin treatment, solvent exchange of the treatment solution, anion exchanger treatment, removal of polymer, Filtration using a porous membrane with an average pore size of 1 to 100 nm is performed, and at least the steps after solvent exchange are performed under conditions of pH 7.5 or higher. Purification method

 2) The purification method according to 1), in which ultrafiltration is further performed before the porous membrane treatment.

 3) Using culture supernatant containing rAT, immobilized heparin treatment, solvent exchange of the treatment solution, anion exchanger treatment, removal of polymer, porosity with average pore diameter of 1 to 100 nm A method for purifying rAT from the culture supernatant, which performs filtration using a membrane.

4) A method for purifying rAT, wherein all or a part of the purification step is performed under conditions of pH 7.5 or more.

 5) A method for purifying rAT, which improves the specific activity of rAT by treating the rAT-containing solution with a pH of 7.5 or more.

6) A method for purifying rAT, wherein turbidity is removed from the rAT-containing solution by filtering the solution using a porous membrane having an average pore diameter of 1 to 100 nm.

AT gene, host expression system

(Hit) Known AT genes can be used. The AT gene may be a mutant type other than the wild type. The mutant type is not particularly limited as long as a part of the amino acid sequence of the wild type is deleted / substituted / added and has at least the same physiological activity as the wild type. Reaction site, amino acid at heparin binding site replaced with another amino acid, amino acid, oligopeptide added at C-terminal, position 135 from asparagine to glutamine Examples are those that have been replaced (all related documents are as described above). As a host expression system, those known in AT can be used. For example, yeast [for example, Saccharomyces Genus (eg, Saccharomyces cerevisiae), Pichia (eg, Pichia pastoris), Kluyveromyces, etc.), animal cells (eg, CHO cells, BHK cells, COS-7 cells, Vero cells) ), Insect cells, capi (such as Aspergillus) and the like. These may be auxotrophs, antibiotic susceptible strains, or certain mutants (strains deficient in certain genes). In addition, transgenic non-human animals (eg, geese, goats, sheep, etc.) can be used. Preferably, animal cells are used as hosts. More preferably, CHO cells are used as a host. r Preparation of AT

 It can be prepared by a known method. That is, a transformant is prepared by incorporating the AT gene in a suitable expression plasmid and incorporating it into a host expression system.

 The transformant is further cultured to produce rAT. The culture conditions (cell density, medium composition, pH, temperature, time, dissolved oxygen amount, carbon dioxide amount, stirring speed, medium flow rate, etc.) may be appropriately selected according to the host expression system. The pH, temperature and time are generally about pH 5 to 8 (preferably pH 6 to 7.3), about 10 to 45 ° C, and about 10 to 500 hours. The culture method may be any of patch culture, fed-batch culture, and continuous culture.

The rAT (containing fraction) produced (or in the culture supernatant) by culturing under the above-mentioned normal conditions is used to determine the AT protein concentration (for example, can be quantified by reversed-phase chromatography). When used to calculate specific activity, the specific activity is lower than the original level (for example, about 6.5 to 7.5 UZ mg for purified plasma-derived products). . Specifically, specific activity 3 to 6 UZ m g. The specific activity of the rAT (containing fraction) is increased (recovered) by treatment with an alkaline pH of 7.5 or more, and after the treatment, the rAT is acidified. When placed under pH conditions, it has the property that the specific activity recovered by the alkaline pH treatment is reduced. The rAT of the present invention is highly purified by the following processing steps. Preprocessing

 The host cells are removed from the culture solution, and only the culture supernatant is collected. If necessary, concentrate to about 1 to 10 U / mL. Also, 0.

A filtration treatment of about 45 x m may be performed. Parin treatment for immobilization

 This treatment is performed to purify rAT, and is a step in which the rAT-containing solution is brought into contact with immobilized parin to immobilize it and eluted after A A is adsorbed. Immobilized heparin is obtained by binding heparin to an insoluble carrier. Examples of the insoluble carrier include agarose (trade name: Sepharose), dextran (trade name: Sephadex), hydrophilic pinyl polymer (trade name: Toyopearl), and the like. The method of binding heparin to the insoluble carrier can be performed according to a known method. Also, commercially available products can be used.

 Examples of the contact conditions between r AT and the immobilized heparin include a pH of about 6 to 8 and a conductivity of about 10 to 40 mS / cm. After the contact, it is preferable to wash the parin once to the immobilized rAT. The washing conditions were PH 6 to 8, salt concentration 0.15 to 0.6.

An example is about 5M. Specifically, 0.15 to 0.65 M sodium chloride or the like is used. In addition, elution conditions pH 6 to 8 and salt concentration of about 1 to 3 M are exemplified. Specifically, sodium chloride of 2.5 to 3.5 M is used. Solvent exchange

 This treatment is a step performed to change the solvent composition of the rAT-containing solution. Specific examples include hydroxypatite treatment, dialysis, and ultrafiltration. In each case, change the solvent so that the pH is at least 7.5. Specifically, the pH is preferably about pH 7.5 to 10 and more preferably about pH 7.5 to 9. The salt concentration is not particularly limited, but is preferably about 0.01 to 0.5M. For example, 0.1 M sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, phosphate buffer, An example is a tris-phosphate buffer (PH 8).

 When performing hydroxyapatite treatment, the rAT-containing solution may be brought into contact with the hydroxyapatite, and then the adsorbed rAT may be eluted and recovered with a solvent to be exchanged. When performing hydroxypropyl treatment, the solvent to be replaced is sodium hydrogen phosphate, dicalcium hydrogen phosphate, sodium dihydrogen phosphate, dihydrogen phosphate. Use potassium hydrogen, phosphate buffer or the like. When performing ultrafiltration and dialysis, the rAT-containing solution may be treated using a solvent to be exchanged as the external solution. Anion exchanger treatment

 This treatment is a step mainly performed to remove DNA by bringing the rAT-containing solution into contact with an anion exchanger.

Anion exchangers are those in which anion exchange groups are bound to an insoluble carrier. Examples of anion exchange groups include DEAE (getyl aminoethyl), QAE (quaternary aminoethyl), and Q (quaternary amino). (Monitor) and the like. Examples of the insoluble carrier include agarose (trade name of Sepharose), dextran (trade name of Sephadex), and hydrophilic vinyl polymer (trade name of Toyopearl). The method of binding the anion exchange group to the insoluble carrier can be performed according to a known method. Also, commercially available products can be used.

 Examples of the mode of this treatment include a method of recovering a non-adsorbed (pass) fraction, a method of once adsorbing rAT, and then eluting and recovering it, and a method of using both together. In any case, the contact condition may be such that the pH is 7.5 or more. Specifically, the pH is preferably about 7.5 to 10 and more preferably about pH 7.5 to 9. The salt concentration is not particularly limited, but is preferably about 0.01 to 0.5M. For example, 0.1 M sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium dihydrogen phosphate, dihydrogen phosphate phosphate, phosphate buffer Solution, Tris-HCl buffer (pH 8) and the like.

 In the case of the first method, the non-adsorbed fraction is collected while maintaining the contact conditions. In the case of the second method, set the contact conditions at an appropriate time

(at a time when rAT has not yet eluted), and elute and collect rAT. Examples of the elution conditions include a pH of about 5 to 7 and a salt concentration of about 0.01 to 0.5 M. For example, physiological saline (0.15 M sodium chloride) is exemplified. In the case of the third method, pass fraction collection method (first method) and elution method

(Second method) may be performed first. Polymer removal

This is a step of removing the polymer (meaning that the polymer has a higher molecular weight than AT. R The polymer of AT is also conceptually included). Specifically, treatment with hydrophobic carrier, re-treatment of immobilized parin, etc. Is exemplified. When performing the treatment with a hydrophobic carrier, the rAT-containing solution is brought into contact with a carrier for treating the hydrophobic carrier, and the polymer is removed by collecting the non-adsorbed fraction.

 The carrier for treating the hydrophobic carrier is one in which a hydrophobic group is bonded to an insoluble carrier. Examples of the hydrophobic group include an alkyl group (having 4 to 18 carbon atoms, for example, a butyl group, an octyl group, an octadecyl group) and a phenyl group. Examples of the insoluble carrier include agarose (trade name: Cepharose), dextran (trade name: Cephadex), hydrophilic vinyl polymer (trade name: Toyopar), and the like. You. The method for binding the hydrophobic group to the insoluble carrier can be performed according to a known method. Also, commercially available products can be used.

 The condition for contacting the carrier for treating the hydrophobic carrier may be such that PH is 7.5 or more. Specifically, the pH is preferably about 7.5 to 10 and more preferably about pH 7.5 to 9. The salt concentration is not particularly limited, but is preferably about 0.5 to 3M. For example, about 0.01 to 0.5 M (specifically, 0.1 M) of phosphoric acid to which inorganic salts such as about 0.5 to 3 M of ammonium sulfate and sodium sulfate are added. Sodium hydrogen hydride, sodium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, phosphate buffer, Tris-HCl buffer (pH 8 ) Is used.

When the hydrophobic carrier treatment is performed, the solvent is exchanged after the treatment. The pH may be 7.5 or more. Specifically, the pH is preferably about 7.5 to 10 and more preferably about pH 7.5 to 9. The salt concentration is not particularly limited, but is preferably about 0.01 to 0.5M. For example, 0.1 M sodium phosphate dibasic, sodium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, phosphate buffer, Tris Hydrochloric acid buffer (PH 8) is exemplified. When parin is reprocessed for immobilization, it may be performed according to the first operation described above. Ultrafiltration

 In the present invention, ultrafiltration is preferably performed before performing the porous membrane treatment. This treatment is for the purpose of removing endotoxin and preventing clogging during the subsequent porous membrane treatment. Ultrafiltration is performed using an ultrafiltration membrane having a molecular weight cut-off of about 100 to 500 kilodaltons (kD). Examples of the material of the ultrafiltration membrane include a polysulfone-based material and a cellulose-based material. Alternatively, commercially available products may be used. An example is Sartorius Zartcon (trade name).

 Also, two or more ultrafiltration membranes having different molecular weight cut-offs may be used in combination. In this case, the above-mentioned ultrafiltration membrane having a molecular weight cut-off of about 100 to 500: 0 and an ultrafiltration membrane having a molecular weight cut-off of about 1 to 50 kD are combined. Can be used. Porous membrane treatment

 This step is performed not only for the purpose of removing the original virus, but also for removing the turbidity contained in the rAT-containing solution. The material of the porous membrane used in the present invention is not particularly limited, but preferably includes regenerated cellulose. Examples of the shape include a hollow fiber shape and a sheet shape, and a hollow fiber shape is preferable. For example, the porous hollow fiber of the regenerated cellulose is preferably prepared by a microphase separation method from a cellulose copper ammonia solution (Non-Patent Document 18).

The average pore size of the porous membrane is between 1 and 100 nm, preferably between 10 and 100 nm. 75 nm, more preferably 10 to 50 nm, particularly preferably 35 earth 2 nm or 15 ± 2 nm, and the film thickness is preferably 35 ± 3.5 mm. m or 2 7 ± 3!丄 m, and the film is preferably of a multilayer structure. When the porous membrane has a hollow fiber shape, the inner diameter is preferably 330 ± 30 m.

When the porous membrane has a hollow fiber shape, it is preferably used in the form of a module. The module is properly preferred membrane area 0. 0 0 1 ~ 1. O m is ² porous hollow fiber membrane and the container and Ri構formed by the adhesive to integrate them for filling this Is done.

 The filtration treatment using the porous membrane is performed, for example, as follows. First, an rAT-containing solution is prepared. It is sufficient that ρ 7 is at least 7.5. Specifically, it is preferable that ρΗ7.5 to about 10, more preferably, ρΗ7.5 to about 9. The salt concentration is, for example, about 0.01 to 0.5Μ. The protein concentration is, for example, about 1 to 10 Omg ZmL.

In carrying out this treatment, pharmacologically acceptable additives (eg, carriers, excipients, diluents, etc.), stabilizing agents, Components may be added. For example, sugars (monosaccharides such as glucose and fructose, disaccharides such as sucrose, lactose and maltose, sugar alcohols such as mannitol and sorbitol, etc.), inorganic salts (such as sodium chloride), organic Acids (such as citric acid, lingic acid, and tartaric acid) or their salts (such as alkaline earth metal salts such as sodium salts and potassium salts, and alkaline metal salts such as calcium salts), nonionic -Based surfactants such as poly (ethylene glycol), poly (oxyethylene) / poly (oxypropylene) copolymer (trade name: Nick), polyoxyethylene sorbitan fatty acid ester (trade name: Tween), etc. Is shown. The added amount is about 0.1 to 40% (w / V) of sugar, about 0.1 to 10% (WZV) of inorganic salt, organic acid or its salt, and about 0.1 to 0.1% of nonionic surfactant. About 1% (wZV).

The above-mentioned rAT-containing solution is subjected to a filtration treatment using a porous membrane. The filtration pressure at this time is preferably about 0.1 to about L kgf Z cm ² . The processing temperature is preferably about 4 to 50 ° C.

 As a mode of the filtration treatment, there are a cross-flow filtration method (circulation type) in which the liquid is filtered while giving a strain rate, and a dead-end filtration method (non-circulation type) in which the filtration is performed without giving a strain rate. It can be either. Alkaline pH treatment

In the present invention, rAT having a low specific activity (for example, specific activity of about 3 to 6 UZ mg) can be recovered by treating it with a pH of 7.5 or more. Specifically, the pH condition is preferably about 7.5 to 10, more preferably about pH 7.5 to 9. Specifically, sodium hydroxide solution, potassium hydroxide solution, sodium phosphate solution, diphosphate phosphate solution, phosphate buffer solution, and tris-HCl buffer solution Etc. should be used. The conductivity (salt concentration) is, for example, about 0.01 to 300 mSZcm (0.01 to 0.5 M). The protein concentration is, for example, about 0.1 to 350 mg / mL, preferably about 1 to 10 Omg ZmL. The processing time is about 1 to 10 hours. Further, the treatment may be performed alone as one treatment step, but may be an embodiment in which the treatment is consequently performed during the treatment step (purification step) of rAT. For example, when rAT is processed by column chromatography, it is eroded under alkaline pH conditions, or May be eluted and developed. The treatment with the alkaline pH of the present invention may be carried out in a state in which rAT has just been produced (that is, in a culture solution or a culture supernatant), in a crude purification step in which they have been purified to some extent, or It may be carried out in the purification step after further purification, or in any of the steps. Preferably, the effect of the recovery of specific activity can be more exerted in the crude purification step or in the purification step than in the unpurified step. Virus inactivation process

 In the present invention, a known virus inactivation treatment can be performed, if necessary. For example, liquid heating (pastry) treatment, dry heating treatment, SD (detergent) treatment, etc. may be used alone or in combination. The treatment may be performed in the presence of a known stabilizer. Examples of the stabilizer include sugars (monosaccharides, disaccharides, sugar alcohols, etc.), amino acids or salts thereof, neutral salts, organic acids or salts thereof, and surfactants. The addition concentration may be appropriately selected within a known range. In addition, the treatment conditions may be any conditions under which the virus is substantially inactivated. Specifically, in the case of liquid heating, it takes about 10 minutes to 20 hours at 50 to 80 ° C, and in the case of dry heat treatment, it takes 10 minutes to 100 hours at 50 to 100 ° C. For example, in the case of SD processing, about 10 minutes to 10 hours at 10 to 50 ° C is exemplified. The stage at which the virus inactivation treatment is performed in the purification process can be arbitrarily selected. Properties of the purified product obtained

The purified product (rAT) prepared according to the present invention has a highly purified specific activity of 6.5 U / mg or more, and is free from other contaminants. The turbidity has been removed by the porous membrane treatment.

(Production method)

 In the present invention, by combining the above-mentioned rAT production method and purification method, the production amount and specific activity can be improved, and further highly purified rAT can be produced. In this production method, at least 1 g of rAT per 1 L of medium (at most about 2 g) and a specific activity of 6.5 U / mg or more are used as the production amount. realizable. Regarding the advanced purification, when the purity is 9.9% or more and the rAT is a solution of 100 to 20 O UZmL as the contaminant, the host-derived protein content is less than IngZmL, A DNA content of less than 10 ng Zml can be achieved. The rAT of the present invention is aimed at inhibiting blood coagulation and correcting hypercoagulability, which are indications for AT preparations, and specifically for treating thrombosis tendency and generalized intravascular coagulation syndrome (DIC). In addition to clinical use, it can be applied to known pharmaceutical uses such as, for example, preeclampsia, placental blood flow, anti-inflammation, sepsis, and severe sepsis. Example

 The advantages, features and possible scope of the present invention will be described in detail below with reference to exemplary embodiments, but the present invention is not limited by the following examples.

(Example of production method)

AT activity was measured using heparin factor activity (U) as an index. 1 U is the amount of AT that is present in Equivalent to. The activity was measured using a commercially available assay kit (Testteam AT-2 kit, Daiichi Kagaku) using a synthetic chromogenic substrate (trade name: S-228). The protein concentration as AT was determined by reversed phase chromatography (RPC) using HPLC (High Performance Liquid Chromatography). The reversed-phase chromatography was performed using a reversed-phase column under the following conditions. Reversed phase column is R 2/10 column (2.1 x 50 mm, Poros), HPLC is Beckman-Coulter HPLC (Beckman), mobile phase is 0.1% TFA. The linear gradient method of water-acetonitrile containing (trifluoroacetic acid) was used, and the absorbance at 220 nm was used for detection. As a control, plasma-derived AT (neutral trade name, Mitsubishi Electric Pharma Co., Ltd.) was used. The value obtained by dividing the activity value by the protein concentration was shown as a specific activity (U mg). The same applies hereinafter. Reference example

 The nucleotide sequence disclosed in Patent Document 11 was used as the cDNA of human AT. A plasmid was constructed that expresses human AT cDNA under the control of the hCMV-MIE promoter. This plasmid was transfused into CHO-K1 cells. Clones (strains A, B, and C) having a high AT-producing ability were selected from the obtained transformants and subjected to the following examples. Example 1

Viable cell density in two 3 L culture tanks 2.2 X 10 ⁵ ce 11 s

The AT-producing CH〇 cell A strain was inoculated so as to have a culture volume of 2 ml / ml and cultured. This one is of the culture tank culture period in p H in this is 7.0 to maintain 0 or more. 5 mo 1 / LN a HC 0 3 was adjusted by the addition of the other one is p H 6 . 6 or more Nourished. In both culture tanks, the culture temperature was controlled at 37 ° C, the dissolved oxygen concentration was 80 mmHg or more, the dissolved carbon dioxide concentration was 38 mmHg, and the stirring speed was 60 rpm. The medium used was a commercially available serum-free medium EXC e1132 medium (JRHBiosciences) with the following components added. Aranine 9 mg / L, asparagine monohydrate 85.2 2 mg / L, aspartic acid 13 mg / L, glutamate 75 mg ZL, proline 11.5 mg / L , Serine 10 m / L guanosine 7 mg / L, cytidine 7 mg / L, guanosine 7 mg / L, thymidine 0.24 mg / L, peridine 7 mg / L, penicillin 1 0,000 units ZL, strobtomycin 10 O mg / L were added.

 The solution was sampled once a day, the sampled culture was centrifuged, and AT activity was measured using the obtained culture supernatant.

 In both culture tanks, cultivation was started at the initial pH of 7.14. pH 6.

実施例 2 3 L容培養槽 4台に生細胞密度 1 . 5 X l 0 ⁵ c e l l s /m L、 培養液量 1 . 4 5 L となるよう に A T産生 C H O細 胞 B株を播種して培養を行った。培養 p Hをそれぞれ 6 . 6 、 6 . 8 、 7 . 0 および 7 . 2 に設定し、 培養期間中 p Hが設 定値 ± 0 . 0 7 になるよ う に 1 m o 1 / L塩酸または 0 . 5 m o 1 / L N a H C 〇 ₃を用いて p Hを調節した （第 3 図）。 いずれの培養槽も培養温度、 溶存酸素濃度、 溶存二酸化炭素 濃度、 撹拌速度は、 実施例 1 と同一条件で制御した。 培地は 市販の無血清培地である E x C e 1 1 3 0 2 G S培地 （ J R H B i o s c i e n c e s 社、 # 6 1 2 1 5 ) にぺニシリ ン 1 0 0 , 0 0 0単位 Z L、 ス ト レプトマイ シン 1 0 O m g Z L を添加して用いた。 この培地は遊離のグルタ ミ ンを含ま ず、 約 1 m Mのグルタミ ン酸を含んでいる。 以下特に記述し ない場合はこの培地を用いた。 Under the conditions cultivated at 6 or more, the pH dropped to pH 6.63 at 73 hours of culture, and then increased to pH 7.2 at 188 hours of culture (Fig. 1). Under the condition of culturing at pH 7.0 or higher, the pH decreased to pH 7.02 at 44 hours of culture, and increased to pH 7.6 at 144 hours of culture. The AT production concentration was more than 4 times higher when cultured at pH 6.6 or higher than when cultured at pH 7.0 or higher (Fig. 2). The results are summarized in Table 1. table 1

Example 2 An AT-producing CHO cell B strain was seeded and cultured in four 3 L culture tanks so that the viable cell density was 1.5 X 10 ⁵ cells / mL and the culture volume was 1.45 L. . The culture pH was set to 6.6, 6.8, 7.0 and 7.2, respectively, and 1 mo 1 / L hydrochloric acid or 0 mol / l was adjusted so that the pH reached the set value ± 0.07 during the culture period. . the 5 mo 1 / LN a HC 〇 ₃ was adjusted p H using (Figure 3). The culturing temperature, dissolved oxygen concentration, dissolved carbon dioxide concentration, and stirring speed of all the culture tanks were controlled under the same conditions as in Example 1. The culture medium was excerin 100,000 units ZL, Streptomycin, and E x Ce 11 32 GS medium (JRHBiosciences, # 61 2 15), a commercially available serum-free medium. 10 O mg ZL was used. This medium is free of free glutamin and contains about 1 mM glutamate. This medium was used unless otherwise specified.

 The feed medium was continuously added so that the glucose concentration was maintained at 0.7 g L during the culture period. The feed medium was modified from that disclosed in Non-Patent Document 19 and used. The modification of the feed medium was performed according to Non-Patent Document 20 and the like.

The culture was terminated when the viability was less than 90%. The culture was terminated at pH 7.0 and 7.2 at 21.6 hours, the culture at pH 6.6 was cultured at 240 hours, and the culture at pH 6.8 was cultured at 288 hours. finished. The AT production concentration reached the highest value when the culture was controlled at pH 6.8 (Fig. 4). AT production at pH 6.8 was 20% higher than at PH 7.0. Table 2 summarizes the results. M 2

 Ρ Η /-,, / song rte

 Production intensity

 "

 Ό · Ό ό 5

 Ό, Ο 丄 U

 7.0 (conventional) 100

 7.2 (Conventional) 800 Example 3

Inoculation of AT-producing CH〇 cell C strain was started in three 3 L culture tanks at a viable cell density of 2.6 X 10 ⁵ ce 11 s / mL and a culture volume of 1 L. . The same serum-free medium as used in Example 2 was used here. In addition, sodium dalmatinate was added so that the initial daltamic acid concentration in the culture solution was 1, 4 and 8 mM, respectively. The pH was set to 6.8 for all the culture tanks, and the culture temperature, dissolved oxygen concentration, dissolved carbon dioxide concentration, and stirring speed were operated under the same conditions as in Example 1.

 The liquid was sampled once a day, the sampled culture was centrifuged, and the glutamate concentration and AT activity were measured using the obtained culture supernatant.

A fed batch medium was added continuously so that the glucose concentration in the solution was maintained at 2 g L. The same feed medium as in Example 2 was used. By using the fed-batch medium, amino acids other than alanine did not die during the culture period. Further, the glutamate concentration in the feed medium was adjusted so that the glutamate concentration in the culture solution was maintained at 1 and 8 mM, respectively. Specifically, the amount of daltamic acid in the feed medium is contained in a ratio of 0.075 mol, 0.181 mol, and 0.265 mol per mol of dalcos, respectively. With such adjustment, the glutamate concentration in the culture solution could be maintained at 1, 4 and 8 ml (Fig. 5). Glutamine Acid concentration had no effect on cell growth. The AT production (AT activity X culture volume) was measured, and the AT production in the case of 1 mM glutamate is shown in FIG. 6 as 100%. At 4 mM and 8 mM daltamate, AT production was increased by 18% and 22%, respectively, compared to lmM daltamate. Table 3 summarizes the results. Table 3

実施例 4

Example 4

3 L in fermentor two viable cell ^{density. 9 X 1 0 0 ce 1} 1 s / m L, KoTsuta cultures were seeded AT producing CH_〇 cell C lines so that the culture volume 1 L. Each culture tank was operated at 35 and 37 ° C. Culture pH was set to 6.8 for all culture tanks, and the dissolved oxygen concentration, dissolved carbon dioxide concentration, and stirring speed were changed under the same conditions as in Example 1. The same serum-free medium as in Example 2 was used. Then, ten

 Then, sodium glutamate was added so that the initial concentration of glutamate in the three nutrient solutions was 7.5 mM. The feed medium was added continuously so that the concentration of Dar-3-s in the culture solution was maintained at 2 g ZL. The same feed medium as in Example 2 was used. The liquid was sampled once a day, the sampled culture was centrifuged, and the AT supernatant was measured using the obtained culture supernatant.

実施例 5 The results are shown in Figure 7 and Table 4. AT production (AT activity X culture volume) increased until the 33rd hour of culture (Fig. 7). According to the present invention, the production of AT is lower than that of the conventional method (culture temperature 37 Table 4

Example 5

 An experiment was performed according to Example 4. Using three 3 L culture vessels, each culture vessel was operated at 33 ° C, 35 ° C and 37 ° C. As a result of measuring the concentration of AT protein in the culture supernatant at 335 hours after culture by reverse phase chromatography and examining the specific activity, the specific activity was higher as the culture temperature was lower (Table 5). Table 5

(Part of Example on Purification Method)

AT activity was measured using heparin factor activity (U) as an index. 1 U corresponds to the amount of AT activity present in 1 mL of plasma of a healthy individual. The activity was measured using a commercially available measurement kit using a synthetic chromogenic substrate S-228, a test kit AT-2 kit, Daiichi Kagaku. The protein amount (weight, mg) as AT was measured by ELISA or reverse phase chromatography. The ELISA method uses anti-human AT polyclonal antibody derived from rabbits (DAKO), anti-human ATI gG derived from horseradish P-D-labeled hidge (affinity purified product, CEDALANE) and a coloring reagent. It was conducted. The activity value The value obtained by dividing by the amount of protein was expressed as specific activity (U / mg). The same shall apply hereinafter. Example 6

A transformant prepared by introducing an AT expression plasmid into CHO cells was cultured in a serum-free medium to produce rAT. The culture conditions were pH 7.2, 37 ° C, and 300 hours. The culture condensate was filtered through a 0.45 jm filter, and then treated with immobilized heparin [a heparin immobilized on hydrophilic pinyl polymer (trade name: Toyopearl)]. The rAT was purified. That is, it was washed with 0.15 M sodium chloride and eluted with 3 M sodium chloride. The eluate was applied to a hydroxyapatite column, and eluted with a 0.1 M diphosphate solution (PH8). The eluate was applied to an anion exchanger (DEAE-agarose, trade name DEAE-Sepharose) column, and the non-adsorbed fraction was collected. To this fraction, ammonium sulfate was added to a concentration of 1 M, and the mixture was applied to a hydrophobic chromatographic (Fuenrou hydrophilic vinyl polymer, trade name, Fuenrut Yopal) column. Collected. The fraction was subjected to ultrafiltration using an ultrafiltration membrane (trade name: Sartorius) having a molecular weight cut off of 30 kD, and the solvent was reduced to 0.52 containing 0.5% sodium chloride. The solution was replaced with a% sodium citrate solution (pH 7.75). Further, the pass fraction was collected by filtration with an ultrafiltration membrane (product name: Sarcon, Sartorius) having a molecular weight cut-off of 100 kD. Filtration was performed using a porous membrane with an average pore size of 15 nm (brand name: Blanova 15; Asahi Kasei). This is a porous hollow fiber flat Hitoshiana径1 5 ± 2 nm, a membrane area 0. 0 0 1 ~ 1. 0 m 2, hollow fiber inner diameter 3 3 0 ± 3 0 m, thickness 2 7 ± 3 xm, multi-layer structure of 150 layers or more This is a BMM module that is a modularized porous hollow fiber (BEMBERG MICROPOLAR MEMBRANE, BMM). This BMM module is integrated in a plastic container made of polycarbonate that can be sterilized by high-pressure steam using a polyurethane adhesive, and the module is filled with distilled water for injection. ing. The safety of various materials composing Branova has been confirmed by the method specified by the Japanese Pharmacopoeia (Non-Patent Document 21).

The rAT-containing solution concentrated to 100 to 20 OU Zml was adjusted to pH 7.75. Membrane filtration (dead-end filtration using air pressure) was performed at a filtration pressure of 0.5 kgf Z cm ² for 1 to 5 hours. After cooling, the solution was filtered through a 0.45 ^ m filter and dispensed into storage containers (purified product A). Example 7

 Example 6 was prepared in the same manner as in Example 6 except that the porous membrane treatment was not performed (purified product B). Example 8

 A cation exchanger treatment was performed instead of the anion exchanger of Example 6. That is, the rAT-containing solution was applied to a hide-open xyanotite column, and eluted with a 0.1 M dicalcium phosphate solution (ρΗ7.5). The eluate is adjusted to ρΗ6.5, applied to a cation exchanger (sulfopropyl pyrogalrose, trade name: SP-Sepharose) column, and eluted with 0.15 M sodium chloride solution. Collected. After that, it was concentrated by ultrafiltration (purified product C). Example 9

The purified product C of Example 8 was diluted with a buffer of ρρ7.75, and Example 10

 RAT was produced according to the culture method of Example 2 (pH 6.8), and rAT was purified using the obtained culture medium containing rAT according to the purification method of Example 6. Example 11

 In Example 10, dialysis was performed instead of the hydroxyapatite treatment during the purification step, and the solvent to be exchanged (ie, during anion exchange) was replaced with the dicarboxyl phosphate solution. Purification was carried out according to Example 10 except that the sodium phosphate solution was used, and instead of hydrophobic chromatography, immobilization was carried out again to immobilization (the same conditions were used for the first time). Then, a purified product of rAT was prepared. Example 1 2

 The rAT was produced by culturing according to Example 4 (culture temperature of 35 ° C), and rAT was purified using the obtained culture medium containing rAT according to the purification method of Example 6. did. Example 13

 Perform a liquid heat treatment of the culture solution at 60 ° C for 10 hours in the presence of 5% (wZv) citrate, 2M sodium chloride and 30% (w / V) sorbitol. Purification was performed in the same manner as in Example 11 except for the addition of the above, and a purified rAT product was prepared. Example 14

0.3% (wZv) tree N-butyl phosphate (TNBP) and 1% (w / V) polyoxygen Except for the addition of a 6-hour SD (degent) treatment at 30 ° C in the presence of Tylenesorbitan fatty acid ester (trade name: Tween 80), all procedures were the same as in Example 11. Purification was performed to prepare a purified product of rAT. Experimental example 1

 The properties of each purified product (purified products A, B, C) prepared in the examples were analyzed. Analytical methods are as follows: Purity was determined by gel filtration analysis (GPC), SDS-PAGE, Western blotting, and recovery were measured by reversed-phase HPLC (RPC). In terms of the percentage of impurities, the CHO-derived protein was measured by ELISA (Cygnus), the DNA was measured by Picogreen (Moleculara Probe), and the specific activity was measured by the heparin factor activity. The appearance was visually observed as a value obtained by dividing the activity by the yield. Table 6 shows the results. Table 6

実験例 2

Experimental example 2

The purified product C (specific activity: about 4.8 U / mg) was diluted in a buffer at pH 5.0 and 7.75, and left at room temperature for 4 hours. result Is shown in Table 7.

実験例 3

Experiment 3

 Fluctuations in the specific activity of rAT (purified product C) between pH 7.4 and 8.0 of the solution were examined. Table 8 shows the results. Table 8

実験例 4

Experimental example 4

When the low specific activity rAT was analyzed by analytical column, it was divided into three peaks. When the specific activities of the respective peaks were measured, they were 6.1 to 6.5, 3.3 to 4.4, and 0.2 to 0.6 (all in U / mg) in the order of elution. In this condition, the composition ratio of each peak (peaks I, Π, and m) is measured, and the rAT is treated with pH 8 or pH 6.5, and similarly analyzed with phenyl column. Then, the composition ratio of each peak was similarly measured. Table 9 shows the results. Table 9

ρ Η 8 処理ではピーク I の組成比が上昇し、 ピーク Π の組 成比は逆に低下した。 ピーク ΠΙは変化しなかった。 また p H 6 . 5処理ではピーク I の組成比は低下し、 ピーク Πおよび mの組成比が上昇した。 これらの結果から、 ピーク I および

In the ρ Η 8 treatment, the composition ratio of peak I increased, while the composition ratio of peak Π decreased. Peak ΠΙ did not change. In the case of pH 6.5 treatment, the composition ratio of peak I decreased, and the composition ratio of peaks Π and m increased. From these results, peak I and peak I

Π indicates that the specific activity reversibly changes depending on pH, peak Π does not depend on ρH, and that peak Π includes those inactivated by pH 6.5 treatment. Was done. Taking into account the specific activity, it was presumed that these three peaks would correspond to the active form of r AT, peak 半 to the semi-active form, and peak m to the inactive form. Experimental example 5

 When purified product A (specific activity: 7.3 U / mg) was dissolved in a buffer solution of pH 6.5 and left at room temperature for 4 hours, the specific activity was reduced to 6.0 UZmg. To this was added a sodium hydroxide solution, the pH was raised to 8.0, and the mixture was allowed to stand at room temperature for 4 hours. As a result, the specific activity was 7.0 UZmg. From these results, it was found that, among the prepared rATs, those having a reversible property (activity) depending on pH affect the specific activity of rAT in the present invention. Example 15

A culture experiment was performed according to Example 4. pH 6.8, Dal Evening The cells were cultured under conditions of 7 mM sodium phosphate and 35 ° C. At 360 hours in the culture, the amount of AT produced reached about 1.4 g / L of medium. Industrial applicability

 According to the production method of the present invention, when culturing animal cells capable of producing AT, it is possible to optimize the culture conditions, and to produce rAT with improved production and specific activity. it can. Therefore, by using the production method of the present invention, it is expected that rAT derived from animal cells can be stably provided to medical sites. Further, according to the purification method of the present invention, it is possible to highly purify low specific activity AT prepared by recombinant technology after restoring the specific activity to the original level. Therefore, if the technology of the present invention is applied, it is possible to stably provide rAT with superior properties as a pharmaceutical to a medical care setting. It was filed with priority claiming 3-1 1 2 2 3 6 and Japanese Patent Application No. 2 0 0 3-1 7 5 6 9 4.

Claims

The scope of the claims 1. Recombinant antithrombin (characterized by culturing animal cells capable of producing antithrombin, which has been transformed by genetic recombination technology, in one of the following modes: The production method of the following r AT):  1) Culture under a combination of pH 6.9 or less, glutamate 4 mM or more, and 35 ° C or less.  2) Culture under pH 6.9 or less, glutamate 4 mM or more, and at least 35 ° C or less under the combination of at least two of them. 3) Improve the production of rAT by culturing in the step of contacting with a culture solution having a pH of 6.9 or less.  4) Improve the production of rAT by performing fed-batch cultivation in the process of contacting with a culture solution containing 4 mM or more glutamate.  5) Improve r AT production and Z or specific activity by fed-batch cultivation at 35 ° C or lower. 6) Culture under conditions that achieve at least 1 g of rAT per 1 L of medium. 2. Culture AT-producing animal cells transformed using genetic recombination techniques under conditions combining pH 6.9 or less, glutamate 4 mM or more, and 35 ° C or less. 2. The production method according to claim 1, wherein the production method is performed. 3. Culturing the cells in a step of contacting them with a culture solution having a pH of 6.9 or less, culturing them in a step of contacting them with a culture solution containing 4 mM or more glutamate, and culturing at 35 ° C or less. The production method according to claim 2. 4. The culture mode is that cells in culture are suspended in culture medium, cells in culture are in serum-free medium, and cells in culture are in medium that does not contain mammalian proteins. 3. The production method according to claim 2, wherein the culture method is fed-batch culture. 5. At least two of the animal cells capable of producing AT, which have been transformed by genetic recombination technology and have a pH of 6.9 or less, glutamate of 4 mM or more, and 35 ° C or less, are obtained. The method according to claim 1, wherein the cells are cultured under combined conditions. 6. Production of rAT by culturing an animal cell capable of producing AT, which has been transformed using a genetic recombination technique, into a culture solution having a pH of 6.9 or less. 2. The production method according to claim 1, wherein the amount is increased. 7. Fed-patch culturing is performed by contacting an animal cell capable of producing AT, which has been transformed using a genetic recombination technique, with a culture medium containing 4 mM or more of glutamate. 2. The production method according to claim 1, wherein the production of AT is improved. 8. The production method according to claim 7, wherein the animal cell has a glutamine synthetase activity. 9. AT batch-producing animal cells transformed by genetic recombination technology that are capable of producing AT can be cultured in a batch at 35 t or less to produce rAT and produce Z or specific activity. Improve The production method according to claim 1, wherein 2. The production method according to claim 1, wherein the 10.rAT may be a mutant. 11.The culture conditions of 1H6.9 or less, glutamate 4 mM or more and 35 ° C or less must be met during the culture period, always or temporarily (or only for a certain period of time). The production method according to claim 1, wherein the production method is any of the following. 1 2. The mode of culturing in the step of contacting with a culture solution having a pH of 6.9 or less 6.7 to 6.9, or set only part of the culture period to pH 6.8 or lower, and in any case, always during the culture period 2. The production method according to claim 1, which has a pH of 6.6 or more. 13. The production method according to claim, wherein the glutamic acid may be in the form of a salt. The method of culturing in the step of contacting with a culture solution containing 14.4 mM or more of glutamate is as follows. (Always during the culture period) Glutamate is set to 3 to 5 mM. 2. The production method according to claim 1, wherein the concentration is set to 4 mM or more, or (during the cultivation period), dartamic acid is set to 6 to 8 mM. 15. A method for purifying rAT, which is performed by any one of the following embodiments: 1) Using a culture supernatant containing rAT, immobilized heparin treatment, solvent exchange of the treatment solution, anion exchanger treatment, removal of macromolecules, porosity with an average pore size of 1 to 100 nm Perform filtration using a membrane, and perform at least the steps after solvent exchange under conditions of pH 7.5 or more to purify rAT from the culture supernatant. 2) Using culture supernatant containing rAT, immobilized heparin treatment, solvent exchange of the treatment solution, anion exchanger treatment, removal of polymer, porosity with average pore diameter of 1 to 100 nm Purifying rAT from the culture supernatant by filtration using a permeable membrane,  3) performing all or a part of the purification step under conditions of pH 7.5 or more;  4) improving the specific activity of rAT by treating the rAT-containing solution with pH 7.5 or more;  5) removing the turbidity from the solution by filtering the rAT-containing solution using a porous membrane having an average pore diameter of 1 to L;  6) Purify rAT to a purity of at least 99%. 16. Using the culture supernatant containing rAT, immobilized heparin treatment, solvent exchange of the treatment solution, anion exchanger treatment, removal of the polymer, removal of the average pore diameter of 1 to 10 O nm 16. The method for purifying from a culture supernatant according to claim 15, wherein a filtration treatment using a porous membrane is performed, and at least steps after the solvent exchange are performed under conditions of pH 7.5 or more. 17. The method for purifying from a culture supernatant according to claim 16, wherein ultrafiltration is further performed before performing the porous membrane treatment. 18.Parin treatment to immobilization using culture supernatant containing rAT 16. The method according to claim 15, wherein the treatment solution is subjected to solvent exchange, anion-exchanger treatment, removal of a polymer, and filtration using a porous membrane having an average pore diameter of 1 to 100 nm. Purification method from pure. 19. The purification method according to claim 15, wherein all or a part of the purification step is performed under conditions of pH 7.5 or more. The purification method according to claim 15, wherein the specific activity of rAT is improved by treating the solution containing 20.rAT with a pH of 7.5 or more. 21. The purification method according to claim 15, wherein turbidity is removed from the rAT-containing solution by filtering the solution using a porous membrane having an average pore diameter of 1 to 10 O nm. . 22. The purification method according to claim 15, wherein r AT may be a mutant. 23. The purification method according to claim 15, further comprising performing a virus inactivating treatment. 24. A method for producing rAT comprising a step of producing rAT by culturing an animal cell capable of producing AT transformed with a gene recombination technique and a step of purifying the produced rAT. And a production method according to any one of claims 1 to 14 or a purification method according to any one of claims 15 to 23. 25. The production method according to any one of claims 1 to 14 is implemented. 24. The production method according to claim 24, wherein 26. The production method according to claim 24, wherein the purification method according to any one of claims 15 to 23 is performed. 27. The production method according to claim 24, wherein the production method according to any one of claims 1 to 14 is performed, and thereafter, the purification method according to any one of claims 15 to 23 is performed. 28. The production method according to claim 24, wherein rAT may be a mutant. 29. rAT with a purity of at least 99%, prepared from rAT produced by culturing animal cells capable of producing AT, which has been transformed using genetic recombination technology. The method according to claim 29, wherein the host-derived protein content is not more than Ing Zml and the DNA content is not more than 10 ng Zml when the solution has a concentration of 30.r AT of 100 to 200 UZml. R AT. 31. The rAT of claim 29, wherein the rAT may be a mutant. 32. A pharmaceutical composition comprising the rAT of claim 29 and a pharmacologically acceptable carrier. 33. The pharmaceutical composition according to claim 32, wherein rAT may be a mutant.