WO2004092218A1 - Procede d'elaboration d'antithrombine de recombinaison - Google Patents

Procede d'elaboration d'antithrombine de recombinaison Download PDF

Info

Publication number
WO2004092218A1
WO2004092218A1 PCT/JP2004/005454 JP2004005454W WO2004092218A1 WO 2004092218 A1 WO2004092218 A1 WO 2004092218A1 JP 2004005454 W JP2004005454 W JP 2004005454W WO 2004092218 A1 WO2004092218 A1 WO 2004092218A1
Authority
WO
WIPO (PCT)
Prior art keywords
rat
culture
treatment
production method
solution
Prior art date
Application number
PCT/JP2004/005454
Other languages
English (en)
Japanese (ja)
Inventor
Kenmi Miyano
Masaaki Hirose
Shinobu Mochizuki
Shinobu Kuwae
Toyoo Ohda
Hiroshi Tamashima
Kaoru Kobayashi
Masahide Kondou
Hideyuki Ohi
Original Assignee
Mitsubishi Pharma Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mitsubishi Pharma Corporation filed Critical Mitsubishi Pharma Corporation
Priority to JP2005505466A priority Critical patent/JP4651536B2/ja
Publication of WO2004092218A1 publication Critical patent/WO2004092218A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8128—Antithrombin III

Definitions

  • 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.
  • 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.
  • AT is composed of 432 amino acid residues, three disulfide bonds, and three or four N-type sugar chains.
  • 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.
  • 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.
  • animal cells 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.
  • 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.
  • 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.
  • Non-Patent Document 4 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).
  • 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).
  • basal media widely used in mammalian cell culture include Dulbecco's Modified Eag1e Medium (DME), Hams F—12, RPMI 1640, and Iscove's
  • DME Dulbecco's Modified Eag1e Medium
  • Hams F—12 Hams F—12
  • RPMI 1640 RPMI 1640
  • 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.
  • 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).
  • the concentration of glutamate in the medium used for culturing animal cells is usually less than or equal to I mM.
  • Patent Document 1 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.
  • Patent Documents 7 and 10 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.
  • Non-Patent Document 11 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.
  • Non-patent Document 13 a method using yeast as a host
  • Patent Document 2 a method using animal cells [especially Chinese / Pemster follicle (CHO) cells]
  • Patent Document 3 non-human mammals
  • Patent Document 3 a method using animal cells [especially Chinese / Pemster follicle (CHO) cells]
  • 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
  • Non-patent Documents 2, 14, and 15 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.
  • 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).
  • 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) .
  • 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. .
  • Non-Patent Document 16 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.
  • 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.
  • Patent Document 10 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 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 Japanese Patent Application Laid-Open No. 5-333992
  • 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
  • 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 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.
  • the present inventors conducted research using animal cells capable of producing AT transformed using genetic recombination technology.
  • p H 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.
  • 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.
  • 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.
  • immobilization is performed using a culture supernatant containing rAT.
  • 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.
  • FIG. 1 shows the time course of the culture pH. Culture pH 7.0 or more
  • 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 ⁇ 6. 6 (+), Culture pH 6.8 ( ⁇ ), Culture pH 7.0 ( ⁇ ), Culture pH 7.0
  • FIG. 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 ( ⁇ ), 4 mM ( ⁇ ), 8 mM (mouth).
  • Figure 6 shows the time course of AT production (heparin factor activator X culture volume).
  • 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 ( ⁇ ), 37 ° C (ten). BEST MODE FOR CARRYING OUT THE INVENTION
  • the method for producing rAT of the present invention originally has the following claims.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the AT gene 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.
  • 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.
  • animal cell a host known in gene recombination technology can be used.
  • 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.
  • those having glutamin synthetase activity can be used.
  • an expression system 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).
  • serum-free protein-free medium which may contain protein hydrolysates
  • sugars, amino acids those with the addition of only low-molecular-weight synthetic products (sugars, amino acids).
  • sucrose amino acids
  • 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.
  • 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 may be mentioned.
  • a pH of 6.9 or less preferably pH 6.8 or less
  • 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.
  • 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.
  • a culture solution containing 4 mM or more daltamate Preferably, 7 mM or more is exemplified.
  • Glutamic acid may be in the form of a salt such as a sodium salt and a calcium salt.
  • 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.
  • rAT 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.
  • 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.
  • 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.
  • 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 eg, capi (such as Aspergillus) and the like.
  • BHK cells, COS-7 cells, Vero cells may be auxotrophs, antibiotic susceptible strains, or certain mutants (strains deficient in certain genes).
  • transgenic non-human animals eg, geese, goats, sheep, etc.
  • animal cells are used as hosts. More preferably, CHO cells are used as a host.
  • 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).
  • the specific activity is lower than the original level (for example, about 6.5 to 7.5 UZ mg for purified plasma-derived products). .
  • 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.
  • 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.
  • change the solvent so that the pH is at least 7.5.
  • 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.
  • An example is a tris-phosphate buffer (PH 8).
  • the rAT-containing solution 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.
  • 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.
  • the rAT-containing solution When performing ultrafiltration and dialysis, the rAT-containing solution may be treated using a solvent to be exchanged as the external solution.
  • 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.
  • anion exchange groups include DEAE (getyl aminoethyl), QAE (quaternary aminoethyl), and Q (quaternary amino). (Monitor) and the like.
  • 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.
  • the mode of this treatment examples 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.
  • the contact condition may be such that the pH is 7.5 or more.
  • 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.
  • the non-adsorbed fraction is collected while maintaining the contact conditions.
  • the contact conditions in the case of the second method, set the contact conditions at an appropriate time
  • elute and collect rAT examples include a pH of about 5 to 7 and a salt concentration of about 0.01 to 0.5 M.
  • physiological saline 0.15 M sodium chloride
  • the polymer of AT is also conceptually included).
  • treatment with hydrophobic carrier, re-treatment of immobilized parin, etc. Is exemplified.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • parin is reprocessed for immobilization, it may be performed according to the first operation described above. Ultrafiltration
  • 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).
  • 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).
  • ultrafiltration membranes having different molecular weight cut-offs may be used in combination.
  • 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.
  • the material of the porous membrane used in the present invention is not particularly limited, but preferably includes regenerated cellulose.
  • the shape include a hollow fiber shape and a sheet shape, and a hollow fiber shape is preferable.
  • 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! ⁇ m, and the film is preferably of a multilayer structure.
  • the inner diameter is preferably 330 ⁇ 30 m.
  • the porous membrane 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 ⁇ 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 ⁇ 7 is at least 7.5. Specifically, it is preferable that ⁇ 7.5 to about 10, more preferably, ⁇ 7.5 to about 9.
  • the salt concentration is, for example, about 0.01 to 0.5 ⁇ .
  • the protein concentration is, for example, about 1 to 10 Omg ZmL.
  • pharmacologically acceptable additives eg, carriers, excipients, diluents, etc.
  • stabilizing agents e.g., 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.
  • sugars monosaccharides such as glucose and fructose, disaccharides such as suc
  • 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.
  • 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.
  • Cirulation type cross-flow filtration method
  • non-circulation type dead-end filtration method
  • rAT having a low specific activity can be recovered by treating it with a pH of 7.5 or more.
  • the pH condition is preferably about 7.5 to 10, more preferably about pH 7.5 to 9.
  • 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.
  • 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.
  • rAT 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.
  • 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.
  • a known virus inactivation treatment can be performed, if necessary.
  • 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.
  • 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.
  • the treatment conditions may be any conditions under which the virus is substantially inactivated.
  • 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.
  • the production amount and specific activity can be improved, and further highly purified rAT can be produced.
  • 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.
  • 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).
  • DIC generalized intravascular coagulation syndrome
  • it can be applied to known pharmaceutical uses such as, for example, preeclampsia, placental blood flow, anti-inflammation, sepsis, and severe sepsis.
  • 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 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.
  • the AT-producing CH ⁇ 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.
  • 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
  • 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.
  • the solution was sampled once a day, the sampled culture was centrifuged, and AT activity was measured using the obtained culture supernatant.
  • 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 ⁇ 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
  • Inoculation of AT-producing CH ⁇ 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.
  • 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.
  • amino acids other than alanine did not die during the culture period.
  • 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.
  • 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
  • Example 2 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.
  • 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.
  • 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.
  • anion exchanger DEAE-agarose, trade name DEAE-Sepharose
  • 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).
  • 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 ⁇ 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 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).
  • 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 ( ⁇ 7.5). The eluate is adjusted to ⁇ 6.5, 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).
  • a cation exchanger sulfopropyl pyrogalrose, trade name: SP-Sepharose
  • Example 10 The purified product C of Example 8 was diluted with a buffer of ⁇ 7.75, 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
  • 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 14 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
  • 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
  • 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.
  • 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
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Communicable Diseases (AREA)
  • Diabetes (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un procédé d'élaboration d'antithrombine (AT) de recombinaison reposant sur une technique de génie génétique, et un procédé de purification correspondant, en particulier pour de l'antithrombine de recombinaison (rAT) avec amélioration du volume de production et de l'activité spécifique, produite par culture de cellules animales qui peuvent elles-mêmes produire de l'AT transformée par technique de génie générique dans les conditions suivantes : acide glutamique en quantité supérieure ou égale à 4 nM, pH inférieur ou égal à 6,9, et température inférieure ou égale à 35° C. On peut aussi augmenter l'activité spécifique de rAT, établir une purification de degré élevé et éliminer la turbidité en soumettant un surnageant de culture qui renferme la rAT ou autre à un traitement à l'héparine immobilisée, un échange de solvant, un traitement d'échangeur d'anion, un retrait de polymère, une filtration par membrane poreuse et un traitement à un pH supérieur ou égal à 7,5.
PCT/JP2004/005454 2003-04-17 2004-04-16 Procede d'elaboration d'antithrombine de recombinaison WO2004092218A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2005505466A JP4651536B2 (ja) 2003-04-17 2004-04-16 組換えアンチトロンビンの製造方法

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2003-112236 2003-04-17
JP2003112236 2003-04-17
JP2003175694 2003-06-20
JP2003-175694 2003-06-20

Publications (1)

Publication Number Publication Date
WO2004092218A1 true WO2004092218A1 (fr) 2004-10-28

Family

ID=33302220

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2004/005454 WO2004092218A1 (fr) 2003-04-17 2004-04-16 Procede d'elaboration d'antithrombine de recombinaison

Country Status (2)

Country Link
JP (2) JP4651536B2 (fr)
WO (1) WO2004092218A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014100148A (ja) * 2007-12-27 2014-06-05 Baxter Internatl Inc 細胞培養プロセス

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09157184A (ja) * 1995-12-14 1997-06-17 Green Cross Corp:The アンチトロンビンｉｉｉ含有組成物およびその製造方法
JPH10215875A (ja) * 1997-02-06 1998-08-18 Mitsubishi Chem Corp 蛋白質の精製方法
SE9902388D0 (sv) * 1999-06-23 1999-06-23 Pharmacia & Upjohn Ab Method for purification of proteins
JP3595466B2 (ja) * 1999-07-23 2004-12-02 日本製薬株式会社 精製アンチトロンビン−ｉｉｉおよびその製法
SE0000178D0 (sv) * 2000-01-21 2000-01-21 Pharmacia & Upjohn Ab Protein purification I

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ALTAMIRANO C. ET AL.: "Improvement of CHO Cell Culture Medium Formulation: Simultaneous Substitution of Glucose and Glutamine", BIOTECHNOL., vol. 16, no. 1, 2000, pages 69 - 75, XP002201424 *
ROESSLER B. ET AL.: "Temperature: A simple parameter for process optimization in fed-batch cultures of recombinant Chinese hamster ovary cells", ENZYME AND MICROBIAL TECHNOLOGY, vol. 18, 1996, pages 423 - 427, XP002238255 *
ZETTLMEISSL G. ET AL.: "Characterization of Recombinant Human Antithrombin III Synthesized in Chinese Hamster Ovary Cells", J. BIOL. CHEM., vol. 264, no. 35, 1989, pages 21153 - 21159, XP002980960 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014100148A (ja) * 2007-12-27 2014-06-05 Baxter Internatl Inc 細胞培養プロセス

Also Published As

Publication number Publication date
JP5274529B2 (ja) 2013-08-28
JP2011042667A (ja) 2011-03-03
JP4651536B2 (ja) 2011-03-16
JPWO2004092218A1 (ja) 2006-09-07

Similar Documents

Publication Publication Date Title
KR100496356B1 (ko) 포유동물세포배양으로생산되는단백질의시알릴화조절방법
EP0363126B2 (fr) Méthode pour la purification de protéines dépendantes de la vitamine K
EP1707634A1 (fr) Procédé pour l'isolation de protéines recombinantes
JP5117486B2 (ja) 高純度可溶性トロンボモジュリンの製造方法
JP2024059641A (ja) 糖タンパク質の製造
JP4772905B2 (ja) アンチトロンビン組成物の製造方法
EP2523971B1 (fr) Structure thérapeutiques d'apyrase, agents de type apyrase et méthodes de production
JPH11225760A (ja) 機能性組換え組織因子の製造法
JP2013515474A (ja) 組換え体ｈ因子ならびにそのバリアントおよびコンジュゲート
RU2698671C2 (ru) КОЛИЧЕСТВЕННАЯ ОЦЕНКА НЕПРАВИЛЬНО СВЕРНУТОГО TNFR2:Fc
JP4157644B2 (ja) 高純度可溶性トロンボモジュリンの製造方法
JP5274529B2 (ja) 組換えアンチトロンビンおよびその製造方法
KR101877352B1 (ko) 세포 배양물 유래 알파1 프로테아제 억제제의 정제
NO322527B1 (no) Fremgangsmate for rensing av rekombinant Desmodus rotundus-spyttplasminogenaktivator (rDSPA α 1) fra et biologisk medium.
JP5086093B2 (ja) 組換え型ヒトｘｉｉｉ因子の精製
US6723536B2 (en) Method of producing and purifying angiostatin
CN116670270A (zh) 控制碱性磷酸酶制造期间的总唾液酸含量(tsac)的方法
KR100708403B1 (ko) 항혈전 항체의 정제방법
US20110230645A1 (en) Purification of factor v
CA2796452C (fr) Thrombomoduline soluble de purete elevee et sa methode de production

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2005505466

Country of ref document: JP

122 Ep: pct application non-entry in european phase