WO2017038986A1 - Procédé de production d'une protéine recombinante - Google Patents

Procédé de production d'une protéine recombinante Download PDF

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Publication number
WO2017038986A1
WO2017038986A1 PCT/JP2016/075848 JP2016075848W WO2017038986A1 WO 2017038986 A1 WO2017038986 A1 WO 2017038986A1 JP 2016075848 W JP2016075848 W JP 2016075848W WO 2017038986 A1 WO2017038986 A1 WO 2017038986A1
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tocilizumab
medium
culture
recombinant protein
protein
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PCT/JP2016/075848
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English (en)
Japanese (ja)
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和彦 野々村
克紀 内藤
二郎 広瀬
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持田製薬株式会社
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Publication of WO2017038986A1 publication Critical patent/WO2017038986A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a method for producing a recombinant protein, an animal cell culture medium, and a method for purifying the recombinant protein.
  • Tocilizumab is an antibody against human interleukin 6 (IL-6) receptor and is a therapeutic agent for diseases involving IL-6 such as rheumatoid arthritis. Tocilizumab is obtained by culturing animal cells that secrete tocilizumab and purifying the culture.
  • IL-6 human interleukin 6
  • Known media used for cultivating tocilizumab and the like include a medium containing an enzyme degradation product of fish meat (Patent Document 1), a medium in which soybean protein hydrolyzate and yeast extract are mixed at a specific ratio (Patent Document 2), and the like. It has been. Further, as a method for isolating tocilizumab from the culture solution, a method using affinity chromatography is known.
  • Patent Documents 3 and 4 A method for forcibly precipitating DNA contaminants to purify tocilizumab (Patent Documents 3 and 4) is known.
  • sugar chains present in the molecule of the recombinant protein have an important influence on the pharmacokinetics, activity or immunogenicity of the recombinant protein.
  • an antibody having an N-linked sugar chain at the asparagine residue of the Fc part of the heavy chain has a pharmacokinetics, safety, etc. depending on the structure of the N-linked sugar chain. Is known to fluctuate (Non-Patent Document 1).
  • An object of the present invention is to provide a method for producing a recombinant protein, including a method for culturing animal cells using a medium that improves the degree of galactosylation of sugar chains of the recombinant protein.
  • Another object of the present invention is to provide tocilizumab having a specific sugar chain constituent ratio expected to exhibit the same pharmacokinetic behavior as that of commercially available Actemra (registered trademark). Moreover, it makes it a subject to provide the new use of the soybean hydrolyzate which can be utilized as a culture medium component as another subject.
  • another object is to provide a method for easily purifying a recombinant protein secreted from animal cells and recovering it in a high yield.
  • the present invention is as follows.
  • ⁇ 1> A method for producing tocilizumab comprising culturing animal cells secreting tocilizumab in a medium containing soybean hydrolysate and isolating tocilizumab from a culture solution obtained by culturing animal cells .
  • Tocilizumab has 26% to 56% G (0), 17% to 37% G (1) -1, 0% to 19% G (1) -2, and 0% to 18% G.
  • the production method according to ⁇ 1> which is tocilizumab showing the sugar chain constituent ratio of (2).
  • ⁇ 3> The production method according to ⁇ 1> or ⁇ 2>, wherein the medium does not contain a protein hydrolyzate or yeast extract other than soybean hydrolysate.
  • Isolating tocilizumab from a culture solution obtained by culturing animal cells includes subjecting the culture solution to an affinity chromatography column of protein A, eluting tocilizumab adsorbed from the affinity chromatography column, and tocilizumab
  • ⁇ 5> The production method according to ⁇ 4>, wherein the salt concentration of the eluate is adjusted to 115 mM to 160 mM.
  • ⁇ 9> Applying the culture solution obtained by culturing animal cells secreting the recombinant protein to an affinity chromatography column of protein A, eluting the adsorbed recombinant protein from the affinity chromatography column, Adjusting the salt concentration of the eluate containing to 110 mM to 170 mM, and a method for purifying the recombinant protein.
  • the manufacturing method of recombinant protein including the method of culturing an animal cell using the culture medium which improves the galactosylation degree of the sugar chain of recombinant protein can be provided.
  • the soybean hydrolyzate which can be utilized as a culture medium component can provide the new use that the galactosylation degree of the sugar chain of the recombinant protein which an animal cell produces improves.
  • the production method in the present invention is characterized by culturing animal cells secreting tocilizumab in a medium containing soybean hydrolyzate (culturing step).
  • the method includes a culturing step and isolating tocilizumab from a culture solution obtained by culturing animal cells (isolation step).
  • the manufacturing method in this invention may include the other process besides the culture
  • the production method of the present invention can produce an effect that the degree of galactosylation of the sugar chain of the recombinant protein can be improved.
  • conditions such as the temperature of the culture solution when culturing animal cells, the pH of the culture solution, the dissolved oxygen concentration in the culture solution, the number of stirring of the culture solution, and the number of days of culture can be set according to the type of animal cell Good.
  • a temperature of 30 ° C. to 40 ° C. a temperature of 30 ° C. to 40 ° C., a pH of 6.5 to 7.7, a 10% to 80% dissolved oxygen concentration, and a stirring of 20 rpm to 150 rpm.
  • the culture may be performed for 10 to 28 days, but is not limited to this condition.
  • Examples of the culture temperature include 30 ° C to 40 ° C, preferably 32 ° C to 39 ° C, and more preferably 33 ° C to 38 ° C.
  • Examples of the pH during culture include a pH of 6.5 to 7.7, a pH of 6.7 to 7.5 is preferable, and a pH of 7.0 to 7.5 is more preferable.
  • Examples of the culture days include 10 to 28 days, preferably 11 to 21 days, and more preferably 13 to 15 days. It is preferable to inoculate the animal cell density to be 1 ⁇ 10 5 cells / mL to 5 ⁇ 10 5 cells / mL, and to inoculate to be 2 ⁇ 10 5 cells / mL to 4 ⁇ 10 5 cells / mL. More preferably.
  • the cell density temporarily increases and then decreases, but for example, when the animal cell density decreases to 1 ⁇ 10 6 cells / mL, the culture can be stopped. It is preferable to stop the culture when it is reduced to 2 ⁇ 10 6 cells / mL, and it is more preferable to stop the culture when it is reduced to 3 ⁇ 10 6 cells / mL.
  • Animal cell culture methods are classified into a batch culture method, a continuous culture method, and a fed-batch culture method.
  • any culture method may be used, but a fed-batch culture method or a continuous culture method is preferably used, and a fed-batch culture method is particularly preferably used.
  • the batch culture method is a culture method in which a small amount of a seed culture solution is added to a medium, and cells are grown without newly adding a medium or discharging the culture solution during the culture.
  • the continuous culture method is a culture method in which a small amount of a seed culture solution is added to a medium, and then the medium is continuously added during the culture and continuously discharged. Note that the continuous method includes perfusion culture.
  • the fed-batch culture method is also called a semi-batch culture method, and a medium is added continuously or sequentially during the culture. However, the continuous culture solution is discharged as in the continuous culture method. This culture method is not broken.
  • fed-batch medium The medium added in the fed-batch culture (hereinafter also referred to as “fed-batch medium”) need not be the same as the medium already used for the culture (hereinafter also referred to as “initial medium”). Different media may be added, or only specific components may be added.
  • the initial culture medium refers to a medium used in the first stage of cell culture.
  • the medium before adding the fed-batch medium can also be referred to as an initial medium.
  • a medium containing soybean hydrolyzate hereinafter also referred to as “medium hydrolyzate-containing medium” is used as the initial medium. It is preferable to use it.
  • a soybean hydrolyzate-containing medium is used as an initial medium, and at a temperature of 33 to 38 ° C.
  • a pH of 7.0 to 7.5 at least 3 days, preferably 5 days or more, more preferably Is cultured for 10 days or more, more preferably 13 to 15 days.
  • content of the soybean hydrolyzate in an initial culture medium is mentioned later, it should just be 0.5 g / L to 30 g / L, for example.
  • the culture solution is fractionated on the third, fifth, seventh, ninth, and eleventh days from the start of culture and an appropriate amount of Feed medium is added. Furthermore, when the glucose concentration in the collected culture solution is low, a culture method in which glucose (Roche etc.) is appropriately added is preferable.
  • a medium containing soybean hydrolyzate may be used as a medium to be added during the culture, or a medium not containing soybean hydrolyzate is used. May be.
  • a medium not containing soybean hydrolyzate it is more preferable to use a medium not containing soybean hydrolyzate as the medium added during the culture.
  • Tocilizumab has only to have the same amino acid sequence as the amino acid sequence (Gln1-Gly448) and L chain amino acid sequence (Asp1-Cys214) of Actemra (registered trademark), which are generally commercially available.
  • the amino acid sequence of Actemura H chain (Glu1-Gly448) and L chain amino acid sequence (Asp1-Cys214) are described in the sequence listing attached to International Publication No. 2005/090405.
  • the N-terminal residue of the H chain may be pyroglutamic acid (pGlu) instead of glutamic acid.
  • the C-terminal residue of the H chain may be up to 447 proline (Pro) instead of the 448 amino acid residue, or 449 amino acid residue in which lysine (Lys) is added to the 448th glycine (Gly). Also good.
  • Tocilizumab is represented by G (0), G (1) -1, G (1) -2 and G (2) shown in Table 1 below asparagine residue (Asn) at position 299 of the heavy chain. It is sufficient that at least any two of the sugar chains are bound per antibody molecule.
  • the sugar chain that binds to Asn at position 299 of the heavy chain of tocilizumab is cultivated in a medium containing soybean hydrolyzate, compared to when culturing animal cells in a medium not containing soybean hydrolyzate. Increase in the abundance of G (1) -1 or G (1) -2 in which one galactose is present, or the abundance in G (2) in which two galactoses are present To do.
  • sugar chains are known to affect metabolism, neutralization activity, effector function, etc. in the body. When the sugar chain composition ratios are different, it is the case where antibodies having the same amino acid sequence are used. However, the pharmacokinetic behavior may be different.
  • tocilizumab has 26% to 56% G (0), 17% to 37% G (1) from the viewpoint that it can exhibit pharmacokinetic behavior similar to that of Actemra that is generally commercially available. ) -1, 0% to 19% G (1) -2 and 0% to 18% G (2) sugar chain composition ratios are preferable.
  • the sugar chain constituent ratio of tocilizumab may be measured by Synapt G2 (Waters), PA800 plus (Beckman Coulter), or the like. Analysis by PA800 Plus revealed that the sugar chain composition ratio of commercially available Actemra was 41% G (0), 27% G (1) -1, 9% G (1) -2 and 8% G ( 2).
  • the animal cell is not particularly limited as long as it can secrete tocilizumab.
  • Specific examples include animal cells transformed with an expression vector containing a nucleic acid molecule encoding tocilizumab.
  • the production method and transformation method of an expression vector containing a nucleic acid molecule encoding tocilizumab may be in accordance with, for example, the methods described in International Publication No. 92/019759 and International Publication No. 2005/090405.
  • the animal cell is preferably a cell line, particularly a mammalian cell.
  • a CHO cell line established from Chinese hamster ovary tissue and its subspecies such as a CHO-K1 cell line (Kao F, Chasin L) , Puck TT.
  • a dihydrofolate reductase (DHFR) deficient strain such as the CHO-DG44 cell line (Urlab, G. and Chasin).
  • DHFR dihydrofolate reductase
  • CHO-DG44 cell line Urlab, G. and Chasin.
  • CHO cells ATCC No. CRL-9618
  • COS-1 cells ATCC No. CRL-1650
  • 293 cells ATCC No. CRL-1573
  • BHK-21 cells ATCC No. CCL- 10
  • CHO DG44 (Thermo) etc. can also be used as a commercial item.
  • the culture medium only needs to contain soybean hydrolyzate.
  • the soybean hydrolyzate may be any component obtained by hydrolyzing soybean, defatted soybean, soybean components or the like (hereinafter also referred to as “soybeans”) with an enzyme such as protease or an acid.
  • soy hydrolyzate is also referred to as soy protein hydrolysate, hydrolysed soy protein, etc., but in the present invention, the components obtained by hydrolyzing soy are generally referred to as soy hydrolysate. .
  • the soybean hydrolyzate may be solid, powder, or liquid, but is preferably powder or liquid from the viewpoint of ease of handling. Although it does not specifically limit as a soybean hydrolysate, Soy Hydrosylate UF solution (50X) (The product made by SAFC, catalog number # 58903C), HyClone HyQ Soy Hydrosylate Solution (made by GE Healthcare, SH30357.01), etc. Is mentioned.
  • the content of soybean hydrolyzate in the medium is not particularly limited, but from the viewpoint of improving the degree of galactosylation, it is preferably 0.5 g / L to 30 g / L with respect to 1 L of the medium.
  • soybean hydrolyzate in the initial culture medium is preferably 0.5 g / L to 30 g / L with respect to 1 L of the medium from the viewpoint of improving the degree of galactosylation, and is 1 g / L to 20 g / L. More preferably, it is more preferably 3 g / L to 10 g / L.
  • the medium preferably contains a complete synthetic medium (Chemical-defined medium) as a basal medium.
  • the complete synthetic medium is not particularly limited, but CD FortiCHO (registered trademark) Medium (GIBCO, catalog number A1148-01), CD OptiCHO (GIBCO, catalog number 12681-111), CD DG44 Medium ( GIBCO, catalog number 12610-010), HyCell CHO (GE Healthcare, catalog number SH30934.01), BalanCD (registered trademark) CHO GROWTH A (Irvine, catalog number 91128), S CHO-CD XP (registered trademark) (Irvine, catalog number 91120), JX G016 (Irvine) and the like.
  • the complete synthetic medium one type of completely synthetic medium may be used, or two or more types of fully synthetic media may be used in combination.
  • the type of Feed medium is not particularly limited.
  • JX Feed001 (Irvine catalog number JX F001), JX Feed002 (Irvine catalog number JX F002), JX Feed003 (Irvine company) Catalog Number JX F003), JX Feed004 (Irvine Catalog Number JX F004), EffectiveFeed (registered trademark) A + AGT (registered trademark) Supplement (GIBCO, catalog number A2502304), EffectiveFeed (registered trademark) B + mGTp registered trademark (GIBCO, catalog number A2503004), EfficientFeed (registered trademark) C + AGT (registered trademark) S supplement (GIBCO, catalog number A2503104), Cell Boost 1 (R05.2) Supplement (GE Healthcare, catalog number SH30584.02), Cell Boost 2 (R15.4) Supplement (GE Health01, catalog number GE Health01).
  • Feed media may be used alone or in combination of two or more.
  • the medium preferably contains no protein hydrolyzate or yeast extract other than soy hydrolyzate from the viewpoint of reducing unknown components and reducing the risk of different protein structures to be produced. It is more preferable not to contain protein hydrolyzate and yeast extract other than the product.
  • the medium may contain other components in addition to the soybean hydrolysate. Examples of other components include amino acids, vitamins, lipids, saccharides, and trace metals.
  • the isolation step is for isolating tocilizumab from a culture solution obtained by culturing animal cells, and subjecting the culture solution in which animal cells are cultured to a protein A affinity chromatography column. (Adsorption step), eluting tocilizumab adsorbed from the affinity chromatography column (elution step), and adjusting the salt concentration of the eluate containing tocilizumab to a molar concentration of 110 mM to 170 mM (adjustment step). You may go out. Furthermore, the isolation step may include other steps. Here, the unit is M (molar) and mM is 10 ⁇ 3 mol / L. Since the isolation step in the present invention does not cause precipitation including DNA contaminants, the precipitate removal step is unnecessary, and tocilizumab can be easily purified and recovered in high yield.
  • the adsorption step is not limited as long as the culture solution is supplied to the protein A affinity chromatography column.
  • the supply conditions such as an impurity.
  • the adsorption step may include measuring the tocilizumab concentration in the culture solution before subjecting the culture solution to the affinity chromatography column.
  • the tocilizumab concentration during the culture may be adjusted to be, for example, a 50 mg / mL column to a 100 mg / mL column.
  • the range of the concentration of the culture solution supplied to the column may be set according to the type of column, and is not particularly limited.
  • the tocilizumab concentration in the culture solution can be determined by measuring the human IgG concentration by ELISA, surface plasmon resonance, Cedex Bio HT (Roche) or the like.
  • the adsorption step may include washing the unadsorbed protein after subjecting the culture solution to an affinity chromatography column.
  • the washing solution used when washing the non-adsorbed protein can be appropriately set depending on the type of column.
  • the cleaning solution may be set to a condition of 1 mol / L NaCl.
  • the adsorption step may include washing impurities that are weakly bound to the column.
  • the cleaning solution used for cleaning impurities that are weakly bound to the column can be appropriately set depending on the type of the column.
  • the elution step is not limited as long as it elutes tocilizumab adsorbed from the affinity chromatography column.
  • the type of eluate, collection of the eluted fraction, conditions for washing impurities that are weakly bound to the column, etc. Can be set.
  • an elution solution having a pH of 4.5 or less can be used. The adsorbed tocilizumab can be eluted.
  • the eluate used in the elution step for example, 100 mM Gly-HCl buffer (pH 3.5), 100 mM citrate buffer (pH 3.5), and phosphate citrate buffer (MacIlvaine) can be used.
  • the eluate may further contain a denaturant, an organic solvent, and the like.
  • the adjustment step may be any one that adjusts the salt concentration of the eluate containing tocilizumab to 110 mM to 170 mM.
  • the salt concentration of the eluate is preferably adjusted to 115 mM to 160 mM, more preferably 120 mM to 150 mM.
  • the salt concentration is higher than 170 mM, the content of impurities (host cell-derived protein, DNA, lipopolypolysaccharide, aggregates, etc.) after the treatment is performed after treatment by ion exchange chromatography in the steps after the adjustment step. May increase.
  • the salt concentration of the eluate can be adjusted using a buffer such as 1.5 mol / L Tris-HCl (pH 8.0), HEPES, MOPS, Tricine, HEPPSO, and TAPS.
  • a buffer such as 1.5 mol / L Tris-HCl (pH 8.0), HEPES, MOPS, Tricine, HEPPSO, and TAPS.
  • pH of the eluate after adjustment is near neutrality.
  • the pH of the effluent after adjustment is preferably in the range of pH 6 to pH 8, more preferably in the range of pH 7 to pH 8, still more preferably in the range of pH 7.5 to pH 8, from the viewpoint of preventing denaturation in addition to isoelectric point and impurity removal. It is mentioned to adjust to.
  • the type of protein A affinity chromatography column that can be used in the isolation step is not limited, and commercially available products can be used.
  • commercially available products include MabSelectSuRe LX, (GE Healthcare Japan, catalog number 17-5474-02), and Prosep Ultra Plus (Merck Millipore, catalog number 17518822).
  • the production method in the present invention for example, inactivates viruses by heating treatment (virus inactivation step), and removes impurities by ion exchange chromatography (impurity removal step). I), removal of impurities by hydrophobic chromatography, ion exchange chromatography, gel filtration, mixed mode chromatography (impurity removal step II), removal of virus, filter sterilization (virus removal sterilization step), solvent replacement (Concentration step) may be further included (concentration step).
  • viruses inactivation step inactivates viruses by heating treatment
  • impurity removal step II removal of impurities by hydrophobic chromatography, ion exchange chromatography, gel filtration, mixed mode chromatography
  • concentration step concentration step
  • the animal cell culture medium for producing tocilizumab in the present invention contains a soybean hydrolyzate and a completely synthetic medium.
  • the medium for producing tocilizumab in the present invention may contain other components in addition to the soybean hydrolyzate and the completely synthetic medium.
  • tocilizumab means 26% to 56% G (0), 17% to 37% G (1) -1, 0% to 19% G (1) -2 and 0%. It means tocilizumab showing a sugar chain composition ratio of G (2) of% to 18%.
  • the medium for producing tocilizumab in the present invention has the effect of producing tocilizumab having a specific sugar chain constituent ratio expected to show excellent pharmacokinetic behavior, similar to commercially available Actemra.
  • the medium for tocilizumab production only needs to contain soybean hydrolyzate and completely synthetic medium, but from the viewpoint of reducing risks such as reducing unknown components and reducing the structure of the protein produced, other than soy hydrolyzate It is preferable not to contain protein hydrolyzate or yeast extract, and it is more preferable not to contain protein hydrolyzate other than soybean hydrolyzate and yeast extract.
  • the medium for producing tocilizumab may contain other components such as amino acids, vitamins, lipids, sugars, and trace metals. As for other matters, the matters described above in the section of the method for producing tocilizumab can be applied.
  • the recombinant protein is a protein produced by cells transformed by applying recombinant DNA technology.
  • the recombinant protein may be any recombinant protein expressed in animal cells and is preferably a recombinant protein secreted from animal cells, but the type of the recombinant protein is not particularly limited.
  • a recombinant protein that can be used as a pharmaceutical is preferable.
  • the recombinant protein is preferably a recombinant protein having a sugar chain.
  • the antibody which has a sugar chain may be sufficient and the recombinant protein which contains the antibody which has a sugar chain in a molecule
  • numerator may be sufficient.
  • Recombinant proteins that can be used as pharmaceuticals include, for example, darbopoietin beta, trastuzumab, rituximab, palivizumab, infliximab, basiliximab, gemtuzumab ozogamicin, bevacizumab, ibritumomab tiuxetane, tocilizumab, adalimumab celizumab, , Panitumumab, ustekinumab, golimumab, canakinumab, denosumab, mogamulizumab, ofatumumab, pertuzumab, trastuzumab emtansin, brentuximab It is not limited to these.
  • Recombinant proteins include trastuzumab, rituximab, palizumab, infliximab, infliximab, basiliximab, gemtuzumab ozogamicin, bevacizumab, ibritumomab tiuxetane, tocilizumab, adalimumab, cetuximab, ranimumab, parizumab Denosumab, mogum lizumab, ofatumumab, pertuzumab, trastuzumab emtansine, brentuximab vedotin, natalizumab, nivolumab, alemtuzumab, secukinumab, ramcilmab, ipilimumab, etanercept, abatacept, abatacept, abatacept, abatacept Denosumab Ri preferred.
  • sugar chain examples include an O-linked sugar chain and an N-linked sugar chain.
  • the recombinant protein preferably has at least an N-linked sugar chain.
  • the sugar chain is more preferably an N-linked sugar chain that binds to the Fc portion of the antibody molecule.
  • the animal cell culture medium in the present invention contains soybean hydrolyzate.
  • the animal cell culture medium may contain a completely synthetic medium or other components.
  • the animal cell culture medium according to the present invention contains the soy hydrolyzate, and thus has a new effect of improving the degree of galactosylation of the sugar chain of the recombinant protein produced by the animal cells.
  • improving the degree of galactosylation means that the proportion of galactose expressed at the non-reducing end of the N-linked sugar chain bound to the asparagine residue in the recombinant protein is improved.
  • galactose is more cultivated when animal cells are cultured in a medium containing soybean hydrolysate than when animal cells are cultured in a medium not containing soybean hydrolyzate.
  • the ratio of G (1) -1 or G (1) -2 in which one galactose is present, or the ratio of G (2) in which two galactoses are present, compared to the ratio of G (0) in which no galactose exists means that any one of them improves.
  • the presence or absence of improvement in the degree of galactosylation is confirmed using a medium containing no protein hydrolyzate other than soybean hydrolyzate and yeast extract.
  • the improvement of the degree of galactosylation can be achieved by, for example, G (1) -1 when the total amount of G (0), G (1) -1, G (1) -2 and G (2) is 100%.
  • G (1) -2 and G (2) may be 30% to 70% (hereinafter also referred to as “total existence ratio”). % To 60%, preferably 45% to 55%, more preferably 50% to 55%.
  • the effect of controlling the degree of galactosylation of sugar chains can be achieved by using the animal cell culture medium of the present invention.
  • the animal cell culture medium has the effect that the degree of galactosylation can be improved without being affected by changes in the culture environment, regardless of the capacity of the culture apparatus used for culture, as detailed in the Examples. Can play.
  • the animal cell culture medium of the present invention only needs to contain a soybean hydrolyzate and a completely synthetic medium, but from the viewpoint of reducing the risk of reducing unknown components and reducing the structure of the protein produced, soy hydrolyzate. It is preferable not to contain a protein hydrolyzate or yeast extract other than the degradation product, and it is more preferable not to contain a protein hydrolyzate other than the soybean hydrolyzate and a yeast extract.
  • the animal cell culture medium of the present invention can be suitably used for culturing the above-mentioned recombinant protein.
  • the animal cell is not particularly limited as long as it can express a recombinant protein. Specific examples include animal cells transformed with an expression vector containing a nucleic acid molecule encoding a recombinant protein. Examples of other components contained in the animal cell culture medium include amino acids, vitamins, lipids, saccharides, and trace metals.
  • the matters described above in the section of the method for producing tocilizumab can be applied as they are.
  • the sugar chain of the recombinant protein produced by animal cells can be measured by the above-mentioned Synapt G2 (Waters), PA800 plus (Beckman Coulter).
  • the matters described above in the section of the method for producing tocilizumab can be applied mutatis mutandis.
  • the method for purifying recombinant protein in the present invention comprises subjecting a culture solution of animal cells to a protein A affinity chromatography column (adsorption step) and eluting the adsorbed recombinant protein from the affinity chromatography column (elution step). And adjusting the salt concentration of the eluate containing the recombinant protein to 110 mM to 170 mM (adjustment step). Furthermore, the purification method may include other steps.
  • the purification method in the present invention can produce an effect that a recombinant protein secreted from animal cells can be easily purified and recovered in a high yield.
  • the recombinant protein purification method of the present invention can be suitably used for the purification of the above-mentioned recombinant protein.
  • the salt concentration of the eluate is preferably adjusted to 115 mM to 160 mM, more preferably 120 mM to 150 mM, from the viewpoints of isoelectric point, impurity removal ability, recovery rate, and stability.
  • the animal cell is preferably an animal cell transformed with an expression vector containing a nucleic acid molecule encoding a recombinant protein.
  • a method for producing an expression vector containing a nucleic acid molecule encoding a recombinant protein and a method for transformation may be in accordance with, for example, the method described in WO 92/019759.
  • the method for purifying a recombinant protein of the present invention is a method for producing a recombinant protein, further comprising culturing animal cells secreting the recombinant protein in a medium containing soybean hydrolyzate (culturing step). May be.
  • the recombinant protein culture method is a culture process, an adsorption process, an elution process, a pH adjustment process, and other processes, except for culturing animal cells that express the above-mentioned recombinant protein containing tocilizumab or other recombinant proteins.
  • the above-mentioned matters in the section of the method for producing tocilizumab can be applied mutatis mutandis.
  • Example 1 30 mL culture
  • FuGENE (registered trademark) 6 Transfection Reagent Promega was used as a transformant preparation reagent.
  • the prepared tocilizumab-producing CHO cells were cultured in JX G016 (available from Irvine) medium containing 8 mM L-Glutamine, Penicillin, and streptomycin.
  • soybean hydrolyzate has a new effect of improving the degree of sugar chain galactosylation.
  • Example 2 10 L culture
  • transformation was carried out according to the methods described in WO 92/09759 and WO 2005/090405.
  • Three tocilizumab-producing CHO cell lines (A, B, C) prepared using FuGENE (registered trademark) 6 Transfection Reagent (Promega) were cultured in a 15 L bioreactor as a body preparation reagent.
  • the cells were cultured in a JX G016 (Irvine) medium containing 2% Soy Hydrosylate UF solution (50X) (manufactured by SAFC, catalog number # 58903C), 8 mM L-Glutamine, Penicillin, and streptomycin.
  • Three tocilizumab-producing CHO cells were seeded in each medium to 3 ⁇ 10 5 cells / mL, and stirred in a total volume of 15 L culture tank (manufactured by ABLE) with an initial culture volume of 7.5 L (37 ° C., 50% DO, 25-30 rpm, pH 7.05-7.35).
  • 30 mL of the culture solution was collected, and then Feed medium (JX Feed003) was added.
  • the Feed medium was added so that the concentration after adding the Feed medium was 6% of the total culture volume.
  • the collected culture solution was used for analysis of various components, and when the glucose concentration in the culture solution was lowered, glucose was appropriately added.
  • the cell solution on the 14th day of culture was used for purification.
  • Tocilizumab contained in the culture supernatant was purified by the method described in detail in Example 3, and the sugar chain analysis of the purified protein was performed. The results are shown in Table 3.
  • the sugar chain of the purified protein was analyzed by PA800 Plus (Beckman Coulter). When analyzed by PA800 Plus, the sugar chain composition ratio of commercially available Actemra was 41% G (0), 27% G (1) -1, 9% G (1) -2 and 8%. G (2).
  • Example 3 Purification method Tocilizumab was purified by the following procedure using the culture supernatant obtained by the method described in detail in Example 2. The purification was performed at room temperature using AKTA Avant 150 (GE Healthcare Japan). The resulting culture supernatant was expressed using Millistak + Pod (Merk Millipore, catalog # MD0HC027H1 or MD0HC054H1) as a pre-filter, Express SHF OpticapXL3 capsule (Merck Millipore, catalog # KGEPA03HNT cartridge or PE # 03CAP cartridge) The culture solution was clarified using CCS-020-E1H) as a sterilizing filter.
  • the obtained culture supernatant was preliminarily equilibrated with 1 ⁇ D-PBS (Sigma-Aldrich, catalog # D1408).
  • Protein A affinity chromatography column (MabSelect SuRe LX (GE Healthcare Japan, catalog # 17-) 5474-02) column ( ⁇ 4.4 cm ⁇ 19.4 cm)).
  • tocilizumab was eluted with 100 mmol / L Gly-HCl buffer (pH 3.5) and pooled.
  • a 1.5 mol / L Tris-HCl buffer solution (pH 8.0) was added to the obtained eluate pool to adjust the salt concentration to 120 mmol / L.
  • the virus was inactivated by heat treatment at 40 ° C. for 4 hours. After the heat treatment, the pool of virus inactivation solution was previously equilibrated with 120 mmol / L Tris-HCl buffer (pH 8.0), Q-Sepharose FF (GE Healthcare Japan, catalog # 17-0510-01) The sample was applied to a column ( ⁇ 4.4 cm ⁇ 19.2 cm), and non-adsorbed components were washed with the same buffer and pooled as an intermediate purified solution (tocilizumab fraction).
  • the solvent of the pool of this intermediate purified solution was replaced with 20 mmol / L sodium phosphate buffer (pH 6.5), and ceramic hydroxyapatite Type II, 80 ⁇ m (Bio-Rad Laboratories, catalog # 157-8000) column ( ⁇ 5 cm ⁇ 21
  • the non-adsorbed components were washed with the same buffer, and tocilizumab was eluted with a 20 mmol / L phosphate buffer solution (pH 6.5) containing 260 mmol / L NaCl and pooled as an impurity removal solution.
  • the resulting pool of the impurity removal solution was concentrated using a hydrosalt zaltocon slice cassette membrane (Saltorius Stedim, catalog # 3051445901E-SG) having a molecular weight cut off of 30 kDa, and then a physiological saline solution (Otsuka Pharmaceutical Factory Co., Ltd., The solvent was replaced with catalog # 1760), and sterilized filtration was performed using a sterilcup GP filter (Merck Millipore, catalog # SCGPU05RE) to obtain a final purified solution.
  • the monomer purity and the remaining DNA amount of the final purified solution obtained by this purification method were measured. The results are shown in Table 4.
  • the monomer purity was measured using SEC (size exclusion chromatography) (Tosoh Corporation column).
  • the amount of remaining DNA was measured according to the protocol using HCDNA (Certal CHO Detection Kit) (manufactured by QIAGEN).

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Abstract

L'invention concerne un procédé de production de tocilizumab, lequel procédé consiste : à mettre en culture des cellules animales qui sécrètent du tocilizumab dans un milieu contenant un hydrolysat de soja, ce qui améliore le degré de galactosylation des chaînes de sucre d'une protéine recombinante ; et à isoler le tocilizumab du bouillon de culture obtenu par la mise en culture des cellules animales.
PCT/JP2016/075848 2015-09-03 2016-09-02 Procédé de production d'une protéine recombinante WO2017038986A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011530606A (ja) * 2008-08-14 2011-12-22 メルク・シャープ・エンド・ドーム・コーポレイション プロテインａアフィニティークロマトグラフィーを用いる抗体の精製方法
JP2013507979A (ja) * 2009-10-26 2013-03-07 エフ．ホフマン−ラ ロシュ アーゲー グリコシル化された免疫グロブリンの調製方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011530606A (ja) * 2008-08-14 2011-12-22 メルク・シャープ・エンド・ドーム・コーポレイション プロテインａアフィニティークロマトグラフィーを用いる抗体の精製方法
JP2013507979A (ja) * 2009-10-26 2013-03-07 エフ．ホフマン−ラ ロシュ アーゲー グリコシル化された免疫グロブリンの調製方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GUPTA A.J. ET AL.: "Influence of protein and carbohydrate contents of soy protein hydrolysates on cell density and IgG production in animal cell cultures.", BIOTECHNOL. PROG., vol. 31, no. 5, June 2015 (2015-06-01), pages 1396 - 1405 *

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