TW201723174A - Method for manufacturing recombinant proteins - Google Patents

Abstract

Provided is a method for producing tocilizumab, the method including: the culture of animal cells that secrete tocilizumab in a medium containing soybean hydrolysate, which improves the degree of galactosylation of the sugar chains of a recombinant protein; and the isolation of tocilizumab from the culture broth obtained by culturing the animal cells.

Description

Method for producing recombinant protein

The present invention relates to a method for producing a recombinant protein, a medium for animal cell culture, and a method for purifying a recombinant protein.

Many recombinant proteins such as antibodies or physiologically active substances have been produced by genetic recombination and developed and sold as pharmaceuticals.

The recombinant protein differs depending on the type, culture method or purification method, and is not easy to manufacture. A series of tocilizumabs are described as an example of a method for producing a recombinant protein.

Tocilizumab is an antibody against human interleukin 6 (IL-6) receptor, and is a therapeutic drug for diseases involving IL-6 such as rheumatoid arthritis.

Totuzumab is obtained by culturing animal cells secreting tocilizumab and purifying the culture solution. As a culture medium for the culture of tocilizumab or the like, a medium containing an enzyme decomposition product of fish meat (Patent Document 1) or a medium obtained by mixing a soybean protein hydrolyzate and a yeast extract at a specific ratio is known (Patent Document 2) )Wait. Further, as a method of separating the tocilizumab from the culture solution, a method using affinity chromatography is known. With The molar concentration of the acidic eluate used to dissolve the tombizumab bound to the affinity chromatography column, and the molar concentration of the neutral solution obtained by neutralizing the eluate are known. A method of purifying a beaduzumab by adjusting a specific range to forcibly precipitate DNA inclusions (Patent Documents 3 and 4).

Furthermore, it is known that a sugar chain present in a molecule of a recombinant protein has an important influence on the in vivo dynamics, activity or immunogenicity of the recombinant protein. For example, it is known that among recombinant proteins, an antibody having an N-binding type sugar chain of an aspartic acid residue in the Fc portion of the heavy chain depends on the composition of the N-binding type sugar chain, and the in vivo dynamics Security, etc. may change (Non-Patent Document 1).

[Previous Technical Literature] [Patent Document]

[Patent Document 1] International Publication No. 99/063058

[Patent Document 2] Japanese Patent Laid-Open Publication No. 2002-520014

[Patent Document 3] International Publication No. 2002/072615

[Patent Document 4] Japanese Patent Laid-Open Publication No. 2009-62380

[Non-patent literature]

[Non-Patent Document 1] Eon-Duval, A. et. Al. Biotechnol. Prog. 2012, 28, 608-622

On the other hand, it has been known that a degree of galactosylation of a sugar chain added to a recombinant protein by a culture condition is different.

Further, if the recombinant protein is purified by forcibly precipitating the DNA inclusions, the content of the finally obtained recombinant protein is reduced.

As such, further technological development is required for the production method of recombinant proteins.

An object of the present invention is to provide a method for producing a recombinant protein comprising a method of culturing an animal cell using a medium for increasing the degree of galactosylation of a sugar chain of a recombinant protein.

In addition, another object of the present invention is to provide a tombuzumab which exhibits a specific in vivo dynamic behavior comparable to that of the commercially available Actemra (registered trademark) and which exhibits a specific sugar chain composition ratio.

Further, another object of the present invention is to provide a new use of a soybean hydrolyzate which can be used as a medium component.

Further, another object of the present invention is to provide a method for easily purifying a recombinant protein secreted by an animal cell and recovering it at a high yield.

The present invention is as follows.

<1> A method for producing tocilizumab, which comprises culturing animal cells secreting tocilizumab in a medium containing soybean hydrolysate Raise; and remove the tocilizumab from the culture solution obtained from the culture of the animal cells.

<2> The production method according to <1>, wherein the tocilizumab exhibits 26% to 56% G(0), 17% to 37% G(1)-1, 0% to 19%G. The sugar chains of (1)-2 and 0%~18%G(2) constitute a ratio of tombuzumab.

<3> The production method according to <1> or <2> wherein the culture medium does not contain a protein hydrolyzate or a yeast extract other than the soybean hydrolyzate.

The production method according to any one of <1> to <3> wherein the single-extraction of the tocilizumab from the culture solution obtained by culturing the animal cell comprises: supplying the culture solution to the protein A The affinity chromatography column; the self-affinity chromatography column dissolves the adsorbed tocilizumab; and the salt concentration of the solution containing the tocilizumab is adjusted to 110 mM to 170 mM.

<5> The production method according to <4>, wherein the salt concentration of the eluate is adjusted to 115 mM to 160 mM.

<6> A culture medium for animal cell culture, which is used for production to show 26% to 56% G(0), 17% to 37% G(1)-1, 0% to 19% G(1)-2 And the 0%~18% G(2) sugar chain constitutes a mixture of the soybean hydrolysate and the complete synthetic medium.

<7> The medium according to <6>, which does not contain a protein hydrolyzate or a yeast extract other than the soybean hydrolyzate.

<8> A culture medium for animal cell culture which is used for enhancing the degree of galactosylation of a sugar chain of a recombinant protein produced by animal cells, which comprises a soybean hydrolyzate.

<9> A method for purifying a recombinant protein, comprising: supplying a culture solution obtained by culturing an animal cell secreting a recombinant protein to an affinity chromatography column of protein A; and dissolving the adsorbed recombinant protein from a affinity chromatography column And adjusting the salt concentration of the resolving solution containing the recombinant protein to 110 mM to 170 mM.

<10> The purification method according to <9>, wherein the salt concentration of the elution solution is adjusted to 115 mM to 160 mM.

According to the present invention, any of the following effects is provided.

According to the present invention, a method for producing a recombinant protein comprising a method of culturing an animal cell using a medium for increasing the degree of galactosylation of a sugar chain of a recombinant protein can be provided.

Further, according to the present invention, it is possible to provide tolipizumab which is expected to exhibit the same in vivo dynamic behavior as the commercially available Actemra and which exhibits a specific sugar chain composition ratio.

Further, according to the present invention, it is possible to provide a new use of a degree of galactosylation capable of enhancing a sugar chain of a recombinant protein produced by an animal cell by using a soybean hydrolyzate as a medium component.

Further, according to the present invention, a method of easily purifying a recombinant protein secreted by an animal cell and recovering it in a high yield can be provided.

Hereinafter, the present invention will be described in detail.

<Method for producing tocilizumab>

The manufacturing method of the present invention is characterized in that animal cells secreting tocilizumab are cultured in a medium containing soybean hydrolyzate (culture step). Preferably, the method comprises the step of culturing, and separately removing the tocilizumab (single-off step) from the culture solution obtained by culturing the animal cells. Further, the manufacturing method in the present invention may include other steps in addition to the culturing step and the detaching step.

The production method of the present invention can exhibit an effect of increasing the degree of galactosylation of a sugar chain of a recombinant protein.

(cultivation step)

In the culturing step, conditions such as the temperature of the culture solution, the pH of the culture solution, the concentration of dissolved oxygen in the culture solution, the number of stirring of the culture solution, and the number of culture days in the case of culturing the animal cells are set according to the type of animal cells. can. For example, in the case of using CHO cells as animal cells secreting tocilizumab, as long as it is at a temperature of 30 ° C to 40 ° C, a pH of 6.5 to 7.7, a concentration of 10% to 80% dissolved oxygen, and a concentration of 20 rpm to 150 rpm. The culture may be carried out for 10 to 28 days under stirring, but is not limited thereto.

The culture temperature is, for example, 30 ° C to 40 ° C, preferably 32 ° C to 39 ° C, more preferably 33 ° C to 38 ° C.

The pH at the time of culture may, for example, be a pH of 6.5 to 7.7, preferably a pH of 6.7 to 7.5, more preferably a pH of 7.0 to 7.5. value.

The number of culture days may, for example, be 10 to 28 days, preferably 11 to 21 days, and more preferably 13 to 15 days.

Preferably, the animal cell density is 1×10 ⁵ cells/mL to 5×10 ⁵ cells/mL, and more preferably 2×10 ⁵ cells/mL to 4×10 ⁵ cells. Inoculate in the form of /mL. Further, by, for example, culturing for 10 to 28 days, the cell density is temporarily increased, and then decreased, and when the animal cell density is, for example, 1 × 10 ⁶ cells/mL, the culture can be stopped, preferably lowered. The culture was stopped at 2 × 10 ⁶ cells/mL, and more preferably, the culture was stopped when the cells were reduced to 3 × 10 ⁶ cells/mL.

The culture method of the animal cells can be classified into a batch culture, a continuous culture, and a fed-batch culture. In the present invention, any culture method may be used, and a feed batch culture method or a continuous culture method is preferably used, and a feed batch culture method is particularly preferred.

The batch culture method is a culture method in which a small amount of the culture solution is added to the culture medium, and a new medium is not added to the culture, and the culture medium is discharged.

The continuous culture method is a culture method in which a small amount of the culture medium is added to the culture medium, the medium is continuously added to the culture, and the medium is continuously discharged. In addition, in the continuous method, a perfusion culture is also included.

The feed batch culture method is also called semi-batch culture, which is added to the culture medium continuously or sequentially in the culture, but does not perform continuous culture liquid discharge as continuous culture method. Cultivator law. The medium to be added at the time of the feed batch culture (hereinafter also referred to as "feed batch medium") need not be the same medium as the medium used in the previous culture (hereinafter, also referred to as "initial medium"). Different media can be added, or only specific ingredients can be added.

In the present invention, the initial medium refers to the medium used in the initial stage of cell culture. However, in the case where the feed batch medium is divided into a plurality of additions, the medium before the respective feed batch medium is added may also be referred to as an initial medium.

In the present invention, from the viewpoint of being able to increase the degree of galactosylation of the sugar chain of the recombinant protein, it is preferred to use a medium containing soybean hydrolyzate (hereinafter also referred to as "soybean hydrolyzate-containing medium") as an initial Medium.

In the present invention, the soybean-containing hydrolyzate medium is used as the initial medium, and cultured at a temperature of 33 ° C to 38 ° C and a pH of 7.0 to 7.5 for at least 3 days, preferably 5 days or more, more preferably 10 times. More than the day, and then the best for the 13th to the 15th. In addition, the content of the soybean hydrolyzate in the initial culture medium may be, for example, 0.5 g/L to 30 g/L as described later. In addition, it is preferred to inoculate the cell density of the animal cells to a cell density of 1 × 10 ⁵ cells/mL to 5 × 10 ⁵ cells/mL, and culture using the initial medium. Further, in the present invention, it is preferred to carry out a feed batch culture in which a culture solution is separated and obtained, for example, on the third, fifth, seventh, ninth, and eleventh days from the start of the culture, and an appropriate amount of the feed medium is added. In addition, it is preferable to add a method of culturing glucose (Roche, etc.) when the concentration of glucose in the culture solution obtained by the separation is lowered.

In the present invention, in the case of using the feed batch culture method or the continuous culture method, as the medium to be added during the culture, a soybean-containing hydrolyzate-containing medium or a medium containing no soy hydrolyzate may be used. Further, from the viewpoint of the osmotic pressure in the culture solution, it is more preferable to use a medium containing no soy hydrolyzate as the medium to be added during the cultivation.

The tocilizumab is an amino acid sequence having the same amino acid sequence as the amino acid sequence of the H chain of the commonly available Actemra (registered trademark) (Gln1-Gly448) and the amino acid sequence of the L chain (Asp1-Cys214). Just fine. Further, the amino acid sequence of the H chain of Actemra (Glu1-Gly448) and the amino acid sequence of the L chain (Asp1-Cys214) are described in the Sequence Listing attached to International Publication No. 2005/090405.

Further, the N-terminal residue of the H chain may also be pyroglutamic acid (pGlu) instead of glutamic acid. The C-terminal residue of the H chain may also be substituted for 448 amino acid residues to the 447 proline (Pro), or may be attached with lysine (Lys) at the 448th glycine (Gly). 449 amino acid residues.

Further, the tocilizumab is at least two of the sugar chains represented by G(0), G(1)-1, G(1)-2, and G(2) shown in Table 1 below. Each of the antibody molecules binds to the aspartate residue (Asn) at position 299 of the heavy chain. Regarding the sugar chain of Asn 299 which binds to the heavy chain of tocilizumab, by culturing animal cells in a medium containing soybean hydrolysate, compared to when the animal cells are cultured in a medium containing no soy hydrolyzate, There is one galactose of G(1)-1 or G(1)-2 The amount of presence or the presence of at least one of G(2) in which two galactose is present increases.

It is known that a sugar chain affects metabolism, neutralizing activity, and effector function in the body. In the case where the sugar chain composition ratio is different, even in the case of using an antibody having the same amino acid sequence, there is an in vivo body. Dynamic behavior is different.

As a preferred aspect of the present invention, tolizumab preferably exhibits 26% to 56% G(0), 17 from the viewpoint of exhibiting the same in vivo dynamic behavior as the commercially available Actemra. The sugar chain composition ratio of %~37%G(1)-1, 0%~19%G(1)-2 and 0%~18%G(2). In addition, the better system shows 30%~50%G(0), 19%~35%G(1)-1, 1%~17%G(1)-2 and 0%~16%G(2) The sugar chain constitutes a ratio.

In addition, the sugar chain composition ratio of tocilizumab is only by Synapt G2 (Waters), PA800 Plus (Beckman Coulter). It can be measured.

After analysis by PA800 Plus, the commercially available Actemra has a sugar chain composition ratio of 41% G(0), 27% G(1)-1, 9% G(1)-2, and 8% G(2).

As the animal cell, there is no particular limitation as long as it can secrete tocilizumab. Specifically, an animal cell obtained by transforming a expression vector comprising a nucleic acid molecule encoding a tocilizumab can be mentioned. Here, the method of producing the expression vector containing the nucleic acid molecule encoding the tocilizumab and the method of transformation can be carried out, for example, according to the methods described in, for example, International Publication No. 92/019759 and International Publication No. 2005/090405.

Further, as the animal cell, a strained cell is preferred, and a mammalian cell is particularly preferred, and for example, a CHO cell strain and a subspecies thereof, such as a CHO-K1 cell strain (Kao F), which are established by Chinese hamster ovarian tissue, may be mentioned. , Chasin L, Puck TT. Proc Natl Acad Sci USA. 1969 Dec; 64(4): 1284-91), specifically, dihydrofolate-reducing enzyme (DHFR)-deficient strain, such as CHO-DG44 cell line (Urlaub, G. and Chasin, LA: Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)) and the like. Specific examples thereof include CHO cells (ATCC No. CRL-9618), COS-1 cells (ATCC No. CRL-1650), 293 cells (ATCC No. CRL-1573), and BHK-21 cells (ATCC No. CCL). -10). Further, as a commercial item, CHO DG44 (Thermo Co., Ltd.) or the like can also be used.

The medium is only required to contain soy hydrolyzate.

Soy hydrolyzate as long as it is soybean, defatted soybean or soybean (hereinafter, also referred to as "soybean, etc.") A component obtained by hydrolysis with an enzyme such as a protease or an acid. Further, the soybean hydrolyzate is also called soy protein hydrolyzate, hydrolyzed soy protein, etc. In the present invention, the components obtained by hydrolyzing soybeans and the like are collectively referred to as soybean hydrolysate.

The soybean hydrolyzate may be a solid, a powder or a liquid, and is preferably a powder or a liquid from the viewpoint of ease of handling.

The soybean hydrolyzate is not particularly limited, and examples thereof include Soy Hydrolysate UF solution (50X) (manufactured by SAFC, catalog number #58903C), HyClone HyQ Soy Hydrolysate Solution (manufactured by GE Healthcare, SH30357.01), and the like.

The content of the soybean hydrolyzate in the medium is not particularly limited, and is preferably from 0.5 g/L to 30 g/L, more preferably 1 g/% from the viewpoint of increasing the degree of galactosylation. L to 20 g/L, more preferably from 3 g/L to 10 g/L. The content of the soybean hydrolyzate in the initial medium is preferably from 0.5 g/L to 30 g/L, more preferably from 1 g/L to 20 g/% from the viewpoint of increasing the degree of galactosylation. L, preferably from 3 g/L to 10 g/L.

Further, the medium preferably contains a complete synthetic medium (Chemical-defined medium) as a basic medium. The fully synthetic medium is not particularly limited, and examples thereof include CD FortiCHO (registered trademark) Medium (GIBCO, catalog number A11483-01), CD OptiCHO (GIBCO, catalog number 12681-011), and CD DG44 Medium (GIBCO). Company, Catalog No. 12610-010), HyCell CHO (GE Healthcare, Catalog Number SH30934.01), BalanCD (registered trademark) CHO GROWTH A (Irvine Corporation, catalog number 91128), S CHO-CD XP (registered trademark) (Irvine Corporation, catalog number 91120), JX G016 (Irvine Corporation), and the like.

As the completely synthetic medium, one type of complete synthesis medium may be used, or two or more types of complete synthesis medium may be used in combination.

When the feed medium is used, the type of the feed medium is not particularly limited, and examples thereof include JX Feed 001 (Irvine catalog number JX F001), JX Feed 002 (Irvine catalog number JX F002), and JX Feed 003 (Irvine catalog number). JX F003), JX Feed004 (Irvine catalog number JX F004), EfficientFeed (registered trademark) A+AGT (registered trademark) Supplement (GIBCO, catalog number A2502304), EfficientFeed (registered trademark) B+AGT (registered trademark) ) Supplement (GIBCO, Catalog No. A2503004), EfficientFeed (registered trademark) C+AGT (registered trademark) Supplement (GIBCO, Catalog No. A2503104), Cell Boost 1 (R05.2) Supplement (GE Healthcare, type) Record number SH30584.02), Cell Boost 2 (R15.4) Supplement (GE Healthcare, catalog number SH30596.01), Cell Boost 3 (JM3.5) Supplement (GE Healthcare, catalog number SH30825.01) , Cell Boost 4 (PS307) Supplement (GE Healthcare, catalog number SH30857.01), Cell Boost 5 (CN-F) Supplement (GE Healthcare, catalogue No. SH30865.01), Cell Boost 6 (CN-T) Supplement (GE Healthcare, catalog number SH30866.01), BalanCD (registered trademark) CHO FEED 1 (Irvine, catalog number 91127-1L), BalanCD ( Registered trademark) CHO FEED 2 (Irvine Corporation, catalog number 91129-1 L), Balan CD (registered trademark) CHO FEED 3 (Irvine Corporation, catalog number 99471-1L).

The feed medium may be used alone or in combination of two or more.

Further, from the viewpoint of reducing the risk of the unknown component and reducing the structural difference of the produced protein, it is preferred that the protein hydrolyzate or yeast extract other than the soybean hydrolyzate is not contained, and more preferably, the soybean hydrolyzate is not contained. Protein hydrolysates and yeast extracts other than those.

The medium may contain other ingredients in addition to the soybean hydrolysate. Examples of other components include amino acids, vitamins, lipids, sugars, and trace metals.

(single step)

In the manufacturing method of the present invention, the isolation step is performed by separately extracting the tocilizumab from the culture solution obtained by culturing the animal cells, which may comprise supplying the culture solution obtained by culturing the animal cells to the affinity layer of the protein A. a column (adsorption step); eluting the adsorbed tocilizumab from the affinity chromatography column (dissolution step); and adjusting the salt concentration of the solution containing the tocilizumab to a molar concentration of 110 mM to 170 mM ( Adjustment steps). Furthermore, the single step can also include other steps. Here, the unit is M (mole concentration) and the mM is 10 ^-3 mol/L.

The single isolation step in the present invention does not require a precipitate removal step for preventing the precipitation of the DNA inclusions from being formed, and it can be visualized that the tocilizumab can be easily purified and carried out in a high yield. The effect of recycling.

The adsorption step may be any step of supplying the culture solution to the affinity chromatography column of the protein A, and it is preferable to set the equilibrium solution of the column, the cleaning solution of the column, the flow rate condition of the culture solution, and the like, depending on the type of the column, and the like. The adsorption conditions of the culture solution, the washing conditions of the culture solution, the supply conditions of impurities, and the like may be used.

For example, in the case of using MabSelect SuRe LX (GE Healthcare Japan, catalog number 17-5474-02) as the affinity chromatography column of protein A, the affinity chromatography column for supplying the culture solution to protein A In the case, it is sufficient to set the conditions such as pH 5 to 8.

The adsorption step may also include determining the concentration of tocilizumab in the culture solution before supplying the culture solution to the affinity chromatography column.

In the adsorption step, when the culture solution is supplied to the affinity chromatography column, it may be adjusted so that the concentration of the tocilizumab in the culture becomes, for example, 50 mg/mL column to 100 mg/mL column. In addition, the range of the concentration of the culture solution supplied to the column is not particularly limited as long as it is set depending on the type of the column.

The concentration of toltuzumab in the culture solution can be determined by measuring the concentration of human IgG by an ELISA method, a surface plasmon resonance method, or Cedex Bio HT (Roche).

The adsorption step can also be carried out by supplying the culture solution to the affinity chromatography. After the column, the unadsorbed protein of the column is washed. The washing solution used for washing the unadsorbed protein of the column can be appropriately set depending on the type of the column. For example, the washing solution may be set to a condition of 1 mol/L NaCl.

Further, the adsorption step may include, in addition to this, washing the impurities which are weakly bonded to the column. The cleaning solution used for the impurities which are weakly bonded to the column can be appropriately set depending on the type of the column.

The dissolving step may be carried out by dissolving the adsorbed bead monoclonal antibody from the affinity chromatography column, and depending on the type of the column, the type of the eluent, the taking of the eluted portion, and the impurity which is weakly bonded to the column are washed. Net conditions, etc.

For example, in the case of using MabSelect SuRe LX (GE Healthcare Japan, catalog number 17-5474-02) as the affinity chromatography column for protein A, self-affinity can be achieved by using an elution solution having a pH of 4.5 or less. The chromatography column dissolves out the adsorbed tocilizumab.

For the dissolution solution used in the dissolution step, for example, 100 mM Gly-HCl buffer (pH 3.5), 100 mM citrate buffer (pH 3.5), and citrate buffer (MacIlvaine) can be used. Further, the dissolving liquid may further contain a denaturant or an organic solvent or the like.

The adjustment step may be such that the salt concentration of the eluate containing tocilizumab is adjusted to 110 mM to 170 mM. Further, the salt concentration of the eluate is preferably adjusted to 115 mM to 160 mM, more preferably 120 mM to 150 mM.

In the case where the salt concentration is made higher than 170 mM, if it is When the treatment by ion exchange chromatography or the like is carried out in the step after the entire step, there is a fear that the content of impurities (proteins derived from host cells, DNA, lipopolysaccharide, aggregates, etc.) increases after the treatment.

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, TAPS, or the like.

Further, the pH of the adjusted eluate is preferably near neutral. The pH of the adjusted eluate is adjusted from the viewpoint of isoelectric point and prevention of denaturation in addition to removal of impurities, and is preferably adjusted to a range of pH 6 to pH 8, more preferably pH 7 to pH 8. It is preferably in the range of pH 7.5 to pH 8.

As the affinity chromatography column of the protein A which can be used in the isolation step, the type and the like are not limited, and a commercially available product can be used.

As a commercial item, for example, MabSelect SuRe LX (GE Healthcare Japan, catalog number 17-5474-02), Prosep Ultra Plus (Merck Millipore, catalog number 175118822) can be used.

The manufacturing method in the present invention may further comprise, for example, deactivation of the virus by warming treatment (virus deactivation step) in addition to the culturing step and the detachment step; removing 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, and filtration sterilization (virus removal sterilization step); replacement of solvent (solvent) The replacement step); the purified liquid obtained by the purification is concentrated (concentration step).

These steps can be appropriately set according to the usual conditions in accordance with conditions such as stability, recovery rate, and removal efficiency of impurities.

<An animal cell medium for producing tocilizumab>

The animal cell culture medium (hereinafter also referred to as "the medium for the production of tocilizumab") which produces the tocilizumab in the present invention contains a soybean hydrolyzate and a completely synthetic medium. Further, the culture medium for the production of tocilizumab in the present invention may contain other components in addition to the soybean hydrolyzate and the completely synthetic medium.

In addition, in the medium for the production of tocilizumab, the tocilizumab line means 26% to 56% G(0), 17% to 37% G(1)-1, 0% to 19% G(1) The glycochain of -2 and 0% to 18% G(2) constitutes a ratio of tocilizumab.

In the culture medium for the production of the tocilizumab of the present invention, it is possible to produce an effect of betozumab which exhibits a desired intrinsic dynamic behavior similar to that of the commercially available Actemra and which exhibits a specific sugar chain composition ratio.

The culture medium for the production of tocilizumab may contain a soybean hydrolyzate and a completely synthetic medium, and is preferably free from soybean hydrolyzate from the viewpoint of reducing the risk of unknown components and reducing the structure of the produced protein. The protein hydrolysate or the yeast extract is more preferably a protein hydrolysate other than the soybean hydrolysate and a yeast extract.

Further, the culture medium for the production of tocilizumab may contain other components such as amino acids, vitamins, lipids, sugars, and trace metals.

For other matters, the paragraphs in the manufacturing method of tocilizumab can be applied. The foregoing matters.

<recombinant protein>

In the culture medium for animal cell culture and the method for producing a recombinant protein, which will be described later, the recombinant protein refers to a protein produced by a cell transformed by a recombinant DNA technique. The recombinant protein is preferably a recombinant protein expressed by an animal cell, and is preferably a recombinant protein secreted by an animal cell, and the type of the recombinant protein is not particularly limited. Preferably, for example, a recombinant protein as a pharmaceutical can be used. Further, as the recombinant protein, a recombinant protein having a sugar chain is preferred. Further, it may be an antibody having a sugar chain or a recombinant protein containing an antibody having a sugar chain in a molecule.

Examples of the recombinant protein that can be used as a pharmaceutical product include darbepoetin, trastuzumab, rituximab, palivizumab, and inflix. Infliximab, basiliximab, gemtuzumab ozogamicin, bevacizumab, ibritumomab tiuxetan, Tocilizumab, adalimumab, cetuximab, ranibizumab, omalizumab, eculizumab, panitux Antibiotic (panitumumab), ustekinumab, golimumab, canakinumab, denosumab, mogmurili Monoclonal (mogamulizumab), ofofumumab (ofatumumab), pertuzumab, trastuzumab emtansine, brentuximab vedotin, that Natalizumab, nivolumab, alemtuzumab, secukinumab, ramucirumab, ipilimumab, yilimumab It is etanercept, abatacept, and ablibercept, but is not limited to this.

Further, as the recombinant protein, trastuzumab, rituximab, palivizumab, infliximab, basiliximab, gemtuzumab, oxazomethoxazole, and beryllium are preferred. Valamycin, temimuzumab, tocilizumab, adalimumab, cetuximab, ranibizumab, omalizumab, eculizumab, panitumumab, excellent Tekmonizumab, golimumab, carnazumab, denosumab, mogmurimumab, orfarizumab, pertuzumab, trastuzumab, star Topazide, natalizumab, navumab, alemtuzumab, sulmenab, remollozumab, ipilimumab, etanercept, abatacept, and a Percyps, more preferably trastuzumab, tocilizumab, adalimumab, utekumab, golimumab, and denosumab.

Examples of the sugar chain referred to herein include an O-binding type sugar chain or an N-binding type sugar chain, and the recombinant protein preferably has at least an N-binding type sugar chain. As the sugar chain, if it is an antibody, it is more preferably an N-binding type sugar chain which binds to the Fc portion of the antibody molecule.

<Animal cell culture medium>

The culture medium for animal cell culture of the present invention contains a soybean hydrolyzate. Further, the medium for animal cell culture may also contain a completely synthetic medium or other ingredients.

The culture medium for animal cell culture of the present invention is capable of exhibiting a novel effect of increasing the degree of galactosylation of a sugar chain of a recombinant protein produced by animal cells by containing a soybean hydrolyzate. Here, increasing the degree of galactosylation means that the proportion of galactose present in the non-reducing end of the N-binding type sugar chain bound to the aspartate residue in the recombinant protein is increased. It is preferred to mean that the proportion of the sugar chain having a galactose at the non-reducing end is increased among the sugar chains constituting the N-binding type sugar chain. Specifically, it means that the animal cell line cultured in the medium containing the soybean hydrolysate is as shown in Table 1 above, compared to the absence of galactose, when the animal cells are cultured in the medium containing no soy hydrolysate. In the ratio of the presence of G(0), there is a ratio of the presence of G(1)-1 or G(1)-2 of one galactose, or the presence ratio of G(2) in which two galactose are present. Any one of them is promoted. The presence or absence of the degree of galactosylation was confirmed using a medium containing no protein hydrolyzate other than soybean hydrolyzate and yeast extract.

In addition, the increase in the degree of galactosylation may be, for example, when the total amount of G(0), G(1)-1, G(1)-2, and G(2) is determined to be 100%, G(1)- 1. The ratio of the total amount of G(1)-2 and G(2) (hereinafter, also referred to as the "total ratio") is 30% to 70%, and the total ratio is preferably 40% to 60%. , better system becomes 45%~55 %, then the best is 50% to 55%.

Thus, by using the animal cell culture medium of the present invention, the effect of controlling the degree of galactosylation of the sugar chain can be exhibited. Further, the animal cell culture medium can be modified as described in the examples, irrespective of the capacity of the culture apparatus for performing the culture, and the degree of galactosylation can be enhanced without being affected by the change of the culture environment. The effect.

The culture medium for animal cell culture of the present invention may contain a soybean hydrolyzate and a completely synthetic medium, and is preferably free from soybean hydrolysate from the viewpoint of reducing the risk of unknown components and reducing the structure of the produced protein. The protein hydrolysate or the yeast extract other than the protein hydrolysate and the yeast extract other than the soybean hydrolyzate are more preferred.

The culture medium for animal cells of the present invention can be suitably used for the culture of the above recombinant protein.

The animal cell is not particularly limited as long as it can express a recombinant protein. Specifically, an animal cell transformed with a expression vector comprising a nucleic acid molecule encoding a recombinant protein can be cited.

Examples of other components contained in the culture medium for animal cell culture include amino acids, vitamins, lipids, sugars, and trace metals.

The above-mentioned items in the paragraph of the method for producing tocilizumab can be applied as appropriate to the soybean hydrolyzate contained in the culture medium for animal cell culture. In addition, the sugar chain of the recombinant protein produced by the animal cell can be measured by the aforementioned Synapt G2 (Waters), PA800 Plus (Beckman Coulter).

For other matters, it can be applied to the paragraph of the manufacturing method of tombuzumab The foregoing matters.

<Method for Purifying Recombinant Protein>

The method for purifying the recombinant protein of the present invention is as long as it comprises a culture chromatography column for supplying the cultured animal cells to the affinity chromatography column of the protein A (adsorption step); and the adsorbed recombinant protein is eluted from the affinity chromatography column (dissolution) Step); and adjusting the salt concentration of the recombination solution containing the recombinant protein to 110 mM to 170 mM (adjustment step). Furthermore, the purification method may also comprise other steps.

The purification method of the present invention can exhibit the effect of easily purifying the recombinant protein secreted by animal cells and recovering it in a high yield.

The method for purifying the recombinant protein of the present invention can be suitably used for the purification of the above recombinant protein.

The adjustment step is preferably such that the salt concentration of the eluate is adjusted to 115 mM to 160 mM, and 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 comprising a nucleic acid molecule encoding the recombinant protein.

The method for producing a expression vector comprising a nucleic acid molecule encoding a recombinant protein and the method for transformation can be carried out, for example, according to the method described in International Publication No. 92/019759.

The method for purifying the recombinant protein of the present invention may further comprise: the animal cell secreting the recombinant protein is contained in the soybean hydrolyzate. A method for producing a recombinant protein which is cultured (culture step) in a medium. The method for culturing the recombinant protein is applicable to the culture step, the adsorption step, the dissolution step, the pH adjustment step, and other steps, in addition to the culture of the animal cell which expresses the recombinant protein comprising tocilizumab or other recombinant protein. The foregoing matters in the paragraph of the method for producing benzumab.

The invention is further illustrated by the following examples, but the invention should not be construed as limited. In the present specification, the % mark is referred to as % by mass unless otherwise specified.

[Examples]

(Example 1) 30 mL culture

According to the method described in International Publication No. 92/019759 and International Publication No. 2005/090405, the use of FuGENE (registered trademark) 6 Transfection Reagent (Promega) as a transformant to prepare a test drug to produce a tocilizumab The CHO cells were cultured in a medium containing 8 mM L-glutamic acid and penicillin, streptomycin, JX G016 (purchased by Irvine).

In order to confirm the effect of the soybean hydrolysate on the expressed recombinant protein, a medium containing no soy hydrolyzate was prepared, and Soy Hydrolysate UF solution (50X) was added in such a manner that the amount of all the culture liquid became 2% (5 g/L) (SAFC Corporation) System, catalog number # 58903C) Nutrient.

The CHO cell line producing the tocilizumab was inoculated to 3 × 10 ⁵ cells/mL in each medium, and vibrated (125 rpm) was performed in a 125 mL Erlenmeyer flask at a scale of 30 mL (37). °C, 5% CO _{2 in the} presence of).

On the third, fifth, seventh, ninth and eleventh days of culture, 1.8 mL of the culture solution was separated, and 1.8 mL of Feed medium (JX Feed 003 (Irvine Co., catalogue number JX F003)) was added. The culture solution obtained by the separation is used for the analysis of various components, and it is preferable to add glucose in the case where the glucose concentration in the culture solution is lowered.

The cell liquid of the 14th day of culture was subjected to a centrifugation operation (2000 g × 10 min), and a filter filtration operation, and the tocilizumab contained in the culture supernatant was purified by the method detailed in Example 3, And conduct analysis. The sugar chain of the purified protein was analyzed by Synapt G2 (Waters). The results are shown in Table 2. In addition, the sugar chain lines labeled G0F, G1F, G1F', and G2F in the table each have G(0), G(1)-1, G(1)-2, and G(2) shown in Table 1. Sugar chain structure.

From the results of Table 2, it can be confirmed that compared to the unadded soybean water In the medium of the solution, the degree of galactosylation of the sugar chain is increased by adding the soybean hydrolyzate to the medium.

Further, the same results were obtained in the case where CD FortiCHO Medium (Gibco, catalog number A11483-01) was used as the base medium instead of JX G016 (Irvine) medium.

In addition, the degree of galactosylation of the sugar chain in each of the medium containing soybean hydrolysate 0%, 1% (2.5 g/L), 2% (5 g/L), and 4% (10 g/L) As a result of the review, it was confirmed that the medium was cultured at 2% and 4%, and the galactosylation degree of the sugar chain was improved compared with the medium containing 0% and 1% of the soybean hydrolyzate. . Further, after comparing the medium having a soybean hydrolyzate content of 2% and the medium having a content of 4%, it was confirmed that the degree of galactosylation of the sugar chain was substantially equal.

As described above, the soybean hydrolyzate exhibits a novel effect of enhancing the degree of galactosylation of the sugar chain.

(Example 2) 10L culture

In order to confirm whether the effect of the soybean hydrolysate can also be adapted to large-scale culture, FuGENE (registered trademark) 6 Transfection Reagent (Promega) will be used according to the method described in International Publication No. 92/019759 and International Publication No. 2005/090405. Three strains of CHO cells (A, B, and C) which were produced as a transformant and prepared to prepare a betuzumab were cultured in a bioreactor having a full capacity of 15 L.

To contain 2% Soy Hydrolysate UF solution (50X) (SAFC The company system, catalog number # 58903C), 8 mM L-glutamic acid and penicillin, streptomycin JX G016 (Irvine) medium was cultured.

3 strains of CHO cells producing tocilizumab were inoculated to 3 × 10 ⁵ cells/mL in each medium, and were administered in a full-capacity 15 L culture tank (manufactured by ABLE) at an initial culture volume of 7.5 L. Stir culture (37 ° C, 50% DO, 25-30 rpm, pH 7.05 ~ 7.35).

30 mL of the culture solution was isolated on the 3rd, 5th, 7th, 9th and 11th day of the culture, and then the Feed medium (JX Feed003) was added. The feed medium was added in such a manner that the concentration after the addition of the feed medium became 6% of the amount of all the culture liquid.

The culture solution obtained by the separation is used for the analysis of various components, and it is preferable to add glucose in the case where the glucose concentration in the culture solution is lowered.

The cell fluid of the 14th day of culture was used for purification. The tocilizumab contained in the culture supernatant was purified by the method detailed in Example 3, and subjected to sugar chain analysis of the purified protein. The results are shown in Table 3.

The sugar chain of the purified protein was analyzed by PA800 Plus (Beckman Coulter). In addition, after analysis by PA800 Plus, the commercially available Actemra has a sugar chain composition ratio of 41% G(0), 27% G(1)-1, 9%G(1)-2 and 8%G(2). ).

From the results of Table 3, it was confirmed that the ratio of the sugar chain of the tocilizumab produced by the cell lines A to C was substantially the same as the ratio of the sugar chain of the commercially available Actemra.

As described above, by using the production method according to the present invention, it is possible to produce a tocilizumab which exhibits a sugar chain composition ratio equivalent to that of Actemra.

(Example 3) Purification method

The culture supernatant obtained by the method detailed in Example 2 was subjected to purification of tocilizumab in the following order. Further, the purification was carried out at room temperature using AKTA Avant 150 (GE Healthcare Japan).

For the culture supernatant obtained, Millistak+Pod (Merk Millipore, catalog #MD0HC027H1 or MD0HC054H1) was used as the pre-filter, and the Express SHF Opticap XL3 capsule (Merck Millipore, catalog # KGEPA03HH3) or PES The capsule sputum filter (ADVANTEC, catalog # CCS-020-E1H) clarified the culture solution as a sterilizing filter.

The obtained culture supernatant was supplied to an affinity chromatography column of Protein A previously equilibrated with 1 × D-PBS (Sigma-Aldrich, catalog # D1408) (MabSelect SuRe LX (GE Healthcare Japan, type) Record # 17-5474-02) pipe column ( 4.4cm × 19.4cm)). Then, the unadsorbed components were washed with the same buffer, and then the tocilizumab was eluted and pooled in a 100 mmol/L Gly-HCl buffer (pH 3.5). To the pool of the obtained elution liquid, 1.5 mol/L Tris-HCl buffer (pH 8.0) was added, and the salt concentration was adjusted to 120 mmol/L. The virus was deactivated by performing heat treatment at 40 ° C for 4 hours.

After the heat treatment, the pool of the virus deactivating solution was supplied to Q-Sepharose FF (GE Healthcare Japan, catalogue # 17-0510-01) previously equilibrated with 120 mmol/L Tris-HCl buffer (pH 8.0). Pillar 4.4 cm × 19.2 cm), the unadsorbed components were washed with the same buffer and pooled into an intermediate purification solution (tocilizumab fraction). The solvent of the pool of the intermediate purification solution was replaced with a 20 mmol/L sodium phosphate buffer (pH 6.5) and supplied to a ceramic hydroxyapatite Type II 80 μm (Bio-Rad Laboratories, catalogue #157-8000) column. ( 5 cm × 21.6 cm), after washing the unadsorbed components with the same buffer, the tocilizumab was eluted with 20 mmol/L phosphate buffer (pH 6.5) containing 260 mmol/L NaCl and pooled as an impurity removal liquid. . The concentrate of the obtained impurity-removing liquid was concentrated using a Hydrosart Sartocon Slice Cassette membrane (Saltorius Stedim, catalogue # 3051445901E-SG) having a molecular weight cut-off of 30 kDa, and then the solvent was replaced with a physiological saline solution (Otsuka Pharmaceutical Co., Ltd.) , Catalog # 1760), sterilized and filtered using a Stericup GP filter (Merck Millipore, catalog # SCGPU05RE) to obtain a final purified solution.

The monomer purity and residual DNA amount of the final purification liquid obtained by the purification method were measured. The results are shown in Table 4. The monomer purity was measured by SEC (size exclusion chromatography) (manufactured by Tosoh Corporation). The amount of residual DNA was measured by using an HCG (Certal CHO Detection Kit) (manufactured by QIAGEN) in accordance with an experimental procedure.

During the separation step, by adjusting the salt concentration of the eluate to 120 mM, no precipitation of particles containing DNA inclusions was produced. In addition, it was confirmed that the toltuzumab contained in the final purification liquid obtained by the single separation step was not contaminated with DNA inclusions, and the like, and there was no problem in terms of practicality.

Further, in the above procedure, the same result was obtained by adding 1.5 mol/L of Tris-HCl buffer (pH 8.0) to the pool of the eluted solution and adjusting the salt concentration to 150 mmol/L.

As described above, by using the manufacturing method of the present invention, it is possible to In the case of sediment removal, tocilizumab was easily purified and recovered in a higher yield.

Claims (10)

A method for producing a tocilizumab comprising: cultivating an animal cell secreting tocilizumab in a medium containing soybean hydrolysate; and separately ejecting the culture solution obtained from the cultured animal cell Bead monoclonal antibody. The manufacturing method of claim 1, wherein the tocilizumab exhibits 26% to 56% G(0), 17% to 37% G(1)-1, 0% to 19% G(1)-2 And the sugar chain of 0%~18% G(2) constitutes a ratio of tombuzumab. The production method of claim 1 or 2, wherein the medium does not contain a protein hydrolysate or a yeast extract other than the soybean hydrolyzate. The manufacturing method according to any one of claims 1 to 3, wherein the single exfoliation of the tocilizumab from the culture solution obtained by culturing the animal cells comprises: supplying the culture solution to the affinity chromatography column of protein A; The self-affining chromatography column dissolves the adsorbed tocilizumab; and the salt concentration of the eluate containing tocilizumab is adjusted to 110 mM to 170 mM. The method of claim 4, wherein the salt concentration of the eluate is adjusted to 115 mM to 160 mM. A culture medium for animal cell culture, which is used for production to show 26% to 56% G(0), 17% to 37% G(1)-1, 0% to 19% G(1)-2 and 0% The ~18% G(2) sugar chain constitutes a mixture of soybean hydrolysate and complete synthetic medium. The medium of claim 6, which is free of protein hydrolysate or yeast extract other than soy hydrolysate. A culture medium for animal cell culture which is used for enhancing the degree of galactosylation of a sugar chain of a recombinant protein produced by animal cells, which comprises a soybean hydrolyzate. A method for purifying a recombinant protein, comprising: supplying a culture solution obtained by culturing an animal cell secreting a recombinant protein to an affinity chromatography column of protein A; dissolving the adsorbed recombinant protein from a affinity chromatography column; and The salt concentration of the resolving solution containing the recombinant protein was adjusted to 110 mM to 170 mM. The purification method of claim 9, wherein the salt concentration of the eluate is adjusted to 115 mM to 160 mM.