WO2017038986A1 - Method for producing recombinant protein - 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, an animal cell culture medium, and a method for purifying the recombinant protein.

Many recombinant proteins such as antibodies and physiologically active substances are produced by genetic recombination, and are developed and sold as pharmaceuticals.
Depending on the type of recombinant protein, the culture method and the purification method differ, and production is not easy. A method for producing a recombinant protein will be described by taking tocilizumab as an example.
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. 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. Specifically, the molar concentration of the acidic eluate for eluting tocilizumab bound to the affinity chromatography column and the molar concentration of the neutral solution obtained by neutralizing the eluate are adjusted to a specific range, A method for forcibly precipitating DNA contaminants to purify tocilizumab (Patent Documents 3 and 4) is known.
In addition, it is known that sugar chains present in the molecule of the recombinant protein have an important influence on the pharmacokinetics, activity or immunogenicity of the recombinant protein. For example, among recombinant proteins, 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).

International Publication No. 99/063058 Special Table 2002-520014 International Publication No. 2002/072615 JP 2009-62380 A

Eon-Duval, A.M. et. al. Biotechnol. Prog. 2012, 28, 608-622

On the other hand, it has been clarified that a situation occurs in which the degree of galactosylation of sugar chains added to the recombinant protein varies depending on the culture conditions.
In addition, when the recombinant protein is purified by forcibly precipitating DNA contaminants, there arises a problem that the content of the finally obtained recombinant protein is reduced.
Thus, further technical development is required for the method for producing a recombinant protein.

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.
Furthermore, 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 .
<2> 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.
<4> 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 The method according to any one of <1> to <3>, comprising adjusting the salt concentration of the eluate containing the solution 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> 26% to 56% G (0), 17% to 37% G (1) -1, 0% to 19% G (1) containing soybean hydrolyzate and fully synthetic medium ) A medium for animal cell culture for producing tocilizumab having a sugar chain composition ratio of -2 and 0 to 18% G (2).
<7> The medium according to <6>, which contains no protein hydrolyzate or yeast extract other than soybean hydrolysate.
<8> A culture medium for animal cell culture containing soybean hydrolyzate for improving the degree of galactosylation of sugar chains of recombinant proteins produced by animal cells.
<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.
<10> The purification method according to <9>, wherein the salt concentration of the eluate is adjusted to 115 mM to 160 mM.

According to the present invention, any of the following effects is provided.
ADVANTAGE OF THE INVENTION According to this invention, 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.
In addition, according to the present invention, it is possible to provide tocilizumab exhibiting a specific sugar chain constituent ratio that is expected to exhibit the same pharmacokinetic behavior as that of commercially available Actemra.
Moreover, according to this invention, 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.
Furthermore, according to the present invention, it is possible to provide a method for easily purifying a recombinant protein secreted from animal cells and recovering it in a high yield.

Hereinafter, the present invention will be described in detail.
<Method for producing tocilizumab>
The production method in the present invention is characterized by culturing animal cells secreting tocilizumab in a medium containing soybean hydrolyzate (culturing step). Preferably, the method includes a culturing step and isolating tocilizumab from a culture solution obtained by culturing animal cells (isolation step). Moreover, the manufacturing method in this invention may include the other process besides the culture | cultivation process and the isolation process.
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.

(Culture process)
In the culturing process, 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. For example, when CHO cells are used as animal cells that secrete tocilizumab, 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. In addition, for example, by culturing for 10 to 28 days, 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. In the present invention, 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. 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.

In the present invention, the initial culture medium refers to a medium used in the first stage of cell culture. However, when the fed-batch medium is added in a plurality of times, the medium before adding the fed-batch medium can also be referred to as an initial medium.
In the present invention, from the viewpoint that the degree of galactosylation of the sugar chain of the recombinant protein can be improved, 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.
In the present invention, a soybean hydrolyzate-containing medium is used as an initial medium, and at a temperature of 33 to 38 ° C. and 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. Moreover, although 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. In addition, it is preferable to inoculate the animal cells so that the cell density is 1 × 10 ⁵ cells / mL to 5 × 10 ⁵ cells / mL and culture using the initial medium. In the present invention, it is preferable to carry out fed-batch culture in which 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.

In the present invention, when a fed-batch culture method or a continuous culture method is used, 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. In addition, from the viewpoint of osmotic pressure in the culture solution, 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.
Further, 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.
In a preferred embodiment of the present invention, 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. Also, 30% to 50% G (0), 19% to 35% G (1) -1, 1% to 17% G (1) -2 and 0% to 16% G (2) It is more preferable to show the sugar chain constituent ratio.
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. Here, 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. For example, 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. Proc Natl Acad Sci USA 1969 Dec; 64 (4): 1284-91), in particular, a dihydrofolate reductase (DHFR) deficient strain, such as the CHO-DG44 cell line (Urlab, G. and Chasin). L. A .: Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)). Specifically, 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). Moreover, 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. In addition, the 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. It is more preferably 1 g / L to 20 g / L, and further preferably 3 g / L to 10 g / L. The content of 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.
As 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.

When the Feed medium is used, the type of Feed medium is not particularly limited. For example, 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). ), Cell Boost 3 (JM3.5) Supplement (GE Healthcare, catalog number SH30825.01), Cell Boost 4 (PS307) Supplement (GE Healthcare, catalog number SH30857.01), Cell 5BoN Supplement (GE Healthcare, catalog number SH3 865.01), 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, catalog number 91129-1L), BalanCD (registered trademark) CHO FEED 3 (Irvine, catalog number 99471-1L).
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.

(Isolation process)
In the production method of the present invention, 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. Depending on the type of column, the column equilibration solution, the column washing solution, the culture solution flow rate condition, the culture solution adsorption condition, What is necessary is just to set suitably the washing | cleaning conditions of a culture solution, the supply conditions, such as an impurity.
For example, when MabSelectSuRe LX, (GE Healthcare Japan, Catalog No. 17-5474-02) is used as an affinity chromatography column for protein A, when the culture solution is applied to an affinity chromatography column for protein A, pH 5 It may be set to a condition of ~ 8.

The adsorption step may include measuring the tocilizumab concentration in the culture solution before subjecting the culture solution to the affinity chromatography column.
In the adsorption step, when the culture solution is supplied 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. In addition, 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. For example, the cleaning solution may be set to a condition of 1 mol / L NaCl.
In addition, 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. Depending on the type of column, etc., the type of eluate, collection of the eluted fraction, conditions for washing impurities that are weakly bound to the column, etc. Can be set.
For example, when MabSelectSuRe LX, (GE Healthcare Japan, catalog number 17-5474-02) is used as an affinity chromatography column for protein A, an elution solution having a pH of 4.5 or less can be used. The adsorbed tocilizumab can be eluted.

As 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.
When 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.
In addition, it is preferable that 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.
Examples of commercially available products that can be used include MabSelectSuRe LX, (GE Healthcare Japan, catalog number 17-5474-02), and Prosep Ultra Plus (Merck Millipore, catalog number 17518822).

In addition to the culturing step and the isolation step, 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).
These steps can be appropriately set in accordance with conventional methods according to conditions such as stability, recovery rate, and impurity removal efficiency.

<Medium for animal cells producing tocilizumab>
The animal cell culture medium for producing tocilizumab in the present invention (hereinafter also referred to as “tocilizumab production medium”) 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.
In the tocilizumab production 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.

<Recombinant protein>
In the animal cell culture medium and the method for producing a recombinant protein, which will be described later, 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. For example, a recombinant protein that can be used as a pharmaceutical is preferable. The recombinant protein is preferably a recombinant protein having a sugar chain. Moreover, 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.
Examples of the sugar chain herein include an O-linked sugar chain and an N-linked sugar chain. The recombinant protein preferably has at least an N-linked sugar chain. In the case of an antibody, the sugar chain is more preferably an N-linked sugar chain that binds to the Fc portion of the antibody molecule.

<Animal cell culture medium>
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. Here, 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. Preferably, this means that the proportion of sugar chains having galactose at the non-reducing end in the sugar chains constituting the N-linked sugar chain increases. Specifically, as shown in Table 1 above, 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 It 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.
Further, 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%.
Thus, 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. In addition, 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.

For the soybean hydrolyzate contained in the animal cell culture medium, the matters described above in the section of the method for producing tocilizumab can be applied as they are. In addition, 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).
For the other matters, the matters described above in the section of the method for producing tocilizumab can be applied mutatis mutandis.

<Method for purifying recombinant protein>
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.

In the adjustment step, 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. With regard to, the above-mentioned matters in the section of the method for producing tocilizumab can be applied mutatis mutandis.

The present invention will be described in further detail with reference to the following examples, but the present invention should not be construed as being limited to these examples. In this specification, the indication of% is mass% unless otherwise specified.

(Example 1) 30 mL culture In accordance with the method described in International Publication No. WO 92/075959 and International Publication No. 2005/090405, 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.
In order to confirm the effect of soybean hydrolyzate on the expressed recombinant protein, medium containing no soy hydrolyzate and Soy Hydrohydrate UF solution (50X) (manufactured by SAFC, catalog number # 58903C) were added to the total culture volume. A medium added to 2% (5 g / L) was prepared.
Tocilizumab-producing CHO cells were seeded at 3 × 10 ⁵ cells / mL in each medium, and cultured with shaking (125 rpm) on a 30 mL scale in 125 mL of Erlenmeyer flash (37 ° C., in the presence of 5% CO ₂ ). .
On the third, fifth, seventh, ninth and eleventh days of culture, 1.8 mL of the culture solution was collected, and 1.8 mL of Feed medium (JX Feed003 (Irvine, catalog number JX F003)) was added. 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 subjected to centrifugation (2000 g × 10 min) and filter filtration, and tocilizumab contained in the culture supernatant was purified and analyzed by the method described in detail in Example 3. . The sugar chain of the purified protein was analyzed by SynaptG2 (Waters). The results are shown in Table 2. In the table, the sugar chains represented as G0F, G1F, G1F ′ and G2F are G (0), G (1) -1, G (1) -2 and G (2) shown in Table 1, respectively. ) Sugar chain structure.

From the results shown in Table 2, it was confirmed that the degree of galactosylation of sugar chains was improved by adding soybean hydrolyzate to the medium as compared to the medium without adding soybean hydrolyzate.
Similar results were obtained when CD FortiCHO Medium (Gibco, catalog number A11483-1) was used as the basal medium instead of JX G016 (Irvine) medium.

Separately, the degree of galactosylation of sugar chains in each medium containing 0%, 1% (2.5 g / L), 2% (5 g / L) and 4% (10 g / L) of soybean hydrolyzate As a result of examining the influence, it was confirmed that the degree of galactosylation of sugar chains was improved by culturing in a medium with 2% and 4% compared to a medium with 0% and 1% soy hydrolyzate content. It was done. In addition, when the culture medium with the content of soybean hydrolyzate of 2% was compared with the culture medium with 4%, it was confirmed that the degree of galactosylation of sugar chains was almost the same.

As described above, soybean hydrolyzate has a new effect of improving the degree of sugar chain galactosylation.

(Example 2) 10 L culture In order to confirm whether the effect of soybean hydrolyzate can be applied to large-scale 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).
On the third, fifth, seventh, ninth and eleventh days of culture, 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).

From the results in Table 3, it was confirmed that the sugar chain composition ratio of tocilizumab produced by cell lines A to C was almost the same as the sugar chain composition ratio of Actemra, which is commercially available.
As described above, tocilizumab having a sugar chain constituent ratio equivalent to that of Actemra can be manufactured by using the manufacturing method according to the present invention.

(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 (φ4.4 cm × 19.4 cm)). Thereafter, non-adsorbed components were washed with the same buffer, and then 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 (φ4.4 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 μm (Bio-Rad Laboratories, catalog # 157-8000) column (φ5 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).

In the course of the isolation step, the precipitation of DNA contaminant-containing particles did not occur by adjusting the salt concentration of the eluate to 120 mM. In addition, it was confirmed that tocilizumab contained in the final purified solution obtained by this isolation step had no problem of contamination with DNA contaminants, and that there was no problem in quality in practical use.
The same applies when 1.5 mol / L Tris-HCl buffer (pH 8.0) is added to the eluate pool to adjust the salt concentration to 150 mmol / L. Results were obtained.
As described above, by using the production method of the present invention, it is not necessary to remove the precipitate, and tocilizumab can be easily purified and recovered with a high yield.

Claims (10)

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 (2) The production method according to claim 1, which is tocilizumab showing the sugar chain constituent ratio. The production method according to claim 1 or claim 2, wherein the medium does not contain a protein hydrolyzate or yeast extract other than soybean hydrolysate. Isolating tocilizumab from the culture medium obtained by culturing animal cells,
Subjecting the culture to an affinity chromatography column for protein A;
Eluting the adsorbed tocilizumab from the affinity chromatography column;
The production method according to any one of claims 1 to 3, comprising adjusting the salt concentration of the eluate containing tocilizumab to 110 mM to 170 mM. The production method according to claim 4, wherein the salt concentration of the eluate is adjusted to 115 mM to 160 mM. A soy hydrolyzate and a fully synthetic medium,
26% -56% G (0), 17% -37% G (1) -1, 0% -19% G (1) -2 and 0% -18% G (2) sugar chains An animal cell culture medium for producing tocilizumab showing a composition ratio. The medium according to claim 6, which contains no protein hydrolyzate or yeast extract other than soybean hydrolysate. Contains soy hydrolyzate,
An animal cell culture medium for improving the degree of galactosylation of sugar chains of recombinant proteins produced by animal cells. Subjecting the culture solution obtained by culturing animal cells secreting the recombinant protein to a protein A affinity chromatography column;
Eluting the adsorbed recombinant protein from the affinity chromatography column;
Adjusting the salt concentration of the eluate containing the recombinant protein to 110 mM to 170 mM, and a method for purifying the recombinant protein. The purification method according to claim 9, wherein the salt concentration of the eluate is adjusted to 115 mM to 160 mM.