KR102653666B1 - Method for producing darbepoetin composition and method for cultivating darbepoetin producing cells - Google Patents

Abstract

The present invention relates to a method for producing a darbepoetin composition by culturing darbepoetin-producing cells, in which the total production of the darbepoetin composition is improved, and the sialic acid in one molecule of darbepoetin secreted from the cell into the culture medium is improved. The purpose is to provide a method for producing a darbepoetin composition that improves the content. The present invention includes a step 1 of culturing darbepoetin-producing cells in a medium containing a plant-derived protein hydrolyzate and obtaining a culture medium, and a step 2 of recovering the darbepoetin composition from the culture medium obtained in step 1. It relates to a method for producing a darbepoetin composition.

Description

Method for producing darbepoetin composition and method for cultivating darbepoetin producing cells

The present invention relates to a method for producing a darbepoetin composition, a method for cultivating animal cells having the ability to produce darbepoetin, etc.

In recent years, the production of useful substances such as genetically recombinant proteins using animal cells has been actively conducted. However, in order to obtain the maximum amount of product, it is necessary to not only increase the expression ability of the relevant animal cells for the relevant protein, but also to obtain the maximum amount of product. There is a need to improve the proliferation of .

Meanwhile, even though genetic recombination technology has advanced, glycoprotein is one of the genetic recombinant proteins that is still difficult to produce. Most proteins exist as glycoproteins with added sugar chains, and it is known that differences in their sugar chain structures have a significant impact on the function of the protein itself.

Biopharmaceuticals using genetic recombination technology are produced by introducing human protein genes into animal cells. However, when a glycoprotein is produced using animal cells, its sugar chain structure becomes non-uniform due to sugar chain decomposition enzymes such as sialidase generated from the production cells, and therefore, it may not exhibit sufficient medicinal efficacy.

Erythropoietin (EPO), a hematopoietic factor, has three N-linked sugar chains and one O-linked sugar chain. Its blood half-life changes depending on the number of sialic acids at the ends of the sugar chains, and when the number of sialic acids at the ends of the sugar chains decreases, It is known that the half-life in the blood is reduced and shows almost no physiological activity (Non-patent Document 1). In addition, darbepoetin was discovered in which part of the amino acid sequence of EPO was modified and the number of sialic acid additions was further increased for the purpose of further extending the blood half-life of EPO (Non-patent Document 2, Patent Document 1).

Darbepoetin has 5 N-linked sugar chains and 1 O-linked sugar chain, has a sustained red blood cell increase effect, and makes it possible to reduce the frequency of administration compared to conventional EPO preparations, reducing the burden on patients. It can be reduced.

However, for the above-mentioned reasons, in order to efficiently produce uniform darbepoetin having more sialic acid-containing sugar chains than EPO, a more advanced sugar chain control technology as well as improvement in production volume was required.

As a sugar chain control method, to inhibit the detachment of sialic acid from glycoproteins by sugar chain decomposition enzymes secreted in the culture medium, for example, 2-deoxy-2,3-dehydro-N-, an inhibitor of sialidase, A method of culturing by adding low-molecular-weight compounds such as acetylneuraminic acid (NeuAc2en) to the medium is known.

On the other hand, in order to improve the proliferation of animal cells and the production of substances produced by the animal cells, the medium used conventionally to culture the animal cells contains serum or serum-derived substances such as albumin, transferrin, or insulin, or animal Derived protein has been added. The addition of serum or serum-derived substances or animal-derived proteins to the medium carries the risk of contamination of the cell culture and the products obtained therefrom with hepatitis, viruses, or bovine spongiform encephalopathy (BSE). Development of a completely synthetic medium (chemically defined medium) that does not contain derived substances and animal-derived proteins has been undertaken.

Components of a typical fully synthetic medium include amino acids, organic or inorganic salts, vitamins, minerals, trace metals, sugars, lipids, and nucleic acids. However, protein hydrolysates derived from plants such as soybeans are added to the fully synthetic medium. It is known that by doing so, the proliferation of animal cells and the production of products obtained from the animal cells can be improved, equivalent to a serum-containing medium (Patent Documents 2 and 3).

Japanese Patent No. 2938572 Specification International Publication No. 2001/023527 International Publication No. 2006/045438

Glycoconjugate J., 1993;10;263 Nephrol. Dial. Transplant., 2001;16;3-13

However, there is no known method for improving the total production of the darbepoetin composition while also improving the sialic acid content in one darbepoetin molecule secreted from the relevant cells into the culture medium.

Therefore, the present invention relates to a method for producing a darbepoetin composition with a high sialic acid addition number by culturing darbepoetin-producing cells, which improves the total production amount of the darbepoetin composition secreted from the cells in the culture medium, and further The object is to provide a method for producing a darbepoetin composition that improves the sialic acid content in one molecule of darbepoetin secreted from the relevant cells into the culture medium.

The present inventors have found that by cultivating darbepoetin-producing cells using a medium containing a plant-derived protein hydrolyzate, the total production of darbepoetin composition secreted from the cells in the culture medium is improved, and the sialic acid secreted in the culture medium is also improved. It was discovered that by inhibiting lidase, the sialic acid addition rate in one darbepoetin molecule secreted from the cell in the culture medium was improved, and a darbepoetin composition with a high sialic acid addition number could be produced significantly, and the present invention was completed. .

That is, the present invention provides the following (1) to (19).

(1) A method for producing a darbepoetin composition comprising the following steps 1 and 2.

Process 1: Process of obtaining a culture medium by culturing darbepoetin-producing cells in a medium containing plant-derived protein hydrolyzate.

Process 2: Process of recovering darbepoetin composition from the culture medium obtained in Process 1

(2) A method for producing a darbepoetin composition in which detachment of sialic acid from darbepoetin molecules is inhibited, comprising the following steps 1 and 2.

Process 1: Process of obtaining a culture medium by culturing darbepoetin-producing cells in a medium containing plant-derived protein hydrolyzate.

Process 2: Process of recovering darbepoetin composition from the culture medium obtained in Process 1

(3) More than 67% of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule in the culture solution obtained in Step 1 is darbepoetin containing 18 to 22 sialic acids per molecule. The production method according to (1) or (2), which is a bepoetin composition.

(4) At least 55% of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule in the culture medium obtained in Step 1 is darbepoetin containing 19 to 22 sialic acids per molecule. The production method according to any one of (1) to (3), which is a bepoetin composition.

(5) At least 44% of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule in the culture medium obtained in Step 1 is darbepoetin containing 20 to 22 sialic acids per molecule. The production method according to any one of (1) to (4), which is a bepoetin composition.

(6) At least 18% of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule in the culture solution obtained in Step 1 is darbepoetin containing 22 sialic acids per darbepoetin molecule. The production method according to any one of (1) to (5), which is a composition.

(7) The production method according to any one of (1) to (6), wherein the total production amount of the darbepoetin composition in the culture medium obtained in Step 1 is higher than when cultured in a medium containing no plant-derived protein hydrolyzate.

(8) The production method according to any one of (1) to (7), wherein the darbepoetin-producing cell is a transformed cell into which a vector containing a gene encoding darbepoetin has been introduced into a host cell.

(9) The production method according to (8), wherein the host cell is an animal cell.

(10) The production method according to (9), wherein the animal cells are Chinese hamster ovary (CHO) cells, mouse melanoma (NS0) cells, or mouse myeloma (SP2/0) cells, or cells derived from these cells.

(11) The production method according to any one of (1) to (10), wherein the medium to which the plant-derived protein hydrolyzate is added is a completely synthetic medium.

(12) The production method according to any one of (1) to (11), wherein the concentration of plant-derived protein hydrolyzate added to the medium is 1 to 5 g/l.

(13) The production method according to any one of (1) to (12), wherein the plant-derived protein hydrolyzate is a soybean-derived protein hydrolyzate.

(14) The production method according to any one of (1) to (13), further comprising a purification step of purifying the darbepoetin composition.

(15) The production method according to (14), wherein the purification process is at least one selected from anion exchange chromatography, reverse phase chromatography, or gel filtration.

(16) The production method according to any one of (1) to (15), wherein the obtained darbepoetin composition is darbepoetin containing an average of 18 or more sialic acids per molecule.

(17) A method of cultivating darbepoetin-producing cells, comprising culturing the darbepoetin-producing cells in a medium supplemented with a plant-derived protein hydrolyzate and secreting a darbepoetin composition into the culture medium.

(18) A method of cultivating darbepoetin-producing cells, comprising culturing darbepoetin-producing cells in a medium supplemented with a plant-derived protein hydrolyzate and inhibiting the detachment of sialic acid from darbepoetin molecules in the culture. .

(19) A method for inhibiting the detachment of sialic acid from darbepoetin molecules in a stored culture medium, comprising culturing darbepoetin-producing cells in a medium supplemented with a plant-derived protein hydrolyzate to obtain a culture medium.

In the production method of the present invention, by culturing darbepoetin-producing cells in a medium supplemented with a plant-derived protein hydrolyzate, the total production of darbepoetin composition secreted from the cells into the culture medium is improved, and further, the darbepoetin composition is secreted from the cells. It can improve the sialic acid content in one molecule of darbepoetin secreted in the culture medium.

Figure 1 is a diagram showing the structure of darbepoetin.
Figure 2 is a diagram showing the structures of the N-linked sugar chain and O-linked sugar chain of darbepoetin. In Figure 2, NeuAc represents N-acetylneuraminic acid, one of sialic acids.
Figure 3 is a diagram showing changes over time in the distribution of sialic acid isoforms of darbepoetin in the culture supernatant cultured in a medium containing soy hydrolyzate and in the soy hydrolyzate removal liquid. Lanes 1 and 14 represent darbepoetin with sialic acid addition numbers of 18 to 22. Lane 2 stores the soy hydrolyzate removal solution at 8℃ for 0 hours, Lane 3 stores the soy hydrolyzate removal solution at 8℃ for 24 hours, Lane 4 stores the soy hydrolyzate removal solution at 8℃ for 48 hours, and Lane 5 stores the soy hydrolyzate removal solution at 8℃ for 48 hours. The distribution of sialic acid isoforms when the decomposed product removal solution was stored at 8°C for 72 hours is shown. Lane 6 is the culture supernatant grown in a soy hydrolyzate-containing medium and stored at 8°C for 0 hours. Lane 7 is a culture supernatant grown in a soy hydrolyzate-containing medium and stored at 8°C for 24 hours. Lane 8 is a soy hydrolyzate-containing medium. Lane 9 shows the distribution of sialic acid isoforms when the culture supernatant cultured in medium containing soy hydrolyzate was stored at 8°C for 72 hours. Lane 10 is a solution in which the sialidase inhibitor NeuAc2en was added to the soy hydrolyzate solution and stored at 8°C for 0 hours. Lane 11 was a solution in which the sialidase inhibitor NeuAc2en was added to the soy hydrolyzate solution and stored at 8°C for 24 hours. Lane 12 Lane 13 is sialic acid obtained when the sialidase inhibitor NeuAc2en was added to the soy hydrolyzate removal solution and stored at 8°C for 48 hours. Lane 13 is sialic acid obtained when the sialidase inhibitor NeuAc2en was added to the soy hydrolyzate removal solution and stored at 8°C for 72 hours. The isoform distribution is shown, respectively.
Figure 4 is a bar graph of the results in Table 1 measuring the ratio (%) of the area value of the darbepoetin composition of each sialic acid adduct number to the total area value of the darbepoetin composition of 12 to 22 sialic acid adduct numbers. It is expressed as . The vertical axis represents the ratio (%) of the area value of the darbepoetin composition for each sialic acid adduct. The ratio of the area values of the 22 sialic acid addition numbers in Table 1 is the ratio of the area values of the isoforms 22, 22-1S and 22-2S, which represents the darbepoetin composition with the 22 sialic acid addition numbers in Figure 4 ( It is expressed as the sum of %).

The method for producing the darbepoetin composition of the present invention includes the following steps 1 and 2.

Process 1: Process of obtaining a culture medium by culturing darbepoetin-producing cells in a medium containing plant-derived protein hydrolyzate.

Process 2: Process of recovering darbepoetin composition from the culture medium obtained in Process 1

Below, each process is explained.

[Process 1]

Step 1 is a step of culturing cells capable of producing darbepoetin in a medium containing a plant-derived protein hydrolyzate and secreting the darbepoetin composition into the medium to obtain a culture medium.

In the present invention, darbepoetin is prepared in International Publication No. 95/05465, wherein the amino acid residues at positions 30, 32, 87, 88, and 90 in the amino acid sequence of human erythropoietin are Asn, Thr, Val, Asn, and It is a human erythropoietin analog represented as [Asn ³⁰ Thr ³² Val ⁸⁷ Asn ⁸⁸ Thr ⁹⁰ ]EPO, substituted with Thr, and has the structure shown in FIG. 1.

In darbepoetin, an N-linked sugar chain is bound to the Asn residues at positions 24, 30, 38, 83, and 88, and an O-linked sugar chain is bound to the Ser residue at position 126, respectively. The N-linked sugar chain and the O- The main structure of the bonded sugar chain includes the structure shown in FIG. 2.

The N-linked sugar chain bound to the darbepoetin molecule contains up to 4 sialic acids, the end of the O-linked sugar chain contains up to 2 sialic acids, and one darbepoetin molecule contains up to 22 sialic acids.

In the present invention, darbepoetin includes, for example, darbepoetin alfa, darbepoetin alfa (genetic recombination), and bio-successors thereof. In addition, the novel erythropoiesis stimulating protein and NESP (trademark) or Aranesp (trademark) disclosed in International Publication No. 2003/020299, or their bio-successors, can also be used in the darbepoetin of the present invention. Included.

As the host cell for the darbepoetin-producing cells used in the present invention, any animal cell belonging to any of mammals, birds, reptiles, amphibians, fish, and insects may be used, but animal cells belonging to mammals are preferably used. More preferably, animal cells derived from primates such as humans or monkeys or animal cells derived from rodents such as mice, rats, or hamsters are used.

Cells belonging to mammals include, for example, myeloma cells or cells derived from myeloma cells, ovarian cells, kidney cells, blood cells, uterine cells, connective tissue cells, mammary cells, or embryonic retinoblasts. In particular, cells selected from myeloma cells or cells derived from myeloma cells and ovarian cells are preferred, specifically, for example, Chinese hamster ovary (CHO) cells, mouse melanoma (NS0) cells or mouse myeloma (SP2) cells. /0) cells, or cells derived from these cells.

Cells belonging to mammals include, for example, human cell lines HL-60 (ATCC CCL-240), HT-1080 (ATCC CCL-121), HeLa (ATCC CCL-2), 293 (ECACC 85120602), and Namalwa. ) (ATCC CRL-1432), Namalva KJM-1 (Cytotechnology, 1, 151 (1988)), NM-F9 (DSM ACC2605, International Publication No. 05/17130) or PER.C6 (ECACC No.96022940, USA) Patent No. 6855544 (specification), monkey cell lines VERO (ATCC CCL-1651) or COS-7 (ATCC CRL-1651), mouse cell lines C127I (ATCC CRL-1616), Sp2/0-Ag14 (ATCC CRL-1581) , NIH3T3 (ATCCCRL-1658), NS0 (ATCC CRL-1827), a rat cell line, Y3 Ag1.2.3. (ATCC CRL 1631), YO (ECACCNo: 85110501) and YB2/0 (ATCC CRL-1662), a hamster cell line. CHO-K1 (ATCC CCL-61), CHO/dhfr- (ATCC CRL-9096), CHO/DG44 [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)] or BHK21 (ATCC CRL-10) or canine MDCK (ATCC CCL-34).

Cells belonging to birds, for example, chicken cell line SL-29 (ATCC CRL-29), cells belonging to fish, for example, zebrafish cell line ZF4 (ATCC CRL-2050), etc. Cells belonging to insects, for example, For example, the moth (Spodoptera frugiperda) cell line Sf9 (ATCC CRL-1711) can be mentioned. Additionally, examples of primary cultured cells include primary monkey kidney cells, primary rabbit kidney cells, primary chicken embryo cells, and primary quail embryo cells.

Examples of myeloma cells or cells derived from myeloma cells include Sp2/0-Ag14, NS0, Y3 Ag1.2.3., YO, or YB2/0. Examples of ovarian cells or cells derived from ovarian cells include CHO-K1, CHO/dhfr-, or CHO/DG44.

Additionally, kidney cells include, for example, 293, VERO, COS-7, BHK21 or MDCK, blood cells include HL-60, Namalba, Namalba KJM-1 or NM-F9, and uterine cells include, for example, For example, HeLa, etc. can be mentioned, examples of connective tissue cells include HT-1080 or NIH3T3, examples of mammary cells include C1271I, and embryonic retinoblasts include, for example, PER.C6. .

Examples of darbepoetin-producing cells include, for example, transformed cells into which a vector containing a gene encoding darbepoetin is introduced into the host cell described above, cells that undergo mutation treatment to produce darbepoetin, or darbepoetin. Examples include hybridomas, which are fusion cells of cells that produce ethyne and myeloma cells. In addition, the cells described above that have been subjected to mutation treatment to increase the expression level of darbepoetin are also included in the darbepoetin-producing cells of the present invention.

The transformed cell into which the vector containing the gene encoding darbepoetin is introduced is a recombinant vector containing DNA and a promoter involved in the production of darbepoetin, etc., to the host cell used in the present invention described above. It is obtained by introducing

As DNA involved in the production of darbepoetin, for example, DNA encoding darbepoetin, DNA encoding enzymes or proteins involved in the biosynthesis of darbepoetin, etc. can all be used.

Vectors used to produce the recombinant vector include, for example, pcDNAI, pcDM8 (manufactured by Funakoshi Corporation), pAGE107 (Japanese Patent Laid-Open No. 3-22979, Cytotechnology, 3, 133 (1990)], pAS3-3. (Japanese Patent Laid-Open No. 2-227075), pcDM8 [Nature, 329, 840 (1987)], pcDNAI/Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochem., 101, 1307 (1987)] or pAGE210.

As the promoter, any one that functions in the animal cells used in the present invention can be used, for example, the promoter of the IE (immediate early) gene of cytomegalovirus (CMV), the early promoter of SV40, the promoter of retrovirus, and the promoter of metallovirus (IE) gene. Examples include onein promoter, heat shock promoter, or SRα promoter. Additionally, an enhancer of the IE gene of human CMV, etc. may be used together with a promoter.

As a method for introducing a recombinant vector into a host cell, any method that introduces DNA into the cell can be used, for example, electroporation method [Cytotechnology, 3, 133 (1990)], calcium phosphate method. (Japanese Patent Publication No. 2-227075) or lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987) or Virology, 52, 456 (1973)].

As the plant-derived protein hydrolyzate, any protein hydrolyzate can be used as long as it is a plant-derived protein hydrolyzate such as wheat, rice, or soybean, but soybean-derived protein hydrolyzate is particularly preferable.

Plant-derived protein hydrolyzate is a mixture containing, as main components, short-chain peptides such as amino acids and dipeptides or tripeptides obtained by decomposing proteins obtained from plants as raw materials, as well as trace metals and the like. In the production of plant-derived protein hydrolysates, acid digestion using an acid such as hydrochloric acid or an enzymatic digestion using an enzyme such as protease may be used, and membrane treatment is performed as necessary.

The average molecular weight of the plant-derived protein hydrolyzate is preferably 800 Da or less, and in steps below, more preferably 600 Da or less, further preferably 450 Da or less, and particularly preferably 300 Da or less.

The molecular weight distribution of the plant-derived protein hydrolyzate is preferably 45 to 80% for <500 Da, preferably 15 to 30% for 500 to 1000 Da, preferably 5 to 15% for 1000 to 2000 Da, and preferably 5 to 15% for 2000 to 5000 Da. Preferably it is 2 to 10%. Alternatively, <2000Da is more preferably 90 to 98%, and <2000Da is more preferably 95 to 98%.

Protein hydrolysates derived from soybeans include, for example, Amysoy, Hy-Soy, N-Z-Soy, HyPep (above, Quest International), Soy peptone (Gibco), Bac-Soyton (Difco), and SE50MK (DMV International Nutritions). Inc.), Peptone Hy-Soy T or Soy Hydrolysate UF (above, manufactured by Sigma-Aldrich), etc., and HyPep (Quest International) as a protein hydrolyzate derived from wheat or rice.

There is no particular limitation on the timing of adding the plant-derived protein hydrolyzate to the culture medium, whether at the start of the culture or during the culture, but it is preferable to add it to the medium at the start of the culture. When added during cultivation as a feed medium in fed-batch culture or perfusion culture, etc., the plant-derived protein hydrolyzate may be added alone as a feed medium, or as a feed medium mixed in advance with other medium components. You may add it.

There is no particular limitation on the culture period, but the final main culture period is preferably 8 days or more. In the fed-batch culture method, the main culture period is preferably 8 days to 1 month, more preferably 10 days to 21 days, and particularly preferably 10 days to 14 days.

The concentration of the plant-derived protein hydrolyzate added to the medium may be appropriately selected depending on the type of cells used for culture, the time of addition of the plant-derived protein hydrolyzate, etc., but is preferably 0.1 to 100 g/L, more preferably 0.1 to 100 g/L. is 1 to 10 g/L, particularly preferably 1 to 5 g/L.

The medium used in the present invention may be any medium as long as it can be used for culturing darbepoetin-producing cells, but a completely synthetic medium (chemically defined medium) that does not contain serum or serum-derived substances or animal-derived proteins is used. It is desirable.

When the host cell of the darbepoetin-producing cell is an animal cell, a basal medium used for culturing normal animal cells is used as the basal medium. Basal media used for culturing common animal cells include, for example, RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM (DMEM) medium [Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], F12 medium (LTI) [Proc. Natl. Acad. Sci. USA, 53, 288 (1965)], Iscove's modified Dulbecco's medium (IMDM medium) [J. Experimental Medicine, 147, 923 (1978)] or EX-CELL325PF medium (manufactured by JRH) or modified or mixed media thereof, but preferably RPMI1640 medium, DMEM medium, F12 medium, IMDM medium or EX- CELL 325PF badge, etc. are used.

As the medium used in the present invention, nutritional factors or physiologically active substances necessary for the growth of animal cells are added to the basal medium as necessary. It is preferable to include these additives in the medium in advance before culturing.

Examples of nutritional factors include sugars such as glucose, amino acids, and vitamins.

As amino acids, for example, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L -Includes lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, or L-valine, and is used 1 type or in combination of 2 or more types. .

Examples of vitamins include d-biotin, D-pantothenic acid, choline, folic acid, myo-inositol, niacinamide, pyridoxal, riboflavin, thiamine, cyanocobalamin, or DL-α-tocopherol, and one or Two or more types are used in combination.

In addition, in the fully synthetic medium that does not contain animal derivatives, substances added in place of animal derivatives include, for example, physiologically active substances manufactured by genetic recombination, hydrolysates, or lipids that do not contain animal derivatives. , minerals, trace metals, or nucleic acids. Examples of physiologically active substances produced by genetic recombination include genetically modified insulin, genetically modified transferrin, genetically modified albumin, and genetically modified growth factors.

Examples of lipids that do not contain animal origin include cholesterol, linoleic acid, or linolenic acid.

Completely synthetic media include, for example, ADPF medium (Animal derived protein free medium; manufactured by HyClone), CD-hybridoma medium (manufactured by Invitrogen), CD-CHO medium (manufactured by Invitrogen), and IS CD-CHO medium. or KINSM-10 medium (manufactured by Irvine Scientific).

For long-term or high-density cultivation, medium containing high concentrations of amino acids and vitamins, for example, RPMI1640 medium, DMEM medium, and F12 medium mixed at a ratio of 1:1:1, DMEM medium, and F12 medium. A medium mixed at a ratio of 1:1, Hybridoma SFM medium (manufactured by Invitrogen), etc. are suitably used.

In the present invention, the improvement in the total production of the darbepoetin composition in the culture medium obtained in Step 1 means that the darbepoetin composition in the unit culture amount at the end of the culture when cultured in a medium containing the above-mentioned plant-derived protein hydrolyzate This means that the concentration of the bepoetin composition is improved compared to when cultured in the above-mentioned plant-derived hydrolyzate-free medium.

The concentration of the darbepoetin composition in the unit culture volume can be determined using any known protein concentration measurement method such as ELISA or HPLC. For example, the protein concentration measurement method using Biacore (manufactured by GE Healthcare) can be mentioned.

As for the darbepoetin composition in the culture medium obtained in Step 1, the number of sialic acids per molecule of darbepoetin is preferably 18 to 22, more preferably 19 to 22, particularly preferably 20 to 22, and most preferably It has 22 additional structures.

As for the darbepoetin composition in the culture medium obtained in Step 1, preferably at least 67% of the darbepoetin composition contains 12 to 22 sialic acids per darbepoetin molecule, and 18 to 22 sialic acids per darbepoetin molecule. It is a darbepoetin composition containing sialic acid. Also, preferably, at least 55% of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule is a darbepoetin composition containing 19 to 22 sialic acids per darbepoetin molecule. . Also, preferably, at least 44% of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule is a darbepoetin composition containing 20 to 22 sialic acids per darbepoetin molecule. . Also, preferably, 18% or more of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule is a darbepoetin composition containing 22 sialic acids per darbepoetin molecule.

The darbepoetin composition in the culture medium obtained in step 1 is specifically, for example, darbepoetin containing 12 to 22 sialic acids per molecule of darbepoetin, preferably on the 10th to 14th day after the start of culture. Examples include darbepoetin compositions in which at least 67% of the composition contains 18 to 22 sialic acids per darbepoetin molecule. Furthermore, specifically, for example, preferably, 55% or more of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule at 10 to 14 days after the start of culture is darbepoetin. and a darbepoetin composition containing 19 to 22 sialic acids per molecule. Furthermore, specifically, for example, preferably, 44% or more of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule at 10 to 14 days after the start of culture is darbepoetin. and a darbepoetin composition containing 20 to 22 sialic acids per molecule. Furthermore, specifically, for example, preferably, 18% or more of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule at 10 to 14 days after the start of culture is darbepoetin. and a darbepoetin composition containing 22 sialic acids per molecule.

The number of sialic acids in the darbepoetin composition can be determined by measuring the distribution of sialic acid isoforms of darbepoetin by isoelectric point electrophoresis using a capillary electrophoresis device (manufactured by Beckman Coulter) or the like.

[Process 2]

Step 2 is a step of recovering the darbepoetin composition from the culture medium obtained in Step 1. The darbepoetin composition secreted into the culture medium by the culture in Step 1 can be purified according to a known method, and the production method of the present invention may further include a purification step of purifying the darbepoetin composition.

For example, a culture supernatant is obtained from the culture medium obtained in step 1 by a method such as centrifugation, and the culture supernatant is isolated and purified from the culture supernatant by conventional methods of isolation and purification of genetic recombinant proteins, such as solvent extraction, salting out by oil, etc., desalting, organic Precipitation using a solvent, anion exchange chromatography using a resin such as Q-Sepharose or DIAION HPA-75 (manufactured by Mitsubishi Chemical), and cation exchange using a resin such as S-Sepharose FF (manufactured by Pharmacia). Chromatographic methods, hydrophobic chromatography methods using resins such as butylsepharose or phenylsepharose, gel filtration methods using molecular sieves, reversed-phase chromatography methods, chromatofocusing methods, electrophoretic methods such as isoelectric point electrophoresis, MF A darbepoetin composition can be obtained by using a filtration method using a membrane such as UF/DF, virus removal membrane, concentration, or solvent exchange method alone or in combination.

The darbepoetin composition is preferably darbepoetin containing an average of 18 or more sialic acids per molecule, and more preferably an average of 18.5 or more sialic acids per molecule. The darbepoetin composition specifically contains, for example, an average of 18 or more sialic acids per molecule, more preferably an average of 18.5 or more sialic acids per molecule, preferably obtained from the culture medium 10 to 14 days after the start of culture. One example is Darbepoetin. Similarly to the above, the number of sialic acids in the darbepoetin composition can be determined by measuring the distribution of sialic acid isoforms of darbepoetin using isoelectric point electrophoresis using a capillary electrophoresis device (manufactured by Beckman Coulter), etc. there is.

The present invention relates to a method of cultivating darbepoetin-producing cells, comprising culturing the darbepoetin-producing cells in a medium supplemented with a plant-derived protein hydrolyzate and secreting a darbepoetin composition into the culture medium. Cultivation of darbepoetin-producing cells in the culture method is the same as Step 1 described above.

In addition, the present invention provides a darbepoetin-producing cell comprising culturing darbepoetin-producing cells in a medium supplemented with a plant-derived protein hydrolyzate and inhibiting the detachment of sialic acid from darbepoetin molecules in the culture. It relates to a culture method. Cultivation of darbepoetin-producing cells in the culture method is the same as Step 1 described above.

Additionally, the present invention relates to a method for inhibiting the detachment of sialic acid from darbepoetin molecules in storage in the culture medium, comprising culturing darbepoetin-producing cells in a medium supplemented with plant-derived protein hydrolyzate to obtain a culture medium. It's about. According to the method of the present invention, the detachment of sialic acid from the darbepoetin molecule by sialidase is inhibited by the action of the plant-derived protein hydrolyzate contained in the culture medium, and from the end of the culture until the start of purification. It is possible to improve the rate of addition of sialic acid to darbepoetin molecules during storage of the culture medium. Storage conditions for the culture medium are not particularly limited, but it is preferably stored at 8°C for 72 hours.

The present invention will be described in more detail through the following examples, but the examples are merely illustrative of the present invention and do not limit the scope of the present invention.

Example

[Example 1] Comparison of sialic acid addition rates of darbepoetin compositions produced in medium containing and without soy hydrolyzate

Using CHO cells producing darbepoetin, fed-batch culture was performed in a 1L Erlenmeyer flask, and the sialic acid addition rate of darbepoetin was compared in media containing and not containing soy hydrolyzate. Darbepoetin-producing CHO cells were expanded from a 125 mL Erlenmeyer flask to a 500 mL Erlenmeyer flask using EX-CELL325PF medium (manufactured by Sigma-Aldrich), and then further expanded into a 1 L Erlenmeyer flask (manufactured by Corning). Seed culture medium required for main culture was obtained.

Expansion culture was performed by seeding cells at 2 × 10 ⁵ cells/mL in a medium volume of approximately 10 to 30% of the volume of each flask for 3 or 4 days at 37°C. In a medium based on KINSM-10 medium (manufactured by Irvine Scientific), 3 g/L of soy hydrolysate (Soy Hydrolysate UF manufactured by Sigma-Aldrich; molecular weight distribution was 66% for <500 Da, 22% for 500 to 1000 Da, 1000 to 1000 Da). In a 1 L Erlenmeyer flask filled with 200 mL of production medium adjusted with (with Soy) or without (without Soy) added (9% for 2000 Da and 3% for 2000 to 5000 Da), the seed culture was centrifuged (1000 rpm, 25°C, for 5 minutes), and the cells from which the supernatant was removed were each seeded at 2.0 × 10 ⁵ cells/mL. The cell density immediately after seeding was 2.4×10 ⁵ cells/mL in the medium containing soy hydrolyzate and 1.8×10 ⁵ cells/mL in the medium without soy hydrolyzate.

After that, the main culture was started by blowing in 5% CO ₂ at 37°C, 100 rpm. The soy hydrolyzate-containing medium was supplemented with feed medium [FMDF-7 medium (manufactured by Irvine Scientific)] at 2% of the culture volume on days 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 after the start of culture. A medium containing 2 g/L of soy hydrolyzate (Soy Hydrolysate UF manufactured by Sigma-Aldrich) was added.

On the other hand, the soy hydrolyzate-free medium was supplemented with 2% of the culture medium amount of feed medium [FMDF-7 medium (Irvine) at 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 days after the start of culture. A medium containing 1.2 g/L potassium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) was added.

The culture medium was collected on the 7th, 10th, and 14th days after the start of the culture, and the total production of the darbepoetin composition in the culture medium was measured using Biacore (manufactured by GE Healthcare). Additionally, approximately 600 μg of the darbepoetin composition in the collected samples was separated and obtained using a separation device (AKTA explorer (manufactured by GE Healthcare)) having an anti-darbepoetin monoclonal antibody column.

The sialic acid isoform distribution of the obtained darbepoetin composition was measured using a capillary electrophoresis device (manufactured by Beckman Coulter). For the measurement of sialic acid isoform distribution, the cartridge temperature was set to 25°C, the voltage value was set to 6kV, and 10 mmol/L Tricine, 10 mmol/L NaCl, 10 mmol/L sodium acetate, 7 mmol/L urea (UREA), 2.5 This was carried out using a buffer containing mmol/L Putrescine, pH 4.7. The results of measuring the ratio (%) of the area value of the darbepoetin composition of each sialic acid adduct number to the total area value of the darbepoetin composition with 12 to 22 sialic acid adduct numbers are shown in Table 1 and Figure 4.

As shown in Table 1 and Figure 4, the proportion of darbepoetin with 18 to 22 sialic acid additions increased in the medium containing soy hydrolyzate (with Soy) compared to the medium without soy hydrolyzate (without Soy). On the 14th day of culture, the ratio was 12.3% for darbepoetin with a sialic acid addition number of 18, 11.2% for darbepoetin with a sialic acid addition number of 19, and 12.4% for darbepoetin with a sialic acid addition number of 20. For darbepoetin with 21 sialic acid additions, it was 13.6%, and for darbepoetin with 22 sialic acid additions, it was 18.6%. In particular, the proportion of darbepoetin with 22 sialic acid additions increased significantly.

As a result, as shown in Table 1, the average number of sialic acid additions per molecule in the medium containing soy hydrolyzate was 18.7. In addition, the total production of the darbepoetin composition containing 12 to 22 sialic acid additions increased to about 1.7 to 2.2 times in the medium containing soy hydrolyzate compared to the medium without soy hydrolyzate.

From the above results, it was confirmed that soybean hydrolyzate is effective as a medium composition for producing a darbepoetin composition with a large number of sialic acid additions.

[Example 2] Inhibition of sialic acid detachment from darbepoetin molecules by soy hydrolyzate in medium

The culture supernatant cultured in a medium containing soy hydrolyzate prepared in the same manner as in Example 1 was stored at 8°C for 0 to 72 hours and a stability test was performed. In addition, the culture supernatant cultured in the soy hydrolyzate-containing medium prepared in the same manner as in Example 1 was concentrated using a concentration membrane (Amicon Ultra-4 30000MWCO, manufactured by Millipore) and then replaced with MilliQ water to prepare a soy hydrolyzate removal solution. and similarly stored at 8°C for 0 to 72 hours. In addition, 0.5mM of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en), a known sialidase inhibitor, was added to the prepared soy hydrolyzate removal solution, and similarly, the temperature was 0 to 8°C at 8°C. Stored for 72 hours.

As a result of analyzing the samples after each storage using isoelectric electrophoresis, it was observed that in the soy hydrolyzate removed solution, detachment of sialic acid from darbepoetin molecules of high sialic acid addition over time was observed (Figure 3, lanes 2 to 2). 5). On the other hand, when NeuAc2en, an inhibitor of sialidase, was added to the culture supernatant (FIG. 3, lanes 6 to 9) cultured in a medium containing soy hydrolyzate and the soy hydrolyzate removal liquid and stored, darbepoetin, a high sialic acid hydrolyzate, was added and stored. Detachment of sialic acid from the molecule was not observed (Figure 3, lanes 10 to 13).

The above results suggest that the soybean hydrolyzate in the medium inhibits the detachment of sialic acid from the darbepoetin molecule of high sialic acid addition by sialidase.

Although the present invention has been described in detail using specific embodiments, it is clear to those skilled in the art that various changes and modifications can be made without departing from the intent and scope of the present invention. In addition, this application is based on the Japanese patent application (Japanese patent application 2017-040747) filed on March 3, 2017, which is incorporated by reference in its entirety.

Claims (19)

Step 1: Obtaining a culture medium by culturing darbepoetin-producing cells in a medium containing 1 to 5 g/l of soy-derived protein hydrolyzate, and culturing for at least 10 days; and
Process 2: Process of recovering darbepoetin composition from the culture medium obtained in Process 1
Includes,
A darbepoetin composition containing 18 to 22 sialic acids per darbepoetin molecule, at least 67% of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule in the culture medium obtained in Step 1. Phosphorus, method for producing darbepoetin composition. Step 1: Obtaining a culture medium by culturing darbepoetin-producing cells in a medium containing 1 to 5 g/l of soy-derived protein hydrolyzate, and culturing for at least 10 days; and
Process 2: Process of recovering darbepoetin composition from the culture medium obtained in Process 1
Includes,
A darbepoetin composition containing 18 to 22 sialic acids per darbepoetin molecule, at least 67% of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule in the culture medium obtained in Step 1. A method for producing a darbepoetin composition in which detachment of sialic acid from darbepoetin molecules is inhibited. The method according to claim 1 or 2, wherein at least 55% of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule in the culture solution obtained in step 1 is 19 to 22 sialic acids per darbepoetin molecule. A method of producing a darbepoetin composition containing canine sialic acid. The method according to claim 1 or 2, wherein at least 44% of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule in the culture solution obtained in step 1 is 20 to 22 sialic acids per darbepoetin molecule. A method of producing a darbepoetin composition containing canine sialic acid. The method according to claim 1 or 2, wherein at least 18% of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule in the culture solution obtained in step 1 is 22 sialic acids per darbepoetin molecule. A method of producing a darbepoetin composition containing an acid. The production method according to claim 1 or 2, wherein the total production amount of the darbepoetin composition in the culture medium obtained in step 1 is higher than when cultured in a medium containing no hydrolyzate of soybean-derived protein. The production method according to claim 1 or 2, wherein the darbepoetin-producing cell is a transformed cell into which a vector containing a gene encoding darbepoetin has been introduced into a host cell. The production method according to claim 7, wherein the host cells are animal cells. The production method according to claim 8, wherein the animal cells are Chinese hamster ovary (CHO) cells, mouse melanoma (NS0) cells, or mouse myeloma (SP2/0) cells, or cells derived from these cells. The production method according to claim 1 or 2, wherein the medium to which the soy-derived protein hydrolyzate is added is a completely synthetic medium. The production method according to claim 1 or 2, further comprising a purification process for purifying the darbepoetin composition. 12. The process according to claim 11, wherein the purification process is at least one selected from anion exchange chromatography, reverse phase chromatography or gel filtration. The production method according to claim 1 or 2, wherein the darbepoetin composition obtained is darbepoetin containing an average of 18 or more sialic acids per molecule. The method includes culturing darbepoetin-producing cells in a medium supplemented with 1 to 5 g/l of soy-derived protein hydrolyzate, culturing the cells for at least 10 days, and secreting the darbepoetin composition into the culture medium. A darbepoetin producing cell, wherein at least 67% of the darbepoetin composition containing 12 to 22 sialic acids per molecule of darbepoetin is a darbepoetin composition containing 18 to 22 sialic acids per molecule of darbepoetin. Cultivation method. Darbepoetin-producing cells are cultured in a medium containing 1 to 5 g/l of soy-derived protein hydrolyzate, the number of days of culture is at least 10 days, and the method includes inhibiting the detachment of sialic acid from the darbepoetin molecule in culture. And, more than 67% of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule in the culture medium is a darbepoetin composition containing 18 to 22 sialic acids per darbepoetin molecule. , Method for culturing darbepoetin producing cells. It involves obtaining a culture medium by culturing darbepoetin-producing cells in a medium containing 1 to 5 g/l of soy-derived protein hydrolyzate, the number of days of culture is at least 10 days, and 12 to 12 darbepoetin molecules per molecule in the culture medium. At least 67% of the darbepoetin composition containing 22 sialic acids is the sialic acid from darbepoetin molecules stored in the culture medium, which is a darbepoetin composition containing 18 to 22 sialic acids per darbepoetin molecule. How to inhibit tally. delete delete delete