本發明之達貝泊汀組合物之製造方法包括以下之步驟1及步驟2。 步驟1：於添加有源自植物之蛋白質水解物之培養基中培養達貝泊汀生產細胞而獲得培養液之步驟 步驟2：自步驟1中所獲得之培養液回收達貝泊汀組合物之步驟 以下，對各步驟進行說明。 [步驟1] 步驟1係於添加有源自植物之蛋白質水解物之培養基中培養具有生產達貝泊汀之能力之細胞，使達貝泊汀組合物分泌至培養基中而獲得培養液的步驟。 於本發明中，達貝泊汀係於國際公開第95/05465號中，人紅血球生成素之胺基酸序列中30、32、87、88及90位之胺基酸殘基分別被置換為Asn、Thr、Val、Asn及Thr的以[Asn30
Thr32
Val87
Asn88
Thr90
]EPO表示之人紅血球生成素類似物，具有圖1所示之結構。 於達貝泊汀之24、30、38、83及88位之Asn殘基鍵結有N-鍵結型糖鏈，於126位之Ser殘基鍵結有O-鍵結型糖鏈，作為N-鍵結型糖鏈及O-鍵結型糖鏈之主要結構，可列舉圖2所示之結構。 鍵結於達貝泊汀分子上之N-鍵結型糖鏈中最多包含4個唾液酸，於O-鍵結型糖鏈之末端最多包含2個唾液酸，於達貝泊汀1分子中最多包含22個唾液酸。 於本發明中，作為達貝泊汀，例如亦包含達貝泊汀α或達貝泊汀α(基因重組)或該等之生物相似性藥品。又，國際公開第2003/020299號所揭示之新紅細胞生成刺激蛋白(novel erythropoiesis stimulating protein)及NESP(商標)或Aranesp(商標)、或者該等之生物相似性藥品亦包括在本發明之達貝泊汀中。 作為本發明所使用之達貝泊汀生產細胞之宿主細胞，可任意地使用屬於哺乳類、鳥類、爬蟲類、兩棲類、魚類及昆蟲類等任一者之動物細胞等，較佳為使用屬於哺乳類之動物細胞，更佳為使用源自人或猴等靈長類之動物細胞或源自小鼠、大鼠或倉鼠等嚙齒類之動物細胞。 作為屬於哺乳類之細胞，例如可列舉骨髓瘤細胞或源自骨髓瘤細胞之細胞、卵巢細胞、腎臟細胞、血球細胞、子宮細胞、結締組織細胞、乳腺細胞或胚胎視網膜母細胞等。特別是較佳為選自骨髓瘤細胞或源自骨髓瘤細胞之細胞及卵巢細胞之細胞，具體而言，例如可列舉中國倉鼠卵巢(CHO)細胞、小鼠黑色素瘤(NS0)細胞或小鼠骨髓瘤(SP2/0)細胞、或者源自該等細胞之細胞。 作為屬於哺乳類之細胞，例如可列舉作為人細胞株之HL-60(ATCC CCL-240)、HT-1080(ATCC CCL-121)、HeLa(ATCC CCL-2)、293(ECACC 85120602)、Namalwa(ATCC CRL-1432)、Namalwa KJM-1(Cytotechnology, 1, 151(1988))、NM-F9(DSM ACC2605、國際公開第05/17130號)或PER.C6(ECACC No.96022940、美國專利第6855544號說明書)，作為猴細胞株之VERO(ATCC CCL-1651)或COS-7(ATCC CRL-1651)，作為小鼠細胞株之C127I(ATCC CRL-1616)、Sp2/0-Ag14(ATCC CRL-1581)、NIH3T3(ATCCCRL-1658)、NS0(ATCC CRL-1827)，作為大鼠細胞株之Y3 Ag1.2.3.(ATCC CRL 1631)、YO(ECACCNo：85110501)及YB2/0(ATCC CRL-1662)，作為倉鼠細胞株之CHO-K1(ATCC CCL-61)、CHO/dhfr-
(ATCC CRL-9096)、CHO/DG44[Proc. Natl. Acad. Sci. USA, 77, 4216(1980)]或BHK21(ATCC CRL-10)，或者作為狗細胞之MDCK(ATCC CCL-34)等。 作為屬於鳥類之細胞，例如可列舉雞細胞株SL-29(ATCC CRL-29)等，作為屬於魚類之細胞，例如可列舉斑馬魚細胞株ZF4(ATCC CRL-2050)等，作為屬於昆蟲類之細胞，例如可列舉蛾(Spodoptera frugiperda)細胞株Sf9(ATCC CRL-1711)等。又，作為例如初代培養細胞，可列舉初代猴腎細胞、初代兔腎細胞、初代雞母細胞或初代鵪鶉母細胞等。 作為骨髓瘤細胞或源自骨髓瘤細胞之細胞，例如可列舉Sp2/0-Ag14、NS0、Y3 Ag1.2.3.、YO或YB2/0等。作為卵巢細胞或源自卵巢細胞之細胞，例如可列舉CHO-K1、CHO/dhfr-
或CHO/DG44等。 又，作為腎臟細胞，例如可列舉293、VERO、COS-7、BHK21或MDCK等，作為血球細胞，可列舉HL-60、Namalwa、Namalwa KJM-1或NM-F9等，作為子宮細胞，例如可列舉HeLa等，作為結締組織細胞，例如可列舉HT-1080或NIH3T3等，作為乳腺細胞，例如可列舉C1271I等，作為胚胎視網膜母細胞，例如可列舉PER.C6等。 作為達貝泊汀生產細胞，例如可列舉於上述宿主細胞中導入含有編碼達貝泊汀之基因的載體而成之轉形細胞、實施過突變處理而可產生達貝泊汀之細胞、或作為產生達貝泊汀之細胞與骨髓瘤細胞之融合細胞的融合瘤等。進而，對上述細胞實施過提昇達貝泊汀之表現量之突變處理而成之細胞等亦包括在本發明之達貝泊汀生產細胞中。 導入含有編碼達貝泊汀之基因的載體而成之轉形細胞係藉由將包含參與達貝泊汀之生產之DNA(deoxyribonucleic acid，去氧核糖核酸)與啟動子等之重組體載體等導入至上述本發明所使用之宿主細胞中而獲得。 作為參與達貝泊汀之生產之DNA，例如可使用編碼達貝泊汀之DNA、編碼與達貝泊汀之生物合成有關之酶或蛋白質之DNA等之任一者。 作為用於製備該重組體載體之載體，例如可列舉pcDNAI、pcDM8(Funakoshi公司製造)、pAGE107[日本專利特開平3-22979號公報、Cytotechnology, 3, 133(1990)]、pAS3-3(日本專利特開平2-227075號公報)、pcDM8[Nature, 329, 840(1987)]、pcDNAI/Amp(Invitrogen公司製造)、pREP4(Invitrogen公司製造)、pAGE103[J. Biochem., 101, 1307(1987)]、或pAGE210等。 作為啟動子，只要為於本發明中使用之動物細胞中發揮功能者，則可使用任一者，例如可列舉巨細胞病毒(CMV)之IE(immediate early，即刻早期)基因之啟動子、SV40之早期啟動子、反轉錄病毒之啟動子、金屬硫蛋白啟動子、熱休克啟動子或SRα啟動子等。又，亦可將人CMV之IE基因之促進子等與啟動子一同使用。 作為向宿主細胞導入重組體載體之方法，例如只要為向該細胞中導入DNA之方法，則可使用任意方法，例如可列舉電穿孔法[Cytotechnology, 3, 133(1990)]、磷酸鈣法(日本專利特開平2-227075號公報)、或脂質體轉染法[Proc. Natl. Acad. Sci. USA, 84, 7413(1987)、或Virology, 52, 456(1973)]等。 作為源自植物之蛋白質水解物，只要為源自小麥、稻或大豆等植物之蛋白質水解物，則可使用任意之蛋白質水解物，尤佳為源自大豆之蛋白質水解物。 源自植物之蛋白質水解物係以將由成為原料之植物所獲得之蛋白質分解而獲得之胺基酸及二肽或三肽等短鏈肽作為主成分，此外亦包含微量金屬等的混合物。關於源自植物之蛋白質水解物之製造，可列舉使用鹽酸等酸之酸分解法或使用蛋白酶等酶之酶分解法，視需要進行膜處理。 源自植物之蛋白質水解物之平均分子量較佳為800 Da以下，以下階梯性地更佳為600 Da以下，進而較佳為450 Da以下，尤佳為300 Da以下。 作為源自植物之蛋白質水解物之分子量分佈，＜500 Da較佳為45～80%，500～1000 Da較佳為15～30%，1000～2000 Da較佳為5～15%，2000～5000 Da較佳為2～10%。或者，＜2000 Da更佳為90～98%，＜2000 Da進而較佳為95～98%。 作為源自大豆之蛋白質水解物，例如可列舉Amysoy、Hy-Soy、N-Z-Soy、HyPep(以上為Quest International公司)、Soy peptone(Gibco公司)、Bac-Soyton(Difco公司)、SE50MK(DMV International Nutritions公司)、Peptone Hy-Soy T、或Soy Hydrolysate UF(以上為Sigma-Aldrich公司製造)等，作為源自小麥或稻之蛋白質水解物，可列舉HyPep(Quest International公司)等。 將源自植物之蛋白質水解物添加至培養液之時期可為培養開始時，亦可為培養中途，並無特別限制，較佳為添加至培養開始時之培養基中並使用。於作為分批補料培養法或灌注培養法等中之補料培養基而在培養中途添加之情形時，源自植物之蛋白質水解物可單獨作為補料培養基而添加至培養基中，亦可作為與其他培養基成分預先混合之補料培養基而添加至培養基中。 作為培養時間，並無特別限制，作為最終之正式培養之時間，較佳為8天以上。於分批補料培養法中，作為正式培養之時間，較佳為8天～1個月，更佳為10天～21天，尤佳為10天～14天。 添加至培養基中之源自植物之蛋白質水解物之濃度可根據用於培養之細胞之種類、源自植物之蛋白質水解物之添加時期等而適當選擇，較佳為0.1～100 g/L，進而較佳為1～10 g/L，尤佳為1～5 g/L。 作為本發明所使用之培養基，只要可用於達貝泊汀生產細胞之培養，則可為任意者，較佳為使用不含血清或源自血清之物質或源自動物之蛋白質之完全合成培養基(chemically defined medium)。 於達貝泊汀生產細胞之宿主細胞為動物細胞之情形時，作為基礎培養基，使用通常用於動物細胞之培養的基礎培養基。作為通常用於動物細胞之培養的基礎培養基，例如可列舉RPMI1640培養基[The Journal of the American Medical Association、199, 519(1967)]、伊格爾之MEM(minimum Eagle’s medium，低限量伊格爾培養基)培養基[Science, 122, 501(1952)]、杜爾貝科改良MEM(DMEM)培養基[Virology, 8, 396(1959)]、199培養基[Proceeding of the Society for the Biological Medicine, 73, 1(1950)]、F12培養基(LTI公司製造)[Proc. Natl. Acad. Sci. USA, 53, 288(1965)]、伊思科夫改良杜爾貝科培養基(IMDM培養基)[J. Experimental Medicine, 147, 923(1978)]或EX-CELL325PF培養基(JRH公司製造)或該等之改良培養基或混合培養基等，較佳為使用RPMI1640培養基、DMEM培養基、F12培養基、IMDM培養基或EX-CELL 325PF培養基等。 作為本發明所使用之培養基，係於基礎培養基中視需要添加動物細胞之生長所必需之營養因子、或生理活性物質等。該等添加物較佳為於培養前預先含有於培養基中。 作為營養因子，例如可列舉葡萄糖等糖、胺基酸、或維生素等。 作為胺基酸，例如可列舉L-丙胺酸、L-精胺酸、L-天冬醯胺、L-天冬胺酸、L-胱胺酸、L-麩胺酸、L-麩醯胺、甘胺酸、L-組胺酸、L-異白胺酸、L-白胺酸、L-離胺酸、L-甲硫胺酸、L-苯丙胺酸、L-脯胺酸、L-絲胺酸、L-蘇胺酸、L-色胺酸、L-酪胺酸或L-纈胺酸等，可使用1種或將2種以上組合而使用。 作為維生素，例如可列舉d-生物素、D-泛酸、膽鹼、葉酸、myo-肌醇、菸鹼醯胺、吡哆醛、核黃素、硫胺素、氰鈷胺或DL-α-生育酚等，可使用1種或將2種以上組合而使用。 此外，於不含源自動物之物質之完全合成培養基中，作為代替源自動物之物質而添加之物質，例如可列舉藉由基因重組法所製造之生理活性物質、水解物或不含源自動物之物質之脂質、礦物質、微量金屬或核酸等。作為藉由基因重組法所製造之生理活性物質，例如可列舉基因重組胰島素、基因重組運鐵蛋白、基因重組白蛋白或基因重組增生因子等。 作為不含源自動物之物質之脂質，例如可列舉膽固醇、亞麻油酸、或次亞麻油酸等。 作為完全合成培養基，例如可列舉ADPF培養基(Animal derived proteinfree medium，無動物源蛋白培養基；HyClone公司製造)、CD-融合瘤培養基(Invitrogen公司製造)、CD-CHO培養基(Invitrogen公司製造)、IS CD-CHO培養基、或KINSM-10培養基(Irvine Scientific公司製造)等。 於以長時間或高密度進行培養之情形時，可適宜地使用以高濃度含有胺基酸類及維生素類之培養基、例如將RPMI1640培養基、DMEM培養基及F12培養基以1：1：1之比率加以混合而成之培養基、將DMEM培養基及F12培養基以1：1之比率加以混合而成之培養基、融合瘤SFM(serum free medium，無血清培養基)培養基(Invitrogen公司製造)等。 於本發明中，步驟1中所獲得之培養液中之達貝泊汀組合物之總生產量提高意指利用含有上述源自植物之蛋白質水解物之培養基進行培養時，培養結束時點之單位培養液量中之達貝泊汀組合物之濃度與利用不含上述源自植物之水解物之培養基培養時相比提昇。 關於單位培養液量中之達貝泊汀組合物之濃度，只要使用ELISA(enzyme linked immunosorbent assay，酶結合免疫吸附分析)法或HPLC(high performance liquid chromatography，高效液相層析)法等公知之蛋白質濃度測定方法求出即可，可使用任意方法求出，例如可列舉使用Biacore(GE Healthcare公司製造)之蛋白質濃度測定方法。 作為步驟1中所獲得之培養液中之達貝泊汀組合物，具有達貝泊汀每1分子加成有較佳為18～22個、更佳為19～22個、尤佳為20～22個、最佳為22個唾液酸之結構。 作為步驟1中所獲得之培養液中之達貝泊汀組合物，較佳為達貝泊汀每1分子含有12～22個唾液酸之達貝泊汀組合物之67%以上為達貝泊汀每1分子含有18～22個唾液酸之達貝泊汀組合物。又，較佳為達貝泊汀每1分子含有12～22個唾液酸之達貝泊汀組合物之55%以上為達貝泊汀每1分子含有19～22個唾液酸之達貝泊汀組合物。又，較佳為達貝泊汀每1分子含有12～22個唾液酸之達貝泊汀組合物之44%以上為達貝泊汀每1分子含有20～22個唾液酸之達貝泊汀組合物。又，較佳為達貝泊汀每1分子含有12～22個唾液酸之達貝泊汀組合物之18%以上為達貝泊汀每1分子含有22個唾液酸之達貝泊汀組合物。 作為步驟1中所獲得之培養液中之達貝泊汀組合物，具體而言，例如較佳可列舉培養開始後第10～14天，達貝泊汀每1分子含有12～22個唾液酸之達貝泊汀組合物之67%以上為達貝泊汀每1分子含有18～22個唾液酸之達貝泊汀組合物。又，具體而言，例如較佳可列舉培養開始後第10～14天，達貝泊汀每1分子含有12～22個唾液酸之達貝泊汀組合物之55%以上為達貝泊汀每1分子含有19～22個唾液酸之達貝泊汀組合物。又，具體而言，例如較佳可列舉培養開始後第10～14天，達貝泊汀每1分子含有12～22個唾液酸之達貝泊汀組合物之44%以上為達貝泊汀每1分子含有20～22個唾液酸之達貝泊汀組合物。又，具體而言，例如較佳可列舉培養開始後第10～14天，達貝泊汀每1分子含有12～22個唾液酸之達貝泊汀組合物之18%以上為達貝泊汀每1分子含有22個唾液酸之達貝泊汀組合物。 達貝泊汀組合物中之唾液酸數可藉由利用使用毛細管電泳裝置(Beckman Coulter公司製造)等之等電點電泳法等測定達貝泊汀之唾液酸異構物分佈而求出。 [步驟2] 步驟2係自步驟1中所獲得之培養液回收達貝泊汀組合物之步驟。藉由步驟1中之培養而分泌至培養液中之達貝泊汀組合物可依據公知之方法進行純化，本發明之製造方法亦可進而包括純化達貝泊汀組合物之純化步驟。 例如，可藉由自步驟1中所獲得之培養液藉由離心分離等方法獲得培養上清液，單獨使用或組合使用如下方法自該培養上清液獲得達貝泊汀組合物，上述方法為：通常之基因重組蛋白質之單離純化法、即溶劑萃取法、利用硫酸銨等之鹽析法、脫鹽法、利用有機溶劑之沈澱法、使用Q-Sepharose或DIAION HPA-75(Mitsubishi Chemical公司製造)等樹脂之陰離子交換層析法、使用S-Sepharose FF(Pharmacia公司製造)等樹脂之陽離子交換層析法、使用丁基瓊脂糖凝膠(butyl Sepharose)或苯基瓊脂糖凝膠(phenyl Sepharose)等樹脂之疏水性層析法、使用分子篩之凝膠過濾法、逆相層析法、層析聚焦法、等電點電泳等電泳法、MF(microfiltration，微濾)、UF/DF(ultrafiltration/diafiltration，超濾/滲濾)、使用病毒去除膜等膜之過濾法或者濃縮或溶劑交換法等。 作為達貝泊汀組合物，較佳為每1分子平均含有18個以上、更佳為每1分子平均含有18.5個以上之唾液酸之達貝泊汀。作為達貝泊汀組合物，具體而言，例如較佳可列舉自培養開始後第10～14天之培養液獲得之每1分子平均含有18個以上之唾液酸之達貝泊汀，更佳為列舉每1分子平均含有18.5個以上之唾液酸之達貝泊汀。與上述同樣地，達貝泊汀組合物中之唾液酸數可藉由使用毛細管電泳裝置(Beckman Coulter公司製造)等之等電點電泳法等測定達貝泊汀之唾液酸異構物分佈而求出。 本發明係關於一種達貝泊汀生產細胞之培養方法，其包括於添加有源自植物之蛋白質水解物之培養基中培養達貝泊汀生產細胞，使達貝泊汀組合物分泌至培養液中。關於該培養方法中之達貝泊汀生產細胞之培養，與上述步驟1相同。 又，本發明係關於一種達貝泊汀生產細胞之培養方法，其包括於添加有源自植物之蛋白質水解物之培養基中培養達貝泊汀生產細胞，抑制培養中唾液酸自達貝泊汀分子脫離。關於該培養方法中之達貝泊汀生產細胞之培養，與上述步驟1相同。 又，本發明係關於一種抑制培養液保存中唾液酸自達貝泊汀分子脫離之方法，其包括於添加有源自植物之蛋白質水解物之培養基中培養達貝泊汀生產細胞而獲得培養液。根據本發明之方法，藉由含有於培養液之源自植物之蛋白質水解物之作用，能夠抑制因唾液酸酶引起之唾液酸自達貝泊汀分子之脫離，提高培養結束後至開始純化前之培養液之保存中唾液酸對達貝泊汀分子之加成率。培養液之保存條件並無特別限定，較佳為可於8℃下保存72小時。 藉由以下之實施例對本發明更具體地進行說明，但實施例僅為本發明之例示，並非限定本發明之範圍。實施例
[實施例1]利用含大豆水解物之培養基與不含大豆水解物之培養基所生產之達貝泊汀組合物之唾液酸加成率之比較 使用生產達貝泊汀之CHO細胞，於1 L錐形燒瓶中進行分批補料培養，比較含大豆水解物之培養基與不含大豆水解物之培養基中達貝泊汀之唾液酸加成率。對達貝泊汀產生CHO細胞使用EX-CELL325PF培養基(Sigma-Aldrich公司製造)，自125 mL容量錐形燒瓶換至500 mL容量錐形燒瓶進行擴大培養，然後進而利用1 L容量錐形燒瓶(Corning公司製造)進行擴大培養，而獲得正式培養所必需之種培養液。 擴大培養係以各燒瓶容量之約10～30%之培養基量以成為2×105
cell/mL之方式接種細胞，並於37℃下培養3天或4天。向以KINSM-10培養基(Irvine Scientific公司製造)為基礎之培養基中添加3 g/L之大豆水解物(Soy Hydrolysate UF，Sigma-Aldrich公司製造；關於分子量分佈，＜500 Da為66%，500～1000 Da為22%，1000～2000 Da為9%，2000～5000 Da為3%)(有大豆)或不進行添加(無大豆)，向裝有所調整之生產培養基200 mL之1 L錐形燒瓶中以成為2.0×105
cell/mL之方式分別接種對種培養液進行離心分離(1000 rpm、25℃、5分鐘)並去除上清液所獲得之細胞。剛接種後之細胞密度於含大豆水解物之培養基中為2.4×105
cell/mL，於不含大豆水解物之培養基中為1.8×105
cell/mL。 其後，通入37℃、100 rpm、5%之CO2
，開始正式培養。於含大豆水解物之培養基中，於培養開始後第4、5、6、7、8、9、10、11、12及13天分別添加培養液量之2%之補料培養基[向FMDF-7培養基(Irvine Scientific公司製造)中添加2 g/L之大豆水解物(Soy Hydrolysate UF，Sigma-Aldrich公司製造)而成之培養基]。 另一方面，於不含大豆水解物之培養基中，於培養開始後第4、5、6、7、8、9、10、11、12及13天分別添加培養液量之2%之補料培養基[向FMDF-7培養基(Irvine Scientific公司製造)中添加1.2 g/L之氯化鉀(和光純藥工業公司製造)而成之培養基]。 於培養開始後第7天、第10天及第14天採集培養液，使用Biacore(GE Healthcare公司製造)測定培養液中之達貝泊汀組合物之總生產量。進而對該等採集樣本使用具有抗達貝泊汀單株抗體管柱之分離裝置[AKTA explorer(GE Healthcare公司製造)]將採集樣本中之達貝泊汀組合物每次分離約600 μg而獲取。 使用毛細管電泳裝置(Beckman Coulter公司製造)測定所獲得之達貝泊汀組合物之唾液酸異構物分佈。唾液酸異構物分佈測定係將藥筒(Cartridge)溫度設定為25℃、將電壓值設定為6 kV，使用包含10 mmol/L之N-三(羥甲基)甲基甘胺酸(Tricine)、10 mmol/L之NaCl、10 mmol/L之乙酸鈉、7 mmol/L之尿素、2.5 mmol/L之腐胺(Putrescine)、pH值4.7之緩衝液而實施。將測定各唾液酸加成數之達貝泊汀組合物之面積值相對於唾液酸加成數12～22個之達貝泊汀組合物之總面積值之比率(%)所得的結果示於表1及圖4。 [表1]
如表1及圖4所示，含大豆水解物之培養基(有大豆)與不含大豆水解物之培養基(無大豆)相比，唾液酸加成數為18個～22個之達貝泊汀之比率增加。於培養第14天，關於其比率，唾液酸加成數為18個之達貝泊汀為12.3%，唾液酸加成數為19個之達貝泊汀為11.2%，唾液酸加成數為20個之達貝泊汀為12.4%，唾液酸加成數為21個之達貝泊汀為13.6%，唾液酸加成數為22個之達貝泊汀為18.6%，特別是唾液酸加成數為22個之達貝泊汀之比率明顯增加。 作為其結果，如表1所示，於含大豆水解物之培養基中，每1分子之平均唾液酸加成數為18.7個。又，關於唾液酸加成數12～22個之達貝泊汀組合物之總生產量，利用含大豆水解物之培養基時與利用不含大豆水解物之培養基時相比增加至約1.7～2.2倍。 根據以上結果，確認大豆水解物作為用以製造唾液酸加成數較多之達貝泊汀組合物之培養基組合物有效。 [實施例2]利用培養基中之大豆水解物抑制唾液酸自達貝泊汀分子脫離 將在藉由與實施例1相同之方法所製備之含大豆水解物之培養基中進行培養而獲得之培養上清液於8℃下保管0～72小時而進行穩定性試驗。又，將在藉由與實施例1相同之方法所製備之含大豆水解物之培養基中進行培養而獲得之培養上清液使用濃縮膜(Millipore公司製造、Amicon Ultra-4 30000MWCO)進行濃縮後，利用MilliQ水進行置換而製備大豆水解物去除液，同樣地於8℃下保管0～72小時。進而，向所製備之大豆水解物去除液中添加作為公知之唾液酸酶之抑制劑的2-脫氧-2,3-脫氫-N-乙醯神經胺酸(NeuAc2en)0.5 mM，同樣地於8℃下保管0～72小時。 使用等電點電泳法對各保管後之試樣進行分析，結果於大豆水解物去除液中確認到唾液酸經時性地自唾液酸高加成數之達貝泊汀分子脫離(圖3、條帶2～5)。另一方面，於利用含大豆水解物之培養基進行培養所獲得之培養上清液(圖3、條帶6～9)及於大豆水解物去除液中添加作為唾液酸酶之抑制劑的NeuAc2en並進行保管之情形時，未確認到唾液酸自唾液酸高加成數之達貝泊汀分子脫離(圖3、條帶10～13)。 根據以上結果，提示培養基中之大豆水解物會抑制因唾液酸酶引起之唾液酸自唾液酸高加成數之達貝泊汀分子之脫離。 以上，使用特定之態樣對本發明進行了詳細說明，但從業者明瞭可於不脫離本發明之意圖與範圍之情況下施加各種變更及變化。再者，本申請係基於2017年3月3日提出申請之日本專利申請(日本專利特願2017-040747)，藉由引用而援用其全文。The method for producing the darbepoetin composition of the present invention comprises the following steps 1 and 2. Step 1: culturing darbepoetin-producing cells in a culture medium supplemented with a plant-derived protein hydrolysate to obtain a culture solution Step 2: recovering the darbepoetin composition from the culture solution obtained in step 1 The following describes each step. [Step 1] Step 1 is a step of culturing cells capable of producing darbepoetin in a culture medium supplemented with a plant-derived protein hydrolysate, allowing the darbepoetin composition to be secreted into the culture medium to obtain a culture solution. In the present invention, darbepoetin is a human erythropoietin analogue represented by [Asn 30 Thr 32 Val 87 Asn 88 Thr 90 ]EPO in which the amino acid residues at positions 30 , 32, 87, 88 and 90 in the amino acid sequence of human erythropoietin in International Publication No. 95/05465 are replaced with Asn, Thr , Val , Asn and Thr , respectively, and has a structure shown in FIG. 1 . The Asn residues at positions 24, 30, 38, 83 and 88 of darbepoetin are bonded to an N-bonded sugar chain, and the Ser residue at position 126 is bonded to an O-bonded sugar chain. As the main structures of the N-bonded sugar chain and the O-bonded sugar chain, the structure shown in FIG2 can be cited. The N-bonded sugar chain bonded to the darbepoetin molecule contains up to 4 sialic acids, the end of the O-bonded sugar chain contains up to 2 sialic acids, and the darbepoetin 1 molecule contains up to 22 sialic acids. In the present invention, darbepoetin includes, for example, darbepoetin α or darbepoetin α (recombinant) or biosimilar drugs thereof. In addition, novel erythropoiesis stimulating protein and NESP (trademark) or Aranesp (trademark) disclosed in International Publication No. 2003/020299, or biosimilar drugs thereof are also included in the darbepoetin of the present invention. As host cells of the darbepoetin-producing cells used in the present invention, animal cells belonging to any of mammals, birds, reptiles, amphibians, fish and insects can be used arbitrarily, preferably animal cells belonging to mammals, more preferably animal cells derived from primates such as humans or monkeys, or animal cells derived from rodents such as mice, rats or hamsters. Examples of mammalian cells include myeloma cells or cells derived from myeloma cells, ovarian cells, kidney cells, blood cells, uterine cells, connective tissue cells, breast cells, and embryonic retinal progenitor cells. In particular, cells selected from myeloma cells or cells derived from myeloma cells and ovarian cells are preferred, and specifically, examples include Chinese hamster ovary (CHO) cells, mouse melanoma (NS0) cells, or mouse myeloma (SP2/0) cells, or cells derived from these cells. Examples of mammalian cells include HL-60 (ATCC CCL-240), HT-1080 (ATCC CCL-121), HeLa (ATCC CCL-2), 293 (ECACC 85120602), Namalwa (ATCC CRL-1432), Namalwa KJM-1 (Cytotechnology, 1, 151 (1988)), NM-F9 (DSM ACC2605, International Publication No. 05/17130) or PER.C6 (ECACC No. 96022940, U.S. Patent No. 6855544) as human cell lines, VERO (ATCC CCL-1651) or COS-7 (ATCC CRL-1651), C127I (ATCC CRL-1616), Sp2/0-Ag14 (ATCC CRL-1581), NIH3T3 (ATCC CRL-1658), NS0 (ATCC CRL-1827) as mouse cell lines, Y3 Ag1.2.3. (ATCC CRL 1631), YO (ECACC No: 85110501) and YB2/0 (ATCC CRL-1662) as rat cell lines, 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 dog cells such as MDCK (ATCC CCL-34). Examples of bird cells include chicken cell strain SL-29 (ATCC CRL-29), fish cells include zebrafish cell strain ZF4 (ATCC CRL-2050), and insect cells include moth (Spodoptera frugiperda) cell strain Sf9 (ATCC CRL-1711). Examples of primary culture cells include primary monkey kidney cells, primary rabbit kidney cells, primary chicken mother cells, or primary quail mother 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. In addition, examples of kidney cells include 293, VERO, COS-7, BHK21, or MDCK, examples of blood cells include HL-60, Namalwa, Namalwa KJM-1, or NM-F9, examples of uterine cells include HeLa, examples of connective tissue cells include HT-1080 or NIH3T3, examples of breast cells include C1271I, and examples of embryonic retinal progenitor cells include PER.C6. Examples of darbepoetin-producing cells include transformed cells obtained by introducing a vector containing a gene encoding darbepoetin into the above-mentioned host cells, cells that have been subjected to mutation treatment to produce darbepoetin, or fusion tumors that are fusion cells of darbepoetin-producing cells and myeloma cells. Furthermore, cells obtained by subjecting the above-mentioned cells to mutation treatment to increase the expression of darbepoetin are also included in the darbepoetin-producing cells of the present invention. Transformed cells into which a vector containing a gene encoding darbepoetin is introduced are obtained by introducing a recombinant vector containing a DNA (deoxyribonucleic acid) involved in the production of darbepoetin and a promoter into the host cells used in the present invention. As the DNA involved in the production of darbepoetin, for example, any one of a DNA encoding darbepoetin, a DNA encoding an enzyme or a protein involved in the biosynthesis of darbepoetin, etc. can be used. Examples of the vector used for preparing the recombinant vector include pcDNAI, pcDM8 (manufactured by Funakoshi Co., Ltd.), 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 Co., Ltd.), pREP4 (manufactured by Invitrogen Co., Ltd.), pAGE103 [J. Biochem., 101, 1307 (1987)], and pAGE210. As a promoter, any promoter may be used as long as it functions in the animal cells used in the present invention, for example, the promoter of the IE (immediate early) gene of cytomegalovirus (CMV), the early promoter of SV40, the promoter of retrovirus, the metallothionein promoter, the heat shock promoter, or the SRα promoter, etc. In addition, a promoter of the IE gene of human CMV, etc. may be used together with the promoter. As a method for introducing a recombinant vector into a host cell, any method can be used as long as it is a method for introducing DNA into the cell, for example, electroporation method [Cytotechnology, 3, 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), or liposome transfection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987), or Virology, 52, 456 (1973)], etc. As a plant-derived protein hydrolyzate, any protein hydrolyzate can be used as long as it is derived from a plant such as wheat, rice or soybean, and a soybean-derived protein hydrolyzate is particularly preferred. Plant-derived protein hydrolysates are mainly composed of amino acids and short-chain peptides such as dipeptides or tripeptides obtained by decomposing proteins obtained from plants as raw materials, and also contain a mixture of trace metals. The production of plant-derived protein hydrolysates can be exemplified by acid decomposition using acids such as hydrochloric acid or enzymatic decomposition using enzymes such as proteases, and membrane treatment is performed as needed. The average molecular weight of plant-derived protein hydrolysates is preferably 800 Da or less, and more preferably 600 Da or less, further preferably 450 Da or less, and most preferably 300 Da or less. As for the molecular weight distribution of plant-derived protein hydrolysates, <500 Da is preferably 45-80%, 500-1000 Da is preferably 15-30%, 1000-2000 Da is preferably 5-15%, and 2000-5000 Da is preferably 2-10%. Alternatively, <2000 Da is more preferably 90-98%, and <2000 Da is further preferably 95-98%. As the protein hydrolysate derived from soybean, for example, Amysoy, Hy-Soy, NZ-Soy, HyPep (all from Quest International), Soy peptone (Gibco), Bac-Soyton (Difco), SE50MK (DMV International Nutritions), Peptone Hy-Soy T, or Soy Hydrolysate UF (all from Sigma-Aldrich), etc. can be cited, and as the protein hydrolysate derived from wheat or rice, HyPep (Quest International) can be cited, etc. The time when the plant-derived protein hydrolysate is added to the culture solution may be at the beginning of the culture or in the middle of the culture, and there is no particular limitation, but it is preferably added to the culture medium at the beginning of the culture and used. When used as a feed medium in a batch feeding culture method or a perfusion culture method and added during the culture, the plant-derived protein hydrolyzate can be added to the culture medium as a feed medium alone, or can be added to the culture medium as a feed medium pre-mixed with other culture medium components. There is no particular restriction on the culture time, but the final formal culture time is preferably 8 days or more. In the batch feeding culture method, the formal culture time is preferably 8 days to 1 month, more preferably 10 days to 21 days, and even more preferably 10 days to 14 days. The concentration of the plant-derived protein hydrolysate added to the culture medium can be appropriately selected according to the type of cells used for culture, the period of addition of the plant-derived protein hydrolysate, etc., and is preferably 0.1 to 100 g/L, more preferably 1 to 10 g/L, and particularly preferably 1 to 5 g/L. The culture medium used in the present invention may be any medium as long as it can be used for culturing darbepoetin-producing cells, but preferably a completely synthetic medium (chemically defined medium) that does not contain serum or serum-derived substances or animal-derived proteins is used. When the host cells of the darbepoetin-producing cells are animal cells, a basal medium commonly used for culturing animal cells is used as the basal medium. Examples of basal media commonly used for culturing animal cells include RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM (minimum Eagle's medium) 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)], and F12 medium (manufactured by LTI Corporation) [Proc. Natl. Acad. Sci. USA, 53, 53 (1967)]. 288 (1965)], Iskoff's modified Dulbecco's medium (IMDM medium) [J. Experimental Medicine, 147, 923 (1978)] or EX-CELL325PF medium (manufactured by JRH) or their modified medium or mixed medium, preferably RPMI1640 medium, DMEM medium, F12 medium, IMDM medium or EX-CELL 325PF medium. The medium used in the present invention is a basic medium to which nutritional factors or physiologically active substances necessary for the growth of animal cells are added as needed. These additives are preferably contained in the medium before culture. Examples of nutritional factors include sugars such as glucose, amino acids, or vitamins. Examples of amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, or L-valine, and one or more of these may be used alone or in combination. 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 more of them may be used in combination. In addition, in a completely synthetic medium that does not contain animal-derived substances, examples of substances added to replace animal-derived substances include physiologically active substances produced by gene recombination, hydrolyzates, or lipids that do not contain animal-derived substances, minerals, trace metals, or nucleic acids, etc. Examples of physiologically active substances produced by gene recombination include recombinant insulin, recombinant transferrin, recombinant albumin, or recombinant growth factor, etc. Examples of lipids that do not contain animal-derived substances include cholesterol, linolenic acid, or linolenic acid. Examples of completely synthetic culture media include ADPF medium (Animal derived protein free medium, animal-derived protein free medium; manufactured by HyClone), CD-fusion tumor medium (manufactured by Invitrogen), CD-CHO medium (manufactured by Invitrogen), IS CD-CHO medium, or KINSM-10 medium (manufactured by Irvine Scientific). When culturing is performed for a long time or at a high density, a medium containing amino acids and vitamins at a high concentration, for example, a medium obtained by mixing RPMI1640 medium, DMEM medium and F12 medium at a ratio of 1:1:1, a medium obtained by mixing DMEM medium and F12 medium at a ratio of 1:1, Fusionoma SFM (serum free medium) medium (manufactured by Invitrogen), etc. can be suitably used. In the present invention, the total production amount of the darbepoetin composition in the culture solution obtained in step 1 is increased, which means that when the culture medium containing the plant-derived protein hydrolysate is used for culture, the concentration of the darbepoetin composition per unit volume of the culture solution at the end of the culture is increased compared with the case of culture using a culture medium not containing the plant-derived protein hydrolysate. The concentration of the darbepoetin composition per unit volume of the culture solution can be determined by a known protein concentration measurement method such as ELISA (enzyme linked immunosorbent assay) or HPLC (high performance liquid chromatography). Any method can be used for determination, and for example, a protein concentration measurement method using Biacore (manufactured by GE Healthcare) can be cited. The darbepoetin composition in the culture medium obtained in step 1 has a structure in which 18 to 22, more preferably 19 to 22, particularly preferably 20 to 22, and most preferably 22 sialic acids are added to each darbepoetin molecule. The darbepoetin composition in the culture medium obtained in step 1 is preferably a darbepoetin composition containing 18 to 22 sialic acids per darbepoetin molecule in an amount of 67% or more. In addition, it is preferred that a darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule in an amount of 55% or more is a darbepoetin composition containing 19 to 22 sialic acids per darbepoetin molecule. Furthermore, preferably, 44% or more 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. Furthermore, 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. As the darbepoetin composition in the culture solution obtained in step 1, specifically, for example, preferably, on the 10th to 14th day after the start of culture, 67% or more of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule is a darbepoetin composition containing 18 to 22 sialic acids per darbepoetin molecule. Furthermore, specifically, for example, it is preferred that on the 10th to 14th day after the start of culture, 55% or more 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. Furthermore, specifically, for example, it is preferred that on the 10th to 14th day after the start of culture, 44% or more 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. Specifically, for example, it is preferable that 18% or more of the darbepoetin composition containing 12 to 22 sialic acids per darbepoetin molecule on the 10th to 14th day after the start of culture is a darbepoetin composition containing 22 sialic acids per darbepoetin molecule. The number of sialic acids in the darbepoetin composition can be determined by measuring the sialic acid isomer distribution of darbepoetin using an isoelectric point electrophoresis method using a capillary electrophoresis apparatus (manufactured by Beckman Coulter). [Step 2] Step 2 is a step of recovering the darbepoetin composition from the culture solution 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. The production method of the present invention may further include a purification step of purifying the darbepoetin composition. For example, the culture supernatant obtained from the culture solution obtained in step 1 can be obtained by centrifugation or the like, and the darbepoetin composition can be obtained from the culture supernatant by using the following methods alone or in combination: conventional gene recombinant protein isolation purification method, solvent extraction method, salt precipitation method using ammonium sulfate or the like, desalination method, precipitation method using an organic solvent, anion exchange chromatography method using a resin such as Q-Sepharose or DIAION HPA-75 (manufactured by Mitsubishi Chemical Co., Ltd.), cation exchange chromatography method using a resin such as S-Sepharose FF (manufactured by Pharmacia Co., Ltd.), butyl agarose gel (butyl agarose gel) or the like. Sepharose) or phenyl Sepharose, gel filtration using molecular sieves, reverse phase chromatography, chromatography focusing, electrophoresis such as isoelectric electrophoresis, MF (microfiltration), UF/DF (ultrafiltration/diafiltration), filtration using a virus removal membrane or other membrane, or concentration or solvent exchange method. As the darbepoetin composition, preferably, one containing an average of 18 or more sialic acids per molecule, more preferably, one containing an average of 18.5 or more sialic acids per molecule. Specifically, for example, a darbepoetin composition preferably contains 18 or more sialic acids per molecule in the culture medium obtained on the 10th to 14th day after the start of culture, and more preferably contains 18.5 or more sialic acids per molecule. Similarly to the above, the number of sialic acids in the darbepoetin composition can be determined by measuring the sialic acid isomer distribution of darbepoetin using an isoelectric point electrophoresis method such as a capillary electrophoresis apparatus (manufactured by Beckman Coulter). The present invention relates to a method for culturing darbepoetin-producing cells, which comprises culturing darbepoetin-producing cells in a culture medium supplemented with a plant-derived protein hydrolysate, so that a darbepoetin composition is secreted into the culture medium. The culturing of the darbepoetin-producing cells in the culturing method is the same as the above-mentioned step 1. In addition, the present invention relates to a method for culturing darbepoetin-producing cells, which comprises culturing the darbepoetin-producing cells in a culture medium supplemented with a plant-derived protein hydrolysate, and inhibiting the dissociation of sialic acid from darbepoetin molecules during the culture. The culturing of the darbepoetin-producing cells in the culturing method is the same as the above-mentioned step 1. In addition, the present invention relates to a method for inhibiting the dissociation of sialic acid from darbepoetin molecules during storage of a culture medium, which comprises culturing darbepoetin-producing cells in a culture medium supplemented with a plant-derived protein hydrolysate to obtain a culture medium. According to the method of the present invention, the dissociation of sialic acid from darbepoetin molecules caused by sialidase can be inhibited by the action of the plant-derived protein hydrolysate contained in the culture medium, thereby increasing the addition rate of sialic acid to darbepoetin molecules during storage of the culture medium after the end of the culture and before the start of purification. The storage conditions of the culture medium are not particularly limited, and preferably can be stored at 8°C for 72 hours. The present invention is described in more detail by the following examples, but the examples are merely illustrative of the present invention and do not limit the scope of the present invention. Examples [Example 1] Comparison of sialic acid addition rates of darbepoetin compositions produced using a culture medium containing soybean hydrolysate and a culture medium not containing soybean hydrolysate Using CHO cells producing darbepoetin, batch feeding culture was performed in a 1 L Erlenmeyer flask to compare the sialic acid addition rates of darbepoetin in a culture medium containing soybean hydrolysate and a culture medium not containing soybean hydrolysate. Darbepoetin-producing CHO cells were cultured in EX-CELL325PF medium (manufactured by Sigma-Aldrich) from a 125 mL Erlenmeyer flask to a 500 mL Erlenmeyer flask, and then cultured in a 1 L Erlenmeyer flask (manufactured by Corning) to obtain the seed culture solution required for formal culture. The cells were inoculated with about 10-30% of the volume of the medium in each flask to a concentration of 2×10 5 cells/mL and cultured at 37°C for 3 or 4 days. 3 g/L soybean hydrolysate (Soy Hydrolysate UF, Sigma-Aldrich; molecular weight distribution: <500 Da: 66%, 500-1000 Da: 22%, 1000-2000 Da: 9%, 2000-5000 Da: 3%) was added to a culture medium based on KINSM-10 medium (Irvine Scientific) (with soybean) or without addition (without soybean). 200 mL of the adjusted production medium was inoculated into a 1 L Erlenmeyer flask, and the seed culture solution was centrifuged (1000 rpm, 25°C, 5 minutes) to obtain cells by removing the supernatant. The cell density immediately after inoculation was 2.4×10 5 cells/mL in the medium containing soybean hydrolysate and 1.8×10 5 cells/mL in the medium without soybean hydrolysate. After that, the medium was aerated at 37°C, 100 rpm, and 5% CO 2 to start the formal culture. In the medium containing soybean hydrolysate, a feed medium (a medium prepared by adding 2 g/L soybean hydrolysate (Soy Hydrolysate UF, manufactured by Sigma-Aldrich) to FMDF-7 medium (manufactured by Irvine Scientific) at 2% of the volume of the culture solution was added on the 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, and 13th day after the start of the culture. On the other hand, in the medium not containing soybean hydrolyzate, a feed medium (a medium prepared by adding 1.2 g/L potassium chloride (manufactured by Wako Junyaku Industries) to FMDF-7 medium (manufactured by Irvine Scientific) at 2% of the volume of the culture medium was added on days 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 after the start of culture. The culture medium was collected on days 7, 10, and 14 after the start of culture, and the total production of the darbepoetin composition in the culture medium was measured using Biacore (manufactured by GE Healthcare). The collected samples were then separated using a separation device [AKTA explorer (manufactured by GE Healthcare)] with an anti-darbepoetin monoclonal antibody column to obtain about 600 μg of the darbepoetin composition in each sample. The sialic acid isomer distribution of the obtained darbepoetin composition was measured using a capillary electrophoresis device (manufactured by Beckman Coulter). The distribution of sialic acid isomers was measured at a cartridge temperature of 25°C and a voltage of 6 kV using a buffer solution containing 10 mmol/L N-tris(hydroxymethyl)methylglycine, 10 mmol/L NaCl, 10 mmol/L sodium acetate, 7 mmol/L urea, 2.5 mmol/L putrescine, and a pH of 4.7. The results of measuring the ratio (%) of the area value of the darbepoetin composition with each sialic acid addition number to the total area value of the darbepoetin composition with 12 to 22 sialic acid addition numbers are shown in Table 1 and Figure 4. [Table 1] As shown in Table 1 and FIG4 , the ratio of darbepoetin with sialic acid addition numbers of 18 to 22 increased in the medium containing soybean hydrolysate (with soybean) compared to the medium without soybean hydrolysate (without soybean). On the 14th day of culture, the ratio of darbepoetin with sialic acid addition numbers of 18 to 22 was 12.3%, darbepoetin with sialic acid addition numbers of 19 to 11.2%, darbepoetin with sialic acid addition numbers of 20 to 12.4%, darbepoetin with sialic acid addition numbers of 21 to 13.6%, and darbepoetin with sialic acid addition numbers of 22 to 18.6%. In particular, the ratio of darbepoetin with sialic acid addition numbers of 22 increased significantly. As a result, as shown in Table 1, the average number of sialic acid additions per molecule in the culture medium containing soybean hydrolysate was 18.7. In addition, the total production of darbepoetin compositions having sialic acid additions of 12 to 22 increased by about 1.7 to 2.2 times when using a culture medium containing soybean hydrolysate compared to when using a culture medium not containing soybean hydrolysate. Based on the above results, it was confirmed that soybean hydrolysate is effective as a culture medium composition for producing darbepoetin compositions having a large number of sialic acid additions. [Example 2] Inhibition of sialic acid dissociation from dabepoetin molecules by using soybean hydrolysate in culture medium The culture supernatant obtained by culturing in a culture medium containing soybean hydrolysate prepared by the same method as in Example 1 was stored at 8°C for 0 to 72 hours to conduct a stability test. In addition, the culture supernatant obtained by culturing in a culture medium containing soybean hydrolysate prepared by the same method as in Example 1 was concentrated using a concentration membrane (Amicon Ultra-4 30000MWCO manufactured by Millipore) and replaced with MilliQ water to prepare a soybean hydrolysate-removed solution, which was similarly stored at 8°C for 0 to 72 hours. Furthermore, 0.5 mM of 2-deoxy-2,3-dehydro-N-acetylneuramine (NeuAc2en), a known inhibitor of sialidase, was added to the prepared soybean hydrolysate-removed solution, and the solution was stored at 8°C for 0 to 72 hours. The samples after each storage were analyzed by isoelectric point electrophoresis, and the results showed that sialic acid was dissociated from the darbepoetin molecules with a high number of sialic acid additions over time in the soybean hydrolysate-removed solution (Figure 3, bands 2 to 5). On the other hand, in the culture supernatant obtained by culturing with a culture medium containing soybean hydrolysate (Figure 3, bands 6-9) and in the case where NeuAc2en as a sialidase inhibitor was added to the soybean hydrolysate removal solution and stored, no sialic acid was confirmed to be separated from the sialic acid high addition number darbepoetin molecule (Figure 3, bands 10-13). Based on the above results, it is suggested that the soybean hydrolysate in the culture medium will inhibit the separation of sialic acid from the sialic acid high addition number darbepoetin molecule caused by sialidase. The above, the present invention is described in detail using a specific embodiment, but practitioners understand that various changes and modifications can be applied 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 No. 2017-040747) filed on March 3, 2017, the entire content of which is incorporated by reference.