JPH06292592A - Production of glycoprotein - Google Patents
Production of glycoproteinInfo
- Publication number
- JPH06292592A JPH06292592A JP6031019A JP3101994A JPH06292592A JP H06292592 A JPH06292592 A JP H06292592A JP 6031019 A JP6031019 A JP 6031019A JP 3101994 A JP3101994 A JP 3101994A JP H06292592 A JPH06292592 A JP H06292592A
- Authority
- JP
- Japan
- Prior art keywords
- sugar
- medium
- glycoprotein
- sugar chain
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000003886 Glycoproteins Human genes 0.000 title claims abstract description 48
- 108090000288 Glycoproteins Proteins 0.000 title claims abstract description 48
- 238000004519 manufacturing process Methods 0.000 title claims description 11
- 235000000346 sugar Nutrition 0.000 claims abstract description 104
- 210000004102 animal cell Anatomy 0.000 claims abstract description 13
- 239000000203 mixture Substances 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 15
- 238000012258 culturing Methods 0.000 claims description 14
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 238000000034 method Methods 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 8
- 229930182830 galactose Natural products 0.000 abstract description 7
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 abstract description 6
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 abstract description 6
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 abstract description 6
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 37
- 239000002609 medium Substances 0.000 description 26
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 25
- 239000008103 glucose Substances 0.000 description 16
- 239000000427 antigen Substances 0.000 description 12
- 102000036639 antigens Human genes 0.000 description 12
- 108091007433 antigens Proteins 0.000 description 12
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 12
- 210000004408 hybridoma Anatomy 0.000 description 10
- 235000010443 alginic acid Nutrition 0.000 description 8
- 229920000615 alginic acid Polymers 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- 229920001661 Chitosan Polymers 0.000 description 7
- 239000000783 alginic acid Substances 0.000 description 7
- 229960001126 alginic acid Drugs 0.000 description 7
- 150000004781 alginic acids Chemical class 0.000 description 7
- 229960000633 dextran sulfate Drugs 0.000 description 7
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 6
- SRBFZHDQGSBBOR-SOOFDHNKSA-N D-ribopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@@H]1O SRBFZHDQGSBBOR-SOOFDHNKSA-N 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 229920001282 polysaccharide Polymers 0.000 description 6
- 239000005017 polysaccharide Substances 0.000 description 6
- 150000004804 polysaccharides Chemical class 0.000 description 6
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 5
- -1 N-acetylglucosamine Chemical compound 0.000 description 5
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 229930091371 Fructose Natural products 0.000 description 4
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 4
- 239000005715 Fructose Substances 0.000 description 4
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 229940045110 chitosan Drugs 0.000 description 4
- 239000012894 fetal calf serum Substances 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 150000002772 monosaccharides Chemical class 0.000 description 4
- 229950006780 n-acetylglucosamine Drugs 0.000 description 4
- 230000009257 reactivity Effects 0.000 description 4
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 3
- 102000003951 Erythropoietin Human genes 0.000 description 3
- 108090000394 Erythropoietin Proteins 0.000 description 3
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229940105423 erythropoietin Drugs 0.000 description 3
- 229960002442 glucosamine Drugs 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000012679 serum free medium Substances 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 2
- 108700023372 Glycosyltransferases Proteins 0.000 description 2
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 2
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 2
- 229930182474 N-glycoside Natural products 0.000 description 2
- YJQCOFNZVFGCAF-UHFFFAOYSA-N Tunicamycin II Natural products O1C(CC(O)C2C(C(O)C(O2)N2C(NC(=O)C=C2)=O)O)C(O)C(O)C(NC(=O)C=CCCCCCCCCC(C)C)C1OC1OC(CO)C(O)C(O)C1NC(C)=O YJQCOFNZVFGCAF-UHFFFAOYSA-N 0.000 description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- ZHSGGJXRNHWHRS-VIDYELAYSA-N tunicamycin Chemical compound O([C@H]1[C@@H]([C@H]([C@@H](O)[C@@H](CC(O)[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C(NC(=O)C=C2)=O)O)O1)O)NC(=O)/C=C/CC(C)C)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1NC(C)=O ZHSGGJXRNHWHRS-VIDYELAYSA-N 0.000 description 2
- MEYZYGMYMLNUHJ-UHFFFAOYSA-N tunicamycin Natural products CC(C)CCCCCCCCCC=CC(=O)NC1C(O)C(O)C(CC(O)C2OC(C(O)C2O)N3C=CC(=O)NC3=O)OC1OC4OC(CO)C(O)C(O)C4NC(=O)C MEYZYGMYMLNUHJ-UHFFFAOYSA-N 0.000 description 2
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 101100068867 Caenorhabditis elegans glc-1 gene Proteins 0.000 description 1
- 101100068866 Caenorhabditis elegans glc-2 gene Proteins 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 102000005367 Carboxypeptidases Human genes 0.000 description 1
- 108010006303 Carboxypeptidases Proteins 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 229940121672 Glycosylation inhibitor Drugs 0.000 description 1
- 102000051366 Glycosyltransferases Human genes 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- DUKURNFHYQXCJG-UHFFFAOYSA-N Lewis A pentasaccharide Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(C(O)C(O)C(CO)O2)O)C(NC(C)=O)C(OC2C(C(OC3C(OC(O)C(O)C3O)CO)OC(CO)C2O)O)OC1CO DUKURNFHYQXCJG-UHFFFAOYSA-N 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 229920000057 Mannan Polymers 0.000 description 1
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 description 1
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- MKXKFYHWDHIYRV-UHFFFAOYSA-N flutamide Chemical compound CC(C)C(=O)NC1=CC=C([N+]([O-])=O)C(C(F)(F)F)=C1 MKXKFYHWDHIYRV-UHFFFAOYSA-N 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000005861 gene abnormality Effects 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 102000045442 glycosyltransferase activity proteins Human genes 0.000 description 1
- 108700014210 glycosyltransferase activity proteins Proteins 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 150000002972 pentoses Chemical class 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000013630 prepared media Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 102220201851 rs143406017 Human genes 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、培地中で動物細胞を培
養して糖蛋白質を生産する際に、細胞の培養条件、特に
培地条件を制御することにより、蛋白質に結合する糖鎖
の種類あるいは分子量を改変し、特性の変化した糖蛋白
質を生産する方法に関する。TECHNICAL FIELD The present invention relates to the type of sugar chain that binds to a protein by controlling the cell culture conditions, particularly the medium conditions, when animal cells are cultured in a medium to produce glycoproteins. Alternatively, the present invention relates to a method for producing a glycoprotein having altered properties by modifying its molecular weight.
【０００２】[0002]
【従来の技術】リンホカイン、サイトカイン、ホルモ
ン、免疫グロブリンなど生体内で重要な役割を果たす生
理活性蛋白質は、殆どが、糖鎖の結合した糖蛋白質であ
る。これらの糖蛋白質のポリペプチド部分は、その遺伝
子がクローニングされさえすれば、遺伝子組み換え法に
より大量生産が可能となっている。しかし糖蛋白質の生
産は、大腸菌などのような原核細胞では糖鎖が結合しな
いため、通常は哺乳動物細胞を用いた生産がおこなわれ
ている。糖蛋白質の糖鎖のはたす役割は完全には解明さ
れていないが、大腸菌により生産された糖鎖の結合しな
い蛋白質の研究から、糖鎖が欠失した蛋白質は生体内の
半減期が、もとの糖蛋白質に比較して著しく短縮するこ
とが知られている（村上他、農化、62巻、1498-1510 、
1988) 。また抗体においてはＦｃ部分に結合する糖鎖
は、Ｆｃ活性の発現に必須であること、造血ホルモンで
あるエリスロポエチンでは、in vivo の活性発現には必
須であることが判明している。さらに糖鎖は、抗体の抗
原特異性にも関与していることが明らかにされている。
村上らは、抗体のＬ鎖に糖鎖を有するモノクローナル抗
体を生産するハイブリドーマを得て、この抗原認識性を
詳細に検討した結果、Ｌ鎖結合糖鎖の有無で抗原認識性
が大きくかわることを確認している（Murakami H.,et a
l.,Animal Cell Technology:Basic & Applied Aspects,
547-551,Kluver Academic Publishers,1992)。このよう
に生理活性糖蛋白質の活性発現には、その結合糖鎖の有
無が重要な機能を果たすことが明らかとなった。Most of physiologically active proteins such as lymphokines, cytokines, hormones and immunoglobulins which play important roles in the living body are glycoproteins to which sugar chains are bound. The polypeptide portion of these glycoproteins can be mass-produced by the gene recombination method as long as the gene is cloned. However, glycoproteins are usually produced using mammalian cells because sugar chains do not bind to prokaryotic cells such as Escherichia coli. Although the role of sugar chains in glycoproteins has not been completely elucidated, studies of proteins produced by E. coli that do not bind to sugar chains indicate that proteins lacking sugar chains have a short half-life in vivo. It is known to be remarkably shortened in comparison with the glycoprotein of Murakami et al. (Murakami et al., Vol. 62, 1498-1510,
1988). In addition, it has been revealed that in the antibody, the sugar chain that binds to the Fc portion is essential for the expression of Fc activity, and that the erythropoietin, which is a hematopoietic hormone, is essential for the expression of activity in vivo. Furthermore, it has been clarified that the sugar chain is also involved in the antigen specificity of the antibody.
Murakami et al. Obtained a hybridoma that produces a monoclonal antibody having a sugar chain in the L chain of the antibody, and examined the antigen recognizability in detail. Confirmed (Murakami H., et a
l., Animal Cell Technology: Basic & Applied Aspects,
547-551, Kluver Academic Publishers, 1992). Thus, it was revealed that the presence or absence of the attached sugar chain plays an important role in the activity expression of the bioactive glycoprotein.
【０００３】糖鎖は、真核細胞においては、細胞核内に
おいてペプチド部分が合成され、その後、ゴルジ中で糖
鎖が付加されると考えられている。糖鎖を構成する糖
は、グルコース、ガラクトース、マンノース、N-アセチ
ルグルコサミンなどのヘキソース、L-アラビノースなど
のペントースなど11種類の単糖から構成されているが、
これら糖種の組み合わせによって無限に近い種類の糖鎖
を構成することができる。この糖鎖の結合は細胞の持つ
糖転移酵素によって変化する。癌細胞など遺伝子の異常
がある細胞は、これらの糖転移酵素の発現が狂い、正常
な細胞と異なる糖鎖が合成される。糖鎖の結合はその細
胞株特有の糖鎖が結合するものとこれまでは考えられて
きた。また糖鎖を結合させないようにするために、細胞
内でのグリコシル化阻害剤であるツニカマイシン１０μ
ｇ／ｍｌ程度を培地中に加えて培養することにより糖鎖
の結合しない蛋白質が得られることは良く知られてい
る。糖鎖の結合はN-グリコシド結合の場合、アスパラギ
ン（Asn 以下アミノ酸の表記は３文字表記で示す) にN-
アセチルグルコサミンが結合する。この場合、Asn-X-Se
r(またはThr)(Xは任意のアミノ酸を示す) の配列を有す
るアミノ酸配列のAsnに糖鎖が結合する。これまでは、
このN-グリコシド糖鎖の結合に関与するアミノ酸を遺伝
子レベルで変換して糖鎖の結合を変えるか、あるいは遺
伝子組み換えの際の宿主細胞を変えることによって糖鎖
を変更することが行われてきた。後者の代表的な例とし
てエリスロポエチンの生産例を挙げることができる。エ
リスロポエチンは、分子量約34000 の糖蛋白質であり、
この蛋白質は166 個のアミノ酸からなる。このアミノ酸
配列をコードする遺伝子をＣＨＯ細胞とφ２細胞に導入
し発現させた場合その糖鎖構造が異なることが知られて
いる。このように宿主細胞を変えて、糖鎖構造の異なる
糖蛋白質を得る方法が知られている。また糖蛋白質をグ
ルカナーゼなどの酵素を用いて処理を行い、糖鎖を部分
的に切断したりする方法も試みられているが大量生産に
はあまり向いていない。糖蛋白質の糖鎖を改変して、あ
らたな機能を有する糖鎖を持つ糖蛋白質を得る方法は、
糖鎖工学として最近新たに開発された技術であるが、ま
だ目的とする糖鎖を自由に得ることは出来ないのが現状
である。It is considered that in eukaryotic cells, a peptide part is synthesized in the cell nucleus of a sugar chain, and then the sugar chain is added in the Golgi. Glucose, galactose, mannose, hexoses such as N-acetylglucosamine, and pentoses such as L-arabinose are composed of 11 types of monosaccharides.
By combining these sugar species, almost infinite kinds of sugar chains can be constructed. The binding of this sugar chain is changed by the glycosyltransferase possessed by the cell. In a cell having a gene abnormality such as a cancer cell, expression of these glycosyltransferases is disordered, and a different sugar chain from that in a normal cell is synthesized. Up to now, it has been considered that sugar chains are bound by sugar chains specific to the cell line. In addition, in order to prevent the sugar chain from binding, tunicamycin 10 μ, which is an intracellular glycosylation inhibitor, is used.
It is well known that a protein to which sugar chains are not bound can be obtained by adding about g / ml to a medium and culturing. When the sugar chain is N-glycoside, the N-glycosidic bond is added to asparagine (Asn and the amino acid notation is shown in three letter notation).
Acetylglucosamine binds. In this case, Asn-X-Se
A sugar chain binds to Asn having an amino acid sequence having a sequence of r (or Thr) (X represents an arbitrary amino acid). until now,
Amino acids involved in the binding of N-glycoside sugar chains have been altered at the gene level to change the sugar chain binding, or the sugar chain has been altered by changing the host cell during gene recombination. . As a typical example of the latter, an example of erythropoietin production can be mentioned. Erythropoietin is a glycoprotein with a molecular weight of about 34,000,
This protein consists of 166 amino acids. It is known that when a gene encoding this amino acid sequence is introduced into and expressed in CHO cells and φ2 cells, their sugar chain structures are different. Thus, a method is known in which host cells are changed to obtain glycoproteins having different sugar chain structures. Further, a method of treating glycoprotein with an enzyme such as glucanase to partially cleave the sugar chain has been tried, but it is not suitable for mass production. A method for modifying a sugar chain of a glycoprotein to obtain a glycoprotein having a sugar chain having a new function is
Although it is a technology newly developed as sugar chain engineering recently, it is the current situation that the desired sugar chain cannot be obtained freely.
【０００４】[0004]
【発明が解決しようとする課題】本発明者らは糖蛋白質
の糖鎖構造に関する研究を進める過程において、これま
で報告されてきた糖蛋白質の生産と全く異なる現象を見
出した。これまでの報告によれば、糖蛋白質の糖鎖は上
述したように、宿主細胞など糖蛋白質を発現する細胞に
固有のものであり、癌化などの遺伝子の変異が起こらな
い限り糖鎖は変わらないものと考えられてきた。ところ
が本発明者らは、抗体生産ハイブリドーマの培養を検討
したところ、細胞固有であると考えられていた糖鎖が、
細胞培養条件に応じて変化してくることを見出した。本
発明は、このような知見に基づいてなされたもので、動
物細胞を培養し、糖蛋白質を生産する際に培地中の糖組
成および／または糖濃度を変えることにより、結合糖鎖
の種類あるいは分子量を改変する糖蛋白質の生産方法を
提供することを課題とする。糖鎖の種類あるいは分子量
を改変することにより、得られる糖蛋白質の活性や安定
性の異なる物質を得ることが可能となる。DISCLOSURE OF THE INVENTION In the course of research on the sugar chain structure of glycoproteins, the present inventors have found a phenomenon completely different from the glycoprotein production reported so far. According to the reports so far, as described above, the sugar chain of a glycoprotein is unique to cells expressing the glycoprotein such as a host cell, and the sugar chain remains unchanged unless gene mutation such as canceration occurs. It has been considered to be absent. However, the present inventors examined the culture of antibody-producing hybridomas, and found that the sugar chains that were considered to be cell-specific were
It was found that it changes depending on the cell culture conditions. The present invention has been made based on such findings, and when culturing animal cells and producing a glycoprotein, by changing the sugar composition and / or the sugar concentration in the medium, the kind of bound sugar chain or It is an object of the present invention to provide a method for producing a glycoprotein whose molecular weight is modified. By modifying the type or molecular weight of the sugar chain, it becomes possible to obtain substances with different activity and stability of the obtained glycoprotein.
【０００５】[0005]
【課題を解決するための手段】これまで動物細胞の培養
にあたっては、細胞の生育に最も適した培地組成を選択
し、その培養条件を変更しないことが原則であった。従
って細胞培養において最大の課題は、細胞の消費する各
種成分をモニターし、それぞれの成分の減少分を補給
し、最適条件を維持することであった。本発明は、細胞
の生育の最適条件といわれる糖濃度、あるいは糖組成と
異なる条件で、糖蛋白質を生産する動物細胞を培養する
ところにある。すなわち、本発明は、培地中で動物細胞
を培養し糖蛋白質を生産するに当り、培地中の糖組成物
および／または糖濃度を変えることにより蛋白質に結合
する糖鎖の種類あるいは分子量を改変することを特徴と
する糖蛋白質の製造法である。動物細胞のなかでも哺乳
動物細胞はその成育条件が良く知られており、本発明を
実施するに最適である。このような哺乳動物細胞として
は、抗体生産ハイブリドーマ、ＣＨＯ細胞、Ｃ１２７細
胞等を挙げることができる。動物細胞を培養する場合の
培養液中の糖は通常グルコースが用いられるが、まれ
に、培養液中の乳酸の生成を抑制する目的でフラクトー
スが添加される。本発明によれば、糖としてグルコース
を用いる場合はその濃度を０〜４０mMの範囲で変化させ
るか、あるいはグルコースに代えて培養液中の糖質をフ
ラクトース、マンノース、ガラクトース、N-アセチルグ
ルコサミン、リボース、フコース、N-アセチルガラクト
サミンなど、糖蛋白質の糖鎖構造中に存在する糖質に交
換することで糖鎖を変換することが可能である。あるい
はキトサン、デキストラン硫酸、アルギン酸など糖鎖を
構成する糖などを含有する多糖を培養中に添加すること
で所望する効果を得ることができる。これは糖蛋白質の
糖鎖を変更する方法としては、全く新しい方法である。In culturing animal cells, it has been a general rule to select a medium composition most suitable for cell growth and not change the culture conditions. Therefore, the biggest challenge in cell culture was to monitor various components consumed by cells, supplement the depleted amount of each component, and maintain optimal conditions. The present invention resides in culturing an animal cell that produces a glycoprotein under conditions that differ from the sugar concentration or sugar composition, which is said to be the optimum condition for cell growth. That is, in the present invention, when an animal cell is cultured in a medium to produce a glycoprotein, the kind or molecular weight of the sugar chain bound to the protein is modified by changing the sugar composition and / or the sugar concentration in the medium. A method for producing a glycoprotein characterized by the following. Among the animal cells, mammalian cells are well known for their growth conditions, and are most suitable for carrying out the present invention. Examples of such mammalian cells include antibody-producing hybridomas, CHO cells, C127 cells and the like. Glucose is usually used as the sugar in the culture medium when culturing animal cells, but rarely fructose is added for the purpose of suppressing the production of lactic acid in the culture medium. According to the present invention, when glucose is used as the sugar, the concentration thereof is changed in the range of 0 to 40 mM, or the sugar in the culture medium is replaced with glucose to replace fructose, mannose, galactose, N-acetylglucosamine, ribose. It is possible to convert a sugar chain by exchanging it with a sugar present in the sugar chain structure of glycoprotein, such as fucose, fucose or N-acetylgalactosamine. Alternatively, a desired effect can be obtained by adding a polysaccharide containing a sugar constituting a sugar chain such as chitosan, dextran sulfate, and alginic acid to the culture. This is a completely new method for changing the sugar chain of a glycoprotein.
【０００６】目的とする糖蛋白質を生産する細胞、特に
遺伝子組み換えにより形質転換させた細胞株を、その細
胞の生育に適した培地組成を選択して培養を行う。糖鎖
の結合状態を変えるためには、最適な糖濃度よりも低い
糖濃度で培養を行うことが、好ましい場合が多い。この
場合の培地は無血清培地が好ましいが、血清含有培地で
あっても差し支えない。ハイブリドーマなど哺乳動物細
胞の培養に優れた特性を示す培地として、ＭＥＭ、ハム
Ｆ−１０培地、ハムＦ−１２培地、ＲＰＭＩ１６４０培
地、ＥＲＤＦ培地などが例示できるが、これらの培地に
使用する最適糖濃度の０〜５０％濃度にすることによ
り、糖蛋白質の糖鎖を変更することができる。実施例に
例示するように、本発明による糖鎖の変更は糖濃度によ
る影響が大きいため、血清が生育に必須な細胞株を用い
る場合には、血清を透析して糖を除去しておくことが好
ましい。グルコース以外の糖質を使用する場合は、細胞
の生育のための最適濃度のグルコース含量全てあるいは
一部を、他の糖質に置き換えることにより、糖鎖の変更
された糖蛋白質を得ることができる。とくにこのような
効果を示す糖質としては、フラクトース、マンノース、
ガラクトース、グルコサミン、リボース等の単糖を例示
することができるが、特にガラクトース、グルコサミ
ン、リボースの使用が好ましい。また多糖としては、キ
トサン、デキストラン硫酸、アルギン酸、ヘパリン、キ
チン、マンナンなどを例示することができ、特にキトサ
ン、デキストラン硫酸、アルギン酸が好ましい。アルギ
ン酸は塩であっても良いが、特にアルギン酸ナトリウム
が好ましい。A cell producing the desired glycoprotein, particularly a cell line transformed by gene recombination, is cultured by selecting a medium composition suitable for the growth of the cell. In order to change the binding state of sugar chains, it is often preferable to carry out the culture at a sugar concentration lower than the optimum sugar concentration. The medium in this case is preferably a serum-free medium, but a serum-containing medium may be used. Examples of the medium exhibiting excellent characteristics for culturing mammalian cells such as hybridoma include MEM, Ham's F-10 medium, Ham's F-12 medium, RPMI1640 medium, ERDF medium, etc., but the optimum sugar concentration to be used in these mediums. The sugar chain of the glycoprotein can be changed by adjusting the concentration to 0 to 50%. As illustrated in the examples, the sugar chain change according to the present invention is greatly influenced by the sugar concentration. Therefore, when using a cell line in which serum is essential for growth, the serum should be dialyzed to remove the sugar. Is preferred. When a sugar other than glucose is used, a glycoprotein having a modified sugar chain can be obtained by replacing all or part of the glucose content at the optimum concentration for cell growth with another sugar. . In particular, sugars showing such effects include fructose, mannose,
Although monosaccharides such as galactose, glucosamine and ribose can be exemplified, use of galactose, glucosamine and ribose is particularly preferable. Examples of polysaccharides include chitosan, dextran sulfate, alginic acid, heparin, chitin, mannan and the like, with chitosan, dextran sulfate and alginic acid being particularly preferred. Although alginic acid may be a salt, sodium alginate is particularly preferable.
【０００７】細胞の培養は、細胞に適した培養方法であ
れば、どのような培養方法であっても使用可能である。
例えば、タンクでの浮遊培養、マイクロキャリアーやウ
レタン素材への接着培養、ホローファイバーによる培養
等が例示できる。また細胞が一定密度になるまでは、そ
の細胞に適した従来のグルコース濃度の培養液で培養
し、糖鎖を変換させた糖蛋白質を生産する段階に至っ
て、糖の種類あるいは濃度を変更した培地に交換しても
良い。多糖の場合は、多糖の溶解液を通常の培養液中に
１〜1000μｇ／mlの濃度になるように添加してもよい。
培養液からの糖蛋白質は通常の精製、回収方法により分
離精製できる。しかし、糖鎖の変化は糖蛋白質の分子量
の差として検出できるため、ＳＤＳゲル電気泳動や、ゲ
ル濾過方法など分子量の差による分離精製方法が適して
いる。以下に実施例を示しさらに本発明を詳細に説明す
る。Any culturing method suitable for the cells can be used for culturing the cells.
For example, suspension culture in tanks, adhesion culture to microcarriers and urethane materials, culture with hollow fibers, etc. can be exemplified. In addition, until the cells reach a certain density, they are cultured in a conventional culture medium with a glucose concentration suitable for the cells, and at the stage of producing glycoproteins with converted sugar chains, the medium in which the type or concentration of sugar is changed You may replace it with. In the case of a polysaccharide, a solution of the polysaccharide may be added to an ordinary culture medium at a concentration of 1 to 1000 μg / ml.
Glycoproteins from the culture broth can be separated and purified by ordinary purification and recovery methods. However, since changes in sugar chains can be detected as differences in the molecular weight of glycoproteins, SDS gel electrophoresis, gel filtration methods and other separation and purification methods based on the difference in molecular weight are suitable. Hereinafter, the present invention will be described in more detail with reference to examples.
【０００８】[0008]
【実施例１】本実施例は、抗体生産ハイブリドーマの培
養によって生産される糖蛋白質である抗体の糖鎖を変更
する方法について説明する。特に本実施例により得られ
た糖蛋白質は糖鎖のちがいによって親和性が異なる抗体
である。 （１）糖濃度、糖組成の異なる培地の調製 村上らによって開発された無血清培地であるＥＲＤＦ培
地（特開平３−１８０１７５号公報参照）を基本培地と
してこの培地のグルコース濃度を０、１、２、５、１
０、２０、３６ｍＭの濃度にした培地を調製した。なお
ＥＲＤＦ培地のグルコース（Ｇｌｃ）濃度は、細胞培養
時には２０ｍＭが最適とされている。またこのＥＲＤＦ
培地の糖をフラクトース（Ｆｒｕ）、マンノース（Ｍａ
ｎ）、ガラクトース（Ｇａｌ）、Ｎ−アセチルグルコサ
ミン（ＧｌｃＮＡｃ）、リボース（Ｒｉｂ）に変えた培
地を調製した。さらに牛胎児血清（ＦＣＳ）をリン酸緩
衝生理食塩水（ＰＢＳ）に対して透析を行い、グルコー
スなどの単糖を含まないＦＣＳ（透析ＦＣＳ）を調製
し、各培地に５％濃度となるように添加した。Example 1 This example describes a method for changing the sugar chain of an antibody, which is a glycoprotein produced by culturing an antibody-producing hybridoma. In particular, the glycoprotein obtained in this example is an antibody having different affinity depending on the difference in sugar chain. (1) Preparation of medium having different sugar concentration and sugar composition ERDF medium (see Japanese Patent Application Laid-Open No. 3-180175), which is a serum-free medium developed by Murakami et al. 2, 5, 1
A medium having a concentration of 0, 20, 36 mM was prepared. The glucose (Glc) concentration in the ERDF medium is optimally 20 mM during cell culture. Also this ERDF
Fructose (Fru), mannose (Ma)
n), galactose (Gal), N-acetylglucosamine (GlcNAc), and ribose (Rib) were used in the prepared medium. Furthermore, fetal calf serum (FCS) is dialyzed against phosphate buffered saline (PBS) to prepare FCS (dialyzed FCS) that does not contain monosaccharides such as glucose, so that each medium has a concentration of 5%. Was added to.
【０００９】（２）糖蛋白質の生産 糖蛋白質生産細胞として、ヒト−ヒトハイブリドーマＣ
５ＴＮを用いた。Ｃ５ＴＮはハイブリドーマＨＢ４Ｃ５
(Murakami et al., In Vitro Cell. Develop.Biol., vo
l. 21, 593-596, 1985)の亜株でありカルボキシペプチ
ダーゼ（以下ＣＰＡ）、二本鎖ＤＮＡ（以下ＤＮＡ）、
カンジダシトクロームＣ（以下ＣｙｔＣ）に対して親和
性を有するヒトＩｇＭ型抗体を分泌する細胞株である(T
achibana, H., et al., Biochem. Biophys. Res. Commu
n., vol. 189, 625-632, 1992)。このハイブリドーマが
分泌する抗体（以下Ｃ５ＴＮ抗体）のＬ鎖はλ鎖であ
り、本発明者らは可変領域（ＶＬドメイン）のＣＤＲ１
領域に-Ser-Gly-Asn-Ser-Ser-Asn-Ile-Gly- という糖鎖
結合部位を持つことを明らかにしている。尚この細胞株
は九州大学農学部大学院細胞工学教室から分与をうける
ことができる。このＣ５ＴＮを各培地５ｍｌに５×１０
4 細胞／ｍｌの密度でまきこみ、３７℃、５％炭酸ガス
雰囲気下で２４時間培養し、細胞を回収し、さらに同じ
条件で４８時間培養した培養上清を回収した。(2) Production of glycoprotein As a glycoprotein producing cell, human-human hybridoma C
5TN was used. C5TN is the hybridoma HB4C5
(Murakami et al., In Vitro Cell. Develop. Biol., Vo
l. 21, 593-596, 1985), a carboxypeptidase (hereinafter, CPA), double-stranded DNA (hereinafter, DNA),
It is a cell line that secretes a human IgM type antibody having an affinity for Candida cytochrome C (hereinafter CytC) (T
achibana, H., et al., Biochem. Biophys. Res. Commu
n., vol. 189, 625-632, 1992). The L chain of the antibody secreted by this hybridoma (hereinafter referred to as C5TN antibody) is a λ chain, and the present inventors have used CDR1 of the variable region (VL domain).
It has been clarified that the region has a sugar chain binding site called -Ser-Gly-Asn-Ser-Ser-Asn-Ile-Gly-. This cell line can be distributed from the Department of Cell Engineering, Graduate School of Agriculture, Kyushu University. This C5TN was added to 5 ml of each medium at 5 × 10 5.
The cells were collected at a density of 4 cells / ml, cultured at 37 ° C. in a 5% carbon dioxide atmosphere for 24 hours, cells were collected, and the culture supernatant was further cultured under the same conditions for 48 hours.
【００１０】（３）糖鎖の異なる糖蛋白質の分離 各培地で培養した培養上清を還元条件下でＳＤＳ−ポリ
アクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）を行
った。各λ鎖は、抗λ抗体を用いたウエスタンブロット
法により確認を行った（図１、図２）。Ｌ鎖は４種類検
出されたが、泳動の位置から判断して分子量の大きいほ
うからＬ１、Ｌ２、Ｌ３、Ｌ４と名付けた。また糖鎖合
成阻害剤であるツニカマイシン存在下ではＬ４のみが合
成された。Ｌ４は糖鎖の結合しない抗体であることが推
定された。各培地での抗体の生産を表１に示した。(3) Separation of glycoproteins having different sugar chains The culture supernatants cultured in each medium were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Each λ chain was confirmed by Western blotting using an anti-λ antibody (Figs. 1 and 2). Although four types of L chains were detected, they were named L1, L2, L3, and L4 from the one having the largest molecular weight, judging from the position of migration. Further, only L4 was synthesized in the presence of tunicamycin, which is a sugar chain synthesis inhibitor. It was presumed that L4 is an antibody to which no sugar chain is bound. The production of antibody in each medium is shown in Table 1.
【００１１】[0011]
【表１】 ────────────────────────────── 培地糖組成 （ｍＭ） Ｌ鎖のタイプ ────────────────────────────── Ｇｌｃ ０ Ｌ１，Ｌ３，Ｌ４ Ｇｌｃ １ Ｌ１，Ｌ３，Ｌ４ Ｇｌｃ ２ Ｌ１，Ｌ３，Ｌ４ Ｇｌｃ ５ Ｌ１，Ｌ２，Ｌ３，Ｌ４ Ｇｌｃ １０ Ｌ１，Ｌ２ Ｇｌｃ ２０ Ｌ１，Ｌ２ Ｇｌｃ ３６ Ｌ１，Ｌ２ Ｆｒｕ ２０ Ｌ１，Ｌ２ Ｍａｎ ２０ Ｌ１，Ｌ２ Ｇａｌ ２０ Ｌ１，Ｌ２，Ｌ４ ＧｌｃＮＡｃ ２０ Ｌ１，Ｌ３，Ｌ４ Ｒｉｂ ２０ Ｌ１，Ｌ３，Ｌ４ ───────────────────────────────[Table 1] ────────────────────────────── Medium sugar composition (mM) L chain type ────── ──────────────────────── Glc 0 L1, L3, L4 Glc 1 L1, L3, L4 Glc 2 L1, L3, L4 Glc 5 L1, L2 , L3, L4 Glc 10 L1, L2 Glc 20 L1, L2 Glc 36 L1, L2 Fru 20 L1, L2 Man 20 L1, L2 Gal 20 L1, L2, L4 GlcNAc 20 L1, L3, L4 RibL3 L1, L2. ───────────────────────────────
【００１２】培地中のグルコース濃度、あるいは糖質の
変化により通常の培養条件では得られないＬ２の抗体を
得ることができた。この抗体はＳＤＳ−ＰＡＧＥにより
他の抗体から分離することができた。本細胞は上述した
ように４種類の分子量を持つλ鎖（Ｌ鎖）を含む抗体を
生産しており、このλ鎖（Ｌ鎖）分子量の差は、糖鎖の
分子量差によるものである。It was possible to obtain an L2 antibody which could not be obtained under usual culture conditions, due to changes in glucose concentration or sugar in the medium. This antibody could be separated from other antibodies by SDS-PAGE. As described above, this cell produces an antibody containing a λ chain (L chain) having four kinds of molecular weights, and the difference in the λ chain (L chain) molecular weights is due to the difference in the sugar chain molecular weights.
【００１３】[0013]
【実施例２】糖鎖の違いによる抗体の特性変化 Ｃ５ＴＮ抗体はＬ鎖に糖鎖が結合しており、本発明によ
りこの糖鎖を（３）のような分子量の異なる糖鎖とする
ことができた。この糖鎖の変更によって抗体特異性が変
化することを以下のように確認した。抗体の抗原特異性
は、抗原をコートしたマイクロプレートを用いた酵素免
疫測定法（ＥＬＩＳＡ）で測定できる（H.Murakami et
al.,Animal Cell Technology:Basic & Applied Aspect
s,547-551,1992,Kluwer Acdemic Publishers)。各培地
で培養したＣ４ＴＮ抗体２００ｎｇ／ｍｌの溶液を調製
し、この抗体溶液の抗原特異性を測定した。測定値は、
Ｇｌｃ ２０ｍＭで培養して得た抗体の反応性を１００
％とした百分率で表した。結果を図３および図４に示し
た。Ｇｌｃの含量を変化させた場合、Ｌ３、Ｌ４の増加
とともにＣＰＡ親和性およびＣｙｔｃ親和性は低下した
が、ＤＮＡ親和性は２ｍＭでピークに達した。また培地
中の糖組成を変えた場合は、抗体の親和性はＧａｌ、Ｇ
ｌｃＮＡｃ、Ｒｉｂ添加により、Ｇｌｃで培養して得た
抗体と全く異なる結果を示した（図３、図４）。これ
は、抗体に結合する糖鎖の構造が変化したためと推定さ
れた。Example 2 Change in Antibody Properties Due to Difference in Sugar Chain The sugar chain is bound to the L chain in the C5TN antibody. According to the present invention, this sugar chain can be a sugar chain having a different molecular weight as in (3). did it. It was confirmed that the antibody specificity was changed by the change of the sugar chain as follows. The antigen specificity of an antibody can be measured by enzyme-linked immunosorbent assay (ELISA) using a microplate coated with an antigen (H. Murakami et al.
al., Animal Cell Technology: Basic & Applied Aspect
s, 547-551, 1992, Kluwer Acdemic Publishers). A solution of C4TN antibody 200 ng / ml cultured in each medium was prepared, and the antigen specificity of this antibody solution was measured. The measured value is
The reactivity of the antibody obtained by culturing with Glc 20 mM was 100%.
It was expressed as a percentage. The results are shown in FIGS. 3 and 4. When the content of Glc was changed, the CPA affinity and Cytc affinity decreased with the increase of L3 and L4, but the DNA affinity peaked at 2 mM. When the sugar composition in the medium is changed, the affinity of the antibody is Gal, G
The addition of lcNAc and Rib showed a completely different result from the antibody obtained by culturing with Glc (FIGS. 3 and 4). This was presumably because the structure of the sugar chain that binds to the antibody was changed.
【００１４】[0014]
【実施例３】グルコース濃度を１０ｍＭに調整したＥＲ
ＤＦ基本無血清培地を用いて、実施例１と同様の条件で
ハイブリドーマＣ５ＴＮ細胞の培養を行った。すなわ
ち、ハイブリドーマＣ５ＴＮを５×１０4 細胞／mlの密
度でまきこみ、６時間培養を行った。その後細胞を回収
し、キトサン添加（１μｇ／ｍｌ）、デキストラン硫酸
添加（１００μｇ／ml）あるいはアルギン酸添加（１０
０μｇ／ｍｌ）培養液（ＥＲＤＦ）に移し、さらに48時
間培養を行った後、培養上清を回収し、これを透析濃縮
した。培養条件は３７℃、５％ＣＯ2 雰囲気下で実施例
１と同様におこなった。又対照は、ＥＲＤＦのみで培養
をおこなった。この濃縮液を還元条件でＳＤＳ−ＰＡＧ
Ｅにより電気泳動を行い、抗ヒトλＬ鎖抗体で、ウエス
タンブロット法により抗体のＬ鎖を特異的に検出した。
結果を図５に示した。各多糖類を添加することにより、
グルコースのみの培養と比較して２８ＫＤおよび３０Ｋ
ＤのＬ鎖が増加した。これは抗体の糖鎖が変化している
ことを裏付ける結果であった。また得られた抗体はいず
れもＩｇＭ型の抗体のみであった。Example 3 ER with glucose concentration adjusted to 10 mM
Hybridoma C5TN cells were cultured under the same conditions as in Example 1 using a DF basic serum-free medium. That is, hybridoma C5TN was seeded at a density of 5 × 10 4 cells / ml and cultured for 6 hours. After that, cells were collected and added with chitosan (1 μg / ml), dextran sulfate (100 μg / ml) or alginate (10).
(0 μg / ml) culture solution (ERDF) and further cultured for 48 hours, and then the culture supernatant was recovered and concentrated by dialysis. Culture conditions were the same as in Example 1 at 37 ° C. and 5% CO 2 atmosphere. As a control, ERDF alone was used for culturing. This concentrated solution is subjected to SDS-PAG under reducing conditions.
Electrophoresis was performed with E, and the anti-human λ L chain antibody was used to specifically detect the L chain of the antibody by Western blotting.
The results are shown in Fig. 5. By adding each polysaccharide,
28 KD and 30 K compared to glucose only culture
The L chain of D was increased. This was the result to prove that the sugar chain of the antibody is changed. The obtained antibodies were all IgM type antibodies.
【００１５】[0015]
【実施例４】実施例３で生産された糖鎖が変化した抗体
の抗原親和性の変化を、実施例２に開示した方法により
確認した。抗原をコートしたマイクロプレートを用いて
同様に測定を行い、ＥＬＩＳＡ法により各抗原に対する
反応性を測定した。各抗原に対する反応性はＥＬＩＳＡ
法による発色の変化を４０５ｎｍの吸光度変化により観
察した。結果を図６〜８に示した。抗体は糖鎖の変化に
より、抗原との反応性が変化していることが確認され
た。Example 4 Changes in the antigen affinity of the antibodies produced in Example 3 with altered sugar chains were confirmed by the method disclosed in Example 2. The same measurement was performed using a microplate coated with an antigen, and the reactivity to each antigen was measured by the ELISA method. ELISA for reactivity to each antigen
The change in color development by the method was observed by the change in absorbance at 405 nm. The results are shown in FIGS. It was confirmed that the reactivity of the antibody with the antigen was changed due to the change of the sugar chain.
【００１６】[0016]
【発明の効果】本発明により、動物細胞を培養して糖蛋
白質を生産する際に、培地中のリボース、ガラクトー
ス、グルコサミン等の単糖の組成あるいは糖濃度等の培
養条件を制御することにより、蛋白質に付加する糖鎖の
種類あるいは分子量を改変し、特性の変化した糖蛋白質
を生産することができる。また培地中にアルギン酸、デ
キストラン硫酸、キトサンのような多糖を添加して糖鎖
の種類、あるいは分子量を改変し、特性の変化した糖蛋
白質を生産することができる。この方法により糖鎖の種
類あるいは分子量の異なる糖蛋白質を得ることができ
る。また糖鎖の改変によって糖蛋白質の生物活性をも変
更することができる。EFFECTS OF THE INVENTION According to the present invention, when a glycoprotein is produced by culturing animal cells, by controlling culture conditions such as ribose, galactose and glucosamine in the medium, composition of monosaccharide or sugar concentration, The type or molecular weight of sugar chains added to proteins can be modified to produce glycoproteins with altered properties. Further, a polysaccharide such as alginic acid, dextran sulfate or chitosan may be added to the medium to modify the type or molecular weight of the sugar chain to produce a glycoprotein with altered properties. By this method, glycoproteins having different sugar chains or different molecular weights can be obtained. The biological activity of glycoprotein can also be changed by modifying the sugar chain.
【図１】実施例１により、グルコース濃度を変えて培養
したＣ５ＴＮ細胞より得られるλ型抗体のＳＤＳ−ＰＡ
ＧＥウエスタンブロットによる検出パターンを示す。FIG. 1 SDS-PA of λ type antibody obtained from C5TN cells cultured with varying glucose concentrations according to Example 1.
The detection pattern by GE Western blot is shown.
【図２】実施例１により、グルコースを他の糖質に変え
て培養したＣ５ＴＮ細胞より得られるλ型抗体のＳＤＳ
−ＰＡＧＥウエスタンブロットによる検出パターンを示
す。FIG. 2 is the SDS of λ-type antibody obtained from C5TN cells cultured by changing glucose to another sugar according to Example 1.
-The detection pattern by PAGE Western blot is shown.
【図３】実施例２により、グルコース濃度を変えて培養
したＣ５ＴＮ細胞より得られる抗体の抗原親和性の変化
を示す。FIG. 3 shows changes in the antigen affinity of antibodies obtained from C5TN cells cultured with varying glucose concentrations according to Example 2.
【図４】実施例２により、グルコースを他の糖質に変え
て培養したＣ５ＴＮ細胞より得られる抗体の抗原親和性
を示す。FIG. 4 shows the antigen affinity of an antibody obtained from C5TN cells cultured by changing glucose to another sugar according to Example 2.
【図５】実施例３より得たヒトλ型抗体のＳＤＳ−ＰＡ
ＧＥウエスタンブロットによる検出パターンを示す。FIG. 5: SDS-PA of human λ type antibody obtained from Example 3
The detection pattern by GE Western blot is shown.
レーン１：分子サイズマーカー レーン２：対照 レーン３：キトサン添加（１μｇ／ml） レーン４：デキストラン硫酸添加（100 μｇ／ml） レーン５：アルギン酸添加（100 μｇ／ml） Lane 1: Molecular size marker Lane 2: Control Lane 3: Chitosan addition (1 μg / ml) Lane 4: Dextran sulfate addition (100 μg / ml) Lane 5: Alginic acid addition (100 μg / ml)
【図６】実施例３により得られた抗体のＣＰＡに対する
親和性の変化を示す。FIG. 6 shows changes in the affinity of the antibody obtained in Example 3 for CPA.
【図７】実施例３により得られた抗体のＣｙｔＣに対す
る親和性の変化を示す。FIG. 7 shows changes in the affinity of the antibody obtained in Example 3 for CytC.
【図８】実施例３により得られた抗体のｄｓＤＮＡに対
する親和性の変化を示す。FIG. 8 shows changes in the affinity of the antibody obtained in Example 3 for dsDNA.
−○− 対照 −□− キトサン添加（１μｇ／ml） −●− デキストラン硫酸添加（100 μｇ／ml） −△− アルギン酸添加（100 μｇ／ml） -○-Control- □-Add chitosan (1 μg / ml)-●-Add dextran sulfate (100 μg / ml)-△-Add alginic acid (100 μg / ml)
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｒ 1:91） ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification code Office reference number FI technical display location C12R 1:91)
Claims (1)
産するに当り、培地中の糖組成および／または糖濃度を
変えることにより、蛋白質に結合する糖鎖の種類あるい
は分子量を改変することを特徴とする糖蛋白質の生産方
法。1. When culturing an animal cell in a medium to produce a glycoprotein, the kind or molecular weight of the sugar chain bound to the protein is modified by changing the sugar composition and / or the sugar concentration in the medium. A method for producing a glycoprotein characterized by:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6031019A JPH06292592A (en) | 1993-02-09 | 1994-02-02 | Production of glycoprotein |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5-44587 | 1993-02-09 | ||
| JP4458793 | 1993-02-09 | ||
| JP6031019A JPH06292592A (en) | 1993-02-09 | 1994-02-02 | Production of glycoprotein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH06292592A true JPH06292592A (en) | 1994-10-21 |
Family
ID=26369476
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6031019A Pending JPH06292592A (en) | 1993-02-09 | 1994-02-02 | Production of glycoprotein |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH06292592A (en) |
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