JPH0361652B2 - - Google Patents
Info
- Publication number
- JPH0361652B2 JPH0361652B2 JP59071568A JP7156884A JPH0361652B2 JP H0361652 B2 JPH0361652 B2 JP H0361652B2 JP 59071568 A JP59071568 A JP 59071568A JP 7156884 A JP7156884 A JP 7156884A JP H0361652 B2 JPH0361652 B2 JP H0361652B2
- Authority
- JP
- Japan
- Prior art keywords
- aqueous solution
- composition
- freeze
- human
- acid
- 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.)
- Expired - Lifetime
Links
- 108010002350 Interleukin-2 Proteins 0.000 claims description 61
- 102000000588 Interleukin-2 Human genes 0.000 claims description 61
- 239000000203 mixture Substances 0.000 claims description 38
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims description 28
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 22
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 16
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 16
- 108010024636 Glutathione Proteins 0.000 claims description 14
- 229960003180 glutathione Drugs 0.000 claims description 14
- 229960005070 ascorbic acid Drugs 0.000 claims description 11
- 235000010323 ascorbic acid Nutrition 0.000 claims description 11
- 239000011668 ascorbic acid Substances 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 description 45
- 239000000047 product Substances 0.000 description 18
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 description 17
- 239000000243 solution Substances 0.000 description 17
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 16
- 230000000694 effects Effects 0.000 description 15
- 238000002347 injection Methods 0.000 description 15
- 239000007924 injection Substances 0.000 description 15
- 229940090044 injection Drugs 0.000 description 14
- 230000001954 sterilising effect Effects 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 239000012153 distilled water Substances 0.000 description 12
- 235000002639 sodium chloride Nutrition 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 11
- 238000004108 freeze drying Methods 0.000 description 11
- 239000011550 stock solution Substances 0.000 description 11
- 238000007865 diluting Methods 0.000 description 10
- 238000003860 storage Methods 0.000 description 9
- 239000004471 Glycine Substances 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 235000001014 amino acid Nutrition 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 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 7
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 238000007710 freezing Methods 0.000 description 6
- 230000008014 freezing Effects 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 5
- -1 mannitrate Chemical class 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 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 4
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 4
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 4
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 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 4
- 239000003638 chemical reducing agent Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 150000002772 monosaccharides Chemical class 0.000 description 4
- 235000013930 proline Nutrition 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 3
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 3
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
- YZCKVEUIGOORGS-NJFSPNSNSA-N Tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000010981 drying operation Methods 0.000 description 3
- 239000012156 elution solvent Substances 0.000 description 3
- 235000013922 glutamic acid Nutrition 0.000 description 3
- 239000004220 glutamic acid Substances 0.000 description 3
- 102000055277 human IL2 Human genes 0.000 description 3
- 239000007951 isotonicity adjuster Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000600 sorbitol Substances 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 229940104230 thymidine Drugs 0.000 description 3
- 229910052722 tritium Inorganic materials 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 2
- 241001452028 Escherichia coli DH1 Species 0.000 description 2
- 206010061598 Immunodeficiency Diseases 0.000 description 2
- 208000029462 Immunodeficiency disease Diseases 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
- 239000006172 buffering agent Substances 0.000 description 2
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 230000007813 immunodeficiency Effects 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000011975 tartaric acid Substances 0.000 description 2
- 235000002906 tartaric acid Nutrition 0.000 description 2
- 229960002180 tetracycline Drugs 0.000 description 2
- 229930101283 tetracycline Natural products 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 150000003522 tetracyclines Chemical class 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- IQFYYKKMVGJFEH-OFKYTIFKSA-N 1-[(2r,4s,5r)-4-hydroxy-5-(tritiooxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound C1[C@H](O)[C@@H](CO[3H])O[C@H]1N1C(=O)NC(=O)C(C)=C1 IQFYYKKMVGJFEH-OFKYTIFKSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- FXEDIXLHKQINFP-UHFFFAOYSA-N 12-O-tetradecanoylphorbol-13-acetate Natural products CCCCCCCCCCCCCC(=O)OC1CC2(O)C(C=C(CO)CC3(O)C2C=C(C)C3=O)C4C(C)(C)C14OC(=O)C FXEDIXLHKQINFP-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 108010062580 Concanavalin A Proteins 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 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 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical group [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- HTWFXPCUFWKXOP-UHFFFAOYSA-N Tertatalol Chemical compound C1CCSC2=C1C=CC=C2OCC(O)CNC(C)(C)C HTWFXPCUFWKXOP-UHFFFAOYSA-N 0.000 description 1
- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 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 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
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- 239000006285 cell suspension Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000000326 densiometry Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
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- 239000000284 extract Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229940093181 glucose injection Drugs 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 210000005104 human peripheral blood lymphocyte Anatomy 0.000 description 1
- 229910052739 hydrogen Chemical group 0.000 description 1
- 239000001257 hydrogen Chemical group 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
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- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
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- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 description 1
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Description
産業上の利用分野
本発明は医薬品などとして有用なインターロイ
キン−２組成物に関する。
従来の技術
インターロイキン−２（以下IL−２と略称する
ことがある）は生体内で免疫調節に中心的な役割
をはたしており直接的あるいは関接的に癌の排除
や免疫失調からの回復やその改善に働くと考えら
れているＴ細胞やナチユラルキラー細胞の増殖因
子としての機能を有する蛋白質である〔ネイチヤ
ー、第302巻、305−310頁（1983）〕。かかる生理
作用を有するためみIL−２は新しい制癌剤ある
いは免疫失調治療剤としての応用が強く期待され
ている。
発明が解決しようとする問題点
本発明者らは、IL−２が不安定であつて水溶
液の保存、凍結あるいは凍結乾燥の操作および凍
結乾燥後の保存において、とりわけ凍結乾燥を行
う際の乾燥操作において容易に活性が減じ、また
凍結乾燥品の再溶解した液に濁りを認める等の問
題点を有し、医療用等に用いる上ではなはだ不都
合があることを見い出した。
かかる事実に鑑み、本発明者らは鋭意研究を進
めた結果、安定なIL−２組成物の製造に成功し、
本発明を完成した。
問題点を解決するための手段
本発明は、グルタチオンまたはアスコルビン酸
を配合したIL−２組成物を提供するものである。
本発明のIL−２は、哺乳動物のものであれば
いかなるものでもよいが、ヒトのものが好まし
い。またIL−２は、天然の、あるいは遺伝子組
み換え技術で得られるいずれのものでもよく、遺
伝子組み換え技術で得られるものが有利に用いら
れ、通常IL−２水溶液として用いる。
好ましいIL−２の例として式
INDUSTRIAL APPLICATION FIELD The present invention relates to an interleukin-2 composition useful as a pharmaceutical or the like. Prior Art Interleukin-2 (hereinafter sometimes abbreviated as IL-2) plays a central role in immune regulation in vivo, and directly or indirectly helps eliminate cancer, recover from immunodeficiency, etc. It is a protein that functions as a growth factor for T cells and natural killer cells, which is thought to work to improve this condition [Nature, Vol. 302, pp. 305-310 (1983)]. Because IL-2 has such physiological effects, there are strong expectations for its application as a new anticancer agent or a therapeutic agent for immunodeficiency. Problems to be Solved by the Invention The present inventors have discovered that IL-2 is unstable, and in the storage of aqueous solutions, freezing or freeze-drying operations, and storage after freeze-drying, especially drying operations during freeze-drying. It has been found that the activity of the lyophilized product is easily reduced, and that the redissolved solution of the lyophilized product becomes cloudy, which is very inconvenient when used for medical purposes. In view of this fact, the present inventors conducted intensive research and succeeded in producing a stable IL-2 composition.
The invention has been completed. Means for Solving the Problems The present invention provides IL-2 compositions containing glutathione or ascorbic acid. The IL-2 of the present invention may be any mammalian IL-2, but human IL-2 is preferred. Further, IL-2 may be either natural or obtained by genetic recombination technology, and IL-2 obtained by genetic recombination technology is advantageously used, and is usually used as an IL-2 aqueous solution. As an example of a preferred IL-2 formula
【表】【table】
【表】
〔式中、ＸはMetまたは水素を示す〕で表わさ
れる遺伝子組み換え技術で製造される非グリコシ
ル化ヒトIL−２を挙げることができ、これらの
混合物でもよい。
なお式（）においてアミノ酸残基は、
IUPAC−IUB コミツシヨン オン バイオケ
ミカル ノメンクレイチヤによる略号で示した。
またIL−２の比活性は20000〜80000単位／mg
であることが望ましく、IL−２水溶液として１
〜80000単位／ml、とりわけ10〜50000単位／mlの
活性を有するものが有利に用いられる。
また上記還元性物質であるグルタチオンまたは
アスコルビン酸は、１種または２種以上併用する
こともできる。
これら還元性物質は、上記濃度IL−２水溶液
に対し、水溶液１ml当り0.01mg以上、とりわけ
0.05〜20mg含有させることが好ましい。
本発明のIL−２組成物には前記した還元性物
質に加え人血清アルブミン（以下HSAと略記す
る）、ブドウ糖、果糖、マンノース、ガラクトー
ス等の単糖類、シヨ糖、マルトース、乳糖等の二
糖類、マンニツト、イノシツト、ソルビツト等の
糖アルコール、グリシン、アスパラギン酸、グル
タミン酸、プロリン等のアミノ酸とりわけモノア
ミノ脂肪族アミノ酸もしくは環状アミノ酸または
これらの生理学的に許容しうる塩もしくは誘導体
のうち１種あるいは２種以上を加えることによ
り、一層の安定性、凍結乾燥品とした場合のその
外観やそれを溶解した液の溶状、容器への吸着防
止等を向上させることができるためこれらを適宜
配合することができる。上記配合剤のうちでも
HSA、ブドウ糖、マンノース、マンニツト、ソ
ルビツト、プロリン、グリシンが好ましく、これ
らを配合する場合には、例えば前記濃度のIL−
２水溶液で１ml当りHSAの場合01〜50mg、好ま
しくは0.5〜10mg、マンニツト、ソルビツト、ブ
ドウ糖またはマンノースの場合10〜100mg、グリ
シンまたはプロリンの場合５〜50mg加えることが
好ましい。
さらに本発明のIL−２組成物は食塩などの等
張化剤、コハク酸、酒石酸、クエン酸等の緩衝
剤、界面活性剤などを含有していてもよい。
また上記IL−２組成物の容器の空間部を配合
した還元性物質の酸化を防止するため真空にする
か窒素ガス置換することによりIL−２組成物の
安定性をさらに高めることができる。
本発明のIL−２組成物は、水溶液、凍結品、
凍結乾燥品等の形態が好ましく、とりわけ凍結乾
燥品が好ましい。
本発明の組成物は、たとえば以下の方法により
製造することができる。
IL−２を１〜80000単位／mlを含有する水溶液
に、還元性物質を0.01mg／ml以上、好ましくは
0.05〜20mg／ml（２種以上の還元性物質を用いる
場合は合計量として）の濃度になるように加え
る。さらに前記したHSA、ブドウ糖、マンニツ
ト、ソルビツト、プロリン、グリシンなどもそこ
に記載した濃度として加えることもできる。また
所望により等張化剤、界面活性剤なども加えるこ
とができる。かくして得られる水溶液としての
IL−２組成物は、下記の凍結および凍結乾燥品
の原料としても用いることができる。
凍結品としてのIL−２組成物は、たとえば上
記水溶液を通常−80〜−20℃で凍結することによ
り製造できる。該凍結組成物は−80゜〜−10℃で
保管することが好ましい。
凍結乾燥品としてのIL−２組成物は、例えば
上記凍結組成物を常法により減圧乾燥するか上記
水溶液または上記凍結組成物の融解により得られ
る水溶液を、所望により小分けし、上記同様凍結
した後、常法により減圧乾燥することにより製造
することができる。
注射用製剤としての本発明の凍結乾燥したIL
−２組成物を製造する場合は、IL−２を含有す
る水溶液および配合剤含有水溶液をそれぞれ除菌
過して混合するか、これらの混合液を小分けす
る前に除菌過等により精製し、無菌操作により
バイアル瓶等に分注小分けした後上記凍結乾燥処
理に付すことが好ましい。
上記した還元性物質を配合したIL−２組成物
とは別途、本発明は下記のIL−２組成物をも提
供するものである。
すなわち、グリシン、グルタミン酸、アスパラ
ギン酸、アラニンなどのアミノ酸とりわけモノア
ミノ脂肪族アミノ酸、ブドウ糖、マンノースなど
の単糖類、ヒト血清アルブミン（HSA）および
これらの生理学的に許容できる塩もしくは誘導体
の１種または２種以上を配合したIL−２組成物
である。
上記IL−２に関しては、前記したIL−２と同
様のものが挙げられ、IL−２水溶液が好ましい。
上記単糖類に関しては10〜100mg／ml、アミノ酸
に関しては５〜50mg／ml、HSAに関しては0.1〜
５mg／ml、好ましくは0.5〜10mg／ml配合するこ
とが好ましい。
上記IL−２組成物は、いずれも水溶液の保存、
凍結あるいは凍結乾燥の操作および凍結乾燥後の
保存において、とりわけ凍結乾燥を行う際の乾燥
操作においてIL−２活性の低下が少ないという
特徴を有する。
上記安定化効果に加えて、アミノ酸を配合した
組成物は凍結乾燥品として場合にその外観が向上
し、注射剤として投与する場合の疼通を軽減する
効果をも奏する。
また単糖類を配合した組成物は注射剤として投
与する場合の疼痛を軽減する効果をも奏する。
HSAを配合した組成物、とりわけその凍結乾
燥品は、その外観が向上し、その器壁への吸着が
防止される効果をも奏するものである。
さらに上記IL−２組成物には食塩などの等張
化剤、コハク酸、酒石酸、クエン酸等の緩衝剤、
界面活性剤なども配合することができる。
上記IL−２組成物は、水溶液、凍結品、凍結
乾燥品等の形態が好ましく、とりわけ凍結乾燥品
が好ましい。
上記IL−２組成物は、前記した還元性物質を
配合したIL−２組成物の製造法に準じて製造す
ることができ、本IL−２組成物においては凍結
する前に、グルタミン酸など酸性アミノ酸を配合
する場合は該物質で、または塩酸、リン酸等の鉱
塩もしくは、上記緩衝剤で、酸性（PH３〜６）に
調製することが好ましい。
作 用
本発明のIL−２組成物は、保存中および凍結
や凍結乾燥操作におけるIL−２活性の低下が少
なく、また凍結乾燥品においてはその再溶解時の
溶状が澄明である点等に優れた特徴を有するもの
であり、とりわけ凍結乾燥を行う時の乾燥操作時
のIL−２活性の低下が少ないことに特徴を有す
る。また凍結乾燥品は、安定化されたIL−２の
粉末として得られ、とりわけ非経口投与製剤とし
て有利に用いることができる。注射用製剤として
用いる場合は、通常用時、凍結乾燥組成物をバイ
アル当り0.5〜100mlの注射用蒸留水、生理食塩液
またはブドウ糖注射液に溶解し、筋肉内あるいは
静脈内に投与する。また適当な担体、賦型剤、希
釈剤を用いて口腔内、眼、耳、鼻内投与用の剤形
として用いることができる。
本発明のIL−２組成物は、低毒性で、公知の
IL−２と同様の目的に同様の用法により使用す
ることができる。
本願明細書中IL−２の活性としての単位（Ｕ）
の算出方法は以下のようにして行つた。
すなわち、IL−２濃度に依存して増殖するマ
ウス細胞株を浮遊した培地にIL−２を含む検体
を加えて培養し、該細胞株の増殖をトリチウムチ
ミジンの取込を指標として求めた。目的とする検
体中のユニツト（Ｕ）算出のためには、常に標準
IL−２（1U／ml）を並べてアツセイを実施して、
その比率からユニツトを算出した。
具体的には、ヒトIL−２を含有するコンデイ
シヨンドメジウムを含む20％FCS加RPMI1640培
地中で、37℃で５％Co2の存在下に継代維持され
たIL−２依存性マウス細胞株〔（NKC3）、
Hinumaら、バイオケミカル・バイオフイジカ
ル・リサーチ・コミユニケイシヨンズ、第109巻、
363頁（1982年）〕を無血清RPMI1640培地を用い
て２回洗浄し、20％FCS加RPMI1640培地に６×
105個／mlになるように再浮遊する。
IL−２を含む資料50μlを96穴平底マイクロタイ
タープレート（ヌンク社、デンマーク）の第１列
目の穴に入れ、50μlずつの20％FCS加RPMI1640
培地を用いて第12列目まで順次２倍段階希釈系列
を作成後、上記NKC3細胞浮遊液を50μlずつ各穴
に分注し、37℃で５％Co2の存在下に24時間培養
する。培養20時間目に、各穴に1μCiずつトリチ
ウムチミジン（アマルシヤム社、イギリス）を添
加してさらに４時間培養を継続後、セルハーベス
ター（フロー社、アメリカ）を使用して細胞をガ
ラスフイルター上に回収し、液体シンチレーシヨ
ンカウンターを用いてトリチウムチミジンの取込
を測定する。測定に際しては標準IL−２標品に
ついて資料と同一の操作を行い、トリチウムチミ
ジンの取込を測定する。
ユニツト（Ｕ）の計算はジヤーナル・オブ・イ
ムノロジー、第120巻、2027頁（1978年）に準じ
てプロビツト変換法により行う。すなわち、標準
IL−２標品（ヒト末梢血リンパ球を５×106個／
mlとなるように10％FCS加RPMI1640培地に浮遊
し、コンカナバリン−A40μgおよび12−Ｏ−テト
ラデカノイルホルボール−13−アセテート
15ng／mlを添加して、37℃で５％CO2の存在下に
48時間培養した培養液の遠心上清を1U／mlと定
める）の希釈系列のうち最大値の取込を100％と
して、各希釈段階の取込値の割合（％）を計算す
る。得られた数値を正規確率紙にプロツトし、50
％の取込を示す希釈倍数を作図から求める。同様
にしてIL−２を含む各資料についても50％の取
込を示す希釈倍数を求める。
資料のIL−２濃度（Ｕ／ml）は次式に従つて
計算される：
資料が50％取込を示す希釈倍数／標準IL−２標品が50
％の取込を示す希釈倍数
下記参考例１(i)に開示した形質転換体エシエリ
ヒア コリ（Escherichia coli）DH1／pTF4
（IFO−14299）は、昭和59年４月６日から通商産
業省工業技術院微生物工業技術研究所（FRI）に
受託番号FERM Ｐ−7578として寄託され、該寄
託がブタペスト条約に基づく寄託に切換えられ
て、受託番号FERM BP−628として同研究所に
保管されている。
実施例
以下の実施例および参考例によつて本発明を具
体的に説明するが、本発明はこれらに限定される
ものではない。
なお実施例および参考例２，３において用いた
原液は、参考例１に記載の方法で得られた非グリ
コシル化ヒトIL−２蛋白質溶液である。
実施例 １
原液を注射用蒸留水で希釈し除菌過して得た
ヒトIL−２ 2450単位を含有する水溶液それぞ
れ0.5mlに、グルタチオン２mgまたはアスコルビ
ン酸２mgを含有する除菌過液0.5mlを加え、グ
ルタチオン２mgまたはアスコルビン酸２mgを含有
する除菌過液0.5mlを加え、得られた２種の水
溶液おのおの１mlをバイアルに分注して−40℃で
凍結し、乾燥後バイアル空間部をN2ガスで置換
し施栓巻締した。
対照としてグルタチオンおよびアスコルビン酸
を含まない同量の水溶液およびグルタチオンおよ
びアスコルビン酸のかわりに凍結乾燥製剤に繁用
されるマンニツト25mgを含有する同量の水溶液を
同様に凍結乾燥した。
これらの凍結乾燥品をいずれも注射用蒸留水１
mlで再溶解し、これらの溶液の溶状（澄明度）及
び力価を調べた。力価については凍結乾燥前の水
溶液力価を100％とした時の残存率を計算した。
その結果は、第１表に示されるとおり、本発明の
IL−２組成物は対照と比較し溶状、力価残存率
共に有意に優れていた。[Table] Examples include non-glycosylated human IL-2 produced by genetic recombination technology represented by the formula [wherein X represents Met or hydrogen], and mixtures thereof may also be used. In formula (), the amino acid residue is
IUPAC-IUB Commission on Biochemical Nomenclature. In addition, the specific activity of IL-2 is 20,000 to 80,000 units/mg.
It is desirable that the IL-2 aqueous solution is 1
Those having an activity of ˜80,000 units/ml, especially 10-50,000 units/ml are advantageously used. Further, the above-mentioned reducing substances such as glutathione or ascorbic acid can be used alone or in combination of two or more. These reducing substances should be 0.01 mg or more per ml of the above concentration IL-2 aqueous solution, especially
It is preferable to contain 0.05 to 20 mg. In addition to the above-mentioned reducing substances, the IL-2 composition of the present invention contains human serum albumin (hereinafter abbreviated as HSA), monosaccharides such as glucose, fructose, mannose, and galactose, and disaccharides such as sucrose, maltose, and lactose. , sugar alcohols such as mannitrate, inosite, and sorbitate, amino acids such as glycine, aspartic acid, glutamic acid, and proline, especially monoamino aliphatic amino acids or cyclic amino acids, or one or more of their physiologically acceptable salts or derivatives. By adding these, it is possible to further improve the stability, the appearance of the freeze-dried product, the solubility of the liquid in which it is dissolved, the prevention of adsorption to the container, etc., so these can be blended as appropriate. Among the above combination agents
HSA, glucose, mannose, mannite, sorbitol, proline, and glycine are preferred, and when these are blended, for example, IL-
It is preferable to add 0.1 to 50 mg, preferably 0.5 to 10 mg, of HSA, 10 to 100 mg of mannitrate, sorbitol, glucose or mannose, and 5 to 50 mg of glycine or proline per ml of the 2 aqueous solution. Furthermore, the IL-2 composition of the present invention may contain an isotonic agent such as common salt, a buffering agent such as succinic acid, tartaric acid, citric acid, a surfactant, and the like. Further, the stability of the IL-2 composition can be further enhanced by evacuating the space in the container of the IL-2 composition or replacing the container with nitrogen gas to prevent oxidation of the reducing substance contained therein. The IL-2 composition of the present invention can be an aqueous solution, a frozen product,
A form such as a freeze-dried product is preferred, and a freeze-dried product is particularly preferred. The composition of the present invention can be produced, for example, by the following method. Add a reducing substance to an aqueous solution containing 1 to 80,000 units/ml of IL-2, preferably 0.01 mg/ml or more.
Add to a concentration of 0.05 to 20 mg/ml (if two or more reducing substances are used, as the total amount). Furthermore, the aforementioned HSA, glucose, mannitrate, sorbitol, proline, glycine, etc. can also be added at the concentrations described therein. Further, an isotonic agent, a surfactant, etc. can be added if desired. As an aqueous solution obtained in this way,
The IL-2 composition can also be used as a raw material for the frozen and lyophilized products described below. The IL-2 composition as a frozen product can be produced, for example, by freezing the above aqueous solution usually at -80 to -20°C. Preferably, the frozen composition is stored at -80° to -10°C. The IL-2 composition as a lyophilized product can be prepared, for example, by drying the above-mentioned frozen composition under reduced pressure by a conventional method, or by dividing the above-mentioned aqueous solution or an aqueous solution obtained by melting the above-mentioned frozen composition into small portions, and freezing them in the same manner as above. , can be produced by drying under reduced pressure by a conventional method. Lyophilized IL of the invention as an injectable formulation
-2 composition, the aqueous solution containing IL-2 and the aqueous solution containing the compounding agent are sterilized and mixed, or the mixed solution is purified by sterilization or the like before being divided into portions. It is preferable to dispense and divide into vials or the like by aseptic operation and then subject to the above-mentioned freeze-drying treatment. Apart from the IL-2 composition containing the above-mentioned reducing substance, the present invention also provides the following IL-2 composition. Namely, amino acids such as glycine, glutamic acid, aspartic acid, alanine, in particular monoamino aliphatic amino acids, monosaccharides such as glucose, mannose, human serum albumin (HSA) and one or two of their physiologically acceptable salts or derivatives. This is an IL-2 composition containing the above ingredients. Regarding the above-mentioned IL-2, the same ones as the above-mentioned IL-2 can be mentioned, and an aqueous solution of IL-2 is preferable.
10-100mg/ml for the above monosaccharides, 5-50mg/ml for amino acids, and 0.1-100mg/ml for HSA.
It is preferable to mix 5 mg/ml, preferably 0.5 to 10 mg/ml. All of the above IL-2 compositions require storage of an aqueous solution,
It is characterized by a small decrease in IL-2 activity during freezing or freeze-drying operations and storage after freeze-drying, particularly during drying operations during freeze-drying. In addition to the above-mentioned stabilizing effect, compositions containing amino acids improve the appearance when used as a lyophilized product, and also have the effect of reducing pain when administered as an injection. Furthermore, compositions containing monosaccharides also have the effect of reducing pain when administered as injections. Compositions containing HSA, especially freeze-dried products thereof, have the effect of improving their appearance and preventing their adsorption to vessel walls. Furthermore, the above IL-2 composition includes an isotonic agent such as common salt, a buffering agent such as succinic acid, tartaric acid, citric acid, etc.
Surfactants and the like can also be blended. The above IL-2 composition is preferably in the form of an aqueous solution, a frozen product, a lyophilized product, etc., and a lyophilized product is particularly preferred. The above-mentioned IL-2 composition can be manufactured according to the manufacturing method of the IL-2 composition containing a reducing substance as described above, and in this IL-2 composition, acidic amino acids such as glutamic acid are When blending, it is preferable to adjust the composition to be acidic (PH 3 to 6) using the substance, a mineral salt such as hydrochloric acid or phosphoric acid, or the above-mentioned buffer. Effect The IL-2 composition of the present invention has excellent properties such as less decrease in IL-2 activity during storage and during freezing and freeze-drying operations, and in the case of a freeze-dried product, the solution state is clear when redissolved. In particular, it is characterized by a small decrease in IL-2 activity during the drying operation during freeze-drying. Further, the lyophilized product is obtained as a stabilized IL-2 powder and can be particularly advantageously used as a parenteral preparation. When used as an injection preparation, the lyophilized composition is dissolved in 0.5 to 100 ml of distilled water for injection, physiological saline, or glucose injection per vial and administered intramuscularly or intravenously. Furthermore, by using appropriate carriers, excipients, and diluents, it can be used as a dosage form for intraoral, ophthalmic, otic, or intranasal administration. The IL-2 composition of the present invention has low toxicity and
It can be used for the same purposes and in the same manner as IL-2. Unit (U) as the activity of IL-2 herein
The calculation method was as follows. That is, a mouse cell line that proliferated depending on the IL-2 concentration was cultured in a suspended medium by adding a specimen containing IL-2, and the proliferation of the cell line was determined using tritium thymidine incorporation as an index. To calculate the unit (U) in the target sample, always use the standard
Perform the assay by arranging IL-2 (1U/ml),
The unit was calculated from that ratio. Specifically, IL-2-dependent mouse cells were subcultured in RPMI1640 medium supplemented with 20% FCS and conditioned medium containing human IL-2 at 37°C in the presence of 5% Co2 . Stock [(NKC3),
Hinuma et al., Biochemical and Biophysical Research Communications, Vol. 109,
363 (1982)] twice with serum-free RPMI1640 medium, and 6x in RPMI1640 medium supplemented with 20% FCS.
10 Resuspend to 5 cells/ml. 50 μl of the material containing IL-2 was placed in the first row of wells of a 96-well flat-bottomed microtiter plate (Nunc, Denmark), and 50 μl of RPMI1640 with 20% FCS was added to each well.
After preparing a 2-fold serial dilution series up to the 12th column using the medium, 50 μl of the above NKC3 cell suspension is dispensed into each well and cultured at 37° C. in the presence of 5% Co 2 for 24 hours. At 20 hours of culture, add 1 μCi of tritium thymidine (Amarsyam, UK) to each well, continue culturing for another 4 hours, and collect cells on a glass filter using a cell harvester (Flow, USA). and measure the uptake of tritiated thymidine using a liquid scintillation counter. During the measurement, the same procedure as in the data is performed on the standard IL-2 specimen, and the uptake of tritium thymidine is measured. The unit (U) is calculated using the probit transformation method according to Journal of Immunology, Vol. 120, p. 2027 (1978). i.e. standard
IL-2 preparation (5 x 106 human peripheral blood lymphocytes/
ml of concanavalin-A and 12-O-tetradecanoylphorbol-13-acetate.
15ng/ml at 37°C in the presence of 5% CO2 .
Calculate the ratio (%) of the uptake value for each dilution step, assuming that the maximum uptake of the dilution series of the centrifuged supernatant of the culture solution after 48 hours of culture is defined as 1 U/ml is 100%. Plot the obtained values on normal probability paper and calculate 50
Determine the dilution factor indicating the % uptake from the plot. Similarly, for each material containing IL-2, find the dilution factor that indicates 50% uptake. The IL-2 concentration (U/ml) of the sample is calculated according to the following formula: Dilution factor for which the sample shows 50% uptake/50 for the standard IL-2 preparation.
Dilution factor showing % uptake Transformant Escherichia coli DH1/pTF4 disclosed in Reference Example 1(i) below
(IFO-14299) was deposited with the Microbial Research Institute (FRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry on April 6, 1982, under accession number FERM P-7578, and the deposit was changed to a deposit based on the Budapest Treaty. It is stored at the same institute under the accession number FERM BP-628. EXAMPLES The present invention will be specifically explained using the following Examples and Reference Examples, but the present invention is not limited thereto. The stock solution used in Examples and Reference Examples 2 and 3 was a non-glycosylated human IL-2 protein solution obtained by the method described in Reference Example 1. Example 1 0.5 ml of a sterilized solution containing 2 mg of glutathione or 2 mg of ascorbic acid was added to 0.5 ml of each aqueous solution containing 2450 units of human IL-2 obtained by diluting the stock solution with distilled water for injection and sterilizing it. In addition, 0.5 ml of sterilizing solution containing 2 mg of glutathione or 2 mg of ascorbic acid was added, and 1 ml of each of the two resulting aqueous solutions was dispensed into vials and frozen at -40°C. After drying, the vial space was sealed with N. The gas was replaced with 2 gases and the plug was tightened. As a control, the same amount of an aqueous solution without glutathione and ascorbic acid and the same amount of an aqueous solution containing 25 mg of mannite, which is commonly used in lyophilized preparations instead of glutathione and ascorbic acid, were similarly freeze-dried. Both of these freeze-dried products are mixed with 1 part of distilled water for injection.
ml, and the solubility (clarity) and potency of these solutions were examined. Regarding the titer, the residual rate was calculated based on the aqueous solution titer before freeze-drying as 100%.
As shown in Table 1, the results are as follows:
The IL-2 composition was significantly superior to the control in terms of solubility and residual potency.
【表】
実施例 ２
原液を注射用蒸留水で希釈し除菌過して得た
ヒトIL−２ 7680単位を含有する水溶液それぞ
れ0.5mlに、グルタチオン２mgまたはアスコルビ
ン酸２mgを含有する除菌過液0.5mlを加え得ら
れた２種の水溶液おのおの１mlを実施例１と同様
に凍結乾燥し、実施例１と同様の方法で製造直後
に溶状を、25℃で１カ月保存後に溶状及び力価残
存率を調べた。
その結果は第２表に示す通りであつた。[Table] Example 2 A sterilized solution containing 2 mg of glutathione or 2 mg of ascorbic acid in 0.5 ml of each aqueous solution containing 7680 units of human IL-2 obtained by diluting the stock solution with distilled water for injection and sterilizing it. 1 ml of each of the two types of aqueous solutions obtained by adding 0.5 ml was freeze-dried in the same manner as in Example 1, and the solution state was determined immediately after production in the same manner as in Example 1, and the solution state and titer remained after storage at 25°C for 1 month. I checked the rate. The results were as shown in Table 2.
【表】
実施例 ３
原液を注射用蒸留水で希釈し除菌過して得た
ヒトIL−２ 7680単位を含有する水溶液それぞ
れ0.5mlに、HSA5mgとグルタチオン２mgもしく
はアスコルビン酸２mgとを含有する除菌過液
0.5mlを加え、得られた２種の水溶液おのおの１
mlを実施例１と同様に凍結乾燥し、実施例２と同
様に溶状および力価残存率を調べた。
その結果は第３表に示す通りであつた。[Table] Example 3 A diluent containing 5 mg of HSA and 2 mg of glutathione or 2 mg of ascorbic acid was added to 0.5 ml of each aqueous solution containing 7680 units of human IL-2 obtained by diluting the stock solution with distilled water for injection and sterilizing it. Bacterial superfluous fluid
Add 0.5ml to each of the two resulting aqueous solutions.
ml was freeze-dried in the same manner as in Example 1, and the solubility and residual titer were examined in the same manner as in Example 2. The results were as shown in Table 3.
【表】
実施例 ４
原液を注射用蒸留水で希釈し除菌過して得た
ヒトIL−２ 7680単位を含有する水溶液それぞ
れ0.5mlに、HSA5mgおよび食塩９mgとグルタチ
オン２mgもしくはアスコルビン酸２mgとを含有す
る除菌過液0.5mlを加え、得られた２種の水溶
液おのおの１mlを実施例１と同様に凍結乾燥し、
実施例２と同様に溶状および力価残存率を調べ
た。
その結果は第４表に示す通りであつた。[Table] Example 4 5 mg of HSA, 9 mg of common salt, and 2 mg of glutathione or 2 mg of ascorbic acid were added to 0.5 ml of each aqueous solution containing 7680 units of human IL-2 obtained by diluting the stock solution with distilled water for injection and sterilizing it. Add 0.5 ml of the sterilizing supernatant solution, freeze-dry 1 ml of each of the two obtained aqueous solutions in the same manner as in Example 1,
In the same manner as in Example 2, the solubility and residual potency were examined. The results were as shown in Table 4.
【表】
実施例 ５
原液を注射用蒸留水で希釈し除菌過して得た
ヒトIL−２ 7680単位を含有する水溶液0.5mlに、
マンニツト50mgとグルタチオン２mgもしくはアス
コルビン酸２mgとを含有する除菌過液0.5mlを
加え、得られた２種の水溶液おのおの１mlを実施
例１と同様に凍結乾燥し、実施例２と同様に溶状
および力価残存率を調べた。
その結果は第５表に示す通りであつた。[Table] Example 5 To 0.5 ml of an aqueous solution containing 7680 units of human IL-2 obtained by diluting the stock solution with distilled water for injection and sterilizing it,
Add 0.5 ml of a sterilizing filtrate containing 50 mg of mannitrate and 2 mg of glutathione or 2 mg of ascorbic acid, freeze-dry 1 ml of each of the two resulting aqueous solutions in the same manner as in Example 1, and dry the solution and The residual titer rate was investigated. The results were as shown in Table 5.
【表】
実施例 ６
原液を注射用蒸留水で希釈し除菌過して得た
ヒトIL−２ 23350単位を含有する水溶液0.5ml
に、グルタチオン５mgおよびグリシン23mgを含有
する除菌過した水溶液0.5mlを加え、得られた
水溶液１mlを実施例１と同様に凍結乾燥し実施例
１と同様の方法で製造直後及び40℃、３週間保存
後に溶状及び力価残存率を調べた。
その結果は第６表に示す通りであつた。[Table] Example 6 0.5 ml of an aqueous solution containing 23,350 units of human IL-2 obtained by diluting the stock solution with distilled water for injection and sterilizing it.
0.5 ml of a sterilized aqueous solution containing 5 mg of glutathione and 23 mg of glycine was added to the solution, and 1 ml of the resulting aqueous solution was freeze-dried in the same manner as in Example 1. After storage for a week, the solubility and residual potency were examined. The results were as shown in Table 6.
【表】
実施例 ７
原液を注射用蒸留水で希釈し除菌過して得た
ヒトIL−２を1790単位または130単位を含有する
水溶液それぞれ0.5mlに、グルタチオン２mg、
HSA5mgおよび食塩９mgを含有する除菌過した
水溶液0.5mlを加えて、得られた２種の水溶液お
のおの１mlを実施例１と同様に凍結乾燥し実施例
１と同様の方法で製造直後および40℃１週間保存
後に溶状及び力価残存率を調べた。
その結果は第７表に示す通りであつた。[Table] Example 7 0.5 ml of each aqueous solution containing 1790 units or 130 units of human IL-2 obtained by diluting the stock solution with distilled water for injection and sterilization was added with 2 mg of glutathione,
Add 0.5 ml of a sterilized aqueous solution containing 5 mg of HSA and 9 mg of common salt, and freeze-dry 1 ml of each of the two resulting aqueous solutions in the same manner as in Example 1. After storage for one week, the solubility and residual potency were examined. The results were as shown in Table 7.
【表】
実施例 ８
原液を注射用蒸留水で希釈し除菌過して得た
ヒトIL−２を1860単位または116単位を含有する
水溶液それぞれ0.5mlにグルタチオン２mg、
HSA1mgおよびグリシン23mgを含有する除菌過
した水溶液0.5mlを加えて、得られた２種の水溶
液おのおの１mlを実施例１と同様に凍結乾燥し製
造直後および40℃１週間保存後に溶状および力価
残存率を調べた。
その結果は第８表に示す通りであつた。[Table] Example 8 0.5 ml of each aqueous solution containing 1860 units or 116 units of human IL-2 obtained by diluting the stock solution with distilled water for injection and sterilization was added with 2 mg of glutathione,
Add 0.5 ml of a sterilized aqueous solution containing 1 mg of HSA and 23 mg of glycine, and freeze-dry 1 ml of each of the two resulting aqueous solutions in the same manner as in Example 1. The survival rate was investigated. The results were as shown in Table 8.
【表】
参考例１非グリコシル化ヒトIL−２蛋白質溶液
の製造
(i) 特願昭58−225079号（昭和58年11月28日出
願）明細書の実施例３で得たヒトIL−２遺伝
子を含有する形質転換体エシエリヒア コリ
（E.coli）DH1／pTF4を250ml容三角フラスコ
内のバクト・トリプトン（デイフコ・ラボラト
リーズ、アメリカ）１％、バクト・イーストエ
キス（デイフコ・ラボラトリーズ、アメリカ）
0.5％、食塩0.5％およびテトラサイクリン
7μg／mlを含む液体培地（PH7.0）50mlに接種し
て37℃で１晩回転振盪培養した。この培養液を
カザミノ酸0.5％、グルコース0.5％およびテト
ラサイクリン7μg／mlを含むM9培地2.5の入
つた５容ジヤーフアーメンターに移し37℃で
４時間、ついで３−β−インドリルアクリル酸
（25μg／ml）を添加して、さらに４時間通気撹
拌培養して培養液2.5を得た。この培養液を
遠心分離し、菌体を集め、−80℃で凍結して保
存した。
(ii) 上記(i)で得た凍結保存菌体37.5ｇを7M塩酸
グアニジン、0.1M Tris・HClを含む抽出液
（PH7.0）500mlに均一に懸濁し、４℃で１時間
撹拌した。この溶菌液を28000×ｇで20分間遠
心分離して上清453mlを得た。
(iii) 上記(ii)で得た上清を0.01M Tris・HCl緩衝
液（PH8.5）に対して透析後19000×ｇで10分間
遠心分離して透析上清458mlを得た。この透析
上清を0.01M Tris・HCl緩衝液（PH8.5）で平
衡化したDE52（DEAE−セルロース、ワツトマ
ン社製、イギリス）カラム（50ml容）に通して
蛋白を吸着させ、NaCl濃度直線勾配（０〜
0.15M NaCl、１）を作成してIL−２を溶出
させた。活性画分105mlをYM−５メンブラン
（アミコン社製、アメリカ）を用いて10.2mlに
濃縮し、0.1M Tris・HCl（PH8.0）−1M NaCl
緩衝液で平衡化したセフアクリルＳ−200（フア
ルマシア社製、スエーデン）カラム（500ml容）
を用いてゲル過を行つた。活性画分56mlを
YM−５メンブランで4.9mlに濃縮した。得ら
れた濃縮液をウルトラポアRPSC（アルテツク
ス社製、アメリカ）カラムに吸着させ、トリフ
ルオロ酢酸−アセトニトリル系を溶出溶媒とす
る高速液体クロマトグラフイーを行つた。
カラム、ウルトラポアRPSC（4.6×75mm）；カ
ラム温度、30℃；溶出溶媒Ａ、0.1％トリフル
オロ酢酸−99.9％水；溶出溶媒Ｂ、0.1％トリ
フルオロ酢酸−99.9％アセトニトリル；溶出プ
ログラム、０分（68％Ａ＋32％Ｂ）−25分（55
％Ａ＋45％Ｂ）−35分（45％Ａ＋55％Ｂ）−45分
（30％Ａ＋70％Ｂ）−48分（100％Ｂ）；溶出速
度、0.8ml／min；検出波長、230nm。本条件下
で保持時間約39分の活性画分を集め、非グリコ
シル化ヒトIL−２蛋白質7.5mg〔比活性、
30000U／mg、出発材料からの活性回収率、
48.2％；蛋白質の純度、99％（デンシトメトリ
ーによる）〕を含む溶液15mlを得た。
参考例 ２
原液を注射用蒸留水で希釈し除菌過して得た
ヒトIL−２ 17600単位を含有する水溶液それぞ
れ0.5mlに、HSA5mgを含有し塩酸でPH４に調整
し除菌過した水溶液またはHSA5mgおよび食塩
９mgを含有し塩酸でPH４に調整し除菌過した水
溶液0.5mlを加え、得られた２種の水溶液を実施
例１と同様に凍結乾燥し、実施例２と同様の方法
で製造直後及び40℃0.5カ月後に溶状及び力価残
存率を調査した。
その結果は第９表に示す通りであつた。[Table] Reference Example 1 Production of non-glycosylated human IL-2 protein solution (i) Human IL-2 obtained in Example 3 of the specification of Japanese Patent Application No. 1983-225079 (filed on November 28, 1988) Transformant E. coli DH1/pTF4 containing the gene was mixed with 1% Bacto tryptone (Difco Laboratories, USA) and Bacto yeast extract (Difco Laboratories, USA) in a 250 ml Erlenmeyer flask.
0.5%, salt 0.5% and tetracycline
It was inoculated into 50 ml of liquid medium (PH 7.0) containing 7 μg/ml and cultured overnight at 37° C. with rotational shaking. The culture was transferred to a 5-volume jar fermenter containing 2.5 volumes of M9 medium containing 0.5% casamino acids, 0.5% glucose, and 7 μg/ml of tetracycline at 37°C for 4 hours, and then incubated with 3-β-indolyl acrylic acid (25 μg/ml). /ml) and cultured with aeration for another 4 hours to obtain a culture solution 2.5. This culture solution was centrifuged, and the bacterial cells were collected, frozen at -80°C, and stored. (ii) 37.5 g of cryopreserved bacterial cells obtained in (i) above were uniformly suspended in 500 ml of an extract solution (PH7.0) containing 7 M guanidine hydrochloride and 0.1 M Tris.HCl, and stirred at 4°C for 1 hour. This lysate was centrifuged at 28,000 xg for 20 minutes to obtain 453 ml of supernatant. (iii) The supernatant obtained in (ii) above was dialyzed against 0.01M Tris.HCl buffer (PH8.5) and centrifuged at 19,000 xg for 10 minutes to obtain 458 ml of dialysis supernatant. This dialysis supernatant was passed through a DE52 (DEAE-Cellulose, Watmann, UK) column (50 ml volume) equilibrated with 0.01M Tris/HCl buffer (PH8.5) to adsorb protein, and the NaCl concentration linear gradient was (0~
0.15M NaCl, 1) was prepared to elute IL-2. 105 ml of the active fraction was concentrated to 10.2 ml using a YM-5 membrane (manufactured by Amicon, USA), and mixed with 0.1 M Tris・HCl (PH8.0) - 1 M NaCl.
Sephacryl S-200 (Pharmacia, Sweden) column (500 ml volume) equilibrated with buffer solution
Gel filtration was performed using 56ml of active fraction
It was concentrated to 4.9 ml using a YM-5 membrane. The obtained concentrated solution was adsorbed on an Ultrapore RPSC (manufactured by Artex, USA) column, and high performance liquid chromatography was performed using trifluoroacetic acid-acetonitrile as an elution solvent. Column, Ultrapore RPSC (4.6 x 75 mm); Column temperature, 30°C; Elution solvent A, 0.1% trifluoroacetic acid - 99.9% water; Elution solvent B, 0.1% trifluoroacetic acid - 99.9% acetonitrile; Elution program, 0 minutes (68% A + 32% B) - 25 minutes (55
% A + 45% B) - 35 minutes (45% A + 55% B) - 45 minutes (30% A + 70% B) - 48 minutes (100% B); Elution rate, 0.8 ml/min; Detection wavelength, 230 nm. The active fraction with a retention time of approximately 39 minutes under these conditions was collected, and 7.5 mg of non-glycosylated human IL-2 protein [specific activity,
30000U/mg, activity recovery from starting material,
15 ml of a solution containing 48.2%; protein purity, 99% (according to densitometry) was obtained. Reference Example 2 An aqueous solution containing 17,600 units of human IL-2 obtained by diluting the stock solution with distilled water for injection and sterilization, and an aqueous solution containing 5 mg of HSA, adjusted to pH 4 with hydrochloric acid and sterilized, or 0.5 ml of an aqueous solution containing 5 mg of HSA and 9 mg of common salt, adjusted to pH 4 with hydrochloric acid and sterilized, was added, and the resulting two types of aqueous solutions were freeze-dried in the same manner as in Example 1, and produced in the same manner as in Example 2. Immediately after and after 0.5 months at 40°C, the solubility and residual titer were investigated. The results were as shown in Table 9.
【表】
参考例 ３
原液を注射用蒸留水で希釈し無菌過して得た
ヒトIL−２を17600単位を含有する水溶液0.5mlに
グリシン23mgを含有しコハク酸でPH４に調整し除
菌過した水溶液0.5mlを加えて、得られた水溶
液１mlを実施例１と同様に凍結乾燥し、実施例２
と同様の方法で製造直後及び40℃0.5カ月後に溶
状及び力価残存率を調べた。
その結果は第10表に示す通りであつた。[Table] Reference Example 3 Human IL-2 obtained by diluting the stock solution with distilled water for injection and sterilizing it was sterilized by adding 23 mg of glycine to 0.5 ml of an aqueous solution containing 17,600 units and adjusting the pH to 4 with succinic acid. 0.5 ml of the obtained aqueous solution was added, and 1 ml of the obtained aqueous solution was freeze-dried in the same manner as in Example 1.
Immediately after production and after 0.5 months at 40°C, the solubility and residual titer were examined in the same manner as above. The results were as shown in Table 10.
【表】
発明の効果
本発明のIL−２組成物は、保存中および凍結
や凍結乾燥操作におけるIL−２活性の低下が少
なく、また凍結乾燥品においてはその再溶解時の
溶状が澄明である点等の優れた特徴を有し、医薬
品製剤等とりわけ非経口投与製剤として有利に用
いることができる。[Table] Effects of the Invention The IL-2 composition of the present invention shows little decrease in IL-2 activity during storage and during freezing and lyophilization operations, and the lyophilized product has a clear solution state when redissolved. It can be advantageously used as pharmaceutical preparations, especially parenteral preparations.
Claims (1)
たインターロイキン−２組成物。 ２ さらにヒト血清アルブミンを配合した特許請
求の範囲第１項記載の組成物。 ３ 凍結乾燥品である特許請求の範囲第１項また
は第２項記載の組成物。[Scope of Claims] 1. An interleukin-2 composition containing glutathione or ascorbic acid. 2. The composition according to claim 1, further comprising human serum albumin. 3. The composition according to claim 1 or 2, which is a lyophilized product.
Priority Applications (17)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59071568A JPS60215631A (en) | 1984-04-09 | 1984-04-09 | Interleukin-2 composition |
| ZA852302A ZA852302B (en) | 1984-04-09 | 1985-03-27 | Stable composition of interleukin-2 |
| AT85302176T ATE66612T1 (en) | 1984-04-09 | 1985-03-28 | STABLE INTERLEUKIN-2 COMPOSITION. |
| EP19850302176 EP0158487B1 (en) | 1984-04-09 | 1985-03-28 | Stable composition of interleukin-2 |
| DE8585302176T DE3583880D1 (en) | 1984-04-09 | 1985-03-28 | STABLE INTERLEUKIN-2 COMPOSITION. |
| PH32069A PH22897A (en) | 1984-04-09 | 1985-03-29 | Stable composition of interleukin-2 |
| DK148885A DK148885A (en) | 1984-04-09 | 1985-04-02 | STABLE PREPARATION OF INTERLEUKIN-2 |
| NZ211702A NZ211702A (en) | 1984-04-09 | 1985-04-04 | Interleukin composition |
| IL74823A IL74823A (en) | 1984-04-09 | 1985-04-04 | Stable composition of interleukin-2 |
| CA000478351A CA1285478C (en) | 1984-04-09 | 1985-04-04 | Stable composition of interleukin-2 |
| AU40839/85A AU579359B2 (en) | 1984-04-09 | 1985-04-04 | Stable composition of interleukin-2 |
| US06/720,754 US4645830A (en) | 1984-04-09 | 1985-04-08 | Stable composition of interleukin-2 and albumin |
| PT80247A PT80247B (en) | 1984-04-09 | 1985-04-08 | METHOD FOR THE PREPARATION OF A STABLE INTERLEUKIN-2 COMPOSITION |
| KR1019850002345A KR920005048B1 (en) | 1984-04-09 | 1985-04-08 | Process for preparing stable composition of interleukin-2 |
| ES542028A ES542028A0 (en) | 1984-04-09 | 1985-04-08 | A METHOD OF PRODUCING AN INTERLEUKIN-2 COMPOSITION |
| IE90285A IE58161B1 (en) | 1984-04-09 | 1985-04-10 | Stable composition of interleukin-2 |
| US06/931,704 US4812557A (en) | 1984-04-09 | 1986-11-17 | Stable composition of interleukin-2 and human serum albumin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59071568A JPS60215631A (en) | 1984-04-09 | 1984-04-09 | Interleukin-2 composition |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP60013226A Division JPS60222424A (en) | 1984-04-09 | 1985-01-25 | Interleukin 2 composition |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS60215631A JPS60215631A (en) | 1985-10-29 |
| JPH0361652B2 true JPH0361652B2 (en) | 1991-09-20 |
Family
ID=13464438
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP59071568A Granted JPS60215631A (en) | 1984-04-09 | 1984-04-09 | Interleukin-2 composition |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPS60215631A (en) |
| ZA (1) | ZA852302B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3621828A1 (en) * | 1986-06-28 | 1988-01-14 | Biotest Pharma Gmbh | STABILIZATION OF A THERAPEUTIC PURPOSE, ESPECIALLY FOR HUMANS, CERTAIN INTERLEUKIN-2-PREPARATES AND STABILIZED AQUEOUS SOLUTION OR SOLID CONTAINING THIS PREPARATION |
| JP2577744B2 (en) * | 1986-07-18 | 1997-02-05 | 中外製薬株式会社 | Stable granulocyte colony-stimulating factor containing preparation |
| JP2799483B2 (en) * | 1988-03-09 | 1998-09-17 | 大塚製薬株式会社 | Method for stabilizing interleukin-1β composition |
| HUP0203133A3 (en) * | 1999-10-04 | 2005-07-28 | Chiron Corp Emeryville | Stabilized liquid polypeptide-containing pharmaceutical compositions |
| US6689353B1 (en) * | 2000-06-28 | 2004-02-10 | Bayer Pharmaceuticals Corporation | Stabilized interleukin 2 |
| ATE477810T1 (en) * | 2002-02-28 | 2010-09-15 | Nipro Corp | STABILIZED ALBUMIN PREPARATIONS |
-
1984
- 1984-04-09 JP JP59071568A patent/JPS60215631A/en active Granted
-
1985
- 1985-03-27 ZA ZA852302A patent/ZA852302B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| JPS60215631A (en) | 1985-10-29 |
| ZA852302B (en) | 1986-11-26 |
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