WO2016117341A1 - Method for purification of recombinant human alpha-galactosidase a from material containing contaminant host cell proteins - Google Patents
Method for purification of recombinant human alpha-galactosidase a from material containing contaminant host cell proteins Download PDFInfo
- Publication number
- WO2016117341A1 WO2016117341A1 PCT/JP2016/000296 JP2016000296W WO2016117341A1 WO 2016117341 A1 WO2016117341 A1 WO 2016117341A1 JP 2016000296 W JP2016000296 W JP 2016000296W WO 2016117341 A1 WO2016117341 A1 WO 2016117341A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gal
- alpha
- column chromatography
- recombinant human
- human alpha
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 44
- 101000718525 Homo sapiens Alpha-galactosidase A Proteins 0.000 title claims abstract description 40
- 102000043404 human GLA Human genes 0.000 title claims abstract description 40
- 239000000463 material Substances 0.000 title claims abstract description 15
- 238000000746 purification Methods 0.000 title description 42
- 108090000623 proteins and genes Proteins 0.000 title description 35
- 102000004169 proteins and genes Human genes 0.000 title description 23
- 239000000356 contaminant Substances 0.000 title description 18
- 101000718529 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) Alpha-galactosidase Proteins 0.000 claims abstract description 151
- NNISLDGFPWIBDF-MPRBLYSKSA-N alpha-D-Gal-(1->3)-beta-D-Gal-(1->4)-D-GlcNAc Chemical compound O[C@@H]1[C@@H](NC(=O)C)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@@H](CO)O1 NNISLDGFPWIBDF-MPRBLYSKSA-N 0.000 claims abstract description 151
- 238000004440 column chromatography Methods 0.000 claims abstract description 63
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 21
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 18
- 239000012228 culture supernatant Substances 0.000 claims abstract description 16
- 210000004962 mammalian cell Anatomy 0.000 claims abstract description 15
- 238000005341 cation exchange Methods 0.000 claims abstract description 14
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 claims abstract description 11
- 238000005349 anion exchange Methods 0.000 claims abstract description 10
- 238000002523 gelfiltration Methods 0.000 claims abstract description 10
- 239000012679 serum free medium Substances 0.000 claims abstract description 10
- 239000007790 solid phase Substances 0.000 claims abstract description 9
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 57
- 239000002609 medium Substances 0.000 claims description 29
- 239000003814 drug Substances 0.000 claims description 23
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 12
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 12
- 150000001768 cations Chemical class 0.000 claims description 11
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 9
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 239000013604 expression vector Substances 0.000 claims description 7
- 230000003993 interaction Effects 0.000 claims description 6
- 238000006664 bond formation reaction Methods 0.000 claims description 5
- 239000001257 hydrogen Substances 0.000 claims description 5
- 229910052739 hydrogen Inorganic materials 0.000 claims description 5
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical compound C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 claims description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 4
- 229910052587 fluorapatite Inorganic materials 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 230000033228 biological regulation Effects 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 64
- 239000008363 phosphate buffer Substances 0.000 description 38
- 239000011780 sodium chloride Substances 0.000 description 32
- 235000018102 proteins Nutrition 0.000 description 22
- 239000000243 solution Substances 0.000 description 20
- 150000003839 salts Chemical class 0.000 description 18
- 239000013598 vector Substances 0.000 description 17
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 16
- SXTAYKAGBXMACB-UHFFFAOYSA-N methionine sulfoximine Chemical compound CS(=N)(=O)CCC(N)C(O)=O SXTAYKAGBXMACB-UHFFFAOYSA-N 0.000 description 16
- 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 12
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 12
- 208000024720 Fabry Disease Diseases 0.000 description 10
- 239000000872 buffer Substances 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 229920002684 Sepharose Polymers 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 8
- 239000001103 potassium chloride Substances 0.000 description 8
- 235000011164 potassium chloride Nutrition 0.000 description 8
- 108091026890 Coding region Proteins 0.000 description 7
- 102000004877 Insulin Human genes 0.000 description 7
- 108090001061 Insulin Proteins 0.000 description 7
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 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 6
- 108020004414 DNA Proteins 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 229940024606 amino acid Drugs 0.000 description 6
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 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 6
- 239000012634 fragment Substances 0.000 description 6
- 229940125396 insulin Drugs 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 239000011347 resin Substances 0.000 description 6
- 229920005989 resin Polymers 0.000 description 6
- 229940104230 thymidine Drugs 0.000 description 6
- 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 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 108010076504 Protein Sorting Signals Proteins 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 108010030074 endodeoxyribonuclease MluI Proteins 0.000 description 5
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 5
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 5
- 229920000053 polysorbate 80 Polymers 0.000 description 5
- 229940068968 polysorbate 80 Drugs 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 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 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- 238000007792 addition Methods 0.000 description 4
- 102000005840 alpha-Galactosidase Human genes 0.000 description 4
- 108010030291 alpha-Galactosidase Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000012298 atmosphere Substances 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 238000002641 enzyme replacement therapy Methods 0.000 description 4
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 4
- 230000002779 inactivation Effects 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000007758 minimum essential medium Substances 0.000 description 4
- 239000002736 nonionic surfactant Substances 0.000 description 4
- 150000002482 oligosaccharides Polymers 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- STCOOQWBFONSKY-UHFFFAOYSA-N tributyl phosphate Chemical compound CCCCOP(=O)(OCCCC)OCCCC STCOOQWBFONSKY-UHFFFAOYSA-N 0.000 description 4
- 108091006522 Anion exchangers Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 239000012564 Q sepharose fast flow resin Substances 0.000 description 3
- 239000012505 Superdex™ Substances 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000008351 acetate buffer Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000013024 dilution buffer Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000009256 replacement therapy Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- HFQKBOPMAOTAIR-TZSVBWBLSA-N α-d-galactosyl-(1->4)-β-d-galactosyl-(1->4)-β-d-glucosylceramide Chemical compound O[C@@H]1[C@@H](O)[C@H](OC[C@@H]([C@H](O)/C=C/CCCCCCCCCCCCC)NC(C)=O)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](CO)O1 HFQKBOPMAOTAIR-TZSVBWBLSA-N 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000212384 Bifora Species 0.000 description 2
- NBSCHQHZLSJFNQ-QTVWNMPRSA-N D-Mannose-6-phosphate Chemical group OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@@H]1O NBSCHQHZLSJFNQ-QTVWNMPRSA-N 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 101100364969 Dictyostelium discoideum scai gene Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- 102000019218 Mannose-6-phosphate receptors Human genes 0.000 description 2
- 101100364971 Mus musculus Scai gene Proteins 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000000919 ceramic Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 150000002402 hexoses Chemical group 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000012510 hollow fiber Substances 0.000 description 2
- 229960000890 hydrocortisone Drugs 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000003169 placental effect Effects 0.000 description 2
- 229940068977 polysorbate 20 Drugs 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- KIDHWZJUCRJVML-UHFFFAOYSA-N putrescine Chemical compound NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 201000003624 spinocerebellar ataxia type 1 Diseases 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- YUDPTGPSBJVHCN-CHUNWDLHSA-N 4-methylumbelliferyl alpha-D-galactoside Chemical compound C1=CC=2C(C)=CC(=O)OC=2C=C1O[C@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O YUDPTGPSBJVHCN-CHUNWDLHSA-N 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 208000004652 Cardiovascular Abnormalities Diseases 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins 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
- 241000238631 Hexapoda Species 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 108010031792 IGF Type 2 Receptor Proteins 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- SXTAYKAGBXMACB-DPVSGNNYSA-N L-methionine sulfoximine Chemical compound CS(=N)(=O)CC[C@H](N)C(O)=O SXTAYKAGBXMACB-DPVSGNNYSA-N 0.000 description 1
- 208000015439 Lysosomal storage disease Diseases 0.000 description 1
- 108050006616 Mannose-6-phosphate receptors Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 102000029797 Prion Human genes 0.000 description 1
- 108091000054 Prion Proteins 0.000 description 1
- 239000005700 Putrescine Substances 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 206010062237 Renal impairment Diseases 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000036783 anaphylactic response Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 201000009431 angiokeratoma Diseases 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- 235000003704 aspartic acid Nutrition 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
- 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 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- GHWVXCQZPNWFRO-UHFFFAOYSA-N butane-2,3-diamine Chemical compound CC(N)C(C)N GHWVXCQZPNWFRO-UHFFFAOYSA-N 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000011545 carbonate/bicarbonate buffer Substances 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012527 feed solution Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 229960001031 glucose Drugs 0.000 description 1
- 229960002743 glutamine Drugs 0.000 description 1
- 102000005396 glutamine synthetase Human genes 0.000 description 1
- 108020002326 glutamine synthetase Proteins 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 150000002506 iron compounds Chemical class 0.000 description 1
- 230000013190 lipid storage Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 208000005907 mitral valve insufficiency Diseases 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- NRNCYVBFPDDJNE-UHFFFAOYSA-N pemoline Chemical compound O1C(N)=NC(=O)C1C1=CC=CC=C1 NRNCYVBFPDDJNE-UHFFFAOYSA-N 0.000 description 1
- 239000007793 ph indicator Substances 0.000 description 1
- 239000007981 phosphate-citrate buffer Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920002492 poly(sulfone) Polymers 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229950008882 polysorbate Drugs 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 229940071643 prefilled syringe Drugs 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 235000004400 serine Nutrition 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 239000011781 sodium selenite Substances 0.000 description 1
- 235000015921 sodium selenite Nutrition 0.000 description 1
- 229960001471 sodium selenite Drugs 0.000 description 1
- CIJQGPVMMRXSQW-UHFFFAOYSA-M sodium;2-aminoacetic acid;hydroxide Chemical compound O.[Na+].NCC([O-])=O CIJQGPVMMRXSQW-UHFFFAOYSA-M 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 229960000281 trometamol Drugs 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000002861 ventricular Effects 0.000 description 1
- 239000013621 viresolve pro solution Substances 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01022—Alpha-galactosidase (3.2.1.22)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- oligosaccharide chain means a chain of oligosaccharides covalently bound to the peptide chain of alpha-Gal A, including an asparagine type sugar chain, which is covalently bound to an asparagine residue of alpha-Gal A.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Cardiology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Heart & Thoracic Surgery (AREA)
- Urology & Nephrology (AREA)
- Hospice & Palliative Care (AREA)
- Vascular Medicine (AREA)
- Obesity (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Dermatology (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
Abstract
Disclosed is a method for production of recombinant human alpha-galactosidase A (rh alpha-Gal A) in a large scale, with a high purity. The method comprises the steps of (a) culturing rh alpha-Gal A-producing mammalian cells in a serum-free medium, (b) collecting culture supernatant, (c) subjecting the culture supernatant to anion-exchange column chromatography, (d) to hydrophobic column chromatography, (e) to a column chromatography employing as solid phase a material having affinity for phosphate group, (f) to cation-exchange column chromatography, (g) to dye-affinity column chromatography, and (h) to gel filtration column chromatography, in the order.
Description
The present invention generally relates to a method for production of human alpha-galactosidase A (rh alpha-Gal A) using host cells, and to a method for purification of the rh alpha-Gal A produced thereby from a material containing contaminant host cell proteins. More specifically the present invention relates to a process for production of rh alpha-Gal A by culturing rh alpha-Gal A-producing mammalian cells in a serum-free medium, as well as to a process for purification of thus produced rh alpha-Gal A from the culture supernatant using column chromatography including dye-affinity column chromatography, in high yield and to such a high purity as permitting direct use of the purified protein as a medical drug.
Alpha-galactosidase A (alpha-Gal A) is a lysosomal enzyme having an activity to hydrolyze the terminal alpha-galactosyl bond of glycolipids and glycoproteins. Trihexosylceramide, which contains three hexose moieties, is one of the substrates for alpha-galactosidase A and receives hydrolysis at its terminal hexose moiety by the enzyme. Human alpha-Gal A is expressed as a precursor peptide comprising 429 amino acids, of which N-terminal 31 amino acids constitute a signal peptide. The precursor is processed to the mature peptide, which consists of 398 amino acids left behind after the removal of the signal peptide, and a pair of the mature peptide molecules readily form a homodimer, wherever condition allows, which is the enzymatically active form of human alpha-Gal A. Human alpha-Gal A possesses oligosaccharide chains containing mannose-6-phosphate residues and can be targeted to the lysosome via a mannose-6-phosphate receptor.
Fabry's disease is an X-linked inherited lysosomal storage disease, which is caused by inherited abnormality of gene encoding alpha-Gal A. Lack of the activity of this enzyme causes accumulation of trihexosylceramide in various tissues, resulting in renal impairment, angiokeratomas, and cardiovascular abnormalities, including ventricular enlargement and mitral valve insufficiency.
It had already been known in the 1960's that the enzymatic activity of alpha-galactosidase A is scarcely or only weakly detected in the tissues of patients with Fabry's disease. It thus had been speculated that some inherited abnormality of the gene encoding alpha-Gal A should be the cause of Fabry's disease. Based on this speculation, alpha-Gal A replacement therapy for Fabry's disease had been conducted by infusing alpha-Gal A extracted and purified from human plasma or spleen to the patient, resulting in the confirmation that alpha-Gal A replacement therapy could lower the levels of trihexosylceramide in the blood of the patients suffering from Fabry's disease (see NPL 1, NPL 2, NPL 3). Therefore it had been considered that alpha-Gal A enzyme replacement therapy must be promising for Fabry's disease. Practical application of alpha-Gal A replacement therapy, however, has been extremely limited due to lack of supply of this enzyme.
In 1986, the gene encoding human alpha-Gal A was isolated (see NPL 4), and further, in 1988, a method was reported for expression and production of rh alpha-Gal A by using CHO cells transformed with a human alpha-Gal A expression vector (see NPL 5). Utilizing recombinant technology, these finding made it possible to produce human alpha-Gal A in a large scale, and to use the enzyme as a medicament for the enzyme replacement therapy for Fabry's disease. The rh alpha-Gal A has also been expressed in insect cells (PTL 1).
Further, some methods for purification of rh alpha-Gal A were reported comprising employment of hydrophobic column chromatography, heparin sepharose column chromatography, hydroxylapatite column chromatography, anion-exchange column chromatography, and gel filtration column chromatography (PTL 2, PTL 3). Although the purity of the rh alpha-Gal A obtained by the method for purification described in PTL 2 or PTL 3 has shown to be sufficiently high, it was not considered to be fully acceptable for human use. The reason is that while any medicaments to be infused to patients are required to be free of contaminants, such as, in particular, proteins derived from the host cells employed, either PTL 2 or PTL 3 fails to show whether such contaminants remain in the purified rh alpha-Gal A or not. And further a method for purification of rh alpha-Gal A were reported comprising employment of anion-exchange column chromatography, hydrophobic column chromatography, column chromatography employing as solid phase a material having affinity for phosphate group, cation-exchange column chromatography, and gel filtration column chromatography, in the order (PTL 4). However the PTLs are silent on contamination of rh alpha-Gal A with host cell proteins.
Currently, medical drugs for Fabry's disease containing rh alpha-Gal A as an active ingredient have been on the market under the trade names such as ReplagalTM and FabrazymeTM.
Mapes et al: Science 169: 987-9 (1970)
Brady et al: N. Engl. J. Med. 289: 9-14 (1973)
Desnick et al: Proc Natl Acad Sci USA 76: 5326-30 (1979).
Bishop et al: Proc Natl Acad Sci USA 83: 4859-63 (1986)
Bishop et al: Lipid Storage Disorders - Biological and Medical Aspects, NY Plenum Press P809-823 (1988)
Against the above background, it is an objective of the present invention to provide a method for production and purification of recombinant human alpha-galactosidase A (rh alpha-Gal A) starting with culturing rh alpha-Gal A producing mammalian cells in a serum-free medium.
It is another objective of the present invention to provide a process for purification of rh alpha-Gal A from a culture supernatant containing contaminant host cell proteins, through column chromatography including dye-affinity column chromatography, in high yield and to such a high purity as permits direct use of the purified protein as a medical drug.
The present inventors found that rh alpha-Gal A contained in the supernatant of a culture of rh alpha-Gal A-producing cells in a serum-free medium, can be purified from the supernatant which contains contaminant host cell proteins to a very high purity, and in a very high yield as well, by subjecting the rh alpha-Gal A to a purification process comprising a combination of anion-exchange column chromatography, hydrophobic column chromatography, chromatography using a column having affinity for phosphate group, cation-exchange column chromatography, dye-affinity column chromatography, and gel filtration column chromatography. The present invention was completed through further studies based on the finding.
Thus, the present invention provides what follows:
1. A method for production of a recombinant human alpha-Gal A comprising the steps of:
(a) culturing recombinant human alpha-Gal A-producing mammalian cells in a serum-free medium to let them secrete the recombinant human alpha-Gal A in the medium,
(b) collecting culture supernatant by removing the cells from the culture that is obtained in step (a) above,
(c) subjecting the culture supernatant collected in step (b) above to anion-exchange column chromatography to collect the recombinant human alpha-Gal A -active fractions,
(d) subjecting the fractions collected in step (c) above to hydrophobic column chromatography to collect recombinant human alpha-Gal A-active fractions,
(e) subjecting the fractions collected in step (d) above to a column chromatography employing as solid phase a material having affinity for phosphate group to collect the recombinant human alpha-Gal A-active fractions,
(f) subjecting the fractions collected in step (e) above to cation-exchange column chromatography to collect the recombinant human alpha-Gal A -active fractions,
(g) subjecting the fractions collected in step (f) above to dye-affinity column chromatography to collect the recombinant human alpha-Gal A -active fractions, and
(h) subjecting the fractions collected in step (g) above to gel filtration column chromatography to collect the recombinant human alpha-Gal A-active fractions, in the order.
2. The method according to (1) above, wherein the cation exchanger employed in the cation-exchange column chromatography is a weak cation exchanger.
3. The method according to (2) above, wherein the weak cation exchanger has a selectivity based on both hydrophobic interaction and hydrogen bond formation.
4. The method according to (2) or (3) above, wherein the weak cation exchanger has phenyl groups, amide bonds and carboxyl groups.
5. The method according to one of (1) to (4) above, wherein the dye employed in the dye-affinity column chromatography is a blue triazine dye.
6. The method according to one of (1) to (5) above, wherein the material having affinity for phosphate group is selected from the group consisting of hydroxyapatite and fluoroapatite.
7. The method according to (6) above, wherein the material having affinity for phosphate group is hydroxyapatite.
8. The method according to one of (1) to (7) above, wherein the mammalian cells are CHO cells transfected with an expression vector which is designed to express the recombinant human alpha-Gal A under the regulation of EF-1(alpha) promoter.
9. A recombinant human alpha-Gal A composition produced by the method according to one of (1) to (8) above.
10. A medicament comprising the composition according to (9) above administered periodically to a patient.
11. The medicament according to (10) above, wherein the medicament is administered to the patient by injection.
1. A method for production of a recombinant human alpha-Gal A comprising the steps of:
(a) culturing recombinant human alpha-Gal A-producing mammalian cells in a serum-free medium to let them secrete the recombinant human alpha-Gal A in the medium,
(b) collecting culture supernatant by removing the cells from the culture that is obtained in step (a) above,
(c) subjecting the culture supernatant collected in step (b) above to anion-exchange column chromatography to collect the recombinant human alpha-Gal A -active fractions,
(d) subjecting the fractions collected in step (c) above to hydrophobic column chromatography to collect recombinant human alpha-Gal A-active fractions,
(e) subjecting the fractions collected in step (d) above to a column chromatography employing as solid phase a material having affinity for phosphate group to collect the recombinant human alpha-Gal A-active fractions,
(f) subjecting the fractions collected in step (e) above to cation-exchange column chromatography to collect the recombinant human alpha-Gal A -active fractions,
(g) subjecting the fractions collected in step (f) above to dye-affinity column chromatography to collect the recombinant human alpha-Gal A -active fractions, and
(h) subjecting the fractions collected in step (g) above to gel filtration column chromatography to collect the recombinant human alpha-Gal A-active fractions, in the order.
2. The method according to (1) above, wherein the cation exchanger employed in the cation-exchange column chromatography is a weak cation exchanger.
3. The method according to (2) above, wherein the weak cation exchanger has a selectivity based on both hydrophobic interaction and hydrogen bond formation.
4. The method according to (2) or (3) above, wherein the weak cation exchanger has phenyl groups, amide bonds and carboxyl groups.
5. The method according to one of (1) to (4) above, wherein the dye employed in the dye-affinity column chromatography is a blue triazine dye.
6. The method according to one of (1) to (5) above, wherein the material having affinity for phosphate group is selected from the group consisting of hydroxyapatite and fluoroapatite.
7. The method according to (6) above, wherein the material having affinity for phosphate group is hydroxyapatite.
8. The method according to one of (1) to (7) above, wherein the mammalian cells are CHO cells transfected with an expression vector which is designed to express the recombinant human alpha-Gal A under the regulation of EF-1(alpha) promoter.
9. A recombinant human alpha-Gal A composition produced by the method according to one of (1) to (8) above.
10. A medicament comprising the composition according to (9) above administered periodically to a patient.
11. The medicament according to (10) above, wherein the medicament is administered to the patient by injection.
The present invention enables purification of rh alpha-Gal A from a material containing contaminant host cell proteins. Recombinant human alpha-Gal A thus purified is suitable for use as a medicament in enzyme replacement therapy (ERT) for Fabry's disease. In this therapy, rh alpha-Gal A must be administered periodically to the patient suffering from Fabry's disease. Therefore if an alpha-Gal A medicament contains host cell proteins, ERT using the medicament could have a potential risk to induce a severe antigen-antibody reaction in patients (i.e. anaphylaxis) against host cell proteins. Such a risk can be minimized by using rh alpha-Gal A purified according to the present invention as the medicament. Further, as it has enabled to produce rh alpha-Gal A starting with serum-free culturing of cells, the present invention provides rh alpha-Gal A which is free of any serum-derived contaminants, including pathogenic agents such as viruses or prions. Therefore, the rh alpha-Gal A obtained according to the present invention can be administered into a human body as a safe medicament substantially without any risks of exposure to such pathogenic agents. Further, as it has enabled purification of rh alpha-Gal A substantially avoiding any use of organic solvents, the present invention eliminates the risk of denaturation of rh alpha-Gal A which otherwise might be caused by the exposure to an organic solvent employed. Furthermore, the present invention is favorable to the environment, for the waste fluid left after performing the purification process according to it does not contain an organic solvent, and in the economic sense as well, for it requires no facility to dispose of organic solvents which would otherwise be contained in the waste fluid.
Further the present invention enables highly selective purification of rh alpha-Gal A having mannose-6-phosphate residue in its oligosaccharide chain. To exert its enzymatic activity after administration to a human body, the rh alpha-Gal A must be taken up by relevant cells through mannose-6-phosphate receptors expressed on their surface. Therefore, the highly selective purification achieved according to the present invention will give an rh alpha-Gal A product as a medicament with dramatically increased efficacy.
In the present invention, while the recombinant human alpha-galactosidase A (rh alpha-Gal A) is preferably a recombinant protein of human wild-type alpha-Gal A, it is not excluded that the rh alpha-Gal A is a mutant-type protein having one or more substitution, deletion, addition, and/or insertion of one or more amino acids as compared with the amino acid sequence of human wild-type alpha-Gal A. The DNA sequence for human wild-type alpha-Gal A and amino acid sequence encoded by it, including N-terminal signal sequence, are shown as SEQ ID NO:13 and SEQ ID NO:14, respectively. The N-terminal signal sequence consists of 31 amino acids and is removed post-translationally.
In the present invention, the term "recombinant human alpha-Gal A-producing mammalian cells" or "rh alpha-Gal A-producing mammalian cells" means mammalian cells which have been artificially manipulated to express, or strongly express, the gene encoding human alpha-Gal A. Though in general the gene to be strongly expressed is one which is introduced to the mammalian cells (transformation) using an expression vector in which the gene is incorporated, it may also be an intrinsic gene which has been artificially modified to be strongly expressed. Examples of the means for artificially modifying an intrinsic gene to make it strongly express itself include, but not limited to, replacement of the promoter upstream of the intrinsic gene with a promoter which strongly induces expression of the gene. Such methods have been disclosed in several literatures (i.e. WO94/12650, WO95/31560). Though there is no particular limitation as to which mammalian cells are to be employed, preferred are those of human-, mouse- or Chinese hamster-origin, and, among others, CHO cells, which originate from Chinese hamster ovary cells, are particularly preferred.
In the present invention, the term "recombinant human alpha-galactosidase A" or "rh alpha-Gal A" means the human alpha-galactosidase A which is secreted by the above-mentioned recombinant human alpha-Gal A-producing mammalian cells in the medium during culture.
In the present invention, the term "oligosaccharide chain" means a chain of oligosaccharides covalently bound to the peptide chain of alpha-Gal A, including an asparagine type sugar chain, which is covalently bound to an asparagine residue of alpha-Gal A.
In the present invention, an example of preferable serum-free media in which the rh alpha-Gal A-producing mammalian cells are to be cultured is the following medium which comprises; 3-700 mg/mL of amino acids, 0.001-50 mg/L of vitamins, 0.3-10 g/L of monosaccharides, 0.1-10000 mg/L of inorganic salts, 0.001-0.1 mg/L of trace elements, 0.1-50 mg/L of nucleosides, 0.001-10 mg/L of fatty acids, 0.01-1 mg/L of biotin, 0.1-20 micrograms/L of hydrocortisone, 0.1-20 mg/L of insulin, 0.0-10 mg/L of vitamin B12, 0.01-1 mg/L of putrescine, 10-500 mg/L of sodium pyruvate, and water-soluble iron compounds. As desired, it may also include thymidine, hypoxanthine, a conventional pH indicator, and antibiotics.
Further, DMEM/F12 medium, a mixed medium consisting of DMEM and F12, may also be used as a basic serum-free medium. Both of these media are well known to those skilled in the art. As a serum-free medium, DMEM(HG)HAM modified (R5) medium may be used, too, which contains sodium hydrogen carbonate, L-glutamine, D-glucose, insulin, sodium selenite, diaminobutane, hydrocortisone, ferric (II) sulfate, asparagine, aspartic acid, serine, and polyvinyl alcohol. Furthermore, a commercially available serum-free medium may also be used as a basic medium, such as CDoptiCHOTM, CHO-S-SFM II or CD CHO (Invitrogen), IS CHO-VTM or IS CHO-V-GSTM (Irvine Scientific), EX-CELLTM 325 PF CHO or EX-CELLTM CD (SIGMA), or the like.
In the present invention, rh alpha-Gal A-producing mammalian cells are cultured for rh alpha-Gal A production in the culture medium for at least 5 days, more preferably for 8 to 14 days. Then the culture supernatant containing rh alpha-Gal A is collected and provided to the purification process for rh alpha-Gal A.
Each of the chromatography procedures for purification of the rh alpha-Gal A from a culture supernatant containing contaminant host cell proteins may, when needed, be carried out in the presence of a nonionic surfactant in order to prevent nonspecific binding of the protein. Though there is no particular limitation as to which nonionic surfactant is to be employed, a polysorbate-based surfactant is preferably employed, and more preferably polysorbate 80 or polysorbate 20. The concentration of such a nonionic surfactant is preferably 0.005% (w/v) to 0.05% (w/v), more preferably about 0.01% (w/v).
The process for purification of the rh alpha-Gal A may be carried out at room temperature or at lower temperatures, but preferably at lower temperatures, particularly at 1-10 deg C.
In the first chromatography step for the purification process, the pH of the culture supernatant is adjusted preferably to 7.0-8.0, more preferably to pH 7.2-7.8, and further preferably to about pH 7.5. Then the culture supernatant is applied to the anion-exchange column that has been equilibrated with a phosphate buffer supplemented with a salt to let rh alpha-Gal A bind to the column. The pH of this phosphate buffer is preferably to 7.0-8.0, more preferably about 7.2-7.8, and still more preferably about 7.5. Though there is no particular limitation as to which salt is to be added to the phosphate buffer, sodium chloride and potassium chloride are preferred. In the case where sodium chloride is used, its concentration is preferably in the range of 5-100 mM, more preferably in the range of 10-60 mM, and still more preferably about 50 mM.
After the anion-exchanger column to which rh alpha-Gal A is bound is washed, the rh alpha-Gal A is eluted using a phosphate buffer with increased salt concentration. Though there is no particular limitation as to which salt is to be used, sodium chloride and potassium chloride are preferred. In the case where sodium chloride is used, its concentration is preferably in the range of 125-500 mM, more preferably 175-350 mM, and still more preferably about 225 mM.
Further, though there is no particular limitation as to which anion-exchanger resin is to be employed, strong anion-exchanger resin may be used preferably. Commercially available resins, such as Q Sepharose Fast Flow (GE Healthcare), may be used preferably.
The hydrophobic column chromatography, the second step of the purification process, is a step for removing contaminants making use of the hydrophobic interaction between rh alpha-Gal A and hydrophobic ligand attached to the resin. Though there is no particular limitation as to which hydrophobic ligand is to be employed in the hydrophobic column chromatography, preferable is a ligand containing phenyl group, and more preferable a ligand containing phenyl group attached to the resin via a spacer arm, such as represented by the formula R-O-CH2-CH(OH)-CH2-O-C6H5 where R denotes the resin. A particularly preferred column material for this is Phenyl Sepharose 6 Fast Flow (GE Healthcare) in which phenyl group is covalently immobilized to Sepharose 6 Fast Flow matrix via a certain spacer arm.
The hydrophobic column chromatography column is equilibrated with a phosphate buffer supplemented with a salt. The pH of this phosphate buffer is preferably 6.5-7.5, more preferably about 6.7-7.3, and still more preferably about 7.0. Though there is no particular limitation as to which salt is to be added to the phosphate buffer, sodium chloride and potassium chloride are preferred. In the case where sodium chloride is used, its concentration is preferably in the range of 500 mM -1 M, and more preferably in the range of 700-800 mM, and still more preferably about 750 mM. Concurrently, Tris buffer may be added to the phosphate buffer at the concentration of preferably 30-70 mM, more preferably about 50 mM.
Then, rh alpha-Gal A-containing fractions of the eluate obtained in the first step are applied to the column. After the column to which rh alpha-Gal A is bound is washed, the rh alpha-Gal A is eluted. Elution may be done by a phosphate buffer not supplemented with salt, where the concentration of phosphate buffer is preferably 3-10 mM, and still more preferably about 5mM. Concurrently, Tris buffer may be added to the phosphate buffer at the concentration of preferably 30-70 mM, more preferably about 50 mM. Alternatively elution may be done by pure water.
The column chromatography employing a solid phase having affinity for phosphate group, the third step of the purification, is a step to eliminate contaminant proteins, such as carboxyl peptidases. Though there is no particular limitation as to which solid phase is to be used having affinity for phosphate group, hydroxyapatite and fluoroapatite may preferably be used, and hydroxyapatite is particularly preferred.
When a hydroxyapatite column is used, it is equilibrated with a phosphate buffer supplemented with a salt. The pH of this phosphate buffer is preferably 6.5-7.5, more preferably about 6.8-7.2, and still more preferably about 7.0, and the concentration of phosphate preferably 3.0-20 mM, more preferably about 3-15 mM, and further preferably about 10 mM. Though there is no particular limitation as to which salt is to be added to the phosphate buffer, sodium chloride and potassium chloride are preferred. In the case where sodium chloride is used, its concentration is preferably lower than 800 mM, and more preferably in the range of 10-200 mM, and further preferably about 50mM. Concurrently, Tris buffer may be added to the phosphate buffer at the concentration of preferably 30-70 mM, more preferably about 50 mM.
Then rh alpha-Gal A-containing fractions of the eluate obtained in the second step are applied to the column. After the column to which rh alpha-Gal A is bound is washed, the rh alpha-Gal A is eluted. Elution may be done by a phosphate buffer containing a salt. The pH of this phosphate buffer is preferably 6.5-7.5, more preferably about 6.7-7.3, and still more preferably about 7.0, and the concentration of phosphate is preferably in the range of 30-80 mM, more preferably in the range of 40-60 mM, and still more preferably about 45 mM. Though there is no particular limitation as to which salt is to be used, sodium chloride and potassium chloride are preferred. In the case where sodium chloride is used, its concentration is preferably lower than 300 mM, more preferably in the range of 10-200 mM, and still more preferably of about 50mM.
The cation-exchange column chromatography, the fourth step of the purification process, is a step to eliminate contaminant proteins. Though there is no particular limitation as to which cation-exchanger is to be employed in the cation-exchange column chromatography, a weak cation exchanger is preferred, and more preferred is a weak cation exchanger having a selectivity based on both hydrophobic interaction and hydrogen bond formation. For example, a weak cation exchanger having phenyl groups, amide bonds and carboxyl groups and having a selectivity based on both hydrophobic interaction and hydrogen bond formation, such as Capto MMC (GE Healthcare), etc., may be employed
In the cation-exchange column chromatography, the column is equilibrated with an acetate buffer supplemented with a salt. The pH of this acetate buffer is preferably 4.5-6.5, more preferably about 5.0-6.0, and still more preferably about 5.5. Though there is no particular limitation as to which salt is to be added to the phosphate buffer, sodium chloride, potassium chloride and calcium chloride are preferred. In the case where sodium chloride is used, its concentration is preferably in the range of 50-250 mM, and more preferably in the range of 100-200 mM, and still more preferably about 150 mM.
Then rh alpha-Gal A-containing fractions of the eluate obtained in the third step are applied to the column. After the column to which rh alpha-Gal A is bound is washed, the rh alpha-Gal A is eluted using a phosphate buffer with an increased pH, which is preferably in the range of about 5.5-7.5, more preferably in the range of about 6.0-7.0 and still more preferably about 6.5.
The dye-affinity chromatography, the fifth step of the purification, is a process for removing contaminants, i.e. host cell protein (HCP). Blue triazine dye is preferably used, but other triazine dyes are also suitable. Particularly preferred is Blue Sepharose 6 FF, Fast Flow (GE Healthcare) shown in the following schematic formula, which consists of Sepharose 6 Fast Flow matrix to which a dye, CibacronTM Blue F3GA, is attached by a covalent bond.
The dye-affinity chromatography column is equilibrated at or near the neutral pH, preferably pH6.8-7.2, more preferably about pH 7.0 with phosphate buffer supplemented with a salt. Though there is no particular limitation as to which salt is to be added to the phosphate buffer, sodium chloride, and potassium chloride are preferred. In the case where sodium chloride is used, its concentration is preferably in the range of 50-150 mM, and more preferably in the range of 100-150 mM, and still more preferably about 125 mM.
Then, fractions of the eluate obtained in the fourth step are applied to the column. After the column to which rh alpha-Gal A is bound is washed, the rh alpha-Gal A is eluted using phosphate buffer containing an increased concentration of a salt. Though there is no particular limitation as to which salt is to be added to the phosphate buffer, sodium chloride, and potassium chloride are preferred. In the case where sodium chloride is used, its concentration is preferably in the range of 400-750 mM, and more preferably in the range of 400-500 mM, and still more preferably about 450 mM. Trometamol (tris(hydroxymethyl)aminomethane) may concurrently be added to the buffer at the concentration of preferably 40-60 mM, and more preferably at about 50 mM.
The gel filtration column chromatography, the sixth step of the purification process, is a step for elimination of low molecular-weight impurities, such as endotoxins, as well as multimeric complexes or decomposition products of rh alpha-Gal A. Thus, substantially pure rh alpha-Gal A is obtained through the first to sixth steps of the purification process.
Additionally one more column chromatography may be incorporated into the purification process mentioned above, comprising anion-exchange chromatography, dye-affinity column chromatography, hydrophobic column chromatography, column chromatography employing a solid phase having affinity for phosphate group, cation-exchange column chromatography, and gel filtration column chromatography. Such an additional column chromatography is preferably dye-affinity column chromatography, which may be employed between two adjacent steps of the purification process mentioned above as desired.
A step for virus inactivation may optionally be added to the purification process of the present invention. Though there is no particular limitation as to which process for virus inactivation is to be applied, a solvent-detergent method may preferably be applied. For this, a nonionic surfactant is added to a solution containing rh alpha-Gal A, and the resulting mixture is incubated for more than 3 hours. Though there is no particular limitation as to which surfactant is to be used, polysorbate 20, polysorbate 80, triton X-100, and tri(n-butyl)phosphate may preferably be used alone or in any combinations, and more preferably the combination of polysorbate 80 and tri(n-butyl)phosphate may be used.
Such an additional step for virus inactivation may be employed either prior to the purification process or between any two adjacent steps of the purification process mentioned above.
Further, the present invention also provides a method for purification of rh alpha-Gal A from a material containing contaminant host cell proteins. The method includes a step in which a solution containing rh alpha-Gal A and proteins inherent in host cells which were employed to produce rh alpha-Gal A is subjected to dye-affinity column chromatography. The major purpose of this step is to eliminate contaminant host cell proteins. The step can be combined with at least one other purification step selected from cation-exchange column chromatography, hydrophobic column chromatography, anion-exchange column chromatography, column chromatography employing a solid phase having affinity for phosphate group (i.e. hydroxyapatite column chromatography) and gel filtration column chromatography, in any order. Preferably, the combination is anion-exchange column chromatography, hydrophobic column chromatography, column chromatography employing a solid phase having affinity for phosphate group, cation-exchange column chromatography, dye-affinity column chromatography, and gel filtration column chromatography in the order.
A recombinant human alpha-Gal A composition produced by the method according to the present invention is suitable for use in preparation of a medicament to be administered periodically to a patient. The medicament is to be administered to the patient preferably by intravenous injection, more preferably by intravenous infusion, and may be administered to the patient preferably once weekly to once every 2 to 4 weeks, more preferably once every two weeks. The amount of rh alpha-Gal A in one administration to a patient may be preferably 0.1 to 2 mg per kg of body weight, more preferably 0.1 to 2 mg per kg of body weight. The medicament may be supplied in the form of prefilled syringe.
The present invention is described in further detail below with reference to examples. However, it is not intended that the present invention be limited to the examples.
Construction of pE-gs/hGHpA(alpha-GalA), a rh alpha-Gal A expression vector
pEF/myc/nuc vector (Invitrogen) was digested with KpnI and NcoI to cut out a DNA fragment including the EF-1(alpha) promoter and its first intron, which then was blunt-ended with T4 DNA polymerase. Then the DNA fragment thus prepared was inserted into pC1-neo vector (Invitrogen) which had been digested with BglII and EcoRI and then blunt-ended with T4 DNA polymerase. Inserted into this was the above-mentioned blunt-ended fragment including the EF-1(alpha) promoter and its first intron to give pE-neo7 vector (Fig. 1).
pEF/myc/nuc vector (Invitrogen) was digested with KpnI and NcoI to cut out a DNA fragment including the EF-1(alpha) promoter and its first intron, which then was blunt-ended with T4 DNA polymerase. Then the DNA fragment thus prepared was inserted into pC1-neo vector (Invitrogen) which had been digested with BglII and EcoRI and then blunt-ended with T4 DNA polymerase. Inserted into this was the above-mentioned blunt-ended fragment including the EF-1(alpha) promoter and its first intron to give pE-neo7 vector (Fig. 1).
A DNA fragment containing poly adenylation signal sequence of human growth hormone gene was synthesized by annealing four synthetic oligonucleotides: (a) hGH-f1 (ID NO:1), (b) hGH-r1 (SEQ ID NO:2), (c) hGH-f2 (SEQ ID NO:3), and (d) hGH-r2 (SEQ ID NO:4). The DNA fragment thus prepared was inserted between the MluI and KpnI sites of pE-neo7 to give pE-neo/hGHpA vector (Fig. 1).
A 5' half of Chinese hamster Glutamine synthetase ("Ch GS" or "GS") coding region was amplified by PCR using a primer set, GS-1 (SEQ ID NO:5) and GS-3 (SEQ ID NO:6), and, as a template, ssDNA prepared from RNA extracted from CHO cells. A 3' half of Ch GS coding region was amplified by PCR using a primer set, GS-2 (SEQ ID NO:7) and GS-4 (SEQ ID NO:8), and, as a template, ssDNA prepared from RNA extracted from CHO cells. Then the amplified 5' half of Ch GS coding region was ligated into pT7blue-T vector (EMD Millipore). The vector thus obtained was digested with EcoRV and BamHI, and then the amplified 3' half of CH GS coding region, after digestion with EcoRV and BamHI, was inserted into the digested vector to obtain a plasmid vector containing the full length cDNA for Ch-GS. The plasmid obtained was designated pT7blue(GS) (Fig. 2-1).
A 5' half of human alpha-GalA coding region was amplified by PCR using a primer set, GAL-Mlu (SEQ ID NO:9) and GAL-Sca2 (SEQ ID NO:10), and, as a template, human placental Quick-CloneTM cDNA (Clontech). A 3' half of human alpha-GalA coding region was amplified by PCR using a primer set, GAL-Sca1 (SEQ ID NO:11) and GAL-Xba (SEQ ID NO:12), and, as a template, human placental Quick-CloneTM cDNA (Clontech). Then both PCR products were ligated into the EcoRV site of pBluescriptSK(+) vector. The 5' half of human alpha-GalA coding region then was excised from the vector thus obtained, by digestion with MluI and ScaI, and the 3' half of human alpha-GalA coding region was excised from the vector by digestion with ScaI and NotI Then the excised fragments were concomitantly inserted into the MluI and NotI sites of the multi cloning site in pE-neo vector to obtain full length cDNA for human alpha-GalA. The plasmid thus obtained was designated pE-neo(alpha-GalA) (Fig.2-2).
pE-neo/hGHpA was digested with AfIII and BstXI, and into the vector thus digested was inserted Ch-GS cDNA which had been excised out from pT7blue(GS) by digestion with AfIII and BstXI. The plasmid obtained was designated pE-gs/hGHpA (Fig. 2-1). pE-gs/hGHpA vector was digested with MluI and NotI, and into the vector thus digested was inserted human alpha-GalA cDNA which had been excised out from pE-neo(alpha-GalA) by digestion with MluI and NotI. The plasmid obtained, designated pE-gs/hGHpA(alpha-GalA), was used as rh alpha-Gal A expression vector in further experiments (Fig.2-2).
Production of Recombinant Cells for Expression of rh alpha-Gal A
CHO cells (CHO-K1: purchased from American Type Culture Collection) was transfected with the above-mentioned expression vector pE-gs/hGHpA(alpha-Gal A) using LipofectamineTM 2000 (Invitrogen) according to the following method. Briefly, two days before transfection, 2.5 x 105 CHO-K1 cells were seeded in a 3.5-cm culture dish containing 3 mL of D-MEM/F12 medium containing 5 % FCS (D-MEM/F12/5%FCS), and the cells were cultured at 37 deg C in a humidified atmosphere of 5% CO2 and 95% air. After two days of culture, the cells were washed with PBS, and 1 mL of fresh serum-free D-MEM/F12 medium was added. Then the cells were transfected by addition of 200 microliters of a 1:1 mixture solution consisting of LipofectamineTM 2000 solution diluted 20 times with Opti-MEMTM I medium (Life Technologies) and a pE-gs/hGHpA(alpha-GalA) solution diluted to 20 micrograms/mL with Opti-MEMTM I medium, followed by incubation at 37 deg C in a humidified atmosphere of 5% CO2 and 95% air for 5 hours. After transfection, the medium was replaced with D-MEM/F12 / 5 % FCS and cultivation followed for 24 hours.
CHO cells (CHO-K1: purchased from American Type Culture Collection) was transfected with the above-mentioned expression vector pE-gs/hGHpA(alpha-Gal A) using LipofectamineTM 2000 (Invitrogen) according to the following method. Briefly, two days before transfection, 2.5 x 105 CHO-K1 cells were seeded in a 3.5-cm culture dish containing 3 mL of D-MEM/F12 medium containing 5 % FCS (D-MEM/F12/5%FCS), and the cells were cultured at 37 deg C in a humidified atmosphere of 5% CO2 and 95% air. After two days of culture, the cells were washed with PBS, and 1 mL of fresh serum-free D-MEM/F12 medium was added. Then the cells were transfected by addition of 200 microliters of a 1:1 mixture solution consisting of LipofectamineTM 2000 solution diluted 20 times with Opti-MEMTM I medium (Life Technologies) and a pE-gs/hGHpA(alpha-GalA) solution diluted to 20 micrograms/mL with Opti-MEMTM I medium, followed by incubation at 37 deg C in a humidified atmosphere of 5% CO2 and 95% air for 5 hours. After transfection, the medium was replaced with D-MEM/F12 / 5 % FCS and cultivation followed for 24 hours.
Then, the medium was replaced with glutamine-free GMEM-S medium (SIGMA) supplemented with 1 x GS supplement (SIGMA), 10% dialyzed FBS and 30 micromole/L methionine sulfoximine (MSX), and a selective culture was carried out at 37 deg C in a humidified atmosphere of 5% CO2 and 95% air. Cells that had grown in the medium for selective culture were subjected to several successive rounds of subculture in the medium to give recombinant cells. In the course of the selective culture, concentration of MSX in the medium was elevated step by step to amplify the inserted recombinant genes. Finally bulk cells that stably grew in the medium with 300 micromole/L MSX were obtained after two steps subculture at elevated MSX concentrations (100 and 300 micromole/L MSX).
Then, according to the limiting dilution technique, the cells having the amplified recombinant gene were seeded on a 96-well plate in such a manner that not more than one cell might be seeded per well. The cells then were cultured for about 10 days to let each of them form a monoclonal colony. The culture supernatant in the wells where a monoclonal colony was formed was each sampled and examined for human alpha-Gal A activity as described below, and cell lines which were found expressing a high activity for human alpha-Gal A were selected.
For adaptation to serum-free suspension cell culture, the selected cell lines were each cultured in a commercially available serum-free medium, CD-OptiCHOTM medium (Invitrogen) supplemented with 10 mg/L insulin, 40 mg/L thymidine, 100 mg/L hypoxantine and 100 micromole/L MSX, at 37 deg C in a humidified atmosphere of 5% CO2 and 95% air until the cells stably grew. The cells then were suspended in CD-OptiCHOTM medium (Invitrogen) supplemented with 10 mg/L insulin, 40 mg/L thymidine, 100 mg/L hypoxantine, 100 micromole/L MSX, and 10% DMSO, and stored as seed cells in liquid nitrogen.
Culture of Recombinant Cells for Expression of rh alpha-Gal A
The above seed cells were thawed and diluted to a density of 4 x 105 cells/mL and cultured for 3 to 4 days with CD-OptiCHOTM medium supplemented with 10 mg/L insulin, 40 mg/L thymidine, 100 mg/L hypoxantine and 100 micromole/L MSX (CD medium), and then diluted to a density of 2 x 105 cells/mL with CD medium and cultured for 3 to 4 days. These cells were again diluted to a density of 2 x 105 cells/mL with CD medium and further cultured for 3 to 4 days.
The above seed cells were thawed and diluted to a density of 4 x 105 cells/mL and cultured for 3 to 4 days with CD-OptiCHOTM medium supplemented with 10 mg/L insulin, 40 mg/L thymidine, 100 mg/L hypoxantine and 100 micromole/L MSX (CD medium), and then diluted to a density of 2 x 105 cells/mL with CD medium and cultured for 3 to 4 days. These cells were again diluted to a density of 2 x 105 cells/mL with CD medium and further cultured for 3 to 4 days.
The number of the cells thus cultured was counted, and the cell culture was diluted with CD medium to a density of 2 x 105 cells/mL, and then the cells in 1L of the diluted culture were cultured with shaking for a few days. The scale of culture was escalated until the culture volume reached 200 L.
Then the number of the cells was counted, and the cells were diluted to a density of 2 x 105 cells/mL with 1800 L of CD-OptiCHOTM medium supplemented with 10 mg/L insulin, 40 mg/L thymidine, 100 mg/L hypoxantine and 0.1% HY soy (Invitrogen), and cultured for 11 days. 22.4 L of feed solution which contained 8.84 g insulin, 35.2 mmol hypoxantine, 5.6 mmol thymidine and 1.8 kg HY soy in CD-OptiCHOTM medium was added on the 3rd, 5th, 7th, and 9th days. Sampling was made every day during the culture, and measurement was performed for cell number, survival rate, glucose concentration, and amount of expressed rh alpha-Gal A. In the case where glucose concentration became lower than 3 g/L, 30% glucose solution was added so that the concentration reached 3.5 g/L. During the 11 days of culture, the viable cell density and the concentration of rh alpha-Gal A were daily monitored. The viable cell density reached about 6 x 106 cells/mL on days 6-7 of culture, indicating that high-density cell culture was successfully achieved (Fig 3). As measured by ELISA as described below, the concentration in the medium of rh alpha-Gal A secreted by the cells increased over time and reached almost 140mg/L on day 6 (Fig 3).
After completion of the culture above, the cell culture was collected and filtered successively through Millistak+TM HC Pod Filter grade D0HC, (Millipore), Millistak+TM HC Pod Filter grade X0HC (Millipore), and then Durapore OpticapTM XLT20 (0.22 micrometer, Millipore), to give a culture supernatant. The obtained culture supernatant was concentrated by a hollow fiber module equipped with PS membrane (polysulfone membrane) to one 13th of the original volume.
Virus Inactivation
Tri-n-butyl phosphate (TNBP) andpolysorbate 80 were added to the approximately 75 L of the concentrated culture supernatant described above so that their final concentrations would be 0.3 %(v/v) and 1 %(w/v), respectively, and this mixture solution then was gently stirred for 3 to 4 hours at room temperature.
Tri-n-butyl phosphate (TNBP) and
Method for Purification of rh alpha-Gal A
The pH of the above virus-inactivated solution then was adjusted to 7.5 by addition of 1 M Tris-buffer (pH 8.8), and subsequently the conductivity was adjusted to that of the 10 mM phosphate buffer (pH7.5) containing 50 mM NaCl, by addition of 50 mM phosphate buffer (pH 7.0) containing 1 M NaCl. Then the solution was filtered through Durapore OpticapTM XL10 (Millipore), and then was loaded on a Q Sepharose Fast Flow column (column volume: about 19.2 L, bed height: about 20 cm, GE Healthcare), an anion-exchange column, which had been equilibrated with 10 mM phosphate buffer (pH 7.5) containing 50 mM NaCl at a linear flow rate of 150 cm/hr to allow rh alpha-Gal A to be adsorbed by the column. Then, the column was washed with a threefold column volume of the same buffer supplied at the same flow rate, and the adsorbed rh alpha-Gal A then was eluted with a sixfold column volume of 10 mM phosphate buffer (pH 7.5) containing 225 mM NaCl, subsequently with a threefold column volume of 50 mM phosphate buffer (pH 7.0) containing 1 M NaCl, and the fractions containing rh alpha-Gal A were collected.
The pH of the above virus-inactivated solution then was adjusted to 7.5 by addition of 1 M Tris-buffer (pH 8.8), and subsequently the conductivity was adjusted to that of the 10 mM phosphate buffer (pH7.5) containing 50 mM NaCl, by addition of 50 mM phosphate buffer (pH 7.0) containing 1 M NaCl. Then the solution was filtered through Durapore OpticapTM XL10 (Millipore), and then was loaded on a Q Sepharose Fast Flow column (column volume: about 19.2 L, bed height: about 20 cm, GE Healthcare), an anion-exchange column, which had been equilibrated with 10 mM phosphate buffer (pH 7.5) containing 50 mM NaCl at a linear flow rate of 150 cm/hr to allow rh alpha-Gal A to be adsorbed by the column. Then, the column was washed with a threefold column volume of the same buffer supplied at the same flow rate, and the adsorbed rh alpha-Gal A then was eluted with a sixfold column volume of 10 mM phosphate buffer (pH 7.5) containing 225 mM NaCl, subsequently with a threefold column volume of 50 mM phosphate buffer (pH 7.0) containing 1 M NaCl, and the fractions containing rh alpha-Gal A were collected.
To the fractions collected at the step of Q Sepharose Fast Flow column chromatography above, one fourth volume of 2 M NaCl was added to adjust the concentration of NaCl to about 500 mM, and subsequently acetic acid was added to adjust the pH to 7.0. The solution obtained was loaded on a Phenyl Sepharose 6 Fast Flow column (column volume: 19.2 L, bed height: about 20 cm, GE Healthcare), a hydrophobic column, which had been equilibrated with 50 mM Tris-HCl, 5 mM phosphate buffer (pH 7.0) containing 750 mM NaCl, at a linear flow rate of 150 cm/hr to allow rh alpha-Gal A to be adsorbed by the column. Then after the column was washed with a threefold column volume of the same buffer supplied at the same flow rate, the adsorbed rh alpha-Gal A was eluted with a ninefold column volume of a buffer consisting of 50 mM Tris-HCl and 5 mM phosphate (pH 7.0), and subsequently with a threefold column volume of pure water, and the fractions containing rh alpha-Gal A were collected.
The fractions collected at the step of Phenyl Sepharose 6 Fast Flow column chromatography above, was loaded on a CHT-I ceramic hydroxyapatite column (column volume: 19.2 L, bed height: about 20 cm, Bio-Rad Laboratories), a hydroxyapatite column, which had been equilibrated with 50 mM Tris-HCl, 10 mM phosphate buffer (pH 7.0) containing 50 mM NaCl, at a linear flow rate of 150 cm/hr to allow rh alpha-Gal A to be adsorbed by the column. Then, the column was washed with a fivefold column volume of the same buffer supplied at the same flow rate, and the adsorbed rh alpha-Gal A then was eluted with a ninefold column volume of a buffer containing 50 mM Tris-HCl, 45 mM sodium phosphate and 50 mM NaCl (pH 7.0), and subsequently with a threefold column volume of 200 mM phosphate buffer (pH7.0) and the fractions containing rh alpha-Gal A were collected.
To the fractions collected at the step of CHT-I ceramic hydroxyapatite column chromatography above, acetic acid was added to adjust the pH to 5.5. A precipitate formed by this was removed by filtration through OpticapTM SHC XL5 (0.22 micrometer, Millipore). The solution obtained was loaded on a CaptoTM MMC column (column volume: 6.3 L, bed height: about 20 cm, GE Healthcare), a cation-exchange column having a selectivity based both on hydrophobic interaction and on hydrogen bond formation, which had been equilibrated with 50 mM acetate buffer (pH 5.5) containing 150 mM NaCl, at a linear flow rate of 300 cm/hr to allow rh alpha-Gal A to be adsorbed by the column. Then after the column was washed with a fourfold column volume of the same buffer supplied at the same flow rate, the adsorbed rh alpha-Gal A was eluted with a fivefold column volume of 50 mM phosphate buffer (pH6.5), and subsequently with a threefold column volume of 50 mM phosphate buffer (pH7.0) containing 1 M NaCl, and the fractions containing rh alpha-Gal A were collected.
To the fractions collected at the step of CaptoTM MMC column chromatography above, 1M Tris buffer (pH8.8) was added to adjust the pH to 7.0. Half of the solution obtained was loaded on Blue Sepharose 6FF column (column volume: about 19.2 L, bed height: about 20 cm, GE Healthcare) equilibrated with 20 mM phosphate buffer (pH 7.0) containing 125 mM NaCl, supplied at a linear flow rate of 150 cm/hr to allow rh alpha-Gal A to be adsorbed by the column. And then, the column was washed with a threefold column volume of 20 mM phosphate buffer (pH 7.0) containing 125 mM NaCl supplied at the same flow rate, and the adsorbed rh alpha-Gal A then was eluted with a fourfold column volume of 50mM Tris-HCl / 5 mM phosphate buffer (pH 7.0) containing 450 mM NaCl supplied at the same flow rate, and the fractions containing rh alpha-Gal A were collected. The other half of the solution was sequentially processed by dye-affinity column chromatography in the same manner as described above. The collected fractions containing rh alpha-Gal A were combined for further purification.
The fractions collected at the step of dye-affinity column chromatography above was concentrated using Pellicon TM 3 Cassettes equipped with Biomax TM 30 membrane (Millipore) until the total volume became less than 1 L. One third of the concentrated solution was loaded on a SuperdexTM 200 prep grade column (column volume: about 58 L, bed height: about 30 cm x 2, GE Healthcare) which had been equilibrated with 20 mM phosphate buffer (pH 5.8) containing 137 mM NaCl. The same buffer then was supplied at a linear flow rate of 25 cm/hr, and the fractions which corresponded to the main peak observed by absorbance at 280 nm were collected as fractions containing purified rh alpha-Gal A. This chromatography was carried out three times to purify all the concentrated solution above, and the fractions collected by each run were combined
The fractions pooled at the step of SuperdexTM 200 prep grade column chromatography above were concentrated by Hollow fiber module equipped with PS membrane, and to the concentrated solution, polysorbate 80 was added at the final concentration of 0.02%. The obtained solution was filtered through ViresolveTM Pro Modus1.3 (0.07 m2 size, Millipore) to avoid any possible contamination with viruses and microorganisms in the final product.
In each step of purification, the amount of rh alpha-Gal A contained in the loaded material and that of recovered rh alpha-Gal A in the collected fractions were quantified using ELISA as described below. The results are shown in Table 1. In the table, "rh alpha-Gal A recovery rate/step" means the proportion of the amount of recovered rh alpha-Gal A in the step to that of rh alpha-Gal A loaded in the step, and "rh alpha-Gal A recovery rate/total" the proportion of the amount of recovered rh alpha-Gal A in the step to the initial amount of rh alpha-Gal A subjected to the purification process. The initial amount of rh alpha-Gal A subjected to the purification process was 140.6 g, of which 91.4 g was finally recovered, thus giving the rh alpha-Gal A recovery rate/total as high as 65.0%. These results show that the method for purification described above enables purification of rh alpha-Gal A in very high yield and in a large production scale.
Analysis of Human alpha-Gal A by ELISA
To each well of a 96-well microtiter plate (Nunc) was added 100 microliters of mouse anti-human alpha-Gal A monoclonal antibody diluted to 0.25 micrograms/mL with 0.05 M carbonate-bicarbonate buffer (pH 9.6), and the plate was left untouched for at least 1 hour at room temperature or overnight at 2-8 deg C to allow the antibody to be absorbed by the wells. Then, after each well was washed with phosphate buffered saline, pH 7.4 (PBS) containing 0.05% Tween 20 (PBS-T), 300 microliters of PBS-T containing 1% BSA was added, and the plate was left untouched for at least one hour at room temperature. Then, after each well was washed three times with PBS-T, 100 microliters of the test sample or human alpha-Gal A standard, which had been diluted as desired with PBS containing 0.5% BSA and 0.05% Tween 20 (PBS-BT), was added to the well, and the plate was left untouched for at least one hour at room temperature. Then, after each well was washed three times with PBS-T, 100 microliters of horseradish peroxidase-labeled rabbit anti-human alpha-Gal A polyclonal antibody diluted with PBS-BT was added and the plate was left untouched for at least one hour. Then, after each well was washed three times with PBS-T, 100 microliters of 0.4 mg/mL o-phenylenediamine with phosphate-citrate buffer (pH 5.0) containing 0.009% hydrogen peroxide was added to the well, and the plate was left untouched for 10 to 25 minutes at room temperature. Then 0.1 mL of 1 mol/L H2SO4 was added to each well to stop the reaction, and the optical density at 490 nm was measured for the well on a 96-well plate reader.
To each well of a 96-well microtiter plate (Nunc) was added 100 microliters of mouse anti-human alpha-Gal A monoclonal antibody diluted to 0.25 micrograms/mL with 0.05 M carbonate-bicarbonate buffer (pH 9.6), and the plate was left untouched for at least 1 hour at room temperature or overnight at 2-8 deg C to allow the antibody to be absorbed by the wells. Then, after each well was washed with phosphate buffered saline, pH 7.4 (PBS) containing 0.05% Tween 20 (PBS-T), 300 microliters of PBS-T containing 1% BSA was added, and the plate was left untouched for at least one hour at room temperature. Then, after each well was washed three times with PBS-T, 100 microliters of the test sample or human alpha-Gal A standard, which had been diluted as desired with PBS containing 0.5% BSA and 0.05% Tween 20 (PBS-BT), was added to the well, and the plate was left untouched for at least one hour at room temperature. Then, after each well was washed three times with PBS-T, 100 microliters of horseradish peroxidase-labeled rabbit anti-human alpha-Gal A polyclonal antibody diluted with PBS-BT was added and the plate was left untouched for at least one hour. Then, after each well was washed three times with PBS-T, 100 microliters of 0.4 mg/mL o-phenylenediamine with phosphate-citrate buffer (pH 5.0) containing 0.009% hydrogen peroxide was added to the well, and the plate was left untouched for 10 to 25 minutes at room temperature. Then 0.1 mL of 1 mol/L H2SO4 was added to each well to stop the reaction, and the optical density at 490 nm was measured for the well on a 96-well plate reader.
Measurement of the Activity of rh alpha-Gal A
Standard solutions were prepared by dissolving 4-methyumbelliferone in Dilution Buffer (26.7mM Citrate-44.8 mM phosphate buffer, pH 4.6 containing 0.1 mg/mL BSA) at concentrations of 0 to 200 micromole/L. A substrate solution was prepared by dissolving 4-methylumbelliferyl-alpha-D-galactopyranoside (SIGMA) in Dilution Buffer at the final concentration of 5 mM. 10 microliters of each of the standard solutions or the test sample diluted with Dilution Buffer was added to each well of a 96-well microtiter plate (FluoroNunc Plate, Nunc). 75 microliters of the substrate solution was added to each well containing the diluted test sample or any of the standard solutions, and the plate was left untouched for one hour at 37 deg C in the dark. After this incubation, 200 microliters of Stop Buffer (0.2 M glycine-NaOH, pH 10.6) was added to each well. Then fluorescent intensity of the solution in each well was measured by a 96-well plate reader with excitation light at the wavelength of 335 nm and fluorescent light at the wavelength of 460 nm.
Standard solutions were prepared by dissolving 4-methyumbelliferone in Dilution Buffer (26.7mM Citrate-44.8 mM phosphate buffer, pH 4.6 containing 0.1 mg/mL BSA) at concentrations of 0 to 200 micromole/L. A substrate solution was prepared by dissolving 4-methylumbelliferyl-alpha-D-galactopyranoside (SIGMA) in Dilution Buffer at the final concentration of 5 mM. 10 microliters of each of the standard solutions or the test sample diluted with Dilution Buffer was added to each well of a 96-well microtiter plate (FluoroNunc Plate, Nunc). 75 microliters of the substrate solution was added to each well containing the diluted test sample or any of the standard solutions, and the plate was left untouched for one hour at 37 deg C in the dark. After this incubation, 200 microliters of Stop Buffer (0.2 M glycine-NaOH, pH 10.6) was added to each well. Then fluorescent intensity of the solution in each well was measured by a 96-well plate reader with excitation light at the wavelength of 335 nm and fluorescent light at the wavelength of 460 nm.
A standard curve was produced by plotting fluorescence intensity measured of the standard solutions and interpolating between the plotted data points. The fluorescence intensity of each sample was corresponded to the concentration of the released 4-MUF using the standard curve. The specific activity of human alpha-Gal A was calculated as Units/mg, where one Unit of activity was equal to one micromole of 4-MUF produced per minute at 37 deg C. A published US patent application (publication No. 2004-0229250) was referred to for conducting the above measurement of the activity of rh alpha-Gal A. The specific activity of rh alpha-Gal A purified by the process described above was found to be about 50 U/mg (48.3 to 53.7 U/mg).
Analysis of purity of rh alpha-Gal A
Five micrograms of the rh alpha-Gal A purified above was subjected to SDS-PAGE electrophoresis under a reductive, heating (70deg C 10 min.) condition. The gel-stained with Coomassie brilliant blue revealed a single band corresponding to rh alpha-Gal A in SuperdexTM 200pg fraction (Fig. 4).
Five micrograms of the rh alpha-Gal A purified above was subjected to SDS-PAGE electrophoresis under a reductive, heating (70
Further, rh alpha-Gal A purified above was analyzed by Size-elimination HPLC (SE-HPLC). HPLC was performed using LC-20A System, SPD-20AV UV/VIS Detector (Shimazu Corp.). For SE-HPLC analysis, 10 microliters of a sample solution containing 2 mg/mL of rh alpha-Gal A purified above was applied onto TSKgel G3000SWXL column (7.8 mm I.D. x 30 cm, TOSOH) equilibrated with 25 mM phosphate buffered saline (PBS) at a flow rate of 0.5 ml/min. Elution profile was produced by monitoring absorbance at 215 nm.
The SE-HPLC analysis of the above purified rh alpha-Gal A revealed a single peak alone (Fig. 5). The results showed that the rh alpha-Gal A obtained above was highly purified and free of any detectable contaminants. This result demonstrates that the rh alpha-Gal A purified above is of such a high purity as may be directly used as a medical drug.
Measurement of Host Cell Proteins (HCPs)
The amount of contaminant host cell proteins (HCPs) present in the pool of collected rh alpha-Gal A-active fractions in each of thepurification steps 1 to 6 was quantified by ELISA in a conventional manner and compared with the amount of rh alpha-Gal A contained in the pool. The results are presented in Table 1 as "HCP/rh alpha-Gal A (ppm)", which is the ratio in mass of HCPs to rh alpha-Gal A. As shown in Table 1, the ratio, after the final step of purification, was as low as 20 ppm, a level which is low enough to allow the rh alpha-Gal A thus purified to be used as a medicament administered to a human repeatedly by injection.
The amount of contaminant host cell proteins (HCPs) present in the pool of collected rh alpha-Gal A-active fractions in each of the
Among the steps of the purification process, the fifth, the dye-affinity chromatography, was most effective to remove HCPs from the rh alpha-Gal A contaminated with it. Namely, in this step, almost 97 % of HCPs were removed and the rate of HCPs/rh alpha-Gal A decreased from 8.6 x 103 ppm to 2.5 x 102 ppm. The result shows that dye-affinity chromatography is a remarkably effective way to remove HCPs from rh alpha-Gal A preparation.
The present invention is utilized for production of rh alpha-galactosidase A (rh alpha-Gal A) in a large scale, with a sufficiently high purity to be administered as a medicament to a patient suffering from Fabry's syndrome.
SEQ ID NO:1 = hGH-f1, synthetic sequence
SEQ ID NO:2 = hGH-r1, synthetic sequence
SEQ ID NO:3 = hGH-f2, synthetic sequence
SEQ ID NO:4 = hGH-r2, synthetic sequence
SEQ ID NO:5 = Primer GS-1, synthetic sequence
SEQ ID NO:6 = Primer GS-3, synthetic sequence
SEQ ID NO:7 = Primer GS-2, synthetic sequence
SEQ ID NO:8 = Primer GS-4, synthetic sequence
SEQ ID NO:9 = Primer GAL-Mlu, synthetic sequence
SEQ ID NO:10 = Primer GAL-Sca2, synthetic sequence
SEQ ID NO:11 = Primer GAL-Sca1, synthetic sequence
SEQ ID NO:12 = Primer GAL-Xba, synthetic sequence
SEQ ID NO:2 = hGH-r1, synthetic sequence
SEQ ID NO:3 = hGH-f2, synthetic sequence
SEQ ID NO:4 = hGH-r2, synthetic sequence
SEQ ID NO:5 = Primer GS-1, synthetic sequence
SEQ ID NO:6 = Primer GS-3, synthetic sequence
SEQ ID NO:7 = Primer GS-2, synthetic sequence
SEQ ID NO:8 = Primer GS-4, synthetic sequence
SEQ ID NO:9 = Primer GAL-Mlu, synthetic sequence
SEQ ID NO:10 = Primer GAL-Sca2, synthetic sequence
SEQ ID NO:11 = Primer GAL-Sca1, synthetic sequence
SEQ ID NO:12 = Primer GAL-Xba, synthetic sequence
Claims (11)
- A method for production of a recombinant human alpha-Gal A comprising the steps of:
(a) culturing recombinant human alpha-Gal A-producing mammalian cells in a serum-free medium to let them secrete the recombinant human alpha-Gal A in the medium,
(b) collecting culture supernatant by removing the cells from the culture that is obtained in step (a) above,
(c) subjecting the culture supernatant collected in step (b) above to anion-exchange column chromatography to collect the recombinant human alpha-Gal A -active fractions,
(d) subjecting the fractions collected in step (c) above to hydrophobic column chromatography to collect recombinant human alpha-Gal A-active fractions,
(e) subjecting the fractions collected in step (d) above to a column chromatography employing as solid phase a material having affinity for phosphate group to collect the recombinant human alpha-Gal A-active fractions,
(f) subjecting the fractions collected in step (e) above to cation-exchange column chromatography to collect the recombinant human alpha-Gal A -active fractions,
(g) subjecting the fractions collected in step (f) above to dye-affinity column chromatography to collect the recombinant human alpha-Gal A -active fractions, and
(h) subjecting the fractions collected in step (g) above to gel filtration column chromatography to collect the recombinant human alpha-Gal A-active fractions, in the order. - The method according to claim 1, wherein the cation exchanger employed in the cation-exchange column chromatography is a weak cation exchanger.
- The method according to claim 2, wherein the weak cation exchanger has a selectivity based on both hydrophobic interaction and hydrogen bond formation.
- The method according to claim 2 or 3, wherein the weak cation exchanger has phenyl groups, amide bonds and carboxyl groups.
- The method according to one of claims 1 to 4, wherein the dye employed in the dye-affinity column chromatography is a blue triazine dye.
- The method according to one of claims 1 to 5, wherein the material having affinity for phosphate group is selected from the group consisting of hydroxyapatite and fluoroapatite.
- The method according to claim 6, wherein the material having affinity for phosphate group is hydroxyapatite.
- The method according to one of claims 1 to 7, wherein the mammalian cells are CHO cells transfected with an expression vector which is designed to express the recombinant human alpha-Gal A under the regulation of EF-1(alpha) promoter.
- A recombinant human alpha-Gal A composition produced by the method according to one of claims 1 to 8.
- A medicament comprising the composition according to claim 9 administered periodically to a patient.
- The medicament according to claim 10, wherein the medicament is administered to the patient by injection.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020177022178A KR102288264B1 (en) | 2015-01-22 | 2016-01-21 | Method for purification of recombinant human alpha-galactosidase a from material containing contaminant host cell proteins |
| CN201680006854.8A CN107208081B (en) | 2015-01-22 | 2016-01-21 | Method for purifying recombinant human α -galactosidase a from material comprising contaminating host cell proteins |
| US15/545,194 US10385325B2 (en) | 2015-01-22 | 2016-01-21 | Method for production of recombinant human alpha-galactosidase A from material containing contaminant host cell proteins by chromatography |
| JP2017538733A JP6749042B2 (en) | 2015-01-22 | 2016-01-21 | Method for purifying recombinant human α-galactosidase A from material containing host cell protein as contaminant |
| EP16739950.0A EP3247794B1 (en) | 2015-01-22 | 2016-01-21 | Method for purification of recombinant human alpha-galactosidase a from material containing contaminant host cell proteins |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/JP2015/000290 WO2016116966A1 (en) | 2015-01-22 | 2015-01-22 | Method for purification of recombinant human alpha-galactosidase a from material containing contaminant host cell proteins |
| JPPCT/JP2015/000290 | 2015-01-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2016117341A1 true WO2016117341A1 (en) | 2016-07-28 |
Family
ID=56416538
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP2015/000290 WO2016116966A1 (en) | 2015-01-22 | 2015-01-22 | Method for purification of recombinant human alpha-galactosidase a from material containing contaminant host cell proteins |
| PCT/JP2016/000296 WO2016117341A1 (en) | 2015-01-22 | 2016-01-21 | Method for purification of recombinant human alpha-galactosidase a from material containing contaminant host cell proteins |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP2015/000290 WO2016116966A1 (en) | 2015-01-22 | 2015-01-22 | Method for purification of recombinant human alpha-galactosidase a from material containing contaminant host cell proteins |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US10385325B2 (en) |
| EP (1) | EP3247794B1 (en) |
| JP (1) | JP6749042B2 (en) |
| KR (1) | KR102288264B1 (en) |
| CN (1) | CN107208081B (en) |
| WO (2) | WO2016116966A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20190040040A (en) | 2016-08-25 | 2019-04-16 | 제이씨알 파마 가부시키가이샤 | Method for producing antibody fusion protein |
| WO2022191158A1 (en) | 2021-03-09 | 2022-09-15 | Ｊｃｒファーマ株式会社 | Method for producing antibody-lysosomal enzyme fusion protein |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990011353A1 (en) | 1989-03-24 | 1990-10-04 | Research Corporation Technologies, Inc. | Recombinant alpha-galactosidase, a therapy for fabry disease |
| WO1994012650A2 (en) | 1992-12-03 | 1994-06-09 | Transkaryotic Therapies, Inc. | Activating expression of an amplifying endogenous gene by homologous recombination |
| WO1994012628A1 (en) * | 1992-11-30 | 1994-06-09 | The Mount Sinai School Of Medicine Of The City University Of New York | Cloning and expression of biologically active alpha-galactosidase a |
| WO1995031560A1 (en) | 1994-05-13 | 1995-11-23 | Transkaryotic Therapies, Inc. | Dna construct for effecting homologous recombination and uses thereof |
| WO1998011206A2 (en) | 1996-09-13 | 1998-03-19 | Transkaryotic Therapies, Inc. | THERAPY FOR α-GALACTOSIDASE A DEFICIENCY |
| WO2000053730A2 (en) | 1999-03-11 | 2000-09-14 | Transkaryotic Therapies, Inc. | Medical preparations for the treatment of alpha-galactosidase a deficiency |
| US20020146771A1 (en) * | 1995-05-11 | 2002-10-10 | Roche Diagnostics Gmbh | Process for producing erythropoietin containing no animal proteins |
| US20040229250A1 (en) | 2003-02-11 | 2004-11-18 | Transkaryotic Therapies, Inc. | Diagnosis and treatment of multiple sulfatase deficiency and other sulfatase deficiencies |
| WO2006051070A1 (en) * | 2004-11-09 | 2006-05-18 | Ares Trading S.A. | Method for purifying fsh |
| WO2011108451A1 (en) * | 2010-03-01 | 2011-09-09 | 日本ケミカルリサーチ株式会社 | Method for producing recombinant lysosomal enzymes using gene knockout cells |
| WO2014017088A1 (en) | 2012-07-26 | 2014-01-30 | Jcr Pharmaceuticals Co., Ltd. | Method for production of recombinant human alpha-galactosidase a |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5356804A (en) * | 1990-10-24 | 1994-10-18 | Mount Sinai School Of Medicine Of The City Of New York | Cloning and expression of biologically active human α-galactosidase A |
| HUE030959T2 (en) | 2010-03-02 | 2017-06-28 | Protalix Ltd | Stabilized alpha-galactosidase and uses thereof |
-
2015
- 2015-01-22 WO PCT/JP2015/000290 patent/WO2016116966A1/en active Application Filing
-
2016
- 2016-01-21 WO PCT/JP2016/000296 patent/WO2016117341A1/en active Application Filing
- 2016-01-21 KR KR1020177022178A patent/KR102288264B1/en active IP Right Grant
- 2016-01-21 JP JP2017538733A patent/JP6749042B2/en active Active
- 2016-01-21 US US15/545,194 patent/US10385325B2/en active Active
- 2016-01-21 CN CN201680006854.8A patent/CN107208081B/en active Active
- 2016-01-21 EP EP16739950.0A patent/EP3247794B1/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990011353A1 (en) | 1989-03-24 | 1990-10-04 | Research Corporation Technologies, Inc. | Recombinant alpha-galactosidase, a therapy for fabry disease |
| WO1994012628A1 (en) * | 1992-11-30 | 1994-06-09 | The Mount Sinai School Of Medicine Of The City University Of New York | Cloning and expression of biologically active alpha-galactosidase a |
| WO1994012650A2 (en) | 1992-12-03 | 1994-06-09 | Transkaryotic Therapies, Inc. | Activating expression of an amplifying endogenous gene by homologous recombination |
| WO1995031560A1 (en) | 1994-05-13 | 1995-11-23 | Transkaryotic Therapies, Inc. | Dna construct for effecting homologous recombination and uses thereof |
| US20020146771A1 (en) * | 1995-05-11 | 2002-10-10 | Roche Diagnostics Gmbh | Process for producing erythropoietin containing no animal proteins |
| WO1998011206A2 (en) | 1996-09-13 | 1998-03-19 | Transkaryotic Therapies, Inc. | THERAPY FOR α-GALACTOSIDASE A DEFICIENCY |
| WO2000053730A2 (en) | 1999-03-11 | 2000-09-14 | Transkaryotic Therapies, Inc. | Medical preparations for the treatment of alpha-galactosidase a deficiency |
| US20040229250A1 (en) | 2003-02-11 | 2004-11-18 | Transkaryotic Therapies, Inc. | Diagnosis and treatment of multiple sulfatase deficiency and other sulfatase deficiencies |
| WO2006051070A1 (en) * | 2004-11-09 | 2006-05-18 | Ares Trading S.A. | Method for purifying fsh |
| WO2011108451A1 (en) * | 2010-03-01 | 2011-09-09 | 日本ケミカルリサーチ株式会社 | Method for producing recombinant lysosomal enzymes using gene knockout cells |
| WO2014017088A1 (en) | 2012-07-26 | 2014-01-30 | Jcr Pharmaceuticals Co., Ltd. | Method for production of recombinant human alpha-galactosidase a |
| WO2014016873A1 (en) | 2012-07-26 | 2014-01-30 | Jcr Pharmaceuticals Co., Ltd. | Method for production of recombinant human alpha-galactosidase a |
Non-Patent Citations (6)
| Title |
|---|
| BISHOP ET AL., PROC NATL ACAD SCI USA, vol. 83, 1986, pages 4859 - 63 |
| BISHOP ET AL.: "Lipid Storage Disorders - Biological and Medical Aspects", 1988, PLENUM PRESS, pages: 809 - 823 |
| BRADY ET AL., N. ENGL. J. MED., vol. 289, 1973, pages 9 - 14 |
| DESNICK ET AL., PROC NATL ACAD SCI USA, vol. 76, 1979, pages 5326 - 30 |
| MAPES ET AL., SCIENCE, vol. 169, 1970, pages 987 - 9 |
| See also references of EP3247794A4 |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20190040040A (en) | 2016-08-25 | 2019-04-16 | 제이씨알 파마 가부시키가이샤 | Method for producing antibody fusion protein |
| US11512135B2 (en) | 2016-08-25 | 2022-11-29 | Jcr Pharmaceuticals Co., Ltd. | Method for producing antibody fusion protein |
| WO2022191158A1 (en) | 2021-03-09 | 2022-09-15 | Ｊｃｒファーマ株式会社 | Method for producing antibody-lysosomal enzyme fusion protein |
| KR20230154043A (en) | 2021-03-09 | 2023-11-07 | 제이씨알 파마 가부시키가이샤 | Method for preparing antibody-lysosomal enzyme fusion protein |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2018504121A (en) | 2018-02-15 |
| CN107208081A (en) | 2017-09-26 |
| EP3247794A1 (en) | 2017-11-29 |
| US10385325B2 (en) | 2019-08-20 |
| EP3247794B1 (en) | 2020-03-11 |
| KR102288264B1 (en) | 2021-08-09 |
| JP6749042B2 (en) | 2020-09-02 |
| WO2016116966A1 (en) | 2016-07-28 |
| CN107208081B (en) | 2020-07-14 |
| KR20170099406A (en) | 2017-08-31 |
| US20180016564A1 (en) | 2018-01-18 |
| EP3247794A4 (en) | 2018-06-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2668272B1 (en) | Method for production of recombinant human iduronate 2-sulfatase | |
| KR101489028B1 (en) | Large-scale production of soluble hyaluronidase | |
| US9617529B2 (en) | Method for production of recombinant human alpha-galactosidase A | |
| BR112013001558A2 (en) | method for purifying recombinant nacetylgalactosamine-6-sulfatase (galns) enzyme, composition comprising said enzyme and its use in the treatment of iva-type mucopolysaccharidosis (mps iva) or morquio a syndrome, as well as formulation | |
| WO2011108451A1 (en) | Method for producing recombinant lysosomal enzymes using gene knockout cells | |
| US10385325B2 (en) | Method for production of recombinant human alpha-galactosidase A from material containing contaminant host cell proteins by chromatography | |
| JP6021647B2 (en) | Method for production and purification of recombinant lysosomal alpha-mannosidase | |
| JP6018572B2 (en) | Method for producing recombinant human iduronate-2-sulfatase | |
| CN106459900B (en) | Culture medium comprising uridine and N-acetyl-D-mannosamine | |
| JP6226435B2 (en) | Method for producing recombinant human α-galactosidase A | |
| KR101426459B1 (en) | Method for effective purification of recombinant trypsin produced in transgenic rice cell suspension culture |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 16739950 Country of ref document: EP Kind code of ref document: A1 |
|
| REEP | Request for entry into the european phase |
Ref document number: 2016739950 Country of ref document: EP |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 15545194 Country of ref document: US |
|
| ENP | Entry into the national phase |
Ref document number: 2017538733 Country of ref document: JP Kind code of ref document: A |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| ENP | Entry into the national phase |
Ref document number: 20177022178 Country of ref document: KR Kind code of ref document: A |