WO2011001963A1 - 多孔質基材に固定されたグラフト鎖に結合しているアミノ基及びアルキル基を有する多孔膜を用いた抗体の精製方法 - Google Patents
多孔質基材に固定されたグラフト鎖に結合しているアミノ基及びアルキル基を有する多孔膜を用いた抗体の精製方法 Download PDFInfo
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- WO2011001963A1 WO2011001963A1 PCT/JP2010/061031 JP2010061031W WO2011001963A1 WO 2011001963 A1 WO2011001963 A1 WO 2011001963A1 JP 2010061031 W JP2010061031 W JP 2010061031W WO 2011001963 A1 WO2011001963 A1 WO 2011001963A1
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- Prior art keywords
- porous membrane
- antibody
- group
- hollow fiber
- solution
- Prior art date
Links
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- 125000003277 amino group Chemical group 0.000 title claims description 50
- 238000000746 purification Methods 0.000 title description 27
- 239000000178 monomer Substances 0.000 claims abstract description 110
- 239000011148 porous material Substances 0.000 claims abstract description 34
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- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 10
- 125000004433 nitrogen atom Chemical group N* 0.000 claims abstract description 6
- 125000004432 carbon atom Chemical group C* 0.000 claims abstract description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 123
- 108090000623 proteins and genes Proteins 0.000 claims description 123
- 239000012535 impurity Substances 0.000 claims description 66
- 239000000758 substrate Substances 0.000 claims description 56
- 239000012466 permeate Substances 0.000 claims description 49
- 238000011084 recovery Methods 0.000 claims description 42
- 150000003839 salts Chemical class 0.000 claims description 41
- -1 isopropylamino group Chemical group 0.000 claims description 36
- 239000000356 contaminant Substances 0.000 claims description 34
- 239000000539 dimer Substances 0.000 claims description 31
- 230000003993 interaction Effects 0.000 claims description 18
- 102000053602 DNA Human genes 0.000 claims description 15
- 108020004414 DNA Proteins 0.000 claims description 15
- 125000001664 diethylamino group Chemical group [H]C([H])([H])C([H])([H])N(*)C([H])([H])C([H])([H])[H] 0.000 claims description 15
- 125000006308 propyl amino group Chemical group 0.000 claims description 12
- 241000700605 Viruses Species 0.000 claims description 11
- 239000002158 endotoxin Substances 0.000 claims description 9
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims description 7
- 150000002009 diols Chemical group 0.000 claims description 7
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 claims description 7
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 7
- 125000000524 functional group Chemical group 0.000 claims description 6
- 229920000642 polymer Polymers 0.000 claims description 6
- 239000004698 Polyethylene Substances 0.000 claims description 5
- 229920000573 polyethylene Polymers 0.000 claims description 5
- 230000009881 electrostatic interaction Effects 0.000 claims description 4
- 239000001257 hydrogen Substances 0.000 claims description 4
- 229910052739 hydrogen Inorganic materials 0.000 claims description 4
- 108091005804 Peptidases Proteins 0.000 claims description 3
- 239000004365 Protease Substances 0.000 claims description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 3
- WLQXEFXDBYHMRG-UPHRSURJSA-N (z)-4-(oxiran-2-ylmethoxy)-4-oxobut-2-enoic acid Chemical compound OC(=O)\C=C/C(=O)OCC1CO1 WLQXEFXDBYHMRG-UPHRSURJSA-N 0.000 claims description 2
- 239000002033 PVDF binder Substances 0.000 claims description 2
- 239000004695 Polyether sulfone Substances 0.000 claims description 2
- 125000003055 glycidyl group Chemical group C(C1CO1)* 0.000 claims description 2
- AVNANMSIFNUHNY-MQQKCMAXSA-N oxiran-2-ylmethyl (2e,4e)-hexa-2,4-dienoate Chemical compound C\C=C\C=C\C(=O)OCC1CO1 AVNANMSIFNUHNY-MQQKCMAXSA-N 0.000 claims description 2
- RPQRDASANLAFCM-UHFFFAOYSA-N oxiran-2-ylmethyl prop-2-enoate Chemical compound C=CC(=O)OCC1CO1 RPQRDASANLAFCM-UHFFFAOYSA-N 0.000 claims description 2
- 229920006393 polyether sulfone Polymers 0.000 claims description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 claims description 2
- 239000003463 adsorbent Substances 0.000 claims 5
- 239000004743 Polypropylene Substances 0.000 claims 1
- 230000015271 coagulation Effects 0.000 claims 1
- 238000005345 coagulation Methods 0.000 claims 1
- 229920000620 organic polymer Polymers 0.000 claims 1
- 229920002492 poly(sulfone) Polymers 0.000 claims 1
- 229920001155 polypropylene Polymers 0.000 claims 1
- 238000001179 sorption measurement Methods 0.000 abstract description 59
- 150000002433 hydrophilic molecules Chemical group 0.000 abstract 1
- 239000012510 hollow fiber Substances 0.000 description 187
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 152
- 239000000243 solution Substances 0.000 description 133
- 235000002639 sodium chloride Nutrition 0.000 description 116
- 239000011780 sodium chloride Substances 0.000 description 76
- 238000011156 evaluation Methods 0.000 description 62
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 50
- 229940098773 bovine serum albumin Drugs 0.000 description 50
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 45
- 238000006243 chemical reaction Methods 0.000 description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 39
- 125000003700 epoxy group Chemical group 0.000 description 29
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- 239000011259 mixed solution Substances 0.000 description 18
- 230000000052 comparative effect Effects 0.000 description 15
- 239000003446 ligand Substances 0.000 description 15
- 238000005349 anion exchange Methods 0.000 description 14
- 238000004113 cell culture Methods 0.000 description 14
- 239000012460 protein solution Substances 0.000 description 14
- 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 13
- 238000004587 chromatography analysis Methods 0.000 description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 238000010828 elution Methods 0.000 description 12
- 238000006467 substitution reaction Methods 0.000 description 11
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- 238000001742 protein purification Methods 0.000 description 10
- 239000012527 feed solution Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical class OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 239000008213 purified water Substances 0.000 description 8
- 125000000467 secondary amino group Chemical group [H]N([*:1])[*:2] 0.000 description 8
- 238000003756 stirring Methods 0.000 description 8
- 125000001302 tertiary amino group Chemical group 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 7
- 238000001042 affinity chromatography Methods 0.000 description 7
- 239000012295 chemical reaction liquid Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- 101710120037 Toxin CcdB Proteins 0.000 description 6
- 229940125644 antibody drug Drugs 0.000 description 6
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 6
- 150000001336 alkenes Chemical class 0.000 description 5
- 150000001412 amines Chemical class 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 239000006143 cell culture medium Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000013636 protein dimer Substances 0.000 description 5
- ROSDSFDQCJNGOL-UHFFFAOYSA-N Dimethylamine Chemical compound CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 4
- 238000005571 anion exchange chromatography Methods 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 125000006309 butyl amino group Chemical group 0.000 description 4
- 238000005341 cation exchange Methods 0.000 description 4
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 description 4
- 229910001873 dinitrogen Inorganic materials 0.000 description 4
- 125000004914 dipropylamino group Chemical group C(CC)N(CCC)* 0.000 description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 4
- WGYKZJWCGVVSQN-UHFFFAOYSA-N propylamine Chemical compound CCCN WGYKZJWCGVVSQN-UHFFFAOYSA-N 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 238000011091 antibody purification Methods 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- HQABUPZFAYXKJW-UHFFFAOYSA-N butan-1-amine Chemical compound CCCCN HQABUPZFAYXKJW-UHFFFAOYSA-N 0.000 description 3
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- 238000004440 column chromatography Methods 0.000 description 3
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- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 3
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- 239000004593 Epoxy Substances 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical class NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
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- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 238000005277 cation exchange chromatography Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- WEHWNAOGRSTTBQ-UHFFFAOYSA-N dipropylamine Chemical compound CCCNCCC WEHWNAOGRSTTBQ-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000010574 gas phase reaction Methods 0.000 description 2
- 125000001165 hydrophobic group Chemical group 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 2
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 2
- 238000001471 micro-filtration Methods 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 238000010979 pH adjustment Methods 0.000 description 2
- 229920000098 polyolefin Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000007142 ring opening reaction Methods 0.000 description 2
- 239000008174 sterile solution Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000013638 trimer Substances 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- BQCIDUSAKPWEOX-UHFFFAOYSA-N 1,1-Difluoroethene Chemical compound FC(F)=C BQCIDUSAKPWEOX-UHFFFAOYSA-N 0.000 description 1
- DOUBAFNWVFAWEC-UHFFFAOYSA-N 3-hydroxypropyl acetate Chemical compound CC(=O)OCCCO DOUBAFNWVFAWEC-UHFFFAOYSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
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- 238000009010 Bradford assay Methods 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Chemical class 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical class [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical class [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 239000005703 Trimethylamine hydrochloride Substances 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- XTXRWKRVRITETP-UHFFFAOYSA-N Vinyl acetate Chemical compound CC(=O)OC=C XTXRWKRVRITETP-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000036783 anaphylactic response Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
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- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 238000012832 cell culture technique Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- UUAGAQFQZIEFAH-UHFFFAOYSA-N chlorotrifluoroethylene Chemical group FC(F)=C(F)Cl UUAGAQFQZIEFAH-UHFFFAOYSA-N 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
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- 235000013305 food Nutrition 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000012787 harvest procedure Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 238000012527 host cell protein-ELISA Methods 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 125000005647 linker group Chemical group 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000004880 lymph fluid Anatomy 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 229910052751 metal Chemical class 0.000 description 1
- 239000002184 metal Chemical class 0.000 description 1
- 238000012434 mixed-mode chromatography Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012433 multimodal chromatography Methods 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 239000004745 nonwoven fabric Substances 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 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 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000004382 potting Methods 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000001632 sodium acetate Chemical class 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- BFKJFAAPBSQJPD-UHFFFAOYSA-N tetrafluoroethene Chemical group FC(F)=C(F)F BFKJFAAPBSQJPD-UHFFFAOYSA-N 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
- ILWRPSCZWQJDMK-UHFFFAOYSA-N triethylazanium;chloride Chemical compound Cl.CCN(CC)CC ILWRPSCZWQJDMK-UHFFFAOYSA-N 0.000 description 1
- SZYJELPVAFJOGJ-UHFFFAOYSA-N trimethylamine hydrochloride Chemical compound Cl.CN(C)C SZYJELPVAFJOGJ-UHFFFAOYSA-N 0.000 description 1
- YFTHZRPMJXBUME-UHFFFAOYSA-N tripropylamine Chemical compound CCCN(CCC)CCC YFTHZRPMJXBUME-UHFFFAOYSA-N 0.000 description 1
- BMXILUZRCXPKOI-UHFFFAOYSA-N tripropylazanium;chloride Chemical compound Cl.CCCN(CCC)CCC BMXILUZRCXPKOI-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
- B01D67/0081—After-treatment of organic or inorganic membranes
- B01D67/0093—Chemical modification
- B01D67/00931—Chemical modification by introduction of specific groups after membrane formation, e.g. by grafting
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
- B01D69/08—Hollow fibre membranes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
- B01D71/06—Organic material
- B01D71/76—Macromolecular material not specifically provided for in a single one of groups B01D71/08 - B01D71/74
- B01D71/78—Graft polymers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J47/00—Ion-exchange processes in general; Apparatus therefor
- B01J47/12—Ion-exchange processes in general; Apparatus therefor characterised by the use of ion-exchange material in the form of ribbons, filaments, fibres or sheets, e.g. membranes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2323/00—Details relating to membrane preparation
- B01D2323/38—Graft polymerization
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
- B01D71/06—Organic material
- B01D71/24—Rubbers
Definitions
- the present invention relates to a porous membrane having an amino group and an alkyl group bonded to a graft chain fixed to a porous substrate, and a protein purification method using the porous membrane.
- proteins are produced by cell culture using animal-derived cell lines.
- the cell culture solution is centrifuged to remove sediment components.
- cell debris of about 1 ⁇ m or less that cannot be removed by centrifugation is removed by size filtration using a microfiltration membrane.
- sterilization filtration is performed using a filtration membrane having a maximum pore size of 0.22 ⁇ m or less to obtain a sterile solution containing the target protein (harvest process).
- HCP Host Cell Protein
- DNA deoxyribonucleic acid
- HCP host cell derived protein
- chromatographic techniques typically including affinity chromatography using protein A.
- Contaminants such as aggregates of target protein, endotoxin, virus, protein A desorbed from the column, and aggregates of protein A and antibody are removed from this sterile solution, and the target protein is separated and purified (downstream) Process).
- the concentration of the target protein in the cell culture medium that is the target of the conventional protein purification method described above is usually about 1 g / L at present. Further, the concentration of impurities is considered to be approximately the same as or lower than the concentration of the target protein. At such a concentration, the conventional protein purification method including the harvesting step and the downstream step may be effective.
- the concentration of the target protein in the cell culture medium may reach 10 g / L or more.
- the concentration of contaminants in the cell culture medium increases as well, and the purification of the target protein is becoming difficult with the conventional protein purification methods.
- the concentration of the target antibody protein in the cell culture medium increases, the concentration of monomer aggregates such as dimers and trimers tends to increase remarkably. Aggregates can result in complement activity or anaphylaxis when administered in vivo. For this reason, it has been pointed out that aggregates can have a detrimental effect on the safety of antibody pharmaceuticals, and in recent years there has been a demand for an effective removal method.
- the purpose is to effectively remove various contaminants including aggregates and purify antibody proteins used as antibody drugs, ie, monoclonal antibodies, polyclonal antibodies, humanized antibodies, human antibodies and immunoglobulins. Many chromatographic processes have been reported.
- Ion exchange chromatography is a method of separating these using the difference in isoelectric point between an antibody and a contaminant.
- anion exchange chromatography is commonly used to remove contaminants such as HCP, DNA, and viruses, which generally have an isoelectric point lower than that of antibody proteins.
- Patent Documents 1 and 2). As a method for removing general impurities such as HCP, in recent years, protein adsorption membranes have been developed in which ion exchange groups are introduced into porous membranes to impart protein adsorption ability (for example, Patent Documents 1 and 2). reference).
- Patent Document 3 discloses that two types of protein adsorption membranes, a cellulose porous membrane introduced with an anion exchange group and a cellulose porous membrane introduced with a cation exchange group, are used for lymph fluid. Disclosed is a method for separating albumin from the same.
- Patent Document 4 discloses a method for separating nucleic acid and endotoxin using a cellulose porous membrane into which an anion exchange group is introduced.
- Patent Documents 5 and 6 disclose protein adsorption membranes in which a cation exchange group and an anion exchange group are introduced into a polyethersulfone porous membrane.
- Patent Document 7 discloses a porous membrane having a swollen gel layer in which a primary amine is fixed as an anion exchange group.
- a mixed solution of an antibody monomer and an aggregate is adjusted to a pH in the vicinity of the isoelectric point of the antibody, and applied to an anion exchange chromatography column.
- a method for purifying an antibody monomer is disclosed in which a permeate is collected by passing the solution, a washing solution is collected by passing a buffer solution having the same pH, and the recovered solution is used as a purified solution of the antibody monomer.
- This purification method is based on the principle that aggregates have a larger number of charge points than monomers and thus are easily fixed by anion exchange groups.
- Patent Document 9 discloses that an antibody monomer and an aggregate are both adsorbed on an anion exchange chromatography column, and then the antibody monomer eluted first by gradient elution in which the salt concentration of the eluate is gradually increased. A method for purifying antibody monomers that recovers the elution peak of is disclosed.
- Patent Document 10 discloses a method for recovering antibody monomers by adsorbing impurities by a flow-through mode using a chromatography of a multimodal ligand consisting of an anion exchange group and a hydrophobic group, particularly a free protein A ligand. And a method for adsorbing and removing aggregates composed of antibody monomers.
- Patent Document 11 adsorbs most contaminants such as DNA, viruses, endotoxins, aggregates, and HCP using multimodal chromatography having a quaternary ammonium group, a hydrogen bonding group, and a hydrophobic group.
- a method for recovering antibody monomers by flow-through is described, and in particular, it is described that HCP is almost completely removed.
- Patent Document 12 discloses that a mixed mode chromatography having a mercapto group and an aromatic pyridine ring is used to adsorb only the antibody monomer and remove the aggregate as a non-adsorbed fraction, thereby binding the antibody in the binding mode. A method for purifying monomers is described.
- Patent Document 13 Hydroxyapatite chromatography is applied to Patent Document 13 as a method for purifying and recovering antibody monomers in a flow-through mode for the purpose of selectively adsorbing aggregates to a column and removing aggregates more effectively.
- An example is described.
- a porous membrane in which an anion exchange group by an amino group is immobilized on the surface of a substrate through a graft chain is described in Patent Document 7, and a limited amino group immobilization method by a gas phase reaction is disclosed. Yes.
- Patent Document 8 describes a method for separating egg white protein using a porous membrane in which a diethylamino group and a 2-hydroxyethylamino group are fixed via a graft chain.
- the protein adsorption membranes disclosed in Patent Documents 1 to 6 have a pore diameter of 1 ⁇ m or more, they have a poor ability to remove cell debris that is a turbid impurity. Moreover, since the adsorption capacity of dissolved protein is small, a large amount of protein cannot be adsorbed. Further, the cell culture solution usually contains a salt, but if the cell culture solution contains a salt of 0.1 mol / L or more, the protein of the protein adsorption membrane disclosed in Patent Documents 1 to 6 The amount of adsorbed is significantly reduced. Therefore, it is not practical to remove the impurity protein from the cell culture solution having high electrical conductivity or the eluate of the cation exchange chromatography step using the protein adsorption membrane disclosed in Patent Documents 1 to 6.
- the porous membrane disclosed in Patent Document 7 exhibits remarkable protein adsorptivity to a solution having a high electrical conductivity containing a salt.
- the adsorption performance is too high, it is difficult to selectively remove only the impurity protein, and the target protein is also easily adsorbed. Therefore, the subject that the recovery rate of the target protein falls arises. Further, since the adsorbed impurities cannot be sufficiently washed and eluted, there is a practical problem that they cannot be used repeatedly.
- Patent Document 14 A porous membrane in which an anion exchange group based on an amino group is immobilized on the surface of a substrate via a graft chain is described in Patent Document 14, but there is no description regarding a protein purification method using the obtained porous membrane.
- Patent Document 15 describes a method for separating egg white protein using a porous membrane in which a diethylamino group and a 2-hydroxyethylamino group are fixed via a graft chain. It is limited to proteins, and there is no description regarding a method for purifying antibody monomers.
- the difficulty of purifying antibodies with effective and rapid removal of all contaminants is effective pH, salt concentration, and solution to effectively separate and purify antibodies in both flow-through and binding modes. This is also due to the fact that conditions such as composition, that is, the process window of the conventional purification technique is narrow and it is not easy to determine stable and versatile purification conditions. This situation is similar in mixed-mode ligand chromatography aimed at more efficiently removing contaminants.
- the process for removing contaminants in the intermediate purification requires high precision, and therefore a process using a chromatography column with high resolution is directed.
- impurities such as HCP that need to be further removed from the state that has been removed to a low concentration by affinity chromatography
- it is more difficult to set conditions because the process window is still narrower. It becomes.
- the aggregate has an interaction property similar to that of an antibody monomer, it is particularly difficult to set conditions, and until now it has not been possible to remove rapidly using an adsorption film or the like. For this reason, no method has been reported so far to remove impurities from antibody monomers using a porous membrane.
- one of the problems to be solved by the present invention is a simple, high-speed and wide process window from a solution having a high electrical conductivity containing proteins and contaminants such as antibody monomers at a high concentration. It is an object of the present invention to provide a protein purification method using a porous membrane capable of effectively and rapidly removing contaminants such as HCP, DNA, endotoxin, lipid, virus, and protein aggregates including a target protein.
- One of the further problems to be solved by the present invention is to provide a protein purification method using a reusable porous membrane by washing impurities adsorbed by appropriate washing after trapping impurities. It is.
- the present inventors have found that the porous substrate, the amino group bonded to the side chain of the graft chain fixed to the surface of the porous substrate, and the amino group It was found that using a porous membrane having an alkyl group bonded to a dimer is effective in removing contaminants that have been difficult to remove, including aggregates including dimers.
- the present invention has been completed.
- the aspect of the present invention includes a hydrophobic porous substrate, a molecular chain having a hydrophilic property different from that of the porous substrate, which is fixed to the pore surface of the porous substrate, and the number of carbon atoms.
- a porous membrane comprising a side chain of a molecular chain containing a nitrogen atom to which 1 to 3 alkyl groups of 1 to 3 are bonded, an antibody solution containing an antibody aggregate of a dimer or higher is permeated through the porous membrane.
- the gist of the present invention is a method for purifying antibody monomers, in which antibody aggregates are recovered in a permeate by adsorbing antibody aggregates to a porous membrane.
- Another aspect of the present invention is a method for purifying an antibody monomer solution having a salt concentration of 0.3 mol / L or less, and the antibody monomer solution is filtered through a porous membrane so that it is contained in at least the antibody monomer solution.
- a method for purifying an antibody monomer solution comprising a hydrophilic molecular chain of a material different from that of a porous substrate and a side chain including a nitrogen atom, a hydroxyl group and a carbonyl group to which 1 to 3 alkyl groups are bonded. This is the gist.
- impurities such as HCP, DNA, endotoxin, virus, and protein aggregates are obtained from a solution having a relatively high salt concentration containing proteins such as antibodies and contaminants. Can be easily and rapidly removed, and protein monomers such as antibodies can be purified effectively and rapidly.
- the present embodiment a mode for carrying out the present invention (hereinafter referred to as “the present embodiment”) will be described in detail.
- the present invention is not limited to the following embodiment, and can be implemented with various modifications within the scope of the gist.
- the porous membrane according to the present embodiment includes a hydrophobic porous substrate, a molecular chain having hydrophilicity of a material different from the porous substrate, which is fixed to the surface including the pores of the porous substrate, And a side chain of a molecular chain containing at least one amino group selected from a propylamino group, an isopropylamino group, a diethylamino group, a triethylamino group, and a tripropylamino group.
- the method for producing an antibody monomer according to the present embodiment includes preparing a porous film according to the present embodiment, allowing the antibody monomer solution containing impurities to permeate the porous film, and adsorbing the impurities to the porous film. And recovering the purified antibody monomer in the permeate at a recovery rate of 80% or more.
- Antibody in the present embodiment is a general term for proteins that cause an antigen-antibody reaction with an antigen that has entered the living body and give immunity to the antigen to the living body.
- monoclonal antibodies, polyclonal antibodies, humans It refers to conjugated antibody, human antibody, immunoglobulin and the like.
- an antibody that can be an antibody drug is suitable as a purification target of the purification method according to the present embodiment.
- antibody monomer in the present embodiment refers to the above-mentioned antibody that exists as a monomer.
- contaminants refers to impurities other than antibody monomers contained in a target solution (mixed solution) for purifying antibody monomers.
- a target solution mixed solution
- impurities other than the target antibody monomer that are produced in the culture tank when the antibody is produced by cell culture are listed as “contaminants”. More specifically, antibody monomer aggregates, misfolded antibody protein species, HCP, endotoxin, DNA, protease, free protein A, viruses, bacteria, and the like are mentioned as “contaminants”.
- the “aggregate” in this embodiment is a complex of one or more types of antibody monomers, or a combination of an antibody monomer and another compound such as a protein. It refers to a complex, and includes, for example, a multimer such as a dimer or trimer of an antibody monomer, and a complex of free protein A and an antibody.
- the “mixed solution containing antibody monomers and contaminants” in the present embodiment is not particularly limited as long as it is a solution (or a solution that may be contained) containing the above antibody monomers and contaminants.
- a target solution for purifying antibody monomer therefrom by the method for purifying antibody monomer according to the embodiment examples thereof include a cell culture solution used for antibody production or a turbid solution thereof, or a partially purified solution during or after the chromatography step in the downstream step thereof.
- the turbid solution in the downstream process typically before the affinity chromatography process using protein A, or the partially purified solution after the affinity chromatography process is a clear solution, Further, since the impurities are partially removed, the load in the subsequent purification step is further reduced, and therefore, it is suitable as a purification target of the purification method according to the present embodiment.
- the concentration of the antibody monomer and impurities in the mixed solution is not particularly limited. However, from the viewpoint that the method for purifying antibody monomers in the present embodiment can be carried out efficiently even for a mixed solution containing antibody monomers and impurities at a particularly high concentration, for example, the mixed solution is an antibody. It may contain 2 g / L or more of monomers and impurities. Furthermore, the mixed solution may contain 5 g / L or more of the antibody monomer, and may contain 1 g / L or more of impurities.
- the “porous membrane” used in the present embodiment is a hydrophobic porous substrate and a molecule having hydrophilicity, which is fixed to the surface including the pores of the porous substrate and is made of a material different from the porous substrate. And a side chain of a molecular chain containing at least one amino group selected from a propylamino group, an isopropylamino group, a diethylamino group, a triethylamino group, and a tripropylamino group.
- graft chain the molecular chain having hydrophilicity fixed to the porous substrate
- the entire surface of the porous substrate including the side walls of the pores inside the porous substrate is simply referred to as “surface”, and the surface not including the side walls of the pores inside the porous substrate is referred to as “main”. It is called “surface”.
- the porous membrane used in the present embodiment utilizes the fact that differences in adsorption performance and purification conditions occur depending on the protein due to the combination of an amino group and an alkyl group. Therefore, a porous membrane for protein purification having a wide process window can be obtained by selecting a combination of an amino group and an alkyl group according to the purpose.
- secondary amino groups include a propylamino group (—N—CH 2 CH 2 CH 3 ) and an isopropylamino group (—N—CH (CH 3 ) 2 ). Is preferred.
- a diethylamino group (—N— (CH 2 CH 3 ) 2 ) and a diisopropylamino group (—N— (CH (CH 3 ) 2 ) 2 ) are preferable.
- the quaternary amino group includes a triethylamino group (—N + — (CH 2 CH 3 ) 3 ), a tripropyl amino group (—N + — (CH 2 CH 2 CH 3 ) 3 ), and tridipropyl amino
- the group (—N + — (CH (CH 3 ) 2 ) 3 ) is preferred.
- the secondary amino group, the tertiary amino group, and the quaternary amino group are also referred to as a monoalkyl-substituted amino group, a dialkyl-substituted amino group, and a trialkyl-substituted amino group, respectively.
- the higher the series of amines and the higher the hydrophobicity the higher the protein adsorption at high salt concentrations. This is because the larger the amine series, the stronger the electrostatic interaction between the carboxylic acid of the protein and the amine, and the higher the hydrophobicity, the easier the adsorption due to the hydrophobic interaction occurs.
- the greater the amine series the greater the attenuation of electrostatic interactions when the salt concentration is higher.
- the higher the salt concentration the stronger the hydrophobic interaction. Therefore, it is difficult to theoretically predict a functional group having adsorptivity even at a high salt concentration from only the amine series and the type of alkyl group.
- a combination of an effective electrostatic interaction and a hydrophobic interaction is determined from the results obtained by experimental evaluation.
- the material of the porous substrate is not particularly limited. However, if the material is hydrophobic, the material can have an additional hydrophobic interaction between the protein and the porous substrate, as well as mechanical properties. From the standpoint of holding, it is preferably composed of a polyolefin-based polymer.
- polyolefin polymers include, for example, homopolymers of olefins such as ethylene, propylene, butylene, and vinylidene fluoride, two or more types of copolymers of the olefins, or one or more types of the olefins. Examples thereof include copolymers with perhalogenated olefins. Moreover, the mixture of 2 or more types of these polymers may be sufficient. Examples of perhalogenated olefins include tetrafluoroethylene and chlorotrifluoroethylene.
- polyethylene or polyvinylidene fluoride is preferable as the material of the porous substrate, and polyethylene is more preferable. preferable.
- the method for fixing the alkylated amino group to the porous substrate is not particularly limited, but generally a highly reactive functional group such as epoxy is introduced onto the surface of the porous substrate, and then the functional group is introduced. It can be carried out by a method in which an organic amine compound is bonded to the group.
- a graft chain having an epoxy group is previously fixed on the surface of the porous substrate, and an organic amine compound composed of a combination of a desired amino group and an alkyl group is reacted with the epoxy group.
- an organic amine compound is fixed to the main surface of a porous base material, and the side wall of the pore provided in the porous base material.
- the “graft chain” is made of a material different from the material of the porous substrate bonded to the main surface of the porous substrate and the side walls of the pores provided in the porous substrate. It is a molecular chain and can be present both inside the substrate skeleton forming the substrate and inside the pores forming the porous portion.
- Examples of the graft chain include molecular chains containing glycidyl methacrylate, glycidyl acrylate, glycidyl sorbate, glycidyl itacolate, glycidyl maleate, vinyl acetate, hydroxypropyl acetate, or any two or more polymers thereof.
- a polymer of glycidyl methacrylate is preferable because an amino group can be easily introduced by ring opening of an epoxy group and a hydroxyl group and a carbonyl group can be easily introduced into a side chain.
- the bond rate (graft rate) of the graft chain to the porous substrate can be measured, for example, by using the method described in Examples and the like to be described later, and ensures both higher adsorption capacity and mechanically stable strength. From the viewpoint, it is preferably 10% to 200%, more preferably 20% to 150%, still more preferably 30% to 100%.
- the amino group is preferably introduced in 60% or more and 97% or less, more preferably 70% or more and 96% or less of all side chains of the graft chain.
- the epoxy group into which no amino group has been introduced is preferably ring-opened by alkali solution treatment or the like in order to suppress chemical bonding with protein to form a diol.
- the side chain having a diol group the hydrophilicity of the graft chain is increased, and the graft chain can effectively spread in the pores filled with the solution.
- the side chain of the graft chain having a diol group is preferably 3% to 40%, more preferably 4% to 30% of all side chains. When the ratio of diol groups is higher than 40%, the effect of anion exchange interaction by amino groups tends to be reduced.
- a method for introducing a graft chain into the surface of the porous substrate and the side wall of the pore and further fixing the alkylated amino group to the graft chain is not limited.
- JP-A-2-132132 Examples include the method disclosed in the publication.
- the amine compound in order to follow a gas phase reaction, the amine compound is limited to the one that exists as a gas phase in the reaction environment and can react with an epoxy group.
- a method of reacting and immobilizing an amine compound with an epoxy group immobilized on a graft chain by a liquid phase reaction for example, there is a method described in Journal of Chromatography A, 689 (1995) 211-218.
- this document only discloses a method of reacting diethylamine.
- the method for fixing an arbitrary amine compound by reacting with an epoxy group needs to be appropriately selected depending on the amine compound to be used. For example, the method described in Examples and the like described later is used.
- the porous membrane used in the present embodiment includes a porous substrate, a graft chain fixed to the main surface of the porous substrate and the side walls of the pores provided in the porous substrate, and the side of the graft chain An amino group and an alkyl group bonded to the chain.
- a porous membrane has a structure in which each graft chain fixed to the porous substrate has one or more side chains, and one or more amino groups and alkyl groups are fixed to the side chains. Therefore, amino groups and alkyl groups are sterically distributed in the pore space. Therefore, the number of adsorption points of amino groups is large with respect to proteins having a charge point, the amount of adsorption is increased, and the adsorptivity of proteins having a small interaction is increased.
- the presence of an alkyl group, a hydroxyl group, and a carbonyl group has the effect of realizing a more highly selective protein adsorption.
- proteins that are difficult to purify only by charge interaction such as antibodies, can be effectively separated from the contaminants and purified.
- antibody purification is performed quickly and efficiently by selectively adsorbing only contaminants by passing a solution containing the antibody monomer and contaminants and allowing the antibody to permeate non-adsorbingly. Especially suitable for the purpose.
- the maximum pore size of the porous membrane is the same as that described above before fixing the anion exchange group and introducing the graft chain from the viewpoint of effectively adsorbing antibody monomers and / or impurities in the solution and obtaining a high permeation flow rate.
- the thickness is preferably 0.01 ⁇ m to 5.0 ⁇ m, more preferably 0.1 ⁇ m to 3.0 ⁇ m, and still more preferably 0.1 ⁇ m to 1.0 ⁇ m.
- the porosity which is the ratio of the volume of the pores to the total volume of the porous membrane, is not particularly limited as long as it retains the shape of the porous membrane and the pressure loss at the time of liquid passage is not problematic in practice. Is from 5% to 99%, more preferably from 10% to 95%, still more preferably from 30% to 90%.
- the measurement of the pore diameter and the porosity can be performed by methods known to those skilled in the art as described in “Membrane Technology” by Marcel Mulder (IPC Co., Ltd.) and the like. Examples thereof include measurement methods such as observation with an electron microscope, bubble point method, mercury intrusion method, and transmittance method. For example, for measurement of the maximum pore diameter, the bubble point method described in Examples and the like described later can be appropriately used.
- the form of the porous substrate is not particularly limited as long as the solution can be passed therethrough, and examples thereof include a flat membrane, a nonwoven fabric, a hollow fiber membrane, a monolith, and a capillary.
- a hollow fiber membrane is preferable from the viewpoints of ease of production, scale-up property, and packing property of the membrane when module-molded.
- the shape of the porous substrate may be, for example, a disk or a cylinder, but is not limited thereto.
- the hollow fiber porous membrane is a cylindrical or fibrous porous membrane having a hollow portion, and the inner layer and the outer layer of the hollow fiber communicate with each other through pores that are through holes, It means a porous body having a property that liquid or gas permeates from the inner layer to the outer layer or from the outer layer to the inner layer by the pores.
- the outer diameter and inner diameter of the hollow fiber are not particularly limited as long as the porous membrane can physically hold the shape and can be molded into a module.
- the step of passing the mixed solution containing the antibody monomer and the contaminants through the porous membrane according to the present embodiment described above to adsorb the contaminants and / or the antibody monomer to the porous membrane will be described below.
- An example of a typical method for purifying antibody monomers according to the present embodiment is not particularly limited, but a mixed solution containing antibody monomers and impurities is passed through the porous membrane according to the present embodiment.
- the most convenient and rapid purification method is to remove impurities from the mixture by adsorbing the impurities in the mixture to the porous membrane and recover the permeate as a purified antibody monomer solution. Is preferable.
- the isoelectric point (pI) of antibody monomers is usually in the range of 6.5 to 8.5.
- many pIs of contaminants such as HCP, DNA, and viruses are 6 or less. Therefore, by suitably controlling the pH range and salt concentration (electric conductivity) of the solution passing through the porous membrane, most of the impurities are adsorbed on the positively charged amino groups of the porous membrane, and the permeate Can be recovered as a purified antibody monomer solution.
- the pi of the aggregate is close to or approximately equal to the antibody monomer.
- the difference between an antibody monomer and an aggregate is that the aggregate is a complex of antibody monomers, so even if the pI is similar to that of the antibody monomer, the aggregate has a charge that one molecule has. It is a point with a large number of points. For this reason, the aggregate has a property that it is slightly adsorbed to the porous membrane having a positively charged amino group as compared with the antibody monomer. Therefore, by appropriately adjusting the pH and salt concentration of the solution, the aggregate can be adsorbed on the amino groups of the porous membrane, and the permeate can be recovered as a purified antibody monomer solution.
- each graft chain has one or more side chains and each side chain has an amino group
- the amino group is sterically fixed in the porous membrane.
- proteins such as aggregates are sterically fixed by a plurality of amino groups at the charge point. Therefore, the aggregate tends to be more strongly adsorbed to the porous membrane than the antibody monomer, and the antibody monomer can be easily purified.
- proteins, DNA, viruses, and the like have properties other than anion exchange interactions such as hydrophobic interactions. Therefore, antibody purification can be performed more easily by using a porous membrane having properties in which hydrophobicity, hydrogen bonding properties, and cation exchange properties are appropriately controlled. Therefore, when the graft chain has an appropriate alkyl group, hydroxyl group, and carbonyl group, it is possible to more selectively adsorb and remove impurities.
- antibody monomer aggregates differ in hydrophobic character from antibody monomers. Therefore, when the graft chain has an alkyl group, the aggregate can be more selectively adsorbed and removed.
- the pH and salt concentration of the mixed solution are adjusted to conditions where the property becomes more remarkable than that of the antibody monomer. Specifically, it is preferable to adjust the mixed solution so that the pH is 6 to 9 and the salt concentration is in the range of 0 mol / L to 0.3 mol / L.
- Examples of the salt used for adjusting the salt concentration include sodium chloride, sodium sulfate, sodium acetate, and ammonium sulfate, as well as metal salts of citric acid, phosphoric acid, or glycine, but are not limited thereto. .
- pH adjustment can usually be performed simply by adding hydrochloric acid or sodium hydroxide, it is not limited to this, The pH adjustment method well-known to those skilled in the art can be used suitably.
- the pH and salt concentration of the solution can be measured by using a method known to those skilled in the art, for example, using a commercially available measuring instrument.
- the mixed solution having the above pH and salt concentration By passing the mixed solution having the above pH and salt concentration through the porous membrane in the present embodiment, contaminants such as aggregates, HCP, DNA, endotoxin, and viruses having a pI of 6 or less are adsorbed on the porous membrane. Is done.
- contaminants especially aggregates
- the acidity of the mixed solution is preferably pH 6 to 9, more preferably pH 7 to 8.5, and further preferably pH 7.5 to 8.5. .
- the salt concentration of the mixed solution is preferably 0 mol / L or more, more preferably 0.01 mol / L or more, and further preferably 0.02 mol / L or more. Moreover, 0.3 mol / L or less is preferable, 0.2 mol / L or less is more preferable, 0.1 mol / L or less is more preferable, 0.05 mol / L or less is especially preferable.
- the pH and salt concentration of the mixed solution are set as above. If it is in the range, it is possible to effectively purify the antibody monomer. In addition, it becomes possible to refine
- a contaminant is contained from a solution containing a high concentration of antibody and a high concentration of contaminant, and further containing a salt. It can be removed simply, at high speed and with a wide process window. Therefore, it becomes possible to purify the antibody from the solution effectively and quickly. Therefore, it becomes possible to obtain the purified antibody industrially efficiently.
- the obtained polyethylene-made hollow fiber porous substrate having radicals was placed in a glass reaction tube, and the pressure in the reaction tube was reduced to 200 Pa or less to remove oxygen in the reaction tube.
- a reaction liquid consisting of 2.5 parts by volume of glycidyl methacrylate (GMA) adjusted to 40 ° C. and 97.5 parts by volume of methanol was injected into 20 parts by mass of the hollow fiber-like porous substrate, and then sealed for 12 hours. The mixture was allowed to stand in the state and subjected to a graft polymerization reaction to obtain a hollow fiber porous membrane having graft chains introduced therein.
- the reaction solution composed of GMA and methanol was previously bubbled with nitrogen to replace oxygen in the reaction solution with nitrogen.
- the reaction solution in the reaction tube was discarded.
- dimethyl sulfoxide was placed in the reaction tube to wash the hollow fiber porous membrane, thereby removing the remaining glycidyl methacrylate, its oligomer, and the graft chain not fixed to the hollow fiber porous membrane.
- dimethyl sulfoxide was further added and washing was performed twice.
- washing with methanol was performed three times in the same manner. The washed hollow fiber porous membrane was dried and weighed, and the weight of the hollow fiber porous membrane was 0.209 g of 155% before graft chain introduction.
- the graft ratio defined as the ratio of the weight of the graft chain to the weight of the porous membrane substrate before the introduction of the graft chain was 55%.
- the hollow fiber after the reaction had an outer diameter of 3.35 mm, an inner diameter of 2.15 mm, and a length of 108 mm.
- the epoxy group possessed by the graft chain is substituted with an amine compound by a ring-opening reaction under reaction conditions suitable for each amine compound described in Examples and Comparative Examples described later, and an amino group and an alkyl group are substituted.
- T can be obtained analytically by solving the linear equation (2) below.
- M 1 ⁇ (W 2 ⁇ W 1 ) / (M 1 T + M 0 (1 ⁇ T) ⁇ / ⁇ W 1 (dg / (dg + 100)) / M 2 ⁇
- M 1 is an amine compound
- M 0 porous hollow fiber after the weight of the porous hollow fiber membrane after 18 by the molecular weight of water
- W 1 is graft polymerization reaction
- W 2 is amino group substitution reaction weight of the membrane
- dg is the graft rate
- M 2 is 142 in the molecular weight of GMA.
- Bubble Point Method The maximum pore diameter of the hollow fiber porous membrane was measured using the bubble point method.
- One end of a hollow fiber porous membrane having a length of 8 cm was closed, and a nitrogen gas supply line was connected to the other end via a pressure gauge.
- nitrogen gas was supplied to replace the inside of the line with nitrogen, and then the hollow fiber porous membrane was immersed in ethanol.
- the hollow fiber porous membrane was immersed in ethanol with a slight nitrogen pressure applied so that ethanol did not flow back into the line.
- the pressure of nitrogen gas was slowly increased, and the pressure P at which nitrogen gas bubbles started to stably emerge from the hollow fiber porous membrane was recorded.
- the maximum pore diameter of the hollow fiber porous membrane was calculated according to the following formula (3), where d is the maximum pore diameter and ⁇ is the surface tension.
- d C 1 ⁇ / P (3)
- C 1 is a constant.
- C 1 ⁇ 0.632 (kg / cm) when ethanol was used as the immersion liquid, and the maximum pore diameter d ( ⁇ m) was determined by substituting P (kg / cm 2 ) into the above equation.
- the electrical conductivity of the BSA solution containing 0 mol / L sodium chloride was 1.3 mS / cm.
- the electrical conductivity of the BSA solution containing 0.1 mol / L sodium chloride was 10.2 mS / cm.
- the solution was passed at a flow rate of 3 mL / min from the inside to the outside of the hollow fiber porous membrane in the evaluation module. Evaluation was performed using AKTAexplorer100 manufactured by GE Healthcare Bioscience. Specifically, when the UV absorbance at 280 nm of the permeate obtained in the apparatus becomes 1/10 (15 mAU) of the UV absorbance (150 mAU) at 280 nm of the feed solution, the breakthrough point is reached.
- the dynamic adsorption capacity was calculated from the volume of the BSA solution supplied to.
- the concentration of the BSA solution Q, from the volume V M of the hollow fiber porous membrane according to an embodiment of the volume V B, and evaluation module BSA solution was transmitted by the time evaluation module has breakthrough, the following formula ( Based on 4), the dynamic adsorption capacity A can be calculated.
- A Q ⁇ V B / V M (4)
- the volume of the hollow fiber porous membrane is the volume excluding the hollow portion. Breakthrough refers to a point in time when the BSA concentration in the permeate exceeds 0.1 g / L, which is 10% of the concentration of the supplied BSA solution.
- a buffer containing 0.15 mol / L NaCl in 12 mmol / L hydrochloric acid was used for elution.
- the antibody protein solution with a concentration of 1 mg / mL adjusted in (i) is added as a sample, adsorbed to the protein G column, and further eluted from the protein G column, the peak area is 1 mg / mL antibody protein concentration It was made to correspond.
- the permeate of the hollow fiber porous membrane module was similarly passed through the column to determine the elution peak area, and from the ratio of the elution peak area obtained from the 1 mg / mL antibody protein solution, the hollow fiber porous membrane The antibody protein concentration in the permeate of the module was calculated to obtain the recovery rate.
- the antibody monomer and the aggregate after elution through the column showed separated elution peaks, and the respective abundance ratios in the solution were calculated from the peak area ratio of the obtained antibody monomer and the aggregate.
- the dimer ratio contained in the antibody protein before mixing used in the evaluation and the antibody protein in the solution prepared in (i) was 4.06%.
- an antibody protein (Benesis Co., Ltd., blood donation venoglobulin-IH) was prepared. Furthermore, 0.1 mg / mL of impurities and 10 mg / mL of the prepared antibody protein were dissolved in 20 mmol / L Tris-HCl (pH 7.5) buffer containing 0.15 mol / L NaCl to prepare a solution. did. The solution thus obtained was used as a feed solution, passed through a hollow fiber porous membrane module having various ligands prepared by the method of the production example, and the antibody protein recovery rate and the impurity concentration after permeation were measured.
- a buffer containing 0.15 mol / L NaCl in 12 mmol / L hydrochloric acid was used. Thereafter, the system displayed the elution peak of antibody protein adsorbed and eluted on the protein G column and the elution peak of impurities that flowed out without being adsorbed on the column. Further, the antibody recovery rate in solution and the impurity concentration were calculated from the antibody protein elution peak area and the impurity elution peak area.
- the HCP concentration in the permeate was quantified by an ELISA method using CHO Host Cell Protein ELISA Kit, 3rd Generation, manufactured by CYGNUS. DNA contained in a trace amount was quantified with a Fluorometer using a dsDNA HS Assay Quit Starter Kit manufactured by Invitrogen.
- the HCP concentration in the feed solution determined by the ELISA method was 87 ⁇ g / mL, and the DNA concentration in this solution determined by the Fluorometer method was 1.7 ⁇ g / mL.
- the adsorbed antibody protein was eluted using 50 mmol / L citrate buffer (pH 3.3) to recover 3 ml of the isolated antibody protein solution.
- the solution was immediately neutralized by adding 17 ml of 50 mmol / L Tris-HCl (pH 8.2) buffer solution.
- the obtained antibody protein isolate was evaluated by the method of Evaluation Method Example 3 (ii), and the ratio between the antibody monomer and the aggregate was determined.
- Example 1 Fixation of isopropylamino group (secondary amino group and isopropyl group) to graft chain:
- the hollow fiber porous membrane introduced with the graft chain obtained in Production Example 1 (i) was swollen by immersing in methanol for 10 minutes or more, and then immersed in pure water for water substitution. Further, isopropylamine was added to 20 mL of purified water while stirring until the total volume became 40 mL, thereby preparing a reaction solution. Next, 25 parts by mass of the reaction liquid with respect to the dry weight of the hollow fiber porous membrane into which the graft chain obtained in (i) above was introduced was placed in a glass reaction tube and adjusted to 40 ° C.
- a hollow fiber porous membrane into which a graft chain after immersion in pure water was introduced was inserted into the reaction solution, and allowed to stand for 48 hours to replace the epoxy group of the graft chain with an isopropylamino group, a secondary amino group,
- a hollow fiber porous membrane in which an isopropyl group was fixed via a graft chain was obtained.
- the obtained hollow fiber porous membrane had an outer diameter of 3.43 mm and an inner diameter of 2.10 mm. From the formula (2), 72% of all epoxy groups of the graft chain were substituted with isopropylamino groups.
- a hollow fiber porous membrane module was produced according to Production Example 1 (iii) using a hollow fiber porous membrane having an isopropylamino group.
- the volume of only the porous membrane excluding the hollow portion of the hollow fiber porous membrane in the hollow fiber porous membrane module was 0.53 mL.
- the inner surface area of the hollow fiber porous membrane was 6.1 cm 2 .
- the dynamic adsorption capacity of BSA was determined according to Evaluation Method Example 2.
- the sodium chloride concentration was 0 mol / L, it was 63 mg / mL, and the sodium chloride concentration was 0.00. In the case of 1 mol / L, it was 33 mg / mL.
- the antibody recovery rate was 89% when the sodium chloride concentration was 0 mol / L, and 95% when the sodium chloride concentration was 0.1 mol / L.
- the dimer ratio was 1.03% when the sodium chloride concentration was 0 mol / L, and 1.21% when the sodium chloride concentration was 0.1 mol / L.
- a hollow fiber porous membrane into which a graft chain after immersion in pure water was introduced was inserted into the reaction solution, and allowed to stand for 20 hours to replace the epoxy group of the graft chain with a butylamino group, a secondary amino group, A hollow fiber porous membrane in which a butyl group was fixed via a graft chain was obtained.
- the obtained hollow fiber porous membrane had an outer diameter of 3.49 mm and an inner diameter of 2.16 mm. From the formula (2), 79% of all epoxy groups of the graft chain were substituted with butylamino groups.
- a hollow fiber porous membrane module was produced according to Production Example 1 (iii) using a hollow fiber porous membrane having a butylamino group.
- the volume of only the porous membrane excluding the hollow portion of the hollow fiber porous membrane in the hollow fiber porous membrane module was 0.53 mL.
- the inner surface area of the hollow fiber porous membrane was 6.2 cm 2 .
- the dynamic adsorption capacity of BSA was determined according to Evaluation Method Example 2.
- the sodium chloride concentration was 0 mol / L, it was 15 mg / mL, and the sodium chloride concentration was 0.00. In the case of 1 mol / L, it was 6 mg / mL and the adsorption capacity was small.
- the antibody recovery rate was 92% when the sodium chloride concentration was 0 mol / L, and 97% when the sodium chloride concentration was 0.1 mol / L.
- the dimer ratio is 3.02% when the sodium chloride concentration is 0 mol / L, and 3.32% when the sodium chloride concentration is 0.1 mol / L. It was inferior.
- Example 2 Fixation of propylamino group (secondary amino group and propyl group) to graft chain:
- the hollow fiber porous membrane introduced with the graft chain obtained in Production Example 1 (i) was swollen by immersing in methanol for 10 minutes or more, and then immersed in pure water for water substitution. Further, normal propylamine was added to 20 mL of purified water while stirring until the total volume became 40 mL, to prepare a reaction solution. Next, 25 parts by mass of the reaction liquid with respect to the dry weight of the hollow fiber porous membrane into which the graft chain obtained in (i) above was introduced was placed in a glass reaction tube and adjusted to 40 ° C.
- a hollow fiber porous membrane into which a graft chain after immersion in pure water was introduced was inserted into the reaction solution, and allowed to stand for 24 hours to replace the epoxy group of the graft chain with a propylamino group, a secondary amino group, A hollow fiber porous membrane in which a propyl group was fixed via a graft chain was obtained.
- the obtained hollow fiber porous membrane had an outer diameter of 3.44 mm and an inner diameter of 2.10 mm. From the formula (2), 78% of all epoxy groups of the graft chain were substituted with propylamino groups.
- a hollow fiber porous membrane module was produced according to Production Example 1 (iii) using a hollow fiber porous membrane having a propylamino group.
- the volume of the porous membrane alone excluding the hollow portion of the hollow fiber porous membrane in the hollow fiber porous membrane module was 0.54 mL.
- the inner surface area of the hollow fiber porous membrane was 6.1 cm 2 .
- the dynamic adsorption capacity of BSA was determined according to Evaluation Method Example 2.
- the sodium chloride concentration was 0 mol / L and 0.1 mol / L, 49 mg / mL and It was 21 mg / mL.
- the recovery rate after passing through the antibody protein solution and the dimer ratio were evaluated.
- the antibody recovery rate was 90% when the sodium chloride concentration was 0 mol / L, and 95% when the sodium chloride concentration was 0.1 mol / L.
- the dimer ratio was 1.08% when the sodium chloride concentration was 0 mol / L, and 1.24% when the sodium chloride concentration was 0.1 mol / L.
- Example 3 Fixation of diethylamino group (tertiary amino group and diethylamino group) to graft chain:
- the hollow fiber porous membrane introduced with the graft chain obtained in Production Example 1 (i) was swollen by immersing in methanol for 10 minutes or more, and then immersed in pure water for water substitution.
- diethylamine was added to 20 mL of purified water while stirring until the total volume became 40 mL to prepare a reaction solution.
- 25 parts by mass of the reaction liquid with respect to the dry weight of the hollow fiber porous membrane after introduction of the graft chain obtained in (i) above was placed in a glass reaction tube and adjusted to 30 ° C.
- a hollow fiber porous membrane into which a graft chain after immersion in pure water was introduced was inserted into the reaction solution, and allowed to stand for 24 hours to replace the epoxy group of the graft chain with a diethylamino group, a tertiary amino group, and ethyl
- a hollow fiber porous membrane in which the group was fixed via a graft chain was obtained.
- the obtained hollow fiber porous membrane had an outer diameter of 3.54 mm and an inner diameter of 2.17 mm. From the formula (2), 89% of all epoxy groups of the graft chain were substituted with diethylamino groups.
- a hollow fiber porous membrane module was produced according to Production Example 1 (iii) using a hollow fiber porous membrane having a diethylamino group.
- the volume of only the porous membrane excluding the hollow portion of the hollow fiber porous membrane in the hollow fiber porous membrane module was 0.56 mL.
- the inner surface area of the hollow fiber porous membrane was 6.3 cm 2 .
- the dynamic adsorption capacity of BSA was determined according to Evaluation Method Example 2.
- the sodium chloride concentration was 0 mol / L and 0.1 mol / L, 59 mg / mL and 24 mg, respectively. / ML.
- the antibody recovery rate was 91% when the sodium chloride concentration was 0 mol / L, and 96% when the sodium chloride concentration was 0.1 mol / L.
- the dimer ratio was 0.83% when the sodium chloride concentration was 0 mol / L, and 0.91% when the sodium chloride concentration was 0.1 mol / L.
- a hollow fiber porous membrane into which a graft chain after immersion in pure water was introduced was inserted into the reaction solution, and allowed to stand for 48 hours to replace the epoxy group of the graft chain with a dimethylamino group, a tertiary amino group, A hollow fiber porous membrane in which a methyl group was fixed via a graft chain was obtained.
- the obtained hollow fiber porous membrane had an outer diameter of 3.40 mm and an inner diameter of 2.07 mm. From the formula (2), 69% of all epoxy groups of the graft chain were substituted with dimethylamino groups.
- a hollow fiber porous membrane module was produced according to Production Example 1 (iii) using a hollow fiber porous membrane having a dimethylamino group.
- the volume of only the porous membrane excluding the hollow portion of the hollow fiber porous membrane in the hollow fiber porous membrane module was 0.53 mL.
- the inner surface area of the hollow fiber porous membrane was 6.0 cm 2 .
- the dynamic adsorption capacity of BSA was determined according to Evaluation Method Example 2. When the sodium chloride concentration was 0 mol / L and 0.1 mol / L, 22 mg / mL and It was 11 mg / mL, and the adsorption capacity was small.
- the antibody recovery rate was 93% when the sodium chloride concentration was 0 mol / L, and 97% when the sodium chloride concentration was 0.1 mol / L.
- the dimer ratio was 3.13% when the sodium chloride concentration was 0 mol / L and 3.65% when the sodium chloride concentration was 0.1 mol / L.
- a hollow fiber porous membrane module was produced according to Production Example 1 (iii) using a hollow fiber porous membrane having a dipropylamino group.
- the volume of the porous membrane alone excluding the hollow portion of the hollow fiber porous membrane in the hollow fiber porous membrane module was 0.54 mL.
- the inner surface area of the hollow fiber porous membrane was 6.1 cm 2 .
- the dynamic adsorption capacity of BSA was determined according to Evaluation Method Example 2. When the sodium chloride concentration was 0 mol / L and 0.1 mol / L, 16 mg / mL and 7 mg, respectively. / ML, and the adsorption capacity was small.
- the antibody recovery rate was 94% when the sodium chloride concentration was 0 mol / L, and 98% when the sodium chloride concentration was 0.1 mol / L.
- the dimer ratio was 3.21% when the sodium chloride concentration was 0 mol / L, and 3.54% when the sodium chloride concentration was 0.1 mol / L.
- Example 4 Fixation of triethylamino group (quaternary amino group and ethyl group) to graft chain:
- the hollow fiber porous membrane introduced with the graft chain obtained in Production Example 1 (i) was swollen by immersing in methanol for 10 minutes or more, and then immersed in pure water for water substitution. Further, 17 mL of 1 mol / L NaOH aqueous solution and 17 mL of methanol were mixed and stirred, and 4.81 g of triethylamine hydrochloride was added thereto while stirring to prepare a reaction solution.
- a hollow fiber porous membrane module was produced according to Production Example 1 (iii) using a hollow fiber porous membrane having a triethylamino group.
- the volume of only the porous membrane excluding the hollow portion of the hollow fiber porous membrane in the hollow fiber porous membrane module was 0.56 mL.
- the inner surface area of the hollow fiber porous membrane was 6.2 cm 2 .
- the dynamic adsorption capacity of BSA was determined according to Evaluation Method Example 2.
- the sodium chloride concentration was 0 mol / L and 0.1 mol / L, 67 mg / mL and 31 mg, respectively. / ML.
- the antibody recovery rate was 92% when the sodium chloride concentration was 0 mol / L, and 97% when the sodium chloride concentration was 0.1 mol / L.
- the dimer ratio was 0.71% when the sodium chloride concentration was 0 mol / L, and 0.78% when the sodium chloride concentration was 0.1 mol / L.
- Example 5 Fixation of tripropylamino group (quaternary amino group and propyl group) to graft chain:
- the hollow fiber porous membrane introduced with the graft chain obtained in Production Example 1 (i) was swollen by immersing in methanol for 10 minutes or more, and then immersed in pure water for water substitution. Further, 17 mL of methanol, 7.5 mL of purified water, and 0.84 g of sodium hydroxide were mixed and stirred, and 4.71 g of tripropylamine hydrochloride was added thereto while stirring to prepare a reaction solution.
- a hollow fiber porous membrane module was produced according to Production Example 1 (iii) using a hollow fiber porous membrane having a tripropylamino group.
- the volume of only the porous membrane excluding the hollow portion of the hollow fiber porous membrane in the hollow fiber porous membrane module was 0.53 mL.
- the inner surface area of the hollow fiber porous membrane was 6.0 cm 2 .
- the dynamic adsorption capacity of BSA was determined according to Evaluation Method Example 2.
- the sodium chloride concentration was 0 mol / L and 0.1 mol / L, 53 mg / mL and 27 mg, respectively. / ML.
- the antibody recovery rate was 89% when the sodium chloride concentration was 0 mol / L, and 96% when the sodium chloride concentration was 0.1 mol / L.
- the dimer ratio was 1.05% when the sodium chloride concentration was 0 mol / L, and 1.23% when the sodium chloride concentration was 0.1 mol / L.
- a hollow fiber porous membrane module was produced according to Production Example 1 (iii) using a hollow fiber porous membrane having a trimethylamino group.
- the volume of only the porous membrane excluding the hollow portion of the hollow fiber porous membrane in the hollow fiber porous membrane module was 0.56 mL.
- the inner surface area of the hollow fiber porous membrane was 6.2 cm 2 .
- the dynamic adsorption capacity of BSA was determined according to Evaluation Method Example 2.
- the sodium chloride concentration was 0 mol / L, it was 60 mg / mL. In the case of 0.1 mol / L, it was as low as 6 mg / mL.
- the antibody recovery rate was 88% when the sodium chloride concentration was 0 mol / L, and 98% when the sodium chloride concentration was 0.1 mol / L.
- the dimer ratio was 3.26% when the sodium chloride concentration was 0 mol / L, and 3.45% when the sodium chloride concentration was 0.1 mol / L.
- Example 7 Purification of antibody solution using porous membrane having isopropylamino group
- a supply liquid comprising impurities mainly composed of HCP prepared in Evaluation Method Example 3 and antibody protein was prepared.
- the hollow fiber porous membrane module was passed through. Furthermore, the permeate corresponding to 20 volumes with respect to the membrane volume contained in the hollow fiber porous membrane module was continuously collected as a fraction. Moreover, the recovery rate of the antibody protein with respect to the amount of permeate and the impurity concentration in each permeate fraction were measured. The results are shown in Table 2. As shown in Table 2, impurities in the permeated liquid of the hollow fiber porous membrane module were significantly reduced compared to the supply liquid.
- the antibody protein recovery rate was 90% or more.
- the ratio between the monomer of the antibody protein and the aggregate contained in each permeation fraction was evaluated by the method described in Evaluation Method Example 3 to determine the dimer ratio in the antibody protein. The results are also shown in Table 2. As shown in Table 2, the antibody protein dimer in the hollow fiber porous membrane module permeate was significantly reduced compared to the feed solution.
- Example 8 Purification of antibody solution using porous membrane having propylamino group
- a supply solution consisting of impurities mainly composed of HCP and antibody protein prepared in Evaluation Method Example 3 was prepared.
- the hollow fiber porous membrane module was passed through. Furthermore, the permeate corresponding to 20 volumes with respect to the membrane volume contained in the hollow fiber porous membrane module was continuously collected as a fraction. Moreover, the recovery rate of the antibody protein with respect to the amount of permeate and the impurity concentration in each permeate fraction were measured. The results are shown in Table 2. As shown in Table 2, the impurities in the permeated liquid of the hollow fiber porous membrane module were significantly reduced compared to the supply liquid.
- the antibody protein recovery rate was 90% or more.
- the ratio between the monomer of the antibody protein and the aggregate contained in each permeation fraction was evaluated by the method described in Evaluation Method Example 3 to determine the dimer ratio in the antibody protein. The results are also shown in Table 2. As shown in Table 2, the antibody protein dimer in the hollow fiber porous membrane module permeate was significantly reduced compared to the feed solution.
- Example 9 Purification of antibody solution using porous membrane having diethylamino group A feed solution composed of impurities mainly composed of HCP prepared in Evaluation Method Example 3 and antibody protein was prepared in Example 3. The liquid was passed through the hollow fiber porous membrane module. Furthermore, the permeate corresponding to 20 volumes with respect to the membrane volume contained in the hollow fiber porous membrane module was continuously collected as a fraction. Moreover, the recovery rate of the antibody protein with respect to the amount of permeate and the impurity concentration in each permeate fraction were measured. The results are shown in Table 2. As shown in Table 2, the impurities in the permeated liquid of the hollow fiber porous membrane module were significantly reduced compared to the supply liquid.
- the antibody protein recovery rate was 90% or more.
- the ratio between the monomer of the antibody protein and the aggregate contained in each permeation fraction was evaluated by the method described in Evaluation Method Example 3 to determine the dimer ratio in the antibody protein. The results are also shown in Table 2. As shown in Table 2, the antibody protein dimer in the hollow fiber porous membrane module permeate was significantly reduced compared to the feed solution.
- Example 10 Purification of antibody solution using porous membrane having triethylamino group A supply solution consisting of impurities mainly composed of HCP prepared in Evaluation Method Example 3 and antibody protein was prepared in Example 4.
- the hollow fiber porous membrane module was passed through. Furthermore, the permeate corresponding to 20 volumes with respect to the membrane volume contained in the hollow fiber porous membrane module was continuously collected as a fraction. Moreover, the recovery rate of the antibody protein with respect to the amount of permeate and the impurity concentration in each permeate fraction were measured. The results are shown in Table 2. As shown in Table 2, the impurities in the permeated liquid of the hollow fiber porous membrane module were significantly reduced compared to the supply liquid.
- the antibody protein recovery rate was 90% or more.
- the ratio between the monomer of the antibody protein and the aggregate contained in each permeation fraction was evaluated by the method described in Evaluation Method Example 3 to determine the dimer ratio in the antibody protein. The results are also shown in Table 2. As shown in Table 2, the antibody protein dimer in the permeate of the hollow fiber porous membrane module was significantly reduced as compared with the feed solution.
- Comparative Example 5 Purification of an antibody solution using a porous membrane having a trimethylamino group A comparative solution comprising an HCP-based impurity prepared in Evaluation Method Example 3 and an antibody protein was prepared in Comparative Example 4.
- the hollow fiber porous membrane module was passed through. Furthermore, the permeate corresponding to 20 volumes with respect to the membrane volume contained in the hollow fiber porous membrane module was continuously collected as a fraction. Moreover, the recovery rate of the antibody protein with respect to the amount of permeate and the impurity concentration in each permeate fraction were measured. The results are shown in Table 2.
- the antibody protein recovery rate is 90% or more, but the impurities in the permeate of the hollow fiber porous membrane module according to the comparative example are It was shown that it was reduced only about half compared to the feed solution. Further, the ratio between the antibody protein monomer and the aggregate contained in each permeation fraction was evaluated by the method described in Evaluation Method Example 3 to obtain the dimer ratio in the antibody protein. The results are also shown in Table 2. As shown in Table 2, it was shown that the degree of decrease of the antibody protein dimer in the permeate of the hollow fiber porous membrane module according to the comparative example was small.
- a hydrophobic porous substrate As described above, a hydrophobic porous substrate, a molecular chain having hydrophilicity made of a material different from the porous substrate, fixed to the surface including the pores of the porous substrate, and having 2 or 3 carbon atoms
- a porous film comprising a nitrogen atom, a hydroxyl group, and a side chain containing a carbonyl group to which 1 to 3 alkyl groups are bonded, and a method for producing the same are shown. It was shown that the purification of the antibody by adsorbing and removing impurities from the antibody solution at a high salt concentration was fast and simple.
- the method according to this embodiment can perform processing at higher speed and higher efficiency, and can be easily scaled up. Therefore, the present invention has industrial applicability that it is suitable for antibody purification when producing pharmaceuticals at an industrial level.
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Abstract
Description
また、側鎖に水酸基及びカルボニル基が含まれることにより、アミノ基によるアニオン交換性、アルキル基による疎水性に加えて、親水性、水素結合性、及び適度なカチオン交換性が相乗し、よりプロセスウィンドウの広いタンパク質精製が可能となることから好適である。
本実施の形態による典型的な抗体モノマーの精製方法の例としては、特に限定するものではないが、抗体モノマーと、夾雑物と、を含む混合液を本実施の形態に係る多孔膜に通液し、混合液中の夾雑物を多孔膜に吸着させることより夾雑物を混合液から除去し、透過液を抗体モノマーの精製液として回収する方法が、最も簡便であり速やかに精製を実施することができることから好ましい。
(i)中空糸状の多孔質基材へのグラフト鎖の導入
外径3.1mm、内径2.1mm、長さ100mm、空孔率70%、重量0.135g、後述するバブルポイント法で測定した最大細孔径が0.3μmのポリエチレン製で中空糸状の多孔質基材(旭化成ケミカルズ製、ポリエチレンB膜)を密閉容器に入れて、容器内の空気を窒素で置換した。その後、容器の外側からドライアイスで冷却しながら、γ線200kGyを照射し、ラジカルを発生させた。得られたラジカルを有するポリエチレン製で中空糸状の多孔基材をガラス反応管に入れて、200Pa以下に減圧することにより、反応管内の酸素を除いた。ここに40℃に調整したグリシジルメタクリレート(GMA)2.5体積部、及びメタノール97.5体積部からなる反応液を、中空糸状の多孔質基材の20質量部に注入した後、12時間密閉状態で静置してグラフト重合反応を施し、グラフト鎖が導入された中空糸多孔膜を得た。なお、GMA及びメタノールからなる反応液は予め窒素でバブリングして、反応液内の酸素を窒素置換した。
グラフト鎖を導入した後乾燥させた中空糸多孔膜をメタノールに10分以上浸漬して膨潤させた後、純水に浸漬して水置換した。次に、アミノ基と、アルキル基と、を有するアミン化合物として、イソプロピルアミン、ノルマルプロピルアミン、ブチルアミン、ジメチルアミン、ジエチルアミン、ジプロピルアミン、トリメチルアミン、トリエチルアミン、及びトリプロピルアミンを用意した。さらに、後述する実施例及び比較例に記載する、それぞれのアミン化合物に適した反応条件により、グラフト鎖が有するエポキシ基を開環反応によりアミン化合物に置換し、アミノ基と、アルキル基と、をグラフト鎖に固定した。置換率Tは、エポキシ基のモル数N0のうち、アミン化合物に置換されたモル数をN1として下記式(1)により表される。
T=N1/N0・・・(1)
N1=N0の場合はT=1となり、エポキシ基の100%がアミン化合物に置換された状態を示す。T=1の場合は、グラフト鎖にジオール基は存在しない。
1={(W2-W1)/(M1T+M0(1-T)}/{W1(dg/(dg+100))/M2}・・・(2)
式(2)中、M1はアミン化合物の分子量、M0は水の分子量で18、W1はグラフト重合反応後の中空糸多孔膜の重量、W2はアミノ基置換反応後の中空糸多孔膜の重量、dgはグラフト率、M2はGMAの分子量で142である。
(ii)で得られた、アミノ基と、アルキル基と、がグラフト鎖を介して固定された中空糸多孔膜1本の長手方向の両末端を、中空糸多孔膜の中空部を閉塞しないようにエポキシ系ポッティング剤で、内径0.5cm、有効長9.2cmのポリスルホン酸製モジュールケースに固定して、中空糸多孔膜モジュールを作製した。これを、以下の実施例等において、評価モジュールとして用いた。
中空糸多孔膜の最大細孔径は、バブルポイント法を用いて測定した。長さ8cmの中空糸多孔膜の一方の末端を閉塞し、他方の末端に圧力計を介して窒素ガス供給ラインを接続した。この状態で窒素ガスを供給してライン内部を窒素に置換した後、中空糸多孔膜をエタノールに浸漬した。この時、エタノールがライン内に逆流しないように極僅かに窒素で圧力をかけた状態で、中空糸多孔膜をエタノールに浸漬した。中空糸多孔膜を浸漬した状態で、窒素ガスの圧力をゆっくりと増加させ、中空糸多孔膜から窒素ガスの泡が安定して出始めた圧力Pを記録した。これより、最大細孔径をd、表面張力をγとして、下記式(3)に従って、中空糸多孔膜の最大細孔径を算出した。
d=C1γ/P・・・(3)
式(3)中、C1は定数である。エタノールを浸漬液としたときのC1γ=0.632(kg/cm)であり、上式にP(kg/cm2)を代入することにより、最大細孔径d(μm)を求めた。
0mol/L又は0.1mol/Lの塩化ナトリウムを含む、20mmol/LのTris-HCl(pH8.0)緩衝液に1g/Lの濃度でウシ血清アルブミン(BSA)(Sigma-Aldrich製)を溶解したBSA溶液を用い、破過が開始するまで(iii)で作成した中空糸多孔膜モジュールにBSA溶液を透過させた。なお、BSA溶液を通液する前に、あらかじめ20mmol/LのTris-HCl(pH8.0)緩衝液を20mL通液し、多孔膜を平衡化した。ここで、0mol/Lの塩化ナトリウムを含むBSA溶液の電気伝導度は1.3mS/cmであった。また、0.1mol/Lの塩化ナトリウムを含むBSA溶液の電気伝導度は10.2mS/cmであった。溶液は評価モジュール内の中空糸多孔膜の内側から外側に向かって、流速3mL/minにて通液した。評価はGEヘルスケアバイオサイエンス製AKTAexplorer100を用いて実施した。具体的には、同装置において得られる、透過液の280nmのUV吸光度が、供給液の280nmのUV吸光度(150mAU)の1/10(15mAU)となった時点を破過点とし、その時点までに供給したBSA溶液の体積から、動的吸着容量を算出した。ここで、BSA溶液の濃度Q、評価モジュールが破過した時までに透過させたBSA溶液の体積VB、及び評価モジュール内の実施例に係る中空糸多孔膜の体積VMから、下記式(4)に基づいて動的吸着容量Aは算出可能である。
A=Q×VB/VM ・・・(4)
中空糸多孔膜の体積とは、中空部分を除いた体積である。また破過とは、透過液中のBSA濃度が、供給されたBSA溶液の濃度の10%である0.1g/Lを超えた時点のことをいう。
(i)凝集体を含む抗体たんぱく溶液の調整
0mol/L又は0.1mol/Lの塩化ナトリウムを含む、20mmol/LのTris-HCl(pH8.0)緩衝液に、抗体タンパク質(株式会社ベネシス製、献血ヴェノグロブリン-IH)を添加し、抗体タンパク質1mg/mLが溶解した溶液を作成した。こうして得られた溶液を供給液とし、製造例の方法にて作成した各種リガンドを有する中空糸多孔膜モジュールに、中空糸多孔膜モジュールに含まれる膜体積に対して100体積に相当する供給液を通液し、透過後の抗体タンパク質の回収率と、抗体凝集体である2量体と、の比率を測定した。
中空糸多孔膜モジュール透過液中の抗体タンパク質の回収率は、アフィニティクロマトグラフィーによって評価した。島津製作所株式会社製高速液体クロマトグラフLC-20AシステムにアフィニティカラムとしてAppliedBiosystems製POROS G(ProteinGカラム)を取り付け、50mmol/Lリン酸に0.15mol/L NaClを含むバッファー(pH7.0)を用い、室温において流速2mL/minでカラムに通液し、ここにサンプルを100μL添加した。また、溶出には12mmol/L塩酸に0.15mol/L NaClを含むバッファーを用いた。(i)にて調整した濃度が1mg/mLの抗体タンパク質溶液をサンプルとして添加し、プロテインGカラムに吸着させ、さらにプロテインGカラムから溶出させた場合のピーク面積を、1mg/mLの抗体タンパク質濃度に対応させた。さらに、中空糸多孔膜モジュールの透過液も同様にして、カラムに通液して溶出ピーク面積を求め、1mg/mLの抗体タンパク質溶液より得られた溶出ピーク面積との比率から、中空糸多孔膜モジュールの透過液中の抗体タンパク質濃度を算出し、回収率を得た。
抗体タンパク質に含まれる、抗体モノマーと、凝集体と、の比率は、ゲルろ過クロマトグラフィーによって評価した。島津製作所株式会社製クロマトグラフLC-20Aシステムにゲルろ過カラムとして東ソー株式会社製TSKgel G3000SWXLを取り付け、0.1mol/Lのリン酸及び0.2mol/Lのアルギニン(pH6.8)を含むバッファーを用いて、25℃において流速0.8ml/minでカラムに通液し、ここに評価サンプルを20μL添加した。カラム透過後に抗体モノマーと、凝集体と、はそれぞれ分離した溶出ピークを示し、得られた抗体モノマーと、凝集体と、のピーク面積比から、溶液中でのそれぞれの存在比率を算出した。評価に用いた混合前の抗体タンパク質、及び(i)にて調整した溶液中の抗体タンパク質に含まれる2量体比率は、ともに4.06%であった。
(i)不純物を含む溶液の調整
インビトロジェン社製、CD OptiCHO(登録商標)無血清培地を用いて得られた細胞培養液を、最大孔径0.45μmの精密ろ過膜(旭化成メディカル社製、MF-SL)によりろ過、除濁し、細胞培養液の清澄液を得た。得られた清澄液をGEヘルスケアバイオサイエンス社製、AKTAclossflowを用いて、脱塩濃縮することにより、HCPを主成分とする細胞培養液の不純物濃縮溶液を得た。得られた不純物溶液のタンパク質濃度を、ウシ血清アルブミンを標準タンパク質としたBradford法(novexin社製、Bradford ULTRA kit)により測定したところ、3mg/mLであった。
中空糸多孔膜モジュール透過液中の抗体タンパク質の回収率と、HCPを主成分とする不純物タンパク質の濃度と、は、アフィニティクロマトグラフィーによって評価した。島津製作所株式会社製高速液体クロマトグラフLC-20AシステムにアフィニティカラムとしてAppliedBiosystems製POROS G(ProteinGカラム)を取り付け、50mmol/Lリン酸に0.15mol/L NaClを含むバッファー(pH7.0)を用い、室温において流速2mL/minでカラムに通液し、ここにサンプルを100μL添加した。また溶出には12mmol/L塩酸に0.15mol/L NaClを含むバッファーを用いた。その後、システムに、プロテインGカラムに吸着・溶出した抗体タンパク質の溶出ピークと、カラム吸着せず流出した不純物の溶出ピークと、を表示させた。さらに、抗体タンパク質の溶出ピーク面積と、不純物の溶出ピーク面積と、から、溶液中の抗体回収率と、不純物濃度と、を算出した。
中空糸多孔膜モジュール透過液として得られる抗体タンパク質溶液は、不純物を含むため、抗体タンパク質のみをアフィニティカラムクロマトグラフィーにて単離した後、評価方法例3(ii)に記載した方法により、抗体モノマーと、凝集体と、の比率を求めた。アフィニティカラムはGEヘルスケアバイオサイエンス製、HiTrap Protein G HP 1mlをGEヘルスケアバイオサイエンス製AKTAexplorer100に取り付け、20mmol/L リン酸バッファー(pH7.0)にて平衡化した後、カラムに中空糸多孔膜モジュール透過液2mlを通液して、抗体タンパク質のみをカラムに吸着させた。続いて、カラムを平衡化バッファー10mlで洗浄した後、50mmol/L クエン酸バッファー(pH3.3)を用いて、吸着した抗体タンパク質を溶出して、3mlの単離された抗体たんぱく溶液を回収し、ここに直ちに50mmol/L Tris-HCl(pH8.2)バッファー溶液17mlを加えて中和した。得られた抗体たんぱく単離液を評価方法例3(ii)の方法により評価し、抗体モノマーと、凝集体と、の比率を求めた。
グラフト鎖へのイソプロピルアミノ基(2級アミノ基とイソプロピル基)の固定:
製造例1(i)によって得られた、グラフト鎖を導入した中空糸多孔膜を、メタノールに10分以上浸漬して膨潤させた後、純水に浸漬して水置換した。また、精製水20mLにイソプロピルアミンを全体積が40mLとなるまで攪拌しながら添加し、反応液を作製した。次に、上記(i)で得られたグラフト鎖を導入した中空糸多孔膜の乾燥重量に対して25質量部の反応液をガラス反応管に入れ、40℃に調整した。その後、反応液に、純水浸漬後のグラフト鎖を導入した中空糸多孔膜を挿入し、48時間静置して、グラフト鎖のエポキシ基をイソプロピルアミノ基に置換し、2級アミノ基と、イソプロピル基と、がグラフト鎖を介して固定された中空糸多孔膜を得た。得られた中空糸多孔膜は外径3.43mm、内径2.10mmであり、式(2)より、グラフト鎖が有する全エポキシ基の72%がイソプロピルアミノ基によって置換されていた。
グラフト鎖へのブチルアミノ基(2級アミノ基とブチル基)の固定:
製造例1(i)によって得られた、グラフト鎖を導入した中空糸多孔膜を、メタノールに10分以上浸漬して膨潤させた後、純水に浸漬して水置換した。また、精製水92.7重量部にノルマルブチルアミン7.3重量部を添加し、攪拌溶解する事により、反応液を作製した。次に、上記(i)で得られたグラフト鎖を導入した中空糸多孔膜の乾燥重量に対して25質量部の反応液をガラス反応管に入れ、40℃に調整した。その後、反応液に、純水浸漬後のグラフト鎖を導入した中空糸多孔膜を挿入し、20時間静置して、グラフト鎖のエポキシ基をブチルアミノ基に置換し、2級アミノ基と、ブチル基と、がグラフト鎖を介して固定された中空糸多孔膜を得た。得られた中空糸多孔膜は外径3.49mm、内径2.16mmであり、式(2)より、グラフト鎖が有する全エポキシ基の79%がブチルアミノ基によって置換されていた。
グラフト鎖へのプロピルアミノ基(2級アミノ基とプロピル基)の固定:
製造例1(i)によって得られた、グラフト鎖を導入した中空糸多孔膜を、メタノールに10分以上浸漬して膨潤させた後、純水に浸漬して水置換した。また、精製水20mLにノルマルプロピルアミンを全体積が40mLとなるまで攪拌しながら添加し、反応液を作製した。次に、上記(i)で得られたグラフト鎖を導入した中空糸多孔膜の乾燥重量に対して25質量部の反応液をガラス反応管に入れ、40℃に調整した。その後、反応液に、純水浸漬後のグラフト鎖を導入した中空糸多孔膜を挿入し、24時間静置して、グラフト鎖のエポキシ基をプロピルアミノ基に置換し、2級アミノ基と、プロピル基と、がグラフト鎖を介して固定された中空糸多孔膜を得た。得られた中空糸多孔膜は外径3.44mm、内径2.10mmであり、式(2)より、グラフト鎖が有する全エポキシ基の78%がプロピルアミノ基によって置換されていた。
グラフト鎖へのジエチルアミノ基(3級アミノ基とジエチルアミノ基)の固定:
製造例1(i)によって得られた、グラフト鎖を導入した中空糸多孔膜を、メタノールに10分以上浸漬して膨潤させた後、純水に浸漬して水置換した。また、精製水20mLにジエチルアミンを全体積が40mLとなるまで攪拌しながら添加し、反応液を作製した。次に、上記(i)で得られたグラフト鎖導入後の中空糸多孔膜の乾燥重量に対して25質量部の反応液をガラス反応管に入れ、30℃に調整した。その後、反応液に、純水浸漬後のグラフト鎖を導入した中空糸多孔膜を挿入し、24時間静置して、グラフト鎖のエポキシ基をジエチルアミノ基に置換し、3級アミノ基と、エチル基と、がグラフト鎖を介して固定された中空糸多孔膜を得た。得られた中空糸多孔膜は外径3.54mm、内径2.17mmであり、式(2)より、グラフト鎖が有する全エポキシ基の89%がジエチルアミノ基によって置換されていた。
グラフト鎖へのジメチルアミノ基(3級アミノ基とメチル基)の固定:
製造例1(i)によって得られた、グラフト鎖を導入した中空糸多孔膜を、メタノールに10分以上浸漬して膨潤させた後、純水に浸漬して水置換した。また、精製水25mLにジメチルアミンを全体積が40mLとなるまで攪拌しながら添加し、反応液を作製した。反応液を作製した。次に、上記(i)で得られたグラフト鎖導入後の中空糸多孔膜の乾燥重量に対して25質量部の反応液をガラス反応管に入れ、30℃に調整した。その後、反応液に、純水浸漬後のグラフト鎖を導入した中空糸多孔膜を挿入し、48時間静置して、グラフト鎖のエポキシ基をジメチルアミノ基に置換し、3級アミノ基と、メチル基と、がグラフト鎖を介して固定された中空糸多孔膜を得た。得られた中空糸多孔膜は外径3.40mm、内径2.07mmであり、式(2)より、グラフト鎖が有する全エポキシ基の69%がジメチルアミノ基によって置換されていた。
グラフト鎖へのジプロピルアミノ基(3級アミノ基とプロピル基)の固定:
製造例1(i)によって得られた、グラフト鎖を導入した中空糸多孔膜を、メタノールに10分以上浸漬して膨潤させた後、純水に浸漬して水置換した。また、精製水5mLと、イソプロピルアルコール20mLと、を混合攪拌した後、ジプロピルアミンを全体積が40mLとなるまで攪拌しながら添加し、反応液を作製した。次に、上記(i)で得られたグラフト鎖導入後の中空糸多孔膜の乾燥重量に対して25質量部の反応液をガラス反応管に入れ、30℃に調整した。その後、反応液に、純水浸漬後のグラフト鎖を導入した中空糸多孔膜を挿入し、48時間静置して、グラフト鎖のエポキシ基をジプロピルアミノ基に置換し、3級アミノ基と、プロピル基と、がグラフト鎖を介して固定された中空糸多孔膜を得た。得られた中空糸多孔膜は外径3.46mm、内径2.11mmであり、式(2)より、グラフト鎖が有する全エポキシ基の68%がジプロピルアミノ基によって置換されていた。
グラフト鎖へのトリエチルアミノ基(4級アミノ基とエチル基)の固定:
製造例1(i)によって得られた、グラフト鎖を導入した中空糸多孔膜を、メタノールに10分以上浸漬して膨潤させた後、純水に浸漬して水置換した。また、1mol/LのNaOH水溶液17mLと、メタノール17mLと、を混合攪拌し、ここにトリエチルアミン塩酸塩4.81gを攪拌しながら添加し、反応液を作製した。次に、上記(i)で得られたグラフト鎖導入後の中空糸多孔膜の乾燥重量に対して25質量部の反応液をガラス反応管に入れ、40℃に調整した。その後、調整した反応液に、純水浸漬後のグラフト鎖を導入した中空糸多孔膜を挿入し、36時間静置して、グラフト鎖のエポキシ基をトリエチルアミノ基に置換し、4級アミノ基と、エチル基と、がグラフト鎖を介して固定された中空糸多孔膜を得た。得られた中空糸多孔膜は外径3.51mm、内径2.13mmであり、式(2)より、グラフト鎖が有する全エポキシ基の69%がトリエチルアミノ基によって置換されていた。
グラフト鎖へのトリプロピルアミノ基(4級アミノ基とプロピル基)の固定:
製造例1(i)によって得られた、グラフト鎖を導入した中空糸多孔膜を、メタノールに10分以上浸漬して膨潤させた後、純水に浸漬して水置換した。また、メタノール17mL、精製水7.5mL、及び水酸化ナトリウム0.84gを混合攪拌し、ここにトリプロピルアミン塩酸塩4.71gを攪拌しながら添加し、反応液を作製した。次に、上記(i)で得られたグラフト鎖導入後の中空糸多孔膜の乾燥重量に対して25質量部の反応液をガラス反応管に入れ、40℃に調整した。その後、調整した反応液に、純水浸漬後のグラフト鎖を導入した中空糸多孔膜を挿入し、46時間静置して、グラフト鎖のエポキシ基をトリプロピルアミノ基に置換し、4級アミノ基と、プロピル基と、がグラフト鎖を介して固定された中空糸多孔膜を得た。得られた中空糸多孔膜は外径3.43mm、内径2.09mmであり、式(2)より、グラフト鎖が有する全エポキシ基の63%がトリプロピルアミノ基によって置換されていた。
グラフト鎖へのトリメチルアミノ基(4級アミノ基とメチル基)の固定:
製造例1(i)によって得られた、グラフト鎖を導入した中空糸多孔膜を、メタノールに10分以上浸漬して膨潤させた後、純水に浸漬して水置換した。また、精製水12.5mLと、1mol/L NaOH水溶液12.5mLと、を混合攪拌し、ここに1.19gのトリメチルアミン塩酸塩を加えて、反応液を作製した。次に、上記(i)で得られたグラフト鎖導入後の中空糸多孔膜の乾燥重量に対して25質量部の反応液をガラス反応管に入れ、40℃に調整した。その後、調整した反応液に純水浸漬後のグラフト鎖を導入した中空糸多孔膜を挿入し、6時間静置して、グラフト鎖のエポキシ基をトリメチルアミノ基に置換し、4級アミノ基と、メチル基と、がグラフト鎖を介して固定された中空糸多孔膜を得た。得られた中空糸多孔膜は外径3.53mm、内径2.15mmであり、式(2)より、グラフト鎖が有する全エポキシ基の90%がトリメチルアミノ基によって置換されていた。
評価方法例3にて作成したHCPを主成分とする不純物と、抗体タンパク質と、からなる供給液を、実施例1にて作成した中空糸多孔膜モジュールに通液した。さらに、中空糸多孔膜モジュールに含まれる膜体積に対して20体積に相当する透過液を、フラクションとして連続的に採取した。また、透過液量に対する抗体タンパク質の回収率、及び各透過フラクション中の不純物濃度を測定した。結果を表2に示す。表2に示すように、中空糸多孔膜モジュールの透過液中の不純物は、供給液に比べ大幅に減少した。また、透過液の体積が膜体積の40倍を超えると、抗体タンパク質の回収率は90%以上となった。さらに、各透過フラクションに含まれる抗体タンパク質のモノマーと、凝集体と、の比率を評価方法例3に記載する方法により評価し、抗体タンパク質中の2量体比率を求めた。この結果も表2に示す。表2に示すように、中空糸多孔膜モジュール透過液中の抗体タンパク質の2量体は、供給液に比べ有意に減少していた。
評価方法例3にて作成したHCPを主成分とする不純物と、抗体タンパク質と、からなる供給液を、実施例2にて作成した中空糸多孔膜モジュールに通液した。さらに、中空糸多孔膜モジュールに含まれる膜体積に対して20体積に相当する透過液を、フラクションとして連続的に採取した。また、透過液量に対する抗体タンパク質の回収率、及び各透過フラクション中の不純物濃度を測定した。結果を表2に示す。表2に示すように、中空糸多孔膜モジュールの透過液中の不純物は、供給液に比べ大幅に減少していた。また、透過液の体積が膜体積の40倍を超えると、抗体タンパク質の回収率は90%以上となった。さらに、各透過フラクションに含まれる抗体タンパク質のモノマーと、凝集体と、の比率を評価方法例3に記載する方法により評価し、抗体タンパク質中の2量体比率を求めた。この結果も表2に示す。表2に示すように、中空糸多孔膜モジュール透過液中の抗体タンパク質の2量体は、供給液に比べ有意に減少していた。
評価方法例3にて作成したHCPを主成分とする不純物と、抗体タンパク質と、からなる供給液を、実施例3にて作成した中空糸多孔膜モジュールに通液した。さらに、中空糸多孔膜モジュールに含まれる膜体積に対して20体積に相当する透過液を、フラクションとして連続的に採取した。また、透過液量に対する抗体タンパク質の回収率、及び各透過フラクション中の不純物濃度を測定した。結果を表2に示す。表2に示すように、中空糸多孔膜モジュールの透過液中の不純物は、供給液に比べ大幅に減少していた。また、透過液の体積が膜体積の40倍を超えると、抗体タンパク質の回収率は90%以上となった。さらに、各透過フラクションに含まれる抗体タンパク質のモノマーと、凝集体と、の比率を評価方法例3に記載する方法により評価し、抗体タンパク質中の2量体比率を求めた。この結果も表2に示す。表2に示すように、中空糸多孔膜モジュール透過液中の抗体タンパク質の2量体は、供給液に比べ有意に減少していた。
評価方法例3にて作成したHCPを主成分とする不純物と、抗体タンパク質と、からなる供給液を、実施例4にて作成した中空糸多孔膜モジュールに通液した。さらに、中空糸多孔膜モジュールに含まれる膜体積に対して20体積に相当する透過液を、フラクションとして連続的に採取した。また、透過液量に対する抗体タンパク質の回収率、及び各透過フラクション中の不純物濃度を測定した。結果を表2に示す。表2に示すように、中空糸多孔膜モジュールの透過液中の不純物は、供給液に比べ大幅に減少していた。また、透過液の体積が膜体積の40倍を超えると、抗体タンパク質の回収率は90%以上となった。さらに、各透過フラクションに含まれる抗体タンパク質のモノマーと、凝集体と、の比率を評価方法例3に記載する方法により評価し、抗体タンパク質中の2量体比率を求めた。この結果も表2に示す。表2に示すように、中空糸多孔膜モジュール透過液中の抗体タンパク質の2量体は、供給液に比べ大幅に減少していた。
評価方法例3にて作成したHCPを主成分とする不純物と、抗体タンパク質と、からなる供給液を、比較例4にて作成した中空糸多孔膜モジュールに通液した。さらに、中空糸多孔膜モジュールに含まれる膜体積に対して20体積に相当する透過液を、フラクションとして連続的に採取した。また、透過液量に対する抗体タンパク質の回収率、及び各透過フラクション中の不純物濃度を測定した。結果を表2に示す。表2に示すように、透過液の体積が膜体積の40倍を超えると、抗体タンパク質の回収率は90%以上となるものの、比較例に係る中空糸多孔膜モジュールの透過液中の不純物は、供給液に比べ半分程度にしか減少していないことが示された。また、各透過フラクションに含まれる抗体タンパク質のモノマーと、凝集体と、の比率を評価方法例3に記載する方法により評価し、抗体タンパク質中の2量体比率を求めた。この結果も表2に示す。表2に示すように、比較例に係る中空糸多孔膜モジュールの透過液中の抗体タンパク質の2量体の減少程度は、少ないことが示された。
Claims (9)
- 疎水性多孔質基材と、前記多孔質基材の細孔表面に固定された、前記多孔質基材と異なる材質の親水性を有する分子鎖と、炭素原子数が2又は3のアルキル基が1乃至3個結合している窒素原子を含む前記分子鎖の側鎖と、備える多孔膜を用いて、2量体以上の抗体凝集体を含む抗体溶液を前記多孔膜に透過させ、前記抗体凝集体を前記多孔膜に吸着させる事により、透過液中に精製された抗体モノマーを回収する、抗体モノマーの精製方法。
- 前記抗体溶液が、宿主細胞由来タンパク質、デオキシリボ核酸、エンドトキシン、プロテアーゼ、及びウィルスより選択される少なくとも1種類の夾雑物をさらに含む、請求項1に記載の抗体モノマーの精製方法。
- 前記抗体溶液の塩濃度が0.3mol/L以下であり、
前記側鎖が、水酸基及びカルボニル基をさらに含む、
請求項1又は2に記載の抗体モノマー溶液の精製方法。 - 前記側鎖が、プロピルアミノ基、イソプロピルアミノ基、ジエチルアミノ基、トリエチルアミノ基、及びトリプロピルアミノ基より選択される、少なくとも1種類のアミノ基を含み、
前記抗体溶液が、不純物タンパク質、宿主細胞由来タンパク質、デオキシリボ核酸、エンドトキシン、プロテアーゼ、及びウィルスより選択される少なくとも1種類の不純物をさらに含み、
前記抗体モノマーの回収率が80%以上であり、
さらに、前記抗体溶液を前記多孔膜に透過させた後、40mS/cm以上の溶液を前記多孔膜に通液して前記多孔膜に吸着した不純物を溶出し、前記多孔膜を再利用可能にする、請求項1乃至3のいずれか一項に記載の抗体モノマーの精製方法。 - 前記多孔質基材が、ポリエチレン、ポリプロピレン、ポリスルホン、ポリエーテルスルホン、及びポリフッ化ビニリデンより選択される少なくとも一種からなる、請求項1乃至4のいずれか一項に記載の抗体モノマーの精製方法。
- 前記分子鎖の骨格が、グリシジルメタクリレート、グリシジルアクリレート、グリシジルソルベート、グリシジルイタコレート、及びグリシジルマレエートから選択される少なくとも1種類の単量体よりなる重合体である、請求項1乃至5のいずれか一項に記載の抗体モノマーの精製方法。
- 前記分子鎖の有する全側鎖の60%以上97%以下に、プロピルアミノ基、イソプロピルアミノ基、ジエチルアミノ基、トリエチルアミノ基、及びトリプロピルアミノ基より選択される窒素原子を含む官能基が1個結合している、請求項1乃至6のいずれか一項に記載の、抗体モノマーの精製方法。
- 疎水性多孔質基材と、
前記多孔質基材の細孔表面に固定された、前記多孔質基材と異なる材質の親水性を有する分子鎖と、
を備える多孔膜であって、
前記分子鎖の有する全側鎖の60%以上97%以下に、プロピルアミノ基、イソプロピルアミノ基、ジエチルアミノ基、トリエチルアミノ基、及びトリプロピルアミノ基より選択される官能基が1個結合し、
前記分子鎖の有する全側鎖の3%以上40%以下の側鎖はジオール基を有する、
多孔膜。 - 静電相互作用、疎水性相互作用、及び水素結合相互作用より選ばれる少なくとも一つの相互作用により、タンパク質を含む有機高分子を吸着する吸着体を用いた、抗体モノマーの精製方法であって、
前記吸着体の表面又は該吸着体の有する細孔の表面に、溶液中で負電荷を有する官能基が結合されており、
前記タンパク質を含む溶液を、前記吸着体に透過させることにより、前記タンパク質の凝集体を含む不純物を除去することを含み、
前記吸着体の透過液中の前記タンパク質の濃度は、透過前の前記タンパク質の濃度の1/2以上であり、前記透過液に含まれる前記タンパク質の回収率は70%以上である、タンパク質の精製方法。
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JPWO2011001963A1 (ja) | 2012-12-13 |
US20120123002A1 (en) | 2012-05-17 |
US9441011B2 (en) | 2016-09-13 |
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