CN114058596A - 腺相关病毒分离方法 - Google Patents
腺相关病毒分离方法 Download PDFInfo
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- CN114058596A CN114058596A CN202110695454.5A CN202110695454A CN114058596A CN 114058596 A CN114058596 A CN 114058596A CN 202110695454 A CN202110695454 A CN 202110695454A CN 114058596 A CN114058596 A CN 114058596A
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- aav
- capsids
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- 238000000034 method Methods 0.000 title claims abstract description 52
- 241000702421 Dependoparvovirus Species 0.000 title claims abstract description 9
- 210000000234 capsid Anatomy 0.000 claims abstract description 61
- 239000000872 buffer Substances 0.000 claims abstract description 41
- 239000006167 equilibration buffer Substances 0.000 claims abstract description 36
- 238000005571 anion exchange chromatography Methods 0.000 claims abstract description 14
- 239000000203 mixture Substances 0.000 claims abstract description 14
- 239000012530 fluid Substances 0.000 claims abstract description 11
- 239000012149 elution buffer Substances 0.000 claims abstract description 10
- 150000003839 salts Chemical class 0.000 claims description 30
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- 239000011780 sodium chloride Substances 0.000 abstract description 14
- 239000002245 particle Substances 0.000 description 52
- 235000002639 sodium chloride Nutrition 0.000 description 38
- 241001634120 Adeno-associated virus - 5 Species 0.000 description 31
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- 238000010828 elution Methods 0.000 description 11
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- 238000002835 absorbance Methods 0.000 description 7
- 238000005349 anion exchange Methods 0.000 description 7
- 238000004440 column chromatography Methods 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 6
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- SEQKRHFRPICQDD-UHFFFAOYSA-N N-tris(hydroxymethyl)methylglycine Chemical compound OCC(CO)(CO)[NH2+]CC([O-])=O SEQKRHFRPICQDD-UHFFFAOYSA-N 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
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- YIXJRHPUWRPCBB-UHFFFAOYSA-N magnesium nitrate Chemical compound [Mg+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O YIXJRHPUWRPCBB-UHFFFAOYSA-N 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
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- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 229940058020 2-amino-2-methyl-1-propanol Drugs 0.000 description 2
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- ACERFIHBIWMFOR-UHFFFAOYSA-N 2-hydroxy-3-[(1-hydroxy-2-methylpropan-2-yl)azaniumyl]propane-1-sulfonate Chemical compound OCC(C)(C)NCC(O)CS(O)(=O)=O ACERFIHBIWMFOR-UHFFFAOYSA-N 0.000 description 2
- LVQFQZZGTZFUNF-UHFFFAOYSA-N 2-hydroxy-3-[4-(2-hydroxy-3-sulfonatopropyl)piperazine-1,4-diium-1-yl]propane-1-sulfonate Chemical compound OS(=O)(=O)CC(O)CN1CCN(CC(O)CS(O)(=O)=O)CC1 LVQFQZZGTZFUNF-UHFFFAOYSA-N 0.000 description 2
- INEWUCPYEUEQTN-UHFFFAOYSA-N 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid Chemical compound OS(=O)(=O)CC(O)CNC1CCCCC1 INEWUCPYEUEQTN-UHFFFAOYSA-N 0.000 description 2
- XCBLFURAFHFFJF-UHFFFAOYSA-N 3-[bis(2-hydroxyethyl)azaniumyl]-2-hydroxypropane-1-sulfonate Chemical compound OCCN(CCO)CC(O)CS(O)(=O)=O XCBLFURAFHFFJF-UHFFFAOYSA-N 0.000 description 2
- XNPKNHHFCKSMRV-UHFFFAOYSA-N 4-(cyclohexylamino)butane-1-sulfonic acid Chemical compound OS(=O)(=O)CCCCNC1CCCCC1 XNPKNHHFCKSMRV-UHFFFAOYSA-N 0.000 description 2
- VTOWJTPBPWTSMK-UHFFFAOYSA-N 4-morpholin-4-ylbutane-1-sulfonic acid Chemical compound OS(=O)(=O)CCCCN1CCOCC1 VTOWJTPBPWTSMK-UHFFFAOYSA-N 0.000 description 2
- 241000702423 Adeno-associated virus - 2 Species 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
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- 239000005695 Ammonium acetate Substances 0.000 description 2
- UNMYWSMUMWPJLR-UHFFFAOYSA-L Calcium iodide Chemical compound [Ca+2].[I-].[I-] UNMYWSMUMWPJLR-UHFFFAOYSA-L 0.000 description 2
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- OWXMKDGYPWMGEB-UHFFFAOYSA-N HEPPS Chemical compound OCCN1CCN(CCCS(O)(=O)=O)CC1 OWXMKDGYPWMGEB-UHFFFAOYSA-N 0.000 description 2
- GIZQLVPDAOBAFN-UHFFFAOYSA-N HEPPSO Chemical compound OCCN1CCN(CC(O)CS(O)(=O)=O)CC1 GIZQLVPDAOBAFN-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- YNLCVAQJIKOXER-UHFFFAOYSA-N N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid Chemical compound OCC(CO)(CO)NCCCS(O)(=O)=O YNLCVAQJIKOXER-UHFFFAOYSA-N 0.000 description 2
- MKWKNSIESPFAQN-UHFFFAOYSA-N N-cyclohexyl-2-aminoethanesulfonic acid Chemical compound OS(=O)(=O)CCNC1CCCCC1 MKWKNSIESPFAQN-UHFFFAOYSA-N 0.000 description 2
- JOCBASBOOFNAJA-UHFFFAOYSA-N N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid Chemical compound OCC(CO)(CO)NCCS(O)(=O)=O JOCBASBOOFNAJA-UHFFFAOYSA-N 0.000 description 2
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 description 2
- CBTVGIZVANVGBH-UHFFFAOYSA-N aminomethyl propanol Chemical compound CC(C)(N)CO CBTVGIZVANVGBH-UHFFFAOYSA-N 0.000 description 2
- 229940043376 ammonium acetate Drugs 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
- IWOUKMZUPDVPGQ-UHFFFAOYSA-N barium nitrate Chemical compound [Ba+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O IWOUKMZUPDVPGQ-UHFFFAOYSA-N 0.000 description 2
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 description 2
- KQHXBDOEECKORE-UHFFFAOYSA-L beryllium sulfate Chemical compound [Be+2].[O-]S([O-])(=O)=O KQHXBDOEECKORE-UHFFFAOYSA-L 0.000 description 2
- NLSCHDZTHVNDCP-UHFFFAOYSA-N caesium nitrate Chemical compound [Cs+].[O-][N+]([O-])=O NLSCHDZTHVNDCP-UHFFFAOYSA-N 0.000 description 2
- 229910001640 calcium iodide Inorganic materials 0.000 description 2
- 229940046413 calcium iodide Drugs 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000009295 crossflow filtration Methods 0.000 description 2
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- HSZCZNFXUDYRKD-UHFFFAOYSA-M lithium iodide Chemical compound [Li+].[I-] HSZCZNFXUDYRKD-UHFFFAOYSA-M 0.000 description 2
- BLQJIBCZHWBKSL-UHFFFAOYSA-L magnesium iodide Chemical compound [Mg+2].[I-].[I-] BLQJIBCZHWBKSL-UHFFFAOYSA-L 0.000 description 2
- 229910001641 magnesium iodide Inorganic materials 0.000 description 2
- UYNRPXVNKVAGAN-UHFFFAOYSA-L magnesium;diiodate Chemical compound [Mg+2].[O-]I(=O)=O.[O-]I(=O)=O UYNRPXVNKVAGAN-UHFFFAOYSA-L 0.000 description 2
- RAFRTSDUWORDLA-UHFFFAOYSA-N phenyl 3-chloropropanoate Chemical compound ClCCC(=O)OC1=CC=CC=C1 RAFRTSDUWORDLA-UHFFFAOYSA-N 0.000 description 2
- 235000011056 potassium acetate Nutrition 0.000 description 2
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 2
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- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- JAAGVIUFBAHDMA-UHFFFAOYSA-M rubidium bromide Chemical compound [Br-].[Rb+] JAAGVIUFBAHDMA-UHFFFAOYSA-M 0.000 description 2
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- AHLATJUETSFVIM-UHFFFAOYSA-M rubidium fluoride Chemical compound [F-].[Rb+] AHLATJUETSFVIM-UHFFFAOYSA-M 0.000 description 2
- WFUBYPSJBBQSOU-UHFFFAOYSA-M rubidium iodide Chemical compound [Rb+].[I-] WFUBYPSJBBQSOU-UHFFFAOYSA-M 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- JHJLBTNAGRQEKS-UHFFFAOYSA-M sodium bromide Chemical compound [Na+].[Br-] JHJLBTNAGRQEKS-UHFFFAOYSA-M 0.000 description 2
- DHEQXMRUPNDRPG-UHFFFAOYSA-N strontium nitrate Chemical compound [Sr+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O DHEQXMRUPNDRPG-UHFFFAOYSA-N 0.000 description 2
- UBXAKNTVXQMEAG-UHFFFAOYSA-L strontium sulfate Chemical compound [Sr+2].[O-]S([O-])(=O)=O UBXAKNTVXQMEAG-UHFFFAOYSA-L 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 238000005199 ultracentrifugation Methods 0.000 description 2
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- 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 1
- WBPWDGRYHFQTRC-UHFFFAOYSA-N 2-ethoxycyclohexan-1-one Chemical compound CCOC1CCCCC1=O WBPWDGRYHFQTRC-UHFFFAOYSA-N 0.000 description 1
- BZSXEZOLBIJVQK-UHFFFAOYSA-N 2-methylsulfonylbenzoic acid Chemical compound CS(=O)(=O)C1=CC=CC=C1C(O)=O BZSXEZOLBIJVQK-UHFFFAOYSA-N 0.000 description 1
- LOJNFONOHINEFI-UHFFFAOYSA-N 4-[4-(2-hydroxyethyl)piperazin-1-yl]butane-1-sulfonic acid Chemical compound OCCN1CCN(CCCCS(O)(=O)=O)CC1 LOJNFONOHINEFI-UHFFFAOYSA-N 0.000 description 1
- XWNSFEAWWGGSKJ-UHFFFAOYSA-N 4-acetyl-4-methylheptanedinitrile Chemical compound N#CCCC(C)(C(=O)C)CCC#N XWNSFEAWWGGSKJ-UHFFFAOYSA-N 0.000 description 1
- KWSLGOVYXMQPPX-UHFFFAOYSA-N 5-[3-(trifluoromethyl)phenyl]-2h-tetrazole Chemical compound FC(F)(F)C1=CC=CC(C2=NNN=N2)=C1 KWSLGOVYXMQPPX-UHFFFAOYSA-N 0.000 description 1
- 239000013607 AAV vector Substances 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
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- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- 239000008000 CHES buffer Substances 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 239000004151 Calcium iodate Substances 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- PJWWRFATQTVXHA-UHFFFAOYSA-N Cyclohexylaminopropanesulfonic acid Chemical compound OS(=O)(=O)CCCNC1CCCCC1 PJWWRFATQTVXHA-UHFFFAOYSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
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- FSVCELGFZIQNCK-UHFFFAOYSA-N N,N-bis(2-hydroxyethyl)glycine Chemical compound OCCN(CCO)CC(O)=O FSVCELGFZIQNCK-UHFFFAOYSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
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- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
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- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- UZMAPBJVXOGOFT-UHFFFAOYSA-N Syringetin Natural products COC1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UZMAPBJVXOGOFT-UHFFFAOYSA-N 0.000 description 1
- 239000007997 Tricine buffer Substances 0.000 description 1
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- 238000010521 absorption reaction Methods 0.000 description 1
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- 229910052784 alkaline earth metal Inorganic materials 0.000 description 1
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- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
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- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013103 analytical ultracentrifugation Methods 0.000 description 1
- -1 and the like Chemical compound 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229910052788 barium Inorganic materials 0.000 description 1
- ITHZDDVSAWDQPZ-UHFFFAOYSA-L barium acetate Chemical compound [Ba+2].CC([O-])=O.CC([O-])=O ITHZDDVSAWDQPZ-UHFFFAOYSA-L 0.000 description 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 1
- NKQIMNKPSDEDMO-UHFFFAOYSA-L barium bromide Chemical compound [Br-].[Br-].[Ba+2] NKQIMNKPSDEDMO-UHFFFAOYSA-L 0.000 description 1
- 229910001620 barium bromide Inorganic materials 0.000 description 1
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 description 1
- 229910001626 barium chloride Inorganic materials 0.000 description 1
- SGUXGJPBTNFBAD-UHFFFAOYSA-L barium iodide Chemical compound [I-].[I-].[Ba+2] SGUXGJPBTNFBAD-UHFFFAOYSA-L 0.000 description 1
- 229910001638 barium iodide Inorganic materials 0.000 description 1
- 229940075444 barium iodide Drugs 0.000 description 1
- ONPIOWQPHWNPOQ-UHFFFAOYSA-L barium(2+);dioxido-oxo-sulfanylidene-$l^{6}-sulfane Chemical compound [Ba+2].[O-]S([O-])(=O)=S ONPIOWQPHWNPOQ-UHFFFAOYSA-L 0.000 description 1
- WAKZZMMCDILMEF-UHFFFAOYSA-H barium(2+);diphosphate Chemical compound [Ba+2].[Ba+2].[Ba+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O WAKZZMMCDILMEF-UHFFFAOYSA-H 0.000 description 1
- 229910052790 beryllium Inorganic materials 0.000 description 1
- ATBAMAFKBVZNFJ-UHFFFAOYSA-N beryllium atom Chemical compound [Be] ATBAMAFKBVZNFJ-UHFFFAOYSA-N 0.000 description 1
- 239000007998 bicine buffer Substances 0.000 description 1
- SXDBWCPKPHAZSM-UHFFFAOYSA-M bromate Inorganic materials [O-]Br(=O)=O SXDBWCPKPHAZSM-UHFFFAOYSA-M 0.000 description 1
- SXDBWCPKPHAZSM-UHFFFAOYSA-N bromic acid Chemical compound OBr(=O)=O SXDBWCPKPHAZSM-UHFFFAOYSA-N 0.000 description 1
- 229910052792 caesium Inorganic materials 0.000 description 1
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Abstract
提供了一种从完整腺相关病毒(AAV)衣壳和空AAV衣壳的混合物中富集完整AAV衣壳的方法，所述方法包括提供包含完整AAV衣壳和空AAV衣壳的混合物的样品，使所述样品经受包含洗脱缓冲液的阴离子交换色谱，所述洗脱缓冲液包含提供初始电导率的初始比例的平衡缓冲液和含盐缓冲液，和改变所述平衡缓冲液和所述含盐缓冲液的比例，以提供每个阶梯约0.5‑2.0mS/cm的阶梯梯度电导率增加，以洗脱空AAV衣壳并提供富含完整AAV衣壳的流体。
Description
背景技术
腺相关病毒(AAV)是具有单链DNA基因组的小病毒，其可用作基因治疗的载体。在AAV载体的制造过程中，产生缺乏所需DNA的不完整颗粒。在样品中空AAV颗粒的存在增加了基因治疗所需的剂量，并可能导致不希望的免疫反应。目前分离空的和完整的AAV颗粒的方法包括密度梯度超速离心和柱色谱。然而，此类方法难以以改善的分离度大规模操作。
因此，仍然需要空的和完整的AAV颗粒的改进的分离以提供一致的、更高质量的用于基因治疗的治疗剂。
发明内容
本发明提供了一种从完整腺相关病毒(AAV)衣壳和空AAV衣壳的混合物中富集完整AAV衣壳的方法。所述方法包括提供包含完整AAV衣壳和空AAV衣壳的混合物的样品，使所述样品经受包含洗脱缓冲液的阴离子交换色谱，所述洗脱缓冲液包含提供约0.5mS/cm至约10mS/cm的范围内的初始电导率的初始比例的平衡缓冲液和含盐缓冲液，和改变所述平衡缓冲液和所述含盐缓冲液的比例，以提供每个阶梯约0.5-2.0mS/cm的阶梯梯度电导率增加，以洗脱空AAV衣壳并提供富含完整AAV衣壳的流体。
所述方法提供了优于已知的色谱分离方法的优点，包括例如线性梯度，以产生具有一致富集比率的完整AAV衣壳的产物(流体)。
附图简要说明
图1是使用带正电荷的膜柱的1mS/cm阶梯梯度阴离子交换柱色谱方法分离空AAV5颗粒和完整AAV5颗粒的图。
图2是在对齐表格上方的分离的空AAV5颗粒和完整AAV5颗粒的色谱图，该对齐表格包含关于所生成的每个峰的基因组拷贝与衣壳的比率的数据，表明在后面的峰(峰3和4)中发现了完整的AAV5颗粒。
图3是使用另一种带正电荷的膜柱的1mS/cm阶梯梯度阴离子交换柱色谱方法分离空AAV5颗粒和完整AAV5颗粒的色谱图。
图4是使用带正电荷的整体柱(monolith column)的1mS/cm阶梯梯度阴离子交换柱色谱方法分离空AAV5颗粒和完整AAV5颗粒的色谱图。
图5显示了使用空AAV5标准品利用带正电荷的膜柱产生的峰。与260nm相比，空AAV5颗粒在280nm处具有更高的吸光度，同时以约10mS/cm和11mS/cm的电导率洗脱。
图6显示了使用完整AAV5标准品利用带正电荷的膜柱产生的峰。完整颗粒在280nm和260nm处具有相同的吸光强度，同时以约11-13mS/cm的电导率洗脱。
图7显示了空(图5)和完整(图6)AAV5标准品分离色谱图的叠加。
图8示出了使用带正电荷的膜柱的线性梯度阴离子交换柱色谱方法无法分离空AAV5颗粒和完整AAV5颗粒。
图9示出了使用带正电荷的整体柱的线性梯度阴离子交换柱色谱方法无法分离空AAV5颗粒和完整AAV5颗粒。
发明详述
本发明涉及用于从异质样品中分离空的和完整的腺相关病毒(AAV)衣壳的阴离子交换色谱方法。本发明至少部分基于以下发现：空AAV颗粒以比完整AAV颗粒更低的电导率洗脱。特别地，完整AAV颗粒比空AAV颗粒在260nm处具有更大的UV吸光度(图1)。通过比较280nm和260nm处的UV吸光度也可以观察到这种分离，使得空AAV颗粒产生更高的280/260信号比，而完整AAV颗粒产生接近1的280/260信号比。除了提供空的和完整的AAV颗粒的改进的分离以外，与已知方法(例如超速离心)相比，该方法还允许根据制造需求进行缩放。本发明的方法的其他优点包括例如快速处理、使用与血清型无关的样品以及使用各种类型的色谱介质和缓冲液组合物。
具体地，本发明提供了一种从完整腺相关病毒(AAV)衣壳和空AAV衣壳的混合物中富集完整AAV衣壳的方法，所述方法包括：
提供包含完整AAV衣壳和空AAV衣壳的混合物的样品，
使所述样品经受包含洗脱缓冲液的阴离子交换色谱，所述洗脱缓冲液包含提供约0.5mS/cm至约10mS/cm的范围内的初始电导率的初始比例的平衡缓冲液和含盐缓冲液，和
改变所述平衡缓冲液和所述含盐缓冲液的比例，以提供每个阶梯约0.5-2.0mS/cm的阶梯梯度电导率增加，以洗脱空AAV衣壳并提供富含完整AAV衣壳的流体。
所述方法适用于AAV衣壳的任何血清型、假型和/或变体。合适的血清型包括1、2、3、4、5、6、7、8、9及其组合。在某些实施方案中，样品包含AAV血清型2(AAV2)、AAV血清型5(AAV5)或其组合。也可以使用假型AAV颗粒，其中某种类型的基因组位于不同血清型的衣壳内。例如，样品可以包含AAV2/5，其包括位于血清型5的衣壳中的血清型2的基因组。
待分离的样品可以具有任何合适的浓度。例如，样品可含有至少约1x1011个衣壳/mL(例如，至少约1.5x1011个衣壳/mL，至少约1x1012个衣壳/mL，至少约1.5x1012个衣壳/mL，至少约1x1013个衣壳/mL)的色谱介质。优选地，起始病毒载量是至少约1x1012个衣壳/mL的色谱介质。
阴离子交换色谱可以使用任何合适的分离方法，包括离子交换膜(例如，带正电荷的膜)、离子交换树脂(例如，带正电荷的树脂)或整料(monolith)。在一些实施方案中，使样品经受阴离子交换色谱包括使样品和带正电荷的微孔膜接触。用于阴离子色谱的色谱介质可以是例如MUSTANGTM Q XT ACRODISCTM(Pall,Port Washington,NY)CIMacTM(BIASeparations,Slovenia)、POROS HQ(ThermoFisher,Waltham,MA)或POROS XQ(ThermoFisher,Waltham,MA)。
在某些实施方案中，分离方法的最后一步提供约20mS/cm的电导率。初始电导率是任何所需值，并且将取决于AAV颗粒的血清型和缓冲液组合物。通常初始电导率的范围为0.5mS/cm或更高至10mS/cm或更低(例如，约0.5mS/cm、约0.75mS/cm、约1mS/cm、约1.5mS/cm、约2mS/cm、约2.5mS/cm、约3mS/cm、约3.5mS/cm、约4mS/cm、约4.5mS/cm、约5mS/cm、约5.5mS/cm、约6mS/cm、约6.5mS/cm、约7mS/cm、约7.5mS/cm、约8mS/cm、约8.5mS/cm、约9mS/cm、约9.5mS/cm或约10mS/cm)。例如，初始电导率可以是约1mS/cm，并且电导率随着每个阶梯增加，直到达到约20mS/cm。通常，阶梯梯度包括电导率的增量变化，其范围通常为每个增量约0.5-2.0mS/cm。提供最终电导率增量的数量将根据电导率增量的大小而变化。优选地，每个增量各自是约1mS/cm。方法内的增量可以是相同的长度或不同的长度。在本文所述的任何实施方案中，梯度阶梯的长度允许紫外线(UV)强度在阶梯结束时在基线信号的25％以内(例如，20％以内，15％以内)。优选地，在阶梯结束时UV强度在基线信号的20％以内。
分离方法需要包含平衡缓冲液和含盐缓冲液的洗脱缓冲液的混合物。洗脱缓冲液(包含平衡缓冲液和含盐缓冲液)可以具有任何合适的pH。优选地，洗脱缓冲液的pH(包括平衡缓冲液的pH和含盐缓冲液的pH)是碱性的(例如，pH大于7)。在一些实施方案中，pH为7.5或更高、8或更高、8.5或更高、9或更高、9.5或更高、或10或更高。在一些优选的实施方案中，pH为约7-10、约7-9、约7.5-10.5、约8-10.5、约8-10、约8.5-9.5或约9。
平衡缓冲液是具有约15mS/cm或更低(例如，约12mS/cm或更低、约10mS/cm或更低、约8mS/cm或更低、约6mS/cm或更低、约5mS/cm或更低、约4mS/cm或更低、约3mS/cm或更低、约2mS/cm或更低、约1mS/cm)的电导率的任何合适的缓冲液。在一些实施方案中，平衡缓冲液具有约1mS/cm的电导率。
一般而言，平衡缓冲液将具有碱性pH(例如，至少7)的工作范围，如本文所述的。平衡缓冲液可以是例如双-三丙烷(BTP)、三(羟甲基)氨基甲烷(TRIS)、TRIS-Cl、TRIS-HCl、双-6TRIS丙烷、乙酸铵、2-[[1,3-二羟基-2-(羟甲基)丙烷-2-基]氨基]乙磺酸(TES)、2-[4-(2-羟乙基)哌嗪-1-基]乙磺酸(HEPES)、3-(N,N-双[2-羟乙基]氨基)-2-羟基丙磺酸(DIPSO)、4-(N-吗啉代)丁磺酸(MOBS)、4-(2-羟乙基)哌嗪-1-(2-羟基丙磺酸)水合物(HEPPSO)、哌嗪-N,N'-双(2-羟基丙磺酸)(POPSO)、4-(2-羟乙基)-1-哌嗪丙磺酸(EPPS)、N,N-双(2-羟乙基)甘氨酸(bicine)、N-[三(羟甲基)甲基]甘氨酸(tricine)、二甘氨酸(gly-gly)、N-(2-羟乙基)哌嗪-N'-(4-丁烷磺酸)(HEPBS)、3-{[1,3-二羟基-2-(羟甲基)丙烷-2-基]氨基}丙烷-1-磺酸(TAPS)、2-氨基-2-甲基-1,3-丙二醇(AMPD)、N-[三(羟甲基)甲基]-3-氨基丙磺酸(TAPS)、N-(1,1-二甲基-2-羟乙基)-3-氨基-2-羟基丙磺酸(AMPSO)、2-(环己氨基)乙磺酸(CHES)、3-(环己氨基)-2-羟基-1-丙磺酸(CAPSO)、3-(环己氨基)-1-丙磺酸(CAPS)、4-(环己氨基)-1-丁磺酸(CABS)或2-氨基-2-甲基-1-丙醇(AMP)。
含盐缓冲液(例如，含高盐的缓冲液)是任何合适的平衡缓冲液，例如本文所述的那些，但其中添加了无机盐。在优选实施方案中，含盐缓冲液与平衡缓冲液(例如，BTP)相同。在某些实施方案中，盐以提供约15mS/cm或更高(例如，约20mS/cm或更高、约30mS/cm或更高、约40mS/cm或更高、约50mS/cm或更高、约60mS/cm或更高、约65mS/cm或更高、约70mS/cm或更高、约75mS/cm或更高、约80mS/cm或更高，或约85mS/cm或更高)的电导率的量存在于缓冲液中。在一些优选实施方案中，含盐缓冲液的电导率为约50mS/cm或更高，优选约80mS/cm或更高。通常，缓冲液中盐的浓度为至少100mM(例如，至少200mM、至少300mM、至少500mM、至少600mM、至少800mM、至少900mM、至少1M、至少1.1M、至少1.2M、至少1.3M、至少1.5M、至少1.6M、至少1.8M、至少1.9M)。在一些实施方案中，缓冲液中盐的浓度为2M或更低(例如，1.9M或更低、1.8M或更低、1.6M或更低、1.5M或更低、1.3M或更低、1.2M或更低、1.1M或更低、1M或更低、900mM或更低、800mM或更低、600mM或更低、500mM或更低、300mM或更低或200mM或更低)。上述端点中的任意两个可用于定义封闭范围，或者单个端点可用于定义开放范围。例如，盐浓度可以是200mM至2M、500mM至1.5M、900mM至1.2M或约1M。
盐是任何无机盐，例如包含第I族金属(锂、钠、钾、铷或铯)、第II族金属(铍、镁、钙、锶或钡)、铵或铝的阳离子的任何盐。抗衡阴离子可以是卤化物、碳酸盐、碳酸氢盐、硫酸盐、硫代硫酸盐、磷酸盐、硝酸盐、亚硝酸盐、醋酸盐、溴酸盐、氯酸盐、碘酸盐等。盐的具体示例包括溴化锂、氯化锂、碘酸锂、碘化锂、氢氧化锂、硫酸锂、磷酸锂、溴化钠、氯化钠、乙酸钠、碳酸氢钠、硫酸氢钠、溴酸钠、氯酸钠、硫氢化钠、氢氧化钠、次磷酸钠、碘酸钠、碘化钠、乙酸钾、碳酸氢钾、溴酸钾、溴化钾、氯化钾、碳酸钾、氯酸钾、氢氧化钾、碘化钾、磷酸钾、硫代硫酸钾、溴化铷、氯化铷、氟化铷、碘化铷、硝酸铷、硫酸铷、溴化铯、氯化铯、碳酸铯、硝酸铯、硝酸铍、硫酸铍、醋酸镁、溴化镁、氯化镁、碘酸镁、碘化镁、硝酸镁、磷酸镁、硫酸镁、醋酸钙、溴化钙、氯化钙、碘化钙、碘酸钙、亚硝酸钙、硝酸钙、磷酸钙、硫酸钙、溴化锶、氯化锶、磷酸氢锶、碘化锶、硝酸锶、硫酸锶、乙酸钡、溴化钡、氯化钡、碘化钡、硝酸钡、磷酸钡、硫酸钡、硫代硫酸钡、乙酸铵、碳酸氢铵、溴化铵、氯化铵、硝酸铵、氯化铝、磷酸铝及其任意组合。在一些实施方案中，盐是第I族-卤化物或第I族-乙酸盐，例如氯化钠、乙酸钠、氯化钾或乙酸钾。
平衡缓冲液和含盐缓冲液的浓度以任何合适的浓度使用。在某些实施方案中，平衡缓冲液和含盐缓冲液各自以至少5mM(例如，至少10mM、至少15mM、至少20mM、至少25mM、至少30mM、至少35mM、至少40mM、至少45mM或至少50mM)的浓度使用。通常，平衡缓冲液和含盐缓冲液各自以100mM或更低(例如，90mM或更低、85mM或更低、80mM或更低、75mM或更低、70mM或更低、65mM或更低、60mM或更低、55mM或更低、50mM或更低、45mM或更低、40mM或更低、35mM或更低、30mM或更低、25mM或更低、或20mM或更低)的浓度使用。上述端点中的任意两个可用于定义封闭范围，或者单个端点可用于定义开放范围。平衡缓冲液和含盐缓冲液的浓度通常会有所不同，并且将基于所需的电导率水平而变化。在一些实施方案中，平衡缓冲液和含盐缓冲液的浓度将各自为10-100mM，优选20-50mM。
通过改变平衡缓冲液和含盐缓冲液的比例在分离方法期间递增改变电导率。因此，洗脱缓冲液中缓冲液的比例将根据所需的阶梯梯度增量(例如，约1mS/cm)而变化。缓冲液的混合物可以预先混合或在色谱系统内混合。平衡缓冲液与含盐缓冲液的初始比例是提供所需初始电导率(例如，约1mS/cm)的任何比例，例如在90:10至100:0内的初始比例。在一些实施方案中，平衡缓冲液与含盐缓冲液的初始比例为约98.3:1.7。平衡缓冲液与含盐缓冲液的最终比例是提供所需最终电导率(例如，约20mS/cm)的任何比例，例如在70:30至85:15内的最终比例。在一些实施方案中，平衡缓冲液与含盐缓冲液的最终比例为约81.5:18.5。在一个优选的实施方案中，平衡缓冲液与含盐缓冲液的初始比例为约98.3:1.7，并且平衡缓冲液与含盐缓冲液的最终比例为约81.5:18.5。
如本文所用，术语“约”通常是指值的±1％、值的±5％或值的±10％。
洗脱体积将随样品体积和柱尺寸而变化。梯度的每个阶梯可以产生不同的体积。当洗脱阶梯的体积不足(例如，少于2个色谱装置的体积)时，峰在洗脱阶梯中未完全洗脱，并且分离是达不到标准的。
随着分离方法的进行，色谱峰的分辨率可以通过280nm的紫外波长监测以监测蛋白质浓度(空的和完整的衣壳)和260nm的紫外波长监测以监测DNA浓度(完整衣壳)。在本文的任何实施方案中，收集来自每个电导率阶梯的流体并且进一步分析包含蛋白质和DNA的样品(如通过UV鉴别的)。分析通常通过酶联免疫吸附测定(ELISA)进行以计算样品中衣壳(即蛋白质)的总数和通过可以鉴定基因组拷贝数的聚合酶链反应(PCR)(例如，DROPLETDIGITALTM PCT(ddPCR)或定量PCR(qPCR))进行。ELISA与PCR的比率可以表明完整衣壳的数量，其随后可以通过其他分析方法(例如透射电子显微镜和/或分析性超速离心)进行确认。
分离方法提供了在较低电导率下洗脱空AAV衣壳以提供富含完整AAV衣壳的流体的解决方案。术语“富集/富含”是指与起始样品相比，在执行本发明的方法之后，流体包含相对于空AAV颗粒更多的完整AAV颗粒。在某些实施方案中，分离的样品(流体)富含相对于空AAV衣壳至少50％(例如，至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、或至少85％)的完整AAV衣壳。优选地，所述方法提供了在最终产品(流体)中相对于空AAV衣壳至少70％的完整AAV的富集。
本文所述的方法通常是较大生产过程的一部分。因此，生产过程的其他阶段可以包括例如AAV样品的产生(上游细胞培养)、AAV的初始过滤(澄清/切向流过滤(TFF))和/或AAV的纯化(例如，通过亲和色谱)。这样的步骤将在使用本发明的电导率梯度洗脱来分离空的和完整的AAV颗粒的阴离子交换色谱方法之前。本发明的分离方法还可以包括初始步骤，包括柱(例如膜)的平衡、含有AAV的样品的施加和/或柱(例如膜)的洗涤，然后是电导率阶梯梯度。
通过以下实施方案进一步举例说明本发明。
(实施方案1)一种从完整腺相关病毒(AAV)衣壳和空AAV衣壳的混合物中富集完整AAV衣壳的方法，所述方法包括提供包含完整AAV衣壳和空AAV衣壳的混合物的样品，使所述样品经受包含洗脱缓冲液的阴离子交换色谱，所述洗脱缓冲液包含提供约0.5mS/cm至约10mS/cm的范围内的初始电导率的初始比例的平衡缓冲液和含盐缓冲液，和改变所述平衡缓冲液和所述含盐缓冲液的比例，以提供每个阶梯约0.5-2.0mS/cm的阶梯梯度电导率增加，以洗脱空AAV衣壳并提供富含完整AAV衣壳的流体。
(实施方案2)实施方案(1)的方法，其中使所述样品经受阴离子交换色谱包括使带正电荷的微孔膜与所述样品接触。
(实施方案3)实施方案(1)或(2)的方法，其中最后的阶梯提供约20mS/cm的电导率。
(实施方案4)实施方案(1)-(3)中任一项的方法，其中平衡缓冲液与含盐缓冲液的初始比例为约98.3:1.7。
(实施方案5)实施方案(1)-(4)中任一项的方法，其中平衡缓冲液与含盐缓冲液的最终比例为约81.5:18.5。
以下实施例进一步举例说明本发明，但当然不应解释为以任何方式限制其范围。
实施例
实施例1
此实施例展示了使用电导率阶梯梯度实现空的和完整的AAV颗粒的分离的方案。步骤如表1所示。
表1
实施例2
本实施例描述了用于实现空的和完整的AAV颗粒的分离的AKTATM Avant方案。
获得包含空的和完整的病毒颗粒的亲和纯化的AAV样品溶液，并将其应用于自动快速蛋白质液相色谱(FPLC)系统(PALL MUSTANGTM Q ACRODISCTM(0.86mL CV))上的阴离子交换柱色谱。洗脱缓冲液包括：
低电导率(<5mS/cm)的平衡缓冲液，pH范围为8.5至9.5
更高电导率(>80mS/cm)的高盐缓冲液，pH范围为8.5至9.5
用于实现分离的方法如表2所示。
表2
空衣壳和完整衣壳的分离通过在280nm波长处的吸光度监测蛋白质含量和在260nm波长处的吸光度监测DNA含量。通过执行DROPLET DIGITALTM聚合酶链反应(ddPCR)(Bio-Rad,Hercules,CA)来测量洗脱的病毒颗粒中存在的基因组拷贝数来确认含有更高比例的完整AAV衣壳的最终洗脱液。衣壳酶联免疫吸附测定(ELISA)用于测量洗脱的病毒颗粒中存在的成形的AAV衣壳的总数。图2显示相对于较早的洗脱峰，在较高电导率阶梯处产生的洗脱峰包含更大量的基因组拷贝。
实施例3
本实施例描述了使用带膜的阴离子色谱分离空的和完整的AAV颗粒的方法。
使用装载有包含AAV5颗粒的样品的SARTOBINDTM Q(Sartorius,France)1mL膜柱，使用1mS/cm洗脱方法重复实施例2。结果示于图3。
实施例4
本实施例描述了使用带有整体柱的阴离子色谱分离空的和完整的AAV颗粒的方法。
使用装载有包含AAV5颗粒的样品的CIMacTM(BIA Separations,Slovenia)0.1mL整体柱，使用1mS/cm洗脱方法重复实施例2。结果示于图4。
实施例5
本实施例举例说明了空AAV5颗粒相对完整AAV5颗粒的不同色谱模式。
通过超速离心获得AAV5空的和完整的颗粒标准品，超速离心是分离空的和完整的AAV颗粒的常用工业方法。本发明的电导率阶梯梯度洗脱方法是在空的和完整的AAV5标准品上进行的。图5显示了使用空AAV5标准品生成的峰，其中与260nm相比，空AAV颗粒在280nm处具有更高的吸光度，同时以约10mS/cm和11mS/cm的电导率洗脱。图6显示了使用完整AAV5标准品生成的峰，其中完整颗粒在280nm和260nm处具有相同的吸光强度，同时以约11-13mS/cm的电导率洗脱。
图7显示了空的和完整的标准品分离色谱图的叠加。与完整的AAV5颗粒相比，空的AAV5颗粒以较低的电导率洗脱。这些色谱图表明，使用这种电导率阶梯方法可以实现AAV5空颗粒和完整颗粒的分离。
比较实施例1
本实施例描述了使用线性梯度阴离子交换柱色谱方法分离空的和完整的AAV颗粒的方法。
当使用(a)MUSTANGTM Q XT 0.86mL ACRODISCTM(Pall,Port Washington,NY)(图8)或(b)0.1mL Analytical CIMacTM整体柱(BIA Separations，Slovenia)(图9)时，所述方法使用线性盐梯度洗脱(0–200mM)用于空AAV5颗粒和完整AAV5颗粒的一般性AAV衣壳分离。如图8和9所示，常用的线性梯度洗脱未显示在PALL MUSTANGTM Q膜或CIMacTM整料(BIASeparations)上空的和完整的AAV5病毒载体的分离。
本文引用的所有参考文献，包括出版物、专利申请和专利，均通过引用以相同的程度并入本文，就好像每篇参考文献被单独且具体地指示通过引用并入并在本文中以其整体示出一样。
在描述本发明的上下文中(尤其是在以下权利要求的上下文中)，术语“一个”和“一种”以及“该”和“至少一个/种”以及类似指代词的使用被解释为涵盖单数和复数，除非本文另有说明或与上下文明显矛盾。术语“至少一个/种”后跟一个或多个项目的列表(例如，“至少一个A和B”)的使用应解释为表示从所列项目(A或B)中选择的一个项目或两个或更多个所列项目(A和B)的任何组合，除非本文另有说明或与上下文明显矛盾。除非另有说明，否则术语“包括”、“具有”、“包含”和“含有”应被解释为开放式术语(即，意为“包括但不限于”)。除非本文另有说明，否则本文对数值范围的引用仅旨在作为单独提及落入该范围内的每个单独值的简略表达方法，并且将每个单独值并入说明书中，就好像其在本文中单独叙述一样。除非本文另有说明或与上下文明显矛盾，否则本文所述的所有方法都可以任何合适的顺序进行。除非另有声明，否则本文提供的任何和所有实例或示例性语言(例如，“诸如/例如”)的使用仅旨在更好地阐明本发明而不对本发明的范围构成限制。说明书中的任何语言都不应被解释为表明任何未要求保护的元素对于本发明的实践是必不可少的。
本文描述了本发明的优选实施方案，包括发明人已知的用于实施本发明的最佳模式。在阅读上述描述后，那些优选实施方案的变化对于本领域的普通技术人员来说会变得明显。本发明人期望技术人员适当地采用这种变化，并且本发明人预期了以不同于本文具体描述的方式来实践本发明。因此，本发明包括在适用法律允许的情况下所附权利要求中记载的主题的所有修改和等效物。此外，除非本文另有说明或与上下文明显矛盾，否则本发明涵盖所有可能变化形式的上述元素的任何组合。
Claims (5)
1.一种从完整腺相关病毒(AAV)衣壳和空AAV衣壳的混合物中富集完整AAV衣壳的方法，所述方法包括：
提供包含完整AAV衣壳和空AAV衣壳的混合物的样品，
使所述样品经受包含洗脱缓冲液的阴离子交换色谱，所述洗脱缓冲液包含提供约0.5mS/cm至约10mS/cm的范围内的初始电导率的初始比例的平衡缓冲液和含盐缓冲液，和
改变所述平衡缓冲液和所述含盐缓冲液的比例，以提供每个阶梯约0.5-2.0mS/cm的阶梯梯度电导率增加，以洗脱空AAV衣壳并提供富含完整AAV衣壳的流体。
2.权利要求1的方法，其中使所述样品经受阴离子交换色谱包括使带正电荷的微孔膜与所述样品接触。
3.权利要求1或2的方法，其中最后的阶梯提供约20mS/cm的电导率。
4.权利要求1-3中任一项的方法，其中平衡缓冲液与含盐缓冲液的初始比例为约98.3:1.7。
5.权利要求1-4中任一项的方法，其中平衡缓冲液与含盐缓冲液的最终比例为约81.5:18.5。
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| CN107646052A (zh) * | 2015-01-20 | 2018-01-30 | 建新公司 | 用于表征重组病毒颗粒的分析性超速离心法 |
| EP3054007A1 (en) * | 2015-02-09 | 2016-08-10 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Recombinant adeno-associated virus particle purification comprising an immuno-affinity purification step |
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| OTTO-WILHELM MERTEN等: "Manufacturing of viral vectors: part II. Downstream processing and safety aspects", PHARMACEUTICAL BIOPROCESSING, vol. 2, no. 3, pages 237 - 251, XP055615575, DOI: 10.4155/pbp.14.15 * |
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