CN105579463A - 混合模式配体 - Google Patents
混合模式配体 Download PDFInfo
- Publication number
- CN105579463A CN105579463A CN201580000860.8A CN201580000860A CN105579463A CN 105579463 A CN105579463 A CN 105579463A CN 201580000860 A CN201580000860 A CN 201580000860A CN 105579463 A CN105579463 A CN 105579463A
- Authority
- CN
- China
- Prior art keywords
- ligand
- solid support
- microns
- mixed mode
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000003446 ligand Substances 0.000 title claims abstract description 62
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 42
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 42
- 239000007787 solid Substances 0.000 claims abstract description 38
- 239000011148 porous material Substances 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 22
- 239000002245 particle Substances 0.000 claims abstract description 21
- 150000003839 salts Chemical class 0.000 claims description 17
- 239000012501 chromatography medium Substances 0.000 claims description 11
- 125000006297 carbonyl amino group Chemical group [H]N([*:2])C([*:1])=O 0.000 claims description 7
- 238000012434 mixed-mode chromatography Methods 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 6
- 239000011159 matrix material Substances 0.000 claims description 6
- 229910001508 alkali metal halide Inorganic materials 0.000 claims description 5
- 150000001340 alkali metals Chemical class 0.000 claims description 5
- 229910001615 alkaline earth metal halide Inorganic materials 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 239000007790 solid phase Substances 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 2
- 239000011347 resin Substances 0.000 description 17
- 229920005989 resin Polymers 0.000 description 17
- 239000000243 solution Substances 0.000 description 14
- 150000001412 amines Chemical class 0.000 description 11
- 150000002009 diols Chemical class 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 8
- 239000000872 buffer Substances 0.000 description 7
- 239000000178 monomer Substances 0.000 description 7
- -1 p-toluenesulfonate Acyl chloride Chemical class 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 5
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- 108060003951 Immunoglobulin Proteins 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 102000018358 immunoglobulin Human genes 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 150000001299 aldehydes Chemical class 0.000 description 3
- 239000000356 contaminant Substances 0.000 description 3
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- 238000005859 coupling reaction Methods 0.000 description 3
- 239000003431 cross linking reagent Substances 0.000 description 3
- 229940072221 immunoglobulins Drugs 0.000 description 3
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- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 3
- 125000006850 spacer group Chemical group 0.000 description 3
- XEPXTKKIWBPAEG-UHFFFAOYSA-N 1,1-dichloropropan-1-ol Chemical compound CCC(O)(Cl)Cl XEPXTKKIWBPAEG-UHFFFAOYSA-N 0.000 description 2
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 2
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
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- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
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- 102000040945 Transcription factor Human genes 0.000 description 2
- XTXRWKRVRITETP-UHFFFAOYSA-N Vinyl acetate Chemical compound CC(=O)OC=C XTXRWKRVRITETP-UHFFFAOYSA-N 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
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- 239000003114 blood coagulation factor Substances 0.000 description 2
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- 239000003054 catalyst Substances 0.000 description 2
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- 238000009792 diffusion process Methods 0.000 description 2
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- 238000002474 experimental method Methods 0.000 description 2
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- 239000001257 hydrogen Substances 0.000 description 2
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- 230000000149 penetrating effect Effects 0.000 description 2
- UVBBCQLPTZEDHT-UHFFFAOYSA-N pent-4-en-1-amine Chemical compound NCCCC=C UVBBCQLPTZEDHT-UHFFFAOYSA-N 0.000 description 2
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- CXCHEKCRJQRVNG-UHFFFAOYSA-N 2,2,2-trifluoroethanesulfonyl chloride Chemical compound FC(F)(F)CS(Cl)(=O)=O CXCHEKCRJQRVNG-UHFFFAOYSA-N 0.000 description 1
- STMDPCBYJCIZOD-UHFFFAOYSA-N 2-(2,4-dinitroanilino)-4-methylpentanoic acid Chemical compound CC(C)CC(C(O)=O)NC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O STMDPCBYJCIZOD-UHFFFAOYSA-N 0.000 description 1
- CTPIHHXCACYCIV-UHFFFAOYSA-N 2-(4-aminophenyl)sulfanylacetic acid Chemical compound NC1=CC=C(SCC(O)=O)C=C1 CTPIHHXCACYCIV-UHFFFAOYSA-N 0.000 description 1
- DEFPNMKDESPGBA-UHFFFAOYSA-N 2-Aminohippuric acid Chemical compound NC1=CC=CC=C1C(=O)NCC(O)=O DEFPNMKDESPGBA-UHFFFAOYSA-N 0.000 description 1
- HPILSDOMLLYBQF-UHFFFAOYSA-N 2-[1-(oxiran-2-ylmethoxy)butoxymethyl]oxirane Chemical compound C1OC1COC(CCC)OCC1CO1 HPILSDOMLLYBQF-UHFFFAOYSA-N 0.000 description 1
- HUPFFHLRZXIFMI-UHFFFAOYSA-N 2-amino-2-phenylethanethioic s-acid Chemical compound OC(=S)C(N)C1=CC=CC=C1 HUPFFHLRZXIFMI-UHFFFAOYSA-N 0.000 description 1
- AMGZELLFFNISNA-UHFFFAOYSA-N 2-fluoro-1-methyl-2h-pyridine;4-methylbenzenesulfonic acid Chemical compound CN1C=CC=CC1F.CC1=CC=C(S(O)(=O)=O)C=C1 AMGZELLFFNISNA-UHFFFAOYSA-N 0.000 description 1
- KUDUQBURMYMBIJ-UHFFFAOYSA-N 2-prop-2-enoyloxyethyl prop-2-enoate Chemical compound C=CC(=O)OCCOC(=O)C=C KUDUQBURMYMBIJ-UHFFFAOYSA-N 0.000 description 1
- PAKCOSURAUIXFG-UHFFFAOYSA-N 3-prop-2-enoxypropane-1,2-diol Chemical compound OCC(O)COCC=C PAKCOSURAUIXFG-UHFFFAOYSA-N 0.000 description 1
- RBHLFWNKEWLHBP-UHFFFAOYSA-N 4-(4-aminophenyl)butanoic acid Chemical compound NC1=CC=C(CCCC(O)=O)C=C1 RBHLFWNKEWLHBP-UHFFFAOYSA-N 0.000 description 1
- RTNUTCOTGVKVBR-UHFFFAOYSA-N 4-chlorotriazine Chemical class ClC1=CC=NN=N1 RTNUTCOTGVKVBR-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- CIXADNKCRCBWMX-UHFFFAOYSA-N CN(c1ccc(CCCC(O)=O)cc1)O Chemical compound CN(c1ccc(CCCC(O)=O)cc1)O CIXADNKCRCBWMX-UHFFFAOYSA-N 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 description 1
- 108010054218 Factor VIII Proteins 0.000 description 1
- 102000001690 Factor VIII Human genes 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 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
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- UQDYTUWNBVAHEE-UHFFFAOYSA-N Nc(cccc1)c1SCC(O)=O Chemical compound Nc(cccc1)c1SCC(O)=O UQDYTUWNBVAHEE-UHFFFAOYSA-N 0.000 description 1
- CSEWAUGPAQPMDC-UHFFFAOYSA-N Nc1ccc(CC(O)=O)cc1 Chemical compound Nc1ccc(CC(O)=O)cc1 CSEWAUGPAQPMDC-UHFFFAOYSA-N 0.000 description 1
- UJEKGISTVUPGCZ-UHFFFAOYSA-N OC(CS(c(cc1)ccc1NI)(=O)=O)=O Chemical compound OC(CS(c(cc1)ccc1NI)(=O)=O)=O UJEKGISTVUPGCZ-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 125000001931 aliphatic group Chemical class 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
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- 125000003118 aryl group Chemical group 0.000 description 1
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- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 1
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- 125000001165 hydrophobic group Chemical group 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
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- 230000014759 maintenance of location Effects 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- ZMLXKXHICXTSDM-UHFFFAOYSA-N n-[1,2-dihydroxy-2-(prop-2-enoylamino)ethyl]prop-2-enamide Chemical compound C=CC(=O)NC(O)C(O)NC(=O)C=C ZMLXKXHICXTSDM-UHFFFAOYSA-N 0.000 description 1
- HFGVZFZKVOBHAQ-UHFFFAOYSA-N n-[2-(2-aminoethyldisulfanyl)ethyl]-n-prop-2-enoylprop-2-enamide Chemical compound NCCSSCCN(C(=O)C=C)C(=O)C=C HFGVZFZKVOBHAQ-UHFFFAOYSA-N 0.000 description 1
- RCHKEJKUUXXBSM-UHFFFAOYSA-N n-benzyl-2-(3-formylindol-1-yl)acetamide Chemical compound C12=CC=CC=C2C(C=O)=CN1CC(=O)NCC1=CC=CC=C1 RCHKEJKUUXXBSM-UHFFFAOYSA-N 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical class OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
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- 239000010452 phosphate Substances 0.000 description 1
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- NXGWSWBWEQYMND-UHFFFAOYSA-N piperazine;prop-2-enamide Chemical compound NC(=O)C=C.NC(=O)C=C.C1CNCCN1 NXGWSWBWEQYMND-UHFFFAOYSA-N 0.000 description 1
- 239000000419 plant extract Substances 0.000 description 1
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- 229910052700 potassium Inorganic materials 0.000 description 1
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- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 150000004053 quinones Chemical class 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
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- 230000002829 reductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 150000003457 sulfones Chemical class 0.000 description 1
- YBBRCQOCSYXUOC-UHFFFAOYSA-N sulfuryl dichloride Chemical class ClS(Cl)(=O)=O YBBRCQOCSYXUOC-UHFFFAOYSA-N 0.000 description 1
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- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- PLCFYBDYBCOLSP-UHFFFAOYSA-N tris(prop-2-enyl) 2-hydroxypropane-1,2,3-tricarboxylate Chemical compound C=CCOC(=O)CC(O)(CC(=O)OCC=C)C(=O)OCC=C PLCFYBDYBCOLSP-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- 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
- B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
- B01J39/08—Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
- B01J39/16—Organic material
- B01J39/18—Macromolecular compounds
- B01J39/20—Macromolecular compounds obtained by reactions only involving unsaturated carbon-to-carbon bonds
-
- 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
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28004—Sorbent size or size distribution, e.g. particle size
-
- 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
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
- B01J20/28078—Pore diameter
- B01J20/28085—Pore diameter being more than 50 nm, i.e. macropores
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- 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
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to 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
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
- B01J20/3206—Organic carriers, supports or substrates
- B01J20/3208—Polymeric carriers, supports or substrates
- B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
-
- 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
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
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Abstract
本发明提供混合模式配体，及其纯化蛋白质的用途。一些实施方式中提供的是从源溶液中纯化目标蛋白质的方法。一些实施方式中，所述方法包括接触连接了配体的固相支持物。一些实施方式中，固相支持物有孔，中值孔径0.5微米或以上，可选的是，基本没有孔径0.1微米或以下的孔。一些实施方式中，固相支持物由中值粒径约25至约150微米的颗粒构成。
Description
相关专利申请的交叉引用
本申请要求基于2014年3月24日提交的第61/953,365号美国临时申请的优先权，并通过引用纳入其全部内容用于所有目的。
发明背景
在为诊断、治疗、实验室研究等各种用途获取足够浓或足够纯蛋白质的过程中，从源液—主要是哺乳动物体液或细胞培养收获物—提取免疫球蛋白及其他蛋白质非常重要。纯化天然或重组蛋白质尤其免疫球蛋白经常遭遇的难题有：得率低、所用分离介质昂贵、分离介质浸入产品，以及提取过程所用外来物质处置的安全问题。
发明概述
一些实施方式中，提供的是一种从源溶液中纯化目标蛋白质的方法。一些实施方式中，该方法包括：接触连接了配体的固相支持物，由此使目标蛋白质与配体结合，所述配体是：
其中，n是0、1或2；m是1、2、3或4；X选自S、C(O)-NH、NH-CO、C(O)-NH-CH2-C(O)-NH或SO2，且所述胺可位于苄基环上对位、邻位或间位；或是：
其中，y是1、2、3、4或5，且所述胺可位于苄基环上对位、邻位或间位；
洗脱目标蛋白，由此纯化目标蛋白。
一些实施方式中，结构式I所示配体选自：
一些实施方式中，结构式II所示配体选自：
一些实施方式中，固相支持物有孔，中值孔径0.5微米或以上，可选的是，基本没有孔径0.1微米或以下的孔。一些实施方式中，固相支持物由中值粒径约25至约150微米的颗粒构成。
一些实施方式中，在pH约4.0至约6.0接触，在pH约6.1至约8.5洗脱。
一些实施方式中，固相支持物是膜。一些实施方式中，固相支持物是整料。
一些实施方式中，源溶液含有选自碱金属及碱土金属卤化物的盐，盐的浓度约50mM至约300mM。一些实施方式中，源溶液含有选自碱金属及碱土金属卤化物的盐，盐的浓度约100mM至约150mM。
本发明还提供一种混合模式色谱介质，具有偶联于固相支持物的配体，所述配体选自结构式I或结构式II。一些实施方式中，所述介质与源溶液在一定的条件下接触，所述条件下蛋白质目标物由源溶液中结合到配体上。
一些实施方式中，结构式I所示配体选自：
一些实施方式中，结构式II所示配体选自：
一些实施方式中，固相支持物有孔，中值孔径约0.5微米或以上且基本没有孔径0.1微米或以下的孔。一些实施方式中，固相支持物由中值粒径约25至约150微米的颗粒构成。
一些实施方式中，固相支持物是膜。一些实施方式中，固相支持物是整料。
附图说明
图1-12显示线性梯度上结合蛋白质从固相支持物洗脱的对应氯化钠浓度。横轴显示测试蛋白质。左上角由上至下排列的配体对应于各蛋白质由左及右的数据。
本发明详细说明
I.引言
许多具有疏水性和阴离子基团(阳离子交换)的混合模式配体被发现具有新的蛋白质结合特异性和结合强度。据此，提供连接本文所述混合模式配体的色谱固相支持物，以及用这些混合模式配体纯化蛋白质的方法。
II.配体
用于蛋白质纯化的配体可选自结构式I和II：
一些实施方式中，n是0、1或2；m是1、2、3或4；X选自S、C(O)-NH、NH-C(O)、C(O)-NH-CH2-C(O)-NH或SO2且所述胺可位于苄基环的对位、邻位或间位。一些实施方式中，n是0、1或2；m是1、2、3或4；X选自S、C(O)-NH、NH-C(O)、C(O)-NH-CH2-C(O)-NH或SO2且所述胺可位于苄基环邻位或间位。一些实施方式中，n是0、1或2；m是1、2、3或4；X选自S、NH-C(O)、C(O)-NH-CH2-C(O)-NH或SO2，且所述胺可位于苄基环上对位、邻位或间位。一些实施方式中，n是0、1或2；m是2、3或4；X选自S、C(O)-NH、NH-C(O)、C(O)-NH-CH2-C(O)-NH或SO2，且所述胺可位于苄基环上对位、邻位或间位。一些实施方式中，n是1或2；m是1、2、3或4；X选自S、C(O)-NH、NH-C(O)、C(O)-NH-CH2-C(O)-NH或SO2，且所述胺可位于苄基环上对位、邻位或间位。
一些实施方式中，y是1、2、3、4或5。结构式I中，胺可位于苄基环上对位、邻位或间位。
本文所有结构式中，胺上的波浪线表示配体于此连接到固相基质(本文亦称固相支持物)，其间或有或没有中间连接基团。
一些实施方式中，结构式I所示配体选自以下所示：
一些实施方式中，结构式II所示配体选自以下所示：
可用各种固相支持物连接配体。例如，固相支持物可以是多孔或无孔的，形式上可以是例如基质、珠、颗粒、片(chip)或其他构造，如膜或整料即单块、单颗/粒或单板材料。用作基质的颗粒可以是球形或珠状，表面光滑或粗糙或具有纹理。孔中许多是通孔，有时所有孔都是通孔，通孔穿透颗粒起着通道的作用，所述通道足够大从而允许水动力流或快速扩散从孔中通过。呈球形或珠状时，中值颗粒直径—“直径”指颗粒的最大外部尺寸—在一些实施方式中约25微米至约150微米。如本段所述的基质及其制备方法可见于Hjertén等的美国专利5,645,717、Liao等的美国专利5,647,979、Liao等的美国专利5,935,429以及Liao等的美国专利6,423,666。可聚合成有用基质的单体的例子有：乙酸乙烯酯，乙烯基丙胺，丙烯酸，甲基丙烯酸酯或盐，丙烯酸丁酯，丙烯酰胺，甲基丙烯酰胺，乙烯基吡咯烷酮，某些时候它们带有官能团。
配体可直接(没有间隔基)或通过接头连到固相支持物。与固相支持物的连接形式取决于所用的具体固相支持物。一些实施方式中，固相支持物具有二醇，可转化为醛，例如用NaIO4转化。配体的胺能通过还原胺化反应与固相支持物上的醛相连，配体由此直接偶联到固相支持物。
一些实施方式中，配体通过间隔基连接到固相支持物。可按照各种常规共价偶联法引入间隔基。典型的偶联化学反应可能涉及例如：表氯醇，表溴醇，烯丙基缩水甘油醚，双环氧化物如丁二醇二缩水甘油醚，卤素取代的脂肪族物质如二氯丙醇，二乙烯基砜，羰二咪唑，醛如戊二醛，醌，溴化氰，高碘酸盐例如高碘酸钠(sodium-metaperiodate)，碳二亚胺，氯三嗪，磺酰氯如对甲苯磺酰氯和三氟乙基磺酰氯，N-羟基琥珀酰亚胺，恶唑酮，马来酰亚胺，2-氟-1-甲基吡啶甲苯-4-磺酸盐，吡啶二硫化物和酰肼。
一些实施方式中，支持基质有孔，中值孔径0.5微米或以上，基本没有孔径小于0.1微米的孔。某些实施方式中，中值孔径约0.5微米至约2.0微米。孔容可以不同。一些实施方式中，孔容约0.5至约2.0cc/g.。基质可以是颗粒、膜或整料，“整料(monolith)”即单块、单颗/粒或单板材料。用作基质的颗粒可以是球形或珠状，表面光滑或粗糙或具有纹理。孔中许多是通孔，有时所有孔都是通孔，通孔穿透颗粒起着通道的作用，所述通道足够大从而允许水动力流或快速扩散从孔中通过。呈球形或珠状时，中值颗粒直径—“直径”指颗粒的最大外部尺寸—优选约25微米至约150微米。如本段所述的基质及其制备方法可见于Hjertén等的美国专利5,645,717、Liao等的美国专利5,647,979、Liao等的美国专利5,935,429以及Liao等的美国专利6,423,666。可聚合成有用基质的单体的例子有：乙酸乙烯酯，乙烯基丙胺，丙烯酸，甲基丙烯酸酯或盐，丙烯酸丁酯，丙烯酰胺，甲基丙烯酰胺，乙烯基吡咯烷酮，某些时候它们带有官能团。许多实施方式用到交联剂，如果用到，交联剂与单体总量之摩尔比一般约0.1至约0.7。交联剂的例子是二羟亚乙基二丙烯酰胺、酒石酸双丙烯铣胺、三烯丙基柠檬酸三酰胺、亚乙基二丙烯酸酯、双(丙稀酰)胱胺、N,N'-亚甲基双丙烯酰胺和哌嗪二丙烯酰胺。
为了与配体尤其具有胺基团的配体形成连接，可包含具有邻二醇的单体，例如烯丙氧丙二醇(3-烯丙氧基-1,2-丙二醇)。邻二醇单体与其他单体一起用于制备共聚物。由含二醇单体制得的聚合物中二醇基团的密度范围很宽，例如，约100至1,000μmol/mL(即每毫升充填珠的二醇微摩尔数)，许多情况下约200至300μmol/mL。符合本文所述基质的一个示例是商业来源的UNOsphereTMDiol(Bio-Rad实验室公司，赫拉克勒斯，加利福尼亚州，美国)。为了将悬垂含胺配体偶联到具有外露邻二醇的基质上，可将二醇氧化成醛基，然后由醛基与胺基偶联形成仲胺连接，以上均可采用已知常规化学技术进行。
可用本领域已知技术手段、采用本文所述树脂(即分离介质)完成蛋白质纯化。蛋白质的例子包括但不限于：抗体、酶、生长调节剂、凝血因子、转录因子和磷蛋白。许多此类常规方法中，树脂在用前先用与结合目标蛋白质(如抗体或非抗体蛋白)所用相同pH的缓冲液平衡。适当时，可针对各种会影响结合环境的因素包括离子强度和传导率进行平衡。
一些实施方式中，所述树脂可用于“结合-洗脱”模式从生物样品中纯化目标蛋白质。“结合-洗脱模式”指一种色谱操作方法，其中，形成一定的缓冲条件，该条件下，当样品施加于配体(可以是结合于固相支持物的)时，目标分子(或许还有不需要的污染物体)与配体结合。然后通过改变条件来将目标物从支持物上洗脱，由此实现目标物的分级。一些实施方式中，目标物洗脱后污染物物仍保持结合。一些实施方式中，污染物或者流走或者先结合而后在目标物洗脱之前先洗脱。一些实施方式中，目标蛋白质结合到树脂上后，可通过改变pH或提高盐浓度来洗脱目标蛋白。
一些实施方式中，对树脂进行平衡后，将源液加到树脂上，同时用合适的缓冲液保持源液和用到的各种其他载体液的pH低于6.0，让目标蛋白质结合到树脂上。一些实施方式中，使用盐浓度介于细胞培养物中盐浓度范围(如50-300mM，或约100-150mM)的溶液。因此，一些实施方式中，蛋白质是在上述盐浓度下加到树脂上的。
一些实施方式中，而后用洗涤液洗涤树脂，洗涤pH可以与加样步骤的一样，洗涤是为了洗去源液中可能存在的不结合的生物物质。
接着可洗脱结合的蛋白质(如抗体或非抗体蛋白，根据需要)。一些实施方式中，而后用pH高于6.0的洗脱液洗脱蛋白质。示例性pH范围为，如上所述，用于结合和洗涤步骤的pH4.0-6.0，用于洗脱步骤的pH6.1-8.5。某些实施方式中，进行结合和洗涤步骤时的样品和洗涤液中含盐。为此所用盐的例子是碱金属和碱土金属卤化物，尤其卤化钠和卤化钾，具体如氯化钠。盐的浓度是可变的，大多数情况下，合适的浓度在约10mM至约1M范围内。如后文实施例所示，某些蛋白质的最佳洗脱条件中，缓冲液的盐浓度高于结合缓冲液的，另外一些情况下则低于结合缓冲液的。具体情况下的最佳选择可通过常规试验来确定。.
树脂可以各种常规构造来使用，包括填充柱和流化床或膨胀床，可以各种常规方法来使用，例如分批模式加载、洗涤和洗脱、以及连续或流通模式。流通式充填柱对制备级提取还是分析级提起都尤为方便。一些实施方式中，柱的直径为1cm至1m，高度为1cm至30cm或更高。
“抗体”指免疫球蛋白及其复合物(如融合蛋白)或其片段。抗体一词包括但不限于来自人或其他动物细胞系的IgA、IgD、IgE、IgG和IgM各类多克隆抗体或单克隆抗体，包括天然形式的和基因改造的抗体，例如人源化抗体，人抗体，单链抗体，嵌合抗体，合成抗体，重组抗体，杂合抗体，突变抗体，接枝抗体，以及体外产生的抗体。“抗体”还可包括复合物形式，这包括但不限于含有免疫球蛋白部分的融合蛋白。“抗体”还可包括抗体片段，例如Fab、F(ab')2、Fv、scFv、Fd、dAb、Fcy，以及其他组成，不论它们是否保留有抗原结合功能。
各种抗体制备物均可用于本发明，包括由天然、合成或重组来源纯化或部分纯化的抗体。非纯化抗体可来自多种来源，所述来源包括但不限于血浆、血清、腹水、乳汁、植物提取物、细菌裂解物、酵母裂解物、或经调理细胞培养基。部分纯化的制备物可来自至少经一次色谱、沉淀、其他分级操作或以上之组合处理过的非纯化制备物。一些实施方式中，纯化前，抗体未经蛋白质A亲和纯化。
如前所述，树脂还可用于纯化非抗体蛋白质。治疗用蛋白质的例子包括但不限于：因子VIII血管性血友病因子的酶(FactorVIIIvonWillebrandFactorenzymes)、生长调节剂、凝血因子、转录因子和磷蛋白。
实施例
从商业来源获得(或者合成，例如以下配体P4或P9)了多种可能可用于混合模式色谱的配体(见表1)，将它们连接到固相支持物UNOsphereTMDiol(3-烯丙氧基-1,2-丙二醇与乙烯吡咯烷酮的共聚物)。
表1
一个配体接合的典型偶联方案如下所述。向10ml醛树脂中加10ml0.05M磷酸盐缓冲液(pH7)。将配体4-(4-氨基苯基)丁酸(400mg)一次性加到以上所得悬浮液中。测定反应混合物的pH并根据需要调至5.0-8.5。此反应混合物于37摄氏度搅拌0.5小时。将100mg氰基硼氢化钠一次性加入反应混合物。该最终反应混合物于37摄氏度搅拌3小时。将树脂收集在玻璃料上，用0.1NNaOH(2倍床层体积)和去离子水(7倍床层体积)洗涤。其他配体的偶联基本同上所述。
如下所示，不同配体在树脂上的配体密度有所不同：
实施例2
对连接了配体P1、P2、P3、P4或P5(见表1和图1)的固相支持物(UNOsphere)树脂进行试验，测定其结合一组商业来源蛋白质(Sigma提供)的能力。这组蛋白质经选择覆盖了一系列相关蛋白质特征(例如大小、EP(静电位)和疏水性)。
于pH5.0扫描偶联于UNOsphere配体的方案：
柱:10cmx0.5cmID
·流速:1mL/min
·输注物:90μL溶于缓冲液A(0MNaCl)的2mg/mL蛋白质溶液，
·缓冲液A:20mM乙酸盐，pH5.0
·缓冲液B:20mM乙酸盐，1.5MNaCl，pH5.0
·梯度:1.5％B至100％B，45CV
结果见图1-6。所进行的试验中，盐浓度越高表示蛋白质与配体的结合强度越高。
实施例3
对连接了配体P6、P7、P8或P9(见表1)的固相支持物(UNOsphere)树脂进行试验，测定其结合一组商业来源蛋白质(Sigma提供)的能力。
如下所示，不同配体在树脂上的配体密度有所不同：
于pH6.0扫描偶联于UNOsphere配体的方案：
柱:10cmx0.5cmID
·流速:1mL/min
·输注物:90μL溶于缓冲液A(0MNaCl)的2mg/mL蛋白质溶液
·缓冲液A:20mM磷酸盐，pH6.0
·缓冲液B:20mM磷酸盐，1.5MNaCl，pH6.0
·梯度:1.5％B至100％B，45CV
结果见图7-12。所进行的试验中，盐浓度越高表示蛋白质与配体的结合强度越高。
P8和P9在pH5的结果见图7；，这些原型类似地表现为混合模式介质，但结合弱于cPrime。P8和P9在pH6的结果见图8。
P6在pH5的结果见图9。该砜也类似地表现为混合模式，但结合略弱(对溶菌酶例外)。柱上的星号表示该蛋白质在盐浓梯度末尾没有洗脱。P6在pH6的结果见图10。
P7在pH5的结果见图11。P7，由于具有邻位连接的氨基苯基硫代乙酸且芳族基团与溶剂的接触增加，与cPrime、P4(邻位氨基马尿酸)或P2(对-氨基苯基硫代乙酸)相比，对更高疏水性的蛋白质有更高滞留率。P7在pH6的结果见图12。
实施例4
如下合成配体P9。10％的2-(2-(4-硝基苯甲酰基)乙酰氨基)乙酸DMSO溶液用1当量HCl处理，然后用氢(气球内)和10％Pd/C催化剂还原。对反应进程进行MS监测，根据需要补充氢(气球内)和10％Pd/C催化剂。全部2-(2-(4-硝基苯甲酰基)乙酰氨基)乙酸被还原后，对反应混合物进行过滤。按照本文所述，将所得溶液中的产物，2-(2-(4-氨基苯甲酰基)乙酰氨基)乙酸(MS,M+H，预计值252.09，实测值252.09)，直接偶联到树脂上。
权利要求中，“一”包含复数含义。“包含”、“包括”及类似用语表示除记载于其后的步骤或元素之外还可选包括而不排除其他步骤或元素。文中提到的所有专利、专利申请及其他出版文献均通过引用而被完全纳入。
Claims (22)
1.一种从源溶液纯化目标蛋白质的方法，所述方法包括：
接触连接了配体的固相支持物以使目标蛋白质与配体结合，其中，所述配体是：
其中，n是0、1或2；m是1、2、3或4；X选自S、C(O)-NH、NH-C(O)、C(O)-NH-CH2-C(O)-NH或SO2；或
其中，y是1、2、3、4或5，
其中，波浪线表示配体于此连接到固相基质；和
洗脱所述目标蛋白，由此纯化所述目标蛋白。
2.如权利要求1所述的方法，其中，所述配体如结构式I所示。
3.如权利要求2所述的方法，其中，所述配体选自：
4.如权利要求1所述的方法，其中，所述配体如结构式II所示。
5.如权利要求4所述的方法，其中，所述配体选自：
6.如权利要求1所述的方法，其中，所述固相支持物有孔，中值孔径约0.5微米或以上，基本没有孔径0.1微米或以下的孔。
7.权利要求1-6中任一项所述的方法，其中，所述接触在约4.0至约6.0的pH进行，所述洗脱在约6.1至约8.5的pH进行。
8.权利要求1-7中任一项所述的方法，其中，所述固相支持物由中值粒径约25至约150微米的颗粒构成。
9.如权利要求1所述的方法，其中，所述固相支持物是膜。
10.如权利要求1所述的方法，其中，所述固相支持物是整料。
11.权利要求1-10中任一项所述的方法，其中，所述源溶液含有选自碱金属及碱土金属卤化物的盐，盐的浓度约50mM至约300mM。
12.如权利要求1所述的方法，其中，所述源溶液含有选自碱金属及碱土金属卤化物的盐，盐的浓度约100mM至约150mM。
13.一种混合模式色谱介质，包含偶联于固相支持物的配体，所述配体是：
其中，n是0、1或2；m是1、2、3或4；X选自S、C(O)-NH、NH-C(O)、C(O)-NH-CH2-C(O)-NH或SO2；
或
其中，y是1、2、3、4或5，
其中，波浪线表示配体于此连接到固相基质。
14.如权利要求13所述的混合模式色谱介质，其中，所述介质与源溶液一定条件下接触，所述条件下蛋白质目标物由源溶液中结合到配体上。
15.如权利要求13所述的混合模式色谱介质，其中，所述配体如结构式I所示。
16.如权利要求15所述的混合模式色谱介质，其中，所述配体选自：
17.如权利要求13所述的混合模式色谱介质，其中，所述配体如结构式II所示。
18.如权利要求17所述的混合模式色谱介质，其中，所述配体选自：
19.如权利要求13所述的混合模式色谱介质，其中，所述固相支持物有孔，中值孔径约0.5微米或以上，基本没有孔径0.1微米或以下的孔。
20.如权利要求13所述的混合模式色谱介质，其中，所述固相支持物由中值粒径约25至约150微米的颗粒构成。
21.如权利要求13所述的混合模式色谱介质，其中，所述固相支持物是膜。
22.如权利要求13所述的混合模式色谱介质，其中，所述固相支持物是整料。
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