WO2022227362A1 - 含有还原剂的毛细管凝胶电泳检测试剂盒 - Google Patents
含有还原剂的毛细管凝胶电泳检测试剂盒 Download PDFInfo
- Publication number
- WO2022227362A1 WO2022227362A1 PCT/CN2021/115986 CN2021115986W WO2022227362A1 WO 2022227362 A1 WO2022227362 A1 WO 2022227362A1 CN 2021115986 W CN2021115986 W CN 2021115986W WO 2022227362 A1 WO2022227362 A1 WO 2022227362A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- buffer
- sample buffer
- reducing agent
- sample
- sds
- Prior art date
Links
- 239000003638 chemical reducing agent Substances 0.000 title claims abstract description 77
- 238000001818 capillary gel electrophoresis Methods 0.000 title claims abstract description 23
- 238000001514 detection method Methods 0.000 title claims abstract description 13
- PSYGHMBJXWRQFD-UHFFFAOYSA-N 2-(2-sulfanylacetyl)oxyethyl 2-sulfanylacetate Chemical compound SCC(=O)OCCOC(=O)CS PSYGHMBJXWRQFD-UHFFFAOYSA-N 0.000 claims abstract description 34
- ULGGZAVAARQJCS-UHFFFAOYSA-N 11-sulfanylundecan-1-ol Chemical compound OCCCCCCCCCCCS ULGGZAVAARQJCS-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 14
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 13
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 13
- 238000002347 injection Methods 0.000 claims abstract description 8
- 239000007924 injection Substances 0.000 claims abstract description 8
- 229960001612 trastuzumab emtansine Drugs 0.000 claims abstract description 7
- 239000012723 sample buffer Substances 0.000 claims description 84
- 239000000872 buffer Substances 0.000 claims description 72
- 238000013368 capillary electrophoresis sodium dodecyl sulfate analysis Methods 0.000 claims description 63
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 47
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 34
- 239000008367 deionised water Substances 0.000 claims description 28
- 229910021641 deionized water Inorganic materials 0.000 claims description 28
- 235000019333 sodium laurylsulphate Nutrition 0.000 claims description 25
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 24
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 21
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 16
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 101100476210 Caenorhabditis elegans rnt-1 gene Proteins 0.000 claims description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical group OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 10
- 239000007983 Tris buffer Substances 0.000 claims description 8
- 239000000523 sample Substances 0.000 claims description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical group Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 7
- 239000007981 phosphate-citrate buffer Substances 0.000 claims description 6
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 claims description 5
- 102000057041 human TNF Human genes 0.000 claims description 5
- 102000005962 receptors Human genes 0.000 claims description 5
- 108020003175 receptors Proteins 0.000 claims description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims 1
- 229940049595 antibody-drug conjugate Drugs 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims 1
- 239000010452 phosphate Substances 0.000 claims 1
- 230000009467 reduction Effects 0.000 abstract description 26
- 238000004458 analytical method Methods 0.000 abstract description 8
- 229960000074 biopharmaceutical Drugs 0.000 abstract description 8
- -1 dithiol compound Chemical class 0.000 abstract description 8
- 150000001875 compounds Chemical class 0.000 abstract description 5
- YTPLMLYBLZKORZ-UHFFFAOYSA-N Divinylene sulfide Natural products C=1C=CSC=1 YTPLMLYBLZKORZ-UHFFFAOYSA-N 0.000 abstract description 4
- 238000003912 environmental pollution Methods 0.000 abstract description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 abstract description 2
- 102000003390 tumor necrosis factor Human genes 0.000 abstract description 2
- HCZMHWVFVZAHCR-UHFFFAOYSA-N 2-[2-(2-sulfanylethoxy)ethoxy]ethanethiol Chemical compound SCCOCCOCCS HCZMHWVFVZAHCR-UHFFFAOYSA-N 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 57
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 32
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 32
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 29
- 102000051325 Glucagon Human genes 0.000 description 27
- 108060003199 Glucagon Proteins 0.000 description 27
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 27
- 229960004666 glucagon Drugs 0.000 description 27
- NJRXVEJTAYWCQJ-UHFFFAOYSA-N thiomalic acid Chemical compound OC(=O)CC(S)C(O)=O NJRXVEJTAYWCQJ-UHFFFAOYSA-N 0.000 description 23
- 239000011550 stock solution Substances 0.000 description 6
- 238000009835 boiling Methods 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 239000001488 sodium phosphate Substances 0.000 description 5
- 229910000162 sodium phosphate Inorganic materials 0.000 description 5
- 150000003573 thiols Chemical class 0.000 description 5
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 238000002844 melting Methods 0.000 description 3
- 230000008018 melting Effects 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- VSZSIEBALNXIFG-UHFFFAOYSA-N 2-hydroxyethyl 2,2-bis(sulfanyl)acetate Chemical compound OCCOC(=O)C(S)S VSZSIEBALNXIFG-UHFFFAOYSA-N 0.000 description 2
- XPCTZQVDEJYUGT-UHFFFAOYSA-N 3-hydroxy-2-methyl-4-pyrone Chemical compound CC=1OC=CC(=O)C=1O XPCTZQVDEJYUGT-UHFFFAOYSA-N 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- LJSQFQKUNVCTIA-UHFFFAOYSA-N diethyl sulfide Chemical compound CCSCC LJSQFQKUNVCTIA-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000013545 self-assembled monolayer Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- WVQBLGZPHOPPFO-LBPRGKRZSA-N (S)-metolachlor Chemical compound CCC1=CC=CC(C)=C1N([C@@H](C)COC)C(=O)CCl WVQBLGZPHOPPFO-LBPRGKRZSA-N 0.000 description 1
- KGYYLUNYOCBBME-UHFFFAOYSA-M 4-fluoro-2-phenyl-4-(4-propylcyclohexyl)cyclohexa-1,5-diene-1-carboxylate Chemical compound C1CC(CCC)CCC1C1(F)C=CC(C([O-])=O)=C(C=2C=CC=CC=2)C1 KGYYLUNYOCBBME-UHFFFAOYSA-M 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 229910001218 Gallium arsenide Inorganic materials 0.000 description 1
- HYMLWHLQFGRFIY-UHFFFAOYSA-N Maltol Natural products CC1OC=CC(=O)C1=O HYMLWHLQFGRFIY-UHFFFAOYSA-N 0.000 description 1
- 102000004868 N-Methyl-D-Aspartate Receptors Human genes 0.000 description 1
- 108090001041 N-Methyl-D-Aspartate Receptors Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 206010033546 Pallor Diseases 0.000 description 1
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 description 1
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004964 aerogel Substances 0.000 description 1
- 230000002152 alkylating effect Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000000729 antidote Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012650 click reaction Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 210000003618 cortical neuron Anatomy 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 150000002019 disulfides Chemical class 0.000 description 1
- 150000004662 dithiols Chemical class 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000001808 exosome Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 229910021389 graphene Inorganic materials 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229940043353 maltol Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000012450 pharmaceutical intermediate Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 229940001470 psychoactive drug Drugs 0.000 description 1
- 239000004089 psychotropic agent Substances 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000012429 release testing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000005464 sample preparation method Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000012430 stability testing Methods 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- ICRHORQIUXBEPA-UHFFFAOYSA-N thionitrous acid Chemical class SN=O ICRHORQIUXBEPA-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
- G01N33/561—Immunoelectrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
Definitions
- the invention belongs to the field of biotechnology and relates to the analysis of proteins by reducing capillary gel electrophoresis, in particular to a capillary gel electrophoresis detection kit containing a reducing agent that can accurately detect the purity of biological pharmaceuticals such as antibodies.
- CGE capillary gel electrophoresis
- m/z mass-to-charge ratio
- the actual technical problem to be solved by the present invention is to provide a capillary gel electrophoresis detection kit containing a reducing agent in view of the problem that the existing reducing CE-SDS detection method is more harmful.
- the sample buffer contains a reducing agent, and the kit can reduce the harm of reduced CE-SDS detection to the experimenter and the environment while ensuring the accuracy of the results of antibody molecular size isomers.
- the invention discloses a capillary gel electrophoresis detection kit containing a reducing agent.
- the sample buffer contains at least one reducing agent; the reducing agent is selected from 11-mercapto-1-undecanol, 2, One or more of 2'-(1,2-ethanediyldioxo)bisethanethiol or ethylene glycol bisthioglycolate.
- the concentration of free sulfhydryl groups in the sample buffer is 0.07-700 mM, preferably 0.7-70 mM, more preferably 7-70 mM.
- the sample buffer is selected from Tris buffer or phosphate buffer, and the pH of the sample buffer is between 3.0 and 10.0.
- the sample buffer is selected from Tris-HCl buffer or Phosphate-citrate buffer, pH between 6.5-10.0. More preferably, the sample buffer is selected from Tris-HCl buffer at pH 9.0 or phosphate-citrate buffer at pH 6.5.
- the sample buffer further contains sodium dodecyl sulfate in an amount between 0.1-5% (w/w), preferably 0.5-2% (w/w), more preferably 1% (w/w).
- the sample buffer contains the following components:
- the pH is 9.0.
- sample buffer solution The preparation method of above-mentioned sample buffer solution is: the Tris buffer salt of recipe quantity, the reducing agent of sodium lauryl sulfate of recipe quantity and recipe quantity are dissolved in deionized water, adjust pH to 9.0 with hydrochloric acid, then use deionized water Make up to 50 mL to obtain sample buffer.
- the sample buffer contains the following components:
- the pH is 6.5.
- the preparation method of the above-mentioned sample buffer solution is as follows: the phosphate salt of the recipe quantity, the sodium lauryl sulfate of the recipe quantity and the reducing agent of the recipe quantity are dissolved in an appropriate amount of deionized water, the pH is adjusted to 6.5 with citric acid, and then the The volume of ionized water was adjusted to 50 mL to obtain a sample buffer.
- the present invention also discloses the application of the above-mentioned kit for reducing CE-SDS detection of protein samples, wherein the protein samples are selected from recombinant anti-ricin humanized monoclonal antibody (mAb1), recombinant anti-HER2 humanized Monoclonal antibody (mAb2), T-DM1 (ADC) and recombinant human tumor necrosis factor receptor type II-antibody fusion protein (Fusion protein) for injection.
- mAb1 recombinant anti-ricin humanized monoclonal antibody
- mAb2 recombinant anti-HER2 humanized Monoclonal antibody
- ADC T-DM1
- Fusion protein recombinant human tumor necrosis factor receptor type II-antibody fusion protein
- the capillary gel electrophoresis detection kit with added reducing agent of the present invention according to the sample processing method recommended by CE manufacturer Beckman Company (heating at 70° C. for 15 minutes), the accuracy of biopharmaceuticals such as antibody purity can be accurately characterized, and at the same time, the accuracy of purity can be significantly reduced.
- the safety problems brought by the traditional reduction method reduce the pollution to the environment and the harm to the human body.
- Figure 1 Structures of monothiol compounds such as mercaptoethanol, 11-mercapto-1-undecanol and mercaptosuccinic acid;
- Fig. 3 The effect of different concentrations of mercaptoethanol in the buffer of pH 9.0 on the reduced CE-SDS purity of recombinant anti-ricin humanized monoclonal antibody (mAb1) in Example 1;
- Fig. 8 embodiment 6 different concentrations of ethylene glycol dithioglycolate are in the buffer solution of pH 9.0 on the reduced CE-SDS purity of recombinant anti-ricin humanized monoclonal antibody (mAb1);
- Figure 9 The effect of different reducing agents with a free sulfhydryl concentration of 7 mM in Example 7 on the purity of recombinant anti-HER2 humanized monoclonal antibody (mAb2, batch in 2013) on reduced CE-SDS purity in pH 9.0 buffer; each The spectra are: mercaptoethanol (a); 11-mercapto-1-undecanol (b); mercaptosuccinic acid (c); dithiothreitol (d); 2,2'-(1,2 - ethylenediyldioxo)bisethanethiol (e); ethylene glycol bisthioglycolate (f);
- Figure 10 The effect of different reducing agents with a free sulfhydryl concentration of 7 mM in Example 8 on the reduced CE-SDS purity of recombinant anti-HER2 humanized monoclonal antibody (mAb2, batch in 2016) in buffer at pH 9.0; each The spectra are: mercaptoethanol (a); 11-mercapto-1-undecanol (b); mercaptosuccinic acid (c); dithiothreitol (d); 2,2'-(1,2 - ethylenediyldioxo)bisethanethiol (e); ethylene glycol bisthioglycolate (f);
- Figure 12 Effect of different reducing agents with a free sulfhydryl concentration of 7 mM in pH 9.0 buffer on the purity of reduced CE-SDS of recombinant human tumor necrosis factor receptor type II-antibody fusion protein (Fusion protein) for injection in Example 10 ;
- Each spectrum is: mercaptoethanol (a); 11-mercapto-1-undecanol (b); mercaptosuccinic acid (c); dithiothreitol (d); 2,2'-(1 , 2-ethanediyldioxo) diethanethiol (e); ethylene glycol bisthioglycolate (f);
- Figure 18 Effect of 70 mM mercaptoethanol in pH 9.0 buffer (without SDS) on the hydrophilicity/hydrophobicity of glucagon in Example 16; reducing agent heated at 70°C for 15 minutes in pH 9.0 buffer (a) ; Glucagon in pH 9.0 buffer at 70°C for 15 minutes (b); reducing agent and glucagon in pH 9.0 buffer at 70°C for 15 minutes (c);
- Figure 19 Effect of 70 mM 11-mercapto-1-undecanol on the hydrophilicity/hydrophobicity of glucagon in pH 9.0 buffer in Example 17; reducing agent heated at 70°C for 15 minutes in pH 9.0 buffer (a); glucagon in pH 9.0 buffer heated at 70°C for 15 minutes (b); reducing agent and glucagon in pH 9.0 buffer heated at 70°C for 15 minutes (c);
- Figure 20 The effect of 70 mM mercaptosuccinic acid on the hydrophilicity/hydrophobicity of glucagon in a buffer at pH 9.0 in Example 18; the reducing agent was heated at 70°C for 15 minutes in a buffer at pH 9.0 (a); pancreatic Glucagon was heated in buffer pH 9.0 at 70°C for 15 minutes (b); reducing agent and glucagon were heated at 70°C in buffer pH 9.0 for 15 minutes (c);
- Figure 21 The effect of 35 mM dithiothreitol on the hydrophilicity/hydrophobicity of glucagon in a buffer at pH 9.0 in Example 19; the reducing agent was heated at 70°C for 15 minutes in a buffer at pH 9.0 (a); Glucagon in pH 9.0 buffer at 70°C for 15 minutes (b); reducing agent and glucagon in pH 9.0 buffer at 70°C for 15 minutes (c);
- Figure 22 Effect of 35 mM 2,2'-(1,2-ethanediyldioxo)bisethanethiol in buffer at pH 9.0 on the hydrophilicity/hydrophobicity of glucagon in Example 20; reducing agent Heating at 70°C for 15 minutes in pH 9.0 buffer (a); glucagon at 70°C for 15 minutes in pH 9.0 buffer (b); reducing agent and glucagon in pH 9.0 buffer Heating at 70°C for 15 minutes (c);
- Figure 23 Effect of 35 mM ethylene glycol bisthioglycolate in pH 9.0 buffer on the hydrophilicity/hydrophobicity of glucagon in Example 21; the reducing agent was heated at 70°C for 15 minutes in a pH 9.0 buffer (a ); glucagon in pH 9.0 buffer heated at 70°C for 15 minutes (b); reducing agent and glucagon in pH 9.0 buffer heated at 70°C for 15 minutes (c).
- 11-Mercapto-1-undecanol is a compound with 9 more carbon atoms than mercaptoethanol, and its structure is shown in Figure 1.
- the melting point of 11-mercapto-1-undecanol is 33-37C (lit.), and the density is 0.790g/mL (25C), it is soluble in water to a certain extent, basically non-volatile, and has no irritating odor. It is often used in the two-step synthesis of asymmetric disulfides for the preparation of hydrophilic self-assembled monolayers ( Wang et al. Direct quantification of cancerous exosomes via surface plasmon resonance with dual gold nanoparticle-assisted signal amplification. Biosens Bioelectron.
- Mercaptosuccinic acid generally refers to DL-mercaptosuccinic acid. Its structure is shown in Figure 1. It is a colorless crystal or white powder with a melting point of 155-157C (lit.). It is the main component of cold blanching agents and is also used for complexation concealment. , Biochemical research, heavy metal antidote and rubber industry (Yapor, JP et al. Biodegradable crosslinked polyesters derived from thiomalic acid and S-nitrosothiol analogs for nitric oxide release.
- Dithiothreitol is a small molecule organic reducing agent, the structure of which is shown in Figure 2. Its melting point is 41-44C (lit.), its boiling point is 125°C, its density is 1.04g/mL (20°C), its acidity coefficient (pKa) is 8.9 (25°C), and it is white powder with good water solubility. Its reducibility largely depends on the conformational stability of the six-membered ring (containing disulfide bonds) in the oxidation state, and the reducing power is affected by the pH value, and the reduction effect is better when the pH value is greater than 7.
- dithiothreitol has been widely used in the reduction analysis of proteins (Cherkaoui, S et al. Tracking of antibody reduction fragments by capillary gel electrophoresis during the coupling to microparticles surface. J Pharm Biomed Anal. 2010, 53 (2): 172-8; Santarino, IB et al. Protein reducing agents dithiothreitol and tris(2-carboxyethyl) phosphine anodic oxidation. Electrochem commun. 2012, 23: 114-117; Wiesner, R et al. A comparative study of CE- SDS, SDS-PAGE, and Simple Western: Influences of sample preparation on molecular weight determination of proteins. Electrophoresis. 2021, 42(3):206-218.).
- 2,2'-(1,2-Ethanediyldioxo)bisethanethiol is an alcohol derivative whose structure is shown in Figure 2. Its boiling point is 225°C (lit.), density is 1.12g/mL (25°C) (lit.), easily soluble in organic solvents, commonly used as pharmaceutical intermediates (Du GL, etc. Tough and biocompatible hydrogels based on in situ interpenetrating networks of dithiol-connected graphene oxide and poly(vinyl alcohol). ACS Appl Mater Interfaces. 2015, 7(5): 3003-8; Yun, S et al.
- Ethylene glycol bisthioglycolate the structure of which is shown in Figure 2. Its boiling point is 137-139C 2mm Hg(lit.), its density is 1.313g/mL(25C)(lit.), its solubility is 20g/L(20°C), and it is used for reducing agent and organic synthesis (TANG, LH, etc.).
- TANG reducing agent and organic synthesis
- Odor content mercaptoethanol is a volatile liquid with a strong pungent odor.
- Chemical Book a resource platform dedicated to providing users with the most valuable information in the chemical industry, mercaptoethanol is regulated as a psychotropic drug or precursor chemical goods or regulated products and shall not be sold or purchased illegally.
- the company or unit needs to obtain the license issued by the relevant department and register and record in accordance with the law. Both logistics and warehousing require logistics companies and warehousing companies with corresponding qualifications, and express delivery is strictly prohibited.
- the carbon skeleton of 11-mercapto-1-undecanol is extended by 9 carbon atoms, and the molecular weight is larger.
- 2,2'-(1,2-Ethanediyldioxo)bisethanethiol is an alcohol derivative with a boiling point of 225°C (lit.), with weak volatility, and can be dissolved in the preferred concentration of this patent. In water, there is almost no obvious odor.
- Ethylene glycol bis-mercaptoacetate is an ester compound, its boiling point is 137-139C 2mm Hg (lit.), and its volatility is relatively weak. It can be dissolved in water at the preferred concentration of the present invention, and has almost no obvious odor.
- Example 1 The effect of different concentrations of mercaptoethanol on the purity of recombinant anti-ricin humanized monoclonal antibody (mAb1) in reduced CE-SDS in pH 9.0 buffer
- Tris 0.606g, SDS 0.5g dissolve with 42mL deionized water until clear, add 2.5mL mercaptoethanol stock solution (14M), adjust pH to 9.0 with 3M hydrochloric acid, and then dilute to 50mL with deionized water to obtain a solution containing 700 mM mercaptoethanol in sample buffer. Then, the sample buffer containing no reducing agent and the sample buffer containing 700 mM mercaptoethanol were sequentially diluted to obtain the sample buffer containing 70 mM, 7 mM, 0.7 mM and 0.07 mM mercaptoethanol.
- the recombinant anti-ricin humanized monoclonal antibody (mAb1) was diluted to 1 mg/mL with the above-prepared sample buffer, then denatured in a water bath, and analyzed by capillary gel electrophoresis.
- Example 2 The effect of different concentrations of 11-mercapto-1-undecanol on the purity of recombinant anti-ricin humanized monoclonal antibody (mAb1) in reduced CE-SDS purity in pH 9.0 buffer
- the sample buffer containing 70 mM 11-mercapto-1-undecanol was then diluted sequentially with the sample buffer without reducing agent and the sample buffer containing 7 mM, 0.7 mM and 0.07 mM 11-mercapto-1-undecanol.
- the recombinant anti-ricin humanized monoclonal antibody (mAb1) was diluted to 1 mg/mL with the above-prepared sample buffer, then denatured in a water bath, and analyzed by capillary gel electrophoresis.
- Example 3 Effect of different concentrations of mercaptosuccinic acid on the purity of recombinant anti-ricin humanized monoclonal antibody (mAb1) in reduced CE-SDS purity in pH 9.0 buffer
- mAb1 anti-ricin humanized monoclonal antibody
- Example 4 The effect of different concentrations of dithiothreitol on the purity of recombinant anti-ricin humanized monoclonal antibody (mAb1) in reduced CE-SDS in pH 9.0 buffer
- Tris 0.606g, SDS 0.5g, dithiothreitol (MW: 154.25) 2.699g dissolve with 42mL deionized water until clear, then adjust pH to 9.0 with 3M hydrochloric acid, and then dilute to 50mL with deionized water , to obtain a sample buffer containing 350 mM dithiothreitol. Then, the sample buffer containing no reducing agent and the sample buffer containing 350 mM dithiothreitol were sequentially diluted to obtain the sample buffer containing 35 mM, 3.5 mM, 0.35 mM and 0.035 mM dithiothreitol.
- the recombinant anti-ricin humanized monoclonal antibody (mAb1) was diluted to 1 mg/mL with the above-prepared sample buffer, then denatured in a water bath, and analyzed by capillary gel electrophoresis.
- Tris 0.606g, SDS 0.5g dissolve with 42mL deionized water until clear, add 0.29mL 2,2'-(1,2-ethanediyldioxo)diethanethiol stock solution (6M), and then use The pH was adjusted to 9.0 with 3M hydrochloric acid, followed by making up to 50 mL with deionized water to obtain a sample buffer containing 35 mM 2,2'-(1,2-ethanediyldioxo)bisethanethiol. Due to solubility limitations, a buffer concentration of 350 mM was not available.
- sample buffer without reducing agent and the sample buffer containing 35mM 2,2'-(1,2-ethanediyldioxo)diethanethiol dilute successively to obtain 3.5mM, 0.35mM and 0.035mM mM 2,2'-(1,2-ethanediyldioxo)diethanethiol in sample buffer.
- the recombinant anti-ricin humanized monoclonal antibody (mAb1) was diluted to 1 mg/mL with the above-prepared sample buffer, then denatured in a water bath, and analyzed by capillary gel electrophoresis.
- Example 6 The effect of different concentrations of ethylene glycol bisthioglycolate on the purity of recombinant anti-ricin humanized monoclonal antibody (mAb1) in the buffer at pH 9.0 by reduced CE-SDS
- Tris 0.606g, SDS 0.5g dissolve with 42mL deionized water until clear, add 0.29mL ethylene glycol bis-mercaptoacetate (6M), adjust the pH to 9.0 with 3M hydrochloric acid, and then use deionized water to make up to 50 mL to obtain a sample buffer containing 35 mM ethylene glycol bisthioglycolate. Due to solubility limitations, a buffer concentration of 350 mM was not available.
- sample buffer containing no reducing agent and the sample buffer containing 35 mM ethylene glycol bisthioglycolate were sequentially diluted to obtain the sample buffer containing 3.5 mM, 0.35 mM and 0.035 mM ethylene bisthioglycolate.
- the recombinant anti-ricin humanized monoclonal antibody (mAb1) was diluted to 1 mg/mL with the above-prepared sample buffer, then denatured in a water bath, and analyzed by capillary gel electrophoresis.
- Example 7 Effect of different reducing agents with a free thiol concentration of 7 mM in pH 9.0 buffer on the reduced CE-SDS purity of recombinant anti-HER2 humanized monoclonal antibody (mAb2, batch in 2013)
- Example 8 Effect of different reducing agents with a free thiol concentration of 7 mM in pH 9.0 buffer on the reduced CE-SDS purity of recombinant anti-HER2 humanized monoclonal antibody (mAb2, batch 2016)
- Example 9 Influence of different reducing agents with a free thiol concentration of 7 mM in pH 9.0 buffer on the purity of reduced CE-SDS of T-DM1 (ADC)
- Example 10 Influence of different reducing agents with free sulfhydryl concentration of 7 mM in pH 9.0 buffer on the purity of reduced CE-SDS of recombinant human tumor necrosis factor receptor type II-antibody fusion protein for injection (Fusion protein)
- Example 11 Effect of different concentrations of mercaptoethanol on the purity of recombinant anti-ricin humanized monoclonal antibody (mAb1) in reduced CE-SDS purity in pH 6.5 buffer
- Example 12 The effect of different concentrations of 11-mercapto-1-undecanol on the reduced CE-SDS purity of recombinant anti-ricin humanized monoclonal antibody (mAb1) in pH 6.5 buffer
- mAb1 anti-ricin humanized monoclonal antibody
- Example 13 The effect of different concentrations of dithiothreitol on the purity of recombinant anti-ricin humanized monoclonal antibody (mAb1) on reduced CE-SDS purity in pH 6.5 buffer
- Example 15 The effect of different concentrations of ethylene glycol bisthioglycolate on the purity of recombinant anti-ricin humanized monoclonal antibody (mAb1) in the buffer at pH 6.5 by reduced CE-SDS
- Example 16 Effect of different reducing agents with a free thiol concentration of 70 mM on the hydrophilicity/hydrophobicity of glucagon in pH 9.0 buffer
- Tris-HCl buffer without SDS, pH 9.0 was configured as blank buffer. Dissolve glucagon with blank buffer at a final concentration of 0.5 mg/mL, and perform reverse-phase chromatography analysis after heat incubation to determine whether the hydrophilic/hydrophobicity of glucagon changes with heat treatment. At the same time, a blank buffer containing different reducing agents was prepared, and the final concentration of free sulfhydryl groups was 70 mM. After passing through a water bath, reversed-phase chromatography was performed to serve as a blank control.
- glucagon was dissolved in buffers containing different reducing agents, and reversed-phase chromatography was performed after thermal incubation to determine whether the hydrophilicity/hydrophobicity of glucagon was changed. The results are shown in Figure 18-23.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明涉及一种含有还原剂的毛细管凝胶电泳检测试剂盒,所述还原剂选自单硫醇化合物和双硫醇化合物,其中单硫醇化合物选自11-巯基-1-十一醇,双硫醇化合物选自2,2'-(1,2-乙二基双氧代)双乙硫醇和双巯基乙酸乙二醇酯。本发明的试剂盒可以降低还原型毛细管凝胶电泳分析生物制药如单克隆抗体过程中的对环境的污染和对人体的危害,同时确保还原条件下纯度的准确性,这些生物药包括重组抗蓖麻毒素人源化单克隆抗体、重组抗HER2人源化单克隆抗体、T-DM1和注射用重组人II型肿瘤坏死因子受体-抗体融合蛋白。
Description
本发明属于生物技术领域,涉及还原型毛细管凝胶电泳对蛋白质的分析,尤其涉及一种能够准确检测生物制药如抗体纯度的含有还原剂的毛细管凝胶电泳检测试剂盒。
随着生物制药领域的不断发展,越来越多的技术被应用于治疗性蛋白的异质性表征。其中,毛细管凝胶电泳(CGE)作为一项从传统的板凝胶电泳发展而来的技术,以其自动化、快速、高分辨率和在线定量等优点,逐步成为诸多生物制药尤其是抗体的纯度表征工具之一。以单抗为例,在还原型分析中,蛋白分子会被还原剂还原并与表面活性剂结合,形成均一质荷比(m/z)的蛋白质-去污剂复合物,随后在电场作用下进行分离。该方法能很好地表征单抗样品的轻重链以及非糖基化重链,被广泛应用于生物制药的批量释放和稳定性试验中,以确定样品的纯度和生产一致性。
目前,巯基乙醇已在生物制药领域内被广泛应用于蛋白样品的还原,并以其较强的还原能力成为了还原型CE-SDS分析中常用的还原剂,参考药典的公示方法(2020中华人民共和国药典<3127>单抗分子大小变异体测定法(CE-SDS法))。但是在该药典规定的还原供试品溶液制备方法中,有5μL巯基乙醇原液被添加进95μL供试品溶液(1mg/mL)中,以进行样品的彻底还原。一方面,这是为了保证还原效果以不影响还原型CE-SDS的分析,该样品制备方法是可取的。但是另一方面,考虑到巯基乙醇原液浓度较高(14M),在高温下有引起燃烧的危险,且巯基乙醇极易挥发,具有极强的刺激性气味,具有较高的生物毒性和环境污染风险等等。因此,实验人员在实验过程中被提醒穿实验服,佩戴防毒面具并在通风橱操作,并且妥善丢弃实验垃圾,但是关于巯基乙醇的危害仍旧不容忽视。我们认为需要将还原型CE-SDS方法进行进一步优化,开发出具有等同还原效果的新方法,同时降低对环境的污染和对实验人员实验安全的影响,以便还原型 CE-SDS分析能够更加安全地进行以适应当前蛋白类生物药分析业内的需求。
发明内容
本发明实际所要解决的技术问题,就是针对现有的还原型CE-SDS检测方法的危害性较大的问题,提供了一种含有还原剂的毛细管凝胶电泳检测试剂盒,所述试剂盒的样品缓冲液中含有一种还原剂,该试剂盒能降低还原型CE-SDS检测对实验人员和环境的危害性的同时确保抗体分子大小异构体结果的准确性。
本发明公开了一种含有还原剂的毛细管凝胶电泳检测试剂盒,所述样品缓冲液中含有至少一种还原剂;所述的还原剂选自11-巯基-1-十一醇、2,2’-(1,2-乙二基双氧代)双乙硫醇或双巯基乙酸乙二醇酯中的一种或多种。
优选的,所述样品缓冲液中游离巯基的浓度为0.07-700mM,优选0.7-70mM,更优选7-70mM。
优选的,所述样品缓冲液选自Tris缓冲液或者磷酸盐缓冲液,所述样品缓冲液的pH值在3.0-10.0之间,优选的,所述样品缓冲液选自Tris-HCl缓冲液或者磷酸盐-柠檬酸缓冲液,pH值在6.5-10.0之间。更优选的,所述样品缓冲液选自pH 9.0的Tris-HCl缓冲液或pH 6.5的磷酸盐-柠檬酸缓冲液。
所述样品缓冲液进一步含有十二烷基硫酸钠,其含量在0.1-5%(w/w)之间,优选0.5-2%(w/w),更优选1%(w/w)。
优选的,所述样品缓冲液含有如下组分:
(1)Tris-HCl缓冲液100mM;
(2)十二烷基硫酸钠1%(w/w);
(3)选自11-巯基-1-十一醇、2,2’-(1,2-乙二基双氧代)双乙硫醇或双巯基乙酸乙二醇酯中的一种或多种的还原剂,其总游离巯基的浓度为7mM;
pH值为9.0。
上述样品缓冲液的制备方法为:将处方量的Tris缓冲盐,处方量的十二烷基硫酸钠和处方量的还原剂溶解在去离子水中,用盐酸调节pH至9.0,接着用去离子水定容至50mL,得到样品缓冲液。
优选的,所述样品缓冲液含有如下组分:
(1)磷酸盐-柠檬酸缓冲液20mM;
(2)十二烷基硫酸钠1%(w/w);
(3)选自11-巯基-1-十一醇、2,2’-(1,2-乙二基双氧代)双乙硫醇或双巯基乙酸乙二醇酯中的一种或多种的还原剂,其总游离巯基的浓度为70mM;
pH值为6.5。
上述样品缓冲液的制备方法为:将处方量的磷酸盐,处方量的十二烷基硫酸钠和处方量的还原剂溶解在适量的去离子水中,用柠檬酸调节pH至6.5,接着用去离子水定容至50mL,得到样品缓冲液。
本发明还公开了上述的试剂盒对蛋白样品进行还原型CE-SDS检测的应用,其中,所述蛋白样品选自重组抗蓖麻毒素人源化单克隆抗体(mAb1)、重组抗HER2人源化单克隆抗体(mAb2)、T-DM1(ADC)和注射用重组人II型肿瘤坏死因子受体-抗体融合蛋白(Fusion protein)。
本发明的有益效果为:
使用本发明的添加了还原剂的毛细管凝胶电泳检测试剂盒,根据CE厂商Beckman公司推荐的样品处理方法(70℃加热15分钟),可以准确表征生物制药如抗体纯度的准确性,同时显著降低传统的还原方法带来的安全问题,降低对环境的污染以及对人体的伤害。
图1单硫醇化合物如巯基乙醇,11-巯基-1-十一醇和巯基丁二酸的结构;
图2双硫醇化合物如二硫苏糖醇,2,2’-(1,2-乙二基双氧代)双乙硫醇和双巯基乙酸乙二醇酯的结构;
图3实施例1中不同浓度巯基乙醇在pH 9.0的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响;
图4实施例2中不同浓度11-巯基-1-十一醇在pH 9.0的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响;
图5实施例3中不同浓度巯基丁二酸在pH 9.0的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响;
图6实施例4中不同浓度二硫苏糖醇在pH 9.0的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响;
图7实施例5中不同浓度2,2’-(1,2-乙二基双氧代)双乙硫醇在pH 9.0的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响;
图8实施例6中不同浓度双巯基乙酸乙二醇酯在pH 9.0的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响;
图9实施例7中游离巯基浓度为7mM的不同还原剂在pH 9.0的缓冲液中对重组抗HER2人源化单克隆抗体(mAb2,2013年批次)的还原型CE-SDS纯度影响;各谱图分别为:巯基乙醇(a);11-巯基-1-十一醇(b);巯基丁二酸(c);二硫苏糖醇(d);2,2’-(1,2-乙二基双氧代)双乙硫醇(e);双巯基乙酸乙二醇酯(f);
图10实施例8中游离巯基浓度为7mM的不同还原剂在pH 9.0的缓冲液中对重组抗HER2人源化单克隆抗体(mAb2,2016年批次)的还原型CE-SDS纯度影响;各谱图分别为:巯基乙醇(a);11-巯基-1-十一醇(b);巯基丁二酸(c);二硫苏糖醇(d);2,2’-(1,2-乙二基双氧代)双乙硫醇(e);双巯基乙酸乙二醇酯(f);
图11实施例9中游离巯基浓度为7mM的不同还原剂在pH 9.0的缓冲液中对T-DM1(ADC)的还原型CE-SDS纯度影响;各谱图分别为:巯基乙醇(a);11-巯基-1-十一醇(b);巯基丁二酸(c);二硫苏糖醇(d);2,2’-(1,2-乙二基双氧代)双乙硫醇(e);双巯基乙酸乙二醇酯(f);
图12实施例10中游离巯基浓度为7mM的不同还原剂在pH 9.0的缓冲液中对注射用重组人II型肿瘤坏死因子受体-抗体融合蛋白(Fusion protein)的还原型CE-SDS纯度影响;各谱图分别为:巯基乙醇(a);11-巯基-1-十一醇(b);巯基丁二酸(c);二硫苏糖醇(d);2,2’-(1,2-乙二基双氧代)双乙硫醇(e);双巯基乙酸乙二醇酯(f);
图13实施例11中不同浓度巯基乙醇在pH 6.5的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响;
图14实施例12中不同浓度11-巯基-1-十一醇在pH 6.5的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响;
图15实施例13中不同浓度二硫苏糖醇在pH 6.5的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响;
图16实施例14中不同浓度2,2’-(1,2-乙二基双氧代)双乙硫醇在pH 6.5的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响;
图17实施例15中不同浓度双巯基乙酸乙二醇酯在pH 6.5的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响;
图18实施例16中70mM巯基乙醇在pH 9.0的缓冲液中(无SDS)对胰高血糖素亲水/疏水性的影响;还原剂在pH 9.0的缓冲液中70℃加热15分钟(a);胰高血糖素在pH 9.0的缓冲液中70℃加热15分钟(b);还原剂和胰高血糖素在pH 9.0的缓冲液中70℃加热15分钟(c);
图19实施例17中70mM 11-巯基-1-十一醇在pH 9.0的缓冲液中对胰高血糖素亲水/疏水性的影响;还原剂在pH 9.0的缓冲液中70℃加热15分钟(a);胰高血糖素在pH 9.0的缓冲液中70℃加热15分钟(b);还原剂和胰高血糖素在pH 9.0的缓冲液中70℃加热15分钟(c);
图20实施例18中70mM巯基丁二酸在pH 9.0的缓冲液中对胰高血糖素亲水/疏水性的影响;还原剂在pH 9.0的缓冲液中70℃加热15分钟(a);胰高血糖素在pH 9.0的缓冲液中70℃加热15分钟(b);还原剂和胰高血糖素在pH 9.0的缓冲液中70℃加热15分钟(c);
图21实施例19中35mM二硫苏糖醇在pH 9.0的缓冲液中对胰高血糖素亲水/疏水性的影响;还原剂在pH 9.0的缓冲液中70℃加热15分钟(a);胰高血糖素在pH 9.0的缓冲液中70℃加热15分钟(b);还原剂和胰高血糖素在pH 9.0的缓冲液中70℃加热15分钟(c);
图22实施例20中35mM 2,2’-(1,2-乙二基双氧代)双乙硫醇在pH 9.0的缓冲液中对胰高血糖素亲水/疏水性的影响;还原剂在pH 9.0的缓冲液中70℃加热15分钟(a);胰高血糖素在pH 9.0的缓冲液中70℃加热15分钟(b);还原剂和胰高血糖素在pH 9.0的缓冲液中70℃加热15分钟(c);
图23实施例21中35mM双巯基乙酸乙二醇酯在pH 9.0的缓冲液中对胰高血糖素亲水/疏水性的影响;还原剂在pH 9.0的缓冲液中70℃加热15分钟(a);胰高血糖素在pH 9.0的缓冲液中70℃加热15分钟(b);还原剂和胰高血糖素在pH 9.0的缓冲液中70℃加热15分钟(c)。
以下通过具体实施例对本发明进一步进行说明。必须指出,以下实施例是用于说明本发明,而不是对本发明的限制。
本发明考虑到巯基乙醇具有极强的刺激性气味,具有较高的生物毒性和环境污染风险等等问题,且原方法的巯基乙醇终浓度为700mM,因此考虑寻找替代性的还原剂。并评估了多款硫醇化合物的还原效果,其中单硫醇化合物包括11-巯基-1-十一醇和巯基丁二酸,双硫醇化合物包括二硫苏糖醇,2,2’-(1,2-乙二基双氧代)双乙硫醇和双巯基乙酸乙二醇酯。
11-巯基-1-十一醇是比巯基乙醇多了9个碳原子的化合物,其结构见图1。11-巯基-1-十一醇的熔点为33-37C(lit.),密度为0.790g/mL(25C),其一定程度上溶于水,基本无挥发性,无刺激性气味,常用于两步法合成不对称二硫化物,用于制备亲水性自组装单分子层(Wang等.Direct quantification of cancerous exosomes via surface plasmon resonance with dual gold nanoparticle-assisted signal amplification.Biosens Bioelectron.2019,135:129-136;Lacour等.Formation kinetics of mixed self-assembled monolayers of alkanethiols on GaAs(100).Langmuir.2019,35(13):4415-4427.)
巯基丁二酸一般指DL-巯基琥珀酸,其结构见图1,为无色结晶或白色粉末,熔点为155-157C(lit.),是冷烫剂的主要组分,还用于络合隐蔽、生化研究、重金属解毒剂及橡胶工业(Yapor,JP等.Biodegradable crosslinked polyesters derived from thiomalic acid and S-nitrosothiol analogues for nitric oxide release.J Mater Chem B.2018,6(24):4071-4081;Sheini,A.A paper-based device for the colorimetric determination of ammonia and carbon dioxide using thiomalic acid and maltol functionalized silver nanoparticles:application to the enzymatic determination of urea in saliva and blood.Mikrochim Acta.2020,187(10):565.)。
二硫苏糖醇(Dithiothreitol,简称为DTT)是一种小分子有机还原剂,其结构见图2。其熔点为41-44C(lit.),沸点为125℃,密度为1.04g/mL(20℃),酸度系数(pKa)为8.9(25℃),白色粉末,水溶性较好。其还原性很大程度上取决于氧化状态六元环(含二硫键)的构象稳定性,还原力受pH值的影响,在pH值大于7的情况下还原效果较好。近些年,在蛋白质的还原型分析中,二硫苏糖醇较有应用(Cherkaoui,S等.Tracking of antibody reduction fragments by capillary gel electrophoresis during the coupling to microparticles surface.J Pharm Biomed Anal.2010,53(2):172-8;Santarino,IB等.Protein reducing agents dithiothreitol and tris(2-carboxyethyl)phosphine anodic oxidation.Electrochem commun. 2012,23:114-117;Wiesner,R等.A comparative study of CE-SDS,SDS-PAGE,and Simple Western:Influences of sample preparation on molecular weight determination of proteins.Electrophoresis.2021,42(3):206-218.)。
2,2’-(1,2-乙二基双氧代)双乙硫醇是一种醇类衍生物,其结构见图2。其沸点为225℃(lit.),密度为1.12g/mL(25℃)(lit.),易溶于有机溶剂,常用作医药中间体(Du GL等.Tough and biocompatible hydrogels based on in situ interpenetrating networks of dithiol-connected graphene oxide and poly(vinyl alcohol).ACS Appl Mater Interfaces.2015,7(5):3003-8;Yun,S等.Low-density,hydrophobic,highly flexible ambientpressure-dried monolithic bridged silsesquioxane aerogels.J Mater Chem A Mater.2015,3(7):3390-3398;Elkassih,SA等.Degradable redox-responsive disulfide-based nanogel drug carriers via dithiol oxidation polymerization.Biomater Sci.2019,7(2):607-617.)。
双巯基乙酸乙二醇酯,其结构见图2。其沸点为137-139C 2mm Hg(lit.),密度为1.313g/mL(25C)(lit.),溶解度为20g/L(20℃),用于还原剂和有机合成(TANG,LH等.The modulation of N-methyl-D-aspartate receptors by redox and alkylating reagents in rat cortical neurones in vitro.J Physiol.1993,465:303-23;Yao,Z等.Kinetics based on the base-catalyzed mechanism of a click reaction between glycol dimercaptoacetate and glycidyl phenyl ether.RSC Adv.2017,7(18):10881-10884.)。
气味内容:巯基乙醇为挥发性液体,具有强烈的刺激性气味,在Chemical Book这一致力于为化学行业用户提供最有价值信息的资源平台中,巯基乙醇被规定属于精神药品或易制毒化学品或管制类产品,不得非法销售或购买。公司或单位需取得相关部门发放的许可证并按法律规定进行登记备案。物流、仓储均需具有相应资质的物流公司和仓储公司,严禁使用快递运输。
11-巯基-1-十一醇的相比于巯基乙醇,其碳骨架延长了9个碳原子,分子量更大,一定程度上溶于水并且挥发性显著减弱,几乎无明显恶臭味。
2,2’-(1,2-乙二基双氧代)双乙硫醇是醇类衍生物,沸点为225℃(lit.),挥发性较弱,在本专利的优选浓度下可以溶于水,几乎无明显的恶臭味。
双巯基乙酸乙二醇酯属于酯类化合物,其沸点为137-139C 2mm Hg(lit.),挥发性较弱,在本发明的优选浓度下可以溶于水,几乎无明显的恶臭味。
以下各实施例中的抗体非还原型CE-SDS纯度检测均使用Beckman公司推荐的方法。
实施例1 不同浓度巯基乙醇在pH 9.0的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响
称取Tris 0.606g,SDS 0.5g,用42mL去离子水溶解至清,加入2.5mL巯基乙醇原液(14M),再用3M盐酸调节pH至9.0,接着用去离子水定容至50mL,得到含700mM巯基乙醇的样品缓冲液。接着用不含还原剂的样品缓冲液与含有700mM巯基乙醇的样品缓冲液,依次稀释得到含有70mM、7mM、0.7mM和0.07mM巯基乙醇的样品缓冲液。将重组抗蓖麻毒素人源化单克隆抗体(mAb1)用上述配制好的样品缓冲液稀释至1mg/mL,随后经水浴变性,进行毛细管凝胶电泳分析。
随后比较了不同浓度的巯基乙醇对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度的影响。结果见表1和图3。其中,发现游离巯基浓度在7mM及以上时,还原较彻底。
表1.不同浓度巯基乙醇对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS轻重链纯度影响
实施例2 不同浓度11-巯基-1-十一醇在pH 9.0的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响
称取Tris 0.606g,SDS 0.5g,11-巯基-1-十一醇(MW:204.37)0.7153g,用42mL去离子水溶解至清,再用3M盐酸调节pH至9.0,接着用去离子水定容至50mL,得到含70mM 11-巯基-1-十一醇的样品缓冲液。由于溶解度的限制,无法得到700mM浓度的缓冲液。接着用不含还原剂的样品缓冲液与含有70mM11-巯基-1-十一醇的样品缓冲液,依次稀释得到含有7mM、0.7mM和0.07mM11-巯基-1-十一醇的样品缓冲液。将重组抗蓖麻毒素人源化单克隆抗体(mAb1)用上述配制好的样品缓冲液稀释至1mg/mL,随后经水浴变性,进行毛细管凝胶电泳分析。
随后比较了不同浓度的11-巯基-1-十一醇对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度的影响。结果见表2和图4。其中,发现游离巯基浓度在7mM及以上时,还原较彻底。
表2.不同浓度11-巯基-1-十一醇对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS轻重链纯度影响
实施例3 不同浓度巯基丁二酸在pH 9.0的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响
称取Tris 0.606g,SDS 0.5g,巯基丁二酸(MW:150.15)5.255g,用42mL去离子水溶解至清,再用3M盐酸调节pH至9.0,接着用去离子水定容至50mL,得到含700mM巯基丁二酸的样品缓冲液。接着用不含还原剂的样品缓冲液与含有700mM巯基丁二酸的样品缓冲液,依次稀释得到含有70mM、7mM、0.7mM和0.07mM巯基丁二酸的样品缓冲液。将重组抗蓖麻毒素人源化单克隆抗体 (mAb1)用上述配制好的样品缓冲液稀释至1mg/mL,随后经水浴变性,进行毛细管凝胶电泳分析。
随后比较了不同浓度的巯基丁二酸对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度的影响。结果见图5。其中,发现不论游离巯基浓度如何,还原均不彻底,甚至高浓度的巯基丁二酸存在下,单抗样品的信号受到显著抑制。
实施例4 不同浓度二硫苏糖醇在pH 9.0的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响
称取Tris 0.606g,SDS 0.5g,二硫苏糖醇(MW:154.25)2.699g,用42mL去离子水溶解至清,再用3M盐酸调节pH至9.0,接着用去离子水定容至50mL,得到含350mM二硫苏糖醇的样品缓冲液。接着用不含还原剂的样品缓冲液与含有350mM二硫苏糖醇的样品缓冲液,依次稀释得到含有35mM、3.5mM、0.35mM和0.035mM二硫苏糖醇的样品缓冲液。将重组抗蓖麻毒素人源化单克隆抗体(mAb1)用上述配制好的样品缓冲液稀释至1mg/mL,随后经水浴变性,进行毛细管凝胶电泳分析。
随后比较了不同浓度的二硫苏糖醇对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度的影响。结果见图6。其中,发现游离巯基浓度在0.7mM及以上时,还原较彻底。但是发现二硫苏糖醇的浓度过高会形成干扰峰,因此推测游离巯基的合适浓度不得低于0.7mM,不得高于70mM,优选0.7-7mM。
实施例5 不同浓度2,2’-(1,2-乙二基双氧代)双乙硫醇在pH 9.0的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响
称取Tris 0.606g,SDS 0.5g,用42mL去离子水溶解至清,加入0.29mL 2,2’-(1,2-乙二基双氧代)双乙硫醇原液(6M),再用3M盐酸调节pH至9.0,接着用去离子水定容至50mL,得到含35mM 2,2’-(1,2-乙二基双氧代)双乙硫醇的样品缓冲液。由于溶解度的限制,无法得到350mM浓度的缓冲液。接着用不含还原剂的样品缓冲液与含有35mM 2,2’-(1,2-乙二基双氧代)双乙硫醇的样品缓冲液,依次稀释得到含有3.5mM、0.35mM和0.035mM 2,2’-(1,2-乙二基双氧代)双乙硫醇的样品缓冲液。将重组抗蓖麻毒素人源化单克隆抗体(mAb1)用上述配制好的样品缓冲液稀释至1mg/mL,随后经水浴变性,进行毛细管凝胶电泳分析。
随后比较了不同浓度的2,2’-(1,2-乙二基双氧代)双乙硫醇对重组抗蓖麻毒素 人源化单克隆抗体(mAb1)的还原型CE-SDS纯度的影响。结果见表3和图7。其中,发现游离巯基浓度在0.7mM及以上时,还原较彻底。
表3.不同浓度2,2’-(1,2-乙二基双氧代)双乙硫醇对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS轻重链纯度影响
实施例6 不同浓度双巯基乙酸乙二醇酯在pH 9.0的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响
称取Tris 0.606g,SDS 0.5g,用42mL去离子水溶解至清,加入0.29mL双巯基乙酸乙二醇酯(6M),再用3M盐酸调节pH至9.0,接着用去离子水定容至50mL,得到含35mM双巯基乙酸乙二醇酯的样品缓冲液。由于溶解度的限制,无法得到350mM浓度的缓冲液。接着用不含还原剂的样品缓冲液与含有35mM双巯基乙酸乙二醇酯的样品缓冲液,依次稀释得到含有3.5mM、0.35mM和0.035mM双巯基乙酸乙二醇酯的样品缓冲液。将重组抗蓖麻毒素人源化单克隆抗体(mAb1)用上述配制好的样品缓冲液稀释至1mg/mL,随后经水浴变性,进行毛细管凝胶电泳分析。
随后比较了不同浓度的双巯基乙酸乙二醇酯对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度的影响。结果见表4和图8。其中,发现游离巯基浓度在0.7mM及以上时,还原较彻底。
表4.不同浓度双巯基乙酸乙二醇酯对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS轻重链纯度影响
实施例7 在pH 9.0的缓冲液中游离巯基浓度为7mM的不同还原剂对重组抗HER2人源化单克隆抗体(mAb2,2013年批次)的还原型CE-SDS纯度影响
比较了在pH 9.0的缓冲液中游离巯基浓度为7mM的不同还原剂对重组抗HER2人源化单克隆抗体(mAb2,2013年批次)的还原型CE-SDS纯度影响,结果见表5和图9。发现除了巯基丁二酸以外,其他还原剂在游离巯基浓度为7mM时,还原较彻底。
表5.不同还原剂对重组抗HER2人源化单克隆抗体(mAb2,2013年批次)的还原型CE-SDS纯度影响
实施例8 在pH 9.0的缓冲液中游离巯基浓度为7mM的不同还原剂对重组抗HER2人源化单克隆抗体(mAb2,2016年批次)的还原型CE-SDS纯度影响
比较了在pH 9.0的缓冲液中游离巯基浓度为7mM的不同还原剂对重组抗HER2人源化单克隆抗体(mAb2,2016年批次)的还原型CE-SDS纯度影响,结果见表6和图10。发现除了巯基丁二酸以外,其他还原剂在游离巯基浓度为7mM时,还原较彻底。
表6.不同还原剂对重组抗HER2人源化单克隆抗体(mAb2,2016年批次)的还原型CE-SDS纯度影响
实施例9 在pH 9.0的缓冲液中游离巯基浓度为7mM的不同还原剂对T-DM1(ADC)的还原型CE-SDS纯度影响
比较了在pH 9.0的缓冲液中游离巯基浓度为7mM的不同还原剂对T-DM1(ADC)的还原型CE-SDS纯度影响,结果见表7和图11。发现除了巯基丁二酸以外,其他还原剂在游离巯基浓度为7mM时,还原较彻底。
表7.不同还原剂对T-DM1(ADC)的还原型CE-SDS纯度影响
实施例10 在pH 9.0的缓冲液中游离巯基浓度为7mM的不同还原剂对注射用重组人II型肿瘤坏死因子受体-抗体融合蛋白(Fusion protein)的还原型CE-SDS纯度影响
比较了在pH 9.0的缓冲液中游离巯基浓度为7mM的不同还原剂对注射用 重组人II型肿瘤坏死因子受体-抗体融合蛋白(Fusion protein)的还原型CE-SDS纯度影响,结果见表8和图12。发现除了巯基丁二酸以外,其他还原剂在游离巯基浓度为7mM时,还原较彻底。
表8.不同还原剂对注射用重组人II型肿瘤坏死因子受体-抗体融合蛋白(Fusion protein)的还原型CE-SDS纯度影响
实施例11 不同浓度巯基乙醇在pH 6.5的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响
称取磷酸钠0.164g,SDS 0.5g,用42mL去离子水溶解至清,加入2.5mL巯基乙醇原液(14M),再用1M柠檬酸调节pH至6.5,接着用去离子水定容至50mL,得到含700mM巯基乙醇的样品缓冲液。接着用该不含还原剂的样品缓冲液与含有700mM巯基乙醇的样品缓冲液,依次稀释得到含有70mM和7mM巯基乙醇的样品缓冲液。将重组抗蓖麻毒素人源化单克隆抗体(mAb1)用上述配制好的样品缓冲液稀释至1mg/mL,随后经水浴变性,进行毛细管凝胶电泳分析。
随后比较了不同浓度的巯基乙醇对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度的影响。结果见图13。其中,发现游离巯基浓度在7mM以上时,还原较彻底,优选70mM。
实施例12 不同浓度11-巯基-1-十一醇在pH 6.5的缓冲液中对重组抗蓖麻毒 素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响
称取磷酸钠0.164g,SDS 0.5g,11-巯基-1-十一醇(MW:204.37)0.7153g,用42mL去离子水溶解至清,再用1M柠檬酸调节pH至6.5,接着用去离子水定容至50mL,得到含70mM 11-巯基-1-十一醇的样品缓冲液。由于溶解度的限制,无法得到700mM浓度的缓冲液。接着用不含还原剂的样品缓冲液与含有70mM 11-巯基-1-十一醇的样品缓冲液,依次稀释得到含有7mM和0.7mM 11-巯基-1-十一醇的样品缓冲液。将重组抗蓖麻毒素人源化单克隆抗体(mAb1)用上述配制好的样品缓冲液稀释至1mg/mL,随后经水浴变性,进行毛细管凝胶电泳分析。
随后比较了不同浓度的11-巯基-1-十一醇对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度的影响。结果见图14。结果表明,在三种游离巯基浓度下,还原均未彻底。
实施例13 不同浓度二硫苏糖醇在pH 6.5的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响
称取磷酸钠0.164g,SDS 0.5g,二硫苏糖醇(MW:154.25)2.699g,用42mL去离子水溶解至清,再用1M柠檬酸调节pH至6.5,接着用去离子水定容至50mL,得到含350mM二硫苏糖醇的样品缓冲液。接着用不含还原剂的样品缓冲液与含有350mM二硫苏糖醇的样品缓冲液,依次稀释得到含有35mM、3.5mM和0.35mM二硫苏糖醇的样品缓冲液。将重组抗蓖麻毒素人源化单克隆抗体(mAb1)用上述配制好的样品缓冲液稀释至1mg/mL,随后经水浴变性,进行毛细管凝胶电泳分析。
随后比较了不同浓度的二硫苏糖醇对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度的影响。结果见图15。其中,发现游离巯基浓度在7mM及以上时,还原较彻底。但是发现游离巯基的浓度过高如达到70mM或者700mM时,会形成干扰峰,因此推测游离巯基的合适浓度不得低于7mM,不得高于70mM,优选7mM。
实施例14 不同浓度2,2’-(1,2-乙二基双氧代)双乙硫醇在pH 6.5的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响
称取磷酸钠0.164g,SDS 0.5g,用42mL去离子水溶解至清,加入0.29mL2,2’-(1,2-乙二基双氧代)双乙硫醇原液(6M),再用1M柠檬酸调节pH至6.5, 接着用去离子水定容至50mL,得到含35mM 2,2’-(1,2-乙二基双氧代)双乙硫醇的样品缓冲液。由于溶解度的限制,无法得到350mM浓度的缓冲液。接着用不含还原剂的样品缓冲液与含有35mM 2,2’-(1,2-乙二基双氧代)双乙硫醇的样品缓冲液,依次稀释得到含有3.5mM和0.35mM 2,2’-(1,2-乙二基双氧代)双乙硫醇的样品缓冲液。将重组抗蓖麻毒素人源化单克隆抗体(mAb1)用上述配制好的样品缓冲液稀释至1mg/mL,随后经水浴变性,进行毛细管凝胶电泳分析。
随后比较了不同浓度的2,2’-(1,2-乙二基双氧代)双乙硫醇对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度的影响。结果见图16。其中,发现游离巯基浓度在7mM以上时,还原较彻底,优选70mM。
实施例15 不同浓度双巯基乙酸乙二醇酯在pH 6.5的缓冲液中对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度影响
称取磷酸钠0.164g,SDS 0.5g,用42mL去离子水溶解至清,加入0.29mL双巯基乙酸乙二醇酯(6M),再用1M柠檬酸调节pH至6.5,接着用去离子水定容至50mL,得到含35mM双巯基乙酸乙二醇酯的样品缓冲液。由于溶解度的限制,无法得到350mM浓度的缓冲液。接着用不含还原剂的样品缓冲液与含有35mM双巯基乙酸乙二醇酯的样品缓冲液,依次稀释得到含有3.5mM和0.35mM双巯基乙酸乙二醇酯的样品缓冲液。将重组抗蓖麻毒素人源化单克隆抗体(mAb1)用上述配制好的样品缓冲液稀释至1mg/mL,随后经水浴变性,进行毛细管凝胶电泳分析。
随后比较了不同浓度的双巯基乙酸乙二醇酯对重组抗蓖麻毒素人源化单克隆抗体(mAb1)的还原型CE-SDS纯度的影响。结果见图17。其中,发现游离巯基浓度在0.7mM以上时,还原较彻底,优选70mM。
实施例16 在pH 9.0的缓冲液中游离巯基浓度为70mM的不同还原剂对胰高血糖素亲水/疏水性的影响
为了探究以上几种还原剂潜在的副反应,我们利用胰高血糖素进行了反相色谱的研究。采用Agilent 1200高效液相色谱系统(Agilent Technologies,Santa Clara,California)和Elite supersil C18反相色谱柱(Dalian,China)对从而对样品的疏水/亲水杂质进行了表征。流动相A为水(含0.1%甲酸),流动相B为乙腈(含0.1%甲酸),使用前用0.45μm尼龙膜过滤脱气。检测波长为214nm,柱温37℃下, 流速为0.8mL/min。初始流动相为10%溶剂B,持续5min,然后在25分钟内,B相比例上升至60%。进样量为20μL。
配置了不含SDS,pH 9.0的Tris-HCl缓冲液,为空白缓冲液。利用空白缓冲液溶解胰高血糖素,终浓度为0.5mg/mL,经过热孵育后进行反相色谱分析,以判断胰高血糖素的亲水/疏水性是否随热处理而改变。同时,配置了含不同还原剂的空白缓冲液,游离巯基终浓度为70mM,将其经过水浴后,进行反相色谱分析,以作为空白对照。最后利用含有不同还原剂的缓冲液分别溶解胰高血糖素,经过热孵育后进行反相色谱分析,以判断胰高血糖素的亲水/疏水性是否改变。结果如图18-23所示。
结果表明,胰高血糖素自身较为稳定,并且当还原剂与胰高血糖素同时存在并加热处理后,其反相色谱图几乎不变,表明还原剂未与胰高血糖素发生反应,表明以上探讨的各还原剂较为安全。
Claims (10)
- 一种含有还原剂的毛细管凝胶电泳检测试剂盒,其特征在于,所述样品缓冲液中含有至少一种还原剂;所述的还原剂选自11-巯基-1-十一醇、2,2’-(1,2-乙二基双氧代)双乙硫醇或双巯基乙酸乙二醇酯中的一种或多种;所述样品缓冲液中游离巯基的浓度为0.07-700mM;所述样品缓冲液选自Tris缓冲液或者磷酸盐缓冲液,所述样品缓冲液的pH值在3.0-10.0之间;所述样品缓冲液进一步含有十二烷基硫酸钠,其含量在0.1-5%(w/w)之间。
- 根据权利要求1所述的试剂盒,其特征在于,所述样品缓冲液中游离巯基的浓度为0.7-70mM。
- 根据权利要求1所述的试剂盒,其特征在于,所述样品缓冲液选自Tris-HCl缓冲液或者磷酸盐-柠檬酸缓冲液,pH值在6.5-10.0之间。
- 根据权利要求3所述的试剂盒,其特征在于,所述样品缓冲液选自pH 9.0的Tris-HCl缓冲液或pH 6.5的磷酸盐-柠檬酸缓冲液。
- 根据权利要求1-4任一项所述的样试剂盒,其特征在于,所述样品缓冲液十二烷基硫酸钠含量为0.5-2%(w/w)。
- 根据权利要求1-4任一项所述的试剂盒,其特征在于,所述样品缓冲液含有如下组分:(1)Tris-HCl缓冲液100mM;(2)十二烷基硫酸钠1%(w/w);(3)选自11-巯基-1-十一醇、2,2’-(1,2-乙二基双氧代)双乙硫醇或双巯基乙酸乙二醇酯中的一种或多种的还原剂,其总游离巯基的浓度为7mM;pH值为9.0。
- 根据权利要求1-6任一项所述的试剂盒,其特征在于,所述样品缓冲液含有如下组分:(1)磷酸盐-柠檬酸缓冲液20mM;(2)十二烷基硫酸钠1%(w/w);(3)选自11-巯基-1-十一醇、2,2’-(1,2-乙二基双氧代)双乙硫醇或双巯基乙酸乙二醇酯中的一种或多种的还原剂,其总游离巯基的浓度为70mM;pH值为6.5。
- 一种制备权利要求6所述试剂盒中的样品缓冲液的方法,其特征在于,所述方法包括:将处方量的Tris缓冲盐,处方量的十二烷基硫酸钠和处方量的还原剂溶解在去离子水中,用盐酸调节pH至9.0,接着用去离子水定容至50mL,得到样品缓冲液。
- 一种制备权利要求7所述试剂盒中的样品缓冲液的方法,其特征在于,所述方法包括:将处方量的磷酸盐,处方量的十二烷基硫酸钠和处方量的还原剂溶解在适量的去离子水中,用柠檬酸调节pH至6.5,接着用去离子水定容至50mL,得到样品缓冲液。
- 权利要求1-9任一项所述的试剂盒对蛋白样品进行还原型CE-SDS检测的应用,其中,所述蛋白样品选自重组抗蓖麻毒素人源化单克隆抗体(mAb1)、重组抗HER2人源化单克隆抗体(mAb2)、T-DM1(ADC)和注射用重组人II型肿瘤坏死因子受体-抗体融合蛋白(Fusion protein)。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110469256.7 | 2021-04-28 | ||
CN202110469256.7A CN113189183A (zh) | 2021-04-28 | 2021-04-28 | 含有还原剂的毛细管凝胶电泳检测试剂盒 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2022227362A1 true WO2022227362A1 (zh) | 2022-11-03 |
Family
ID=76980113
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2021/115986 WO2022227362A1 (zh) | 2021-04-28 | 2021-09-01 | 含有还原剂的毛细管凝胶电泳检测试剂盒 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN113189183A (zh) |
WO (1) | WO2022227362A1 (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113189183A (zh) * | 2021-04-28 | 2021-07-30 | 浙江大学 | 含有还原剂的毛细管凝胶电泳检测试剂盒 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6074837A (en) * | 1990-08-23 | 2000-06-13 | New York Blood Center, Inc. | Assays using a soluble fibrin-like monomer |
CN110225925A (zh) * | 2016-11-18 | 2019-09-10 | 拜康有限公司 | 快速高效的糖蛋白去糖基化 |
CN111316091A (zh) * | 2017-09-07 | 2020-06-19 | 百时美施贵宝公司 | 通过毛细管电泳进行纯度测定的方法 |
CN113189183A (zh) * | 2021-04-28 | 2021-07-30 | 浙江大学 | 含有还原剂的毛细管凝胶电泳检测试剂盒 |
-
2021
- 2021-04-28 CN CN202110469256.7A patent/CN113189183A/zh active Pending
- 2021-09-01 WO PCT/CN2021/115986 patent/WO2022227362A1/zh active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6074837A (en) * | 1990-08-23 | 2000-06-13 | New York Blood Center, Inc. | Assays using a soluble fibrin-like monomer |
CN110225925A (zh) * | 2016-11-18 | 2019-09-10 | 拜康有限公司 | 快速高效的糖蛋白去糖基化 |
CN111316091A (zh) * | 2017-09-07 | 2020-06-19 | 百时美施贵宝公司 | 通过毛细管电泳进行纯度测定的方法 |
CN113189183A (zh) * | 2021-04-28 | 2021-07-30 | 浙江大学 | 含有还原剂的毛细管凝胶电泳检测试剂盒 |
Non-Patent Citations (1)
Title |
---|
TANG L H, AIZENMAN E: "The modulation of N-methyl-D-aspartate receptors by redox and alkylating reagents in rat cortical neurones in vitro.", THE JOURNAL OF PHYSIOLOGY, vol. 465, no. 1, 1 June 1993 (1993-06-01), GB , pages 303 - 323, XP055981248, ISSN: 0022-3751, DOI: 10.1113/jphysiol.1993.sp019678 * |
Also Published As
Publication number | Publication date |
---|---|
CN113189183A (zh) | 2021-07-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Li et al. | Facile synthesis of red emitting 3-aminophenylboronic acid functionalized copper nanoclusters for rapid, selective and highly sensitive detection of glycoproteins | |
Ackerson et al. | Synthesis and bioconjugation of 2 and 3 nm-diameter gold nanoparticles | |
Santiago Gonzalez et al. | One step synthesis of the smallest photoluminescent and paramagnetic PVP-protected gold atomic clusters | |
Zhan et al. | Multidentate zwitterionic ligands provide compact and highly biocompatible quantum dots | |
Tracy et al. | Poly (ethylene glycol) ligands for high-resolution nanoparticle mass spectrometry | |
Kassam et al. | Place exchange reactions of alkyl thiols on gold nanoparticles | |
Aldeek et al. | Understanding the self-assembly of proteins onto gold nanoparticles and quantum dots driven by metal-histidine coordination | |
Xu et al. | Nitrilotriacetic acid-modified magnetic nanoparticles as a general agent to bind histidine-tagged proteins | |
Gies et al. | Electrospray mass spectrometry study of tiopronin monolayer-protected gold nanoclusters | |
Fabris et al. | Gold nanoclusters protected by conformationally constrained peptides | |
Burt et al. | Noble-metal nanoparticles directly conjugated to globular proteins | |
Shon et al. | Alkanethiolate-protected gold clusters generated from sodium S-dodecylthiosulfate (Bunte salts) | |
Lin et al. | Synthesis, characterization, and bioconjugation of fluorescent gold nanoclusters toward biological labeling applications | |
Zheng et al. | Ethylene glycol monolayer protected nanoparticles: synthesis, characterization, and interactions with biological molecules | |
Chakraborti et al. | Structure and activity of lysozyme on binding to ZnO nanoparticles | |
DeCollo et al. | Effects of Aromatic Thiols on Thiol− Disulfide Interchange Reactions That Occur during Protein Folding | |
Sha et al. | A fluorescence turn-on biosensor based on transferrin encapsulated gold nanoclusters for 5-hydroxytryptamine detection | |
Liu et al. | Surfactant-free synthesis and functionalization of highly fluorescent gold quantum dots | |
Paau et al. | Synthesis of 1.4 nm α-cyclodextrin-protected gold nanoparticles for luminescence sensing of mercury (II) with picomolar detection limit | |
Moser et al. | Ellman’s and aldrithiol assay as versatile and complementary tools for the quantification of thiol groups and ligands on nanomaterials | |
Kwon et al. | Ultrasensitive and selective recognition of peptide hormone using close-packed arrays of hPTHR-conjugated polymer nanoparticles | |
Shamsipur et al. | Novel blue-emitting gold nanoclusters confined in human hemoglobin, and their use as fluorescent probes for copper (II) and histidine | |
Wang et al. | Ultraefficient cap-exchange protocol to compact biofunctional quantum dots for sensitive ratiometric biosensing and cell imaging | |
Huang et al. | Facile preparation of highly blue fluorescent metal nanoclusters in organic media | |
Harkness et al. | A structural mass spectrometry strategy for the relative quantitation of ligands on mixed monolayer-protected gold nanoparticles |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21938826 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 21938826 Country of ref document: EP Kind code of ref document: A1 |