High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides, Proteins, And Monoclonal Antibodies with a Flow-through Microvial Interface
Capillary isoelectric focusing directly coupled to high resolution mass spectrometry (cIEF–MS) provides information on amphoteric molecules, including isoelectric point and accurate mass, which enables structural interrogation of biopolymer pI variants. The coupling of cIEF with MS was facilitated b...
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| Veröffentlicht in: | Analytical chemistry (Washington) 2018-08, Vol.90 (15), p.9495-9503 |
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| creator | Wang, Lingyu Bo, Tao Zhang, Zhengxiang Wang, Guanbo Tong, Wenjun Da Yong Chen, David |
| description | Capillary isoelectric focusing directly coupled to high resolution mass spectrometry (cIEF–MS) provides information on amphoteric molecules, including isoelectric point and accurate mass, which enables structural interrogation of biopolymer pI variants. The coupling of cIEF with MS was facilitated by a flow-through microvial interface, made by stainless steel with high chemical resistance and mechanical robustness. Two on-column electrolyte configurations of cIEF–MS were demonstrated using peptide and protein pI markers. The pI resolution was 0.02 pH unit in the pH range of 5.5 to 7.0, with no anticonvective reagent (glycerol) added. High resolution Orbitrap detector provides mass spectra for midsized proteins ( |
| doi_str_mv | 10.1021/acs.analchem.8b02175 |
| format | Article |
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The coupling of cIEF with MS was facilitated by a flow-through microvial interface, made by stainless steel with high chemical resistance and mechanical robustness. Two on-column electrolyte configurations of cIEF–MS were demonstrated using peptide and protein pI markers. The pI resolution was 0.02 pH unit in the pH range of 5.5 to 7.0, with no anticonvective reagent (glycerol) added. High resolution Orbitrap detector provides mass spectra for midsized proteins (<30 kDa), enabling deconvolution with high accuracy for IEF-focused low abundance species. Charge heterogeneity of therapeutic monoclonal antibodies (mAb) is one of the most important attributes in the biopharmaceutical industry, and it is routinely monitored by IEF and fractionation-based methods. As a proof of concept, the commercial formulation of infliximab was directly analyzed using cIEF–MS for separation and online identification of mAb charge variants. The main intact antibody species along with two basic and one acidic variants were observed, and their accurate molecular weights (M w) recorded by MS detector readily revealed the structural differences of these variants. Variants with 0.1 unit in pI difference and 1 Da difference in molecular weight were readily resolved. The deconvoluted intact M w values showed ppm level accuracy compared to theoretical predictions.</description><identifier>ISSN: 0003-2700</identifier><identifier>EISSN: 1520-6882</identifier><identifier>DOI: 10.1021/acs.analchem.8b02175</identifier><identifier>PMID: 29993237</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Analytical chemistry ; Antibodies, Monoclonal - analysis ; Biopolymers ; Capillary flow ; Chemistry ; Coupling (molecular) ; Electrophoresis, Capillary - instrumentation ; Equipment Design ; Fractionation ; Glycerol - chemistry ; High resolution ; Hydrogen-Ion Concentration ; Infliximab - analysis ; Interrogation ; Isoelectric focusing ; Isoelectric Focusing - instrumentation ; Isoelectric Point ; Mass spectra ; Mass spectrometry ; Mass Spectrometry - instrumentation ; Molecular weight ; Monoclonal antibodies ; Organic chemistry ; Peptides ; Peptides - analysis ; Proteins ; Proteins - analysis ; Reagents ; Spectroscopy ; Stainless steel</subject><ispartof>Analytical chemistry (Washington), 2018-08, Vol.90 (15), p.9495-9503</ispartof><rights>Copyright American Chemical Society Aug 7, 2018</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a413t-f74cc0533004e123416af7d3b31ec20b92fd7a04af3ffde831a30be169615e253</citedby><cites>FETCH-LOGICAL-a413t-f74cc0533004e123416af7d3b31ec20b92fd7a04af3ffde831a30be169615e253</cites><orcidid>0000-0002-5468-8993 ; 0000-0002-3669-6041</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/acs.analchem.8b02175$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/acs.analchem.8b02175$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,780,784,2765,27076,27924,27925,56738,56788</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/29993237$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wang, Lingyu</creatorcontrib><creatorcontrib>Bo, Tao</creatorcontrib><creatorcontrib>Zhang, Zhengxiang</creatorcontrib><creatorcontrib>Wang, Guanbo</creatorcontrib><creatorcontrib>Tong, Wenjun</creatorcontrib><creatorcontrib>Da Yong Chen, David</creatorcontrib><title>High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides, Proteins, And Monoclonal Antibodies with a Flow-through Microvial Interface</title><title>Analytical chemistry (Washington)</title><addtitle>Anal. Chem</addtitle><description>Capillary isoelectric focusing directly coupled to high resolution mass spectrometry (cIEF–MS) provides information on amphoteric molecules, including isoelectric point and accurate mass, which enables structural interrogation of biopolymer pI variants. The coupling of cIEF with MS was facilitated by a flow-through microvial interface, made by stainless steel with high chemical resistance and mechanical robustness. Two on-column electrolyte configurations of cIEF–MS were demonstrated using peptide and protein pI markers. The pI resolution was 0.02 pH unit in the pH range of 5.5 to 7.0, with no anticonvective reagent (glycerol) added. High resolution Orbitrap detector provides mass spectra for midsized proteins (<30 kDa), enabling deconvolution with high accuracy for IEF-focused low abundance species. Charge heterogeneity of therapeutic monoclonal antibodies (mAb) is one of the most important attributes in the biopharmaceutical industry, and it is routinely monitored by IEF and fractionation-based methods. As a proof of concept, the commercial formulation of infliximab was directly analyzed using cIEF–MS for separation and online identification of mAb charge variants. The main intact antibody species along with two basic and one acidic variants were observed, and their accurate molecular weights (M w) recorded by MS detector readily revealed the structural differences of these variants. Variants with 0.1 unit in pI difference and 1 Da difference in molecular weight were readily resolved. The deconvoluted intact M w values showed ppm level accuracy compared to theoretical predictions.</description><subject>Analytical chemistry</subject><subject>Antibodies, Monoclonal - analysis</subject><subject>Biopolymers</subject><subject>Capillary flow</subject><subject>Chemistry</subject><subject>Coupling (molecular)</subject><subject>Electrophoresis, Capillary - instrumentation</subject><subject>Equipment Design</subject><subject>Fractionation</subject><subject>Glycerol - chemistry</subject><subject>High resolution</subject><subject>Hydrogen-Ion Concentration</subject><subject>Infliximab - analysis</subject><subject>Interrogation</subject><subject>Isoelectric focusing</subject><subject>Isoelectric Focusing - instrumentation</subject><subject>Isoelectric Point</subject><subject>Mass spectra</subject><subject>Mass spectrometry</subject><subject>Mass Spectrometry - instrumentation</subject><subject>Molecular weight</subject><subject>Monoclonal antibodies</subject><subject>Organic chemistry</subject><subject>Peptides</subject><subject>Peptides - analysis</subject><subject>Proteins</subject><subject>Proteins - analysis</subject><subject>Reagents</subject><subject>Spectroscopy</subject><subject>Stainless steel</subject><issn>0003-2700</issn><issn>1520-6882</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kU1vEzEQhi0EoqHwDxCyxIUDG8b2fh6jiNBIjaj4OK-83nHjancdbC9V_w6_lImS9sCBk-3R876emZextwKWAqT4pE1c6kkPZo_jsu6oVBXP2EIUErKyruVztgAAlckK4IK9ivEOQAgQ5Ut2IZumUVJVC_bnyt3u-TeMfpiT8xNf64MbBh0e-DZ6HNCk4AzfeDNHN93ynY6Rfz8cy37ERNiKeniILnJv-Q0ekusxfuQ3wSd0E91WU893fvJm8ETSM7nO9w4jv3dpzzXfDP4-S_vgZ-pk50zwvx2B2ylhsNrga_bC6iHim_N5yX5uPv9YX2XXX79s16vrTOdCpcxWuTFQKAWQo5AqF6W2Va86JdBI6Bpp-0pDrq2ytsdaCa2gQ1E2pShQFuqSfTj5HoL_NWNM7eiiQVrGhH6OrYSyVrkSsiL0_T_onZ8DjUeUgLwpZC1qovITRSPFGNC2h-BGWm0roD1m2FKG7WOG7TlDkr07m8_diP2T6DE0AuAEHOVPH__X8y9AVK2t</recordid><startdate>20180807</startdate><enddate>20180807</enddate><creator>Wang, Lingyu</creator><creator>Bo, Tao</creator><creator>Zhang, Zhengxiang</creator><creator>Wang, Guanbo</creator><creator>Tong, Wenjun</creator><creator>Da Yong Chen, David</creator><general>American Chemical Society</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QF</scope><scope>7QO</scope><scope>7QQ</scope><scope>7SC</scope><scope>7SE</scope><scope>7SP</scope><scope>7SR</scope><scope>7TA</scope><scope>7TB</scope><scope>7TM</scope><scope>7U5</scope><scope>7U7</scope><scope>7U9</scope><scope>8BQ</scope><scope>8FD</scope><scope>C1K</scope><scope>F28</scope><scope>FR3</scope><scope>H8D</scope><scope>H8G</scope><scope>H94</scope><scope>JG9</scope><scope>JQ2</scope><scope>KR7</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>P64</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0002-5468-8993</orcidid><orcidid>https://orcid.org/0000-0002-3669-6041</orcidid></search><sort><creationdate>20180807</creationdate><title>High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides, Proteins, And Monoclonal Antibodies with a Flow-through Microvial Interface</title><author>Wang, Lingyu ; Bo, Tao ; Zhang, Zhengxiang ; Wang, Guanbo ; Tong, Wenjun ; Da Yong Chen, David</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a413t-f74cc0533004e123416af7d3b31ec20b92fd7a04af3ffde831a30be169615e253</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Analytical chemistry</topic><topic>Antibodies, Monoclonal - analysis</topic><topic>Biopolymers</topic><topic>Capillary flow</topic><topic>Chemistry</topic><topic>Coupling (molecular)</topic><topic>Electrophoresis, Capillary - instrumentation</topic><topic>Equipment Design</topic><topic>Fractionation</topic><topic>Glycerol - chemistry</topic><topic>High resolution</topic><topic>Hydrogen-Ion Concentration</topic><topic>Infliximab - analysis</topic><topic>Interrogation</topic><topic>Isoelectric focusing</topic><topic>Isoelectric Focusing - instrumentation</topic><topic>Isoelectric Point</topic><topic>Mass spectra</topic><topic>Mass spectrometry</topic><topic>Mass Spectrometry - instrumentation</topic><topic>Molecular weight</topic><topic>Monoclonal antibodies</topic><topic>Organic chemistry</topic><topic>Peptides</topic><topic>Peptides - analysis</topic><topic>Proteins</topic><topic>Proteins - analysis</topic><topic>Reagents</topic><topic>Spectroscopy</topic><topic>Stainless steel</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wang, Lingyu</creatorcontrib><creatorcontrib>Bo, Tao</creatorcontrib><creatorcontrib>Zhang, Zhengxiang</creatorcontrib><creatorcontrib>Wang, Guanbo</creatorcontrib><creatorcontrib>Tong, Wenjun</creatorcontrib><creatorcontrib>Da Yong Chen, David</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Aluminium Industry Abstracts</collection><collection>Biotechnology Research Abstracts</collection><collection>Ceramic Abstracts</collection><collection>Computer and Information Systems Abstracts</collection><collection>Corrosion Abstracts</collection><collection>Electronics & Communications Abstracts</collection><collection>Engineered Materials Abstracts</collection><collection>Materials Business File</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>Toxicology Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ANTE: Abstracts in New Technology & Engineering</collection><collection>Engineering Research Database</collection><collection>Aerospace Database</collection><collection>Copper Technical Reference Library</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Materials Research Database</collection><collection>ProQuest Computer Science Collection</collection><collection>Civil Engineering Abstracts</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>Computer and Information Systems Abstracts Academic</collection><collection>Computer and Information Systems Abstracts Professional</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Analytical chemistry (Washington)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wang, Lingyu</au><au>Bo, Tao</au><au>Zhang, Zhengxiang</au><au>Wang, Guanbo</au><au>Tong, Wenjun</au><au>Da Yong Chen, David</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides, Proteins, And Monoclonal Antibodies with a Flow-through Microvial Interface</atitle><jtitle>Analytical chemistry (Washington)</jtitle><addtitle>Anal. Chem</addtitle><date>2018-08-07</date><risdate>2018</risdate><volume>90</volume><issue>15</issue><spage>9495</spage><epage>9503</epage><pages>9495-9503</pages><issn>0003-2700</issn><eissn>1520-6882</eissn><abstract>Capillary isoelectric focusing directly coupled to high resolution mass spectrometry (cIEF–MS) provides information on amphoteric molecules, including isoelectric point and accurate mass, which enables structural interrogation of biopolymer pI variants. The coupling of cIEF with MS was facilitated by a flow-through microvial interface, made by stainless steel with high chemical resistance and mechanical robustness. Two on-column electrolyte configurations of cIEF–MS were demonstrated using peptide and protein pI markers. The pI resolution was 0.02 pH unit in the pH range of 5.5 to 7.0, with no anticonvective reagent (glycerol) added. High resolution Orbitrap detector provides mass spectra for midsized proteins (<30 kDa), enabling deconvolution with high accuracy for IEF-focused low abundance species. Charge heterogeneity of therapeutic monoclonal antibodies (mAb) is one of the most important attributes in the biopharmaceutical industry, and it is routinely monitored by IEF and fractionation-based methods. As a proof of concept, the commercial formulation of infliximab was directly analyzed using cIEF–MS for separation and online identification of mAb charge variants. The main intact antibody species along with two basic and one acidic variants were observed, and their accurate molecular weights (M w) recorded by MS detector readily revealed the structural differences of these variants. Variants with 0.1 unit in pI difference and 1 Da difference in molecular weight were readily resolved. The deconvoluted intact M w values showed ppm level accuracy compared to theoretical predictions.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>29993237</pmid><doi>10.1021/acs.analchem.8b02175</doi><tpages>9</tpages><orcidid>https://orcid.org/0000-0002-5468-8993</orcidid><orcidid>https://orcid.org/0000-0002-3669-6041</orcidid></addata></record> |
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| subjects | Analytical chemistry Antibodies, Monoclonal - analysis Biopolymers Capillary flow Chemistry Coupling (molecular) Electrophoresis, Capillary - instrumentation Equipment Design Fractionation Glycerol - chemistry High resolution Hydrogen-Ion Concentration Infliximab - analysis Interrogation Isoelectric focusing Isoelectric Focusing - instrumentation Isoelectric Point Mass spectra Mass spectrometry Mass Spectrometry - instrumentation Molecular weight Monoclonal antibodies Organic chemistry Peptides Peptides - analysis Proteins Proteins - analysis Reagents Spectroscopy Stainless steel |
| title | High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides, Proteins, And Monoclonal Antibodies with a Flow-through Microvial Interface |
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