Unveiling a Glycation Hot Spot in a Recombinant Humanized Monoclonal Antibody
Biotechnological companies and regulatory agencies are pursuing the complete characterization of protein therapeutics in every detail as a means to mitigate risks of product quality related safety issues. During the characterization of a recombinant humanized monoclonal antibody (referred to as rhuM...
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| Veröffentlicht in: | Analytical chemistry (Washington) 2008-04, Vol.80 (7), p.2379-2390 |
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| creator | Zhang, Boyan Yang, Yi Yuk, Inn Pai, Roger McKay, Patrick Eigenbrot, Charles Dennis, Mark Katta, Viswanatham Francissen, Kathleen Champion |
| description | Biotechnological companies and regulatory agencies are pursuing the complete characterization of protein therapeutics in every detail as a means to mitigate risks of product quality related safety issues. During the characterization of a recombinant humanized monoclonal antibody (referred to as rhuMAb), electrospray mass spectrometric analysis suggested that the light chain was highly glycated. The glycated and unglycated materials, separated using boronate affinity chromatography, were fully characterized using tryptic peptide mapping and tandem mass spectrometry. Using an automatic SEQUEST search of the single protein database for this antibody and extensive manual investigations of the mass spectra of the matched peptides, multiple tentative glycation sites in the light and heavy chains were observed in the highly glycated (>53%) samples. A predominant glycation site was identified and confirmed to be lysine 49 on the light chain, by performing extensive sequence analysis on an isolated glycated peptide utilizing Edman degradation analysis and MALDI-TOF/TOF mass spectrometry. Sequence alignments of rhuMAb with 12 other recombinant monoclonal antibodies and computer modeling of the Fab part of rhuMAb suggest that the unusually high level of glycation of lysine residue 49, which is located adjacent to the second complementarity-determining region (CDR2) in the light chain, is due to a spatial proximity effect in catalyzing the Amadori rearrangement by aspartic acid residue 31 in the CDR1 on the light chain. |
| doi_str_mv | 10.1021/ac701810q |
| format | Article |
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During the characterization of a recombinant humanized monoclonal antibody (referred to as rhuMAb), electrospray mass spectrometric analysis suggested that the light chain was highly glycated. The glycated and unglycated materials, separated using boronate affinity chromatography, were fully characterized using tryptic peptide mapping and tandem mass spectrometry. Using an automatic SEQUEST search of the single protein database for this antibody and extensive manual investigations of the mass spectra of the matched peptides, multiple tentative glycation sites in the light and heavy chains were observed in the highly glycated (>53%) samples. A predominant glycation site was identified and confirmed to be lysine 49 on the light chain, by performing extensive sequence analysis on an isolated glycated peptide utilizing Edman degradation analysis and MALDI-TOF/TOF mass spectrometry. Sequence alignments of rhuMAb with 12 other recombinant monoclonal antibodies and computer modeling of the Fab part of rhuMAb suggest that the unusually high level of glycation of lysine residue 49, which is located adjacent to the second complementarity-determining region (CDR2) in the light chain, is due to a spatial proximity effect in catalyzing the Amadori rearrangement by aspartic acid residue 31 in the CDR1 on the light chain.</description><identifier>ISSN: 0003-2700</identifier><identifier>EISSN: 1520-6882</identifier><identifier>DOI: 10.1021/ac701810q</identifier><identifier>PMID: 18307322</identifier><identifier>CODEN: ANCHAM</identifier><language>eng</language><publisher>Washington, DC: American Chemical Society</publisher><subject>Amino Acid Sequence ; Analytical chemistry ; Antibodies, immunoglobulins ; Antibodies, Monoclonal - analysis ; Antibodies, Monoclonal - chemistry ; Antibodies, Monoclonal - immunology ; Biochemistry ; Biological and medical sciences ; Chemistry ; Chromatographic methods and physical methods associated with chromatography ; Exact sciences and technology ; Fundamental and applied biological sciences. Psychology ; Fundamental immunology ; Glycosylation ; Humans ; Immune system ; Mass Spectrometry ; Models, Molecular ; Molecular immunology ; Molecular Sequence Data ; Monoclonal antibodies ; Other chromatographic methods ; Protein Structure, Quaternary ; Proteins ; Recombinant Proteins - analysis ; Recombinant Proteins - chemistry ; Recombinant Proteins - immunology ; Spectrometric and optical methods</subject><ispartof>Analytical chemistry (Washington), 2008-04, Vol.80 (7), p.2379-2390</ispartof><rights>Copyright © 2008 American Chemical Society</rights><rights>2008 INIST-CNRS</rights><rights>Copyright American Chemical Society Apr 1, 2008</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a474t-e4289c68a49b869f45b321da46306590ec3e3da0df39b93f2b5d5d1d3ab9c6bd3</citedby><cites>FETCH-LOGICAL-a474t-e4289c68a49b869f45b321da46306590ec3e3da0df39b93f2b5d5d1d3ab9c6bd3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/ac701810q$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/ac701810q$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,776,780,2752,27053,27901,27902,56713,56763</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=20246874$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18307322$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Zhang, Boyan</creatorcontrib><creatorcontrib>Yang, Yi</creatorcontrib><creatorcontrib>Yuk, Inn</creatorcontrib><creatorcontrib>Pai, Roger</creatorcontrib><creatorcontrib>McKay, Patrick</creatorcontrib><creatorcontrib>Eigenbrot, Charles</creatorcontrib><creatorcontrib>Dennis, Mark</creatorcontrib><creatorcontrib>Katta, Viswanatham</creatorcontrib><creatorcontrib>Francissen, Kathleen Champion</creatorcontrib><title>Unveiling a Glycation Hot Spot in a Recombinant Humanized Monoclonal Antibody</title><title>Analytical chemistry (Washington)</title><addtitle>Anal. Chem</addtitle><description>Biotechnological companies and regulatory agencies are pursuing the complete characterization of protein therapeutics in every detail as a means to mitigate risks of product quality related safety issues. During the characterization of a recombinant humanized monoclonal antibody (referred to as rhuMAb), electrospray mass spectrometric analysis suggested that the light chain was highly glycated. The glycated and unglycated materials, separated using boronate affinity chromatography, were fully characterized using tryptic peptide mapping and tandem mass spectrometry. Using an automatic SEQUEST search of the single protein database for this antibody and extensive manual investigations of the mass spectra of the matched peptides, multiple tentative glycation sites in the light and heavy chains were observed in the highly glycated (>53%) samples. A predominant glycation site was identified and confirmed to be lysine 49 on the light chain, by performing extensive sequence analysis on an isolated glycated peptide utilizing Edman degradation analysis and MALDI-TOF/TOF mass spectrometry. Sequence alignments of rhuMAb with 12 other recombinant monoclonal antibodies and computer modeling of the Fab part of rhuMAb suggest that the unusually high level of glycation of lysine residue 49, which is located adjacent to the second complementarity-determining region (CDR2) in the light chain, is due to a spatial proximity effect in catalyzing the Amadori rearrangement by aspartic acid residue 31 in the CDR1 on the light chain.</description><subject>Amino Acid Sequence</subject><subject>Analytical chemistry</subject><subject>Antibodies, immunoglobulins</subject><subject>Antibodies, Monoclonal - analysis</subject><subject>Antibodies, Monoclonal - chemistry</subject><subject>Antibodies, Monoclonal - immunology</subject><subject>Biochemistry</subject><subject>Biological and medical sciences</subject><subject>Chemistry</subject><subject>Chromatographic methods and physical methods associated with chromatography</subject><subject>Exact sciences and technology</subject><subject>Fundamental and applied biological sciences. 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Psychology</topic><topic>Fundamental immunology</topic><topic>Glycosylation</topic><topic>Humans</topic><topic>Immune system</topic><topic>Mass Spectrometry</topic><topic>Models, Molecular</topic><topic>Molecular immunology</topic><topic>Molecular Sequence Data</topic><topic>Monoclonal antibodies</topic><topic>Other chromatographic methods</topic><topic>Protein Structure, Quaternary</topic><topic>Proteins</topic><topic>Recombinant Proteins - analysis</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - immunology</topic><topic>Spectrometric and optical methods</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Zhang, Boyan</creatorcontrib><creatorcontrib>Yang, Yi</creatorcontrib><creatorcontrib>Yuk, Inn</creatorcontrib><creatorcontrib>Pai, Roger</creatorcontrib><creatorcontrib>McKay, Patrick</creatorcontrib><creatorcontrib>Eigenbrot, Charles</creatorcontrib><creatorcontrib>Dennis, Mark</creatorcontrib><creatorcontrib>Katta, Viswanatham</creatorcontrib><creatorcontrib>Francissen, Kathleen Champion</creatorcontrib><collection>Istex</collection><collection>Pascal-Francis</collection><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>Zhang, Boyan</au><au>Yang, Yi</au><au>Yuk, Inn</au><au>Pai, Roger</au><au>McKay, Patrick</au><au>Eigenbrot, Charles</au><au>Dennis, Mark</au><au>Katta, Viswanatham</au><au>Francissen, Kathleen Champion</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Unveiling a Glycation Hot Spot in a Recombinant Humanized Monoclonal Antibody</atitle><jtitle>Analytical chemistry (Washington)</jtitle><addtitle>Anal. Chem</addtitle><date>2008-04-01</date><risdate>2008</risdate><volume>80</volume><issue>7</issue><spage>2379</spage><epage>2390</epage><pages>2379-2390</pages><issn>0003-2700</issn><eissn>1520-6882</eissn><coden>ANCHAM</coden><abstract>Biotechnological companies and regulatory agencies are pursuing the complete characterization of protein therapeutics in every detail as a means to mitigate risks of product quality related safety issues. During the characterization of a recombinant humanized monoclonal antibody (referred to as rhuMAb), electrospray mass spectrometric analysis suggested that the light chain was highly glycated. The glycated and unglycated materials, separated using boronate affinity chromatography, were fully characterized using tryptic peptide mapping and tandem mass spectrometry. Using an automatic SEQUEST search of the single protein database for this antibody and extensive manual investigations of the mass spectra of the matched peptides, multiple tentative glycation sites in the light and heavy chains were observed in the highly glycated (>53%) samples. A predominant glycation site was identified and confirmed to be lysine 49 on the light chain, by performing extensive sequence analysis on an isolated glycated peptide utilizing Edman degradation analysis and MALDI-TOF/TOF mass spectrometry. Sequence alignments of rhuMAb with 12 other recombinant monoclonal antibodies and computer modeling of the Fab part of rhuMAb suggest that the unusually high level of glycation of lysine residue 49, which is located adjacent to the second complementarity-determining region (CDR2) in the light chain, is due to a spatial proximity effect in catalyzing the Amadori rearrangement by aspartic acid residue 31 in the CDR1 on the light chain.</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>18307322</pmid><doi>10.1021/ac701810q</doi><tpages>12</tpages></addata></record> |
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| source | ACS Publications; MEDLINE |
| subjects | Amino Acid Sequence Analytical chemistry Antibodies, immunoglobulins Antibodies, Monoclonal - analysis Antibodies, Monoclonal - chemistry Antibodies, Monoclonal - immunology Biochemistry Biological and medical sciences Chemistry Chromatographic methods and physical methods associated with chromatography Exact sciences and technology Fundamental and applied biological sciences. Psychology Fundamental immunology Glycosylation Humans Immune system Mass Spectrometry Models, Molecular Molecular immunology Molecular Sequence Data Monoclonal antibodies Other chromatographic methods Protein Structure, Quaternary Proteins Recombinant Proteins - analysis Recombinant Proteins - chemistry Recombinant Proteins - immunology Spectrometric and optical methods |
| title | Unveiling a Glycation Hot Spot in a Recombinant Humanized Monoclonal Antibody |
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