HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals
In contrast with conventional drugs, biopharmaceuticals are highly complex molecules with remarkable heterogeneity. Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the gly...
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| Veröffentlicht in: | Analytical and bioanalytical chemistry 2010-09, Vol.398 (2), p.905-914 |
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| creator | Melmer, Michael Stangler, Thomas Schiefermeier, Mark Brunner, Werner Toll, Hansjörg Rupprechter, Alfred Lindner, Wolfgang Premstaller, Andreas |
| description | In contrast with conventional drugs, biopharmaceuticals are highly complex molecules with remarkable heterogeneity. Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the glycan pattern is an important issue for characterization and quality control in the biopharmaceutical industry. In this publication we describe a complete workflow for the analysis of protein N-glycans. The sample-preparation procedure, consisting of the release of the N-glycans by PNGase-F, followed by fluorescence labeling with 2-aminobenzamide and removal of excess label, was optimized to avoid alteration of the glycan sample. Subsequently, labeled glycans were analyzed by hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection. The developed method was validated for analysis of antibody N-glycans. To demonstrate the accuracy of the method an antibody sample was additionally analyzed by an orthogonal method. The antibody was digested with lysyl endopeptidase and the (glyco-)peptides were analyzed by RP-HPLC-MS. The consistency of the results between these two methods demonstrates the reliability of the glycan analysis method introduced herein. |
| doi_str_mv | 10.1007/s00216-010-3988-x |
| format | Article |
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Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the glycan pattern is an important issue for characterization and quality control in the biopharmaceutical industry. In this publication we describe a complete workflow for the analysis of protein N-glycans. The sample-preparation procedure, consisting of the release of the N-glycans by PNGase-F, followed by fluorescence labeling with 2-aminobenzamide and removal of excess label, was optimized to avoid alteration of the glycan sample. Subsequently, labeled glycans were analyzed by hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection. The developed method was validated for analysis of antibody N-glycans. To demonstrate the accuracy of the method an antibody sample was additionally analyzed by an orthogonal method. The antibody was digested with lysyl endopeptidase and the (glyco-)peptides were analyzed by RP-HPLC-MS. The consistency of the results between these two methods demonstrates the reliability of the glycan analysis method introduced herein.</description><identifier>ISSN: 1618-2642</identifier><identifier>EISSN: 1618-2650</identifier><identifier>DOI: 10.1007/s00216-010-3988-x</identifier><identifier>PMID: 20640408</identifier><language>eng</language><publisher>Berlin/Heidelberg: Berlin/Heidelberg : Springer-Verlag</publisher><subject>2-Aminobenzamide ; Analysis ; Analytical Chemistry ; Antibodies ; Biochemistry ; Biopharmaceutical ; Biopharmaceutics ; Carbohydrate Sequence ; Characterization and Evaluation of Materials ; Chemistry ; Chemistry and Materials Science ; Chromatographic methods and physical methods associated with chromatography ; Chromatography, High Pressure Liquid - methods ; Chromatography, Liquid - methods ; Drugs ; Effectiveness ; Exact sciences and technology ; Fluorescence ; Fluorescence labeling ; Fluorescent Dyes - chemistry ; Food Science ; Glycan ; Glycoproteins ; Glycoproteins - chemistry ; Glycoproteins - metabolism ; Health aspects ; Heterogeneity ; High performance liquid chromatography ; HILIC ; Laboratory Medicine ; Liquid chromatography ; Mass Spectrometry ; Molecular Sequence Data ; Monitoring/Environmental Analysis ; N-glycans ; Original Paper ; ortho-Aminobenzoates - chemistry ; Other chromatographic methods ; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - metabolism ; Polysaccharides ; Polysaccharides - analysis ; Polysaccharides - chemistry ; Polysaccharides - metabolism ; Proteins ; Recombinant ; Reproducibility of Results ; Sample preparation ; Sensitivity and Specificity ; Spectrometric and optical methods</subject><ispartof>Analytical and bioanalytical chemistry, 2010-09, Vol.398 (2), p.905-914</ispartof><rights>Springer-Verlag 2010</rights><rights>2015 INIST-CNRS</rights><rights>COPYRIGHT 2010 Springer</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c567t-25b7482c7dd66e17671d6607a198d49b7c690c27cb08a5b80f28bd163a5fc5ab3</citedby><cites>FETCH-LOGICAL-c567t-25b7482c7dd66e17671d6607a198d49b7c690c27cb08a5b80f28bd163a5fc5ab3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00216-010-3988-x$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00216-010-3988-x$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=23248493$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20640408$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Melmer, Michael</creatorcontrib><creatorcontrib>Stangler, Thomas</creatorcontrib><creatorcontrib>Schiefermeier, Mark</creatorcontrib><creatorcontrib>Brunner, Werner</creatorcontrib><creatorcontrib>Toll, Hansjörg</creatorcontrib><creatorcontrib>Rupprechter, Alfred</creatorcontrib><creatorcontrib>Lindner, Wolfgang</creatorcontrib><creatorcontrib>Premstaller, Andreas</creatorcontrib><title>HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals</title><title>Analytical and bioanalytical chemistry</title><addtitle>Anal Bioanal Chem</addtitle><addtitle>Anal Bioanal Chem</addtitle><description>In contrast with conventional drugs, biopharmaceuticals are highly complex molecules with remarkable heterogeneity. Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the glycan pattern is an important issue for characterization and quality control in the biopharmaceutical industry. In this publication we describe a complete workflow for the analysis of protein N-glycans. The sample-preparation procedure, consisting of the release of the N-glycans by PNGase-F, followed by fluorescence labeling with 2-aminobenzamide and removal of excess label, was optimized to avoid alteration of the glycan sample. Subsequently, labeled glycans were analyzed by hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection. The developed method was validated for analysis of antibody N-glycans. To demonstrate the accuracy of the method an antibody sample was additionally analyzed by an orthogonal method. The antibody was digested with lysyl endopeptidase and the (glyco-)peptides were analyzed by RP-HPLC-MS. The consistency of the results between these two methods demonstrates the reliability of the glycan analysis method introduced herein.</description><subject>2-Aminobenzamide</subject><subject>Analysis</subject><subject>Analytical Chemistry</subject><subject>Antibodies</subject><subject>Biochemistry</subject><subject>Biopharmaceutical</subject><subject>Biopharmaceutics</subject><subject>Carbohydrate Sequence</subject><subject>Characterization and Evaluation of Materials</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Chromatographic methods and physical methods associated with chromatography</subject><subject>Chromatography, High Pressure Liquid - methods</subject><subject>Chromatography, Liquid - methods</subject><subject>Drugs</subject><subject>Effectiveness</subject><subject>Exact sciences and technology</subject><subject>Fluorescence</subject><subject>Fluorescence labeling</subject><subject>Fluorescent Dyes - chemistry</subject><subject>Food Science</subject><subject>Glycan</subject><subject>Glycoproteins</subject><subject>Glycoproteins - chemistry</subject><subject>Glycoproteins - metabolism</subject><subject>Health aspects</subject><subject>Heterogeneity</subject><subject>High performance liquid chromatography</subject><subject>HILIC</subject><subject>Laboratory Medicine</subject><subject>Liquid chromatography</subject><subject>Mass Spectrometry</subject><subject>Molecular Sequence Data</subject><subject>Monitoring/Environmental Analysis</subject><subject>N-glycans</subject><subject>Original Paper</subject><subject>ortho-Aminobenzoates - chemistry</subject><subject>Other chromatographic methods</subject><subject>Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - metabolism</subject><subject>Polysaccharides</subject><subject>Polysaccharides - analysis</subject><subject>Polysaccharides - chemistry</subject><subject>Polysaccharides - metabolism</subject><subject>Proteins</subject><subject>Recombinant</subject><subject>Reproducibility of Results</subject><subject>Sample preparation</subject><subject>Sensitivity and Specificity</subject><subject>Spectrometric and optical methods</subject><issn>1618-2642</issn><issn>1618-2650</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1v1DAQhiMEoqXwA7hALgguKTOOv3KsVkBXWsGB9mzZjr24SuLFTqTuv8dVlnIr8mEs-3nHIz9V9RbhEgHE5wxAkDeA0LSdlM39s-ocOcqGcAbPH_eUnFWvcr4DQCaRv6zOCHAKFOR5dXO93W03tZ70cMwh19HXflhictm6ybpm0MYNrq-_N_vhaPWUa5_iWCdn42jCpKe5NiEefuk0auuWOVg95NfVC1-Ke3OqF9Xt1y83m-tm9-PbdnO1ayzjYm4IM4JKYkXfc-5QcIFlA0JjJ3vaGWF5B5YIa0BqZiR4Ik2PvNXMW6ZNe1F9XPseUvy9uDyrMZS5h0FPLi5ZSd4xyVhH_ksKxlCyVtBCfnqSREGpQOBcFvRyRfd6cCpMPs5J27J6NwYbJ-dDOb8qajpkBLEEcA3YFHNOzqtDCqNOR4WgHoyq1agqRtWDUXVfMu9O8yxmdP1j4q_CAnw4ATqXz_dJTzbkf1xLqKRdWziycrlcTXuX1F1cUvGen3z9_RryOiq9T6Xx7U8C2ALKDiiK9g_rD8DQ</recordid><startdate>20100901</startdate><enddate>20100901</enddate><creator>Melmer, Michael</creator><creator>Stangler, Thomas</creator><creator>Schiefermeier, Mark</creator><creator>Brunner, Werner</creator><creator>Toll, Hansjörg</creator><creator>Rupprechter, Alfred</creator><creator>Lindner, Wolfgang</creator><creator>Premstaller, Andreas</creator><general>Berlin/Heidelberg : Springer-Verlag</general><general>Springer-Verlag</general><general>Springer</general><scope>FBQ</scope><scope>IQODW</scope><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>7SR</scope><scope>7U5</scope><scope>8BQ</scope><scope>8FD</scope><scope>JG9</scope><scope>L7M</scope><scope>7X8</scope><scope>7QH</scope><scope>7QO</scope><scope>7UA</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope></search><sort><creationdate>20100901</creationdate><title>HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals</title><author>Melmer, Michael ; Stangler, Thomas ; Schiefermeier, Mark ; Brunner, Werner ; Toll, Hansjörg ; Rupprechter, Alfred ; Lindner, Wolfgang ; Premstaller, Andreas</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c567t-25b7482c7dd66e17671d6607a198d49b7c690c27cb08a5b80f28bd163a5fc5ab3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>2-Aminobenzamide</topic><topic>Analysis</topic><topic>Analytical Chemistry</topic><topic>Antibodies</topic><topic>Biochemistry</topic><topic>Biopharmaceutical</topic><topic>Biopharmaceutics</topic><topic>Carbohydrate Sequence</topic><topic>Characterization and Evaluation of Materials</topic><topic>Chemistry</topic><topic>Chemistry and Materials Science</topic><topic>Chromatographic methods and physical methods associated with chromatography</topic><topic>Chromatography, High Pressure Liquid - methods</topic><topic>Chromatography, Liquid - methods</topic><topic>Drugs</topic><topic>Effectiveness</topic><topic>Exact sciences and technology</topic><topic>Fluorescence</topic><topic>Fluorescence labeling</topic><topic>Fluorescent Dyes - chemistry</topic><topic>Food Science</topic><topic>Glycan</topic><topic>Glycoproteins</topic><topic>Glycoproteins - chemistry</topic><topic>Glycoproteins - metabolism</topic><topic>Health aspects</topic><topic>Heterogeneity</topic><topic>High performance liquid chromatography</topic><topic>HILIC</topic><topic>Laboratory Medicine</topic><topic>Liquid chromatography</topic><topic>Mass Spectrometry</topic><topic>Molecular Sequence Data</topic><topic>Monitoring/Environmental Analysis</topic><topic>N-glycans</topic><topic>Original Paper</topic><topic>ortho-Aminobenzoates - chemistry</topic><topic>Other chromatographic methods</topic><topic>Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - metabolism</topic><topic>Polysaccharides</topic><topic>Polysaccharides - analysis</topic><topic>Polysaccharides - chemistry</topic><topic>Polysaccharides - metabolism</topic><topic>Proteins</topic><topic>Recombinant</topic><topic>Reproducibility of Results</topic><topic>Sample preparation</topic><topic>Sensitivity and Specificity</topic><topic>Spectrometric and optical methods</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Melmer, Michael</creatorcontrib><creatorcontrib>Stangler, Thomas</creatorcontrib><creatorcontrib>Schiefermeier, Mark</creatorcontrib><creatorcontrib>Brunner, Werner</creatorcontrib><creatorcontrib>Toll, Hansjörg</creatorcontrib><creatorcontrib>Rupprechter, Alfred</creatorcontrib><creatorcontrib>Lindner, Wolfgang</creatorcontrib><creatorcontrib>Premstaller, Andreas</creatorcontrib><collection>AGRIS</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>Engineered Materials Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Materials Research Database</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>MEDLINE - Academic</collection><collection>Aqualine</collection><collection>Biotechnology Research Abstracts</collection><collection>Water Resources Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><jtitle>Analytical and bioanalytical chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Melmer, Michael</au><au>Stangler, Thomas</au><au>Schiefermeier, Mark</au><au>Brunner, Werner</au><au>Toll, Hansjörg</au><au>Rupprechter, Alfred</au><au>Lindner, Wolfgang</au><au>Premstaller, Andreas</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals</atitle><jtitle>Analytical and bioanalytical chemistry</jtitle><stitle>Anal Bioanal Chem</stitle><addtitle>Anal Bioanal Chem</addtitle><date>2010-09-01</date><risdate>2010</risdate><volume>398</volume><issue>2</issue><spage>905</spage><epage>914</epage><pages>905-914</pages><issn>1618-2642</issn><eissn>1618-2650</eissn><abstract>In contrast with conventional drugs, biopharmaceuticals are highly complex molecules with remarkable heterogeneity. Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the glycan pattern is an important issue for characterization and quality control in the biopharmaceutical industry. In this publication we describe a complete workflow for the analysis of protein N-glycans. The sample-preparation procedure, consisting of the release of the N-glycans by PNGase-F, followed by fluorescence labeling with 2-aminobenzamide and removal of excess label, was optimized to avoid alteration of the glycan sample. Subsequently, labeled glycans were analyzed by hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection. The developed method was validated for analysis of antibody N-glycans. To demonstrate the accuracy of the method an antibody sample was additionally analyzed by an orthogonal method. The antibody was digested with lysyl endopeptidase and the (glyco-)peptides were analyzed by RP-HPLC-MS. The consistency of the results between these two methods demonstrates the reliability of the glycan analysis method introduced herein.</abstract><cop>Berlin/Heidelberg</cop><pub>Berlin/Heidelberg : Springer-Verlag</pub><pmid>20640408</pmid><doi>10.1007/s00216-010-3988-x</doi><tpages>10</tpages></addata></record> |
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| subjects | 2-Aminobenzamide Analysis Analytical Chemistry Antibodies Biochemistry Biopharmaceutical Biopharmaceutics Carbohydrate Sequence Characterization and Evaluation of Materials Chemistry Chemistry and Materials Science Chromatographic methods and physical methods associated with chromatography Chromatography, High Pressure Liquid - methods Chromatography, Liquid - methods Drugs Effectiveness Exact sciences and technology Fluorescence Fluorescence labeling Fluorescent Dyes - chemistry Food Science Glycan Glycoproteins Glycoproteins - chemistry Glycoproteins - metabolism Health aspects Heterogeneity High performance liquid chromatography HILIC Laboratory Medicine Liquid chromatography Mass Spectrometry Molecular Sequence Data Monitoring/Environmental Analysis N-glycans Original Paper ortho-Aminobenzoates - chemistry Other chromatographic methods Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - metabolism Polysaccharides Polysaccharides - analysis Polysaccharides - chemistry Polysaccharides - metabolism Proteins Recombinant Reproducibility of Results Sample preparation Sensitivity and Specificity Spectrometric and optical methods |
| title | HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals |
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