title: Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry.
abstract: Monoclonal antibodies (mAbs) are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product - and time - consuming. This work presents the characterization of trastuzumab sequence using sheathless capillary electrophoresis (referred as CESI) - tandem mass spectrometry (CESI-MS/MS). Using this bottom-up proteomic-like approach, CESI-MS/MS provided 100% sequence coverage for both heavy and light chain via peptide fragment fingerprinting (PFF) identification. The result was accomplished in a single shot, corresponding to the analysis of 100 fmoles of digest. The same analysis also enabled precise characterization of the post-translational hot spots of trastuzumab, used as a representative widely marketed therapeutic mAb, including the structural confirmation of the five major N-glycoforms.
authors.affiliation: Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS); UDS-CNRS UMR 7140; Université de Strasbourg; Strasbourg, France.
authors.forename: Rabah
authors.initials: R
authors.lastname: Gahoual
authors.affiliation: Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS); UDS-CNRS UMR 7140; Université de Strasbourg; Strasbourg, France.
authors.forename: Alicia
authors.initials: A
authors.lastname: Burr
authors.affiliation: Beckman Coulter Inc.; Brea, CA, USA.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Busnel
authors.affiliation: Plateforme Protéomique; Université de Strasbourg; Institut de Biologie Moléculaire et Cellulaire; Strasbourg, France.
authors.forename: Lauriane
authors.initials: L
authors.lastname: Kuhn
authors.affiliation: Plateforme Protéomique; Université de Strasbourg; Institut de Biologie Moléculaire et Cellulaire; Strasbourg, France.
authors.forename: Phillipe
authors.initials: P
authors.lastname: Hammann
authors.affiliation: Centre d'immunologie Pierre Fabre; Saint-Julien-en-Genevois, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.affiliation: Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS); UDS-CNRS UMR 7140; Université de Strasbourg; Strasbourg, France.
authors.forename: Yannis-Nicolas
authors.initials: YN
authors.lastname: François
authors.affiliation: Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS); UDS-CNRS UMR 7140; Université de Strasbourg; Strasbourg, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Leize-Wagner
mesh_terms: D000595:Amino Acid Sequence; D061067:Antibodies, Monoclonal, Humanized / Q000032:analysis* / Q000302:isolation & purification; D019075:Electrophoresis, Capillary; D006031:Glycosylation; D006801:Humans; D010449:Peptide Mapping; D020033:Protein Isoforms / Q000032:analysis / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D053719:Tandem Mass Spectrometry; D000068878:Trastuzumab
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D020033:Protein Isoforms; D000068878:Trastuzumab
keywords: CESI-MS/MS; N-linked glycosylation; mass spectrometry; microheterogeneity; microvariant; monoclonal antibody; peptide mapping; posttranslational modifications; sheathless capillary electrophoresis
references.citation: J Am Soc Mass Spectrom. 2009 Nov;20(11):2021-33
references.pmid: 19744865
references.citation: Anal Chem. 2012 Jun 19;84(12):5380-6
references.pmid: 22663182
references.citation: Biotechnol Prog. 2005 Jan-Feb;21(1):11-6
references.pmid: 15903235
references.citation: Electrophoresis. 2011 Nov;32(21):3078-84
references.pmid: 22145164
references.citation: Anal Chim Acta. 2011 Jul 18;698(1-2):77-83
references.pmid: 21645662
references.citation: MAbs. 2011 Mar-Apr;3(2):209-17
references.pmid: 21441787
references.citation: Mol Cell Proteomics. 2010 Aug;9(8):1716-28
references.pmid: 20103567
references.citation: Anal Chem. 2012 Jun 5;84(11):4637-46
references.pmid: 22510259
references.citation: J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Sep 1;872(1-2):23-37
references.pmid: 18672411
references.citation: Proc Natl Acad Sci U S A. 1992 May 15;89(10):4285-9
references.pmid: 1350088
references.citation: Anal Biochem. 2009 Sep 15;392(2):145-54
references.pmid: 19497295
references.citation: MAbs. 2012 Nov-Dec;4(6):701-9
references.pmid: 22932441
references.citation: Mass Spectrom Rev. 2009 Jan-Feb;28(1):147-76
references.pmid: 18720354
references.citation: Anal Biochem. 2011 Oct 15;417(2):256-63
references.pmid: 21756870
references.citation: Anal Chem. 2012 Jan 17;84(2):885-92
references.pmid: 22148170
references.citation: MAbs. 2010 Jul-Aug;2(4):379-94
references.pmid: 20458189
references.citation: J Chromatogr B Biomed Sci Appl. 2001 Mar 10;752(2):233-45
references.pmid: 11270864
references.citation: Curr Pharm Biotechnol. 2008 Dec;9(6):482-501
references.pmid: 19075687
references.citation: Expert Opin Drug Discov. 2010 Jan;5(1):95-111
references.pmid: 22823974
references.citation: J Pharm Sci. 2008 Jul;97(7):2426-47
references.pmid: 17828757
references.citation: Nat Rev Immunol. 2010 May;10(5):345-52
references.pmid: 20414207
references.citation: J Chromatogr A. 2010 Nov 26;1217(48):7605-11
references.pmid: 20970804
references.citation: Anal Chem. 2007 Jun 1;79(11):4241-6
references.pmid: 17447730
references.citation: J Chromatogr B Analyt Technol Biomed Life Sci. 2005 May 25;819(2):203-18
references.pmid: 15833284
references.citation: MAbs. 2009 Sep-Oct;1(5):394-416
references.pmid: 20065643
references.citation: Anal Chem. 2011 Oct 1;83(19):7297-305
references.pmid: 21848273
references.citation: MAbs. 2009 Mar-Apr;1(2):142-50
references.pmid: 20061815
references.citation: MAbs. 2012 May-Jun;4(3):413-5
references.pmid: 22531442
references.citation: Anal Biochem. 2011 Dec 1;419(1):17-25
references.pmid: 21867674
references.citation: N Engl J Med. 2007 Jul 5;357(1):39-51
references.pmid: 17611206
references.citation: Anal Chem. 2010 Nov 15;82(22):9476-83
references.pmid: 21028888
references.citation: Electrophoresis. 2012 Jun;33(11):1550-6
references.pmid: 22736356
references.citation: Anal Biochem. 2006 Aug 15;355(2):249-58
references.pmid: 16712766
references.citation: MAbs. 2012 Jul-Aug;4(4):419-25
references.pmid: 22699226