title: Glycoforms of Immunoglobulin G Based Biopharmaceuticals Are Differentially Cleaved by Trypsin Due to the Glycoform Influence on Higher-Order Structure.
abstract: It has been reported that glycosylation can influence the proteolytic cleavage of proteins. A thorough investigation of this phenomenon was conducted for the serine protease trypsin, which is essential in many proteomics workflows. Monoclonal and polyclonal immunoglobulin G biopharmaceuticals were employed as model substances, which are highly relevant for the bioanalytical applications. Relative quantitation of glycopeptides derived from the conserved Fc-glycosylation site allowed resolution of biases on the level of individual glycan compositions. As a result, a strong preferential digestion of high mannose, hybrid, alpha2-3-sialylated and bisected glycoforms was observed over the most abundant neutral, fucosylated glycoforms. Interestingly, this bias was, to a large extent, dependent on the intact higher order structure of the antibodies and, consequently, was drastically reduced in denatured versus intact antibodies. In addition, a cleavage protocol with acidic denaturation was tested, which featured reduced hands-on time and toxicity while showing highly comparable results to a published denaturation, reduction, and alkylation based protocol.
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center , Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
authors.forename: David
authors.initials: D
authors.lastname: Falck
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center , Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
authors.forename: Bas C
authors.initials: BC
authors.lastname: Jansen
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center , Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
authors.forename: Rosina
authors.initials: R
authors.lastname: Plomp
authors.affiliation: Pharma Biotech Development Penzberg, Roche Diagnostics GmbH , 82377 Penzberg, Germany.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Reusch
authors.affiliation: Pharma Biotech Development Penzberg, Roche Diagnostics GmbH , 82377 Penzberg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Haberger
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center , Albinusdreef 2, 2333 ZA Leiden, The Netherlands.|Division of BioAnalytical Chemistry, VU University Amsterdam , De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006020:Glycopeptides / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D040901:Proteomics / Q000379:methods; D013552:Swine; D014357:Trypsin / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D007074:Immunoglobulin G; D014357:Trypsin
keywords: biopharmaceuticals; glycoproteomics; glycosylation; higher-order structure; immunoglobulin G; method development; monoclonal antibodies; proteolytic biases; trypsin substrate specificity; tryptic cleavage