Glycoforms of Immunoglobulin G Based Biopharmaceuticals Are Differentially Cleaved by Trypsin Due to the Glycoform Influence on Higher-Order Structure

D. Falck, B.C. Jansen, R. Plomp, D. Reusch, M. Haberger, M. Wuhrer

Research output: Contribution to JournalArticleAcademicpeer-review

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.
Original languageEnglish
Pages (from-to)4019-4028
JournalJournal of Proteome Research
Volume14
Issue number9
DOIs
Publication statusPublished - 2015

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