title: Evaluation of a glycoengineered monoclonal antibody via LC-MS analysis in combination with multiple enzymatic digestion.
abstract: Glycosylation affects the efficacy, safety and pharmacokinetics/pharmacodynamics properties of therapeutic monoclonal antibodies (mAbs), and glycoengineering is now being used to produce mAbs with improved efficacy. In this work, a glycoengineered version of rituximab was produced by chemoenzymatic modification to generate human-like N-glycosylation with α 2,6 linked sialic acid. This modified rituximab was comprehensively characterized by liquid chromatography-mass spectrometry and compared to commercially available rituximab. As anticipated, the majority of N-glycans were converted to α 2,6 linked sialic acid, in contrast to CHO-produced rituximab, which only contains α 2,3 linked sialic acid. Typical posttranslational modifications, such as pyro-glutamic acid formation at the N-terminus, oxidation at methionine, deamidation at asparagine, and disulfide linkages were also characterized in both the commercial and glycoengineered mAbs using multiple enzymatic digestion and mass spectrometric analysis. The comparative study reveals that the glycoengineering approach does not cause any additional posttranslational modifications in the antibody except the specific transformation of the glycoforms, demonstrating the mildness and efficiency of the chemoenzymatic approach for glycoengineering of therapeutic antibodies.
authors.affiliation: a New England Biolabs Inc. , Ipswich , MA 01938.
authors.forename: Renpeng
authors.initials: R
authors.lastname: Liu
authors.affiliation: b Department of Chemistry & Biochemistry ; University of Maryland , College Park , MD 20742.
authors.forename: John
authors.initials: J
authors.lastname: Giddens
authors.affiliation: a New England Biolabs Inc. , Ipswich , MA 01938.
authors.forename: Colleen M
authors.initials: CM
authors.lastname: McClung
authors.affiliation: a New England Biolabs Inc. , Ipswich , MA 01938.
authors.forename: Paula E
authors.initials: PE
authors.lastname: Magnelli
authors.affiliation: b Department of Chemistry & Biochemistry ; University of Maryland , College Park , MD 20742.
authors.forename: Lai-Xi
authors.initials: LX
authors.lastname: Wang
authors.affiliation: a New England Biolabs Inc. , Ipswich , MA 01938.
authors.forename: Ellen P
authors.initials: EP
authors.lastname: Guthrie
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D000069283:Rituximab / Q000737:chemistry*
chemical_list: D000069283:Rituximab; D019158:N-Acetylneuraminic Acid
keywords: LC-MS; antibody characterization; glycoengineering; glycosylation analysis; procainamide