title: Parallel development of chromatographic and mass-spectrometric methods for quantitative analysis of glycation on an IgG1 monoclonal antibody.
abstract: Monitoring post-translational modifications (PTMs) in biotherapeutics is of paramount importance. In pharmaceutical industry, chromatography with optical detection is the standard choice of quantitation of product related impurities; and mass spectrometry is used only for characterization. Parallel development of a boronate affinity chromatographic (BAC) and a mass spectrometric methods for quantitative measurement of glycation on a monoclonal antibody (mAb) shed light on the importance of certain characteristics of the individual methods. Non-specific interactions in BAC has to be suppressed with the so-called shielding reagent. We have found that excessive amount of shielding reagents in the chromatographic solvents may cause significant underestimation of glycation. Although contamination of the retained peak with the non-glycated isoforms in BAC is unavoidable, our work shows that it can be characterized and quantitated by mass spectrometry. It has been demonstrated that glycation can be measured by mass spectrometry at the intact protein level with an LOQ value of 3.0% and error bar of ±0.5%. The BAC and MS methods have been found to provide equivalent results. These methods have not been compared from these points of view before.
authors.affiliation: Formulation Department, Gedeon Richter Plc. 19-21, Gyomroi St., Budapest 1103, Hungary.
authors.forename: Kornél
authors.initials: K
authors.lastname: Viski
authors.affiliation: Formulation Department, Gedeon Richter Plc. 19-21, Gyomroi St., Budapest 1103, Hungary. Electronic address: gengeliczkizs@richter.hu.
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Gengeliczki
authors.affiliation: Formulation Department, Gedeon Richter Plc. 19-21, Gyomroi St., Budapest 1103, Hungary.
authors.forename: Krisztián
authors.initials: K
authors.lastname: Lenkey
authors.affiliation: Formulation Department, Gedeon Richter Plc. 19-21, Gyomroi St., Budapest 1103, Hungary.
authors.forename: Katalin
authors.initials: K
authors.lastname: Baranyáné Ganzler
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001897:Boronic Acids / Q000737:chemistry; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods*; D003412:Cricetulus; D006031:Glycosylation; D007074:Immunoglobulin G / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D001897:Boronic Acids; D007074:Immunoglobulin G
keywords: Boronate affinity; Glycation; Mass spectrometry; Monoclonal antibody