Identification of an IgG Degradants Detected by Size Exclusion Chromatography and Electrophoretic Techniques Using Electrospray Ionization Mass Spectrometry (ESI-MS)

摘要

Structure-activity relationship (SAR) studies play an important role in biopharmaceutical process development. The main purpose of these studies is to characterize all the major impurities and isoforms in a potential biopharmaceutical, such as charge variants, aggregates and degradants, etc., and evaluate their impact on the biological activity of the biopharmaceutical. This poster will discuss a part of a SAR study performed for an IgG molecule, involving characterization of two low molecular weight (LMW) impurity peaks (LMW1 and LMW2), detected by size exclusion chromatography (SEC). The intact mass analysis and Lys-C peptide mapping of the collected LMW1 and LMW2 fractions led to the identification of LMW1 as Fab-Fc and LMW2 as a Fab and its further degradants. Both LMW1 and LMW2 were found to be heterogeneous mixtures due to the cleavage that occurs at different amino acids in the hinge region of the antibody molecule. Further CE-SDS analyses of the LMW1 and LMW2 fractions collected from SEC and fractions collected from preparative SDS-PAGE led to a clear correlation of the LMW peaks observed in analysis using the three size-based separation techniques.