An improved capillary isoelectric focusing-mass spectrometry method for high-resolution characterization of monoclonal antibody charge variants.

Abstract

Routine and high-resolution characterization of monoclonal antibody (mAb) charge variants is vital for controlling mAb quality as therapeutics. Capillary isoelectric focusing-mass spectrometry (cIEF-MS) has emerged as a powerful tool for characterizing mAb charge variants because it can achieve high-resolution separation and highly sensitive detection of proteins. It provides much better identification of charge variants than the traditionally used cIEF-UV method. However, further improvement of cIEF-MS regarding stability and separation resolution is needed. Here, we improved the stability and enhanced separation resolution of automated cIEF-MS by bettering the quality of capillary neutral coating, reducing catholyte pH to 10 for cIEF-MS for the first time, and systematically optimizing the cIEF separation conditions. The improved cIEF-MS method was applied to characterize charge variants of three previously well characterized mAbs (NISTmAb, cetuximab, trastuzumab) and one tool mAb (mAb1). The charge variants of the studied mAbs were well resolved, and the majority of post-translational modifications (PTMs) found in those mAbs agreed with the literature. cIEF-MS analyses of mAb1 were capable of discovering ten charge variants with various interesting PTMs, such as PGK amidation, incomplete C-terminal lysine clipping, glycosylation, and deamination. cIEF-MS was successfully used for accurately determining the isoelectric points (pIs) of mAb1 charge variants via analyzing the pI markers and spiking in a standard protein (cytochrome c) to samples for migration time normalization, which is beneficial for evaluating pI-related pharmacokinetic properties. Our cIEF-MS agreed with and, in some cases (i.e., cetuximab and mAb1), outperformed cIEF-UV for detecting mAb charge variants.