Separation techniques for intact antibody analysis by mass spectrometry

Abstract

The possibilities to directly couple different separation methods with mass spectrometry (MS) for the analysis of intact proteins has attracted significant attention over the last decade. While sample preparation is critical for MS analyses, coupling an efficient separation method before MS can significantly improve the ability to resolve protein heterogeneity and reveal intact masses or higher-order structure information (e.g., PTM or conformation changes). To that end, this review focuses on common online separation techniques coupled with MS detection to detect antibodies or large proteins with a focus on biopharmaceutical applications. An overview of liquid chromatography modes, such as reversed-phase, size-exclusion, mixed-mode, hydrophobic interaction, and ion-exchange chromatography will be discussed, along with electrophoretic separation, gas-phase separation using ion-mobility and other next-generation tools. Finally, the application space within the biopharmaceutical industry will be discussed along with how improvements for separation techniques before MS detection can ultimately help characterize charge-, size-, or hydrophobic variants of novel biotherapeutics throughout all stages of drug development.

GRAPHICAL ABSTRACT

Keywords:

• HPLC
• mass spectrometry
• monoclonal antibodies
• native separations
• RP-HPLC
• RPLC

Acknowledgments

The authors gratefully acknowledge John Mehl, Naidong Weng, Enric Bertran, and Eric Yang for their support.

Disclosure statement

The authors declare the following competing financial interest(s): The research discussed was supported by GSK. The authors are employees of GSK and may be eligible for stock options or have stock ownership. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.