Issue 3, 2021

Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches

Abstract

Accelerated development of new therapeutics in an increasingly competitive landscape requires the use of high throughput analytical platforms. In addition, the complexity of novel biotherapeutic formats (e.g. fusion proteins, protein-polymer conjugates, co-formulations, etc.) reinforces the need to improve the selectivity and resolution of conventional one-dimensional (1D) liquid chromatography (LC). Liquid chromatography-mass spectrometry (LC-MS)-based technologies such as native LC-MS for intact mass analysis or peptide mapping (also called bottom-up approach)-based multi-attribute methods (MAM) have already demonstrated their potential to complement the conventional analytical toolbox for monoclonal antibody (mAb) characterization. Two-dimensional liquid-chromatography (2D-LC-MS) methods have emerged in the last ten years as promising approaches to address the increasing analytical challenges faced with novel antibody formats. However, off-line sample preparation procedures are still required for conventional 1D and 2D-LC-MS methods for the in-depth variant characterization at the peptide level. Multi-dimensional LC-MS (mD-LC-MS) combine sample preparation and multi-level (i.e. intact, reduced, middle-up and peptide) analysis within the same chromatographic set-up. This review presents an overview of the benefits and limitations of mD-LC-MS approaches in comparison to conventional chromatographic methods (i.e. 1D-LC-UV methods at intact protein level and 1D-LC-MS methods at peptide level). The current analytical trends in antibody characterization by mD-LC-MS approaches, beyond the 2D-LC-MS workhorse, are also reviewed, and our vision on a more integrated multi-level mD-LC-MS characterization platform is shared.

Graphical abstract: Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches

Article information

Article type
Critical Review
Submitted
02 okt 2020
Accepted
10 dec 2020
First published
15 dec 2020
This article is Open Access
Creative Commons BY license

Analyst, 2021,146, 747-769

Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches

J. Camperi, A. Goyon, D. Guillarme, K. Zhang and C. Stella, Analyst, 2021, 146, 747 DOI: 10.1039/D0AN01963A

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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