The Application of Mass Spectrometry for the Characterization of Monoclonal Antibody-Based Therapeutics
Rosie Upton 1, Kamila J. Pacholarz1, David Firth2, Sian Estdale2 and Perdita E. Barran1
- 1 Michael Barber Centre for Collaborative Mass Spectrometry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK
- 2 Covance Laboratories Ltd., Harrogate, UK
Monoclonal antibody (mAb) therapeutics currently represent the fastest- growing class of biopharmaceuticals . With the introduction of several stand-alone immunoglobulin G (IgG) therapies, fragment crystallizable (Fc)- fusion proteins/peptides [2, 3], fragment antigen-binding (Fab) fragments , bispecific antibodies (bsAbs) [2, 5, 6], antibody-drug conjugates (ADCs) [7, 8], radioimmunoconjugates [9, 10], as well generic versions known as biosimilars, it is clear that mAbs are of significant interest to the pharmaceutical industry , to healthcare providers , as well as to potential consumers [13, 14].
Analogous to the development of small-molecule drugs, structural and analytical characterization of biologics is critically required at many points in the pipeline to ensure comparability to the original product . The current limitations in probing the inherent complexities of many biologics, and understanding their potential impact in a safety or clinical setting, presents a substantial challenge. Even very minor alterations to the structure of a biologic can have unintended clinical consequences. mAbs possess specific structural features that affect their function . As a biologic is developed and manufactured, all levels of structure from the primary sequence and post-translational modifications (PTMs) to the quaternary fold must be shown to be highly similar (see ICH Q5E). Effective clinical application relies on the consistency of the formulated product at the molecular level, and hence they must be characterized using sensitive, orthogonal analytical techniques. Effective characterization that provides relevant quantifiable or qualitative information regarding
Analysis of Protein Post-Translational Modifications by Mass Spectrometry, First Edition. Edited by John R. Griffiths and Richard D. Unwin.
© 2017 John Wiley & Sons, Inc. Published 2017 by John Wiley & Sons, Inc.
the intended clinical outcome (or mechanism of action) can greatly enhance understanding and derisking of manufacturing changes and also prevent unnecessary nonclinical or clinical studies.
As patents for many mAb-based therapeutics are expiring, introduction of generic versions - the so-called biosimilars or biobetters - presents even more regulatory challenges. Clinicians are wary of the interchangeability of biosimilars and rightly have questions regarding the transferral and extrapolation of clinical trial data obtained for a particular disease toward different diseases that have not been through the same vigorous testing . Developing confidence in regulatory procedures and acceptance criteria for biosimilars will be enabled by evidence proving consistency of the products, which relies on robust analytical characterization, pharmacokinetic (PK) and pharmacodynamic (PD) analyses, and in vivo efficacy testing .
The rapidly evolving market for mAb-based therapeutics demands comprehensive, analytical characterization of these complex and flexible biological structures. One such technique that has shown dominance in this field is mass spectrometry (MS) [19, 20], including liquid chromatography-mass spectrometry (LC-MS), native MS, and recently developed ion mobility-mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS). This review concentrates on the use of these techniques for the characterization of intact mAbs and ADCs while introducing the concept of biosimilars and their characterization requirements. We first introduce the common structural features of mAbs, with a focus on IgG. We then highlight how the structures can be altered during expression with focus on variations in N-glycosylation patterns, which can affect the mode of action. Certain modifications are deliberately introduced in the case of ADCs, and these will be described, as will biosimilars. We describe common MS approaches for examining mAb structure and present some more advanced examples of the use of MS to provide structural and functional insights.