Approaches to Interchain Cysteine-Linked ADC Characterization by Mass Spectrometry

Abstract

Therapeutic antibody-drug conjugates (ADCs) harness the cell-killing potential of cytotoxic agents and the tumor targeting specificity of monoclonal antibodies to selectively kill tumor cells. Recent years have witnessed the development of several promising modalities that follow the same basic principles of ADC based therapies but which employ unique cytotoxic agents and conjugation strategies in order to realize therapeutic benefit. The complexity and heterogeneity of ADCs present a challenge to some of the conventional analytical methods that industry has relied upon for biologics characterization. This current review will highlight some of the more recent methodological approaches in mass spectrometry that have bridged the gap that is created when conventional analytical techniques provide an incomplete picture of ADC product quality. Specifically, we will discuss mass spectrometric approaches that preserve and/or capture information about the native structure of ADCs and provide unique insights into the higher order structure (HOS) of these therapeutic molecules.

Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine‐linked antibody drug conjugate

“…In consideration of the many benefits described in the literature that native MS offers for the analytical characterization of noncovalent cysteinelinked ADCs, 24,36,37 we used this technique on the covalent ADC T-DM1. As expected, the same analysis performed under nondenaturing conditions (150 mM ammonium acetate at pH 7.4, see the "Materials and Methods") strongly reduces the charge state distribution, such that the different drug load species are more clearly resolved with fewer overlapping ion distributions.…”

Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates

“…As lysine‐conjugated ADCs typically exhibit greater heterogeneity and poorer chromatographic peak shapes than equivalent cysteine‐modified therapeutics, mass spectrometry (MS) is typically deployed for their DAR assessment . Recently, native MS has been increasingly applied for ADC analysis, providing highly accurate DAR values under native conditions, which are especially critical for capturing accurate values produced by cysteine‐linked modification chemistries . Moreover, with advances in high‐resolution instrumentation for native MS experiments, DAR values can often be readily extracted from ADCs prepared at physiologically relevant pH …”

“…Recently, MS‐based techniques have emerged as an important class of tools for protein HOS characterization. Among these, hydrogen–deuterium exchange (HDX)‐MS has been used to compare the overall conformation and flexibility of ADCs to their parent mAbs . Despite its ability to access localized structure information in large therapeutic proteins, HDX‐MS experiments often take a long time to perform and analyze.…”

Multiple heart-cutting and comprehensive two-dimensional liquid chromatography hyphenated to mass spectrometry for the characterization of the antibody-drug conjugate ado-trastuzumab emtansine

“…With 80-100 lysine and only 8 interchain cysteine residues available, lysine conjugation yields a more heterogeneous mixture of species compared to cysteine conjugation this despite the similar overall level of drugs incorporated per antibody (between 0 and 8 with an average of 3-4) [6]. Chromatography and mass spectrometry are at the forefront in ADC analysis and critical characteristics such as drug-to-antibody ratio (DAR), drug distribution and conjugation sites can be extracted out of the generated data [7,[10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28]. 1.…”