Characterizing Antibody-Drug Conjugates Using Mass Spectrometry

Scientists from the National Institutes for Food and Drug Control in Beijing, China, have created a new means of characterizing antibody-drug conjugates (ADCs) using various mass spectrometry (MS)-based techniques. Their findings were published in the Journal of Chromatography A (1).

B cell releasing antibodies | Image Credit: © Artur - stock.adobe.com

Antibody-drug conjugates (ADCs) are typically made of an antibody covalently attached to a cytotoxic drug via either a permanent or labile linker, thus making a highly heterogeneous product. Some of these substances have been approved at different times by the United States Food and Drug Administration (U.S. FDA), though their effectiveness does not come without shortcomings. Drug-to-antibody ratio (DAR) distributions can present issues, as ADCs with DARs greater than 4 can have lower tolerability and efficacy and greater plasma clearance. Third-generation ADCs have allowed for explosive growth by combining factors that fail in the first and second generations. Their greatly improved stability and pharmacokinetics stem from more site-specific payload conjugation technology, which also reduces their toxicity and leads to fewer naked monoclonal antibodies.

Ion-exchange chromatography (IEX) and image capillary isoelectric focusing (icIEF) are important front-end separation techniques for mass spectrometry (MS)-based charge variant analysis of proteins. IEX does not separate analytes based on overall charge, unlike icIEF, but rather on the charge available for interaction with the solid phase. Meanwhile, icIEF has become a key element of therapeutic protein development and manufacturing, mainly due to its high analytical throughput, ease of use, fast method development, and excellent reproducibility. While these techniques are effective by themselves, because ADCs are extremely complex in the presence of antibodies, linkers, and payloads, a single analytical platform cannot achieve accurate characterization of these complex heterogeneities. As such, it is vital to address critical quality attributes (CQAs) by employing a portfolio of comprehensive techniques. Such techniques include ultrahigh-pressure liquid chromatography (UHPLC), high-resolution tandem mass spectrometry (HRMS/MS), and IEX-MS, which are widely utilized for in-depth characterization of complex protein drugs.

In this study, an innovative workflow that employs multiple separation techniques and tandem high-resolution mass spectrometry for comprehensive and in-depth characterization of disitamab vedotin was introduced. This process includes reversed-phase liquid chromatography (RPLC), IEX, and icIEF. RPLC was used for reduced chain analysis, while the other two were utilized for charge heterogeneity analysis.

Disitamab vedotin is a cysteine-linked ADC comprised of a monoclonal antibody against human epidermal growth factor receptor 2 (HER2) conjugated via a cleavable linker to the cytotoxic agent MMAE. In June 2021, the drug received its first Biologics License Application (BLA) approval in China for treating patients with HER2-overexpressing (defined as IHC2+ or 3+) locally advanced or metastatic gastric cancer (including gastroesophageal junction adenocarcinoma) and who have received at least two systemic chemotherapy regimens.

UHPLC–MS and UHPLC–MSMS were used to characterize disitamab vedotin via native intact protein analysis, reduced chains and subunit analysis under denaturing condition, and peptide mapping with post-translational modifications (PTM) identification. With this integrated MS-based workflow, disitamab vedotin was successfully characterized, and the CQAs of the ADC were assessed. This workflow underlines the importance of comprehensive separation techniques based on MS technology in ADC development.

References

(1) Wu, G.; Zhang, X.; Wang, X.; et al. In-Depth Characterization of a Cysteine-Linked ADC Disitamab Vedotin by Multiple LC-MS Analysis Methods and Cutting-Edge Imaged Capillary Isoelectric Focusing Coupled with Native Mass Spectrometry. J. Chromatogr. A 2024, 465353. DOI: 10.1016/j.chroma.2024.465353