Simultaneous monitoring of multiple attributes of pyrrolobenzodiazepine antibody-drug conjugates by size exclusion chromatography – high resolution mass spectrometry

• Published in: Journal of pharmaceutical and biomedical analysis 2021-10, Vol.205, p.114287-114287, Article 114287
• Main Authors: Füssl, Florian, Barry, Conor S., Pugh, Kathryn M., Chooi, K. Phin, Vijayakrishnan, Balakumar, Kang, Gyoung-Dong, von Bulow, Christina, Howard, Philip W., Bones, Jonathan
• Format: Article
• Language: English
• Subjects: Aggregation; Antibody-drug conjugate; Drug-antibody ratio; Fragmentation; Glycosylation; Payload; Pyrrolobenzodiazepine; SEC-MS
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2021.114287

•A LC–MS method for the native, intact analysis of pyrrolobenzodiazepine-based ADCs was developed.•SEC-MS enables the separation of size variants and the generation of clear, native MS spectra.•Quality attributes including average DAR, glycosylation and size heterogeneity are monitored using a single method.•Applicability for ADCs with different PDB-based payloads was demonstrated. Antibody-drug conjugates (ADCs) are an emerging class of oncology treatments combining the unique specificity of monoclonal antibodies with the highly cytotoxic properties of small molecule compounds. Pyrrolobenzodiazepines (PBDs) are highly potent agents capable of inhibiting cellular DNA replication which leads to apoptosis. To ensure efficacy and patient safety upon administration of such toxic and heterogeneous molecules, their structure and quality attributes must be closely monitored. Size exclusion chromatography (SEC) is a powerful, fast and robust tool for the separation of compounds varying in molecular weight. When using volatile components in the chromatographic mobile phase, SEC has also been shown to be amenable for interfacing to mass spectrometry, providing potential for reliable identification of protein isoforms across the size variants present. Here, we present a SEC-MS method developed for the characterisation of PBD-based ADCs on the intact molecular level. We demonstrate that information on ADC monomers such as the glycoform distribution and the average drug-antibody ratio (DAR) can be obtained in 15 minutes of analysis time. Qualitative and quantitative information on low and high molecular weight impurities such as aggregates and fragments, fundamental for critical quality attribute analysis of biopharmaceuticals, can be generated simultaneously. SEC-MS enables the characterisation of multiple product quality attributes of complex biotherapeutics at the same time.