3D-LC-MS with 2 D Multimethod Option for Fully Automated Assessment of Multiple Attributes of Monoclonal Antibodies Directly from Cell Culture Supernatants

• Published in: Analytical chemistry (Washington) Vol. 94; no. 17; pp. 6502 - 6511
• Main Authors: Verscheure, Liesa, Vanhoenacker, Gerd, Schneider, Sonja, Merchiers, Tom, Storms, Julie, Sandra, Pat, Lynen, Frederic, Sandra, Koen
• Format: Journal Article
• Language: English
• Published: United States 03.05.2022
• Subjects: Antibodies, Monoclonal - chemistry; Antineoplastic Agents, Immunological; Cell Culture Techniques; Chromatography, Liquid; Tandem Mass Spectrometry; Trastuzumab
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.1c05461

Fully automated analysis of multiple structural attributes of monoclonal antibodies (mAbs) using three-dimensional liquid chromatography-mass spectrometry (3D-LC-MS) is described. The analyzer combines Protein A affinity chromatography in the first dimension ( D) with a multimethod option in the second dimension ( D) (choice between size exclusion (SEC), cation exchange (CEX), and hydrophobic interaction chromatography (HIC)) and desalting SEC-MS in the third dimension ( D). This innovative 3D-LC-MS setup allows simultaneous and sequential assessment of mAb titer, size/charge/hydrophobic variants, molecular weight (MW), amino acid (AA) sequence, and post-translational modifications (PTMs) directly from cell culture supernatants. The reported methodology that finds multiple uses throughout the biopharmaceutical development trajectory was successfully challenged by the analysis of different trastuzumab and tocilizumab samples originating from biosimilar development programs.