The versatility of heart-cutting and comprehensive two-dimensional liquid chromatography in monoclonal antibody clone selection

• Published in: Journal of Chromatography A 2017-11, Vol.1523, p.283-292
• Main Authors: Sandra, Koen, Steenbeke, Mieke, Vandenheede, Isabel, Vanhoenacker, Gerd, Sandra, Pat
• Format: Article
• Language: English
• Subjects: Biosimilar; Clone selection; Monoclonal antibody; Protein A; Two-dimensional liquid chromatography
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2017.06.052

•Protein A affinity chromatography was combined with SEC, CEX or RPLC–MS in a heart-cutting 2D set-up.•Attributes such as mAb titer and structure can simulatenously be assessed.•Comprehensive LC×LC of trypsinized mAbs provides a next level of structural detail.•Clear decisions can be made for further mAb or biosimilar development. In recent years, two-dimensional liquid chromatography (2D-LC) has seen an enormous evolution and one of the fields where it is being widely adopted is in the analysis of therapeutic monoclonal antibodies (mAbs). We here further add to the many flavours of this powerful technology. Workflows based on heart-cutting (LC-LC) and comprehensive (LC×LC) 2D-LC are described that allow to guide the clone selection process in mAb and biosimilar development. Combining Protein A affinity chromatography in the first dimension with size exclusion (SEC), cation exchange (CEX) or reversed-phase liquid chromatography-mass spectrometry (RPLC–MS) in the second dimension simultaneously allows to assess mAb titer and critical structural aspects such as aggregation, fragmentation, charge heterogeneity, molecular weight (MW), amino acid sequence and glycosylation. Complementing the LC-LC measurements at intact protein level with LC×LC based peptide mapping provides the necessary information to make clear decisions on which clones to take further into development.