Ultrasensitive Characterization of Charge Heterogeneity of Therapeutic Monoclonal Antibodies Using Strong Cation Exchange Chromatography Coupled to Native Mass Spectrometry

Abstract

In therapeutic monoclonal antibody (mAb) development, charge heterogeneity of a mAb molecule is often associated with critical quality attributes and is therefore monitored throughout development and during QC release to ensure product and process consistency. Elucidating the cause of each charge variant species is an involved process that often requires offline fractionation by ion exchange chromatography (IEX) followed by mass spectrometry (MS) analysis, largely due to the incompatibility of conventional IEX buffers for direct MS detection. In this study, we have developed a method that combines a generic strong cation exchange (SCX) chromatography step with ultrasensitive online native MS analysis (SCX-MS) optimized for mAb separation and detection. As demonstrated by analyzing mAb molecules with a wide range of pI (isoelectric point) values, the developed method can consistently achieve both high-resolution IEX separation and ultrasensitive MS detection of low-abundance charge variant species. Using this method, we analyzed the charge heterogeneity of NISTmAb reference material 8671 (NISTmAb) at both whole antibody and subdomain levels. In particular, due to the high sensitivity, a nonconsensus Fab glycosylation site, present at a very low level (<0.1%), was directly detected in the NISTmAb sample without any enrichment. The structure and location of this Fab glycosylation was further characterized by peptide mapping analysis. Despite the extensive characterization of NISTmAb material in previous studies, this is the first time that this Fab-glycosylated variant has been identified in the NISTmAb, demonstrating the value of this new method in achieving a more comprehensive characterization of charge heterogeneity for therapeutic mAbs.

Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches

“…The use of volatile mobile phases allows the hyphenation of IEC (31)(32)(33)(34), SEC (35)(36)(37), HIC (38,39), RPLC (40), and affinity chromatography (AC)(41-44) techniques to native MS. LC-nMS separates mAb variants prior to the entrance into the MS system, significantly improving sensitivity, dynamic range, and thus the detection of minor variants by limiting competition effects during the MS ionization process. For instance, the direct hyphenation of IEC to MS utilizing low ionic strength eluents on a strong cation exchange (SCX) stationary phase enabled MS data generation from minor mAb variants, and intact mass analysis of various PTMs, such as lysine truncation, glycosylation, and deamidation (mass shift of +1 Da relative to the main peak) (33).…”

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

“…29,33 Yan et al recently developed a method that combines a generic strong cation exchange (SCX) chromatography step with ultrasensitive online native MS analysis optimized for mAb separation and detection. 34 Bailey et al demonstrated the online WCX-MS analysis of an mAb using a method that incorporates a wellcontrolled pH gradient with high mass resolution and lower adduct formation. 35 Here, we describe increasing sensitivity and the addition of top-down to CEX-MS to achieve site-specific structural information of charge modifications.…”

Investigation of monoclonal antibody dimers in a final formulated drug by separation techniques coupled to native mass spectrometry

“…Differences in charge state distribution observed for the separated variants can be used to determine their conformation and aggregation state, while UV detection allows their accurate quantification at very low levels. [41][42][43] Hydrophobic interaction chromatography (HIC), which separates proteins based on hydrophobicity and folding state, can also be useful. HIC-MS allowed already the detection of dimers and trimers linked by non-covalent interactions, analyzed under non-denaturing separation and native MS conditions.…”