An Online Two-Dimensional Approach to Characterizing the Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies Using a 2D-CEX–AEX–MS Workflow

Assessment of product quality attributes such as charge heterogeneity is an upmost requisite for the release of a monoclonal antibody (mAb). Analytical techniques, such as cation-exchange chromatography (CEX), accomplish this, causing the mAb to separate into acidic, main species, and basic variants. Here, an online volatile-salt-containing two-dimensional liquid chromatography (2D-LC) method coupled with mass spectrometry (MS) was performed to characterize the charge heterogeneity of mAbs using CEX chromatography in the first dimension (D¹) and anion-exchange chromatography (AEX) in the second dimension (D²). The main peak of the CEX profile of D¹ was transferred through a 2D heart-cut method to D² for further analysis by the AEX-MS method. In the CEX method, mAb A showed 10 distinct variants, while the AEX method resulted in eight variants. However, a total of 13 variants were successfully resolved for mAb A in the 2D method. Similarly, mAb B exhibited seven variants in the CEX method and four variants in the AEX method, but the 2D-LC method revealed a total of nine variants for mAb B. Likewise, mAb C displayed seven variants in CEX and seven variants in AEX, whereas the 2D-LC method unveiled a total of 11 variants for mAb C. Additionally, native MS analysis revealed that the resolved charge variants were identified as amidation, oxidation, and isomerization of Asp variants in the main peak, which were not resolved in stand-alone methods. The present study demonstrates how 2D-LC can assist in identifying minor variations in charge distribution or conformation of mAb variants that would otherwise not be picked up by a single analytical method alone.