Kyutae Kim (Thermofisher Scientific)

Kyutae Kim (Thermofisher Scientific)

Junghyang Lee (Thermofisher Scientific)

Hansun Kwon (Thermofisher Scientific)

Intact mass analysis of monoclonal antibodies (mAbs) is employed to determine their molecular structure, modifications (e.g., glycosylation), and quality attributes by measuring the molecular weight of the antibody. During the biological production process, the produced and purified mAb has structural heterogeneities by the post-translational modifications, necessitating the use of multiple analytical methods for comprehensive structural characterization.1 Recently, the demand for antibody-drug conjugate (ADC) development is increasing because ADCs can more effectively target and kill cancer cells by conjugating highly potent payloads to mAbs. However, due to their structural complexity, ADCs are more challenging to characterize than mAbs.

  The most utilized Reversed Phase (RP) separates proteins based on their hydrophobic interactions, and provides high resolution and sensitivity, as well as LC-MS compatibility under denatured conditions. Size Exclusion Chromatography (SEC) separates proteins based on their size, and can efficiently separate aggregates, monomers, and fragments. Since it is performed on the native form of the protein, it can be applied to protein complexes formed by non-covalent bonds among multiple subunits, and cysteine-linked ADC. Finally, Cation Exchange Chromatography (CEX) separates proteins based on their charge differences and can separate charge variants through ion exchange mechanisms such as salt or pH gradients. CEX via pH gradient provides deeper insights into key CQAs at an intact level by separating charge variants resulting from PTMs such as deamidation, lysine clipping, N-terminal pyroglutamate formation, and glycosylation that can affect protein charge.2 In this poster, I have performed experiments to compare the differences in results from different separation methods using examples of mAb and ADC and to gain deeper insights into the sample.