Orthogonal LCMS Techniques for Lysine-Conjugated ADC Characterization
Orthogonal techniques are used for LCMS characterization of a lysine-conjugated ADC.
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Lysine-based conjugation is a widely used non-specific conjugation strategy for the development of Antibody Drug Conjugates (ADCs). Conjugation at lysine residues results in highly heterogeneous products that pose challenges to current characterization methods. These challenges include:
- Accurate drug to antibody ratio (DAR) calculation.
- Site specific localization.
- Quantification of site occupancy.
The last two are quality attributes which often need to be determined by bottom-up MS approaches and typically use lysine specific proteases (trypsin or LysC). As lysine conjugation results in variable modification, the use of such proteases results in complex mixtures of peptides which can be challenging when quantifying site occupancy. Here we present an analytical strategy utilizing the arginine specific protease RgpB (GingisREX®, Genovis AB). Intact reconstruction from peptide mapping data was used to determine the level of correlation between the orthogonal techniques of intact mass and peptide mapping.
