Fast and Efficient non-reduced Lys-C digest using pressure cycling technology for antibody disulfide mapping by LC–MS

Abstract

Conventional sample preparation for antibody disulfide mapping often requires relatively long digestion time (from several hours to overnight) and relatively high endoproteinase concentration. These conditions are typically necessitated by the fact that antibody molecules are not sufficiently denatured under non-reduced conditions and chaotropic agents are used during digestion to achieve optimal denaturation. Disulfide scrambling can occur as artifacts of digestion as proteins are incubated for extended periods, often at neutral to slightly alkaline pH conditions. Shortening digestion time and lowering the pH during digestion frequently result in incomplete peptide cleavages or variable recoveries. Here, we report the development of a fast and efficient non-reduced Lys-C digestion method based on pressure cycling technology (PCT) and its application in determining disulfide-linkages in monoclonal antibodies (mAbs). Conditions were optimized to ensure complete digestion of the mAb with minimal sample preparation-related disulfide scrambling. The PCT-based method was able to generate up to 10-fold signal increase for some disulfide peptides in a 1h Lys-C digestion compared to the conventional bench-top digestion method. As a result of the shorter digestion time, disulfide scrambling that is seen as a major assay artifact of the conventional method was reduced to less than 0.05% in tested molecules. The results show that the PCT-based method offers fast digestion in a shorter time for all the mAbs tested.