Development of an efficient LC-MS peptide mapping method using accelerated sample preparation for monoclonal antibodies

Peptide mapping by liquid chromatography-mass spectrometry (LC-MS) is a common method for characterizing primary sequences of monoclonal antibodies (mAbs) and their post-translational modifications (PTMs). Most methods prepare digests by incubating samples with proteases from several hours to overnight. This often induces artifacts of some modifications such as deamidation and isomerization, resulting in overestimated product-related modifications levels. Hour-long-digestion can generate complicate chromatographic profiles due to semi-cleavages or other unnecessary reactions, interfering with the quantitation of peaks of interest. On the other hand, shortening digestion-time can cause incomplete peptide cleavages, thus low sequence coverage and poor repeatability. This study applied pressure cycling technology (PCT) to tryptic digestion and PNGase F deglycosyaltion. A 0.5-h PCT assistant tryptic digestion, by alternating cycles of 10-s atmospheric pressure and 50-s high pressure (30 kpsi) at 37 °C, was evaluated and compared with two conventional digestions, 4-h and 18-h (i.e., overnight) incubations at 37 °C under atmospheric pressure. The 0.5-h PCT assistant deglycosylation was also assessed using the same conditions as those by PCT tryptic digestion. The results demonstrated the application of a 0.5-h PCT to tryptic digestion minimized modification artifacts and reduced interference with quantitation by providing a clean chromatographic profile. The 0.5-h PCT assistant deglycosylation completely removed the N-glycans from the Asn301 in the heavy chains of monoclonal antibody with no impact on the chromatographic profile of the tryptic digests.