Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis

Monoclonal antibodies (mAbs) are the fastest growing class of therapeutic drugs, because of their high specificities to target cells. Facile analysis of therapeutic mAbs and their post-translational modifications (PTMs) is essential for quality control, and mass spectrometry (MS) is the most powerful tool for antibody characterization. This study uses pepsin-containing nylon membranes as controlled proteolysis reactors for mAb digestion prior to ultrahigh-resolution Orbitrap MS analysis. Variation of the residence times (from 3 ms to 3 s) of antibody solutions in the membranes yields "bottom-up" (1-2 kDa) to "middle-down" (5-15 kDa) peptide sizes within less than 10 min. These peptides cover the entire sequences of Trastuzumab and a Waters antibody, and a proteolytic peptide comprised of 140 amino acids from the Waters antibody contains all three complementarity determining regions on the light chain. This work compares the performance of "bottom-up" (in-solution tryptic digestion), "top-down" (intact protein fragmentation), and "middle-down" (in-membrane digestion) analysis of an antibody light chain. Data from tandem MS show 99%, 55%, and 99% bond cleavage for "bottom-up", "top-down", and "middle-down" analyses, respectively. In-membrane digestion also facilitates detection of PTMs such as oxidation, deamidation, N-terminal pyroglutamic acid formation, and glycosylation. Compared to "bottom-up" and "top-down" approaches for antibody characterization, in-membrane digestion uses minimal sample preparation time, and this technique also yields high peptide and sequence coverage for the identification of PTMs.