"Monoclonal antibodies are the fastest growing class of therapeutic drugs because of their high specificities to targeQt 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. Conventional antibody characterization workflows contain an in-solution digestion step, which is labor-intensive and time-consuming. Protease-containing membranes are an attractive alternative platform for protein digestion because of their high local enzyme concentrations, short radial diffusion distances, rapid convection in pores, simple fabrication and low cost. Additionally, variation of protein residence time in the membrane gives control over the size of proteolytic peptides. This research focuses on developing workflows for monoclonal antibody characterization using functionalized porous membranes. Sequential adsorption of poly (styrene sulfonate) and pepsin in a porous nylon membrane forms a pepsin membrane reactor. Pepsin is inexpensive and catalyzes proteolysis in acidic solutions, which avoids the need to alkylate cysteine residues and limits antibody deamidation. Variation of the residence times (3 ms to 3 s) of antibody solutions in pepsin-containing membranes yields "bottom-up" (1-2 kDa) to 'middle-down' (5-15 kDa) peptides in less than 10 min. These peptic peptides cover the entire sequences of Herceptin and a WatersTM antibody. Compared with the performance of bottom-up (in-solution tryptic digestion) and top-down (intact protein fragmentation) analysis of an antibody light chain, middle-down (in-membrane peptic digestion) analysis gives the highest bond cleavage (99%). In-membrane digestion also facilitates detection of PTMs such as oxidation, deamidation, N-terminal pyroglutamic acid formation and glycosylation. Recently developed protease-containing spin membranes provide an excellent platform for rapid, membrane-based protein digestion prior to ultrahigh-resolution Orbitrap MS analysis. Centrifugation of 100-200 æL of pretreated protein solutions through the pepsin- or trypsin-containing membranes takes less than 1 min and gives nearly 100% coverage of the protein sequences in subsequent direct infusion MS analysis of digests of apomyoglobin and four commercial monoclonal antibodies (Herceptin, Avastin, Rituxan and Vectibix). MS analysis of peptic and tryptic peptides also reveals mAb PTMs such as N-terminal pyroglutamate formation, C-terminal Lysine clipping and glycosylation. Liquid chromatography coupled to tandem mass spectrometry analysis of tryptic spin digests and subsequent MaxQuant data searching show 100% sequence coverage of all four antibody light chains, and 75.1%-98.4% coverage of the heavy chains. Compared to in-solution tryptic digestion of mAbs, spin digestion yields higher sequence coverage and a larger number of unique peptides. In-membrane digestion also facilitates protein sequence comparison. Rapid peptic in-membrane digestion of two antibodies with direct infusion MS analysis accurately reveals the antibody modification site in less than 1 h. Overall, membrane-based protein digestion uses minimal sample preparation time and yields high peptide and sequence coverages for identification of protein PTMs."--Page ii-iii.
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