title: Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis.
abstract: 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.
authors.affiliation: Department of Chemistry, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Yongle
authors.initials: Y
authors.lastname: Pang
authors.affiliation: Department of Chemistry, University of Virginia , Charlottesville, Virginia 22904, United States.
authors.forename: Wei-Han
authors.initials: WH
authors.lastname: Wang
authors.affiliation: School of Chemistry, Department of Biochemistry and Molecular Biology, Bio21 Molecular Science & Biotechnology Institute, The University of Melbourne , Parkville, Victoria 3010, Australia.
authors.forename: Gavin E
authors.initials: GE
authors.lastname: Reid
authors.affiliation: Department of Chemistry, University of Virginia , Charlottesville, Virginia 22904, United States.|Department of Pathology, Health Sciences Center, University of Virginia , Charlottesville, Virginia 22908, United States.
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
authors.affiliation: Department of Chemistry, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Merlin L
authors.initials: ML
authors.lastname: Bruening
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002853:Chromatography, Liquid; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000737:chemistry*; D059748:Proteolysis; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D008567:Membranes, Artificial; D010434:Pepsin A