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

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• 作者：Yongle Pang ; Wei-Han Wang ; Gavin E. Reid ; Donald F. Hunt ; Merlin L. Bruening
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：November 3, 2015
• 年：2015
• 卷：87
• 期：21
• 页码：10942-10949
• 全文大小：513K
• ISSN：1520-6882

文摘

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 鈥渂ottom-up鈥?(1鈥? kDa) to 鈥渕iddle-down鈥?(5鈥?5 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 鈥渂ottom-up鈥?(in-solution tryptic digestion), 鈥渢op-down鈥?(intact protein fragmentation), and 鈥渕iddle-down鈥?(in-membrane digestion) analysis of an antibody light chain. Data from tandem MS show 99%, 55%, and 99% bond cleavage for 鈥渂ottom-up鈥? 鈥渢op-down鈥? and 鈥渕iddle-down鈥?analyses, respectively. In-membrane digestion also facilitates detection of PTMs such as oxidation, deamidation, N-terminal pyroglutamic acid formation, and glycosylation. Compared to 鈥渂ottom-up鈥?and 鈥渢op-down鈥?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.