Pepsin-Containing Membranes for Controlled Monoclonal Antibody Digestion Prior to Mass Spectrometry Analysis
Yongle Pang, Wei-Han Wang, Gavin E Reid, Donald F Hunt, Merlin L Bruening
Analytical Chemistry | AMER CHEMICAL SOC | Published : 2015
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..View full abstract
Awarded by U.S. National Science Foundation
Awarded by NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
Awarded by Direct For Mathematical & Physical Scien
We gratefully acknowledge the U.S. National Science Foundation (Nos. CHE-1152762 and CHE-1506315) for funding this work. We thank Dr. Mohammad Muhsin Chisti from Michigan State University for providing Trastuzumab. We also thank the Michigan State University Mass Spectrometry Facility and Dr. Todd Lydic (Molecular Metabolism and Disease Collaborative Mass Spectrometry Core) for helping to analyze the samples.