Monoclonal Antibody Disulfde Bond Mapping
by LC-MS/MS

Monoclonal antibodies are increasing in prevalence as biotherapeutic agents. Like many therapeutic proteins, monoclonal antibodies derive their function from their structure. Disulfde bonds between cysteine residues are an important posttranslational modifcation which contribute to a monoclonal antibody’s secondary and tertiary structure. Guidelines from ICH (Q6B) and the FDA (April 2015) require assessment of secondary and tertiary structure, and posttranslational modifcations in the proposed product and the reference product. Shuffled disulfde bond arrangements represent impurities in the therapeutic and can arise from poorly optimized expression systems, processing and improper handling.

Historically, disulfde bond characterization presented a major analytical challenge because typical mass spectrometry-based peptide mapping approaches disrupt cysteines during sample preparation. Problematically, methodologies designed to assess disulfde bond arrangement induce disulfde bond shuffling as sample handling artefacts. Using modern techniques of high resolution mass spectrometry and systematic software analysis, we confrm the presence of expected disulfde bonds from an antibody standard.