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Toward a Comprehensive Bottom-up and Top-down Analysis of the NIST Reference Monoclonal Antibody

Careful characterization of therapeutic proteins is required due to their inherent variability. A reference protein standard can represent a major analytical tool for the industry, but it must be well-characterized in order to serve as a reference. In this study, high resolution mass spectrometric data of the NIST mAb interim reference material (RM 8670, lot 3F1b) was analyzed by new bioinformatics tools to efficiently identify and quantify modifications, including oxidation, deamidation, glycation, glycosylation, and sequence variants.

Heavy and light chain components were measured by both bottom-up and top-down (sometimes called middle-down) approaches by a high resolution Thermo Fisher Orbitrap Elite mass spectrometer employing CID, HCD and ETD fragmentation modes. The data was analyzed by a combination of the search engine Byonic™ and new inspection software Byologic® that combines and compares MS1 and MS2 data streams and performs label-free quantification by taking the ratio of extracted ion chromatograms (XICs) of the modified to unmodified peptide. In addition, new peptide mapping software, Byomap™, was employed to quantify and annotate the peptide map.

Bottom-up analysis with just a few data sets led to extensive site-specific identification and quantification of sequence variants, glycations, glycosylations, oxidations, and deamidations. Glycosylation (both bottom-up and top-down) was measured for the intact molecular forms. The top-down analysis by Byonic provided annotation of fragments spanning the entire sequences, with ETD, at least initially, providing the greatest degree of fragment coverage. Oxidation sites, glycosylation and truncations were observed in the top-down analysis. Overall, with a modest amount of high quality mass spectra, extensive quantitative analysis of the NIST interim reference standard was performed in a rapid fashion.