All Ion Differential Analysis Refines the Detection of Terminal and Internal Diagnostic Fragment Ions for the Characterization of Biologics Product-Related Variants and Impurities by Middle-down Mass Spectrometry
ACS Publications
June 12, 2019
Characterization and monitoring of post-translational modifications (PTMs) are key analytical requirements during the development of biologics. Top- and middle-down (MD) approaches aim at capturing a direct snapshot of all proteoforms with their combinatorial distribution. However, classical MD data analysis is predominantly limited to the interpretation of terminal ion series and PTMs matched by mass. In this study, time-resolved deconvolution (TRD) maps were produced to detect variants and impurities in Fd, Fc/2, and LC subunits of an IgG1 consistently across multiple samples. Classical MD analysis retrieved terminal ions, suggesting a deamidation at a NN motif for a LC+1 Da species, and inconclusive information for a LC+40 Da species. Additionally, we performed differential analysis of all MS2 ions across unmodified and variant subunit spectra to focus data analysis on spectral differences and reveal diagnostic ions (present, absent, enriched, or depleted ions) before fragment assignment. This sensitive methodology was able to detect diagnostic ions in a chimeric spectrum pointing at a proline-to-histidine sequence variant (+40 Da) missed by classical MD analysis. This methodology was pivotal to unravel relevant terminal ions and internal fragments N-terminal to proline as diagnostic ions to confirm the deamidation site. Moreover, different cleavage propensities were revealed at the deamidated DN site compared to the native NN motif for terminal and internal fragments, which may be tracked as a diagnostic behavior. Differential analysis may refine the detection of novel diagnostic ions and leverage the sequence information on internal fragments for the characterization of product-related variants and impurities by MD mass spectrometry.
Genedata Expressionist® supported this research by automating and harmonizing the mass spectrometry data processes, enabling the monitoring of behavior for terminal and internal fragments. This streamlined workflow facilitated and refined the detection of diagnostic ions for deamidation and sequence variants in monoclonal antibodies, increasing sequence coverage and improving the characterization of biologics by middle-down mass spectrometry.