A Novel C-terminal Sequence Variant Discovered in an IgG1 Monoclonal Antibody by LC-MS

Sequence variant analysis is critical for characterizing biologic therapeutics and ensuring product quality, consistency, and safety. During routine tryptic peptide mapping of an IgG1 monoclonal antibody therapeutic, we identified a novel variant with a +113 Da mass shift at the heavy chain C-terminus. This variant was present at an unusually high abundance of up to 1.5%. The initial hypotheses suggested a leucine/isoleucine (Leu/Ile) addition; however, HCD multistage tandem mass spectrometry revealed an unexpected two-amino acid substitution (Val-Ala). A synthetic peptide with the hypothesized sequence confirmed this novel sequence variant, exhibiting identical retention time and fragmentation patterns. Further investigation suggested aberrant mRNA splicing, involving a cryptic splice site near glycine-encoding codons in the expression constructs, as a potential underlying mechanism. These findings demonstrate the importance of high-resolution mass spectrometry and alternate fragmentation methods for resolving isobaric ambiguities and highlight the influence of codon optimization on sequence variant profiles.