Artifactual Sequence Variant Induced by Nonspecific Cleavage-Linked Transpeptidation in Peptide Mapping

Identification of sequence variants that may affect the safety and efficacy is critical in cell line development and process optimization for therapeutic antibodies. Protein-level sequence variant analysis typically employs LC-MS/MS peptide mapping. However, incorrect variant identification often arises from mismatched mass spectral assignments. Furthermore, the sample preparation workflow, particularly the proteolytic digestion steps, may inadvertently generate nonphysiological sequence variants that are not readily distinguishable from authentic variants due to their correct peptide matches. Here we report a type of artifactual sequence variant (e.g., C⁴²⁵S → C⁴²⁵R variant in the antibody Fc region) and elucidate its formation mechanism. Our findings demonstrate that such byproducts originate from digested amino acid transfer to the C-terminus of nonspecifically cleaved peptides, such as the transfer of Arg to W⁴¹⁷QQGNVFSC⁴²⁵ in Trypsin/Lys-C mix-digested antibodies, resulting in W⁴¹⁷QQGNVFSC⁴²⁵R. The significantly higher cleavage efficiency at carbamidomethyl-C⁴²⁵S compared to carbamidomethyl-C⁴²⁵R facilitates byproduct accumulation. This study establishes that such artifacts arise through nonspecific cleavage-associated transpeptidation, enabling their identification as analytical artifacts during data processing.