Expression vector-derived heterogeneity in a therapeutic IgG4 monoclonal antibody

While characterizing a therapeutic IgG4 monoclonal antibody (mAb), we observed a variant with a mass 1177 Da larger than the predominant mAb form that could not be ascribed to previously described modifications. Through successive rounds of experimentation, we localized the mass addition to the C-terminus of the heavy chain (HC). During this process we observed that when the mAb was broken down into separate domains, the Fc and the 1177 Da-modified Fc could be chromatographically separated. Separation allowed collection of native and modified Fc fractions for LC/MS peptide mapping. A unique peptide present in the modified fraction was de novo sequenced and demonstrated to be a modified form of the HC C-terminus lacking two native residues (GK) and gaining twelve additional non-native residues (EAEAASASELFQ). Aware of other mAb variants with genetic origins, we sought to understand whether this modification too had a genetic basis. In silico translation of the expression vector encoding the mAb demonstrated that a normally non-coding section of nucleotides in the + 1 reading frame relative to the HC C-terminal coding region could have led to a transcript with the non-native C-terminal extension. Two potential mechanisms for how this nucleotide sequence might have fused to the native HC coding region and led to expression of the extension product are presented.