Biopharmaceutical C-Terminal Lysine Variants Quantitation
Using nanoLC-MS/MS

Reut Kornasio, Development, BTG-FERRING, Kiryat Malachi, Israel
Yishai Levin, De Botton Institute For Protein Profiling, The Nancy And Stephen Grand Israel National Center For Personalized Medicine, Weizmann Institute Of Science, Rehovot, Israel

The C-terminal lysine variation is commonly observed in biopharmaceutical drugs containing IgG Fc region.

Although human IgG heavy chain gene encodes a C-terminal lysine, this residue is mostly absent from the endogenous antibodies isolated from serum.

This modification can be important since it is found to be sensitive to the production process.

Targeted proteomics was applied to determine the percentage of C-terminal lysine residue present in different batches of a biopharmaceutical drug.

The reduced and alkylated protein was subjected to tryptic digestion. Samples were then spiked with two Aqua stable isotopically labelled peptide standards and analysed using nanoflow UPLC coupled to a tandem quadrupole MS for quantification of the C-terminal lysine.

All drug batches demonstrated the presence of truncated C-terminal lysine in abundance greater than 98%. These results demonstrate batch-to-batch consistency with regard to this post-translational modification . Results correspond to the natural state of the endogenous IgG Fc in serum.

The presented method can be used as a tool for quality control of batch-to-batch consistency of Fc-containing drugs  with respect to C-terminal variants. Such valuable information may facilitate the development and optimization of consistent manufacturing processes.