Observation of Heavy-Chain C-Terminal Amidation in Human Endogenous IgG

Therapeutic IgG mAbs expressed from Chinese hamster ovary (CHO) cells are known to contain three C -ter-minal variants in their heavy chains, namely, the unprocessed C-terminal lysine, the processed C-terminal lysine, and C-terminal amidation. Although the presence of C-terminal amidation in CHO-expressed IgGs is well studied, the biological impact of the variant on the safety and efficacy of biotherapeutics has not been well understood. To further our biological understanding of C-terminal amidation, we analyzed a series of IgG samples, including both endogenous human IgGs as well as recombinant IgGs of different subclasses expressed from both CHO and murine cell lines, for their heavy-chain C-terminal variants by LC-MS/MS based peptide mapping. The results demonstrate that heavy-chain C-terminal amidation is a common variant occurring in IgG of all four subclasses (IgG1, IgG2, IgG3 and IgG4). The variant is generally present in recombi-nant IgG mAbs expressed from CHO cell lines but not in IgG mAbs expressed from murine cell lines, whereas the IgGs expressed from murine cell lines contain a much larger amount of unprocessed C-terminal lysine. Additionally, a significant amount of heavy-chain C-terminal amidation is observed in endogenous human IgGs, indicating that small amount of the variant present in therapeutic IgGs does not pose a safety concern. (c) 2022 The Authors. Published by Elsevier Inc. on behalf of American Pharmacists Association. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Automated summary

Therapeutic IgG monoclonal antibodies expressed from Chinese hamster ovary (CHO) cells contain C-terminal variants in their heavy chains, including C-terminal amidation. These variants are commonly found in IgG of all subclasses and are more prevalent in recombinant IgG expressed from CHO cell lines compared to murine cell lines. The presence of a small amount of C-terminal amidation in therapeutic IgGs does not pose a safety concern.