Brian J. Roberts ¹ Aimee E. Mattei ¹ Kristina E. Howard ² James L. Weaver ² Hao Liu ² Sandra Lelias ¹ William Martin ¹ Daniela Verthelyi ² Eric Pang ² Katie Edwards ³ Anne S. De Groot ^1*

• ¹ EpiVax (United States), Providence, Rhode Island, United States
• ² Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, Maryland, United States
• ³ Calspan-University of Buffalo Research Center (CUBRC), Buffalo, United States

Advances in synthetic peptide synthesis have enabled rapid and cost-effective peptide drug manufacturing. For this reason, peptide drugs that were first produced using recombinant DNA (rDNA) technology are now being produced using solid and liquid phase peptide synthesis. While peptide synthesis has some advantages over rDNA expression methods, new peptide-related impurities may be generated during synthesis that differ from the active pharmaceutical ingredient (API). These impurity byproducts of the original peptide sequence feature amino acid insertions, deletions and side chain modifications that may alter the immunogenicity risk profile of the drug product. Impurities resulting from synthesis have become the special focus of regulatory review and approval for human use as outlined in the FDA’s Center for Drug Evaluation and Research guidance document, “ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs of rDNA Origin”, published in 2021. This case study illustrates how in silico and in vitro methods can be applied to assess the immunogenicity risk of impurities that may be present in synthetic generic versions of the salmon calcitonin (SCT) drug product. Sponsors of generic drug ANDA should consider careful control of these impurities (for example, keeping the concentration of the immunogenic impurities below the cut-off recommended by FDA regulators). Twenty example SCT impurities were analyzed using in silico tools and assessed as having slightly more or less immunogenic risk potential relative to the SCT API peptide. Class II HLA binding assays provided independent confirmation that a 9-mer sequence present in the C-terminus of SCT binds promiscuously to multiple HLA DR alleles, while T cell assays confirmed expected T cell responses to SCT and selected impurities. In silico analysis combined with in vitro assays that directly compare the API to each individual impurity peptide may be a useful approach to assessing the potential immunogenic risk posed by peptide impurities that are present in generic drug products.