(W1130-02-09) Immunogenicity Risk Assessment of Salmon Calcitonin Peptide Impurities Using In Silico and In Vitro Methods

Purpose: Recent advances in synthetic peptide synthesis have enabled more cost-effective peptide drug manufacturing. Accordingly, peptide drugs initially produced using recombinant DNA (rDNA) technology are now 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). Impurity byproducts of the original peptide sequence feature modifications that may alter the immunogenicity risk profile of the drug product. These impurities have become the 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. Here we present a case study illustrates how in silico and in vitro methods can be applied to assess the immunogenicity risk of impurities found in synthetic versions of the salmon calcitonin (SCT) drug product.

Methods: The immunogenicity risk was assessed utilizing three independent (orthogonal) methods: in silico analysis, class II HLA binding assays and naïve T cell assays (De Groot et al., 2023). Using in silico epitope prediction tools, we evaluated the sequences of the salmon calcitonin API peptide and 20 impurities identified within synthetic generic salmon calcitonin. Impurities were ranked for immunogenic risk potential. Following in silico analysis, a subset of impurity peptides were selected for in vitro assays.  An in vitro HLA binding was next employed to assess in HLA DRB1 binding affinity between API and impurity sequences, followed by a comparison of T cell responses to the API and impurities ex vivo.

Results: Consistent with the in silico analysis, the number of donor PBMC samples responding to the peptide impurities was increased, when compared to donor PBMC sample responses to the SCT API peptide, in the naïve CD4 T cell assays.

Conclusion: Taken together, the results indicate that the in vitro class II HLA binding and T cell assays that were performed using the API peptide and selected impurities were generally aligned with in silico risk assessments of the same sequences, suggesting that this approach of combining in silico and in vitro evaluation methods is useful for evaluating the immunogenicity risk of peptide impurities as recommended in the FDA guidance, “ANDAs for Certain Highly Purified Synthetic Drug Products That Refer to Listed Drugs of rDNA Origin”.

References: De Groot, A. S., Roberts, B. J., Mattei, A., Lelias, S., Boyle, C., and Martin, W. D. (2023). Immunogenicity risk assessment of synthetic peptide drugs and their impurities. Drug Discov. Today 28, 103714. doi: 10.1016/j.drudis.2023.103714.
Roberts, BJ, Aimee Mattei, Kristina E. Howard, James L Weaver, Hao Liu Sandra Lelias, William D Martin, Daniella Verthelyi, Eric Pang, Katie Edwards and Anne S. De Groot. Assessing the Immunogenicity Risk of Salmon Calcitonin Peptide Impurities Using In Silico and In Vitro Methods. Submitted to Fronteirs in Pharmacology, manuscript under review.

Acknowledgements: AS De Groot and WD Martin are senior officers and majority shareholders of EpiVax, a privately held immunoinformatics and vaccine design company. Brian J Roberts, Aimee Mattei and Sandra Lelias are current employees. These authors have no other relevant affiliations or financial involvement with any other organization or entity with financial interests in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. The research described in this report was performed by EpiVax in collaboration with CUBRC and the FDA contributors listed in acknowledgements below. The work for this project was supported by an FDA contract HHSF223018186C. We wish to acknowledge the contributions of Dr Kristina E. Howard, Dr James L Weaver, Dr Hao Liu, Dr Daniella Verithelyi and Dr Eric Pang of the US FDA, and Dr Katie Edwards (CUBRC)