(M1330-02-08) In Vitro Assays for the Evaluation of Innate Immunogenicity in Generic Peptide Drug Products

Purpose: The FDA issued a guidance titled “ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs of rDNA Origin” in May 2021, aiming to facilitate submission and approval for generic peptide drugs. This guidance outlines the FDA’s recommendations for demonstrating equivalence of synthetically produced generic peptide drug products referencing recombinantly produced reference listed drugs (RLDs). An essential aspect in developing and approving these generic peptide products involves assessing immunogenicity risks using in vitro assays. When there are differences in impurities, generic applicants may conduct additional non-clinical assays, such as cell-based or functional binding assays, to ensure their proposed test product does not increase the potential immunogenicity risk. In vitro immunogenicity assays can assess both adaptive and innate immune responses and immunogenicity risks. However, there is currently no standardized methods or regulatory guidance for assessing immunogenicity risks, posing challenges for generic applicants in providing necessary data. In order to address this challenge, we conducted a study to analyze the current practices, methods, and regulatory recommendations. We systematically compared innate immunogenicity data submitted by generic applicants while analyzing the approval criteria for U.S. generic peptide drugs. Furthermore, the gaps in current regulatory guidelines are explored. Our study provides the basis for recommending the best practices in conducting various in vitro assays to evaluate innate immunogenicity.

Methods: We conducted an analysis of abbreviated new drug applications (ANDAs) submitted to the U.S. FDA. This involved cross-comparing conditions, optimization, and key parameters assessed during the method development process by ANDA applicants. These parameters include coverage of human leukocyte antigen (HLA) genotypes in the donor set, number of cells, product concentration, incubation time and combined assay readouts. Manual data extraction techniques were employed to analyze the FDA database, with information organized and compiled in an Excel spreadsheet. Furthermore, common deficiencies were collated for the in vitro immunogenicity assays submitted as part of the ANDAs. The specific submission details, including application numbers, applicants, and specific drug names, were intentionally blinded.

Results: Various in vitro cell-based methods have been employed to assess the innate immune response associated with peptide products (Table 1). This includes: 1) the innate immune response modulating impurity assay, commonly utilizing macrophage cell lines such as RAW-BLUE, THP-1 and HEK (hNOD1 and hNOD2); 2) assessment of the innate immune response to stress-induced aggregates, conducted in human monocyte-like cell lines like THP-1 and MM6; 3) an in vitro assay for monitoring dendritic cell (DC) maturation, evaluated in monocyte-derived DCs; and 4) the PBMC stimulation assay using peripheral blood mononuclear cells (PBMC), which is co-cultured with PBMC to assess the immune response. In addition, common deficiencies have been identified and collated in the assessment of the innate immune response (Table 2).  The deficiencies include: 1) failure to investigate the innate immune response in instances where no new impurities are found because the impurities found through innate immune assays are not usually those detected in the impurity profile comparisons; 2) inadequate justification for the selection of cytokine signal readouts; 3) insufficient demonstration of assay sensitivity; 4) lack of a comprehensive description of the methodology employed; and 5) neglect of the examination of potential formulation effects

Conclusion: In assay development, it is important to carefully identify and prioritize key factors for optimization (Table 3). These include sensitivity and specificity of the assay, determination of optimal concentrations for the drug products, assessment of cell viability or metabolic activity, selection of appropriate positive standards, and examination of potential effects of excipient. These essential parameters will facilitate optimization of immune cell-based assays to enhance their reliability and accuracy in assessing immunogenicity. This work highlights the need for optimization and standardization of these in vitro immunogenicity assays, which are becoming an important part of immunogenicity risk assessments for generic peptide products. At the same time, the results and data collected through this study can be used to support the development of regulatory guidance on conducting this type of assays.

References: FDA’s guidance for industry, ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs of rDNA Origin (May 2021) https://www.fda.gov/media/107622/download

Acknowledgements: This research was supported by federal funds from the U.S. Food and Drug Administration, Center for Drug Evaluation and Research/Office of Generic Drugs, under an Inter-Agency Agreement with NCL/NCI/NIH (IAA #224-19-3008S). Dr. Lee was supported by an appointment to the Oak Ridge Institute for Science and Education (ORISE) Research Participation Program at the CDER administered by the ORISE through an agreement between the U. S. Department of Energy and CDER. The views expressed in this poster do not necessarily reflect the official policies of the Department of Health and Human Services; nor does any mention of trade names, commercial practices, or organization imply endorsement by the United States Government.
Disclaimer: This abstract reflects the views of the author and should not be construed to represent FDA’s views or policies


Table 1. Cross-comparison of Common In Vitro Cell-Based Methods for Assessing Innate Immune Responses


Table 2. Immunogenicity Risk

Assessment: Comments and Guidance Across Varied Parameters


Table 3. Key Components for Comprehensive In Vitro Innate Immunogenicity Analysis Report