Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients

Thacker, Seth G.; Her, Cheng; Kelley-Baker, Logan; Ireland, Derek C.; Manangeeswaran, Mohanraj; Pang, Eric S.; Verthelyi, Daniela

doi:10.3389/fimmu.2022.970499

Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients

Journal Article · Tue Sep 06 00:00:00 UTC 2022 · Frontiers in Immunology

DOI:https://doi.org/10.3389/fimmu.2022.970499· OSTI ID:1983088

Thacker, Seth G. ^[1]; Her, Cheng ^[1]; Kelley-Baker, Logan ^[1]; Ireland, Derek C. ^[1]; Manangeeswaran, Mohanraj ^[1]; Pang, Eric S. ^[1]; Verthelyi, Daniela ^[1]

U.S. Food and Drug Administration (FDA), Silver Spring, MD (United States). Center for Drug Evaluation and Research

Unintended immunogenicity can affect the safety and efficacy of therapeutic proteins and peptides, so accurate assessments of immunogenicity risk can aid in the selection, development, and regulation of biologics. Product- and process- related impurities can act as adjuvants that activate the local or systemic innate immune response increasing the likelihood of product immunogenicity. Thus, assessing whether products have innate immune response modulating impurities (IIRMI) is a key component of immunogenicity risk assessments. Identifying trace levels of individual IIRMI can be difficult and testing individually for all potential impurities is not feasible. Therefore, to mitigate the risk, cell-based assays that use human blood cells or monocyte-macrophage reporter cell lines are being developed to detect minute quantities of impurities capable of eliciting innate immune activation. As these are cell-based assays, there is concern that excipients could blunt the cell responses, masking the presence of immunogenic IIRMI. Here, we explore the impact of frequently used excipients (non-ionic detergents, sugars, amino acids, bulking agents) on the sensitivity of reporter cell lines (THP-1- and RAW-Blue cells) and fresh human blood cells to detect purified TLR agonists as model IIRMI. We show that while excipients do not modulate the innate immune response elicited by TLR agonists in vivo, they can impact on the sensitivity of cell-based IIRMI assays. Reduced sensitivity to detect LPS, FSL-1, and other model IIRMI was also evident when testing 3 different recombinant drug products, product A (a representative mAb), B (a representative growth factor), C (a representative peptide), and their corresponding formulations. These results indicate that product formulations need to be considered when developing and validating cell-based assays for assessing clinically relevant levels of IIRMI in therapeutic proteins. Optimization of reporter cells, culture conditions and drug product concentration appear to be critical to minimize the impact of excipients and attain sensitive and reproducible assays.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN (United States); U.S. Food and Drug Administration (FDA), Silver Spring, MD (United States)

Sponsoring Organization:: US Food and Drug Administration (FDA); USDOE Office of Science (SC)

Grant/Contract Number:: SC0014664

OSTI ID:: 1983088

Journal Information:: Frontiers in Immunology, Journal Name: Frontiers in Immunology Vol. 13; ISSN 1664-3224

Publisher:: Frontiers Research FoundationCopyright Statement

Country of Publication:: United States

Language:: English

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in vitro model
masking
reporter cell lines

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