Laboratory of Immunology, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United States.
Division of Therapeutic Performance, Office of Research and Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United States.
Front Immunol. 2022 Sep 6;13:970499. doi: 10.3389/fimmu.2022.970499. eCollection 2022.
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 , 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.
非预期的免疫原性会影响治疗性蛋白和肽的安全性和疗效，因此准确评估免疫原性风险有助于生物制品的选择、开发和监管。产品和工艺相关杂质可以作为佐剂，激活局部或全身固有免疫反应，增加产品免疫原性的可能性。因此，评估产品是否具有固有免疫反应调节杂质（IIRMI）是免疫原性风险评估的关键组成部分。鉴定痕量的个别 IIRMI 可能很困难，并且单独针对所有潜在杂质进行测试是不可行的。因此，为了降低风险，正在开发基于细胞的测定法，该方法使用人血细胞或单核细胞-巨噬细胞报告细胞系来检测能够引发固有免疫激活的痕量杂质。由于这些是基于细胞的测定法，人们担心赋形剂会削弱细胞反应，掩盖存在免疫原性 IIRMI。在这里，我们探讨了常用赋形剂（非离子型清洁剂、糖、氨基酸、增稠剂）对报告细胞系（THP-1 和 RAW-Blue 细胞）和新鲜人血细胞检测纯化 TLR 激动剂作为模型 IIRMI 的敏感性的影响。我们表明，虽然赋形剂不会调节 TLR 激动剂引起的固有免疫反应，但它们会影响基于细胞的 IIRMI 测定的敏感性。当测试 3 种不同的重组药物产品、产品 A（一种代表性的 mAb）、B（一种代表性的生长因子）、C（一种代表性的肽）及其相应的制剂时，也明显降低了检测 LPS、FSL-1 和其他模型 IIRMI 的敏感性。这些结果表明，在开发和验证用于评估治疗性蛋白中临床相关水平的 IIRMI 的基于细胞的测定法时，需要考虑产品配方。优化报告细胞、培养条件和药物产品浓度似乎对于最小化赋形剂的影响并获得敏感和可重复的测定法至关重要。
