Haile Lydia Asrat, Puig Montserrat, Kelley-Baker Logan, Verthelyi Daniela
Laboratory of Immunology, Division of Biotechnology Review and Research III, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.
PLoS One. 2015 Apr 22;10(4):e0125078. doi: 10.1371/journal.pone.0125078. eCollection 2015.
Therapeutic proteins can contain multiple impurities, some of which are variants of the product, while others are derived from the cell substrate and the manufacturing process. Such impurities, even when present at trace levels, have the potential to activate innate immune cells in peripheral blood or embedded in tissues causing expression of cytokines and chemokines, increasing antigen uptake, facilitating processing and presentation by antigen presenting cells, and fostering product immunogenicity. Currently, while products are tested for host cell protein content, assays to control innate immune response modulating impurities (IIRMIs) in products are focused mainly on endotoxin and nucleic acids, however, depending on the cell substrate and the manufacturing process, numerous other IIRMI could be present. In these studies we assess two approaches that allow for the detection of a broader subset of IIRMIs. In the first, we use commercial cell lines transfected with Toll like receptors (TLR) to detect receptor-specific agonists. This method is sensitive to trace levels of IIRMI and provides information of the type of IIRMIs present but is limited by the availability of stably transfected cell lines and requires pre-existing knowledge of the IIRMIs likely to be present in the product. Alternatively, the use of a combination of macrophage cell lines of human and mouse origin allows for the detection of a broader spectrum of impurities, but does not identify the source of the activation. Importantly, for either system the lower limit of detection (LLOD) of impurities was similar to that of PBMC and it was not modified by the therapeutic protein tested, even in settings where the product had inherent immune modulatory properties. Together these data indicate that a cell-based assay approach could be used to screen products for the presence of IIRMIs and inform immunogenicity risk assessments, particularly in the context of comparability exercises.
治疗性蛋白质可能含有多种杂质，其中一些是产品的变体，而其他杂质则源自细胞基质和生产过程。这些杂质即使以痕量水平存在，也有可能激活外周血中或组织内的先天免疫细胞，导致细胞因子和趋化因子表达，增加抗原摄取，促进抗原呈递细胞的加工和呈递，并增强产品的免疫原性。目前，虽然产品会检测宿主细胞蛋白含量，但控制产品中先天免疫反应调节杂质（IIRMI）的检测方法主要集中在内毒素和核酸上，然而，根据细胞基质和生产过程的不同，可能还存在许多其他IIRMI。在这些研究中，我们评估了两种方法，可用于检测更广泛的IIRMI子集。第一种方法是，我们使用转染了Toll样受体（TLR）的商业细胞系来检测受体特异性激动剂。该方法对痕量水平的IIRMI敏感，并能提供存在的IIRMI类型的信息，但受到稳定转染细胞系可用性的限制，并且需要预先了解产品中可能存在的IIRMI。另一种方法是，使用人和小鼠来源的巨噬细胞系组合，可以检测更广泛的杂质谱，但无法确定激活的来源。重要的是，对于这两种系统，杂质的检测下限（LLOD）与外周血单核细胞（PBMC）的检测下限相似，并且不受所测试治疗性蛋白质的影响，即使在产品具有固有免疫调节特性的情况下也是如此。这些数据共同表明，基于细胞的检测方法可用于筛选产品中是否存在IIRMI，并为免疫原性风险评估提供信息，特别是在可比性研究的背景下。
