Key Laboratory of Antibody Engineering of Guangdong Higher Education Institutes, Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China.
Guangzhou Baiyunshan Biologics Co., Ltd, No.1 Wanbao North Street, Panyu District, Guangzhou, China.
J Virol Methods. 2023 Aug;318:114752. doi: 10.1016/j.jviromet.2023.114752. Epub 2023 May 18.
Host cell proteins (HCPs) are the process-specific and inevitable impurities during the manufacture via a host cell, which affect the safety or efficacy of the bio-product. However, the commercial HCP enzyme-linked immunosorbent assay (ELISA) kits may not apply to specific products such as rabies vaccine from Vero cells. More advanced and process-specific assay methods are needed in the quality control of rabies vaccine throughout the whole manufacturing process. Therefore, a novel time-resolved fluoroimmunoassay (TRFIA) for the detection of process-specific HCP of Vero cells in rabies vaccine was established in this study. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) was used during the preparation of HCP antigen. Based on a sandwich-type immunoassay format, analytes in samples were captured by one antibody coating in the wells and "sandwiched" by another antibody labeled with europium chelates. Due to the complex composition of HCP, both the capture and detected antibodies are polyclonal antibodies from the same anti-HCP antibodies pool. Multiple experiments have identified the optimal conditions to allow the valid and reliable detection of HCP in rabies vaccine. The TRFIA had a satisfactory limit of detection value (0.011 μg/ml) under optimal conditions, with the linear range from 0.0375 to 2.4 μg/ml of HCP. The coefficient variations (CVs) were all < 10%, and the recoveries were in the range of 97.00-102.42%. All the test results of Vero cell protein reference substance were included in the expected concentration, which demonstrated that the present method was available for the test of HCP in rabies vaccine. Based on these results, the novel TRFIA to detect HCP appears to be important for application in modern vaccine quality control during the whole manufacturing process.
宿主细胞蛋白（HCPs）是在宿主细胞制造过程中产生的特定工艺杂质和不可避免的杂质，会影响生物制品的安全性或效力。然而，商业 HCP 酶联免疫吸附测定（ELISA）试剂盒可能不适用于特定产品，如来自 Vero 细胞的狂犬病疫苗。在整个制造过程中，狂犬病疫苗的质量控制需要更先进和特定于工艺的检测方法。因此，本研究建立了一种用于检测狂犬病疫苗中 Vero 细胞特定工艺 HCP 的新型时间分辨荧光免疫分析（TRFIA）。在 HCP 抗原制备过程中使用液相色谱串联质谱法（LC-MS/MS）。基于夹心型免疫分析格式，样品中的分析物被孔中的一种抗体捕获，并被另一种标记有铕螯合物的抗体“夹在中间”。由于 HCP 的复杂组成，捕获和检测抗体均来自同一抗 HCP 抗体池中的多克隆抗体。多项实验确定了最佳条件，可允许有效和可靠地检测狂犬病疫苗中的 HCP。在最佳条件下，TRFIA 的检测限（0.011μg/ml）令人满意，HCP 的线性范围为 0.0375 至 2.4μg/ml。变异系数（CV）均<10%，回收率在 97.00%至 102.42%范围内。Vero 细胞蛋白参考物质的所有测试结果均包含在预期浓度内，表明本方法可用于狂犬病疫苗中 HCP 的检测。基于这些结果，新型 TRFIA 检测 HCP 似乎对于整个制造过程中现代疫苗质量控制中的应用非常重要。
