Biologics - Process R&D and Downstream Process, Merck & Co., Inc., Kenilworth, NJ, USA.
Biologics - Process R&D and Downstream Process, Merck & Co., Inc., Kenilworth, NJ, USA.
J Pharm Biomed Anal. 2020 Sep 10;189:113472. doi: 10.1016/j.jpba.2020.113472. Epub 2020 Jul 15.
Chinese hamster ovary (CHO) cells are the host cell of choice for manufacturing biologic drugs, like monoclonal antibody, in the biopharmaceutical industry. Retrovirus-like particles (RVLPs) are made during the manufacturing process with CHO cells and it is incumbent upon the manufacturer to perform risk assessment based on levels of RVLP in unprocessed bulk. Quantification of RVLP using electron microscopy (EM) is the standard method. However, reverse transcription based real-time PCR (RT qPCR) is an alternative method available. This method involves RNase digestion of cell culture fluid to remove free RNA, followed by extraction of total nucleic acid and digestion with DNase to remove extracted DNA molecules, and then finally reverse transcription and PCR. Here we report a method where the nucleic acids extraction step is eliminated prior to qPCR. In this method the cell-free culture supernatant sample is digested with thermolabile DNase and RNase at the same time in a 96-well PCR plate; subsequently the enzymes are heat-denatured; then RT qPCR reagents are added to the wells in the PCR plate along with standards and controls in other wells of the same plate; finally the plate is subjected to RT qPCR for analysis of RVLP RNA in the samples. This direct RT qPCR method for RVLP is sensitive to 10 particles of RVLP with good precision and accuracy and has a wide linear range of quantification. The method has been successfully tested with different production batches, shown to be consistent, and correlates well with the extraction-based method. However, the results are about 1-log higher compared to EM method. This method simplifies the RVLP quantification protocol, reduces time of analysis and leads to increased assay sensitivity and development of automated high-throughput methods. Additionally, the method can be an added tool for viral clearance studies, by testing process-intermediate samples like Protein A column and ion-exchange column eluates, for increased confidence in purification of biologics manufactured in CHO cell culture.
中国仓鼠卵巢（CHO）细胞是生物制药行业生产生物药物（如单克隆抗体）的首选宿主细胞。在制造过程中，CHO 细胞会产生类逆转录病毒样颗粒（RVLP），制造商有责任根据未经处理的大容量 RVLP 水平进行风险评估。使用电子显微镜（EM）定量 RVLP 是标准方法。然而，基于逆转录的实时 PCR（RT-qPCR）是一种可用的替代方法。该方法涉及用 RNase 消化细胞培养液以去除游离 RNA，然后提取总核酸并用 DNase 消化以去除提取的 DNA 分子，最后进行逆转录和 PCR。在这里，我们报告了一种在 qPCR 之前消除核酸提取步骤的方法。在该方法中，无细胞培养上清样品在 96 孔 PCR 板中同时用热敏 DNase 和 RNase 消化；随后酶被热变性；然后将 RT-qPCR 试剂添加到 PCR 板的孔中，同时在同一板的其他孔中加入标准品和对照品；最后将板进行 RT-qPCR 分析样品中的 RVLP RNA。该 RVLP 的直接 RT-qPCR 方法对 10 个 RVLP 颗粒敏感，具有良好的精密度和准确性，并且具有广泛的定量线性范围。该方法已成功用于不同的生产批次测试，结果一致，与基于提取的方法相关性良好。然而，与 EM 方法相比，结果高约 1 个对数。该方法简化了 RVLP 定量方案，减少了分析时间，并提高了测定灵敏度和自动化高通量方法的开发。此外，该方法可以作为病毒清除研究的附加工具，通过测试蛋白 A 柱和离子交换柱洗脱液等过程中间样品，提高对 CHO 细胞培养生产的生物制剂的纯化信心。
