Holland David A, Acevedo-Skrip Jillian, Barton Joshua, Thompson Rachel, Bowman Amy, Dewar Emily A, Miller Danielle V, Zhao Kaixi, Swartz Andrew R, Loughney John W
Analytical Research & Development, Merck & Co., Inc., Rahway, NJ 07065, USA.
Process Research and Development, Merck & Co., Inc., Rahway, NJ 07065, USA.
Vaccines (Basel). 2024 Aug 8;12(8):899. doi: 10.3390/vaccines12080899.
The rise of mRNA as a novel vaccination strategy presents new opportunities to confront global disease. Double-stranded RNA (dsRNA) is an impurity byproduct of the in vitro transcription reaction used to manufacture mRNA that may affect the potency and safety of the mRNA vaccine in patients. Careful quantitation of dsRNA during manufacturing is critical to ensure that residual dsRNA is minimized in purified mRNA drug substances. In this work, we describe the development and implementation of a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) to quantitate nanogram quantities of residual dsRNA contaminants in mRNA process intermediates using readily available commercial reagents. This sandwich ELISA developed in this study follows a standard protocol and can be easily adapted to most research laboratory environments. Additionally, a liquid handler coupled with an automated robotics system was utilized to increase assay throughput, improve precision, and reduce the analyst time requirement. The final automated sandwich ELISA was able to measure <10 ng/mL of dsRNA with a specificity for dsRNA over 2000-fold higher than mRNA, a variability of <15%, and a throughput of 72 samples per day.
信使核糖核酸（mRNA）作为一种新型疫苗接种策略的兴起为应对全球疾病带来了新机遇。双链核糖核酸（dsRNA）是用于制造mRNA的体外转录反应的杂质副产物，可能会影响mRNA疫苗对患者的效力和安全性。在生产过程中仔细定量dsRNA对于确保纯化的mRNA药物中残留的dsRNA降至最低至关重要。在这项工作中，我们描述了一种夹心酶联免疫吸附测定（ELISA）的开发与实施，该方法使用现成的商业试剂来定量mRNA工艺中间体中纳克级数量的残留dsRNA污染物。本研究中开发的这种夹心ELISA遵循标准方案，并且可以轻松适用于大多数研究实验室环境。此外，还使用了液体处理仪与自动机器人系统相结合，以提高检测通量、提高精度并减少分析人员的时间需求。最终的自动化夹心ELISA能够检测到浓度低于10 ng/mL的dsRNA，对dsRNA的特异性比对mRNA高2000倍以上，变异系数小于15%，每天的检测通量为72个样本。
