Silas Dharia Sara, Juneja Bindiya, Kaur Keerat, Narayanareddy Gari Muralikrishna, You Yingjian, Moon Youmi, Chen Yizhuo, Arora Srishti, Hansen Johanna, Muthusamy Kathir, Fu Yue, Palackal Nisha, Pyles Erica A
Protein Biochemistry, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA.
Regeneron Genetic Medicines, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA.
Pharmaceutics. 2024 Sep 19;16(9):1227. doi: 10.3390/pharmaceutics16091227.
In vitro-transcribed (IVT) mRNA has been established as a promising platform for therapeutics and vaccine development. Double-stranded RNA (dsRNA) is a major impurity of IVT mRNA and can trigger unfavored immune responses, potentially causing adverse events in patients. Existing dsRNA detection and quantitation methods, such as gel electrophoresis, ELISA, or homogeneous time-resolved fluorescence (HTRF), have low sensitivity or are time-consuming. A recently published lateral flow immunoassay (LFSA) was shown to be fast, but it lacks the sensitivity for dsRNA with uridine modifications. In this study, we provided a possible explanation for the reduced sensitivity of existing quantitation methods for dsRNA with modified uridines by characterizing the binding affinities of commonly used anti-dsRNA antibodies. Then, a rapid and sensitive biolayer interferometry (BLI) dsRNA detection assay utilizing Flock House Virus (FHV) B2 protein was developed to overcome the challenges in dsRNA detection and the reduced sensitivity. This assay allows the detection of dsRNA with different uridine modifications (ψ, m1ψ, 5 moU) with similar sensitivity as dsRNA without modification. Furthermore, we demonstrated this method can be used to quantify both short and long dsRNA, as well as hairpin-structured dsRNA, providing a more comprehensive detection for dsRNA impurities. Moreover, we applied this assay to monitor dsRNA removal through a purification process. Taken together, this BLI method could enable real-time monitoring of impurities in IVT mRNA production to prevent immunogenicity stemming from dsRNA.
体外转录（IVT）mRNA已成为治疗和疫苗开发的一个有前景的平台。双链RNA（dsRNA）是IVT mRNA的主要杂质，可引发不良免疫反应，可能导致患者出现不良事件。现有的dsRNA检测和定量方法，如凝胶电泳、酶联免疫吸附测定（ELISA）或均相时间分辨荧光（HTRF），灵敏度低或耗时。最近发表的一种侧向流动免疫测定（LFSA）显示速度很快，但对具有尿苷修饰的dsRNA缺乏灵敏度。在本研究中，我们通过表征常用抗dsRNA抗体的结合亲和力，对现有用于修饰尿苷的dsRNA定量方法灵敏度降低的原因给出了一种可能的解释。然后，开发了一种利用禽 flock House病毒（FHV）B2蛋白的快速灵敏的生物层干涉术（BLI）dsRNA检测方法，以克服dsRNA检测中的挑战和灵敏度降低的问题。该检测方法能够以与未修饰的dsRNA相似的灵敏度检测具有不同尿苷修饰（ψ、m1ψ、5 moU）的dsRNA。此外，我们证明了该方法可用于定量短链和长链dsRNA以及发夹结构的dsRNA，为dsRNA杂质提供更全面的检测。此外，我们应用该检测方法监测通过纯化过程去除dsRNA的情况。综上所述，这种BLI方法能够实时监测IVT mRNA生产中的杂质，以防止由dsRNA引起的免疫原性。
