Institute of Bioprocess Engineering and Pharmaceutical Technology, Department Life Science Engineering, University of Applied Sciences Mittelhessen (THM), 35390 Giessen, Germany.
Viruses. 2023 Apr 19;15(4):998. doi: 10.3390/v15040998.
Accurate and rapid quantification of (infectious) virus titers is of paramount importance in the manufacture of viral vectors and vaccines. Reliable quantification data allow efficient process development at a laboratory scale and thorough process monitoring in later production. However, current gold standard applications, such as endpoint dilution assays, are cumbersome and do not provide true process analytical monitoring. Accordingly, flow cytometry and quantitative polymerase chain reaction have attracted increasing interest in recent years, offering various advantages for rapid quantification. Here, we compared different approaches for the assessment of infectious viruses, using a model baculovirus. Firstly, infectivity was estimated by the quantification of viral nucleic acids in infected cells, and secondly, different flow cytometric approaches were investigated regarding analysis times and calibration ranges. The flow cytometry technique included a quantification based on post-infection fluorophore expression and labeling of a viral surface protein using fluorescent antibodies. Additionally, the possibility of viral (m)RNA labeling in infected cells was investigated as a proof of concept. The results confirmed that infectivity assessment based on qPCR is not trivial and requires sophisticated method optimization, whereas staining of viral surface proteins is a fast and feasible approach for enveloped viruses. Finally, labeling of viral (m)RNA in infected cells appears to be a promising opportunity but will require further research.
准确快速地定量（感染性）病毒滴度在病毒载体和疫苗的制造中至关重要。可靠的定量数据可在实验室规模上实现高效的工艺开发，并在后期生产中进行全面的工艺监测。然而，目前的金标准应用，如终点稀释测定，繁琐且无法提供真正的过程分析监测。因此，近年来流式细胞术和定量聚合酶链反应吸引了越来越多的关注，为快速定量提供了各种优势。在这里，我们使用模型杆状病毒比较了评估感染性病毒的不同方法。首先，通过感染细胞中的病毒核酸定量来估计感染性，其次，针对分析时间和校准范围研究了不同的流式细胞术方法。该流式细胞术技术包括基于感染后荧光团表达的定量以及使用荧光抗体标记病毒表面蛋白。此外，还研究了在感染细胞中标记病毒（mRNA）的可能性，作为概念验证。结果证实，基于 qPCR 的感染性评估并非易事，需要进行复杂的方法优化，而标记病毒表面蛋白是一种快速且可行的方法，适用于包膜病毒。最后，标记感染细胞中的病毒（mRNA）似乎是一个很有前途的机会，但需要进一步研究。
