Roldão António, Oliveira Rui, Carrondo Manuel J T, Alves Paula M
ITQB-UNL/IBET, Apartado 12, P-2781-901 Oeiras, Portugal.
J Virol Methods. 2009 Jul;159(1):69-80. doi: 10.1016/j.jviromet.2009.03.007. Epub 2009 Mar 17.
The success of baculovirus/insect cells system in heterologous protein expression depends on the robustness and efficiency of the production workflow. It is essential that process parameters are controlled and include as little variability as possible. The multiplicity of infection (MOI) is the most critical factor since irreproducible MOIs caused by inaccurate estimation of viral titers hinder batch consistency and process optimization. This lack of accuracy is related to intrinsic characteristics of the method such as the inability to distinguish between infectious and non-infectious baculovirus. In this study, several methods for baculovirus titration were compared. The most critical issues identified were the incubation time and cell concentration at the time of infection. These variables influence strongly the accuracy of titers and must be defined for optimal performance of the titration method. Although the standard errors of the methods varied significantly (7-36%), titers were within the same order of magnitude; thus, viral titers can be considered independent of the method of titration. A cost analysis of the baculovirus titration methods used in this study showed that the alamarblue, real time Q-PCR and plaque assays were the most expensive techniques. The remaining methods cost on average 75% less than the former methods. Based on the cost, time and error analysis undertaken in this study, the end-point dilution assay, microculture tetrazolium assay and flow cytometric assay were found to be the techniques that combine all these three main factors better. Nevertheless, it is always recommended to confirm the accuracy of the titration either by comparison with a well characterized baculovirus reference stock or by titration using two different methods and verification of the variability of results.
杆状病毒/昆虫细胞系统在异源蛋白表达方面的成功取决于生产流程的稳健性和效率。控制工艺参数并使其尽可能减少变异性至关重要。感染复数（MOI）是最关键的因素，因为病毒滴度估计不准确导致的MOI不可重复性会阻碍批次一致性和工艺优化。这种准确性的缺乏与该方法的内在特性有关，例如无法区分感染性和非感染性杆状病毒。在本研究中，比较了几种杆状病毒滴定方法。确定的最关键问题是感染时的孵育时间和细胞浓度。这些变量对滴度准确性有很大影响，必须为滴定方法的最佳性能加以定义。尽管这些方法的标准误差差异显著（7 - 36%），但滴度处于同一数量级；因此，可以认为病毒滴度与滴定方法无关。对本研究中使用的杆状病毒滴定方法进行的成本分析表明，alamarblue、实时定量PCR和噬斑测定是最昂贵的技术。其余方法的成本平均比前几种方法低75%。基于本研究进行的成本、时间和误差分析，终点稀释测定、微量培养四氮唑测定和流式细胞术测定被认为是能更好地结合这三个主要因素的技术。然而，始终建议通过与特征明确的杆状病毒参考毒株进行比较，或使用两种不同方法进行滴定并验证结果的变异性来确认滴定的准确性。
