Schulze-Horsel Josef, Genzel Yvonne, Reichl Udo
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany.
BMC Biotechnol. 2008 Apr 30;8:45. doi: 10.1186/1472-6750-8-45.
In cell culture-based influenza vaccine production the monitoring of virus titres and cell physiology during infection is of great importance for process characterisation and optimisation. While conventional virus quantification methods give only virus titres in the culture broth, data obtained by fluorescence labelling of intracellular virus proteins provide additional information on infection dynamics. Flow cytometry represents a valuable tool to investigate the influences of cultivation conditions and process variations on virus replication and virus yields.
In this study, fluorescein-labelled monoclonal antibodies against influenza A virus matrix protein 1 and nucleoprotein were used for monitoring the infection status of adherent Madin-Darby canine kidney cells from bioreactor samples. Monoclonal antibody binding was shown for influenza A virus strains of different subtypes (H1N1, H1N2, H3N8) and host specificity (human, equine, swine). At high multiplicity of infection in a bioreactor, the onset of viral protein accumulation in adherent cells on microcarriers was detected at about 2 to 4 h post infection by flow cytometry. In contrast, a significant increase in titre by hemagglutination assay was detected at the earliest 4 to 6 h post infection.
It is shown that flow cytometry is a sensitive and robust method for the monitoring of viral infection in fixed cells from bioreactor samples. Therefore, it is a valuable addition to other detection methods of influenza virus infection such as immunotitration and RNA hybridisation. Thousands of individual cells are measured per sample. Thus, the presented method is believed to be quite independent of the concentration of infected cells (multiplicity of infection and total cell concentration) in bioreactors. This allows to perform detailed studies on factors relevant for optimization of virus yields in cell cultures. The method could also be used for process characterisation and investigations concerning reproducibility in vaccine manufacturing.
在基于细胞培养的流感疫苗生产中，感染过程中病毒滴度和细胞生理学的监测对于工艺表征和优化至关重要。传统的病毒定量方法只能给出培养液中的病毒滴度，而通过细胞内病毒蛋白荧光标记获得的数据可提供有关感染动态的额外信息。流式细胞术是研究培养条件和工艺变化对病毒复制及病毒产量影响的宝贵工具。
在本研究中，针对甲型流感病毒基质蛋白1和核蛋白的荧光素标记单克隆抗体被用于监测生物反应器样品中贴壁的马-达二氏犬肾细胞的感染状态。不同亚型（H1N1、H1N2、H3N8）和宿主特异性（人、马、猪）的甲型流感病毒株均显示出单克隆抗体结合。在生物反应器中高感染复数时，通过流式细胞术在感染后约2至4小时检测到微载体上贴壁细胞中病毒蛋白开始积累。相比之下，血凝试验最早在感染后4至6小时检测到滴度显著增加。
结果表明，流式细胞术是监测生物反应器样品中固定细胞病毒感染的一种灵敏且可靠的方法。因此，它是流感病毒感染其他检测方法（如免疫滴定和RNA杂交）的宝贵补充。每个样品测量数千个单个细胞。因此，所提出的方法被认为相当独立于生物反应器中感染细胞的浓度（感染复数和总细胞浓度）。这使得能够对细胞培养中与病毒产量优化相关的因素进行详细研究。该方法还可用于工艺表征以及疫苗生产中有关再现性的研究。
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