Vaccine Bioprocess R&D, Merck Research Laboratories, P.O. Box 4, 19486, West Point, PA, USA.
Cytotechnology. 1996 Jan;22(1-3):79-85. doi: 10.1007/BF00353926.
A rapid, in-process assessment of virus replication is disired to quickly investigate the effects of process parameters on virus infection, and to monitor consistency of process in routine manufacturing of viral vaccines. Live virus potency assays are generally based on plaque formation, cytopathic effect, or antigen production (TCID(50)) and can take days to weeks to complete. Interestingly, when infected with viruses, cultured cells undergo changes in cellular metabolism that can be easily measured. These phenomena appear to be common as they has been observed in a variety of virus-host systems, e.g., in insect cells infected with baculovirus, Vero cells infected with Rotavirus, MRC-5 cells infected with Hepatitis A virus, and MRC-5 cells infected with the Varicella Zoster Virus (VZV). In this article, changes in glycolytic metabolism of MRC-5 cells as a result of CVZ infection are described. Both glucose consumption and lactate production in VZV infected MRC-5 cells are significantly elevated in comparison to uninfected cells. Based on this result, a rapid, in-process assay to follow VZV infection has been developed. The relative increase in lactate production in infected cells (α) increases as the infection progresses and then plateaus as the infection peaks. This plateau correlates with time of peak virus titer and could be used as a harvest triggering parameter in a virus production process.X(u) = cell density of uninfected cellsX(i) = cell density of infected cellsX(T) = total cell densityL(i) = cumulative lactate production in infected culturesL(u) = cumulative lactate production in uninfected culturesq(Li) = specific lactate production of infected cellsq(Lu) = specific lactate production of uninfected cellsk(1), K(2) = constants.
人们希望对病毒复制进行快速的过程评估，以便快速研究工艺参数对病毒感染的影响，并监测常规生产病毒疫苗过程的一致性。病毒滴度的活病毒检测通常基于噬菌斑形成、细胞病变效应或抗原产生（TCID(50)），可能需要数天到数周才能完成。有趣的是，当细胞被病毒感染时，细胞代谢会发生变化，这些变化很容易测量。这些现象似乎很常见，因为它们已经在多种病毒-宿主系统中观察到，例如，杆状病毒感染的昆虫细胞、轮状病毒感染的 Vero 细胞、甲型肝炎病毒感染的 MRC-5 细胞和水痘带状疱疹病毒（VZV）感染的 MRC-5 细胞。在本文中，描述了 CVZ 感染导致 MRC-5 细胞糖酵解代谢的变化。与未感染的细胞相比，感染 VZV 的 MRC-5 细胞中的葡萄糖消耗和乳酸生成显著增加。基于这一结果，开发了一种快速的过程检测方法来跟踪 VZV 感染。感染细胞中乳酸生成的相对增加（α）随着感染的进展而增加，然后在感染高峰时达到平台期。这个平台与病毒滴度的峰值时间相关，可作为病毒生产过程中的收获触发参数。X(u) = 未感染细胞的细胞密度 X(i) = 感染细胞的细胞密度 X(T) = 总细胞密度 L(i) = 感染培养物中的累积乳酸产量 L(u) = 未感染培养物中的累积乳酸产量 q(Li) = 感染细胞的比乳酸产量 q(Lu) = 未感染细胞的比乳酸产量 k(1), K(2) = 常数。
