Table_1_Aeration and Shear Stress Are Critical Process Parameters for the Production of Oncolytic Measles Virus.DOCX

Oncolytic Measles virus is a promising candidate for cancer treatment, but clinical studies have shown that extremely high doses (up to 1011 TCID50 per dose) are required to effect a cure. Very high titers of the virus must therefore be achieved during production to ensure an adequate supply. We have previously shown that Measles virus can be produced in Vero cells growing on a Cytodex 1 microcarrier in serum-containing medium using a stirred-tank reactor (STR). However, process optimization and further process transfer or scale up requires the identification of critical process parameters, particularly because the use of STRs increases the risk of cell damage and lower product yields due to shear stress. Using a small-scale STR (0.5 L working volume) we found that Measles virus titers are sensitive to agitator-dependent shear, with shear stress ≥0.25 N m−2 reducing the titer by more than four orders of magnitude. This effect was observed in both serum-containing and serum-free medium. At this scale, virus of titers up to 1010 TCID50 mL−1 could be achieved with an average shear stress of 0.1 N m−2. We also found that the aeration method affected the virus titer. Aeration was necessary to ensure a sufficient oxygen supply to the Vero cells, and CO2 was also needed to regulate the pH of the sodium bicarbonate buffer system. Continuous gassing with air and CO2 reduced the virus titer by four orders of magnitude compared to head-space aeration. The manufacture of oncolytic Measles virus in a STR can therefore be defined as a shear-sensitive process, but high titers can nevertheless be achieved by keeping shear stress levels below 0.25 N m−2 and by avoiding extensive gassing of the medium.

溶瘤麻疹病毒（Oncolytic Measles virus）是一种极具潜力的癌症治疗候选药物，但临床研究显示，实现治疗效果需使用极高剂量（单剂可达10¹¹ TCID₅₀）。因此，生产过程中必须获得极高滴度的病毒以保障充足供应。我们此前已证实，可在搅拌釜式反应器（stirred-tank reactor, STR）中，于含血清培养基内利用贴附于Cytodex 1微载体的Vero细胞生产麻疹病毒。然而，工艺优化及后续工艺转移或放大需明确关键工艺参数——尤其因为搅拌釜式反应器的使用会因剪切应力提升细胞损伤风险，降低产物收率。通过工作体积为0.5 L的小型搅拌釜式反应器实验，我们发现麻疹病毒滴度对搅拌引发的剪切应力敏感：当剪切应力≥0.25 N·m⁻²时，病毒滴度会下降超过四个数量级。该现象在含血清及无血清培养基中均有观测到。在此反应器规模下，当平均剪切应力维持在0.1 N·m⁻²时，可获得最高达10¹⁰ TCID₅₀·mL⁻¹的病毒滴度。我们还发现曝气方式会影响病毒滴度：曝气是为保障Vero细胞获得充足氧气供应，同时需通入二氧化碳以调节碳酸氢钠缓冲体系的pH值。与顶空曝气（head-space aeration）相比，持续通入空气与二氧化碳会使病毒滴度下降四个数量级。综上，在搅拌釜式反应器中生产溶瘤麻疹病毒属于剪切敏感型工艺，但只要将剪切应力控制在0.25 N·m⁻²以下，并避免对培养基进行过度曝气，即可获得较高的病毒滴度。