Abstract：
The aim of this study was the development of a scalable production process for high titer (108 pfu/mL and above) recombinant baculovirus stocks with low cell line-derived impurities for the production of virus-like particles (VLP). To achieve this, we developed a high cell density (HCD) culture for low footprint cell proliferation, compared different infection strategies at multiplicity of infection (MOI) 0.05 and 0.005, different infection strategies and validated generally applicable harvest criteria of cell viability ≤ 80%. We also investigated online measurable parameters to observe the baculovirus production. The infection strategy employing a very low virus inoculum of MOI 0.005 and a 1:2 dilution with fresh medium one day after infection proved to be the most resource efficient. There, we achieved higher cell-specific titers and lower host cell protein concentrations at harvest than other tested infection strategies with the same MOI, while saving half of the virus stock for infecting the culture compared to other tested infection strategies. HCD culture by daily medium exchange was confirmed as suitable for seed train propagation, infection, and baculovirus production, equally efficient as the conventionally propagated seed train. Online measurable parameters for cell concentration and average cell diameter were found to be effective in monitoring the production process. The study concluded that a more efficient VLP production process in large scale can be achieved using this virus stock production strategy, which could also be extended to produce other proteins or extracellular vesicles with the baculovirus expression system.
摘要：
这项研究的目的是开发用于生产病毒样颗粒（VLP）的高滴度（108pfu/mL及以上）具有低细胞系来源杂质的重组杆状病毒储液的可扩展生产方法。为了实现这一点，我们开发了一种用于低足迹细胞增殖的高细胞密度（HCD）培养物，比较了感染复数（MOI）0.05和0.005时的不同感染策略，不同的感染策略并验证了细胞活力≤80％的普遍适用的收获标准。我们还调查了在线可测量参数以观察杆状病毒的产生。使用MOI0.005的极低病毒接种物和感染后一天用新鲜培养基1：2稀释的感染策略被证明是最有效的资源。在那里,我们在收获时获得了更高的细胞特异性滴度和更低的宿主细胞蛋白浓度，比使用相同MOI的其他测试感染策略，与其他测试的感染策略相比，同时节省了一半的病毒库存用于感染培养物。通过每日培养基交换进行的HCD培养被证实适用于种子训练繁殖，感染,和杆状病毒生产，与常规繁殖的种子火车一样有效。发现细胞浓度和平均细胞直径的在线可测量参数在监测生产过程中是有效的。该研究得出的结论是，使用这种病毒库存生产策略可以实现更有效的大规模VLP生产过程，它也可以延伸到产生其他蛋白质或细胞外囊泡与杆状病毒表达系统。
