Lentiviral vectors have proven their great potential to serve as a DNA delivery tool for gene modified cell therapy and gene therapy applications. The downstream processing of these vectors is however still a great challenge, particularly because of the low stability of the virus. Harvesting and clarification are critical and until now insufficiently characterized steps for lentivirus processing. To address this bottleneck, we analyzed whether lentiviral vectors produced by transient transfection of HEK293 T/17 SF suspension cells can be efficiently clarified with a lab-scale method with the filter aid diatomaceous earth (DE) and bioburden reducing membrane filters achieving high lentivirus recoveries.

Using a design of experiment approach we found that higher DE concentrations are advantageous for a higher turbidity reduction and shorter filtration times, but at the same time LV titer decreases with increasing DE concentration. A DE concentration of 9 g/L was identified with a DoE as a robust set-point. Clarification with DE was compared with for lab-scale traditionally employed centrifugation and subsequent bioburden reduction filtration of viral vectors. The use of DE allows to perform a harvest and clarification process, which does not only facilitate faster and safer virus handling, but enables a lower material consumption due to the extremely increased filter capacity, thus representing an efficient and robust lab-scale clarification process.

慢病毒载体已证明其作为基因修饰细胞治疗和基因治疗应用的 DNA 递送工具的巨大潜力。然而，这些载体的下游加工仍然是一个巨大的挑战，特别是因为病毒的稳定性低。收获和澄清是至关重要的，到目前为止，慢病毒处理的特征步骤还不够充分。为了解决这个瓶颈，我们分析了通过瞬时转染 HEK293 T/17 SF 悬浮细胞产生的慢病毒载体是否可以通过实验室规模的方法有效澄清，使用助滤剂硅藻土 (DE) 和生物负载减少膜过滤器实现高慢病毒回收率.

使用实验方法设计，我们发现较高的 DE 浓度有利于更高的浊度降低和更短的过滤时间，但同时 LV 滴度随着 DE 浓度的增加而降低。9 g/L 的 DE 浓度被确定为 DoE 作为可靠的设定点。将 DE 的澄清与实验室规模的传统离心和随后的病毒载体生物负载减少过滤进行了比较。使用 DE 可以进行收获和澄清过程，这不仅有助于更快、更安全地处理病毒，而且由于极大增加的过滤能力，从而降低了材料消耗，从而代表了一种高效而强大的实验室规模澄清过程。