Department of Biochemical Engineering, University College London, London, UK.
Division of Infection and Immunology, University College London, London, UK.
Biotechnol Bioeng. 2024 Aug;121(8):2466-2481. doi: 10.1002/bit.28498. Epub 2023 Aug 1.
Lentiviral vectors (LVs) are used in advanced therapies to transduce recipient cells for long term gene expression for therapeutic benefit. The vector is commonly pseudotyped with alternative viral envelope proteins to improve tropism and is selected for enhanced functional titers. However, their impact on manufacturing and the success of individual bioprocessing unit operations is seldom demonstrated. To the best of our knowledge, this is the first study on the processability of different Lentiviral vector pseudotypes. In this work, we compared three envelope proteins commonly pseudotyped with LVs across manufacturing conditions such as temperature and pump flow and across steps common to downstream processing. We have shown impact of filter membrane chemistry on vector recoveries with differing envelopes during clarification and observed complete vector robustness in high shear manufacturing environments using ultra scale-down technologies. The impact of shear during membrane filtration in a tangential flow filtration-mimic showed the benefit of employing higher shear rates, than currently used in LV production, to increase vector recovery. Likewise, optimized anion exchange chromatography purification in monolith format was determined. The results contradict a common perception that lentiviral vectors are susceptible to shear or high salt concentration (up to 1.7 M). This highlights the prospects of improving LV recovery by evaluating manufacturing conditions that contribute to vector losses for specific production systems.
慢病毒载体 (LVs) 被用于先进的治疗中，以转导受者细胞，实现长期基因表达，从而带来治疗益处。该载体通常通过替代的病毒包膜蛋白进行假型化，以改善亲嗜性，并选择具有增强功能滴度的载体。然而，它们对制造过程和各个生物加工单元操作的成功的影响很少得到证明。据我们所知，这是首次针对不同慢病毒载体假型化的可加工性进行的研究。在这项工作中，我们比较了三种常见的包膜蛋白在不同的制造条件下，如温度和泵流量，以及在下游加工中常见的步骤中的表现。我们已经表明，不同包膜蛋白在澄清过程中，过滤膜化学对载体回收率的影响，并且在使用超缩小技术的高剪切制造环境中观察到载体的完全稳健性。在切向流过滤模拟中膜过滤过程中的剪切影响表明，采用比当前 LV 生产中更高的剪切速率有利于提高载体回收率。同样，确定了优化的阴离子交换色谱纯化在整体柱形式中的应用。研究结果与一种常见的观点相矛盾，即慢病毒载体易受剪切或高盐浓度（高达 1.7M）的影响。这突出了通过评估特定生产系统中导致载体损失的制造条件来提高 LV 回收率的前景。
