PDF(Pubmed)
Abstract：
Large-scale transient transfection has advanced significantly over the last 20 years, enabling the effective production of a diverse range of biopharmaceutical products, including viral vectors. However, a number of challenges specifically related to transfection reagent stability and transfection complex preparation times remain. New developments and improved transfection technologies are required to ensure that transient gene expression-based bioprocesses can meet the growing demand for viral vectors. In this paper, we demonstrate that the growth of cationic lipid-based liposomes, an essential step in many cationic lipid-based transfection processes, can be controlled through adoption of low pH (pH 6.40 to pH 6.75) and in low salt concentration (0.2× PBS) formulations, facilitating improved control over the nanoparticle growth kinetics and enhancing particle stability. Such complexes retain the ability to facilitate efficient transfection for prolonged periods compared with standard preparation methodologies. These findings have significant industrial applications for the large-scale manufacture of lentiviral vectors for two principal reasons. First, the alternative preparation strategy enables longer liposome incubation times to be used, facilitating effective control in a good manufacturing practices setting. Second, the improvement in particle stability facilitates the setting of wider process operating ranges, which will significantly improve process robustness and maximise batch-to-batch control and product consistency.
摘要：
大规模瞬时转染在过去20年中取得了显著进展，能够有效生产各种生物制药产品，包括病毒载体。然而,与转染试剂稳定性和转染复合物制备时间相关的许多挑战仍然存在。需要新的发展和改进的转染技术来确保基于瞬时基因表达的生物过程能够满足对病毒载体日益增长的需求。在本文中,我们证明了阳离子脂质脂质体的生长，在许多基于阳离子脂质的转染过程中，可以通过采用低pH（pH6.40至pH6.75）和低盐浓度（0.2×PBS）配方来控制，促进对纳米颗粒生长动力学的改进控制和增强颗粒稳定性。与标准制备方法相比,此类复合物在延长的时间内保持促进有效转染的能力。由于两个主要原因,这些发现对于大规模制造慢病毒载体具有重要的工业应用。首先,替代制备策略可以使用更长的脂质体孵育时间，在良好的制造实践环境中促进有效的控制。第二,颗粒稳定性的改善有助于设定更宽的工艺操作范围，这将显着提高过程的鲁棒性，并最大限度地提高批次之间的控制和产品的一致性。
