Nie Jianqi, Xu Zhaojing, Sun Yang, Ren He, Song Zichuan, Zhang Yan, Bai Zhonghu
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, China.
Tianjin University of Science and Technology, Tianjin, 300457, China.
Bioprocess Biosyst Eng. 2025 Apr 26. doi: 10.1007/s00449-025-03167-9.
In recent years, recombinant adeno-associated virus (rAAV) vectors are the promising viral transfer tools for gene therapy and clinical trials, thanks to their favorable safety profile and long-term transgene expression. The increasing demand for rAAVs for gene therapy led to a rise in the amount of these vectors required for pre-clinical trials, clinical trials, and approved therapeutic applications. A majority of suspension HEK293 cell-based rAAV production protocols reported rely on a triple transfection at cell density below 2 × 10 cells/mL. However, the low yield of such biomanufacturing challenges bioprocess engineers to develop more efficient strategies capable of increasing volumetric productivity. In this study, we developed a perfusion bioprocess to enable rAAV production efficiently at high cell density. We first optimized three key process parameters (the total DNA amount, ratio of polyethyleneimine (PEI) to DNA, and proportion of the three plasmids) of rAAV production at cell density of 2 × 10 cells/mL by the design of experiment method, from which the robust setpoint (total DNA amount of 1.37 μg/mL, ratio of PEI to DNA of 1.52 μL/μg, the proportion of plasmids pHelper 24%, pRC 46%, pGOI 30%) was explored. We then developed a rAAV production process at a cell density of ~ 8 × 10 cells/mL, with increasing DNA amount on a cell basis and optimizing transfection complex preparation. This approach was confirmed in a 5 L benchtop bioreactor connected with a perfusion system, resulting in a viral genomic titer of 7.28 × 10 vg/mL and a cell-specific viral genomic titer of 4.97 × 10 vg/cell. This study demonstrates that the perfusion process coupled with optimized transfection complex preparation has the potential to improve manufacturing productivity.
近年来，重组腺相关病毒（rAAV）载体因其良好的安全性和长期转基因表达，成为基因治疗和临床试验中颇具前景的病毒转导工具。基因治疗对rAAV的需求不断增加，导致临床前试验、临床试验及获批治疗应用所需的此类载体数量上升。报道的大多数基于悬浮HEK293细胞的rAAV生产方案都依赖于在细胞密度低于2×10⁶个细胞/mL时进行三重转染。然而，这种生物制造的低产量对生物工艺工程师提出了挑战，促使他们开发更高效的策略以提高体积生产力。在本研究中，我们开发了一种灌注生物工艺，以实现高细胞密度下rAAV的高效生产。我们首先通过实验设计方法，在细胞密度为2×10⁶个细胞/mL时优化了rAAV生产的三个关键工艺参数（总DNA量、聚乙烯亚胺（PEI）与DNA的比例以及三种质粒的比例），由此探索出稳健的设定值（总DNA量为1.37μg/mL，PEI与DNA的比例为1.52μL/μg，质粒pHelper占24%、pRC占46%、pGOI占30%）。然后，我们在细胞密度约为8×10⁶个细胞/mL的条件下开发了rAAV生产工艺，在细胞基础上增加DNA量并优化转染复合物制备。这种方法在连接灌注系统的5L台式生物反应器中得到了验证，产生了7.28×10¹²vg/mL的病毒基因组滴度和4.97×10⁶vg/细胞的细胞特异性病毒基因组滴度。本研究表明，灌注工艺与优化的转染复合物制备相结合有提高生产效率的潜力。
