Recombinant adeno-associated virus (rAAV) vectors are a leading gene delivery platform, but vector manufacturing remains a challenge. New methods are needed to increase rAAV yields and reduce costs. Past efforts to improve rAAV production have focused on optimizing a single variable at a time, but this approach does not account for the interactions of multiple factors that contribute to vector generation. Here, we utilized a design-of-experiment (DOE) methodology to optimize rAAV production in a HEK293T suspension cell system. We simultaneously varied the transgene, packaging, and helper plasmid ratios, the total DNA concentration, and the cell density to systematically evaluate the impact of each variable across 52 conditions. The results revealed a unique set of parameters with a lower concentration of transgene plasmid, a higher concentration of packaging plasmid, and a higher cell density than previously described protocols. Using this DOE-optimized protocol, we achieved unpurified yields approaching 3 × 10¹⁴ viral genomes (VGs)/L of cell culture. Additionally, we incorporated polyethylene glycol (PEG)-based virus precipitation, pH-mediated protein removal, and affinity chromatography to our downstream processing, enabling average purified yields of >1 × 10¹⁴ VGs/L for rAAV-EGFPs across 13 serotypes and capsid variants.

重组腺相关病毒（rAAV）载体是领先的基因传递平台，但载体的生产仍然是一个挑战。需要新的方法来提高rAAV的产量并降低成本。过去提高rAAV产量的努力集中在一次优化单个变量上，但是这种方法没有考虑到有助于向量生成的多个因素的相互作用。在这里，我们利用实验设计（DOE）方法来优化HEK293T悬浮细胞系统中的rAAV生产。我们同时改变了转基因，包装和辅助质粒的比例，总DNA浓度和细胞密度，以系统地评估52个条件下每个变量的影响。结果显示一组独特的参数，但转基因质粒的浓度较低，与先前描述的方案相比，包装质粒的浓度更高，细胞密度更高。使用DOE优化的方案，我们获得了接近3×10的未纯化产率细胞培养^14个病毒基因组（VG）/ L。此外，我们将基于聚乙二醇（PEG）的病毒沉淀，pH介导的蛋白质去除和亲和色谱法整合到了我们的下游处理中，从而使13种血清型和衣壳中的rAAV-EGFP的平均纯化产率> 1×10 ¹⁴ VGs / L变体。