Creating an optimized and scalable production platform in suspension adapted serum-free HEK293 cells for rAAV gene delivery using a Design-of-Experime

使用实验设计在悬浮适应的无血清 HEK293 细胞中创建优化且可扩展的生产平台，用于 rAAV 基因递送

• 批准号: 2776049
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2776049
• 关键词: Creating; optimized; scalable; production; platform

The scope of the collaboration between the University College London and Sartorius is to establish a human embryonic kidney (HEK)293 cell line suspension platform for recombinant adeno-associated vector-based gene therapy manufacturing. Recombinant adeno-associated virus (rAAV) vectors are a leading gene delivery platform, and as June 2022, there are two rAAV-based gene therapies approved (Luxturna, in 2017, a rAAV2-based gene therapy and Zolgesma, in 2019, a rAAV9-base gene therapy) and more than seven-hundred active clinical trials. The conventional process for the manufacture of rAAV vectors is the usage of HEK293 cell line, which is transiently transfected with the genetic material required to produce the viral vector (either for research, pre-clinical and early clinical studies purposes) (Escandell et al., 2022). The widespread use of the HEK/transient transfection platform is due to recent developments supporting the design of the molecular constructs and the introduction of the plasmid DNA (pDNA) into HEK293 cells with chemical transfection reagents. However, vector production is a complex process that requires an efficient and optimized method of introducing the necessary virus genes into the cells (Dismuke & Kotin, 2017), suitable analytics and properly defined critical quality attributes (CQAs) for processes monitoring to ensure robustness and quality of the final product (Escandell et al., 2022). Despite advances in the clinic, rAAV vector manufacturing remains a challenging strategy for therapeutic applications requiring high systemic doses due to the high cost of materials (pDNA as GMP starting material), as well as a lack of process scalability and robustness. The recent development of HEK293 cell lines optimized for transfection in suspension culture has already improved the scale of production (Chahal et al., 2014). Also, stable producer cell lines represent a potential alternative to transient manufacturing systems, being more robust and easier to scale-up (Escandell et al., 2022). Although many strategies have been developed, to introduce the virus genes into HEK293 cells and to create serum-free, suspension-adapted cell lines, no rAAV production platform has been fully optimized or standardized (Dismuke & Kotin, 2017). Efficient and scalable production of rAAV is critical to enable lower cost of goods and easier drug commercialization for gene therapy applications. Therefore, the aim of the project is to optimize a transient and stable platform for suspension adapted HEK293 for rAAV production using Design of Experiments (DoE) to study the interaction of multiple factors and its impact on CQAs affecting the product quality. The process conditions and factors that impact rAAV vector production such as temperature, agitation, pH, production cell line, cell density, culture medium, harvest time, and plasmid concentrations (Zhao et al., 2020) will be investigated using rAAV2 as a representative vector to optimize virus production. Another rAAV serotype relevant to specific target tissue will be evaluated and further optimized in a data-driven approach. Sartorius transient and stable producer cell lines will serve as basis for the project. The transient cell was successfully adapted to growth in suspension culture using a proprietary serum-free, chemically defined media by direct adaptation. The PhD thesis will comprise selection of different cell clones based on cell growth and virus titer and the evaluation of different proprietary and benchmarking media and feed supplement / strategy. Analytical methods to measure metabolites, infectious titers, AAV protein identity, vector genome quantification, AAV capsid/titer, empty to full ratio and/or other product qualities were developed in-house, and samples will be retained for analysis (when performed by Sartorius).

伦敦大学学院和赛多利斯之间的合作范围是建立一个人胚胎肾（HEK）293细胞系悬浮平台，用于基于重组腺相关载体的基因治疗制造。重组腺相关病毒 (rAAV) 载体是领先的基因递送平台，截至 2022 年 6 月，有两种基于 rAAV 的基因疗法获得批准（Luxturna，2017 年，一种基于 rAAV2 的基因疗法；Zolgesma，2019 年，一种 rAAV9） -基础基因治疗）和七百多项活跃的临床试验。制造 rAAV 载体的常规过程是使用 HEK293 细胞系，该细胞系用生产病毒载体所需的遗传物质瞬时转染（用于研究、临床前和早期临床研究目的）（Escandell 等，2017）。 ，2022）。 HEK/瞬时转染平台的广泛使用是由于最近的发展支持分子构建体的设计以及使用化学转染试剂将质粒 DNA (pDNA) 引入 HEK293 细胞。然而，载体生产是一个复杂的过程，需要一种有效且优化的方法将必要的病毒基因引入细胞中（Dismuke＆Kotin，2017），适当的分析和正确定义的关键质量属性（CQA）用于过程监控，以确保稳健性和最终产品的质量（Escandell 等人，2022）。尽管临床取得了进展，但由于材料成本高（pDNA 作为 GMP 起始材料），并且缺乏工艺可扩展性和稳健性，rAAV 载体制造对于需要高全身剂量的治疗应用来说仍然是一个具有挑战性的策略。最近开发的针对悬浮培养转染进行优化的 HEK293 细胞系已经提高了生产规模（Chahal 等，2014）。此外，稳定的生产细胞系是瞬时制造系统的潜在替代方案，更稳健且更容易扩大规模（Escandell 等人，2022）。尽管已经开发了许多策略，将病毒基因引入 HEK293 细胞并创建无血清、悬浮适应的细胞系，但尚未完全优化或标准化 rAAV 生产平台（Dismuke & Kotin，2017）。 rAAV 的高效和可扩展生产对于降低基因治疗应用的商品成本和更容易实现药物商业化至关重要。因此，该项目的目的是使用实验设计（DoE）来优化用于rAAV生产的悬浮适应HEK293的瞬态和稳定平台，以研究多种因素的相互作用及其对影响产品质量的CQA的影响。以rAAV2为代表，研究影响rAAV载体生产的工艺条件和因素，如温度、搅拌、pH、生产细胞系、细胞密度、培养基、收获时间和质粒浓度（Zhao et al., 2020）载体以优化病毒生产。另一种与特定靶组织相关的 rAAV 血清型将通过数据驱动的方法进行评估和进一步优化。 Sartorius 瞬时和稳定生产细胞系将作为该项目的基础。通过直接适应，使用专有的无血清、化学成分确定的培养基，瞬时细胞成功地适应了悬浮培养中的生长。博士论文将包括根据细胞生长和病毒滴度选择不同的细胞克隆，以及评估不同的专有和基准培养基和饲料补充剂/策略。内部开发了测量代谢物、传染性滴度、AAV 蛋白质身份、载体基因组定量、AAV 衣壳/滴度、空满比和/或其他产品质量的分析方法，并且将保留样品进行分析（当由 Sartorius 进行时） ）。