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
基本信息
- 批准号:2776049
- 负责人:
- 金额:--
- 依托单位:
- 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:
- 关键词:
项目摘要
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).
伦敦大学学院与Sartorius的合作范围是建立人胚胎肾脏(HEK)293细胞系悬浮平台,用于重组腺相关载体基因治疗制造。重组腺相关病毒(RAAV)载体是领先的基因传递平台,截至2022年6月,已有两种基于rAAV的基因治疗获得批准(Luxturna于2017年批准,一种基于rAAV2的基因治疗;Zolgesma,于2019年批准,一种基于rAAV9的基因治疗)和700多项活跃的临床试验。制造rAAV载体的传统方法是使用HEK293细胞系,瞬时地将产生病毒载体所需的遗传物质(用于研究、临床前和早期临床研究)(Escapell等人,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结构的设计和通过化学转染剂将质粒DNA(PDNA)导入HEK293细胞。然而,载体生产是一个复杂的过程,需要一种将必要的病毒基因导入细胞的高效和优化的方法(Dismuke&Kotin,2017)、适当的分析和适当定义的关键质量属性(CQA)以进行过程监测,以确保最终产品的稳健性和质量(Escobell等人,2022)。尽管在临床上取得了进展,但由于材料成本高(以PDNA为GMP起始材料)以及缺乏过程可伸缩性和稳定性,rAAV载体制造对于需要高系统剂量的治疗应用仍然是一种具有挑战性的策略。针对悬浮培养中的转基因进行优化的HEK293细胞系的最新发展已经提高了生产规模(Chahal等人,2014年)。此外,稳定的生产者细胞系代表了一种替代临时制造系统的潜在选择,它更健壮,更容易扩大(埃斯波贝尔等人,2022年)。虽然已经开发了许多策略,将病毒基因导入HEK293细胞并创建无血清、悬浮适应的细胞系,但还没有完全优化或标准化的rAAV生产平台(Dismuke&Kotin,2017)。高效和可扩展的rAAV生产对于降低产品成本和更容易实现用于基因治疗的药物商业化至关重要。因此,本项目的目标是利用实验设计(DoE)优化一个适用于rAAV生产的HEK293悬浮过渡稳定平台,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为代表载体,研究温度、搅拌、pH、生产细胞系、细胞密度、培养基数、收获时间和质粒浓度等影响rAAV2生产的工艺条件和因素,以优化病毒生产。另一种与特定目标组织相关的rAAV血清型将在数据驱动的方法中进行评估和进一步优化。Sartorius瞬变和稳定的生产细胞系将作为该项目的基础。通过直接适应,瞬时细胞成功地适应了悬浮培养中的生长,使用了专利的无血清、化学定义的培养基。博士论文将包括基于细胞生长和病毒滴度的不同细胞克隆的选择,以及对不同专利和基准培养基及饲料补充/策略的评估。内部开发了测量代谢物、感染性滴度、AAV蛋白同一性、载体基因组量化、AAV衣壳/滴度、空满率和/或其他产品质量的分析方法,并将保留样品进行分析(当由缝纫机进行分析时)。
项目成果
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其他文献
吉治仁志 他: "トランスジェニックマウスによるTIMP-1の線維化促進機序"最新医学. 55. 1781-1787 (2000)
Hitoshi Yoshiji 等:“转基因小鼠中 TIMP-1 的促纤维化机制”现代医学 55. 1781-1787 (2000)。
- DOI:
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LiDAR Implementations for Autonomous Vehicle Applications
- DOI:
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2021 - 期刊:
- 影响因子:0
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吉治仁志 他: "イラスト医学&サイエンスシリーズ血管の分子医学"羊土社(渋谷正史編). 125 (2000)
Hitoshi Yoshiji 等人:“血管医学与科学系列分子医学图解”Yodosha(涉谷正志编辑)125(2000)。
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Effect of manidipine hydrochloride,a calcium antagonist,on isoproterenol-induced left ventricular hypertrophy: "Yoshiyama,M.,Takeuchi,K.,Kim,S.,Hanatani,A.,Omura,T.,Toda,I.,Akioka,K.,Teragaki,M.,Iwao,H.and Yoshikawa,J." Jpn Circ J. 62(1). 47-52 (1998)
钙拮抗剂盐酸马尼地平对异丙肾上腺素引起的左心室肥厚的影响:“Yoshiyama,M.,Takeuchi,K.,Kim,S.,Hanatani,A.,Omura,T.,Toda,I.,Akioka,
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