Development of a Production Method for Scaling Up Lentiviral Vector Manufacture

开发扩大慢病毒载体生产规模的生产方法

• 批准号: 7586742
• 负责人: Madhusudan Viswanath Peshwa
• 金额: $ 39.44万
• 依托单位: MAXCYTE, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7586742
• 关键词: Achievement; Age related macular degeneration; Agitation; Alphavirus; Area; Biological; Biological Assay; Biological Products; Bioreactors; Buffers; Cell Culture Techniques; Cell Density; Cell Line; Cell physiology; Cells; Clinic; Clinical; Clinical Research; Clinical Trials; Contracts; Cyclic GMP; Dental; Dependovirus; Development; Disease; Electroporation; Engineering; FDA approved; Gene Transduction Agent; Goals; HIV Infections; Healthcare; Human; In Situ; Indiana; L Cells; Lentivirus Vector; Marketing; Measurement; Measures; Medical; Methods; Muscular Dystrophies; Operative Surgical Procedures; Parkinson Disease; Pathology; Patients; Perfusion; Phase; Process; Production; Recovery; Research; Running; Serum-Free Culture Media; Services; Sickle Cell Anemia; Sindbis Virus; Small Business Innovation Research Grant; Sterility; Subfamily lentivirinae; Suspension Culture; Suspension substance; Suspensions; System; Technology; Technology Transfer; Testing; Therapeutic; TimeLine; Transfection; Translating; United States; Universities; Viral; Viral Vector; Virus; Work; base; beta Thalassemia; bioprocess; cGMP production; clinical application; cost effective; design; gene therapy; gene therapy clinical trial; innovation; instrumentation; interest; leukemia/lymphoma; manufacturing facility; manufacturing process; meetings; novel; particle; programs; public health relevance; response; scale up; success; vector

DESCRIPTION (provided by applicant): The long-term goal of this research program is to develop an efficient large-scale commercial system for manufacture of lentiviral gene therapy vectors in suspension cells. This proposal is submitted in response to PA-06-013, "Manufacturing Processes of Medical, Dental and Biological Technologies." There is a critical unmet need to develop robust, scalable, closed, cGMP-compliant manufacturing systems for generating and producing lentivirus gene therapy vectors that are suitable for clinical and commercial use. Lentiviral vectors are being developed for a number of therapeutic applications and clinical studies are underway. Unfortunately, lentiviral vectors are difficult to produce in large numbers due to the lack of stable packaging cell lines and due to inefficiencies, lack of robustness, and limited scalabilities associated with standard methods using transient transfection, which is a key step in the manufacturing process. MaxCyte has developed a cell transfection and production system that is an efficient, scalable process for engineering cell function and enabling cell-based manufacturing for clinical applications. In Phase 1 of this project, we demonstrated that this system enables high titer lentivector production in adherent and suspension-adapted cells in both small and large scale transfections. The next challenge is to optimize and scale up an aseptic production system and demonstrate large scale lentivector production technology in a cGMP manufacturing facility. This achievement will enable the first application of an FDA-approved cell loading technology for scalable production of lentiviral vectors by transient transfection in non-adherent cells cultured in serum-free medium. Therefore, the Specific Aims of this Phase 2 project are to: 1) optimize culture conditions for culturing suspension-adapted cells for vector production; 2) achieve sterile integration of the MaxCyte cell processing instrumentation with the Wave cell production technology; 3) optimize viral production capacity by maximizing the number of infectious viral particles that can be produced by and collected from transfected cells; 4) demonstrate feasibility of the completely integrated viral production system at a small commercial scale; and 5) demonstrate large scale lentivector production in a cGMP manufacturing facility. The interest expressed in this application of the MaxCyte cell loading technology by Indiana University Vector Production Facility (IUVPF) and SAFC Pharma emphasizes the importance of the project. These facilities provide contract manufacturing services for lentiviral vectors (and a number of other viral vectors) for gene therapy clinical trials. IUVPF will perform cGMP manufacturing for Aim 5 and thereby demonstrate the ability to transfer the MaxCyte technology to large scale commercial manufacturing. Public Health Relevance: There is a critical unmet need to develop robust, scalable, closed, cGMP-compliant manufacturing systems for producing lentiviral gene therapy vectors that are suitable for clinical and commercial use. The goal of this Phase 2 SBIR project is to optimize and validate a proprietary cell transfection and production system that provides an efficient, large-scale commercial process for developing and manufacturing lentiviral gene therapy vectors for clinical applications. The benefits of this production system will translate into safer, better, cost effective gene therapies on the market more rapidly to benefit patients in many disease areas with unmet need today, such as: Parkinson's disease, age-related macular degeneration, beta-thalassemia, sickle-cell anemia, muscular dystrophy, and a variety of lympho-hematopoetic pathologies including HIV infection, leukemias, and lymphomas.

描述（由申请人提供）：该研究项目的长期目标是开发一种高效的大规模商业系统，用于在悬浮细胞中制造慢病毒基因治疗载体。该提案是针对 PA-06-013“医疗、牙科和生物技术的制造工艺”而提交的。目前迫切需要开发稳健、可扩展、封闭、符合 cGMP 的制造系统，用于生成和生产适合临床和商业用途的慢病毒基因治疗载体。慢病毒载体正在开发用于多种治疗应用，临床研究正在进行中。 不幸的是，由于缺乏稳定的包装细胞系，以及与使用瞬时转染的标准方法相关的效率低下、缺乏稳健性和可扩展性有限，慢病毒载体难以大量生产，瞬时转染是制造过程中的关键步骤。 MaxCyte 开发了一种细胞转染和生产系统，该系统是一种高效、可扩展的流程，可用于设计细胞功能并实现临床应用的细胞制造。在该项目的第一阶段，我们证明该系统能够在小规模和大规模转染中在贴壁细胞和悬浮适应细胞中产生高滴度慢载体。下一个挑战是优化和扩大无菌生产系统，并在 cGMP 制造设施中展示大规模慢病毒生产技术。这一成就将首次应用 FDA 批准的细胞装载技术，通过在无血清培养基中培养的非贴壁细胞中瞬时转染来大规模生产慢病毒载体。因此，该二期项目的具体目标是： 1）优化培养悬浮适应细胞以进行载体生产的培养条件； 2) 实现MaxCyte细胞处理仪器与Wave细胞生产技术的无菌集成； 3) 通过最大化转染细胞可以产生和收集的感染性病毒颗粒的数量来优化病毒生产能力； 4）展示完全集成的病毒生产系统在小规模商业规模下的可行性； 5) 在 cGMP 生产设施中展示大规模慢载体生产。 印第安纳大学载体生产设施 (IUVPF) 和 SAFC Pharma 对 MaxCyte 细胞装载技术的应用表示了兴趣，强调了该项目的重要性。这些设施为基因治疗临床试验的慢病毒载体（以及许多其他病毒载体）提供合同制造服务。 IUVPF 将为 Aim 5 进行 cGMP 制造，从而展示将 MaxCyte 技术转移到大规模商业制造的能力。 公共卫生相关性：开发稳健、可扩展、封闭、符合 cGMP 的制造系统以生产适合临床和商业用途的慢病毒基因治疗载体是一个尚未得到满足的关键需求。该 SBIR 2 期项目的目标是优化和验证专有的细胞转染和生产系统，该系统为开发和制造用于临床应用的慢病毒基因治疗载体提供高效、大规模的商业流程。该生产系统的优势将更快地转化为市场上更安全、更好、更具成本效益的基因疗法，使当今需求未得到满足的许多疾病领域的患者受益，例如：帕金森病、年龄相关性黄斑变性、β-地中海贫血、镰状细胞性贫血、肌营养不良以及各种淋巴造血疾病，包括艾滋病毒感染、白血病和 淋巴瘤。

项目成果

Efficient large volume lentiviral vector production using flow electroporation.

• DOI: 10.1089/hum.2011.088
• 发表时间: 2012-02
• 期刊: Human gene therapy
• 影响因子: 4.2
• 作者: S. Witting;Linhong Li;A. Jasti;C. Allen;K. Cornetta;J. Brady;R. Shivakumar;M. Peshwa