Continuous Production of Viral Vectors using membraneless Perfusion Culture of Host Cells

项目摘要 通过高细胞密度、无膜灌注培养连续生物生产病毒载体 在这个项目中，我们建议通过无膜灌注强化病毒载体生产过程 生物反应器系统，这是由我们在麻省理工学院的团队开发的。基于惯性微流体技术， 在CHO细胞培养中得到验证，该系统与现有的灌注生物反应器相比具有许多关键的优点， 包括高产物回收率和避免膜污染，连续除去死细胞，细胞碎片， 易于维护和自动化。因此，我们的生物反应器可以保持高细胞 即使在细胞活力和培养条件发生显著变化时，也能可靠地保持密度， 控制病毒载体生产过程。与基于HEK 293和Sf 9的生产专家合作 系统，我们将实施长期，高细胞密度培养宿主细胞，并探索和验证几个 创新的载体生产平台，提供对新鲜细胞生长、转染 孵育/生产和产物的回收。惯性微流体细胞操作过程允许 在生物反应器中对宿主细胞的许多新的控制，包括选择性分离转染的 未转染的宿主细胞。最后，将优化连续生物处理系统，以最大限度地提高产量。 和最终病毒载体产品的质量。为此，我们将采用新的微流体电动力学 分离空病毒衣壳与填充的病毒衣壳，这可以提供近实时测定的质量， 产品与补料分批或分批培养相比，对载体质量和数量的连续监测使得能够 快速调整培养方案以恢复/维持载体生产的最佳条件， 延长期限。该项目预计将对病毒载体的生物生产产生更广泛的影响， 特别是在罕见和超罕见疾病方面，低患病率需要相对较小的产量 卷这些疾病并不能证明生物制药行业需要对载体进行大量投资是合理的。 制造业

Project Summary Abstract Continuous Bioproduction of Viral Vectors via High Cell Density, Membrane-less Perfusion Culture In this project, we propose to intensify the viral vector production process by the membrane-less perfusion bioreactor system, which was developed by our team at MIT. Based on the inertial microfluidics and fully validated in CHO cell cultivation, this system has many critical advantages over existing perfusion bioreactors, including high product recovery and avoiding membrane fouling, continuous removal of dead cells, cell debris, and cell aggregates, and easy maintenance and automation. As a result, our bioreactors can maintain high cell density reliably even when the cell viability and culture conditions change significantly, ideally suited for controlling viral vector production processes. Teaming with experts in HEK293 and Sf9 based production systems, we will implement long-term, high cell density cultures of host cells and explore and validate several innovative vector production platforms that provide more accurate control over fresh cell growth, transfection, incubation/production, and recovery of the products. Inertial microfluidic cell manipulation processes allow many novel controls over host cells in bioreactors, including selective separation of transfected from untransfected host cells. Finally, a continuous bioprocessing system will be optimized to maximize the quantity and quality of the finished viral vector products. For this, we will employ novel microfluidic electrokinetic separation of empty from filled viral capsid, which can provide near real-time determination of the quality of the product. In contrast to fed-batch or batch cultures, continuous monitoring of vector quality and quantity enables rapid adjustment of the culture protocols to restore/maintain optimal conditions for vector production over an extended period. This project is expected to have a broader impact on the bioproduction of viral vectors, but especially on rare and ultra-rare diseases where low disease prevalence requires relatively small production volumes. Such diseases do not justify the biopharmaceutical industry’s large investment needed for vector manufacturing.