Magnetic sorting and selection of producer cells based on secretion and growth using nanovial technology

摘要 然而，细胞分泌是生物过程、生物技术和细胞疗法的基础。 根据分泌物快速分离出活细胞群并不是很容易获得的。能够迅速地 根据数量分泌表型对细胞(如B细胞、CHO细胞)进行分类可以加速发现和 产生治疗性或诊断性抗体。此外，许多细胞治疗产品的功能是最好的 由分泌的蛋白质的类型和数量定义的，如细胞因子。强调需要，有几个 已经开发了微流控平台来执行分泌细胞的分选，专注于产生 抗体。然而，这些系统需要专门的专业知识或商业设备，而这些并不广泛 可用并且在吞吐量方面受到限制。基于市场研究表明有必要进行功能选择 减轻克隆产生细胞系的遗传和表观遗传漂移的方法，这项提议旨在支持 一项专注于将Partilion的水凝胶纳米小瓶平台作为基于试剂的解决方案的新计划 分离并在培养中保持高分泌产生细胞(如CHO)亚克隆的富集群 使用磁激活细胞分选(MACS)。我们的纳米瓶技术是基于微尺度的新月形 捕获细胞的水凝胶颗粒被功能化以捕获分泌物，并以模板形成 数以百万计的均匀液滴并行，防止分泌物的损失和串扰。此工作流仅需要 简单的移液和离心步骤。然后，带有捕获的分泌物和相关细胞的纳米瓶可以 用磁性纳米颗粒标记，结合到感兴趣分泌物的特异性抗体上，并使用 MACs基于分泌物的数量。我们将设计我们的纳米瓶产品，并开发一个工作流程 在Mac上表现强劲，使我们的客户能够在每个工作日分拣1000万个电池，至少两个订单 比市场上的竞争技术高出数量级，以丰富关键的生产亚群。 具体目标(1)侧重于开发与磁激活细胞分选兼容的工作流程，以确保 易于被更广泛的客户群采用，并允许选择显著更多的小区。具体目标(2)将 研究基于分泌物和生长的组合进行磁性分类的可能性，并调整 选择阈值，以提高细胞系的生产率。随着我们的目标的成功完成，我们 将为新试剂产品的研制奠定坚实的基础，为CHO等生产者的细胞分选奠定基础 与广泛使用的Mac系统兼容的分泌物。最终，这可以实现更具成本效益的 以及重组产品的快速生产，如单抗疗法、疫苗和诊断 亲和试剂。

ABSTRACT Cell secretions are fundamental to biological processes, biotechnology, and cell therapies, however, approaches to rapidly separate out viable cell populations based on secretions are not widely accessible. The ability to rapidly sort cells (e.g. B cells, CHO cells) based on a quantitative secretion phenotype can accelerate the discovery and production of therapeutic or diagnostic antibodies. Further, the function of many cell therapy products are best defined by the type and quantity of secreted proteins, such as cytokines. Emphasizing the need, several microfluidic platforms have been developed to perform sorting of secreting cells, focusing on cells that produce antibodies. However, these systems require specialized expertise or commercial equipment that is not widely available and are limited in throughput. Based on market research suggesting a need for functional selection approaches to mitigate the genetic and epigenetic drift in clonal producer cell lines, this proposal aims to support a new program focused on engineering Partillion’s hydrogel nanovial platform as a reagent-based solution to separate and maintain in culture enriched populations of highly secreting producer cell (e.g. CHO) sub-clones using magnetic activated cell sorting (MACS). Our nanovial technology is based on microscale crescent-shaped hydrogel particles which capture cells, are functionalized to capture secretions, and template the formation of millions of uniform drops in parallel, preventing the loss and cross-talk of secretions. This workflow only requires simple pipetting and centrifugation steps. Nanovials with captured secretions and associated cells can then be labeled with magnetic nanoparticles, conjugated to antibodies specific to secretions of interest, and sorted using MACS based on the quantity of secretion. We will engineer our nanovial product and develop a workflow to work robustly with MACS, enabling our customers to sort through > 10 million cells per work day, at least two orders of magnitude higher than competing technologies on the market, to enrich key productive sub-populations. Specific Aim (1) focuses on developing workflows for compatibility with magnetic activated cell sorting to ensure ease of adoption by a wider customer base and enable selection of significantly more cells. Specific Aim (2) will investigate the potential to magnetically sort based on a combination of secretion and growth and tune the selection threshold to yield improved cell line productivity. Following the successful completion of our aims we will have laid a strong foundation for a new reagent product for CHO and other producer cell sorting based on secretion that is compatible with widely available MACS systems. Ultimately, this can enable more cost-effective and rapid production of recombinant products such as monoclonal antibody therapies, vaccines, and diagnostic affinity reagents.