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

使用纳米瓶技术基于分泌和生长对生产细胞进行磁分选和选择

• 批准号: 10248280
• 负责人: Joseph de Rutte
• 金额: $ 23.64万
• 依托单位: PARTILLION BIOSCIENCE CORPORATION
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10248280
• 关键词: Address; Adoption; Affect; Affinity; Alpha Particles; Antibodies; Antibody titer measurement; Antibody-Producing Cells; Area; Award; B-Lymphocytes; Binding; Biological; Biological Process; Biological Products; Biomass; Biotechnology; Capital; Cell Line; Cell Separation; Cell Survival; Cell Therapy; Cell secretion; Cells; Centrifugation; Chinese Hamster Ovary Cell; Clone Cells; Cost Savings; Detection; Development; Devices; Diagnostic; Diagnostic tests; Dose; Drops; Engineering; Ensure; Enzyme-Linked Immunosorbent Assay; Epigenetic Process; Equipment; Fluorescence-Activated Cell Sorting; Foundations; Genetic; Growth; Hydrogels; Immunoglobulin-Secreting Cells; Label; Magnetic nanoparticles; Magnetism; Maintenance; Market Research; Microfluidics; Monoclonal Antibody Therapy; National Institute of General Medical Sciences; Particle Size; Phase; Phenotype; Population; Production; Productivity; Proteins; Protocols documentation; Reagent; Recombinants; Small Business Innovation Research Grant; Sorting - Cell Movement; Speed; Surface; System; Technology; Therapeutic; Therapeutic Uses; Time; Vaccines; Work; antibody diagnostic; base; cell growth; cost; cost effective; cytokine; improved; innovation; instrument; instrumentation; interest; magnetic beads; magnetic field; nanolitre scale; novel therapeutics; particle; population based; prevent; programs; response; vaccine evaluation

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.

摘要 细胞分泌物是生物过程、生物技术和细胞疗法的基础，然而， 基于分泌物快速分离出活细胞群并不容易。能够迅速 基于定量分泌表型分选细胞（例如B细胞、CHO细胞）可以加速发现 生产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 由分泌的蛋白质如细胞因子的类型和数量定义。强调需要，若干 已经开发了微流控平台来进行分泌细胞的分选，集中于产生分泌细胞的细胞。 抗体的然而，这些系统需要专门的专业知识或商业设备，这并不广泛。 可用并且在吞吐量上是有限的。根据市场调查，建议需要进行功能选择 减轻克隆生产细胞系中遗传和表观遗传漂移的方法，该提案旨在支持 一个新的计划，重点是工程Partillion的水凝胶纳米管平台作为一个基于试剂的解决方案， 分离并在培养物中保持高度分泌的生产细胞（例如CHO）亚克隆的富集群体 使用磁性激活细胞分选（MACS）。我们的纳米管技术是基于微尺度的新月形 捕获细胞的水凝胶颗粒被官能化以捕获分泌物，并模板化细胞的形成。 数以百万计的均匀液滴并行，防止分泌物的损失和串扰。此工作流只需要 简单的移液和离心步骤。然后可以将具有捕获的分泌物和相关细胞的纳米管 用磁性纳米颗粒标记，与对感兴趣的分泌物特异性的抗体缀合，并使用 MACS基于分泌量。我们将设计我们的nanovial产品，并制定工作流程， MACS的强大功能，使我们的客户能够在每个工作日分拣超过1000万个细胞，至少两个订单 这些技术的重要性高于市场上的竞争技术，以丰富关键的生产亚群体。 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，并能够选择更多的电池。具体目标（2） 研究基于分泌和生长的组合进行磁性分选的潜力，并调整 选择阈值以产生改善的细胞系生产力。在成功实现我们的目标后， 将为CHO和其他生产细胞分选的新试剂产品奠定坚实的基础， 与广泛可用的MACS系统相容的分泌物。最终，这可以实现更具成本效益的 和快速生产重组产品，如单克隆抗体疗法、疫苗和诊断试剂， 亲和试剂