Simplifying stable CHO cell line generation with high probability of monoclonality by using microfluidic dispensing as an alternative to fluorescence activated cell sorting

IF 2.5 3区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Biotechnology Progress Pub Date : 2024-03-10 DOI:10.1002/btpr.3441
Lina Chakrabarti, James Savery, John Patrick Mpindi, Judith Klover, Lina Li, Jie Zhu
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Abstract

Single cell cloning is a critical step for cell line development (CLD) for therapeutic protein production, with proof of monoclonality being compulsorily sought in regulatory filings. Among the different single cell deposition technologies, we found that fluorescence activated cell sorting (FACS) offers high probability of monoclonality and can allow selective enrichment of the producer cells. However, FACS instruments are expensive and resource-intensive, have a large footprint, require highly skilled operators and take hours for setup, thereby complicating the cell line generation process. With the aim of finding an easy-to-use alternative to FACS, we identified a flow cytometry-based microfluidic cell dispenser, which presents a single cell sorting solution for biopharmaceutical CLD. The microfluidic cell dispenser is small, budget-friendly, easy-to-use, requires lower-cost consumables, permits flow cytometry-enabled multiparametric target cell enrichment and offers fast and gentle single cell dispensing into multiwell plates. Following comprehensive evaluation, we found that single cell deposition by the microfluidic cell dispenser resulted in >99% probability of monoclonality for production cell lines. Moreover, the clonally derived producer cell lines generated from the microfluidic cell dispenser demonstrated comparable or improved growth profiles and production capability compared to the FACS derived cell lines. Taken together, microfluidic cell dispensing can serve as a cost-effective, efficient and convenient alternative to FACS, simplifying the biopharmaceutical CLD platform with significant reductions in both scientist time and running costs.

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利用微流体喷点技术替代荧光激活细胞分选技术,简化具有高单克隆概率的稳定 CHO 细胞系的生成。
单细胞克隆是用于生产治疗性蛋白质的细胞系开发(CLD)的关键步骤,而单细胞性证明则是监管部门强制要求的。在不同的单细胞沉积技术中,我们发现荧光激活细胞分拣(FACS)具有很高的单克隆概率,并能选择性地富集生产细胞。然而,荧光激活细胞分选仪价格昂贵、资源密集、占地面积大、需要高技能的操作人员,而且设置时间长,从而使细胞系的生成过程复杂化。为了找到一种易于使用的方法来替代 FACS,我们发现了一种基于流式细胞仪的微流体细胞分注器,它为生物制药 CLD 提供了一种单一的细胞分选解决方案。这种微流体细胞分装机体积小、成本低廉、易于使用、所需耗材成本较低、可通过流式细胞仪进行多参数靶细胞富集,并能快速、温和地将单细胞分装到多孔板中。经过综合评估,我们发现微流体细胞分装机的单细胞沉积使生产细胞系的单克隆概率大于 99%。此外,与 FACS 衍生的细胞系相比,微流体细胞分装机产生的克隆生产细胞系的生长曲线和生产能力与之相当或有所提高。综上所述,微流体细胞分装技术是一种经济、高效、便捷的 FACS 替代技术,它简化了生物制药 CLD 平台,显著减少了科学家的时间和运行成本。
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来源期刊
Biotechnology Progress
Biotechnology Progress 工程技术-生物工程与应用微生物
CiteScore
6.50
自引率
3.40%
发文量
83
审稿时长
4 months
期刊介绍: Biotechnology Progress , an official, bimonthly publication of the American Institute of Chemical Engineers and its technological community, the Society for Biological Engineering, features peer-reviewed research articles, reviews, and descriptions of emerging techniques for the development and design of new processes, products, and devices for the biotechnology, biopharmaceutical and bioprocess industries. Widespread interest includes application of biological and engineering principles in fields such as applied cellular physiology and metabolic engineering, biocatalysis and bioreactor design, bioseparations and downstream processing, cell culture and tissue engineering, biosensors and process control, bioinformatics and systems biology, biomaterials and artificial organs, stem cell biology and genetics, and plant biology and food science. Manuscripts concerning the design of related processes, products, or devices are also encouraged. Four types of manuscripts are printed in the Journal: Research Papers, Topical or Review Papers, Letters to the Editor, and R & D Notes.
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