Clonal selection of high producing, stably transfected HEK293 cell lines utilizing modified, high-throughput FACS screening
✍ Scribed by Michael Song; Kristin Raphaelli; Martina L. Jones; Khosrow Aliabadi-Zadeh; Kar Man Leung; David Crowley; Benjamin Hughes; Stephen Mahler; Peter P. Gray; Edwin P. Huang; David Y. Chin
- Book ID
- 102316661
- Publisher
- Wiley (John Wiley & Sons)
- Year
- 2011
- Tongue
- English
- Weight
- 255 KB
- Volume
- 86
- Category
- Article
- ISSN
- 0268-2575
No coin nor oath required. For personal study only.
✦ Synopsis
Abstract
BACKGROUND: Human embryonic kidney‐293 (HEK‐293) cells are commonly used as a transient expression host but their application in stable therapeutic protein production is limited. This is presumably due to the absence of a suitable amplifiable expression system and hence limited protein output compared with other mammalian cells such as Chinese hamster ovary cells. This paper describes a rapid clonal selection method for isolating HEK293 cell lines with high specific productivity, for a non‐amplifiable expression system, to achieve high‐level, scalable expression of recombinant antibodies.
RESULTS: Flow cytometry utilizing cold capture of secreted protein on the cell surface was applied to isolate high expressing clones from a stable antibiotic resistant pool. The top three isolated clones showed a five‐ to seven‐fold improvement in volumetric outputs compared with the initial resistant pool (∼20 mg L^−1^) under batch conditions. In fed‐batch conditions using commercially available hydrolysate supplements, the final titre was further increased to 500–600 mg L^−1^ in shaker flasks. One clone was scaled up to 25 L bag production using a similar hydrolysate feeding regime. The antibody titre reached 655 mg L^−1^, and 12 g of antibody was recovered after purification, demonstrating scalability of the process. The process of clonal selection through to fed‐batch production of gram quantities was completed within 4 months.
CONCLUSION: HEK‐293 cells can be used as a stable host for the production of biopharmaceuticals, producing gram quantities of recombinant proteins for preclinical development. Copyright © 2011 Society of Chemical Industry