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Clonal selection of high producing, stably transfected HEK293 cell lines utilizing modified, high‐throughput FACS screening
Author(s) -
Song Michael,
Raphaelli Kristin,
Jones Martina L.,
AliabadiZadeh Khosrow,
Leung Kar Man,
Crowley David,
Hughes Benjamin,
Mahler Stephen,
Gray Peter P.,
Huang Edwin P.,
Chin David Y.
Publication year - 2011
Publication title -
journal of chemical technology and biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.64
H-Index - 117
eISSN - 1097-4660
pISSN - 0268-2575
DOI - 10.1002/jctb.2618
Subject(s) - hek 293 cells , chinese hamster ovary cell , recombinant dna , biology , cell culture , flow cytometry , transfection , biopharmaceutical , clone (java method) , antibody , microbiology and biotechnology , laboratory flask , titer , high throughput screening , biochemistry , chemistry , gene , genetics
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