Clonal selection of high producing, stably transfected HEK293 cell lines utilizing modified, high-throughput FACS screening

Song, Michael, Raphaelli, Kristin, Jones, Martina L., Aliabadi-Zadeh, Khosrow, Leung, Kar Man, Crowley, David, Hughes, Benjamin, Mahler, Stephen, Gray, Peter P., Huang, Edwin P. and Chin, David Y. (2011) Clonal selection of high producing, stably transfected HEK293 cell lines utilizing modified, high-throughput FACS screening. Journal of Chemical Technology and Biotechnology, 86 7: 935-941. doi:10.1002/jctb.2618


Author Song, Michael
Raphaelli, Kristin
Jones, Martina L.
Aliabadi-Zadeh, Khosrow
Leung, Kar Man
Crowley, David
Hughes, Benjamin
Mahler, Stephen
Gray, Peter P.
Huang, Edwin P.
Chin, David Y.
Title Clonal selection of high producing, stably transfected HEK293 cell lines utilizing modified, high-throughput FACS screening
Journal name Journal of Chemical Technology and Biotechnology   Check publisher's open access policy
ISSN 0268-2575
1097-4660
Publication date 2011-07-01
Sub-type Article (original research)
DOI 10.1002/jctb.2618
Volume 86
Issue 7
Start page 935
End page 941
Total pages 7
Place of publication Bognor Regis, West Sussex, U.K.
Publisher John Wiley & Sons
Collection year 2012
Language eng
Formatted 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.
Keyword HEK-293
FACS
Monoclonal antibody
Stable transfection
Cold capture
Recombinant CHO-cells
Antibody production
Mammalian-cells
Gene-expression
293E cells
Enhancement
Populations
Culture
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

 
Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 9 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 9 times in Scopus Article | Citations
Google Scholar Search Google Scholar
Created: Mon, 26 Sep 2011, 23:22:12 EST by Dr Martina Jones on behalf of Aust Institute for Bioengineering & Nanotechnology