Clonal selection of high producing, stably transfected HEK293 cell lines utilizing modified, high-throughput FACS screening
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 compar...
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| Published in | Journal of chemical technology and biotechnology (1986) Vol. 86; no. 7; pp. 935 - 941 |
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| Main Authors | , , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Chichester, UK
John Wiley & Sons, Ltd
01.07.2011
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| Subjects | |
| Online Access | Get full text |
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| 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 |
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| AbstractList | 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 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 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. |
| Author | Aliabadi-Zadeh, Khosrow Hughes, Benjamin Song, Michael Raphaelli, Kristin Gray, Peter P. Jones, Martina L. Crowley, David Mahler, Stephen Huang, Edwin P. Chin, David Y. Leung, Kar Man |
| Author_xml | – sequence: 1 givenname: Michael surname: Song fullname: Song, Michael organization: Biologics Facility, AIBN, The University of Queensland, Brisbane, Australia – sequence: 2 givenname: Kristin surname: Raphaelli fullname: Raphaelli, Kristin organization: Biologics Facility, AIBN, The University of Queensland, Brisbane, Australia – sequence: 3 givenname: Martina L. surname: Jones fullname: Jones, Martina L. organization: Biologics Facility, AIBN, The University of Queensland, Brisbane, Australia – sequence: 4 givenname: Khosrow surname: Aliabadi-Zadeh fullname: Aliabadi-Zadeh, Khosrow organization: Biologics Facility, AIBN, The University of Queensland, Brisbane, Australia – sequence: 5 givenname: Kar Man surname: Leung fullname: Leung, Kar Man organization: Biologics Facility, AIBN, The University of Queensland, Brisbane, Australia – sequence: 6 givenname: David surname: Crowley fullname: Crowley, David organization: Biologics Facility, AIBN, The University of Queensland, Brisbane, Australia – sequence: 7 givenname: Benjamin surname: Hughes fullname: Hughes, Benjamin organization: Biologics Facility, AIBN, The University of Queensland, Brisbane, Australia – sequence: 8 givenname: Stephen surname: Mahler fullname: Mahler, Stephen organization: Biologics Facility, AIBN, The University of Queensland, Brisbane, Australia – sequence: 9 givenname: Peter P. surname: Gray fullname: Gray, Peter P. organization: Biologics Facility, AIBN, The University of Queensland, Brisbane, Australia – sequence: 10 givenname: Edwin P. surname: Huang fullname: Huang, Edwin P. organization: Biologics Facility, AIBN, The University of Queensland, Brisbane, Australia – sequence: 11 givenname: David Y. surname: Chin fullname: Chin, David Y. email: david.chin@uq.edu.au organization: Biologics Facility, AIBN, The University of Queensland, Brisbane, Australia |
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| Title | Clonal selection of high producing, stably transfected HEK293 cell lines utilizing modified, high-throughput FACS screening |
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