High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells
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Published:2013-06-26
Issue:1
Volume:13
Page:
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ISSN:1472-6750
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Container-title:BMC Biotechnology
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language:en
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Short-container-title:BMC Biotechnol
Author:
Jäger Volker,Büssow Konrad,Wagner Andreas,Weber Susanne,Hust Michael,Frenzel André,Schirrmann Thomas
Abstract
Abstract
Background
The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility.
Results
In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average.
Conclusion
Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.
Publisher
Springer Science and Business Media LLC
Reference61 articles.
1. Taussig MJ, Stoevesandt O, Borrebaeck CAK, Bradbury AR, Cahill D, Cambillau C, de Daruvar A, Dübel S, Eichler J, Frank R, Gibson TJ, Gloriam D, Gold L, Herberg FW, Hermjakob H, Hoheisel JD, Joos TO, Kallioniemi O, Koegl M, Koegll M, Konthur Z, Korn B, Kremmer E, Krobitsch S, Landegren U, van der Maarel S, McCafferty J, Muyldermans S, Nygren P-A, Palcy S, Plückthun A, Polic B, Przybylski M, Saviranta P, Sawyer A, Sherman DJ, Skerra A, Templin M, Ueffing M, Uhlén M: ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome. Nat Methods. 2007, 4: 13-17. 10.1038/nmeth0107-13. 2. Dübel S, Stoevesandt O, Taussig MJ, Hust M: Generating recombinant antibodies to the complete human proteome. Trends Biotechnol. 2010, 28: 333-339. 10.1016/j.tibtech.2010.05.001. 3. Colwill K, Gräslund S: A roadmap to generate renewable protein binders to the human proteome. Nat Methods. 2011, 8: 551-558. 10.1038/nmeth.1607. 4. Hust M, Meyer T, Voedisch B, Rülker T, Thie H, El-Ghezal A, Kirsch MI, Schütte M, Helmsing S, Meier D, Schirrmann T, Dübel S: A human scFv antibody generation pipeline for proteome research. J Biotechnol. 2011, 152: 159-170. 10.1016/j.jbiotec.2010.09.945. 5. Hust M, Steinwand M, Al-Halabi L, Helmsing S, Schirrmann T, Dübel S: Improved microtitre plate production of single chain Fv fragments in Escherichia coli. N Biotechnol. 2009, 25: 424-428. 10.1016/j.nbt.2009.03.004.
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