Abstract
The generation of stable, high-level monoclonal antibody (mAb) producing cell lines remains a major challenge in biopharmaceutical industry. The commonly used plasmid vectors for mAb expression, which express light chain (LC), heavy chain (HC), and selection marker genes on separate vectors or via multiple promoters on a single vector, are not able to accurately control the ratio of LC over HC expression and tend to result in non-expressing clones. To overcome these issues, we have developed a tricistronic vector using two internal ribosome entry sites (IRES) to express the LC, HC, and dihydrofolate reductase (DHFR) selection marker genes in one transcript. In this tricistronic vector, the three genes are under the control of a hapten-modified human cytomegalovirus (hCMV) promoter containing a core CpG island element (IE) to enhance the production stability. The LC gene is arranged as the first cistron followed by a wild-type IRES to control the HC expression. Such design expresses excess LC polypeptides which enhance mAb expression level and reduce aggregate. A mutated IRES with attenuated strength is applied on DHFR to reduce its expression for enhancing the stringency of selection for high producers. This vector allows easy generation of stable, high mAb producing CHO DG44 pools and clones for antibody development and manufacturing.
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References
Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22(11):1393–1398. https://doi.org/10.1038/nbt1026. nbt1026 [pii]
Ye JX, Kober V, Tellers M, Naji Z, Salmon P, Markusen JF (2009) High-level protein expression in scalable CHO transient transfection. Biotechnol Bioeng 103(3):542–551. https://doi.org/10.1002/bit.22265
Ye JX, Alvin K, Latif H, Hsu A, Parikh V, Whitmer T, Tellers M, Edmonds MCD, Ly J, Salmon P, Markusen JF (2010) Rapid protein production using CHO stable transfection pools. Biotechnol Prog 26(5):1431–1437. https://doi.org/10.1002/btpr.469
Zhang PQ, Woen S, Wang TH, Liau B, Zhao S, Chen C, Yang YS, Song ZW, Wormald MR, Yu CF, Rudd PM (2016) Challenges of glycosylation analysis and control: an integrated approach to producing optimal and consistent therapeutic drugs. Drug Discov Today 21(5):740–765. https://doi.org/10.1016/j.drudis.2016.01.006
Barnes LM, Bentley CM, Dickson AJ (2003) Stability of protein production from recombinant mammalian cells. Biotechnol Bioeng 81(6):631–639. https://doi.org/10.1002/bit.10517
Chusainow J, Yang YS, Yeo JH, Toh PC, Asvadi P, Wong NS, Yap MG (2009) A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng 102(4):1182–1196. https://doi.org/10.1002/bit.22158
Jordan A, Defechereux P, Verdin E (2001) The site of HIV-1 integration in the human genome determines basal transcriptional activity and response to Tat transactivation. EMBO J 20(7):1726–1738. https://doi.org/10.1093/emboj/20.7.1726
Gonzalez R, Andrews BA, Asenjo JA (2002) Kinetic model of BiP- and PDI-mediated protein folding and assembly. J Theor Biol 214(4):529–537. https://doi.org/10.1006/jtbi.2001.2478. S0022519301924786 [pii]
Ho SCL, Bardor M, Feng HT, Mariati TYW, Song ZW, Yap MGS, Yang YS (2012) IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol 157(1):130–139. https://doi.org/10.1016/j.jbiotec.2011.09.023
Ho SCL, Bardor M, Li B, Lee JJ, Song ZW, Tong YW, Goh LT, Yang YS (2013) Comparison of internal ribosome entry site (IRES) and furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells. PLoS One 8(5):e63247. https://doi.org/10.1371/journal.pone.0063247
Ho SCL, Koh EYC, van Beers M, Mueller M, Wan C, Teo G, Song ZW, Tong YW, Bardor M, Yang YS (2013) Control of IgG LC:HC ratio in stably transfected CHO cells and study of the impact on expression, aggregation, glycosylation and conformational stability. J Biotechnol 165(3–4):157–166. https://doi.org/10.1016/j.jbiotec.2013.03.019
Li JD, Zhang CC, Jostock T, Dubel S (2007) Analysis of IgG heavy chain to light chain ratio with mutant Encephalomyocarditis virus internal ribosome entry site. Protein Eng Des Sel 20(10):491–496
Pybus LP, Dean G, West NR, Smith A, Daramola O, Field R, Wilkinson SJ, James DC (2014) Model-directed engineering of “difficult-to-express” monoclonal antibody production by Chinese hamster ovary cells. Biotechnol Bioeng 111(2):372–385. https://doi.org/10.1002/bit.25116
Pybus LP, James DC, Dean G, Slidel T, Hardman C, Smith A, Daramola O, Field R (2014) Predicting the expression of recombinant monoclonal antibodies in Chinese hamster ovary cells based on sequence features of the CDR3 domain. Biotechnol Prog 30(1):188–197. https://doi.org/10.1002/btpr.1839
Schlatter S, Stansfield SH, Dinnis DM, Racher AJ, Birch JR, James DC (2005) On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells. Biotechnol Prog 21(1):122–133. https://doi.org/10.1021/bp049780w
Yang YS, Mariati HSCL, Yap MGS (2009) Mutated polyadenylation signals for controlling expression levels of multiple genes in mammalian cells. Biotechnol Bioeng 102(4):1152–1160. https://doi.org/10.1002/bit.22152
Lee CJ, Seth G, Tsukuda J, Hamilton RW (2009) A clone screening method using mRNA levels to determine specific productivity and product quality for monoclonal antibodies. Biotechnol Bioeng 102(4):1107–1118
Barnes LM, Bentley CM, Moy N, Dickson AJ (2007) Molecular analysis of successful cell line selection in transfected GS-NS0 myeloma cells. Biotechnol Bioeng 96(2):337–348. https://doi.org/10.1002/bit.21119
Ng SK, Lin W, Sachdeva R, Wang DI, Yap MG (2010) Vector fragmentation: characterizing vector integrity in transfected clones by southern blotting. Biotechnol Prog 26(1):11–20. https://doi.org/10.1002/btpr.281
Bailey LA, Hatton D, Field R, Dickson AJ (2012) Determination of Chinese hamster ovary cell line stability and recombinant antibody expression during long-term culture. Biotechnol Bioeng 109(8):2093–2103. https://doi.org/10.1002/bit.24485
Barnes LM, Bentley CM, Dickson AJ (2001) Characterization of the stability of recombinant protein production in the GS-NS0 expression system. Biotechnol Bioeng 73(4):261–270
Dorai H, Corisdeo S, Ellis D, Kinney C, Chomo M, Hawley-Nelson P, Moore G, Betenbaugh MJ, Ganguly S (2012) Early prediction of instability of Chinese hamster ovary cell lines expressing recombinant antibodies and antibody-fusion proteins. Biotechnol Bioeng 109(4):1016–1030. https://doi.org/10.1002/bit.24367
Fann CH, Guirgis F, Chen G, Lao MS, Piret JM (2000) Limitations to the amplification and stability of human tissue-type plasminogen activator expression by Chinese hamster ovary cells. Biotechnol Bioeng 69(2):204–212
He L, Winterrowd C, Kadura I, Frye C (2012) Transgene copy number distribution profiles in recombinant CHO cell lines revealed by single cell analyses. Biotechnol Bioeng 109(7):1713–1722. https://doi.org/10.1002/bit.24428
Jun SC, Kim MS, Hong HJ, Lee GM (2006) Limitations to the development of humanized antibody producing Chinese hamster ovary cells using glutamine synthetase-mediated gene amplification. Biotechnol Prog 22(3):770–780
Kim M, O'Callaghan PM, Droms KA, James DC (2011) A mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies. Biotechnol Bioeng 108(10):2434–2446. https://doi.org/10.1002/bit.23189
Kim SJ, Kim NS, Ryu CJ, Hong HJ, Lee GM (1998) Characterization of chimeric antibody producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure. Biotechnol Bioeng 58(1):73–84
Osterlehner A, Simmeth S, Goepfert U (2011) Promoter methylation and transgene copy numbers predict unstable protein production in recombinant Chinese hamster ovary cell lines. Biotechnol Bioeng 108(11):2670–2681. https://doi.org/10.1002/bit.23216
Strutzenberger K, Borth N, Kunert R, Steinfellner W, Katinger H (1999) Changes during subclone development and ageing of human antibody-producing recombinant CHO cells. J Biotechnol 69(2–3):215–226
Mariati, Koh EYC, Yeo JHM, Ho SCL, Yang YS (2014) Bioengineered 5(5):340–345. https://doi.org/10.4161/bioe.32111
Mariati, Yeo JHM, Koh EYC, Ho SCL, Yang YS (2014) Insertion of core CpG island element into human CMV promoter for enhancing recombinant protein expression stability in CHO cells. Biotechnol Prog 30(3):523–534. https://doi.org/10.1002/btpr.1919
Ng SK, Wang DIC, Yap MGS (2007) Application of destabilizing sequences on selection marker for improved recombinant protein productivity in CHO-DG44. Metab Eng 9(3):304–316
Acknowledgments
This work was supported by the Biomedical Research Council/Science and Engineering Research Council of A*STAR (Agency for Science, Technology and Research), Singapore.
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Yeo, J.H.M., Mariati, Yang, Y. (2018). An IRES-Mediated Tricistronic Vector for Efficient Generation of Stable, High-Level Monoclonal Antibody Producing CHO DG44 Cell Lines. In: Nevoltris, D., Chames, P. (eds) Antibody Engineering. Methods in Molecular Biology, vol 1827. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8648-4_17
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DOI: https://doi.org/10.1007/978-1-4939-8648-4_17
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