Antibody Expression in Eukaryotic Hosts
Since eukaryotic hosts possess complex protein folding and secretory pathways, they can carry out complex post-translational modifications and secretion of rAbs. Their ability to glycosylate [add sugar groups] is absolutely critical for antibody effector functions such as ADCC and CDC. And so they are the most ideal host choice for the production of full length rAbs.
The use of gram positive bacteria such as Bacillus has been reported for rAb expression9,10. Since Gram positive bacteria lack an outer cell wall and have no periplasmic space, secreted proteins accumulate into the culture media. Filamentous fungi such as Aspergillus have been known to express antibody fragments11. Nicotianna tobaccum12, Alfalfa13, rice, wheat14, soya bean15 have been engineered to produce rAbs. Transgenic animals are also known to produce antibodies16. But given its robust expression profile, suitable glycosylation pattern and wide usage, mammalian cells are the best choice for the production of rAbs.
The production of antibodies was once limited to hybridomas. Hybridomas are unsuitable for large scale production due to low expression levels [<100mg/L] and low integrated viable cell count [IVCC] in bioreactors. These days, antibody genes are isolated from individual B-cells which are then cloned into mammalian expression vectors for rAb production. Mammalian cell lines typically used for rAb production include CHO, NS0, BHK, HEK293 and few others with HEK293 and CHO cells being the most popular choices for transient and stable expression respectively.
HEK293: Isolated from human embryonic kidney17. Used as host for small-scale recombinant protein production for over 2 decades. These cells can be efficiently transfected in suspension at large scale using cost-effective methods such as PEI or calcium-phosphate18.
CHO cells: These cells are derived from the ovary of the Chinese hamster. They are the preferred mammalian host for biologics production worldwide, with over 70% approved mAbs expressed in these cells19.
Some of the advantages of CHO cells include:
- Ease of cultivation at large scale
- Adaptability to suspension growth in serum-free media20-22
- Good safety profile with low risk of adventitious viral agent reproduction23
- Glycoforms that mimic human IgG glycoforms24
Factors that affect rAb production in mammalian cells
Expression vector technology is critical to achieve optimal stable or transient rAb expression. Key elements of a vector include the promoter and enhancer elements, kozak sequence, poly A signal, selectable markers, origin of replication and chromatin remodeling elements.
In order to achieve efficient IgG expression, it is essential to control the balance of heavy chain and light chain production. On entry into the endoplasmic reticulum as unfolded polypeptides, both chains are modified and assembled. Only completely assembled molecules can bind antigen and carry out effector functions. Light chains are synthesized 15%-25% faster than heavy chains25 and the isotype of light chain has been shown to influence the kinetics of intracellular IgG assembly26.
Most light chains can be secreted as free monomers or homodimers27. Whereas full length H chains are only exported from the cell when combined with the light chain to form complete antibody molecules28. The expression of both antibody chains is usually achieved either by co-transfecting cells with 2 independent monocistronic constructs or the use of a single vector with the light chain and heavy chain genes linked in series and transcription driven by 2 identical promoters.
The previous section in this series is “Antibody Expression in E. coli". To review, click here
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- Beck, A. et al. Trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and Fc-fusion proteins. Curr Pharm Biotechnol 9, 482-501 (2008).
- Reavy, B. et al. Expression of functional recombinant antibody molecules in insect cell expression systems. Protein Expr Purif 18, 221-228, doi:10.1006/prep.1999.1191 (2000).
- Liang, M. et al. Baculovirus expression cassette vectors for rapid production of complete human IgG from phage display selected antibody fragments. J Immunol Methods 247, 119-130 (2001).
- Shuler M.L, H. D. A., Granados R.R., Wood, H.A. in Baculovirus expression systems and biopesticides 1-12 (Wiley-Liss, New York, 1995).
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- Jin, B. R., Ryu, C. J., Kang, S. K., Han, M. H. & Hong, H. J. Characterization of a murine-human chimeric antibody with specificity for the pre-S2 surface antigen of hepatitis B virus expressed in baculovirus-infected insect cells. Virus Res 38, 269-277 (1995).
- Edelman, L. et al. Obtaining a functional recombinant anti-rhesus (D) antibody using the baculovirus-insect cell expression system. Immunology 91, 13-19 (1997).
- Jordan, E., Al-Halabi, L., Schirrmann, T. & Hust, M. Antibody production by the gram-positive bacterium Bacillus megaterium. Methods Mol Biol 525, 509-516, xiv, doi:10.1007/978-1-59745-554-1_27 (2009).
- Jordan, E. et al. Production of recombinant antibody fragments in Bacillus megaterium. Microb Cell Fact 6, 2, doi:10.1186/1475-2859-6-2 (2007).
- Frenken, L. G., Hessing, J. G., Van den Hondel, C. A. & Verrips, C. T. Recent advances in the large-scale production of antibody fragments using lower eukaryotic microorganisms. Res Immunol 149, 589-599 (1998).
- Valdes, R. et al. Large-scale purification of an antibody directed against hepatitis B surface antigen from transgenic tobacco plants. Biochem Biophys Res Commun 308, 94-100 (2003).
- Bardor, M. et al. Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a N-glycosylation that is homogenous and suitable for glyco-engineering into human-compatible structures. Plant Biotechnol J 1, 451-462, doi:10.1046/j.1467-7652.2003.00041.x (2003).
- Stoger, E. et al. Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Mol Biol 42, 583-590 (2000).
- Zeitlin, L. et al. A humanized monoclonal antibody produced in transgenic plants for immunoprotection of the vagina against genital herpes. Nat Biotechnol 16, 1361-1364, doi:10.1038/4344 (1998).
- Kind, A. & Schnieke, A. Animal pharming, two decades on. Transgenic Res 17, 1025-1033, doi:10.1007/s11248-008-9206-3 (2008).
- Graham, F. L., Smiley, J., Russell, W. C. & Nairn, R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36, 59-74, doi:10.1099/0022-1317-36-1-59 (1977).
- Durocher, Y., Perret, S. & Kamen, A. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res 30, E9 (2002).
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- Jefferis, R. Glycosylation of recombinant antibody therapeutics. Biotechnol Prog 21, 11-16, doi:10.1021/bp040016j (2005).
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