Antibody Expression in Eukaryotic Hosts

Overview

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.

Yeast

Insect

Alternative Hosts

The use of gram positive bacteria such as Bacillus has been reported for rAb expression^9,10. 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.

Mammalian Cells

The production of antibodies was once limited to hybridomas. Hybridomas are unsuitable for large scale production due to low expression levels [<100 mg/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 media^20-22
• 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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References

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