Biotechnology overview: therapeutic glycoprotein production in various expression systems
Abstract
In the past few years, the biopharmaceutical industry has turned to mammalian cell expression systems for the production of biological agents. Of all the mammalian cell expression systems to date, CHO cells are the most widely used, accounting for 70% of recombinant protein production, and most of the proteins are monoclonal antibodies. This article introduces the production of therapeutic glycoproteins by the cell line of mammalian cells, and outlines the screening system and gene expression system of CHO cell lines.
Figure. Host cells of glycoprotein production
- Chinese hamster ovary cells (CHO)
CHO cells are widely used in the production of glycoproteins, which are determined by their numerous advantages, such as high protein production rate, suitable for large-scale industrial suspension culture, and can adapt to various serum-free and chemically defined media. In addition, the sugar chains of recombinant glycoproteins produced by CHO cells are highly similar to humans themselves, and have better compatibility and biological activity in humans. In addition, since many viral genes that enter CHO cells are not expressed, they are highly resistant to human virus-infected CHO cells, thereby minimizing the rate of protein infection by viruses and reducing biosafety risks. In addition, different gene amplification systems have been developed in CHO cells to achieve higher protein yields. More than half of the FDA and EU-approved monoclonal antibodies have been produced by CHO cells in recent years.
Although CHO cells have many advantages in glycoprotein production, they are not catalyzed for certain types of human proteins, such as alpha-2,6-sialylation and alpha-1,3/4-fucosylation. Moreover, for some glycosylated CHO cells that are not expressed by humans, such as hydroxyacetoxy-neuraminic acid (Neu5Gc) and galactose-α1,3-galactose (α-gal), even if the content of both is not more than 2%, but may also be immunogenic to humans. Even after improvement by metabolic engineering, CHO cells have some limitations on the production of γ-carboxylic acid recombinant proteins, such as coagulation factors.
- Human cell lines
One way to produce humanized glycoproteins is to use human cell lines for protein production. It can be guaranteed that even if it is not the ideal glycoprotein type, it at least won’t cause an immune response. The most widely used strains are HEK293 and HT-1080 cells from human embryonic kidney and fibrosarcoma. Xigris is produced by HEK293 and is the first FDA and EMA approved protein synthesized by human cell lines. The production of four proteins (Agalsidase alfa, Epoetin delta, Idursulfase and Velaglucerase alfa) in HT-1080 cells was approved by the FDA or EMA, although these products were withdrawn from the market due to commercial factors. Compared with Velaglucerase alfa produced by HT-1080 cells, CHO cells have similar glycosylation performance, and their in vitro activity, stability and potency are comparable. Many of the therapeutic proteins produced using human cell lines in 2014 were approved by the FDA or EMA, such as rFVIIIFc and rFIXFc for the prevention of hemophilia A and B bleeding episodes. These proteins expressed in the HEK293 cell line have higher levels of tyrosine sulfation and glutamate gamma carboxylation compared to CHO cell lines, and there is no Neu5Gc and a-gal glycosylation.
At present, some human cell lines are also used in preclinical studies and/or recombinant glycoprotein production, such as PER.C6 cells, which can obtain high yield proteins even without amplification of related genes. Both MOR103 and CL184 are therapeutic proteins produced by PER.C6, and clinical 1/2 studies have been conducted. HKB-11 cells obtained by fusion of HEK293S cells and human B cells exhibited high concentrations of protein production and α2,3 and α2,6-sialic acid linkages. The other two cell lines, proteins produced by CAP and HuH-7 cells, are currently in clinical trials and exhibit similar levels of glycosylation as human proteins.
- Other mammalian cell lines
Young hamster kidney cells (BHK) are commonly used in vaccine production. Currently only two recombinant glycoproteins are produced by BHK cells, namely factors VIIa and VIII. These macromolecular proteins are challenging for BHK cells due to their large levels of glycosylation and sulfation.
Murine myeloma cells (NS0 and Sp2/0), derived from tumor cells that no longer synthesize native immunoglobulins, can also be used to produce commercial monoclonal antibodies such as cetuximab and palivizumab. In 2015, the FDA approved three monoclonal antibodies, Dinutuximab, Necitumumab and Elotuzumab, produced by murine cells, for the treatment of different cancers. In addition, the level of Neu5Gc and α-gal glycosylation expressed by murine cells is higher than that of mouse cells, thus increasing the risk of immunogenicity.
- Non-mammalian cell lines and other expression systems
Although the use of mammalian systems to produce recombinant proteins has been a trend for nearly a decade, other expression systems are also available for recombinant protein production. Since these biological cells lack the required related enzymatic mechanism and are not suitable for the production of proteins containing sugar chains, they are often used for protein production without glycosylation.
Bacterial expression systems have the advantage of rapidly proliferating and highly expressed proteins. However, proteins tend to form aggregates, and extraction procedures are necessary because of the loss of chaperone protein. Some commercial proteins, such as asparaginase and collagenase without sugar chains are commonly produced by bacterial expression systems.
Yeast expression system has the same advantages as bacteria, rapid proliferation and high protein expression, but these proteins often have high mannose sugar type, which may be immunogenic and not effective for human body, such as gram plasmin.
For both plant and insect cells, both can produce proteins with complex glycosylation, but their type structure is quite different from that of humans. In fact, plants express sugar chains with α1,3-fructo and β1,2-xylose as cores, which are completely different from humans and may be immunogenic to humans. The N-glycotype produced by insect cells is either a high mannose type or an oligosaccharide type, and sialylation is absent in the glycosylation levels of both plant and insect cells. Glycosylation genetic engineering is also used in plant and insect cells to produce proteins. In 2012, the FDA approved the first therapeutic recombinant protein, taliglucerase alfa, produced by plant cells. The therapeutic proteins approved for adoption in insect systems are the human papillomavirus vaccine, prostate cancer vaccine and influenza vaccine. Recently, some therapeutic proteins have also been obtained in transgenic animals. Like other mammalian expression systems, the glycosylation structure of transgenic animals expressing proteins is somewhat different from that of humans. The therapeutic protein produced by the first transgenic animal in the market is the antithrombotic drug recombinant human antithrombin III, obtained from transgenic goat milk. Two more proteins were subsequently approved, namely recombinant C1-Esterase inhibitors obtained from transgenic rabbit milk and recombinant humanized lysosomal acid lipase obtained from transgenic eggs.