Next-Gen Expression Systems - Pharmaceutical Technology

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Next-Gen Expression Systems
More sophisticated biological expression systems expand the functionality of the traditional systems for protein synthesis.

Pharmaceutical Technology
Volume 35, Issue 6, pp. 36-39

Building better bacteria

While E. coli has been a mainstay of biopharmaceutical manufacturing, other bacteria may offer certain advantages over E. coli. For instance, Corynebacterium glutamicum is a gram-positive soil bacterium that has been used for decades in the food and pharmaceutical industries to produce amino acids, most notably, monosodium glutamate. Unlike the traditional E. coli system, C. glutamicum possesses secretory pathways that can be coopted to secrete engineered proteins into the culture broth. Marketed as the Corynex expression system by Ajinomoto AminoScience, this strain secretes few endogenous proteins, but engineered proteins can be targeted to one of two secretion pathways, the secretion pathway (sec), or the twin arginine translocase pathway (tat). The sec pathway initiates secretion of unfolded proteins that subsequently fold in the broth, while tat is able to transport folded proteins, and thus is able to handle larger proteins. Expression is constituative, and no inducer system is required. Joel White, business manager of biotechnology at Ajinomoto Aminoscience, says that the growth of Corynex is robust: it has a rapid doubling time, only slightly slower than E. coli, and can be grown in inexpensive, defined media in standard bioreactors. The great advantage of the system, according to White, is that the extra steps of cell lysis, centrifiguation, and protein refolding are not necessary, thereby providing a simpler, faster, less expensive purification route. White adds that this strain broadens the spectrum of folded proteins that can be produced in a bacterial expression system.

Other companies have been engineering E. coli to overcome its limitations in producing eukaryotic proteins. New England Biolabs, for example, markets several engineered strains of E. coli that are used primarily for bench-scale protein production. One of the strains, known as SHuffle, has been engineered to allow disulfide bond formation in the cytoplasm. In wild-type E. coli, reductases in the cytoplasm inhibit disulfide bond formation by keeping cysteine residues in their reduced form. Two reductases have been deleted in the SHuffle strain, and a version of a periplasmic disulfide bond isomerase has been targeted to the cytoplasm, which helps correct misoxidized bonds and promotes proper folding. Company data show that the SHuffle strain is capable of producing biologically active truncated tissue plasminogen activator, a protein that contains nine disulfide bonds when correctly folded. According to Dr. James Samuelson, a staff scientist at New England Biolabs, the strain grows well in fermenters and produces high protein yields. While most interest in the strain has come from academic labs, other companies, including manufacturers, have also expressed interest.

Controlling glycosylation

Bacterial systems grow quickly, but can generally not produce large proteins that require extensive posttranslational modification. Mammalian systems grow more slowly, but can deal with posttranslational modifications, and yeast fall somewhere in between. Yeast grow rapidly and can be cultured inexpensively, and unlike bacteria, secrete glycosylated proteins through the Golgi apparatus. However, the pattern of glycosylation in wild-type yeast differs markedly from the mammalian pattern and can alter a drug's potency or create unwanted immunogenic responses.

Developing yeast strains that produce human-like glycosylation patterns required extensive genetic engineering. Researchers had to first suppress the endogenous yeast glycosylation systems, and then provide the necessary enzymes to produce human-like carbohydrate chains. Working in the yeast strain Pichia pastoris, a team of researchers at New-Hampshire based Glycofi and Dartmoth University were able to reproduce the N-glycosylation pattern found on human glycoproteins. This was done by deleting four yeast genes, and inserting 14 heterologous genes, producing a humanized carbohydrate chain that contained sialic acid attached to the terminus of the chain, which is critical for metabolic stability in vivo (3). Glycofi and its humanized P. pastoris platform were acquired by Merck in 2006 specifically to support Merck's biosimilar-development program.

Other companies have since stepped up to provide expression systems where glycosylation patterns can be controlled. Wild-type CHO cells produce proteins with a heterogeneous composition of carbohydrate chains, and the factors that control the extent of glycosylation and type of carbohydrate chains attached to any given protein are not well understood (4). CHO cells have been engineered through a collaboration between BioWa of New Jersey, and Lonza to produce a cell line, called Potelligent, which is a suspension-adapted CHO cell line that, lacking the enzyme fucocyl transferase, produces glycoproteins without fucose. Non-fucosylated antibodies have been found to exhibit more effective antibody-dependent cell-mediated cytotoxicity than fucosylated antibodies (5). Company data from Lonza have confirmed this finding for multiple antibodies. While glycoengineering has not proceeded to the same extent in mammalian cells as it has in yeast cells, a cell line such as Potelligent demonstrates that control of glycosylation patterns can have dramatic effects not only on protein stability and immunogenicity, but also on efficacy.


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