The history of biotechnology has been a tale of continuous improvement. Where the objective is proteins, traditional staples of transfection, media/feed, incubation, and clone selection have worked wonders for improving yields and quality.

Modern molecular techniques, where improvements are made at the genome level, have recently emerged to improve proteins even further.

Genome engineering, including direct gene editing, has become the go-to approach for improving protein yields or quality. “CRISPR/Cas9, in particular, has revolutionized that aspect of protein engineering, particularly in its application to complex mammalian genome with the purpose of eliminating undesirable host cell proteins, altering metabolic pathways, and directing favorable post-translational modifications,” says Jim Samuelson, Ph.D. senior scientist at New England Biolabs (NEB).

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As one would expect the exact method used is cell-type dependent: What works for prokaryotes usually does not apply to eukaryotes, for example. Furthermore monoclonal antibodies, the largest and most lucrative group of therapeutic proteins, are characterized by glycosylation, which is inherently heterogeneous. Thus, a major objective of protein engineering, predictable and uniform glycosylation, has become a priority.

Faster and better

Expression hosts, or more precisely expression systems, are often the target of protein engineering efforts. In January, 2017, Synthetic Genomics subsidiary SGI-DNA launched Vmax™ Express, an electrocompetent bacterial host expression system based on Vibrio natriegens, a gram-negative, nonpathogenic bacterium that exhibits the fastest growth rate of any known organism. Using E.coli-compatible plasmids, antibiotics and growth media, Vmax Express generates larger amounts of recombinant protein faster and more efficiently than E. coli, the most common bacterial protein expression host.

Expression hosts, or more precisely expression systems, are often the target of protein engineering efforts.

“Instead of further tweaking E. coli for incremental improvements, our team began with a clean slate, unconstrained by that organism’s inherent limitations, and designed a novel, advanced, fast-growing expression host for next-generation protein production in research, biopharma, and industrial biotechnology,” says Daniel Gibson, Ph.D., vice president of DNA technology at SGI.

As the production host for 30% of FDA-approved biologic drugs, E. coli is nevertheless relatively slow-growing and prone to low yields of often-insoluble proteins. According to SGI, Vmax Express offers at least double the titer of E. coli, a twofold improvement in doubling time, and improved solubility for some proteins that are difficult to express in E. coli.

SGI has moreover subjected purified proteins from Vmax to rigorous analysis, including mass spectrometry, and has not noted any quality issues, according to Gibson.

While Vibrio production organisms are far less common than E. coli, Gibson does not anticipate any unusual regulatory issues. “We anticipate challenges similar to those faced with E. coli, all of which are surmountable, and some of which we have already addressed through advanced strain engineering.”

Vmax is just one of multiple systems that SGI offers to lower the cost of producing high-value medicines. Cmax™, a eukaryotic organism engineered to produce more-complex glycosylated protein therapeutics, is suitable for manufacturing antigens and antibodies. SGI’s goal with Cmax is to replace legacy production systems like Chinese hamster ovary cells.

SGI is also working on platforms that bypass host strains through automated in vitro protein production. In a proof of concept study published in May, 2017 in Nature Biotechnology, co-authored by J. Craig Venter of human genome fame, SGI reported on a digital-to-biological converter (DBC) prototype that produces proteins on demand without human intervention. The DBC integrates synthetic biology tools developed by SGI with the BioXp System, SGI’s commercially available DNA printer. The BioXp system converts digital DNA sequences into physical genes, which, in turn, serve as instructions for the production of RNA, proteins, synthetic viruses, etc. downstream. The next-generation BioXp System will work with high-fidelity synthetic DNA as well.

The DBC synthesized polypeptides of the monoclonal antibodies abatacept, ranibizumab, and trastuzumab in less than two days, several weeks faster than current approaches starting with only DNA sequence. For the DBC proof of concept Synthetic Genomics used cell-free E. coli extracts to generate protein. Because the system is modular, more advanced cell-free lysates should could work as well.

“Think of the DBC as a rapid prototyper for therapeutic proteins,” Gibson says.

New proteins from new genes

Protein engineering ultimately seeks to improve proteins by making new ones, but that comes with a catch.

“The creation of novel proteins starts with the creation of novel genes,” says Samuelson. Incorporating those genes into expression hosts and harvesting protein is a moot concept unless those genes can be made.

Fusing smaller genes into large, transferrable constructs is often the issue. For this, NEB offers the gene assembly kits Gibson Assembly and NEBuilder HiFi DNA Assembly master mix.

“In contrast to restriction enzyme cloning, state-of-the-art DNA assembly methods allow for scarless fusion of virtually any gene sequences,” Samuellson adds. NEB’s tools join up to five DNA fragments consisting of virtually any gene sequence into an expression vector through a single operation.

Taking protein engineering to the de novo level, Arzeda creates “new life” through genetic engineering of host expression organisms. Arzeda’s platform enables the manufacture of almost any water-soluble small molecule, chemical intermediate, or product by inserting genes coding for the designed enzymes that carry out the relevant chemical reactions.

In April, 2016, Gen9 entered an agreement to supply Arzeda with megabase quantities of synthetic DNA to enable Arzeda to develop novel molecules for itself and commercial partners. Through Gen9’s MAP (Multiple Access Partnership) program, Arzeda will create novel proteins and incorporate genes coding for them into protein expression hosts.

Arzeda’s CEO, Alexandre Zanghellini, Ph.D., believes that combining the partners’ technologies could revolutionize how chemicals are produced through synergy of multiple novel enzymes and proteins within cells, that convert sugar into high-value chemicals and intermediates. “We will not be limited to molecules that nature has thus far created. We have the opportunity to create new molecules.”

MAP is fueled by Gen9’s BioFab® DNA synthesis platform that generates mass quantities of synthetic, low-cost, high-quality, long-length, clonal DNA. Arzeda uses these genes to create enzymes and specialty chemicals for polymers, crops, pharmaceuticals, industrial chemicals, and advanced materials.

Arzeda’s technology is suitable to the direct synthesis of novel, improved versions of therapeutic or industrial proteins as well. The potential for engineering cells with additional enzymes to carry out desirable post-translational modifications, such as glycosylation, creates the opportunity to improve the circulating halflife or other quality attributes, or reduce the immunogenicity, of monoclonal antibodies.

The key to this capability is Arzeda’s expertise in computational protein design and synthetic biology that competes with “natural” production platforms on cost and performance, while in some cases reducing the environmental impact associated with traditional organic synthesis. Replacing dangerous reagents like cyanide and benzene with clean enzymatic reactions could be a boon to the small molecule pharmaceutical industry, which despite its devotion to human health creates one of the highest toxic waste streams of any industry.

In late 2016 Arzeda, working with Amyris, validated its high-throughput protein design platform through the production of chemical intermediates whose synthesis normally involves toxic reagents. Amyris will further optimize these molecules.

The goal, again, is the production through fermentation of chemicals that are currently accessible only through synthetic chemistry. Further optimization is also possible by designing enzymes that synthesize improved, follow-on products that are difficult or impossible to make synthetically.

“The beauty of this system is that as long as the product is soluble in water, not toxic to the production host, and possesses feasible thermodynamics, we can design an enzyme for it,” Zanghellini explains. “Current enzymatic synthesis harnesses a small fraction of available chemistry. We are aiming to address all of it.”

With academic researchers Zanghellini has demonstrated enhancement of protein folding, a key quality attribute of biopharmaceuticals; stereoselective biomolecular addition for forming multiple complex carbon-carbon bonds with stereochemical fidelity; and creation of an enzyme that catalyzes the retro-aldol reaction, another classical organic chemical transformation.

Image: Arzeda founder Alex Zanghellini, Ph.D., is also the inventor of his company's protein engineering technology. Image courtesy of Arzeda.