Plasmids are naturally occurring parasitic extrachromosomal DNA elements that replicate inside microorganisms. They vary in size (some as small as a few thousand base pairs, while others are more than a million base pairs) and shape (some are linear while others are circular). Many of them have developed symbiotic relationships with their host organisms by encoding genes that provide a survival advantage to the host.

Production Of Recombinant Protein

We already discussed Genentech’s use of E. coli to accomplish the cloning and subsequent manufacturing of recombinant human insulin. Recombinant production of insulin in E. coli has continued since the early 1980s, providing a stable supply of the lifesaving drug to diabetic patients around the globe. In addition to insulin, E. coli has been used to produce a variety of other pharmaceutical proteins, including hormones for the treatment of growth hormone deficiency (somatropin), osteoperosis (teriparatide), achondroplasia (vosoritide), lipodystrophy (metreleptin), short bowel syndrome (teduglutide), congestive heart failure (nesiritide), Paget disease (calcitonin), and hypoglycemia (glucagon); therapeutic enzymes for the treatment of methotrexate toxicity (glucarpidase), kidney transplant rejection (imlifidase), phenylketonuria (pegvaliase), severe combined immune deficiency (elapegademase-lvlr), cancer (pegaspargase), and gout (pegloticase); fusion proteins for the treatment of cancer (tebentafusp-tebn, tagraxofusp-erzs) and thrombocytopenia (romiplostim); colony stimulating factors for the treatment of neutropenia (pegfilgrastim); interferons and cytokines for the treatment of hepatitis C (peginterferon alfa-2a), multiple sclerosis (interferon beta-1b), and rheumatoid arthritis (anakinra); antibody fragments for the treatment of inflammatory conditions (certolizumab pegol), blood clotting disorders (caplacizumab), or age-related macular degeneration (ranibizumab); and recombinant vaccines such as the meningococcal group B vaccine (for a comprehensive listing see Reference 1).

Despite its success in the cases listed above, it was determined as early as the 1980s that E. coli has several limitations from a protein production perspective. First, it was found that most eukaryotic proteins of therapeutic interest did not express well in E. coli. Many of these proteins contain complex disulfide bonds or require essential post-translational modifications such as glycosylation that E. coli could not produce. Additionally, E. coli lacks the more complex protein-folding machinery present in eukaryotic cells that help proteins to adopt their correct structure. These deficiencies mean that many of the proteins being expressed in E. coli are produced in small quantities in an inactive or incorrectly folded form.

Genentech experienced these difficulties first hand when they attempted to produce tissue plasminogen activator (tPA), an enzyme that can be administered to patients to break down deadly blood clots. Following the playbook that was employed to produce human insulin and several other simple proteins, the tPA gene was cloned into a suitable plasmid and introduced into E. coli cells[2]. However, the small amount of material that was generated was largely inactive.

Genentech was able to solve the problem by switching to a different organism for protein expression—Chinese Hamster Ovary (CHO) cells. This cell line was chosen because it had been demonstrated to grow robustly in the lab and, being a mammalian cell, it had the cellular machinery necessary to process and fold complex eukaryotic proteins. In 1987, tPA became the first United States FDA-approved recombinant protein to be produced in CHO cells.

As the biopharmaceutical industry set its sights on other complex eukaryotic proteins that require disulfide bonds and post-translational modifications to function (such as antibodies), more and more proteins began to be produced in CHO instead of E. coli. Until 1989, 66% of approved biopharmaceuticals were produced in non-mammalian cell lines (primarily E. coli), whereas today, only 28% of approved biopharmaceuticals are manufactured in non- mammalian cell lines. If we exclude legacy products and focus on recently approved biopharmaceuticals, we find that 85% of protein drugs entering the market over the past few years are manufactured in mammalian systems such as CHO cells, NS0 mouse cells, and baby hamster kidney (BHK) cells1. By far, CHO is the most popular system, owing to its tried-and-true capabilities to reliably produce high-quality antibodies at high-titer, and its ability to secrete these proteins outside the cell (greatly simplifying downstream processing). Given that antibodies are a dominant modality in protein-based pharmaceuticals, the dominance of CHO as a production host is easily understood.

Chemical Production

While microorganisms have been used by humans for thousands of years to produce desired chemicals (such as the ancient art of using wild yeasts to convert sugars into ethanol to produce alcoholic beverages), the advent of modern tools for genetic engineering has supercharged our ability to produce molecules of interest through biological systems. Metabolic engineers have successfully tinkered with microbes to coax them to produce molecules that can be used for diverse applications such as treating cancer, powering combustion engines, flavoring foods, making textiles, or formulating fragrances.

A notable example is the production of 1,3-propanediol (PDO)—a versatile chemical that is used as a building block for producing textiles, composite materials, paints, antifreeze solutions, adhesives, and industrial coatings. Dupont Tate & Lyle successfully commercialized a genetically engineered E. coli strain capable of producing PDO from renewable sugar feedstocks, providing a sustainable biological process to replace the prevalent chemical manufacturing process.[3]

While E. coli is a commonly chosen host organism for metabolic engineering, there are many factors that go into the selection of a host organism, such as that organism’s innate metabolic capabilities, the ability to express certain types of proteins in the host to enable new metabolic capabilities, the expense of the nutrients required for growth, and suitability for the overall process. For example, companies focused on biofuel production typically prefer photosynthetic organisms such as algae to enable the conversion of sunlight and atmospheric CO2 into fuels. Not only does this offset the carbon released during combustion of these fuels, but sunlight and CO2 are abundant and cheap. A similar process in E. coli, even if technically possible, would not offer the cost and environmental benefits of a process relying on algae. Thus, while E. coli continues to play a role as a host for metabolic engineering, it has been joined by a cadre of other microbes such as fungi, algae, and other bacteria.

Plasmid Production

When it comes to the manufacture of plasmid DNA, E. coli is still the dominant production system. Unlike for protein or chemical production, where a wide variety of host organisms can be leveraged depending on the task, for plasmid DNA production, the field continues to use essentially the same E. coli technology developed in the 1970s. As significantly more attention is placed on genetic medicines (cell and gene therapies, nucleic acid vaccines, and RNA-therapeutics) where plasmid DNA is a critical reagent in the manufacturing process, the demand for plasmid DNA production has skyrocketed. Our research has shown that customers who need plasmid DNA are reporting increasing costs, long lead times, and routine failures. These challenges show that there is room for innovation.

Some are experimenting with alternative methods, such as cell-free plasmid amplification technologies using purified enzymes.

These technologies have not seen widespread adoption yet due to

1. Higher costs
2. Incompatibility with existing upstream/downstream manufacturing infrastructure
3. Incompatibility with commonly used plasmid backbones
4. Unresolved regulatory considerations

It will be interesting to see how these technologies develop over the next several years, and whether there is an uptick in adoption in the industry.

E. coli certainly has a legacy to be proud of, and will no doubt continue to play an important role in multiple areas of biotechnology. It has many strengths, including being the most well-studied and characterized organism on the planet; widespread adoption by the research community; robust and forgiving growth in a laboratory setting; existing tools, protocols, and kits for genetic modification; and a history of commercial applications. Its weaknesses include the inability to perform the common post- translational modifications some proteins require to properly function; a known inability to produce many complex proteins of therapeutic interest; high production costs when used for some commodity chemicals; and challenges in the production of complex plasmid DNAs.

References

• Walsh, G and Walsh, E. 2022. Biopharmaceutical benchmarks 2022. Nat. Biotechnol. 40(12):1722–1760
• Pennica, D. et al. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. 1983. Nature 301(5897):214–221
• www.tateandlyle.com/news/dupont-tate-lyle-joint-venture-officially-opens-100-million-bio-pdo- facility-world-s-first