Ode to the Microbe

After decades of development and promise, the COVID-19 pandemic brought mRNA vaccines to the forefront of immunization technology. Compared with other vaccine technologies, mRNA vaccines are fast to develop, quick to modify in response to new variants, and less costly to produce. The cell-free manufacturing process is endorsed as a safer and simpler option compared with existing vaccine technologies that rely on growing and culturing microbes and viruses; some of which are infectious and potentially hazardous (1). However, we should not overlook the fact that many of the raw materials required for the cell-free process of mRNA vaccines are derived from cells – using microbial fermentation to be specific.

The manufacture of mRNA vaccines uses a biochemical in vitro transcription (IVT) reaction. The DNA template that encodes the antigen, RNA polymerase, and nucleic acids are combined under the proper conditions to transcribe the mRNA. Post-transcription capping and tailing is performed by RNA modifying enzymes to improve mRNA stability and protect against degradation. The target mRNA molecule is then purified and encapsulated in a lipid nanoparticle to stabilize and allow for uptake once administered.

Many of the critical raw materials used in the IVT process are produced from microbial fermentation. The DNA template is produced in microbes from plasmids. The enzymes used for the IVT reaction – RNA  polymerase, RNA modifying enzymes including guanylyl transferase, 2’-O-methyltransferase and poly-A polymerase – are also produced in microbes. Even the processing aid DNase, used to remove non-target DNA during purification, is produced via microbial fermentation.

Understanding fermentation
 

Microbial fermentation is described as the use of microbial cells to generate energy and facilitate biochemical reactions. Like microscopic factories, the cells convert nutrients, such as glucose (carbon), peptones (nitrogen), and minerals into energy and building blocks to make important products of interest. Strain improvement techniques and genetic modification strategies, such as recombinant DNA transformation, have birthed an entire bioindustry that uses microbial hosts to manufacture biological molecules at large scale. By cloning foreign DNA in cells, microbes like Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris can be reprogrammed to make molecules in high quantities in an economical and sustainable manner. Insulin, somatropin (human growth hormone), and the aforementioned DNA templates for the SARS-CoV-2 mRNA vaccines are all made using genetic transformation.

Genetically engineered E. coli is one of the most important industrially relevant microbial hosts. First used for DNA cloning back in the 1970s, it is well studied, robust to industrial conditions, and easy to genetically modify (2). Most commercial E. coli strains are descendants of two isolates, K-12 and B. These isolates have been improved through genetic alteration to create strains for specific purposes, including cloning DNA and expressing proteins. In addition to the strains, the design of the DNA vector or plasmid used to genetically modify the cells is an equally important consideration.

To produce plasmid DNA for mRNA vaccines, E. coli strain and vector combinations are chosen to yield high copies of the plasmid. E. coli DH5α, a K-12 derivative, is better suited for DNA plasmid production because it lacks the genes for endonuclease 1 (endA) and DNA repair protein (recA) to increase plasmid yield and stability. Plasmid vectors used for mRNA vaccines often include bacterial sites of origin for high copy amplification in E. coli and a eukaryotic region to express the antigen once administered into the host cell.

For the enzymes used in the IVT process, E. coli BL21 (DE3), a B-strain derivative, is often used. It has been genetically modified to carry a chromosomal copy of the T7 polymerase, which allows for faster RNA transcription and ultimately greater enzyme or protein expression. Additionally, BL21 strains are deficient in two proteases, Lon and Ompt, which help minimize protein degradation during purification. Plasmid vectors for protein production often contain an inducible promoter to “switch on” expression. One example is the Lac promoter under the control of lactose or lactose derivatives (for example, isopropyl β- d-1-thiogalactopyranoside, IPTG). When added to the growth medium, IPTG induces the cells to produce the target protein of interest. Protein expression is metabolically demanding; thus, inducible promoters help fine tune the timing of expression to shorten lag times and increase productivity.

In summary, microbial fermentation plays an important role in the manufacture of mRNA vaccines. Looking ahead, I believe that new technologies, such as cell-free systems and the IVT process, will be increasingly sought after to meet the demands of the ever-burgeoning bio-based economy. But it’s also clear that cell-dependent approaches will continue to play a crucial role in manufacture of mRNA vaccines, gene therapies, and other genetic medicines.