August 16, 2022 PAO-08-022-CL-03
Interest in microbial fermentation in bacterial systems has been increasing recently, because protein expression is easier in these systems than in mammalian cell culture. In addition, the results tend to be better for bacterial proteins and enzymes. It also works well for the production of many different types of modalities, including antibody fragments, peptides, smaller proteins, enzymes, hormones, and so on.
For pharmaceutical manufacturing, Vectron Biosolutions is focused on improving the performance of Escherichia coli systems by using expression vectors based on the xylS/Pm regulator/promoter system from Pseudomonas putida (xylS is the transcription regulator gene) on an RK2 plasmid modified to greatly reduce its size. The Pm promoter does not operate via a repressor mechanism but rather requires a benzoic acid external inducer to bind to the transcription regulator to turn it on.
Notably, the benzoic acid inducer readily and passively moves across bacterial membranes, allowing regulation of the level of expression by simply adding more or less of the inducer. More specifically, this ability to fine-tune the expression rate allows for its balancing with the protein folding rate, enabling all of the synthesized polypeptides to be folded correctly.
VB Evolution technology mimics evolution through the generation of libraries of random expression vectors through mutagenization. Millions of vectors are subjected to high-throughput screening to identify those that are the best for a specific protein, because every protein is unique and poses unique challenges. Consequently, the VB Evolution technology allows Vectron to create tailor-made, bespoke expression vectors for each and every protein we work with by evolving a solution to every protein through an iterative process similar to what occurs in nature.
The ultra-high-throughput screening method is unique in the industry in that it is a universal screen that looks at titers, not activities, and thus works independently of the target protein. All that is necessary is cloning to establish the customer’s gene inside our library of mutagenized vectors before screening can begin. The ultra-high-throughput screening process itself is extremely efficient and allows the screening of millions of candidates in a couple of days.
Overall, therefore, the time from receipt of a client’s gene to generation of an expression vector that produces very high amounts of the client’s protein may be just a few weeks. The actual project timeline will depend on the number of desired iterations. The short timeline makes VB Evolution especially attractive for customers that have unexpected problems in clinical development and are anxious to get back on their planned development schedules.
VB Secretion technology, originally developed by Professor Kelly Hughes at the University of Utah and commercialized through the company T3S Technologies, is based on truncated versions of bacterial flagella, organelles in bacteria that help in locomotion. These flagella have been engineered to act as pores or channels that direct proteins produced by the cell to exit the cell into the surrounding media. This internal process eliminates the need for a cell lysis step before harvest and downstream purification. The product proteins are in a cleaner bioprocess fluid, and downstream purification is much simpler, leading to time and cost savings.
The technology was initially developed for Salmonella, which is not an attractive host for pharmaceutical production processes. Initially, Vectron collaborated with T3S to move the secretion system from Salmonella to E. coli and use it in combination with our expression vectors. Vectron then acquired T3S in the summer of 2021. Final improvements to the technology in E. coli are being made to increase the secretion rate and ensure that the technology is applicable to many different proteins.
While Vectron started with a specific set of expression vectors, we now have the ability to generate novel, unique expression vectors and combine them with engineered secretion technologies. Together, these solutions comprise the most sophisticated technology platform for the production of proteins in E. coli. This approach is particularly advantageous for expressing hard-to-produce proteins, such as those that are prone to aggregation, toxic to the cells, or only produced in low titers. The technology is also effective for easy-to-produce proteins and can achieve titers of more than 60 grams per liter.
Each strain developed by Vectron is tailor-made for each customer. Strains differ from project to project and are unique and optimized to balance production and expression for each and every protein. That is because every protein behaves differently inside the cell, and the expression vector must give the right amount of protein at the right rate to enable proper folding and minimize aggregation.
This ability is particularly relevant today, because the proteins in development are much more difficult to produce in general than proteins brought to the market 10–20 years ago. They may comprise different domains from different proteins and other entirely new modalities with new functions. Many are very complex and can be toxic to the host cells, especially at high titers. The strains required for production of these proteins are therefore necessarily unique and substantially or significantly different from one another.
Vectron’s current business model involves granting exclusive licenses to customers for the specific vectors and strains developed for them. Vectron holds patents on the technology and grants licenses to each customer on the basis of the specific proteins that they are producing. As with the technology itself, which differs from project to project, the details of each license agreement differ. Only aspects of the technology that are being exploited by a customer for the manufacture of a specific protein will be included in the license. Because each license is very specific to the protein and the project requirements, this approach makes it possible to issue infinite exclusive licenses.
Creating bespoke E. coli strains requires significant effort and a great deal of innovation. Unlike other companies offering strain development services, Vectron does not use a limited set of expression systems over and over again. We create novel E. coli strains for which separate patent protection may be available.
Therefore, as a value-add, Vectron will offer customers the potential of obtaining patents on the specific E. coli strains we develop for them. Such a patent would provide an additional layer of intellectual property protection. It may not be possible for all of the strains developed by Vectron to be patented, but a narrow patent should be possible for many. As with the license agreements that we establish, these patents would only cover the specific aspects of the strains related to the specific proteins being produced for the customer.
Each individual strain contains a specific expression vector and possibly genomic alterations that result in unique productivity in terms of how much protein is produced and in what quality. The patent claims would be for a specific sequence or strain (and subsequences) that produces protein X. Any engineering of the chromosome — down to the sequence level — would also be defined.
Such an approach would ensure that the particular combination of changes engineered into a specific strain to provide the expression levels and other attributes for the specific protein would be protected. Even though such a patent would be very narrow, it would cover our customers’ needs.
The potential for providing added value to customers through the patenting of any specific strains developed for them will be discussed at the outset of all collaborations. Typically, a patent for an E. coli strain would last 20 years. Value would be gained for both customers seeking to boost the titers of easier-to-produce proteins and those working with difficult-to-produce products, because patenting the bacterial strain provides additional patent protection for the production process and the direct product of that process. Adding a patent for the production strain could also be quite valuable for customers with products close to losing patent protection as a means for extending market exclusivity.
Our current mission at Vectron Biosolutions is to realize the full potential of our technologies and establish the company as the expert in E. coli protein expression. Within a few years, Vectron will have clients using the VB Evolution and VB Expression technologies to create expression vectors that produce large quantities of high-quality protein inside of E. coli cells. VB Secretion technology will also be leveraged for appropriate proteins.
While Vectron has thus far focused on E. coli, VB expression technology can be used to transform other bacteria as well. Given that the RK2 plasmid can exist in many different gram-negative species, we haven’t yet realized the commercial potential of this broad host range feature. Therefore, part of the future plan at Vectron is to explore the use of VB Expression and VB Evolution technologies in other bacteria and yeast. For any additional unique solutions developed for customers, we will also offer the added value of patent protection.
Dr. Aune is co-founder of Vectron Biosolutions and has led the company as its chief executive officer since its foundation. Under his leadership, Vectron has been branded as a leading provider of cutting-edge technologies and services for microbial production of proteins. Aune has negotiated license deals with customers, suppliers, and vendors, a Series A investment, and the acquisition of innovative technologies from academia and industry. Aune holds a Ph.D. in bacterial gene expression. More importantly, Aune is the proud co-founder of two beautiful daughters who are a continuous source of happiness to him and his wife despite the long runway, unclear exit strategies, and obvious lack of profitability.