March 15, 2023 PAO-03-23-CL-03
Despite the cost-effectiveness of producing many biologic molecules, such as plasmids and proteins, in Escherichia coli bacteria, there is still a large demand in the industry for improved and innovative E. coli strains, including strains that can secrete proteins out of the cell. When proteins are manufactured using conventional methods in E. coli, they are retained inside the bacterial cells. When the cells are ruptured to release the desired protein, thousands of other proteins and biomolecules are also released, greatly contaminating the product and resulting in the need for extensive and costly downstream processing.
Proteins secreted from E. coli can be more simply purified, reducing downstream costs, which are significant for many industrial proteins, most notably therapeutic products. In addition, secretion of proteins foreign to the cell would prevent their buildup at high concentrations within the cell, thereby reducing the potential for aggregation and degradation. In fact, many proteins produced in E. coli end up as inclusion bodies — precipitated aggregated proteins that must be broken apart with subsequent refolding of each protein into its proper structure. In some cases, the refolding efficiency can be as low as 10%, significantly reducing the protein yield. Secretion overcomes these issues, resulting in higher titers.
Efficient secretion of proteins produced in E. coli will dramatically increase the opportunities for application of bacterial fermentation to commercial protein production, both for new and emerging protein products and for many proteins currently manufactured using fungi and yeast. Secretion also significantly reduces levels of host DNA compared with conventional separation methods, which is a priority for other industries requiring recombinant proteins, including the food, feed, and nutrition industries. Indeed, introduction of a technology that reliably and robustly facilitates controlled protein secretion during industrial E. coli fermentation processes should elicit new excitement and interest in this attractive approach to bioprocessing.
Professor Kelly Hughes from the University of Utah in Salt Lake City developed a controlled secretion technology for Salmonella that is based on truncated versions of the bacterial flagellum, an organelle that helps in locomotion. The researchers engineered the bacterial flagella to act as channels linking the insides of cells to the outside, allowing for recombinant proteins produced by the cell to exit into the surrounding media in a controlled manner.
Salmonella is not an ideal host for industrial protein production, however. T3S Technologies, the company founded based on Kelly Hughes’ research, recognized the need to find a partner with strong expertise in E. coli genetics in order to move the technology into this organism and thus make it more applicable for commercial protein manufacturing. Adapting the technology from Salmonella to E. coli is not a simple process and requires in-depth knowledge and expertise in E. coli engineering and gene expression.
In their search for the ideal partner with the capabilities and E. coli engineering expertise needed to adapt the secretion technology — as well as complementary platforms that would synergize optimally with the technology — T3S Technologies selected Vectron Biosolutions. At Vectron, we clearly recognized the strategic fit and the commercial opportunity of uniting these technologies, and we completed the acquisition of T3S Technologies’ assets in the summer of 2021, taking another important step on our journey to a new industry standard for protein production.
There have been attempts in the industry to develop E. coli secretion technology, but the technologies involved often lead to uncontrolled leakage of materials from the bacterial cells or low secreted titers. Our VB Secretion technology is different; it enables the controlled secretion of proteins into the extracellular medium via transport by the truncated flagellum. Because only the protein of interest is secreted into the media, downstream processing is accelerated and simplified compared with existing scenarios.
The engineering is complex and involves extensive chromosomal modifications because many of the E. coli strains used in the industry today do not naturally synthesize the flagellum key to VB Secretion technology. In addition, while Salmonella and E. coli are related organisms, there are sufficient differences between them. Some proprietary solutions had to be developed.
Engineering the new strains with the appropriate flagella was the first milestone. Establishing a means for ensuring that only the desired protein is secreted was another. The third was to demonstrate proof of concept for the secretion technology, with proteins observed in the extracellular medium. The fourth big milestone was secretion of industrially valuable proteins.
For proteins that are secreted effectively, titers are expected to be comparable to those obtained when producing them inside the cell, but with less downstream processing required. Different proteins, however, are secreted at different rates. Current development work is focused on understanding which proteins will be more efficiently secreted and why. This will lead to strategies for improving the system to allow efficient secretion of additional proteins, taking us further on our journey toward a new industry standard for protein production.
Early studies at Vectron Biosolutions have provided evidence for secretion of different types of proteins in E. coli engineered with the VB Secretion technology. These proteins were chosen because of very different properties, including different structures, sizes, folding patterns, and/or commercial applications, and comprise proteins that are used in different industries.
At this stage in the development program, the evidence shows encouraging yields of > 10 mg/L from non-optimized expression strains used in cultivations at the microtiter scale. This would typically correspond to > 100 mg/L from a fed batch process. After a simple purification step that involves separating the medium from the biomass, protein purities of well over 90% are expected.
Different secretory E. coli strains engineered for secretion modified with different expression vectors were used for these studies. Secretion into the surrounding medium was confirmed via SDS-PAGE and Western blot analysis. Figure 1 shows an SDS-PAGE gel of the extracellular fraction from two generations of VB secretion strains that secrete an undisclosed protein. The figure confirms secretion specificity and increased secretion yields from later secretion strain generations.
Two note-worthy examples of proteins successfully secreted in E. coli using VB Secretion are the enzyme beta-lactamase and the therapeutic human growth hormone (sold as somatotropin and under many other biosimilar names). Both of these proteins are well known, understood, and characterized in the industry, and have substantial market values.
With these results in hand, Vectron Biosolutions has made tremendous forward progress toward our journey’s goal of bringing to the biopharma industry a new standard for protein production.
The VB Secretion technology introduces a new paradigm to E. coli applications that, when combined with other state-of-the-art technologies developed by Vectron Biosolutions, will afford the most advanced and best-in-class system in the market.
The VB Expression system allows control of the level of protein expression in E. coli, which is essential for achieving maximum yields of high-quality protein. Fine-tuning the expression rate so that it is balanced with the protein folding rate enables correct protein folding. In addition, this platform contains libraries of signal peptides for efficient translocation to the periplasm and libraries of chaperones for facilitated protein folding.
VB Evolution is an evolutionary approach to identify and create the theoretically best expression vector for each and every protein.
Combining the VB Expression or VB Evolution technologies, which enable very high titer processes, with the VB Secretion technology allows production of bioactive proteins of the right quality and quantity in the extracellular medium — a very powerful solution that simultaneously optimizes and streamlines both upstream and downstream processes. The intent is to develop tailored expression vectors for use in secretory strains, allowing not only maximized titers but also protein secretion for vastly reduced downstream costs. With these advanced capabilities, this platform is expected to become the industry standard for E. coli protein production.
The VB Secretion technology is not yet marketed. Further studies and optimization are underway. In addition to gaining greater insight into which proteins can be successfully secreted and why, our current focus is on increasing yields, demonstrating secretion of other industrially valuable proteins, and establishing a method to remove the protein tags that direct the protein for secretion.
Vectron Biosolutions is partnering with selected companies in the industry that are interested in working with us on further development of the VB Secretion technology and evaluation of its potential. These partnerships serve as a vital feedback loop, providing insights that lead to the development of protein production solutions with optimal market fit. Current customer-based projects are already helping Vectron gain additional extensive experience and data beyond the knowledge and information generated through internal programs, helping us to not only optimize the efficiency and yield of our platforms themselves but to maximize their use in a range of applications to meet the needs of both existing and future customers. We remain open to working with different kinds of companies across industries and evaluate partners on the basis of the fit of our technologies with the right high-value applications rather than the intended market, as well as ensuring the right organizational and collaborative fit.
With the addition of the VB Secretion technology to our existing microbial development and production toolbox — underpinned by our expertise in E. coli genetics and strain engineering — Vectron Biosolutions is well positioned to help realize the promise of E. coli 2.0 and to maximize the opportunity for commercial production of proteins via bacterial fermentation. We have demonstrated initial proof of concept of our VB Secretion and are actively looking for the right collaborative partners seeking to evaluate how VB Secretion can support their objectives while we push the boundaries of what the technology can deliver and optimize the platform for different protein types, sizes, and applications. We are enthusiastic about our part in the journey toward game-changing E. coli production solutions that offer high titers, high purity and quality, and simple, cost-effective downstream purification for industrially relevant and valuable proteins.
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.