SBIR Phase I: Novel Continuous Flow Manufacturing of Recombinant Proteins Secreted Through the Bacterial Type-III Secretion System

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) project is to streamline and reduce the cost of manufacturing biopharmaceuticals by developing a heterologous protein expression system for large scale batch and continuous flow fermentation systems. Currently, many large molecule therapeutics, such as insulin, are made using bacterial host organisms. These cells are genetically engineered to produce large quantities of a desired product, known as a recombinant protein, in the cellular compartments of these single-celled organisms. However, due to the small size of bacteria and its unfavorable physiology, these products often end up interfering with production and require additional costs and effort to then extract, purify and activate the product. The T3S system is the most efficient known protein secretion system in bacteria, and could enable rapid export of recombinant proteins, allowing for these products to be produced in a favorable environment and negate the need for extraction, exhaustive purification, and costly reactivation of the product. This SBIR Phase I project proposes to develop the a protein expression system based on the bacterial Type-III Secretion (T3S) system for use in scalable fermenters to maximize the yield output. The goal is to test different media types as well as different growth and expression conditions, and then quantify the amount of secreted protein through various techniques. Additionally, this Phase I project will develop for the first time a continuous flow manufacturing process with a bacterial production system for greater efficiency, and further reduce the cost and effort associated with recombinant protein production. Due to the T3S systems' rapid and direct secretion of products directly into the supernatant a new filtration system to continually harvest the spent media for product will be employed. In addition, the purity of the secreted products directly from the spent media will be characterized to determine what additional purification steps are required. Finally, the T3S system unfolds proteins as they are secreted into the oxidative media lead by their N-terminus, in the same manner that they are translated by the ribosome. This maximizes the possibility for proteins to form into their native and active confirmation, which will be verified using LC-MS techniques.