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

这个小企业创新研究（SBIR）项目的更广泛的影响/商业潜力是通过开发用于大规模批量和连续流发酵系统的异源蛋白表达系统来简化和降低生产生物制药的成本。目前，许多大分子药物，如胰岛素，都是用细菌宿主生物制成的。这些细胞经过基因工程改造，在这些单细胞生物的细胞隔室中产生大量所需的产物，即重组蛋白。然而，由于细菌体积小及其不利的生理特性，这些产品往往最终会干扰生产，并且需要额外的成本和精力来提取、纯化和激活产品。T3S系统是已知细菌中最有效的蛋白质分泌系统，可以快速输出重组蛋白，允许这些产品在有利的环境中生产，并且不需要提取，彻底的纯化和昂贵的产品再激活。本SBIR一期项目拟开发一种基于细菌iii型分泌（T3S）系统的蛋白表达系统，用于可扩展发酵罐，以最大限度地提高产量。我们的目标是测试不同的培养基类型以及不同的生长和表达条件，然后通过各种技术量化分泌蛋白的数量。此外，该一期项目将首次开发具有细菌生产系统的连续流程制造工艺，以提高效率，并进一步降低重组蛋白生产相关的成本和工作量。由于T3S系统能够快速直接地将产物直接分泌到上清液中，因此将采用一种新的过滤系统来连续地收集用于生产产品的废培养基。此外，将对直接从废培养基中提取的产物的纯度进行表征，以确定需要哪些额外的纯化步骤。最后，当蛋白质分泌到由其n端引导的氧化介质中时，T3S系统展开蛋白质，与它们被核糖体翻译的方式相同。这最大限度地提高了蛋白质形成其天然和活性确认的可能性，这将使用LC-MS技术进行验证。

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.