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

SBIR 第一阶段：通过细菌 III 型分泌系统分泌的重组蛋白的新型连续流程制造

• 批准号: 1622018
• 负责人: Daniel Wee
• 金额: $ 22.5万
• 依托单位: T3S Technologies, Inc.
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1622018&HistoricalAwards=false
• 关键词: SBIR; Phase; Novel; Continuous; Flow

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.

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分批和连续流发酵系统的异源蛋白表达系统来简化和降低生物制药的生产成本。目前，许多大分子治疗剂，如胰岛素，是使用细菌宿主生物体制成的。这些细胞经过基因工程改造，在这些单细胞生物的细胞区室中产生大量所需的产物，称为重组蛋白。然而，由于细菌的小尺寸及其不利的生理学，这些产品通常最终干扰生产，并且需要额外的成本和努力来提取，纯化和活化产品。T3 S系统是细菌中已知的最有效的蛋白质分泌系统，并且能够快速输出重组蛋白质，允许这些产物在有利的环境中产生，并且不需要提取、彻底纯化，该SBIR第一阶段项目建议开发基于细菌III型分泌（T3 S）的蛋白表达系统。该系统用于可扩展的发酵罐中，以最大化产量输出。目的是测试不同的培养基类型以及不同的生长和表达条件，然后通过各种技术定量分泌蛋白的量。此外，该I期项目将首次开发具有细菌生产系统的连续流生产工艺，以提高效率，并进一步降低与重组蛋白生产相关的成本和工作量。由于T3 S系统将产物快速直接分泌到上清液中，因此将采用新的过滤系统来连续收获产物用过的培养基。此外，将表征直接来自废培养基的分泌产物的纯度，以确定需要哪些额外的纯化步骤。最后，T3 S系统解折叠蛋白质，因为它们以与核糖体翻译相同的方式通过其N-末端分泌到氧化介质中。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。