Development of a Novel Method to Enhance Therapeutic Protein Production

开发增强治疗性蛋白质产量的新方法

• 批准号: 7481997
• 负责人: Tauseef R. Butt
• 金额: $ 28.35万
• 依托单位: LIFESENSORS, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7481997
• 关键词: Address; Affinity; Affinity Chromatography; Antibodies; Bacteria; Cell Line; Cells; Chimeric Proteins; Chinese Hamster; Chinese Hamster Ovary Cell; Cleaved cell; Clinical; Complex; Condition; Data; Development; Endopeptidases; Ensure; Eukaryotic Cell; Exercise; Figs - dietary; Genetic Transcription; Goals; In Vitro; Insecta; Laboratories; Mammalian Cell; Marketing; Mediating; Methods; Modeling; Modification; Numbers; Ovary; Peptide Hydrolases; Phase; Pichia; Play; Post-Translational Protein Processing; Procedures; Process; Production; Prokaryotic Cells; Protein Engineering; Protein Family; Proteins; Proteolysis; Public Health; Purpose; Rate; Recombinant Proteins; Role; Saccharomyces cerevisiae; Solubility; System; Technology; Testing; Therapeutic; Therapeutic antibodies; Time; Translations; antibody engineering; base; commercialization; cost; design; desire; improved; novel; numb protein; protein expression; protein folding; secretion process; therapeutic protein; vector

DESCRIPTION (provided by applicant): The first step in any protein therapeutic exercise is expression of the protein. In some cases, this step is not troublesome. For increasing numbers of designer proteins and requirements for post-translation modifications, however, enhanced expression and increasing protein yield have become very challenging tasks. Cost of goods is an important factor. In some cases therapeutic protein projects are abandoned because of an unreasonable cost of goods. It may come as a surprise that companies spend 12-18 months to optimize the cell lines and improve the yield of recombinant proteins. Several approaches have been taken to overcome the expression problems; nevertheless, it is highly likely that no single technology or procedure will be able to overcome the difficult-to-express protein problem. Selection of host, culture conditions and downstream processing can all play a role in the quality and quantity of product protein. LifeSensors has previously described a novel SUMO based fusion system. Attachment of SUMO to N-termini of inactive or poorly expressed proteins has led to a remarkable improvement in the functionality of proteins and 10-500-fold enhancement in protein production. Expression in prokaryotes yields an intact fusion that is purified by virtue of a variety of affinity tags at the N-terminus of SUMO. SUMO-fusions are cleaved in vitro by a highly efficient SUMO protease to generate native protein with desired N-termini. However, a similar system is not available for higher eukaryotic cells ,e.g., CHO that are primarily used for production of therapeutic antibodies and proteins. In this proposal we describe the development of related fusion system that will enhance the expression and secretion of difficult-to-express protein in CHO cells. With the advent of large numbers of engineered antibodies and therapeutic proteins as clinical candidates, protein expression has become a real bottleneck. Development of a system that improves quality and quantity of protein production by 10x fold will be a great advance in protein therapeutics. PUBLIC HEALTH RELEVANCE: Production of recombinant protein is a costly process. Increasing numbers of proteins are engineered to develop therapeutic products. It is becoming difficult to produce some of the proteins at a reasonable cost to the point that clinical projects are abandoned due to high cost of protein production. We have proposed the novel use of our previously demonstrated fusion proteins. Such a strategy would likely increase the quantity and quality of protein expression in therapeutics development.

描述（由申请人提供）：任何蛋白质治疗练习的第一步是蛋白质的表达。在某些情况下，这一步并不麻烦。然而，随着设计蛋白数量的增加和翻译后修饰的要求的增加，增强表达和增加蛋白产量已成为非常具有挑战性的任务。商品成本是一个重要因素。在某些情况下，治疗性蛋白质项目因商品成本不合理而被放弃。令人惊讶的是，公司花费 12-18 个月来优化细胞系并提高重组蛋白的产量。已经采取了多种方法来克服表达问题；然而，很可能没有任何一种技术或程序能够克服难以表达的蛋白质问题。宿主、培养条件和下游加工的选择都会对产物蛋白质的质量和数量产生影响。 LifeSensors 之前描述了一种新颖的基于 SUMO 的融合系统。将 SUMO 连接到无活性或表达不良的蛋白质的 N 末端可显着改善蛋白质的功能，并使蛋白质产量提高 10-500 倍。原核生物中的表达产生完整的融合体，该融合体通过 SUMO N 末端的各种亲和标签进行纯化。 SUMO 融合体在体外被高效 SUMO 蛋白酶切割，生成具有所需 N 末端的天然蛋白质。然而，类似的系统不适用于高等真核细胞，例如主要用于生产治疗性抗体和蛋白质的 CHO。在本提案中，我们描述了相关融合系统的开发，该系统将增强 CHO 细胞中难以表达的蛋白质的表达和分泌。随着大量工程化抗体和治疗性蛋白质作为临床候选物的出现，蛋白质表达已成为真正的瓶颈。开发一种将蛋白质生产的质量和数量提高 10 倍的系统将是蛋白质治疗领域的一大进步。 公共卫生相关性：重组蛋白的生产是一个成本高昂的过程。越来越多的蛋白质被设计用于开发治疗产品。以合理的成本生产某些蛋白质变得越来越困难，以至于临床项目由于蛋白质生产成本高昂而被放弃。我们提出了我们之前展示的融合蛋白的新用途。这种策略可能会提高治疗药物开发中蛋白质表达的数量和质量。