Tailor-made expression hosts depleted in protease activity for recombinant protein production; PRODuCE (PROtease Depleted CEll line)

传统上，小分子药物(如抗生素)一直是人类治疗和治疗的主要手段。然而，在过去的10-20年里，基于蛋白质的药物(例如，经常用于治疗乳腺癌的赫赛汀、胰岛素)已经开发出来，现在这些药物已经成为药物市场中增长最快的部分。以蛋白质为基础的药物有几种，其中许多是从培养的哺乳动物或酵母细胞中工业化生产的。由于生产这种生物治疗药物需要很高的精确度，这种以蛋白质为基础的“重组”药物(生物制药)通常是由在特定条件下培养的细胞生产的。这样做的一个问题是，科学家用来制造治疗用蛋白质的细胞也会制造自己的蛋白质，包括蛋白酶，这些蛋白质的表达可能不利于治疗性蛋白质的生产。这些类型的蛋白质实际上可以降解目标重组蛋白，使它们在临床意义上毫无用处。因此，科学家可能无法生产足够的这些药物，和/或生产成本可能太高，从而使卫生保健提供者无法推荐使用这些药物。这项提案旨在解决支持从培养的哺乳动物和植物细胞中合成重组蛋白质的一个关键领域。我们的目标是全面了解用于生产生物药物的不同细胞系(动物和植物)中的有害蛋白分解活性。我们将使用最先进的方法和技术来定义那些对从这些表达系统中生产重组蛋白不利的蛋白酶，然后利用这一知识来减少或消除宿主细胞中的这些数量。这最终将导致产生的治疗性蛋白质较少或没有蛋白质水解性裂解和损伤。因此，本方案建议解决我们对蛋白酶在确定产品质量和产量方面的重要性的认识上的差距。总体目标是产生新的细胞和系统，利用细胞蛋白分解机制的操纵来提高工业规模重组治疗性蛋白的生产。这一信息与工业具有非常重要的相关性，因为这些有害反应潜在地阻碍了具有商业价值的治疗性蛋白的生产。如果没有改进的表达系统，生物技术/制药行业将无法大量生产这些有价值和有效的药物，以满足需求，价格将使它们能够为所有受益的患者开出处方。

Small molecule drugs (e.g. antibiotics) have traditionally been the mainstay of treatments and therapies in man. However in the last 10-20 years protein based drugs (e.g. Herceptin, often used to treat breast cancer, insulin) have been developed such that these now constitute the fastest growing section of the pharmaceutical market. There are several categories of protein-based drugs, many of which are produced from cultured mammalian or yeast cells at an industrial scale. Due to the high precision required to produce such biotherapeutics, such 'recombinant' protein-based drugs (biopharmaceuticals) are usually produced by cells kept in culture under defined conditions. One problem with this is that the cells scientists use to make proteins for therapeutic uses also make their own proteins including proteases whose expression can be detrimental to the production of therapeutic proteins. These types of proteins can actually degrade the target recombinant protein making them useless in a clinical sense. As a consequence, scientists may not be able to produce enough of these drugs and/or the cost of producing them may be too high, thus precluding health care providers from recommending their use. This proposal sets out to address a key area that underpins recombinant protein synthesis from cultured mammalian and plant cells. We aim to develop a comprehensive knowledge of the detrimental proteolytic activities in different cell lines (animals and plants) used for the production of biopharmaceuticals. We will use state-of-the-art approaches and technologies to define those proteases that are detrimental to recombinant protein production from these expression systems and then utilise this knowledge to reduce or eliminate the amount of these in the host cells. This will ultimately result in less or no proteolytic cleavage and damage of the therapeutic protein being produced. This programme therefore proposes to address the gap in our understanding as to the importance of proteases in defining product quality and yield. The overall aim is to generate new cells and systems that exploit manipulations of the cells proteolytic machinery to enhance the production of recombinant therapeutic proteins at the industrial scale. This information is of very substantial relevance to industry since the production of commercially valuable therapeutic proteins is potentially hindered by these detrimental reactions. Without improved expression systems, the biotechnology/pharmaceutical industries will lack the capability to produce large enough amounts of these valuable and effective drugs to meet the demand at a price that will allow them to be prescribed for all patients who would benefit from them.