Unravelling and engineering the role of trace metals on recombinant therapeutic protein synthesis and heterogeneity from Chinese hamster ovary cells

小分子药物(例如抗生素)传统上一直是人类治疗和治疗的主要药物，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)已经发展到了这样的程度，现在这些药物已经成为药物市场的重要组成部分。有几种基于蛋白质的药物，其中一种是单抗，它构成了使用中或临床试验中一类蛋白质分子中数量最多的。许多基于蛋白质的药物的生产具有挑战性，因为它们(A)需要特定的辅助蛋白质折叠并组装成其最终的活性状态，(B)其表面装饰有糖和其他对其生物活性至关重要的分子。由于生产生物治疗药物所需的精密度很高，这种基于蛋白质的治疗疾病的药物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，我们用来制造治疗用蛋白质的细胞并不像我们希望的那样有效，细胞对生长环境的微小变化做出反应。这可能会影响最终药物--物质药物或蛋白质药物--的一致性和质量。因此，我们可能无法生产足够的这些药物，和/或生产成本太高。这项建议旨在解决支持哺乳动物细胞在培养中合成重组蛋白的一个关键领域，即微量金属(如镁、锰、铁、锌、铜、镍、钴)在哺乳动物细胞生长和治疗性重组蛋白(RP)生产中的作用及其影响。这些微量金属在细胞生长溶液中的浓度可能会影响治疗蛋白药物的质量(特别是这些对药物物质的安全性和有效性以及生产过程的批次到批次的变化/重现性的影响)和异质性。在这个项目中，我们将以申请者的协同专业知识为基础，发展和提供与细胞过程相关的关键金属生物学的新理解，这些过程最终决定了重组蛋白的异质性和中国仓鼠卵巢(CHO)细胞的产量。CHO细胞是目前工业上用于生产治疗性重组蛋白的金标准哺乳动物细胞系。这些研究将首次探讨细胞外和细胞内金属生物学(总金属离子浓度和金属与蛋白质结合时的自由/缓冲)在支持重组CHO细胞系表型方面的作用，并确定如何操纵金属浓度、细胞通量和金属转运体，为培养过程提供更好的过程控制(例如，在筛选原料时要监测哪些金属离子)。这将导致更一致的药物生产，提高安全性和有效性，降低供应链的成本/安全性，并长期与具有增强的工业表型的细胞系合作，例如增加和延长生长，减少RP异质性，改善糖基化情况。如果没有改进的过程控制和表达系统，生物技术/制药行业将没有能力生产足够数量的这些有价值和有效的药物，以满足卫生保健提供者负担得起的价格的需求。

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, which is often used to treat breast cancer) have developed to such a point that these now constitute a significant section of the pharmaceutical market. There are several categories of protein based drugs, one of which, monoclonal antibodies, constitutes the largest number of protein molecules in a class either in use or in clinical trials. Many protein based drugs are challenging to produce because they (a) require particular helper proteins to fold and assemble into their final active state and (b) are decorated on their surfaces by sugars and other molecules that are essential to their bioactivity. Due to the high precision required to produce biotherapeutics, such protein based drugs for the treatment of diseases are usually produced by cells kept in culture under defined conditions. One problem with this is that the cells we use to make proteins for therapeutic uses are not as efficient as we would like them to be and the cells respond to small changes in the environment in which they are grown. This can affect the consistency and quality of the final drug-substance or protein drug. As a consequence, we may not be able to produce enough of these drugs and/or the cost of producing them is too high. This proposal sets out to address a key area that underpins recombinant protein synthesis yields from mammalian cells in culture, the role of trace metals (e.g. magnesium, manganese, iron, zinc, copper, nickel, colbalt) in, and their influence upon, mammalian cell growth and therapeutic recombinant protein (rP) production. The concentrations of such trace metals in the solution in which cells are grown can impact upon the therapeutic protein drug quality (particularly how these impact upon safety and efficacy of the drug substance and batch-to-batch variation/reproducibility of the process used to manufacture it) and heterogeneity. During this project we will build upon the synergistic expertise of the applicants to develop and deliver new understanding of key metal biology related to the cellular processes that ultimately determine recombinant protein heterogeneity and yield from Chinese hamster ovary (CHO) cells. CHO cells are the current gold standard mammalian cell line used in industry to produce therapeutic recombinant proteins. The studies will, for the first time, investigate the role of metal biology extra- and intra-cellularly (both total metal ion concentrations and free/buffered when the metal is bound to proteins) in underpinning the phenotype of recombinant CHO cell lines and determine how metal concentrations, cellular flux, and metal transporters may be manipulated to provide culture processes with better process control (e.g. which metal ions to monitor when screening raw materials). This will lead to more consistent drug substance production, improved safety, efficacy and reduced costs/improved security of the supply chain and longer term with cell lines with enhanced industrial phenotypes e.g. increased and prolonged growth, reduced rP heterogeneity, improved glycosylation profiles. Without improved process control and 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 is affordable for health care providers.