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

揭示和改造微量金属对中国仓鼠卵巢细胞重组治疗性蛋白合成和异质性的作用

• 批准号: BB/K017640/1
• 负责人: Christopher Smales
• 金额: $ 65.91万
• 依托单位: University of Kent
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK017640%2F1
• 关键词: Unravelling; engineering; role; trace; metals

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.

小分子药物（例如抗生素）传统上是人类治疗和疗法的支柱，然而在过去的10-20年中，基于蛋白质的药物（例如赫赛汀，其通常用于治疗乳腺癌）已经发展到这样的程度，使得这些药物现在构成了医药市场的重要部分。有几类基于蛋白质的药物，其中之一，单克隆抗体，在使用或临床试验中构成一类中最大数量的蛋白质分子。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来折叠和组装成它们的最终活性状态，并且（B）在它们的表面上被糖和对其生物活性至关重要的其他分子修饰。由于生产生物治疗剂所需的高精度，用于治疗疾病的此类基于蛋白质的药物通常由在限定条件下保持在培养物中的细胞生产。其中一个问题是，我们用来制造蛋白质用于治疗用途的细胞并不像我们希望的那样有效，细胞会对它们生长环境的微小变化做出反应。这可能会影响最终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/或生产它们的成本太高。该提案旨在解决一个关键领域，该领域支持培养中哺乳动物细胞的重组蛋白合成产量，微量金属（例如镁、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。细胞生长的溶液中此类痕量金属的浓度可影响治疗性蛋白质药物质量（特别是这些如何影响药物物质的安全性和有效性以及用于生产药物的工艺的批次间差异/再现性）和异质性。在本项目期间，我们将利用申请人的协同专业知识，开发并提供与细胞过程相关的关键金属生物学的新理解，这些细胞过程最终决定了重组蛋白质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。这些研究将首次调查金属生物学在细胞外和细胞内的作用（总金属离子浓度和当金属与蛋白质结合时的游离/缓冲）在支持重组CHO细胞系的表型中的作用，并确定金属浓度，细胞通量，并且可以操纵金属转运蛋白以提供具有更好的过程控制的培养过程（例如，当筛选原料时监测哪些金属离子）。这将导致原料药生产更加一致，安全性、有效性提高，成本降低/供应链安全性提高，并且长期使用具有增强的工业表型的细胞系，例如生长增加和延长，rP异质性降低，糖基化谱改善。如果没有改进的过程控制和表达系统，生物技术/制药行业将缺乏以医疗保健提供者负担得起的价格生产足够数量的这些有价值且有效的药物来满足需求的能力。

项目成果

A comparative analysis of recombinant Fab and full-length antibody production in Chinese hamster ovary cells.

中国仓鼠卵巢细胞中重组 Fab 和全长抗体生产的比较分析。

• DOI: 10.1002/bit.27944
• 发表时间: 2021
• 期刊: Biotechnology and bioengineering
• 影响因子: 3.8
• 作者: Hussain H

Engineering of the cellular translational machinery and non-coding RNAs to enhance CHO cell growth, recombinant product yields and quality

• DOI: 10.1016/j.coche.2018.11.002
• 发表时间: 2018-12
• 期刊: Current Opinion in Chemical Engineering
• 影响因子: 6.6
• 作者: Davide Vito;C. Smales

Isolation and characterisation of exosomes from Chinese hamster ovary (CHO) cells.

• DOI: 10.1007/s10529-023-03353-3
• 发表时间: 2023-04
• 期刊: BIOTECHNOLOGY LETTERS
• 影响因子: 2.7
• 作者: Skrika-Alexopoulos, Eleftheria;Smales, C. Mark
• 通讯作者: Smales, C. Mark

The future of host cell protein (HCP) identification during process development and manufacturing linked to a risk-based management for their control.

• DOI: 10.1002/bit.25628
• 发表时间: 2015-09
• 期刊: Biotechnology and bioengineering
• 影响因子: 3.8
• 作者: Bracewell DG;Francis R;Smales CM
• 通讯作者: Smales CM

Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS.

• DOI: 10.1371/journal.pone.0172140
• 发表时间: 2017
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Saintas E;Abrahams L;Ahmad GT;Ajakaiye AM;AlHumaidi AS;Ashmore-Harris C;Clark I;Dura UK;Fixmer CN;Ike-Morris C;Mato Prado M;Mccullough D;Mishra S;Schöler KM;Timur H;Williamson MD;Alatsatianos M;Bahsoun B;Blackburn E;Hogwood CE;Lithgow PE;Rowe M;Yiangou L;Rothweiler F;Cinatl J Jr;Zehner R;Baines AJ;Garrett MD;Gourlay CW;Griffin DK;Gullick WJ;Hargreaves E;Howard MJ;Lloyd DR;Rossman JS;Smales CM;Tsaousis AD;von der Haar T;Wass MN;Michaelis M
• 通讯作者: Michaelis M