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

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.

This application details a novel approach towards understanding and exploiting trace metal biology in underpinning mammalian cell growth and therapeutic recombinant protein (rP) production, quality and heterogeneity in the industrially important Chinese hamster ovary (CHO) cell line. The proposal balances the long-term goals of cell engineering to exploit metal homeostasis with the more immediate deliverables of developing a better process understanding with regard to trace metals that will lead to improved process control and impact upon the consistency and safety of the drug substance. The programme of work will test the hypothesis that 'extra- and intra-cellular metals, including those introduced as impurities in raw materials, and the maintenance of their homeostasis during the culturing of CHO cells engineered to produce rPs is critical in determining cell growth, product yield and product heterogeneity and these can be manipulated directly in the media or indirectly intra-cellularly by engineering approaches to improve cell growth and reduce product heterogeneity'. In addressing this hypothesis we will characterise the variation in total metal ion concentrations in key raw materials and the extra- to intra-cellular flux of trace metal ions, determine the effect of extra- and intra-cellular metal ion concentrations on rP product quality, investigate intracellular metal compartmentalisation and how this relates to cell growth and product quality, determine the effect of manipulating cell culture metal concentrations on the CHO cell proteome, and develop a 'ranking' of total metal ion concentration impurities and impact so that target screening of raw materials can be used to assess risk. The outcomes will be (i) novel process control and engineered CHO cell lines that exploit metal biology underpinning cell growth and rP production, quality and heterogeneity, and (ii) an understanding of how metals impact upon safety and efficacy of the drug substance.

Who will benefit?
In terms of research findings, the primary beneficiaries will be researchers in the academic and biopharmaceutical sectors who are interested in understanding the role of trace metals in Chinese hamster ovary (CHO) cell culture media and how these ultimately influence cell growth, recombinant protein synthesis, batch-to-batch variation of product quality and product efficacy. As such, this proposal is relevant to all those academics and industrialists who are interested in the process and/or manufacturing of proteins and wish to deliver them at increased yield in a functionally active form at less cost. The impacts of this research will therefore be national and international and will benefit the following:
(1) those in the research fields of metal biology, metal homeostasis, mammalian cell culture, metallo-proteins, cell biology, provision of raw materials to the bioprocessing industry, and recombinant protein synthesis;
(2) the academic and industrial bioprocessing and scientific communities;
(3) ultimately the National Health Service (and thus the wider public, its patients) and the UK economy through the development of new methods to produce larger amounts of increasingly important 'bio-drugs' more efficiently and at lower cost.

How will they benefit?
The major impact of this work will be to provide both industry and academia with a much better understanding of the role trace metals play in underpinning mammalian cell growth and therapeutic recombinant protein production, product quality (particularly how trace metals impact upon safety and efficacy of the drug substance and batch-to-batch variation/reproducibility of the process used to manufacture it) and heterogeneity in the industrially important CHO cell line. Metal biologists will also benefit via the generation of new knowledge with regard to buffered concentrations of specific trace metals and how these are controlled during culture of mammalian cells. The potential ability to produce high cost recombinant protein drugs at lower cost will ultimately allow access to these drugs to a wider sector of the population both nationally and internationally, thus contributing to health and quality of life. In order to ensure that these delivered, our results will be published in peer-reviewed high-quality journals and presented at relevant conferences. We will publicise our findings through our own websites, press releases, BBSRC Business and via the local media and our own public engagement activities (e.g. science fairs and outreach with local schools). As the PIs are well-placed to inform the activities of industry and to exploit their own discoveries commercially (Smales, Warren and Robinson have well-established links with industry) we will build on these industrial links to translate our findings into applications in the recombinant protein production field and inform industry of our results. The PIs, together with Kent Innovation and Enterprise, Durham Business and Innovation Services and Lonza Biologics will take the lead in ensuring this is completed in a timely fashion such that the IP is protected and assigned to the correct individuals and institutions. Kent Innovation and Enterprise will have the task of initiating dialogue with additional potential collaborators and parties. Regular teleconferences and meetings between sites will ensure close coordination between the activities at Kent, Durham and Lonza, such that findings in one lab are rapidly conveyed to the other to inform and develop the project. The programme provides opportunities for staff training through (i) the range of approaches and techniques to be used; (ii) the close interactions with members of the applicant's laboratories working on projects in similar areas, (iii) interactions with the bioprocessing and pharma industries, and (iv) the opportunity to undertake public engagement work.