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

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description This project is on-going, and as such progress has been in line with the objectives expected at this stage of the project. The project aims to understand, manipulate and exploit trace metal biology underpinning mammalian cell growth and recombinant biotherapeutic protein production, quality and heterogeneity; particularly how metals impact upon safety and efficacy of the drug substance and batch-to-batch variation of the process used to manufacture it in Chinese hamster ovary (CHO) cells. To date we have demonstrated that over-expression of metal transporters is not tolerated in some cases in CHO cells and well in others. This results in a change to the growth characteristics of the cells that may have industrial relevance. We have also shown that different cell lines utilise trace metals in a way that relates to the growth and recombinant protein product amounts made and potentially the quality of these proteins that are ultimately destined for use in the clinic. We are now using this knowledge to change the amounts of trace metals in the media used to grow these cells to improve their growth and the amount of the protein drug they are making.
Exploitation Route As stated above, this project is on-going and approximately 75% completed. The knowledge generated so far is useful to both academia and industry in terms of defining the role of metal biology extra- and intra-cellularly (both total metal ion concentrations and free/buffered as described below) in underpinning the cellular 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 recombinant protein heterogeneity, improved glycosylation profiles.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description This project is on-going and has had an extension until the end of 2019. There has been limited opportunity to translate the findings into impact yet. However, industry has taken note of our findings to date and used these to help define trace metal requirements in their bioprocess systems for the manufacture of high value recombinant biotherapeutic proteins. We have also had interest from those working in the cancer biology field to investigate metal flux in cancer cells and how this relates to cancer cell line resistance and we are currently investigating potential collaborative projects in this area. We have also related metal flux in CHO cells to fragmentation of biotherapeutics and are developing a collaboration with industry in this area.
First Year Of Impact 2017
Sector Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description BIO)PHARMA IRELAND WHITE PAPER
Geographic Reach Europe 
Policy Influence Type Membership of a guideline committee
 
Description IBCatalyst
Amount £1,700,000 (GBP)
Funding ID BB/N023501/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2016 
End 06/2019
 
Description MC Training Network EU - Horizon 2020 MSCA
Amount € 819,863 (EUR)
Funding ID ITN 642663 
Organisation European Commission 
Department Horizon 2020
Sector Public
Country European Union (EU)
Start 03/2015 
End 03/2019
 
Description Wellcome Trust Collaborative Award
Amount £1,877,553 (GBP)
Funding ID UNS16981 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2016 
End 08/2021
 
Description Lonza Biologics Development of novel CHO cells and systems for expression of difficult to express proteins 
Organisation Lonza Group
Department Lonza Biologics
Country United States 
Sector Private 
PI Contribution Developed new technology for expression of difficult to express proteins
Collaborator Contribution Worked in collaboration to commercialise technology
Impact IP filed.
Start Year 2015
 
Description Metals in CHO media 
Organisation Dublin City University
Country Ireland 
Sector Academic/University 
PI Contribution Analysis of metals in cell culture media and spent medium.
Collaborator Contribution Provision of cell culture samples and media for analysis.
Impact Analysis of metal fluctuations in CHO cell medium.
Start Year 2016
 
Description 7 open days at University 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Describing the research we undertake in the laboratory, covering all aspects of the research and the impact this has/can have. Particular questions around genetic modification of cells to produce recombinant proteins in all sessions and discussions around both the ethical aspects of this and the potential applications of such technology.
Year(s) Of Engagement Activity 2015,2016
 
Description Chartham primary school science day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 150 primary school children got hands-on experience of how science integrates into everyday life. The teachers said the children found it really interesting.
Year(s) Of Engagement Activity 2018
 
Description Open Days At University 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This activity is take take parents and secondary students around my research laboratories to explain the research undertaken and to demonstrate some of the research that we do in the laboratory.

Parents and students asked about engineering of cell lines and therapeutic recombinant protein drugs and how these are made, cost implications and on-going research.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011
 
Description Open day tours of research laboratory and discussions with participants 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact 8 days of open day activities.
Year(s) Of Engagement Activity 2017
 
Description Presentation on Biosimilar protein based biotherapeutic drugs 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Health professionals
Results and Impact The talk stimulated discussion with the nurses and health care professionals present about what biosimilars are and when/how to assess if these are appropriate to administer as opposed to the innovator drug.

Nurses and healthcare professionals asked for further information on biosimilars.
Year(s) Of Engagement Activity 2014
 
Description Royal Society Summer Science Exhibition 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact The Summer Science Exhibition is an annual display of the most cutting-edge science and technology in the UK. This free, week-long festival features exhibits and a series of inspiring talks and activities for all ages. Meet the scientists, discover the exciting research and technology they work on and have fun with great hands-on activities.
Year(s) Of Engagement Activity 2018
URL https://royalsociety.org/science-events-and-lectures/2019/summer-science-exhibition/
 
Description School visit/outreach for several days at Simon Langton Boys Grammar - MBP2 project showing students how to clone, express and purify recombinant proteins in the laboratory and discuss science behind this. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Helping with research activity in school - cloning and expression of recombinant proteins
Year(s) Of Engagement Activity 2015,2016
 
Description Turkey Public Biotechnology Talk 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Presentation to public audience in Turkey on behalf of the British Council around biotechnology and its application. Event was filmed and followed by a question and answer session, answering questions sent in before the talk by social media and then from the audience. The event was filmed and shown on national TV in Turkey. Large range of topics discussed around the application of biotechnology to every day life and issues with long discussion/debate.
Year(s) Of Engagement Activity 2016
URL http://www.britishcouncil.org.tr/en/programmes/education/science-innovation-talks/biotechnology