Modelling cellular processes underpinning recombinant monoclonal antibody production by mammalian cells

Lead Research Organisation: University of Sheffield
Department Name: Chemical & Biological Engineering

Abstract

This proposal is concerned with 'bioprocessing'. Bioprocessing collectively describes the range of manufacturing processes that enable the production of new biological medicines. You may be familiar with the one of the first biological medicines produced by recombinant DNA technology - a small protein called insulin. Insulin is now used very successfully to treat an increasingly common metabolic disease, diabetes. Before insulin, diabetics suffered a short life fraught with serious medical complications. This project is targeted at the production of other high-value therapeutic proteins by genetically engineered mammalian cells in culture, specifically monoclonal antibodies. In the body, natural antibodies present in the blood play an important role in our immune system: They target disease-causing microbes and foreign substances for removal. Recombinant monoclonal antibodies, being almost identical to natural antibodies, are specifically designed to target diseased cells. Unlike traditional small-molecule medicines such as penicillin and paracetamol, monoclonal antibody biopharmaceuticals are large, complex and relatively fragile proteins which have to be produced by living mammalian cells in culture, genetically engineered to produce the recombinant protein product. They are proving to be highly successful treatments for serious diseases such as rheumatoid arthritis and a range of cancers. It is anticipated that within the next five to ten years up to fifty percent of all drugs in development will be biopharmaceuticals; a very substantial proportion recombinant proteins produced by mammalian cells in culture. Since the first recombinant protein medicines produced by genetically engineered mammalian cells in culture were licensed as therapeutics over 25 years ago, we have learnt to substantially increase the productivity of biopharmaceutical manufacturing processes (bioprocesses). However, they are still complicated and expensive, and industry has to undertake time-consuming screening processes to find engineered cells making adequate amounts of recombinant protein. To date, the output of industrial bioprocesses has predominantly been increased by gradually improving the growth of producer cells in culture, and not by engineering each cell to make the product more efficiently. This is important, because if we knew how to instruct or programme the cell factory appropriately, we could substantially improve the productivity of manufacturing processes and decrease the time it takes to generate a productive cell culture. However this is not a simple problem. The cell utilises and coordinates a diverse range of its complex machinery to turn, for example, recombinant monoclonal antibody genes in its nucleus into a fully folded protein which can be secreted out of the cell. How can we understand this cellular 'production line' well enough so that we can rationally implement strategies to improve flux from recombinant genes to protein product? In this project we will implement a novel, multidisciplinary combination of technical approaches to answer this question; mathematical modelling, gene expression, molecular cell biology, protein analysis and cell culture. We believe this is crucial - an integrated mathematical bioscience approach can massively increase the information content and utility of biological measurements and enable us to understand cellular processes from a systems control perspective. This project will, for the first time, provide a quantitative understanding of the cell factory on which to rationally build strategies to increase the productivity of therapeutic monoclonal antibody production systems. Without this knowledge, cell culture engineering will largely remain based on trial and error.

Technical Summary

Recombinant monoclonal antibodies (Mab's) are now the second largest category of biopharmaceutical products in development and are predominantly manufactured by mammalian cells in culture. Cell engineering strategies to increase cell specific Mab production have proved intractable largely because we still do not systematically understand how the host cell coordinates and regulates the diverse variety of cellular processes that contribute to flux from recombinant gene to secreted protein during production processes. This proposal describes a pre-competitive, interdisciplinary research project that combines advanced gene expression technology, molecular cell biology and mathematical modelling to quantitatively describe the process of recombinant Mab production by mammalian cells. In collaboration with our industrial partner, Lonza Biologics, we will create the first empirically derived kinetic and metabolic control analysis of recombinant Mab production. Model construction will utilise dynamic biochemical measurements of Mab mRNA and polypeptide intermediate pools in industrially relevant cell culture formats that employ both stable and controllable gene expression systems. In general, we hypothesise that cellular flux control coefficients will vary through production processes in a broadly predicable manner, from a model where control of flux is initially limited by recombinant gene expression to one where flux is limited primarily by folding and assembly of the product or supply of metabolic precursors. The extent to which control of flux is shared by discrete cellular processes, and the effect of gene expression and production variables will be determined. A main outcome of this research will be an understanding of cellular control of flux through different levels of cellular organisation enabling rational design of engineering strategies to increase the efficiency of recombinant gene utilisation by engineered mammalian cells in a production environment.
 
Description We demonstrated that:

1. Genetic variation between mammalian cell factories used to manufacture complex protein biopharmaceuticals underpins the ability of some cells to function effectively as recombinant protein factories. This can be harnessed to generate second generation "fit-for-purpose" cell factories
2. It is possible to computationally predict, for a given cell factory, which engineering strategies will significantly improve their productivity. This avoids lengthy, labour-intensive, time-consuming trial and error approaches to increase production rate.
3. Discrete cellular mechanisms permit maintenance of recombinant protein production rate through an entire production process even as the host mammalian cell's own capacity for cellular biomass production declines markedly.
Exploitation Route All main objectives were met. Specific outcomes that were taken forward include:

A suite of quantitatively validated analytical methods. These were used to determine intra- and extracellular Mab synthetic intermediate levels (both recombinant mRNAs and polypeptides).

A metabolic control analysis of Mab production. We constructed a kinetic model of rMab synthesis in-silico, and constrained by detailed empirical measurements of cellular intermediates we used this to describe the varying control different intracellular synthetic processes impart over rMab production. We showed how control varies significantly between different CHO cell lines producing the same Mab. This work illustrated, for the first time, that cell engineering to increase qP should be cell line specific.

Applied metabolic control analysis to production system models to determined rational strategies for cell/process engineering. Using our established empirical modelling platform we constructed a whole-process model of Mab production. This permitted quantitative comparison of discrete cell engineering targets via the calculation of a process control coefficient for each synthetic step in silico.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Description Dissemination to bioindustrial community via oral presentations 1. Informa European Cell Line Development and Engineering, Prague, 2008 (Featured Speaker) 2. Informa US Cell Line Development and Engineering, San Diego, 2008 (Keynote Speaker) 3. Japanese Association of Animal Cell Culture Symposium, Fukuoka, 2008. 4. University of Osaka, Osaka, 2008. 5. 12th Annual Cambridge Healthcare Institute Protein Expression Conference, San Diego, 2009 6. UCB Celltech, Slough 2009. 7. Informa US Cell Line Development and Engineering, San Diego, 2009 (Featured Speaker) 8. Bioprocess Technology Education Center at North Carolina State University, Raleigh/Durham, 2009 9. Biogen Idec, Boston, 2010 10. Pfizer Biopharmaceutical Development, St. Louis, 2010 11. Life Technologies, Carlsbad, 2010 12. Informa US Cell Line Development and Engineering, San Francisco, 2010 13. Bioproduction Conference, Barcelona, 2010 (Keynote speaker) 14. Informa European Cell Line Development and Engineering, Munich, 2011 (Keynote Speaker) 15. 21st Annual ESACT-UK meeting, Loughborough University, 12th Jan 2011. "BRIC Bridging Excellence" session. MedImmune, Cambridge (UK), Sept 2010. 16. Cell Culture Engineering XII, Banff, 2010 (one of only two invited speakers from the UK) Contribution to, and stimulation of further industrial funding for collaborative R&D in DCJs laboratory 1. £300,000 from Pfizer Inc (St. Louis, USA). Identification of Molecular Markers of Phenotypic Stability in Production Mammalian Cell Lines. 2008-2011. 2. £74,000 from Lonza Biologics. Contribution towards research funding for postdoctoral research. Design and analysis of mammalian expression vectors. 2007-2010. 3. £160,000 from the BBSRC (Bioprocess Research for Industry Club) plus a consortium of four biotech companies (£103K BBSRC/60K direct industry). Sequencing the CHO cell genome. 2010-2011. 4. £75,560 from Yorkshire Forward and EPSRC KTA. Commercial development of mammalian gene expression technology. 2010-2011. 5. £200,000 from Biogen Idec (Boston, USA). Fully funded studentship. Harnessing genetic/functional heterogeneity in CHO cell populations. 2010-2013. 6. £217,000 from the BBSRC. Targeted Priority Scheme Doctoral Training Grant. An Interdisciplinary Doctoral Training Centre in Bioprocessing for UK Bioindustry. 2008-2012. 7. £48,000 from MedImmune. Support for Industrial CASE studentship. Modelling monoclonal antibody expression by mammalian cells. 2009-2012. 8. £128,000 from Wyeth (Andover, MA, USA). Mechanisms of mammalian cell adaptation to a synthetic environment. 2008-2012. 9. £87,864 from the EPSRC and MedImmune (Cambridge, UK). Industrial CASE studentship: Multiparameter modelling of transient protein production by mammalian cells. 2007-2010. 10. £90,000 from the BBSRC and Lonza Biologics (Slough, UK). Industrial CASE studentship: Controlling the glycosylation of recombinant protein biopharmaceuticals: analysis and exploitation of genetic heterogeneity in cellular glycan processing. 2007-2011. 11. £31,500 from Lonza Biologics. Support for EPSRC DTA studentship: Analysis of genetic heterogeneity in mammalian cell populations. 2007-2010. 12. £75,000 from Pfizer Inc (St. Louis, USA). Design of mammalian expression systems for increased translational efficiency. 2007-2010. 13. £83,000 from the EPSRC and Asterion Ltd (Sheffield, UK). New mammalian cell expression systems utilising GPI anchors. 2007-2010. 14. £110,000 from the BBSRC and Pall Europe Ltd. (Portsmouth, UK). Development of a platform for transient production of recombinant proteins using disposable processing technology. 2008-2012.
First Year Of Impact 2007
Sector Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic
 
Description BBSRC CTP BB/P011608/1
Amount
Funding ID BB/P011608/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2016 
End 10/2020
 
Description Strategic partnership with Biogen 
Organisation Biogen Idec
Country United States of America 
Sector Private 
PI Contribution CHO cell engineering technology
Collaborator Contribution Project management, research materials, datasets
Impact Johari Y, Estes S, Alves C, James DC. (2015) Integrated cell and process engineering strategies for improved production of a difficult-to-express fusion protein by CHO cells. Biotechnology and Bioengineering. In press.
Start Year 2010
 
Description Strategic partnership with Lonza Biologics 
Organisation Lonza Group
Department Lonza Biologics
Country United States of America 
Sector Private 
PI Contribution Genetic vector and cell engineering technology development
Collaborator Contribution Project management, laboratory facilities, research materials.
Impact Grainger RG, James DC (2013). Cell line specific control and prediction of recombinant monoclonal antibody glycosylation. Biotechnology and Bioengineering. 110: 2970-2983. Davies SL, Lovelady CS, Grainger RK, Racher AJ, Young RJ, James DC. (2013) Functional heterogeneity and heritability in CHO cell populations. Biotechnology and Bioengineering 110: 260-274. Highlighted "Spotlight" paper. McLeod J, O'Callaghan PM, Pybus LP, Wilkinson SJ, Root T, Racher AJ, James DC (2011) An empirical modeling platform to evaluate the relative control discrete CHO cell synthetic processes exert over recombinant monoclonal antibody production process titer. Biotechnology and Bioengineering. 108: 2193-2204. Davies SL, McLeod J, O'Callaghan PM, Pybus LP, Sung YH, Wilkinson SJ, Rance J, Racher AJ, Young RJ, James DC. (2011) Impact of gene vector design on the control of recombinant monoclonal antibody production by CHO cells. Biotechnology Progress 27: 1689-1699. O'Callaghan PM, MacLeod J, Pybus L, Lovelady CS, Wilkinson S, Racher AJ, Porter A, James DC. (2010) Cell line specific control of recombinant monoclonal antibody production by CHO cells. Biotechnology and Bioengineering. 106: 937-951.
Start Year 2006
 
Description Strategic partnership with Technopath 
Organisation TechnoPath
Country Ireland, Republic of 
Sector Private 
PI Contribution Development of high-throughput MAb product titre and cell phenotyping technology
Collaborator Contribution Project management, EU funding coordination, research equipment
Impact Inventors: James D, Clifford J, Thompson B. Predicting relative production titer of a panel of clonal producer cells in fed batch culture by simultaneously incubating each producer cell with a chemical cell stressor and determining the production titer response of each cell Patent Number: EP2902493-A1; WO2015113704-A1 Patent Assignee: TECHNO-PATH DISTRIBUTION; VALITACELL LTD Inventors: James D, Davies S, Thompson B. Method for determining e.g. cell identity of mammalian producer of user, involves comparing query cell-specific growth response fingerprint with reference cell-specific growth response fingerprints corresponding to cells Patent Number: WO2014207166-A1 Patent Assignee: TECHNO-PATH DISTRIBUTION Inventors: James D, Clifford J, Thompson B. A method of measuring antibody concentration in a sample. Patent Number: PCT/EP2015/063781. 18 June 2015. Patent Assignee: VALITACELL LTD
Start Year 2011
 
Description Strategic partnership with UCB 
Organisation UCB Pharma
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Private 
PI Contribution Development of synthetic vector sequences
Collaborator Contribution Project management, research material
Impact Brown A, Sweeney B, Mainwaring D, James DC. (2015) NF-?B, CRE and YY1 elements are key functional regulators of CMV promoter driven-transient gene expression in CHO cells. Biotechnology Journal. 10: 1019-1028. Brown A, Sweeney B, Mainwaring D, James DC (2014) Synthetic promoters for CHO Cell Engineering. Biotechnology and Bioengineering 111: 1638-1647. Brown AJ, Mainwaring DO, Sweeney B, James DC (2013) Block-Decoys: Transcription factor decoys designed for in vitro gene regulation studies. Analytical Biochemistry 443: 205-210. Patents/Applications Inventors: James DC, Brown AJ. New chinese hamster ovary cell comprises a synthetic promoter suitable for eliciting recombinant protein expression in it, for use in the biopharmaceutical field. Patent Number: WO2015079053-A2; WO2015079053-A3 Patent Assignee: UCB BIOPHARMA SPRL Inventors: James DC, Brown AJ. Regulating recombinant gene expression in vitro comprises providing a host cell encoding recombinant genes for expression, and contacting the cell with an exonuclease resistant block decoy Patent Number: WO2014202640-A1 Patent Assignee: UCB BIOPHARMA SPRL
Start Year 2010
 
Description Strategic partnership with Wyeth/Pfizer 
Organisation Pfizer Inc
Country United States of America 
Sector Private 
PI Contribution Mechanistic analyses of CHO cell factory function
Collaborator Contribution Project management, research materials and model systems
Impact Walther CG, Whitfield R, James DC. (2015) Importance of interaction between integrin and the actin cytoskeleton in suspension adaptation of CHO cells. Applied Biochemistry and Biotechnology. In press. Kim MS, O'Callaghan PM, Droms K, James DC (2011) A mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies. Biotechnology and Bioengineering. 108: 2434-2446.
Start Year 2007