Translation of Step-changing Bioprocesses and Expression System Technologies for Next Generation Protein Biologics Production in CHO Cells
Lead Research Organisation:
University of Kent
Department Name: Sch of Biosciences
Abstract
Chinese hamster ovary (CHO) cells are the main production host for >US$145billion/yr of protein biologics used as medicines for a range of diseases. The CHO platform is mature when considering production of monoclonal antibodies, but new format non-native molecules such as fusion proteins, antibody fragments and other exotic molecules remain difficult to express (DTE) in this, or any other host. This project builds upon proof of concept work demonstrating that engineering the CHO chassis, together with growth media manipulation, increases both the yield and quality of a number of DTE proteins that are in development for application to unmet clinical needs and diseases with no current treatments. The project will advance the technology readiness level of our preliminary findings beyond proof-of-concept to deliver the commercialization of new CHO cell systems for DTE proteins and associated bioprocesses ready for industrial application to produce these important new medicines.
Technical Summary
This proposal's overall output is a novel integrated pipeline for difficult to express (DTE) biologics production. A 3-fold approach to realising the commercial potential of the technology will be adopted. Firstly, a lipid suplementation strategy will be developed to improve existing CHO processes. Secondly, a suite of expression vectors will be created comprising new marker genes that complement CHO auxotrophies and lipid biosynthesis genes. Thirdly, engineered CHO chassis will be generated that stably express the lipid metabolism genes and/or combinations of these. The final, and preferred, embodiment of the technology will be a combination of new cell chassis and improved processes. This approach will maximise the realisation of value by ensuring early implmentation and broad application (both existing and new cell lines) and also mitigates against the failure of any one approach. WPs 1-4 are based upon innovations that both teams together were involved in developing. Specific milestones: 1. Appointment of staff/training (Q1, y1). 2. 3/4 gene cassette vectors available (Q2, y1). 3. Vectors with novel metabolic selection markers (tyrosine/proline) available (Q4, y1). 4. New lipid supplementation processes for DTE proteins in existing technologies developed (Q4, y1). 5. Initial lipid engineered Xceed host cell line available and validated for enhanced DTE protein yield/quality (Q2, y2). 6. Cell line construction and process development validated for novel lipid engineered Xceed hosts (Q2, y3). 7. cGMP cell banks available (Q2, y3). 8. cGMP cell bank testing complete, novel cell lines and upload of process conditions suitable for cGMP use into Lonza's GMP document management system and release of a process uptake to Lonza's GS Licensees (Q4, y3).
Planned Impact
As described in proposal submitted to IUK
People |
ORCID iD |
| Christopher Smales (Principal Investigator) |
Publications
Vito D
(2018)
Engineering of the cellular translational machinery and non-coding RNAs to enhance CHO cell growth, recombinant product yields and quality
in Current Opinion in Chemical Engineering
Tamošaitis L
(2018)
Meta-Analysis of Publicly Available Chinese Hamster Ovary (CHO) Cell Transcriptomic Datasets for Identifying Engineering Targets to Enhance Recombinant Protein Yields.
in Biotechnology journal
Migani D
(2017)
Effects of lysosomal biotherapeutic recombinant protein expression on cell stress and protease and general host cell protein release in Chinese hamster ovary cells.
in Biotechnology progress
Knight TJ
(2022)
Manipulation of mRNA translation elongation influences the fragmentation of a biotherapeutic Fc-fusion protein produced in CHO cells.
in Biotechnology and bioengineering
Knight TJ
(2021)
Selection of CHO host and recombinant cell pools by inhibition of the proteasome results in enhanced product yields and cell specific productivity.
in Journal of biotechnology
Feary M
(2017)
Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis.
in Biotechnology progress
Budge JD
(2020)
Engineering of Chinese hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant biotherapeutic protein production.
in Metabolic engineering
Budge JD
(2020)
Data for engineering lipid metabolism of Chinese hamster ovary (CHO) cells for enhanced recombinant protein production.
in Data in brief
Budge JD
(2021)
A proline metabolism selection system and its application to the engineering of lipid biosynthesis in Chinese hamster ovary cells.
in Metabolic engineering communications
Budge JD
(2021)
Engineering of Chinese Hamster Ovary Cells With NDPK-A to Enhance DNA Nuclear Delivery Combined With EBNA1 Plasmid Maintenance Gives Improved Exogenous Transient Reporter, mAb and SARS-CoV-2 Spike Protein Expression.
in Frontiers in bioengineering and biotechnology
| Description | We have developed new selection systems for the generation of stable recombinant Chinese hamster ovary cell lines that have been filed to be protected. We have also developed new metabolic selection markers and engineered CHO cell metabolism to overcome industrial challenges associated with metabolism. These new technologies are now being evaluated in the commercial environment. We have also established new synthetic plasmid DNA based vector systems for the expression of multiple genes in mammalian cell systems.The team has also now generated a range of potential new engineered CHO host cell lines to expand the capacity of these cells to produce difficult to express proteins. The impact of these manipulations has been validated in the commercial environment and the commercial partner is now looking to commercialise these new cell lines and reagents. We have thus established methods for manipulating specific processes in CHO cells that results in enhancing their ability to make recombinant protein based medicines at greater yield and quality. Further work beyond the end of the award has demonstrated the capability of these cells and utility of the metabolic selection systems. |
| Exploitation Route | Industrially for the commercial manufacture of protein based medicines. |
| Sectors | Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | Although this project has just completed and the full impacts will be realised in the coming years, we have generated findings that are being validated and applied in an industrial setting for the manufacture of recombinant biotherapeutic proteins. These will be further evaluated and developed as the technology is adopted by the industry. In particular, we have developed CHO cells whose lipid metabolism has been engineered to help overcome some bioprocess challenges and provide a unique solution to these problems. These are now being validated in the commercial environment. We have also developed engineered cell lines to enhance the ability of such cells to produce difficult to express recombinant proteins. This has relied upon new selection technology developed during the project. The industrial partner of the project has had GMP cell banks prepared of the lipid modified clonal host cell lines that will be available for commercialisation. A follow up project with the industrial partner is now utilising knowhow from this project to develop a new suite of vectors for their customers and to allow greater versatility of the offering they have to the client. |
| First Year Of Impact | 2016 |
| Sector | Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Economic |
| Description | Generation of new GS expression systems |
| Amount | £609,000 (GBP) |
| Organisation | Lonza Group |
| Sector | Private |
| Country | Global |
| Start | 01/2020 |
| End | 12/2022 |
| Description | KTP Award |
| Amount | £204,432 (GBP) |
| Funding ID | KTP010320 |
| Organisation | Innovate UK |
| Sector | Public |
| Country | United Kingdom |
| Start | 07/2016 |
| End | 01/2019 |
| Description | UCL Biochemical Engineering CDT PhD Award |
| Amount | £58,212 (GBP) |
| Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 10/2017 |
| End | 09/2021 |
| Title | New engineered CHO cells for expression of biotherapeutics |
| Description | We have developed new lipid metabolism engineered CHO cells for the expression of biopharmaceuticals with our industrial partner that they are now in the process of commercialising. |
| Type Of Material | Cell line |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| Impact | Ability to manufacture enhanced amounts of difficult to express biopharmaceutical proteins in cultured CHO cell systems. |
| Title | Raw data to accompany the manuscript 'Data for Engineering Lipid Metabolism of Chinese Hamster Ovary (CHO) Cells for Enhanced Recombinant Protein Production' published in the Journal Data in Brief |
| Description | This repository consists of the raw western blot, microscopy and mass spectrometry data to accompany the manuscript 'Data for Engineering Lipid Metabolism of Chinese Hamster Ovary (CHO) Cells for Enhanced Recombinant Protein Production' published in the Journal Data in Brief and associated with the article 'Engineering of Chinese hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant biotherapeutic protein production' published in the journal Metabolic Engineering (see DOI: 10.1016/j.ymben.2019.11.007). The western blot raw file is associated with Figure 1a and 1b of the Data in Brief manuscript. The confocal microscopy raw image files (x3) are associated with Figure 1c of the Data in Brief manuscript. The mass spectrometry files are the raw data that refers to the samples presented in Figure 5 of the Data in Brief manuscript. Files are labelled as in the Data in Brief and Metabolic Engineering manuscripts. The file name structures is as follows; CHO-Controlpoolai Where 'a' represents replicate 'a' of three biological replicates and 'i' refers to mass spectrometry technical analysis 1 of 3 technical analyses of each replicate (thus for each cell pool or line there are three biological replicates that are each analysed in triplicate such that there are 9 raw mass spectrometry files for each cell pool or line). All the mass spectrometry files are found in the compressed (zip) file named mass_spectrometry_raw_files_archive.zip |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| URL | https://zenodo.org/record/3610075 |
| Title | Raw data to accompany the manuscript 'Data for Engineering Lipid Metabolism of Chinese Hamster Ovary (CHO) Cells for Enhanced Recombinant Protein Production' published in the Journal Data in Brief |
| Description | This repository consists of the raw western blot, microscopy and mass spectrometry data to accompany the manuscript 'Data for Engineering Lipid Metabolism of Chinese Hamster Ovary (CHO) Cells for Enhanced Recombinant Protein Production' published in the Journal Data in Brief and associated with the article 'Engineering of Chinese hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant biotherapeutic protein production' published in the journal Metabolic Engineering (see DOI: 10.1016/j.ymben.2019.11.007). The western blot raw file is associated with Figure 1a and 1b of the Data in Brief manuscript. The confocal microscopy raw image files (x3) are associated with Figure 1c of the Data in Brief manuscript. The mass spectrometry files are the raw data that refers to the samples presented in Figure 5 of the Data in Brief manuscript. Files are labelled as in the Data in Brief and Metabolic Engineering manuscripts. The file name structures is as follows; CHO-Controlpoolai Where 'a' represents replicate 'a' of three biological replicates and 'i' refers to mass spectrometry technical analysis 1 of 3 technical analyses of each replicate (thus for each cell pool or line there are three biological replicates that are each analysed in triplicate such that there are 9 raw mass spectrometry files for each cell pool or line). All the mass spectrometry files are found in the compressed (zip) file named mass_spectrometry_raw_files_archive.zip |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| URL | https://zenodo.org/record/3610074 |
| Description | IBCatalyst LMM collaboration |
| Organisation | Lonza Group |
| Country | Global |
| Sector | Private |
| PI Contribution | This project has been in partnership with Lonza Biologics, Slough, UK to develop and commercialise new technology for the production of difficult to express recombinant proteins from Chinese hamster ovary (CHO) cells. Both partners have contributed knowledge and knowhow and expertise to deliver the project and objectives. The project is still on-going as of March 2019. |
| Collaborator Contribution | As above. |
| Impact | Commercially confidential at this stage. Patents submitted, publications will arise once these are granted. |
| Start Year | 2016 |
| Title | INHIBITION OF PROTEIN DEGRADATION FOR IMPROVED PRODUCTION |
| Description | Disclosed herein are methods and compositions useful for evaluating, selecting, identifying, or making a cell or cell line that has improved production capacity for generating higher yields of products and/or improved capacity to produce higher quality products. Products, as described herein, can include a polypeptide that is endogenously expressed by the cell, a recombinant polypeptide that is not endogenously expressed, or a non-naturally occurring recombinant polypeptide. The methods described herein include modulating, e.g., inhibiting, the protein degradation pathway by using a proteasome inhibitor, an ER-associated degradation (ERAD) inhibitor, or a ubiquitin pathway inhibitor. |
| IP Reference | WO2017118726 |
| Protection | Patent application published |
| Year Protection Granted | 2017 |
| Licensed | Commercial In Confidence |
| Impact | Understanding of how protein turnover can be used to select for Chinese hamster ovary (CHO) cells with an increased capacity to generate secretory recombinant proteins. |
| Title | METHODS OF CELL SELECTION AND MODIFYING CELL METABOLISM |
| Description | Described herein are compositions and methods for identifying, selecting, or culturing cells comprising a subject nucleic acid sequence of interest. Generally, a nucleic acid comprising a subject nucleic acid and a sequence encoding an enzyme molecule involved in biosynthesis of an amino acid is introduced into a cell. The cell is then grown on media lacking the amino acid, such that cells comprising the introduced nucleic acid are capable of growth. In some instances, the cell further comprises an inhibitor of the enzyme molecule to increase the stringency of the selection. |
| IP Reference | WO2019152876 |
| Protection | Patent application published |
| Year Protection Granted | 2019 |
| Licensed | Commercial In Confidence |
| Impact | Assessment of the IP by Lonza Biologics its on-going. |
| Title | MODULATION OF LIPID METABOLISM FOR PROTEIN PRODUCTION |
| Description | The present disclosure features methods and compositions for modulating lipid metabolism to achieve improved production and quality of recombinant products, such as next generation biologics. Modulation of lipid metabolism as described herein includes, for example, introducing a lipid metabolism modulator described herein to a cell or a cell-free system. Also encompassed by the present disclosure are engineered cells with improved production capacity and improved product quality, methods for engineering such cells, and preparations and mixtures comprising the products from such cells. |
| IP Reference | WO2017191165 |
| Protection | Patent application published |
| Year Protection Granted | 2017 |
| Licensed | Commercial In Confidence |
| Impact | This has contributed towards knowledge around lipid metabolism in recombinant CHO producing cell lines and the importance of this for underpinning recombinant cell growth and productivity. |
| 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 | Conference |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Presentation at international BBSRC NIBB secretion meeting in Glasgow, Scotland. |
| Year(s) Of Engagement Activity | 2017 |
| Description | Langton MBP project |
| 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 | Mentoring and aiding in running of research project at Simon Langton Grammar School for boys to sixth formers. |
| Year(s) Of Engagement Activity | 2019,2020 |
| Description | Open Day Activities |
| 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 | Activity involved practical demonstration of cell culture and gene editing. Also involved demonstration of mass spectrometry to determine post-translational modifications of proteins. Major activity was describing how engineering of cells can be used to produce recombinant biotherapeutic drugs. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Open Day talks and lab tours |
| 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 | Tours of research laboratory, demonstrating of research and talk/presentation on the work that we do. |
| Year(s) Of Engagement Activity | 2019,2020 |
| 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 |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Presentation at 13th Protein Expression in Animal Cells (PDRA on project) |
| Year(s) Of Engagement Activity | 2017 |
| URL | http://www.peace-conference.org |
| 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 | Talk at Simon Langton Girls Grammar School |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Schools |
| Results and Impact | Talk on research of making biopharmaceuticals. |
| Year(s) Of Engagement Activity | 2019 |