Optimisation of CHO for Biotherapeutic Manufacture
Lead Research Organisation:
University of Edinburgh
Department Name: Sch of Biological Sciences
Abstract
Biological drugs (e.g. monoclonal antibodies, MAbs) based on recombinant DNA technology have transformed the treatment of life-limiting diseases including cancer, haemophilia and rheumatoid arthritis. The recent explosive growth in the biologics sector looks set to continue, with growing applications in precision medicine and personalised healthcare, and there are many new complex biologics in the drug discovery pipeline (e.g. bispecific, trispecific, and conjugated MAbs). The intrinsic complexity of these life-saving drugs is too challenging for synthesis by simple chemistry and requires the utilisation of living cells. Forcing cells to produce proteins that they do not naturally express is complex, and often requires a long period of trial and error cell manipulation, making the bio-manufacturing process time-consuming and very expensive and directly impacting on the delivery of transformative medicines to patients. With the recent remarkable development of powerful tools for editing mammalian genomes, new methods and automation for the synthesis of large numbers of DNA constructs, and the context provided by systems biology, the time is now right for using Synthetic Biology to establish a new paradigm for cost-effective manufacture of biologic drugs. In turn this will have a major impact on medicine and the health related industries, and make the biopharmaceutical value chain more cost-efficient.
The scale of the economic opportunity associated with this project is enormous. The UK has one of the most dynamic and innovative healthcare industries in the world and has developed over 20% of the world's top 100 selling drugs. The medical technology sector in the UK consists of around 2,800 companies, employing 52,000 people and generating around £10.6bn of turnover annually. An increasing portion of all medicines, currently estimated at 20%, are biopharmaceuticals. The global biologics market was valued at an estimated $251.5 billion in 2018 and is predicted to reach $319 billion by 2021. The CHO cell is the most widely used industrial expression system, which generates ~70% of approved and marketed therapeutic recombinant proteins, including multiple monoclonal antibodies (mAbs), so any enhancement of production efficiency and quality has a huge economic impact.
The vision of this prosperity partnership is to utilise state of the art investigational tools and synthetic biology approaches to both elucidate the intricacies of the CHO cell manufacturing platform and engineer it to be more predictive, effective, cost-efficient, and competitive for the production of biotherapeutics in the UK.
The scale of the economic opportunity associated with this project is enormous. The UK has one of the most dynamic and innovative healthcare industries in the world and has developed over 20% of the world's top 100 selling drugs. The medical technology sector in the UK consists of around 2,800 companies, employing 52,000 people and generating around £10.6bn of turnover annually. An increasing portion of all medicines, currently estimated at 20%, are biopharmaceuticals. The global biologics market was valued at an estimated $251.5 billion in 2018 and is predicted to reach $319 billion by 2021. The CHO cell is the most widely used industrial expression system, which generates ~70% of approved and marketed therapeutic recombinant proteins, including multiple monoclonal antibodies (mAbs), so any enhancement of production efficiency and quality has a huge economic impact.
The vision of this prosperity partnership is to utilise state of the art investigational tools and synthetic biology approaches to both elucidate the intricacies of the CHO cell manufacturing platform and engineer it to be more predictive, effective, cost-efficient, and competitive for the production of biotherapeutics in the UK.
Publications
Sizer RE
(2023)
Use of ubiquitous chromatin opening elements (UCOE) as tools to maintain transgene expression in biotechnology.
in Computational and structural biotechnology journal
Sizer RE
(2022)
TFIIIC-based chromatin insulators through eukaryotic evolution.
in Gene
Moses T
(2023)
Right in two: capabilities of ion mobility spectrometry for untargeted metabolomics
in Frontiers in Molecular Biosciences
Mentlak DA
(2024)
Dissecting cell death pathways in fed-batch bioreactors.
in Biotechnology journal
Donaldson JS
(2021)
Decoupling Growth and Protein Production in CHO Cells: A Targeted Approach.
in Frontiers in bioengineering and biotechnology
Donaldson J
(2022)
Synthetic biology approaches for dynamic CHO cell engineering.
in Current opinion in biotechnology
Christofi E
(2023)
Ion Mobility Mass Spectrometry (IM-MS) for Structural Biology: Insights Gained by Measuring Mass, Charge, and Collision Cross Section
in Chemical Reviews
| Description | 1. Enhance molecular understanding of CHO biologics production cell lines using a suite of omics technologies. To enhance understanding, cutting-edge analytical tools have been applied. Genomics: Genomes of two industrial Apollo X CHO cell lines (parent and mAb producer) were sequenced to generate over 30GB of highly accurate long-read sequences, which were assembled and annotated to obtain complete genomes. Over 99% of the reads from the mAb producer line mapped to the parent line, allowing comparison of the mAb producer line genome with the parent line genome, identifying several deletions (less than 1,000 bp) and duplications (over 10,000 bp). This is the first time the genome architecture of FDBK's mAb-producing cell line has been investigated. A genome browser website is being developed allowing the easy interrogation of the Apollo X CHO cell genome. This will provide a wealth of information on how cells have adapted to high yield protein production and for further cell line development and engineering strategies to develop efficient mAb-producing CHO cell platforms. Transcriptomics: The transcriptome of a mAb-producing cell line was analysed on different days of a fed-batch fermentation to understand expression profiles of genes during growth and mAb production. This in-depth analysis will monitor native and heterologously introduced gene expression, including the level of gene expression, the phase of growth or production when the gene is expressed, the relative level of gene expression in metabolic pathways, etc, during a bioproduction process. Knowing the relative levels of transcripts will inform engineering strategies to further optimise mAb production in this cell line, as well as other similar mAb lines. The transcriptome analysis also aims to identify genes that are only expressed in late phases of the bioproduction process that could potentially be detrimental to the mAb product. Such insights at the transcriptional level of the CHO cell line, will identify gene targets that can be engineered to reduce or avoid mAb production bottlenecks, optimise expression of heterologous genes and enhance product quality. As part of the Prosperity Partnership, FDBK have developed a RT-PCR assay for transcript levels which can be utilised to identify high expressing clones for products which traditionally have been difficult to quantify during early stages of cell line development. Implementation of this assay into the cell line selection workflow will allow identification of higher expressing cell lines for these typically difficult-to-express products. Viability loss during fed-batch culture has important consequences for antibody titres and product quality. Understanding the pathways involved is a crucial step in trying to mitigate these deleterious effects on therapeutics production. We have recently characterised non-apoptotic death pathways in CHO cell fed-batch bioreactors. Our results challenged the long-standing assumption that apoptosis is the only death pathway activated. Under the settings used, we did not observe apoptosis activation, but additional pathways such as parthanatos and ferroptosis could account for cell death. This data has provided a framework for improving antibody production through targeting appropriate death pathways. Metabolomics: The metabolite levels in mAb-producing cell lines in various fed batch processes were analysed over bioproduction runs to obtain insights into metabolic pathways. Several metabolic bottlenecks were identified suggesting bottlenecks in metabolism and potential targets for cell line engineering. A set of synbio engineering strategies have been initiated and these have exemplified the potential to enhance the efficiency of intacellular metabolism for enhanced cell yield and production of recombinant mAb. We have successfully completed the first recorded complete real time mass spectral analysis of a CHO cell bioprocess. Additionally, proteomics and metabolomics data has provided several targets for growth optimisation, including oxidative stress, short chain organic acid synthesis and single carbon metabolism, that are currently being tested at scale. 2. Optimise the Culture Process - by increased understanding and engineered control of the cellular environment. Next generation bioprocesses are challenged with providing a high-quality product with a reduced cost-of-goods to further democratise biopharmaceutical supply. FDB undertook an iterative, Design of Experiments led approach with multiple cell line and products in an automated microbioreactor system, followed by fine-tuning and scale-up verification at bench scale. This resulted in the development of a new platform bioreactor process which will be implemented into our commercial CDMO offering. The feeding aspects of the process development work is centred on responsiveness to nutrient requirement in the culture, and as such is designed to cater for the variable needs of different cell lines. The resultant upstream process delivered equivalent productivity but with a favourable shift in charge profile from acidic to main peak species. Furthermore, the process was characterised by lower demands for gassing input for oxygen and pH control, minimal base requirement for pH control, reduced foaming and lower levels of lactate, osmolality and pCO2. The introduced new feeds are estimated to yield a reduction in feed costs of greater than 50% and utilising feed with known composition will aid the metabolomic work. One of the biggest challenges in manufacturing biologics is loss of productivity that occurs in CHO cells over time. This phenomenon has been attributed to genome instability, epigenetic gene silencing and changes in stress responses within the cell population. To investigate this, we are using an advanced cell analysis optofluidic platform, the Beacon, developed by Berkeley Lights, which was recently acquired by the Edinburgh Genome Foundry. The Beacon platform allows for automated sub-cloning whereby single cells from a suspension culture are isolated within up to 1750 nanopens, enabling growth and relative levels of antibody secretion to be monitored in real time. Using the Beacon System we have investigated differences between two cell-lines, one of which has been determined previously to be stable in its production of monoclonal antibodies compared with one which appears to lose productivity. We have shown that the Beacon can aid identification of which cultures are likely to be stable and which are not at an earlier stage of cell-line development, ultimately preventing resources being wasted on unproductive cell-lines and improve overall efficiency of the entire process. This will also aid development of cell-lines which exhibit enhanced stability of protein production in culture for longer periods of time, with associated cost and economic impact. 3. Create an in silico predictive CHO cell model Initial work has involved assessing the usability of a published CHO genome scale metabolic model, and how it may need to be refined to represent the ApolloX systems more accurately. The metabolomics team are in close discussion with the modellers and we have both NMR data providing extracellular constraints as well as intracellular metabolite pool data. The modellers are using this data to validate the results of the modelling. The next phase will be incorporating transcriptomics and genomics data to improve the model. 4. Develop an engineering biology toolbox for mammalian cell factory redesign and use it to engineer a new highly productive CHO cell line. An enduring challenge for the biopharmaceutical industry is expression instability, where active transgenes become silenced during the time required for industrial process scale up. Epigenetic silencing of transgenes is most commonly responsible for this affect, which can compromise consistency and regulatory approval, as well as yield. We have developed novel barriers that protect transgenes against silencing and maintain high expression during long-term culture. When tested in parallel, our barriers are substantially more effective at preventing silencing than a widely used barrier (UCOE) that is available commercially. The demand for high yields and, increasingly, for the production of engineered, often difficult-to-express proteins can often result in endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). If the demands of continued (over-)production exceed their ability to restore proteostasis, cells will go into cell death, leading to loss of high-producing cells. To address this issue, we have developed an ER-stress biosensor which will enable stress conditions to be managed to prevent cell death caused by prolonged UPR. development of. The successful construction of a fluorescent ER stress reporter has been extended to build a novel ER stress 'sense-and-respond' module based on stress responsive synthetic transcription factors that is currently being tested aimed at ameliorating stress caused by overexpression of difficult-to-express proteins. A new project was started to determine the optimal configuration of gene cassettes within a single expression vector. We have shown that transcription through a gene can impact expression of neighboring genes, likely via effects on supercoiling, and that the relative orientation of expression cassettes influences output. We are currently investigating how these effects are influenced by the inclusion and positioning of particular genetic elements in the constructs. |
| Exploitation Route | This is a prosperity partnership with Fujifilm Diosynth Biotechnologies. Results from the work will be implemented by Fujifilm in their manufacturing processes. |
| Sectors | Healthcare Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |