Genome engineering and synthetic biology approaches for improving industrial CHO cell production of biologics

Lead Research Organisation: Imperial College London
Department Name: Life Sciences


Biologics are protein-based biopharmaceutical drugs that are used in a variety of diseases. The overall market for biopharmaceuticals is steadily increasing and in 2013 generated sales in excess of $140 billion. The Lonza GS platform is considered industry leading with 18 marketed products, and over 100 in active human trials. Despite this success, there are increasing numbers of "difficult-to-express" biologics reaching the pipeline. The new technologies of rational genome and gene circuit engineering promise new ways of regulating biologic expression to maximise production, while reducing toxicity. The aim is to build "genetic thermostats" to prevent toxic proteins from shutting down cellular production altogether, using genetic feedback. The final goal of this feasibility study is to pave the way for the development of a robust and one-fits-all cell line platform for the production of "difficult-to-express" biologics.

Technical Summary

The production of biopharmaceuticals is part of a growing multi-billion dollar industry. The current standard for mammalian CHO recombinant (rP) production relies on constitutive promoters to control rP transcription. While this has long formed part of a platform process suitable for non-engineered monoclonal antibodies (mAbs), issues regarding productivity and stability are increasingly being faced as the diversity of rP proteins increases.This 12-month project will aim to develop proof-of-principle approaches for an innovative, universal, cell line platform for the expression of recombinant proteins that are characterized as "difficult-to-express" or that exhibit cellular toxicity. Using Lonza's proprietary CHOK1SV cell line and GS Gene Expression system, the project will combine: (i) rational site-targeted genome engineering (ii) gene circuit design, employing elements of genetic feedback regulation and conditional regulation. In this way, difficult-to-express proteins will be tested against a benchmark gold-standard expression protein, with the aim of improving protein production in a self-regulating, widely-applicable manner.

Planned Impact

As described in proposal submitted to IUK


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Description Lay Summary

This was a 1-year proof-of-concept academia/industry joint development grant (2015-16) and we developed ways to improve protein expression for the production of a range of pharmaceutical drugs, known as 'biologics'. These are protein-based drugs, and include artificial antibodies against a range of diseases, such as cancer. The methods we developed are to improve the yield in industrial production and will be applied directly by the co-applicant pharmaceutical company, Lonza.

Detailed Summary of work:

Synthetic gene networks were tested to improve the yield of biologics. They consist of a gene encoding for biologics and genetic elements that induce or inhibit production of biologics, promoters and repressors respectively. The function of each synthetic network was validated by insertion into a commonly used manufacturing cell line (Chinese Hamster Ovary (CHO)) and the protein yield assessed.

Three gene network designs were tested in parallel, each with increasing levels of risk, complexity and novelty. The first gene network used a well-characterised system that has been shown to increase yields compared to standard manufacturing processes (Misaghi et al 2014). It comprises a promoter that is only active in the presence of chemicals called tetracyclines.

The second gene network exploited endogenous promoters that are activated when cell populations are high, i.e. the stationary growth phase. Lonza provided data that characterises all promoters in their proprietary CHO cell line and this was used to successfully identify 10 candidate promoters. During this task it was also evident that phase promoters induce low levels of protein expression. Based on this new result we designed two strategies to enhance expression. First, a synthetic enhancer was incorporated into our gene network to increase production. An alternative gene network used synchronised repressors to control protein expression.

The final gene network is the most exciting design and aimed to provide self-regulation of the industry standard manufacturing process. Current methods use promoters that induce persistent expression of biologics - a process associated with reduced yields. We aimed to use endogenous promoters activated in response to high levels of protein to induce artificial repressors. These molecular sensors inhibit protein expression when levels are high, but become inactive when protein levels decrease. This work was been the most promising part of this pilot project and has continued beyond the funding period. As a result of this work, Lonza filed a patent: On 19-05-2018, Peter O Callaghan at Lonza stated to Mark Isalan by email, "in order to protect any IP we submitted a provisional patent application as per the Imperial-Lonza agreement."
Exploitation Route This is an industry development grant and is awarded 50%-50% with the pharma company Lonza, who will apply the results in their industrial pipeline for the production of biologics (e.g. antibody drugs).
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description Intellectual Property & Licensing: As a direct result of this grant a patent application has been made by Lonza. On 19-05-2018, Peter O Callaghan at Lonza stated to Mark Isalan by email: "in order to protect any IP we submitted a provisional patent application as per the Imperial-Lonza agreement."
First Year Of Impact 2018
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

Title Functional insulator scanning of CpG islands to identify regulatory regions of promoters using CRISPR 
Description Dr. Marbiah used the CRISPR technology that she developed in this Innovate UK grant to develop a new method that she published as joint first-author: Grob, A., Marbiah, M., & Isalan M. Functional insulator scanning of CpG islands to identify regulatory regions of promoters using CRISPR Methods in Mol Biol 1766:285-301 (2018). 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact This work has already begun to be cited by others. 
Description Lonza Biologics PLC 
Organisation Lonza Group
Department Lonza Biologics
Country United States 
Sector Private 
PI Contribution This 1-year catalyst award has allowed us to develop and ongoing collaboration with Lonza. We are working together on a synthetic biology feedback-regulated system to improve the industrial production of difficult-to-express proteins (biologics).
Collaborator Contribution Lonza has provided expertise and cell lines for production of difficult-to-express proteins. Imperial has provided synthetic biology expertise. We are still working on optimising the system.
Impact We are still working on the project (beyond the 1-year funding period, with an extra contribution from Lonza) and will report on IP and publications when they are ready.
Start Year 2015