13 ERA IB: Investigating NOvel VAluable bio-Therapeutics and Expression systems

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

Abstract

The production of IgG's has revolutionized the biotech industry with products used for the treatment of cancer and immune disorders. The success of standard IgG's built over years of optimization, has led to high expectations for novel recombinant proteins (rPs), "third generation" biopharmaceuticals. Timeframes, stakeholders and commercial pressures are different to those of a decade ago and the manufacture of new rPs is challenging. The promise of commercial and clinical returns from new rPs such as fusion proteins (for novel disease targeting), IgA (the most abundant Ig isoform, for treatment of IgA deficiencies, cancer) and secreted mucins (for disease therapy, vaccines) is obvious but these are unexpectedly difficult to manufacture, with unpredictable failures to generate any (or enough) product. Studies accessible to scrutiny indicate unpredictable limitations occur at the level of translation, folding and post-translational processing (especially N- and O-glycosylation). This project will develop new animal (CHO) and plant (tobacco) cell expression technologies, and establish bioprocesses, for the production of difficult to express and novel rPs. Objectives are to (1) define the molecular events linked to production of difficult to express rPs; (2) engineer new expression technologies for enhanced synthesis, folding and assembly of such molecules via manipulation of translational and secretory machinery; (3) engineer CHO cell lines for the efficent N- and O-glycosylation of IgAs; (4) engineer and establish plant cell lines capable of producing industrially relevant amounts of IgAs with bespoke glycosylation; (5) develop an understanding of the metabolic load on the cell expressing challenging rPs; and (6) establish upstream and downstream processes for the new expression technologies and rPs and evaluate the potential for scale-up. The consortium will seek protection of IP for the new expression technologies, as these will be of immense interest to industry.

Technical Summary

The production of IgG's has revolutionized the biotech industry with products used for the treatment of cancer and immune disorders. The success of standard IgG's built over years of optimization, has led to high expectations for novel recombinant proteins (rPs), "third generation" biopharmaceuticals. Timeframes, stakeholders and commercial pressures are different to those of a decade ago and the manufacture of new rPs is challenging. The promise of commercial and clinical returns from new rPs such as fusion proteins (for novel disease targeting), IgA (the most abundant Ig isoform, for treatment of IgA deficiencies, cancer) and secreted mucins (for disease therapy, vaccines) is obvious but these are unexpectedly difficult to manufacture, with unpredictable failures to generate any (or enough) product. Studies accessible to scrutiny indicate unpredictable limitations occur at the level of translation, folding and post-translational processing (especially N- and O-glycosylation). This project will develop new animal (Chinese hamster ovary, CHO) and plant (tobacco) cell expression technologies, and establish bioprocesses, for the production of difficult to express and novel rPs.

Objectives are to
(1) define the molecular events linked to production of difficult to express rPs;
(2) engineer new expression technologies for enhanced synthesis, folding and assembly of such molecules via manipulation of translational and secretory machinery;
(3) engineer CHO cell lines for the efficent N- and O-glycosylation of IgAs;
(4) engineer and establish plant cell lines capable of producing industrially relevant amounts of IgAs with bespoke glycosylation;
(5) develop an understanding of the metabolic load on the cell expressing challenging rPs; and
(6) establish upstream and downstream processes for the new expression technologies and rPs and evaluate the potential for scale-up.

Planned Impact

Who will benefit from this research?
In terms of research findings, the primary beneficiaries will be researchers in the academic and biopharmaceutical sectors who are interested in understanding the limitations upon the production of difficult to express recombinant proteins in mammalian and plant expression systems with respect to product yield and quality in industrially relevant systems. 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 lower cost. The impacts of this research will therefore be national and international. They will benefit the following: (1) those in the research fields of recombinant protein synthesis, purification and development; (2) the academic and industrial bioprocessing and scientific communities; (3) the biopharmaceutical sector; ultimately the National Health Service (and thus the wider public, its patients); (4) the UK economy through the development of new methods to produce novel and larger amounts of increasingly important 'bio-drugs' (i.e. recombinant proteins) more efficiently and thus at lower cost thereby, (v) benefitting health-care providers and their patients.

How will they benefit?
The major impact of this work will be to provide both industry and academia (i) with a much better understanding of the biology underpinning difficult to express proteins (particularly heavily glycosylated proteins) and their control in an industrial sense with respect to cell growth and the production and quality of recombinant proteins in mammalian and plant cells, and (ii) the subsequent application of this information to generate new tools and methodologies (engineered cell lines and downstream processing methodology). This will generate expression systems with an increased capacity for recombinant protein production and facilitate the faster development of cell lines expressing novel recombinant proteins, specifically IgA and mucin type molecules that can then be used for the treatment of novel disease indications. The ability to produce these high cost drugs will ultimately allow the development of new therapies base upon such molecules and 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 this is delivered, our results will be published in peer-reviewed high-quality journals and presented at relevant academic and industrial conferences.

We will publicise our findings through our websites, press releases, BBSRC Business and via the local media and our own public engagement activities, including national and local science fairs and working with local schools. We will build upon our industrial links to translate our findings into applications in the recombinant protein production field. Regular teleconferences and meetings between the applicants and PDRAs, and the use of a sharepoint for all data generated in the project, will ensure close coordination between the activities at the institutions, such that findings in one lab are rapidly conveyed to the others to inform and develop the project in a timely and efficient way.

Publications

10 25 50
 
Description We have developed new expression technology and cell lines making therapeutic IgA molecules and evaluated the effect of expressing these on the cell to further improve the expression and quality of protein yielded. We have developed systems that can express significant amounts on these difficult to manufacture molecules and are evaluating these under industrial conditions. The heavy chains of these molecules tend to accumulate in the cell due to limitations upon assembly and secretion and we are currently investigating methods to elevate this. This will help provide methods for enhancing the yields of recombinant IgA molecules from CHO cells such that these may be made in sufficient quantities to allow assessment of these as potential clinical treatments in humans. We have subsequently found that the addition of chemical chaperones can improve the assembly of IgA molecules and improve the secretory yields. Further, manipulation of culture conditions also leads to enhanced secretory yields, although much material is still retained intracellularly. Such approaches can be used to help enhance the secretory yields of other difficult to express proteins.
Exploitation Route We anticipate publishing our results and presenting these in the next 12 months so that the key findings can be used by others to develop new approaches to expressing difficult to express biotherapeutic proteins ultimately destined for use in the clinic to treat a range of diseases.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description We have produced new expression vectors that can be used by industry to evaluate expression of difficult to express proteins. We are also investigating the glycosylation patterns of these difficult to express proteins and how modification of the cell machinery influences the sugars on the protein. We have developed systems for the expression of commercially viable amounts of IgA molecules and are about to validate these/characterise these in the industrial setting with our industrial partners. We have identified that IgA production is limited by intracellular constraints that result in the retention of the heavy chain in particular and an inability to correctly assemble and secret these molecules. This results in the majority of material being retained intracellularly and not secreted out of the cell. Changing culture conditions can lead to increased amount of secreted material but this is in response to a general increase in IgA production and results in more material being retained intracellularly. This in turn activates stress responses in the cell. We have shown that the variable region on the constant IgA regions can impact the assembly as well. This information is now being used to redesign IgA molecules for recombinant expression.
First Year Of Impact 2016
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description BIO)PHARMA IRELAND WHITE PAPER
Geographic Reach Europe 
Policy Influence Type Membership of a guideline committee
 
Description GCRF/RCUK
Amount £4,090,773 (GBP)
Funding ID BB/P02789X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 12/2021
 
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 Lyophilisation of Factor IX 
Organisation Federal University of Rio de Janeiro
Country Brazil 
Sector Academic/University 
PI Contribution Developed approaches to lyophilise Factor IX produced in CHO cells.
Collaborator Contribution Provided factor IX and PhD student to undertake work.
Impact Multidisciplinary - chemical engineering, biology, biochemistry
Start Year 2015
 
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 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 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 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 day 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 Discussion on recombinant protein expression systems and importance of these as biotherapeutics or potentially for gene therapy applications.

Lots of interest in expression of recombinant proteins. Discussed visiting school to speak to biology club specifically on area in more detail.
Year(s) Of Engagement Activity 2014
 
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