Tailor-made expression hosts depleted in protease activity for recombinant protein production; PRODuCE (PROtease Depleted CEll line)

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

Abstract

Small molecule drugs (e.g. antibiotics) have traditionally been the mainstay of treatments and therapies in man. However in the last 10-20 years protein based drugs (e.g. Herceptin, often used to treat breast cancer, insulin) have been developed such that these now constitute the fastest growing section of the pharmaceutical market. There are several categories of protein-based drugs, many of which are produced from cultured mammalian or yeast cells at an industrial scale. Due to the high precision required to produce such biotherapeutics, such 'recombinant' protein-based drugs (biopharmaceuticals) are usually produced by cells kept in culture under defined conditions. One problem with this is that the cells scientists use to make proteins for therapeutic uses also make their own proteins including proteases whose expression can be detrimental to the production of therapeutic proteins. These types of proteins can actually degrade the target recombinant protein making them useless in a clinical sense. As a consequence, scientists may not be able to produce enough of these drugs and/or the cost of producing them may be too high, thus precluding health care providers from recommending their use. This proposal sets out to address a key area that underpins recombinant protein synthesis from cultured mammalian and plant cells.

We aim to develop a comprehensive knowledge of the detrimental proteolytic activities in different cell lines (animals and plants) used for the production of biopharmaceuticals. We will use state-of-the-art approaches and technologies to define those proteases that are detrimental to recombinant protein production from these expression systems and then utilise this knowledge to reduce or eliminate the amount of these in the host cells. This will ultimately result in less or no proteolytic cleavage and damage of the therapeutic protein being produced. This programme therefore proposes to address the gap in our understanding as to the importance of proteases in defining product quality and yield. The overall aim is to generate new cells and systems that exploit manipulations of the cells proteolytic machinery to enhance the production of recombinant therapeutic proteins at the industrial scale. This information is of very substantial relevance to industry since the production of commercially valuable therapeutic proteins is potentially hindered by these detrimental reactions. Without improved expression systems, the biotechnology/pharmaceutical industries will lack the capability to produce large enough amounts of these valuable and effective drugs to meet the demand at a price that will allow them to be prescribed for all patients who would benefit from them.

Technical Summary

The demand for biopharmaceuticals for the treatment of a wide range of diseases is high and predicted to increase. Depending on the complexity of the target protein, these are either produced in microorganisms or in mammalian cell cultures, predominantly CHO cells. Plant suspension cells are an emerging platform for protein production and the first plant-made biopharmaceutical (Elelyso) has recently received market approval. The protein products must be produced within defined parameters and the potential unintended processing of the target protein by endogenous proteases, both during the fermentation or the downstream process, limits the final yield of the target protein and impacts on its quality and shelf life. There are various reports of protein products being compromised by such processing including those that describe proteolysis of the product during fermentation, matrix degradation during downstream processing and co-purification of proteolytic enzymes with the target protein. However, a systematic survey of proteolytic activities that interfere with the production of biopharmaceuticals has not been described. Within the proposed project we will systematically evaluate the proteolytic activities that hamper the successful production or purification of selected target proteins. The project will include CHO cells as the current industry workhorse for protein production but also plant suspension cells as emerging alternative production platforms. Proteolytic activities will be determined in cells and spent culture medium using activity-based protein profiling (ABPP) probes. The knowledge gained about the nature of the proteases will be utilized to engineer cell lines with reduced endogenous protease activities. We will (1) Knock-out protease genes by gene targeting, (2) Knock-down protease genes by posttransby posttranscriptional gene silencing, (3) Co-express proteinaceous protease inhibitors, and (4) Rationally design of media to suppress protease activity.

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 role and control of proteases and proteolysis 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 proteases and protein synthesis; (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 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 roles of proteases 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 alternative media). This will generate expression systems with an increased capacity for recombinant protein production and facilitate the faster development of cell lines expressing recombinant proteins, lowering the cost of producing such biomedicines. The ability to produce these high cost drugs at lower cost will ultimately allow 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. The applicant at Kent, together with Kent Innovation and Enterprise (KIE) and their counterparts at the partner institutions will take the lead in ensuring this is completed in a timely fashion. 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

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Hogwood CE (2014) Measurement and control of host cell proteins (HCPs) in CHO cell bioprocesses. in Current opinion in biotechnology

 
Description We have been investigating proteases from CHO cells and how these can lead to degradation of high value recombinant biotherapeutic proteins. The aim is to identify what these proteases are and then use strategies to reduce these (or remove completely). Different protein production platforms, Chinese hamster ovary (CHO) cells, tobacco BY-2 suspension cells, Medicago suspension cells, and Nicotiana benthamina plants have been analyzed for their potential to degrade recombinant model proteins. The model proteins used in these studies were the anti-HIV monoclonal antibody 2F5 (mAb 2F5), human serum albumin (HSA) and Protein A (rPA). The model proteins were spiked into cell lysates and/or spent culture medium of the different cell lines and their degradation assessed by immunoblot analyses.

By adding individual compounds of a diverse collection of small molecule inhibitors of proteases, the enzymatic classes of the relevant proteases were determined for each production platform. It has been found that the degradation of the model proteins in CHO cells depends mainly on the activity of serine and cysteine proteases.

We have also used activity-based protein profiling (ABPP) and/or mass-spectrometry based analysis of cell lysates and spent culture medium, to identify individual proteases in CHO cells and culture supernatants.

The findings from these studies are now being applied to generate new cell lines with reduced protease activity for the production of biotherapeutic proteins.
Exploitation Route The identification of these proteases will allow industry and academia to screen for the presence of these to reduce protein loss during manufacture of these high cost drugs before use in the clinic. Their identification also allows for the targeted inhibition or removal of these in novel cell expression systems for use commercially. Finally, target proteins themselves could be manipulated to prevent protease degradation.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description We have had discussions on a project with an industrial partner as to how these results can be used to prevent degradation of a key target biotherapeutic molecule that is currently manufactured and used in the clinic. We are using this information to develope new host cell expression systems that will be utilised/validated industrially. The information has also been used to advise industry on host cell protein assay development and to develop new mass spectrometry based approaches to provide greater certainty of those host cell proteins cleared and remaining in biotherapeutic protein preparations that could be destined for use in the clinic to treat a range of diseases.
First Year Of Impact 2016
Sector Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
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 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 Proteasome activity profiling 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Profiling of proteasome activity in various cell lines using activity probes.
Collaborator Contribution Provision of proteasome activity probes and inhibitors and knowhow to undertake experiments.
Impact Understanding of proteasome activity in CHO cells during bioprocessing.
Start Year 2014
 
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 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 Presentation on Biosimilar protein based biotherapeutic drugs 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Health professionals
Results and Impact The talk stimulated discussion with the nurses and health care professionals present about what biosimilars are and when/how to assess if these are appropriate to administer as opposed to the innovator drug.

Nurses and healthcare professionals asked for further information on biosimilars.
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