Characterization of post-transcriptional constraints that determine rP yield during bioprocessing in mammalian cells

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

Abstract

We all have an in-built defence mechanism to respond to infection when our body recognises a foreign 'invader'. A type of cell known as a B cell responds to infection by changing into an antibody-producing cell. Antibodies are proteins that work by attacking the foreign invader and destroying it, clearing infection by removing the foreign agent. Because antibodies are our body's natural defence against disease, many new antibody type drugs are being developed to help treat a number of human diseases such as cancer. These antibodies are usually produced by cells kept in a culturing solution under defined conditions. The problem is that these antibodies must be in a special shape; otherwise they do not work. The cells used to produce such antibodies have a very complex set of machinery to make the antibodies and put their components together into the right shape. This works very well when the cell is not expected to make much of the antibody in question. However, the cells we use to make antibodies are much less efficient at producing these drugs when we try and produce more of the product. As a result, we are not able to produce enough of these drugs and the cost and demand for them is therefore high e.g. the breast cancer drug Herceptin. The root of the problem is that when cells are asked to produce much more protein to meet our needs the machinery can no longer cope / the cells sometimes die or else don't produce antibodies of the right shape, of no clinical use whatsoever. It is largely agreed that this problem will become even serious as further antibody-based drugs are developed. The research proposed here will investigate how the cell machinery for making proteins works and examine whether, and in what ways, it can be manipulated to produce more antibody. We want to determine the different parts of this machinery that are limiting in terms of making the antibody, and then investigate how these parts work together to ultimately produce the antibody of interest. At present it is unknown if this is possible, and the process is poorly understood in the mammalian cells presently used to produce antibodies. We will employ a combination of new state-of-the-art technologies and approaches to take apart the antibody assembly line in mammalian cells in a step-wise manner, and then using the information gathered will determine the relationship between each step of the assembly process. Ultimately this should enable the manipulation of cells to change the balance of each step in the assembly line to produce more of the target antibody drug at reduced cost and higher quality. As stated above, this is extremely important as it is expected that with an increasing number of protein 'drugs' being developed we will lack the capability of producing large enough amounts to meet the required demand for these new drugs for the majority, as opposed to for those who can afford what must currently remain prohibitively expensive, but very effective, medicines.

Technical Summary

In recent years the capacity of expression systems (e.g. E. coli, animal cells) to produce recombinant proteins (rP) has advanced through improved growth medium, vector systems and bioprocessing. On-the-other-hand, our understandings of those cellular processes that constrain or limit rP production (rPP) during bioprocessing are poorly understood and open to conjecture. The proposed programme of research will utilise a combination of related approaches to investigate the hypothesis that it is the balance between mRNA levels and their regulatory features, mRNA turnover, the translational machinery, key chaperones, protein folding/degradation, and the cell's ability to coordinate and manipulate each of these processes to the demands of heterologous protein production that ultimately determines recombinant protein expression levels. To achieve this 5 related approaches will be employed. The 1st approach will characterise the diversity of translational machinery within cell populations and the effect of 5'-UTRs on mRNA translation. The 2nd approach will investigate those translational/post-translational mechanisms that limit the quantity and quality of rPP, determining the flux throughout the system by measurement of cell division rates, mRNA and protein levels and half-lives in model expression systems. The 3rd is targeted at the functional and physical link between translation and the chaperone CCT. The 4th approach will investigate the coordination and functional relationship between key members of the chaperone network, protein folding/turnover, and the relationship of these events to rPP. The 5th approach is to investigate disulphide bond formation pathways during protein folding. The proposal provides a rational approach towards understand the limitations and processes/molecular response(s) governing rPP in eukaryotic systems (outcome 1) and for identification of rational targets/strategies for enhancing rPP and for novel gene expression technologies (outcome 2).

Publications

10 25 50
 
Description i. Development of mathematical models using experimental data that describe the gene expression pathway for luciferase and an industrially relevant IgG (monoclonal antibody such as those used for the treatment of disease in the clinic) from mammalian expression systems confirming limitations are cell line specific, however translational limitations for the IgG heavy chain results in non-productive light chain/light chain dimmers being secreted in some high producers. These models have identified targets for cell engineering or can be used for screening.

ii. Demonstrating a direct link between the translational machinery and specific chaperones that influence protein synthesis/cell growth and identification of targets for cell engineering/screening strategies. Further, that manipulation of untranslated regions (UTRs) influences recombinant protein synthesis towards the end of culture and may allow for enhanced yields.

iii. An orchestrated panel of chaperones controls folding of recombinant proteins and yeast chaperone over-expression in mammalian cells can aid recombinant protein expression and disaggregation. Screening for the appropriate balance of chaperones in host cells is preferable to manipulation.
Exploitation Route These findings could be utilised and implementation of our strategies developed through the knowledge generated during this programme of work to further improve biotherapeutic recombinant protein (protein based drugs for use in the clinic for the treatment of disease) expression from mammalian cell lines either through cell engineering strategies or the use of novel screening approaches to select either host or recombinant cell lines with enhanced bioprocessing properties (enhanced yields or biomass).
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The findings from these studies are being used by the biopharmaceutical industry and those industries that manufacture biopharmaceutical protein based drugs to enhance the predictability of expression of mammalian cell lines and hence develop more efficient cell lines for use commercially. These findings also formed the basis of a further study funded in whole by industry to develop methods to 'fingerprint' mammalian cell lines, resulting in the protection (via patent) of the resulting technology, using MALDI mass spectrometry to rapidly fingerprint and select recombinant cell lines for commercial use.
First Year Of Impact 2010
Sector Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description Early Career Develoment Award
Amount £86,657 (GBP)
Funding ID ECF-2001-339 
Organisation The Leverhulme Trust 
Sector Academic/University
Country United Kingdom
Start 01/2012 
End 06/2016
 
Description Royal Society Industrial Fellowship
Amount £173,850 (GBP)
Funding ID IF130004 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 11/2013 
End 10/2017
 
Description Lonza Biologics Mass Spec Fingerprinting Project 
Organisation Lonza Group
Country Global 
Sector Private 
PI Contribution Development of a mass spectrometry fingerprinting technology for the identification of highly productive recombinant CHO cell lines.
Collaborator Contribution Cell line development, fermentation and bioreactor runs and validation, provision of reagents and expertise.
Impact IP/Patents, publications.
Start Year 2008
 
Description Pall Case Studentship 
Organisation PALL Europe
Country United Kingdom 
Sector Private 
PI Contribution A PhD CASE Studentship to investigate host cell proteins during bioprocessing including upstream and downstream bioprocesses.
Collaborator Contribution Access to industrial relevant cell lines, culture facilities and materials, consumables. knowhow and knowledge.
Impact PhD thesis, publications.
Start Year 2009
 
Title Rapid method for targeted cell (line) selection 
Description Development of mass spectrometry method to identify highly productive recombinant mammalian cell lines. Ideal came from original BBSRC work and then funded development via industry. 
IP Reference EP2447717 
Protection Patent application published
Year Protection Granted
Licensed No
Impact Invitation to talks.
 
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 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 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