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

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.

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.

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.