Production of difficult to express essential bacterial proteins

Absynth Biologics (http://www.absynthbiologics.co.uk) has a novel platform of protein antigens for prevention of bacterial infections. Absynth's approach is a means of fighting the increase in antibiotic resistance by preventing infections and therefore directly reducing antibiotic use. Having already demonstrated its approach can work in principle, a key step for Absynth is the production of its proteins (or variants of them) efficiently and cost-effectively, so that they can be made to a consistent quality at an economic cost. This is where the expertise of the University of Manchester is important and forms the basis for the shared programme in this FLIP project. The University of Manchester, through the considerable expertise built up by Dr Eddie McKenzie and the Protein Expression Faciility (competencies that have been tested tested by successful optimsiation of expression of proteins in multiple academic and industrial collaborative project,) has proven knowledge and technical skills to develop efficient processes to produce the Absynth proteins, which will be tested to further optimize their use in preventing infection. The research project will see the proteins from Absynth being put through the unique profiling technology developed at the University of Manchester. The FLIP interchanger scientist (Ms Hirra Hussain) will undertake this work at both at the University of Manchester (6-7 months) and at Absynth's laboratories (5-6 months). The programme described presents a mutally beneficial exchange of knowledge and technologies (embedded into the work programme by a scientist who will work at both sites). Success in the project outlined will provide an exemplar approach for Absynth to expand into other projects and for the University of Manchester to apply its improved understanding of fundamental production mechanisms to other related molecules.

Absynth Biologics (http://www.absynthbiologics.co.uk) discovers and develops vaccines to prevent bacterial infections based on a platform for identifying novel protein antigen targets that harness the immune system and that use a dual-action mechanism. The antigens are selected on the basis of being essential and conserved: homologues have been identified in C.difficile, S. aureus and S. pyogenes. From a panel of twelve protein antigens, two (coded Ant2 and Ant3) were selected as the most effective from in vitro and in vivo immunogenicity and protection studies and a substantial body of data has been generated to support product development. However the proteins, which are the extracellular domains of membrane proteins, express at low levels (<30mg/L), tend to be insoluble, requiring use of solubilizing agents, and purification yield is low after dialysis and endotoxin removal. These technical issues must be overcome for successful product development and highlight the need for the project to focus on protein production to generate orders of magnitude higher expression levels and reproducible recovery of high quality protein in a purified and stable form. The expertise that will be applied at the University of Manchester is specifically facilitated through Prof Alan Dickson at the Centre of Excellence in Biopharmaceuticals (focused on understanding the processes that define difficult to express proteins; BBSRC Project Grants BB/M001164/1 and BB/M01701X/1) and the Protein Expression Facility (that has a track record with internal and external collaborations of trouble-shooting and curing low harvest of recombinant proteins in E. coli expression systems). The application focuses on the use of FLIP (and a talented post-doctoral exchanger) to embed a collaboration to solve the problems that are impacting and preventing the commercial development of Absynth's vaccine products, facets that will have wide implications for production of many other protein products.

The biological products that are the focus of this FLIP are one part of the very strong biopharmaceuticals sector in the UK. The UK sector works along the pipeline of manufacture, from drug design/discovery to research and development scale production through to scale up for production processes (bioprocessing) that generates kgs of purified product under GMP conditions for treatment of patients.
The work to be undertaken in this FLIP application integrates academic knowledge and technological know-how with the need to manufacture difficult to express proteins and protein variants that will offer innovative approaches to generate therapeutics. By integrating the FLIP exchanger with staff at Absynth and using the vast expertise developed by the head of the Protein Expression Facility at the MIB (Dr McKenzie) the FLIP will move the production of essential protein reagents to the next stage in the pathway to market, whilst generating data of value to many other related product pipelines.
The data and outputs to be derived from this FLIP programme will benefit
(1) The biopharmaceuticals industrial sector, in general, and Absynth, in particular, by providing insight into the manufacturing events that determine the amount and quality of difficult to express proteins reproducibly. Consequently, such proteins may be made more efficiently, rapidly and, due to predictability of success, at lower cost. Such outcome will strengthen the UK biopharmaceuticals sector.
(2) Patients, through greater certainty and, hence, lower cost of product manufacture. Industrial investment in manufacture of biological products is huge (ca £1Bn) and savings can be passed on to the end user and enable greater access to such life-changing medicines due to lower costs and greater likelihood of approval of such products by regulators.
(3) Bacterial vaccines for livestock should eliminate the need to include antibiotics in feed which is a strategic challenge faced by the farming industry in Europe and the USA.
(4) There are general impacts for international recognition of the UK manufacturing sector (biologicals/biopharmaceuticals, specifically, and Industrial Biotechnology in general). The outcomes of the FLIP (via communication in publications, presentations and patents) will add to the strong network of research leadership given by the UK sector to economic competitiveness in biopharmaceuticals, encouraging inward investment and securing existing jobs in the face of increased internationalization.
(5) Overall direct benefit to society - by potential for cheaper/better medicines and understanding the role of BBSRC support.
The project will involve staff at the University of Manchester (Prof Dickson and Dr McKenzie) and staff at Absynth in the UK (Alderley Park, Biohub). The impact for Prof Dickson and Dr McKenzie will be to provide information on platform screening approaches that can be used to initiate projects with other difficult to express proteins and, which by interpretation may allow the formulation of fundamental understanding of molecular events suitable for synthetic protein design. This will lead to publications and further research. The FLIP PDRA interchanger (Ms Hirra Hussain) will gain direct interaction with commercial activity, great training and the potential to develop his/her career from the experience of working with insight into the industrial and academic worlds. Staff at Absynth will gain the input of the experience of Prof Dickson and Dr McKenzie into the detailed understanding of molecular and cellular events that link to certainty in their product generation and manufacture scale-up. In addition, they will build stronger collaborative links with and gain experience of technologies held at the University of Manchester that have the potential to be incorporated into, and enhance, their standard work programmes. This will set the basis for further collaboration in other areas of R&D between these two organisations.