The World Health Organisation recognises HIV/AIDS as the number one infectious disease in the world. Doctors and scientists know that the best way to stop this disease would be to develop a vaccine to stop the 14,000 new infections with AIDS virus that occur each day worldwide. The difficulty is that scientists do not know whether an AIDS vaccine needs to stimulate the production of antibodies, molecules in the blood that recognise the virus and stop it from infecting new cells, or killer T cells that eliminate virus infected cells before the infection can spread further. Alternatively the vaccine may need to do something different to be fully effective. The team at NIBSC are studying an animal model of HIV and have found that animals vaccinated with a disabled form of the virus first are resistant to disease causing strains. This group think that events that occur within 3 weeks of vaccination are critical for the vaccine and that many of these occur in and around the gut. As a result, they will focus on unravelling these early events to enable a better AIDS vaccine to be designed and developed in the future.

Botulinum toxins, some of the most poisonous naturally occurring substances are proteins produced by the bacterinum Clostridium botulinum. They are neurotoxic, that is toxic to the nerve cells and responsible for causing botulism, most often associated with eating food containing toxin or in hard drug users. The toxins cause respiratory and muscle paralysis, and even death, by blocking the nerve function. Treatment is possible with antitoxins, provided they are given to patients on time. Although highly toxic in extremely small quantities toxin can be administered safety to treat painful muscle spasms and involuntary muscle contractions. It is increasingly applied to many new medical conditions and for cosmetic purposes. Botulism is diagnosed by injecting animals with body fluids from patients and in suspected food samples to confirm presence of toxins. In pharmaceutical industries manufacturers of therapeutic toxins and antitoxins require many animals in severe lethality assay to confirm safety, potency and stability. Alternative methods developed to date have limitations by either still relying on animal or for provision of tissues or for reflecting only one of several factors that contribute to toxin mode of action in animals. One approach to avoid these limitations is to develop in vitro functional assay which offers the potential for entire animal replacement because of involving all key steps of botulinum intoxication. Because botulinum toxin induces paralysis by blocking the release of chemicals (neurotransmitters such as acetylcholine) at the neuromuscular nerve endings, relevant in vitro assays must focus on this activity. Established human neuronal cell lines and human stem cells have not offered the required sensitivity and studies to date have confirmed need for differentiation into neuronal like structures before these cells can be used with neurotoxins. In this work we will apply differentiated cells onto micro-electrode arrays (MEAs) in order to perform measurement of cell function in situ. We will support these studied by also looking at cell recycling activity by staining of the components involved in neurotransmitter release and by selectively detecting key proteins within the cells which are attacked by toxins and are also associated with neurotransmitter release. This new approach reflects the full function of the toxin yet it is entirely non animal orientated and will not rely on any animal experimentation used in other applications and strategies.

By 2025 targeted biological medicines, personalised and stratified, will transform the precision of healthcare prescription, improve patient care and quality of life. Novel manufacturing solutions have to be created if this is to happen. This is the unique challenge we shall tackle. The current "one-size-fits-all" approach to drug development is being challenged by the growing ability to target therapies to only those patients most likely to respond well (stratified medicines), and to even create therapies for each individual (personalised medicines). Over the last ten years our understanding of the nature of disease has been transformed by revolutionary advances in genetics and molecular biology. Increasingly, treatment with drugs that are targeted to specific biomarkers, will be given only to patient populations identified as having those biomarkers, using companion diagnostic or genetic screening tests; thus enabling stratified medicine. For some indications, engineered cell and gene therapies are offering the promise of truly personalised medicine, where the therapy itself is derived at least partly from the individual patient. In the future the need will be to supply many more drug products, each targeted to relatively small patient populations. Presently there is a lack of existing technology and infrastructure to do this, and current methods will be unsustainable. These and other emerging advanced therapies will have a critical role in a new era of precision targeted-medicines. All will have to be made economically for healthcare systems under extreme financial pressure. The implications for health and UK society well-being are profound There are already a small number of targeted therapies on the market including Herceptin for breast cancer patients with the HER2 receptor and engineered T-cell therapies for acute lymphoblastic leukaemia. A much greater number of targeted therapies will be developed in the next decade, with some addressing diseases for which there is not currently a cure. To cope, the industry will need to create smarter systems for production and supply to increasingly fragmented markets, and to learn from other sectors. Concepts will need to address specific challenges presented by complex products, of processes and facilities capable of manufacture at smaller scales, and supply chains with the agility to cope with fluctuating demands and high levels of uncertainty. Innovative bioprocessing modes, not currently feasible for large-scale manufacturing, could potentially replace traditional manufacturing routes for stratified medicines, while simultaneously reducing process development time. Pressure to reduce development costs and time, to improve manufacturing efficiency, and to control the costs of supply, will be significant and will likely become the differentiating factor for commercialisation. We will create the technologies, skill-sets and trained personnel needed to enable UK manufacturers to deliver the promise of advanced medical precision and patient screening. The Future Targeted Healthcare Manufacturing Hub and its research and translational spokes will network with industrial users to create and apply the necessary novel methods of process development and manufacture. Hub tools will transform supply chain economics for targeted healthcare, and novel manufacturing, formulation and control technologies for stratified and personalised medicines. The Hub will herald a shift in manufacturing practice, provide the engineering infrastructure needed for sustainable healthcare. The UK economy and Society Wellbeing will gain from enhanced international competitiveness.

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Investigators: Paul Dalby (Biochemical Engineering, UCL), Paul Matejtschuk (NIBSC, HPA) We aim to establish an automated microscale platform to assess therapeutic protein, vaccine and cell formulations, and identify any correlations (or lack of) between physical property measurements, forced degradation studies, and the long-term shelf life of liquid and freeze-dried formulations Significance: To minimise the chemical and physical degradation of biologics, excipients can be added in complex formulations. The optimisation of freeze-dried or liquid formulations is currently empirical, and requires many time and sample-consuming experiments. To save time and materials, initial formulation screens often measure properties such as melting temperatures (biologic thermostability - Tm & glass transitions - Tg), aggregation propensity (B22 values), or aggregate formed by forced degradation at elevated temperatures (light scattering, SEC). The validity of using such measurements as indicators for long term (1-2 yr) storage stability is still debated as degradation mechanisms can be complex. We recently established accurate microscale methods to subject small quantities of biologics to bioprocess stresses such as protein refolding, agitation and freeze-drying [1,2] (EPSRC studentship with NIBSC), and rapidly evaluate their thermostability [3,4], activity [1,2,5] and propensity to aggregate [1,6]. Recent BBSRC/BRIC and follow-on (BB/FOF/272) projects established microfluidic techniques to measure the thermostability of 85000-fold less protein [7]. We aim to integrate the microscale techniques to simultaneously evaluate multiple bioprocess stresses and molecular properties, obtain a body of data for a range of biologics and formulations, then identify or disprove potential correlations between initial (Tm, Tg, B22 by DLS, activity), medium-term (forced degradation) and long-term (shelf life) properties. Workplan: Year 1 - training in analytical and bioprocess skills at NIBSC. Formulate and freeze dry a range of biologics [8,9] in process vials. Evaluate their biological activity and aggregation. Learn existing microscale and DoE techniques at UCL and NISBC, and validate them in process scale vials. Integrate microscale techniques to allow parallel evaluations of the impact of formulations on liquid and freeze dried sample stability, and sample properties. Year 2 - combine microscale techniques and Design of Experiments (DoE) to derive parallel surface response models for the impact of formulations on product Tm, Tg, B22, aggregation, tolerances to freeze-drying and forced degradation (liquid and freeze-dried), for a wide range of proteins, vaccines and cells. Year 3 - evaluate long-term storage (0-12 months at 70 to 45oC) of optimal and selected sub-optimal liquid and freeze-dried formulations in 10 ml vials. Measure biological activity, aggregates and misfolded forms, oxidation and deamidation by standard HPLC, DLS and LCMS techniques at UCL and NIBSC. Compare data to equivalent short-term property measurements, and medium term forced degradation studies from yr2, to identify or disprove correlations between them. Determine the best microscale DoE, measurement and bioprocess stress strategy for predicting 10 ml vial formulations with long-term stability. 1. Mannall GJ, Myers JP, Liddell J, Titchener-Hooker NJ, Dalby PA 2009 Biotech Bioeng 103:329 2. Grant Y, Matejtschuk P, Dalby PA 2009 Biotech Bioeng 104:957 3. Aucamp JP, Cosme AM, Lye GJ, Dalby PA 2005 Biotech Bioeng 89: 599 4. Aucamp JP, Martinez-Torres RJ, Hibbert EG, Dalby PA 2008 Biotech Bioeng 99:1303 5. Miller OJ, Hibbert EG, Ingram CU, Lye GJ, Dalby PA 2007 Biotech Letts 29:1759 6. Ahmad SS, Dalby PA 2010 Biotech Bioeng 108:322 7. Gaudet M, Remtulla N, Jackson SE, Main ERG, Bracewell DG, Aeppli G, Dalby PA 2010 Protein Science 19:1544 8. Hubbard A, Bevan S, Matejtschuk P 2007 Anal Bioanal Chem 387:2503 9. Matejtschuk P et al 2009 Biologicals 37:1-7