Porject title- An upstream platfrom for the production of high grade heterologous proteins in Pichia pastoris Background and significance Pichia pastoris is finding increasing use as a host for the expression of a wide variety of recombinant proteins in the laboratory and in industry. It is capable of successfully secreting the product of interest thus easing overall processing. However, the product titres obtained are usually not very high. As a result most processes include high cell density fermentations to allow large quantities of the product to be made. On the other hand, high cell densities are known to result in reduced cell viability, increased cell debris, as well as an increase in the concentration of amorphous and colloidal organic constituents, thus posing a challenge to the purification stage. In addition the high biomass requires a dilution step to accommodate most recovery steps such as centrifugation. Challenges There are several challenges (amongst others) currently facing HCD processing 1. Extension of cell viability 2. Enhancement of protein expression 3. Enhancement / maintenance of product quality Cell viability which impacts on the level of contaminants in broth (e.g.cell debris) and protease action is the result of both cell design and processing conditions namely stress exerted by foreign protein production, length of culture, and potentially, pH levels and adaptability of cell to the inducer. Furthermore, the interrelation between the 'quality' (e.g. robustness and physico-chemical characteristics) of the desired molecule and the process conditions is not yet well established. Objectives a) To investigate the interaction between culture conditions, cell viability distribution and product expression with the view to enhance production ,reduce the burden on downstream processing and achieve optimal cell separation. b)To investigate the impact of the upstream conditions on product quality. Where appropriate the project will draw on existing tool box of technology available in the UCL Advanced Centre for Biochemical Engineering

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Biopharmaceutical manufacturing continues to evolve with an increased emphasis on underpinning science and engineering. Effective deployment of contemporary knowledge in science and engineering throughout the product life cycle will facilitate manufacturing efficiencies and regulatory adherence for biopharmaceuticals. Fundamental to this paradigm shift has been the drive to adopt an integrated systems approach based on science and engineering principles for assessing and mitigating risks related to poor product and process quality. Changes have been enabled as a consequence of the regulatory authorities introducing a new risk-based pharmaceutical quality assurance system. The traditional approach to manufacture has been to accommodate product variability into the specifications and fix operational strategies to ensure repeatability. Developments in measurement technology have invited changes in operational strategy. This revised approach is based on the application of Quality by Design (QbD), underpinned by process analytical technology (PAT) to yield products of tighter quality and more assured safety. QbD is defined as the means by which product and process performance characteristics are scientifically designed to meet specific objectives. Practical improvements therefore demand a knowledge base of science and engineering understanding to identify the interrelationship between variables and integrate the learning into different manufacturing scenarios. The focus of the Centre is to address the challenges emerging from this paradigm shift and to train a new generation of students with competencies in all stages of commercial biopharmaceutical process development. Critical to this is to ensure they have the skills to work at the discipline interfaces in the areas of biosystem development, upscaled upstream process engineering, and the engineering and development of downstream processing. The training will be formulated around three elements that form the backbone of achieving an enhanced understanding of the process. The three elements are (i) Measurement, Data and Knowledge Management, (ii) Enhance Available Knowledge and (iii) Use Knowledge More Effectively. The power of the approach being adopted is that it is equally applicable to established bioprocesses based on microbial and animal cell culture, as well as emerging areas including stem cells, marine biotechnology and bio-nanotechnology. The rationale for proposing a Centre in this area is to address a well recognised problem, a lack of appropriately trained personnel, who will deliver the next generation of biopharmaceutical development. These issues have been clearly articulated in a series of reports. SEMTA reported that over a quarter of bioscience companies do not have sufficient science skills. 39% of bioscience/pharmaceutical companies have long-term vacancies; with 22% having skill shortages in the science arena (five times that for other sectors). Lord Sainsbury, concerned at the rapidly changing nature of the bioscience business, set up the BIGT and commissioned Bioscience 2015. One of the strong messages raised was the serious shortfall in trained staff. Furthermore a quantitative assessment of the increase needed of trained people entering the sector was made by bioProcessUK. They estimated an increase of 100 trained personnel was required on top of the current 150 doctoral level candidates graduating per year. It is not simply a matter of increasing the number of trained persons. The Centre will also address the limitations of the current UG training of engineers, chemists and biologists which does not prepare them for the challenge of working in process development distinguished by disciplinary interfaces. The proposed programme will address a strategic shortfall and produce a new generation of graduates with the appropriate inter-disciplinary skills to drive both the research agenda and knowledge transfer of underlying concepts into industry.

Sustainable production of safe chicken is an international priority and it is estimated that in the next 20 years chicken production will have to quadruple to satisfy growing global demand. The key question is whether this can be done in a way that does not increase the public health threat of contaminated chicken meat and preserves chicken health and welfare. Most chicken meat consumed internationally is produced in large-scale intensive (broiler) systems and most birds in the UK (>75%) are Campylobacter-positive at retail, mainly with C. jejuni, posing a huge public health threat. Campylobacter is the most common cause of bacterial diarrhoea in the UK and despite millions of pounds of research funding it is estimated that contaminated chicken caused >500000 human campylobacteriosis cases in the UK in 2016 with around 100 deaths, mainly in elderly people. Infection is characterised by severe abdominal pain and acute (sometimes bloody) diarrhoea and costs the UK economy over £1 billion per year. In addition, Campylobacter are not only major chicken-associated human pathogens, they also compromise the health, welfare and performance of broilers. Campylobacter contamination of chicken takes two forms. First, surface contamination of carcasses, as a result of spillage of gut contents during slaughter, can lead to cross-contamination in the kitchen. Second, and perhaps of greater importance than currently thought, is contamination within muscle and liver tissues, which increases the health risk by facilitating bacterial survival during cooking. Until recently it was believed that Campylobacter only colonised the lower gut of the chicken (the caecum). However, spread from the gut to edible tissues is associated with the ability of certain Campylobacter strains to colonise the upper intestine of the chicken, where the gut lining (mucosa) is more easily damaged. As Campylobacter comprise a diverse population in broilers, with different strains varying in their effects on gut integrity and their ability to spread to edible tissues like liver and muscle, it is important to better understand the host-pathogen interactions of different types if the bacteria are to be controlled in chickens and the public health threat reduced. In particular, it is essential to identify the key host immune responses and the bacterial genes most important in these interactions - and in colonisation of the whole gut and extra-intestinal spread. This information, which is currently not available, is essential for the development of immunity-based and other control measures. This multidisciplinary research programme will enhance understanding of the influence of Campylobacter strain on bird gut health, host innate immune responses and spread to edible tissues and thus the public health threat. The quantitative information and modelling will be used to give direct advice to industry about Campylobacter infection biology in broiler chickens, providing an unprecedented basis for interventions to mitigate the on-going challenge of Campylobacter contamination in chicken meat. These interventions potentially include new vaccines and/or genetically more resistant chickens.

Project title: Rapid microscale evaluation of the impact of fermentation conditions on inclusion body formation, solubilisation and protein refolding yields. Hypothesis: That protein refolding steps can be performed and optimized at a microlitre scale and that the technique, once established, can rapidly evaluate the impact of earlier bioreactor stages on whole bioprocess performance. Significance and Background: Microscale processing techniques offer the potential to speed up the delivery of new drugs to market to reduce development costs and thereby increase patient benefits. We and others have shown that in selected cases the study of bioprocess unit operations in microwell plate formats and the use of automation can significantly enhance experimental throughput and facilitate the parallel evaluation of a large number of process conditions (eg Nealon et al. 2005, Jackson et al., 2006, Lacki, 2007). While the majority of microscale studies have focused on microbial fermentation, by comparison little work has been done on downstream processing operations. This potential bottleneck requires considerable attention if significant step-wise process enhancements are to be gained. In particular the impact of fermentation conditions on whole process performance must be understood if downstream processing is not to become rate limiting (Micheletti and Lye, 2006). A particular example that could benefit from this approach is the refolding of recombinant protein from inclusion bodies (IB). Protein refolding yields at industrially relevant concentrations are restricted by aggregation of protein upon dilution of the denatured form. A number of studies have investigated chemical (Buswell and Middleberg, 2002, Mannall et al., 2007a) as well as physical (Mannall et al., 2005) factors affecting the dilution refolding in small (20-200ml) bioreactors, however for the majority of proteins a large number of refold conditions usually need to be tested in order to optimize this processing step. The use of microwell plates as a format in which to perform protein refold experiments has recently been preliminary investigated (Mannall et al., 2007b). Not only has it been shown that refold reactions scaled well between microwell and bench scale operations but it was also demonstrated that a significant amount of information can be gained in a short period of time using a small amount of the valuable IB-derived protein. In this project we propose to radically enhance the microwell approach for the rapid optimization of the protein refolding step by: 1) adopting a whole process approach to optimization which investigates the effect of fermentation conditions on subsequent refolding yields and product quality 2) establish the automation of the refolding step, both in terms of liquid handling operations and associated analytical methods, to speed up the investigation of multiple variables under different mixing conditions. Research Program: 1) Establish and demonstrate an automated microwell-based dilution-refolding system using an industrially relevant strain provided by Avecia Biologics 2) validate the dilution-refolding system by comparing the yields obtained to standard bench scale operations 3) generate E. coli fermentation broths using different carbon sources, induction time and window and harvest time strategies and then use the microwell format in investigating the effect of the upstream parameters on IB solubilisation and refolding yields. Jackson et al. (2005) J. of Membrane Sci., 276, 31-34. Nealon et al. (2006) Chem. Eng. Sci., 61, 4860-4870. Lacki et al. (2007) ACS National Mtg., Boston BIOT-472. Micheletti and Lye (2006) Curr. Opinion in Biotechnol., 17, 611-618. Buswell and Middleberg (2002) Biotech. Progress, 18, 470-475. Mannall et al. (2007a) Biotech. Bioeng., 97, 1523-1534. Mannall et al. (2006) Biotech. Bioeng., 93, 955-963. Mannall et al. (2007b) Biotech. Bioeng., submitted.