Predominantly protein/peptide based, the development and formulation of biopharmaceutical products is frequently hindered by aggregation of the active biomolecular species. Indeed, often the time-consuming, and thus costly step is the identification of appropriate formulation conditions that minimize/prevent aggregation. Appropriate formulation conditions are currently identified utilizing a range of analytical techniques employed for the detection and quantification of aggregates. The current standard for this is size-exclusion chromatography although, due to a range of reported problems, recent years have seen the application of a range of other approaches, including sedimentation velocity and light scattering. Using these techniques, typically the optimal formulation is identified through evaluation of aggregate level in a wide range of test formulations that have been exposed to different storage conditions. Here we propose to develop a step-change to this strategy and develop novel platforms based on the detection of aggregation at the nanoscale, to allow rapid screening and identification of optimal formulation conditions. Importantly the project will build on the proven track record of the academic supervisors in utilizing biophysical and surface characterization techniques for the investigation of biomolecular interactions. The industrial partner, Molecular Profiles Ltd, will provide invaluable complementary business related experience and training in the application of analytical approaches to solve formulation issues for the pharmaceutical industry. Initial studies will focus on the development of an assay to screen for optimal formulation conditions to prevent aggregation in solution. A potential format to be exploited will build on that developed by the group of Engel for high resolution imaging of membrane proteins (Müller (1999) Biophys.J. 76, 1101). To develop this approach for protein aggregation screening, AFM probes will either be directly functionalized with protein or, to provide an improved control of probe-surface interaction area, with a spherical colloid particle coated with the biomolecule of interest. Forces will then be recorded by bringing the functionalized AFM probe into and out of contact, at frequent spatial location intervals, with a sample surface also functionalized with the biomolecule of interest. We would then seek to 'tune' the surrounding test formulation media (i.e. by changing its composition, pH and/or ionic strength) in order to reduce/eliminate the magnitude of probe-sample, and thus the biomolecule-biomolecule, interaction. The validity of this approach will be confirmed through the use of model biomolecular systems (e.g. monoclonal antibodies and insulin), and formulations conditions, including extreme conditions reported in the literature in which such molecules are known to be prone to aggregation. Data will also be compared with that from parallel experiments performed with techniques currently employed for aggregate detection (e.g. available via Dr Scott). In later studies, we will seek to test the applicability of the assay through studies of molecules with more commercial interest (available through existing collaborative links with Molecular Profiles Ltd.), including systems that have been abandoned during development due to difficulties in aggregation. If time permits within the project we would also seek to extend such studies to investigate other avenues of research, and in particular the use of newly available AFM modules (e.g. Harmonix, Veeco) that will permit the high-speed spatial mapping of tip-sample interactions with nanometre scale resolution. Possible areas of investigation would be to use such methods to explore the aggregation state within lyophilized biopharmaceutical formulations, and also to test the sensitivity of chemically functionalized tips to detect changes in the protein chemistry associated with aggregation (e.g. deamidation, oxidation).

Mutlicellular organisms possess tissues containing various cell types. Each cell type differentiates to perform defined functions. We are interested in processes that occur in specific cells. The aim is to identify mechanisms allowing differential gene expression in particular cells, and the consequences of this differential expression in those cells. For example, Applicant 1 aims to identify differences in methylation of promoter elements between cell-types, applicants 3-5 wish to determine transcript abundance from specific cell-types, and then use that information to generate developmental and circadian networks, or understand responses to pathogen attack or cell signalling. As few as ten plant cells have been collected via LCM prior to analysis of gene expression, and so all the work proposed is feasible and we should make significant advances in each of our fields.

Microscopy and cellular imaging has hitherto been amenable to small-scale experimental samples. New approaches to cell-based analysis are required to meet the demanding needs of cell-biology, genetics and experimental therapeutics. At the core is the need to convert images into numbers, this is a significant challenge which would greatly enhance the ability of the biologist to effectively interrogate and interpret complex data. To achieve large-scale imaging experiments we need to address critical issues of data bottlenecks. High-throughput screening instrumentation has been available in the commercial sector for the last five years, therefore the objective is to bring these capabilities to academia. Such equipment not only greatly augments research capacity but provides new opportunities taking a systems approach to cell-biology.

Molecular compounds, including pharmaceuticals, can often adopt several crystal structures. These crystals, which contain the same molecules with different arrangements of the molecules in space, are known as polymorphs. An example would be for a u shaped molecule, which might be able to form the two polymorphs: uuuuuuu & ununun. Polymorphs are important as they can have different physical properties, despite containing exactly the same molecules. Solubility is one physical property that can differ between polymorphs, and is a critical parameter in controlling drug dosage. The pharmaceutical industry therefore screen all new drugs for polymorphism, and this screen is a requirement for getting the drug onto the market. Current state of the art screening methods require a significant amount of sample and significant amounts of time. This means that screening for polymorphism has to occur at a far later stage in the drug development pipeline than we would like.This research will look at a new method constrained crystallisation for screening compounds for polymorphism which requires far less compound (sub-milligram amounts rather than 10-50 grams) and less time (minutes rather than weeks). We will look at the use of in-situ Raman microscopy to characterise the polymorphs as they form, which will allow us to identify the polymorphs, understand how the molecules are linked together and to understand how one polymorph can transform into another. The recent development of new highly sensitive detectors for Raman microscopy, and the recent investment of 270k by the University of Nottingham in a world-class Raman microscope, are the key factors that enable this research. We will be working closely with Dr Graeme Day (Cambridge) to model the Raman spectra, and with Molecular Profiles (Nottingham) to develop constrained crystallisation to the level where it can be used in an industrial context.