'BRIC DOCTORATE PROGRAMME' - Development of single molecule assays for the detection of aggregation within high concentration protein therapeutics

Protein based biopharmaceuticals comprise a major proportion of the medicines currently under development. Although the number of such products is increasing, they present considerable challenges during development, and preventing unwanted aggregation of the active remains a major issue. Protein aggregation can be unpredictable and result in significant difficulties at any stage of biopharmaceutical development e.g. from formulation, through to manufacture and storage. This is compounded by an increasingly frequent requirement to formulate such medicines at high protein concentration (>50mg/ml); conditions which have a tendency to promote aggregation. Protein aggregates are currently detected and quantified utilizing a range of analytical techniques. The industry standard is size-exclusion chromatography (SEC) although the increased use of orthogonal approaches, such as analytical ultracentrifugation and light scattering is now common. Optimal conditions are identified through a time consuming evaluation of aggregate type and level in a wide range of test formulations, exposed to different storage conditions. For high protein concentration formulations the samples also require dilution prior to analysis, hence the obtained aggregate profile may not fully represent that actually present within the formulation. The development of new approaches to identify aggregation within high concentration formulations, at its earliest possible stages would therefore significantly reduce the time, effort and costs associated with development. In collaboration with Pfizer, this studentship will explore the ability of single molecule force measurements to fulfil the need for new approaches in this area. Such measurements are obtained by recording forces acting on a force transducer (e.g. atomic force microscopy (AFM) cantilever) as its surface is brought into and out of contact with an opposing sample surface. Dr Allen and Professor Williams have considerable experience in utilizing such measurements to unfold single proteins or break a range of biomolecular complexes. Measurements at the single molecule level are attractive for applications early in pharmaceutical research and development, when amounts of material can be small. As the measurements also involve the separation of individual proteins after they are forced into intimate contact, we propose that they provide an experimental system more reflective of the conditions within high protein concentrations. Here we wish to explore this hypothesis, and the use of single molecule forces measurements for the detection and prediction of aggregation with protein based biopharmaceuticals. Initial experiments will focus on simple formats in which model proteins (e.g. a monoclonal antibody) will be immobilized to the transducer and sample surface, and forces recorded in a range of formulation conditions (e.g. those known to exacerbate and prevent aggregation). In later studies, we will aim to extend this format to other biomolecular actives (e.g. peptides), and to test for interaction and aggregation potential with container surfaces, stopper materials etc. A longer-term goal of the project will be to develop and extend the use of protein constructs, as already employed in single molecule protein unfolding studies, for aggregate detection and screening. Such molecules contain repeats of proteins/protein domains, which are mechanically unfolded by the force transducer. The obtained data directly provide information on the propensity of the protein to unfold under force, and may provide information relevant to understanding how proteins would respond to shear stresses during formulation and/or processing. Throughout these studies complementary biophysical data on protein aggregation will be obtained through collaboration with Pfizer; A key goal of the industrial placement will be for the student to obtain such data using conventional aggregate analysis techniques.