Development of nanoscale screening methods for the rapid optimization of biopharmaceutical formulations

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).