Protein aggregation in solution: a colloidal approach

As the number and volume of therapeutic proteins produced commercially has risen rapidly in recent years the problem of protein misfolding and aggregation have come to be more widely recognised. A common biopharmaceutical expression system is E coli which in high productivity systems can frequently express the recombinant protein as an inclusion body. Recovery of native structure requires solubilisation and then refolding of the protein. Due to competition between the processes of refolding and aggregation these can be low yielding processes carried out at high dilutions. Additionally during protein processing and in formulation protein aggregation can be manifest arising from concentration and solution conditions. While there has been much work in this area, much of the current work reported has a largely empirical emphasis due to both the experimental complexities and restricted to individual cases making it difficult to draw general learning and scope for new approaches Despite the enormous number of configurations of a polypeptide chain, often the protein folding into the native state can be rapid and robust. However, subtle and poorly understood variations in the forces between the solvent (as a function of ionic strength and pH) with the different part of the polypeptide backbone chain and it's side chains leads to misfolding and aggregated proteins. Surprising small variations can lead to substantially different outcomes! The pathways by which polypeptides misfold and aggregate, and the stability of these aggregates relative to the natively folded protein are of considerable industrial relevance and academic importance. The research work proposed here will be based on an experimentally focussed plan exploiting improvements in current research instrumentation for following protein aggregation using Dynamic Light Scattering (DLS), Analytical Ultracentrifugation (AUC), Dynamic Surface Tension/Surface Rheology (DSTSR), Fourier Transform Infrared Spectroscopy (FTIR) and Size Exclusion Chromatography (SEC). DLS especially has shown significant improvements in performance in recent years and is well suited for studying both steady state protein size as well as the kinetics of protein aggregation in solution. As a CMO Avecia potentially has access to a number of both model and industrially relevant protein systems and part of the pre-initiation activities of the project will be to determine the most useful set of proteins to include in the study. A suggested initial model is rPA (anthrax protective antigen) a 80kD single subunit protein with a known 3D structure, refolding conditions and known aggregation propensity. This and other model proteins will be experimentally investigated systematically with the plans to : (i) Determine the sensitivity to aggregation based on variations in pH, ionic strength, counter-ions, concentration, additives, surfactants and temperature (ii) Identify common mechanisms which drive aggregation phenomena including the importance of misfolding and partial unfolding. (iii) Apply well established colloidal aggregation (DVLO) and flocculation theories to problems in protein aggregation phenomena. (iv) Consider solution hydration/dehydration behaviour of proteins, exploiting the known like behaviour of water soluble polymers including the complex competition between proteins, counter ions, stabilisers for the water molecules. (v) Identify the role played by surfaces as well as contaminant particles in aggregation phenomena (vi) Propose methods for predicting protein stability to aggregation and well as strategies to mitigate the risks of aggregation.