(386g) Towards Understanding Non-Integer Order Kinetics of Protein Denaturation

Understanding and controlling aggregation and denaturation of proteins is crucial for their downstream processing, including purification, sterilization and storage. Control of protein aggregation state is necessary for improvement of lifetime stability of therapeutic proteins as well as for rational development of novel separation and sensing procedures for bioprocessing. Protein stability is a particularly relevant issue today in the pharmaceutical industry, since protein based therapeutics provide treatments for numerous human diseases, and will continue to gain more importance as the number of therapeutic protein products in development increases. If a therapeutic protein cannot be stabilized adequately, its benefits to human health will be not realized. Achieving this goal is particularly difficult because proteins are often only marginally stable and are highly susceptible to chemical and physical degradation. Non-native aggregation is particularly problematic because it is encountered routinely during refolding, purification, sterilization, shipping and storage processes.

Here we present a detailed analysis of protein denaturation kinetics based on an extended Lumry-Eyring framework of protein denaturation [1] coupled with a general population balance model for protein aggregation. The resulting model is formulated in terms of key dimensionless parameters describing relative importance of folding, unfolding and aggregation kinetics of a protein. We found that there are several well defined denaturation kinetics regime in the space of these dimensionless parameters. Experimental data on denaturation kinetics are typically expressed in terms of a fraction of protein native state measured over the course of time under experimental conditions of interest. Although it is often possible to fit such data with a simple n-th order kinetics, where n is typically around 1.5, it is less clear that this apparent order can be used for reliable prediction of long term time evolution of native protein concentration (e.g., in shelf-life) or extrapolated to other protein concentrations.

Our modelling and analysis of experimental data on enzyme deactivation kinetics shows that additional experimental measurements, such as those obtained from static and dynamic light scattering [2-3], are necessary to uniquely determine mechanisms of protein aggregation and their role in denaturation kinetics. Moreover, we show that only for early stages of simple aggregation of fully unfolded protein there is a single unique order for denaturation kinetics, where it is equal to exactly 1.5. However, interplay of folding/unfolding kinetics with aggregation results in time-dependent profile of denaturation kinetics order which can vary in a wide range between 1 and 2. Such reaction order profiles are distinctly different in each of the denaturation kinetics regimes identified in the space of dimensionless kinetic parameter.

[1] Roberts, C.J. J. Phys. Chem. B 2003, 107, 1194-1207. [2] Sandkuhler, P.; Sefcik, J.; Morbidelli, M. J. Phys. Chem. B 2004, 108, 20105-20121. [3] Lattuada, M.; Wu, H,; Sandkuhler, P.; Sefcik, J.; Morbidelli, M. Chem. Eng. Sci. 2004, 1783-1798.