Mutual diffusion of proteins in cold concentration gradients measured by holographic interferometry

Abstract

Protein mutual diffusion in cold concentration gradients is important for development of processes involving biopharmaceutical formulations. The diffusivities of α-chymotrypsinogen (aCgn), ovalbumin (Ova) and IgG1 were measured from −10 °C to 25 °C, by holographic interferometry at protein concentrations ranging from 5 g/L to 31.5 g/L in multicomponent systems containing sucrose (up to 10% mass). A dialysis step enabled to measure mutual diffusion of the proteins under equi-chemical potential conditions of the other components. The protein could therefore be considered a single component in a continuous fluid, as defined by Stokes-Einstein (SE) assumption. Overall, the SE model provided a reasonable description for mutual diffusion of all the proteins studied, with higher accuracy for aCgn and IgG1 in the full range of temperatures and concentrations. Ova with 10% sucrose, was less accurately described by this model, diffusing 20% faster than predicted. It was also observed a non-linear temperature dependence for the diffusion of Ova in a 10% sucrose medium: this was only apparent at a temperature near −10 °C. The Williams Landel Ferry (WLF) model was used to provide a measure of the non-Arrhenius regime taken the glass transition temperature (Tg) of the mixture Ova+ sucrose as reference (WLF constants obtained are C1,g ~ 18 and C2,g ~ 1). Overall, the present work offers a new approach to obtain experimental diffusion coefficients of relevant protein formulations under sub-zero conditions. The accuracy of classical transport models was explored in this manuscript which can help to gather insight into transport properties of globulins and Mabs under concentration polarization gradients occurring during freezing: deviations to the linear diffusion-temperature relation above Tg – i.e., “continuous medium” diffusion predicted by SE model - can be quantified by the WLF model. Diffusion coefficients may be further related with protein conformational changes and this was proposed for Ova.