Crystallization of proteins on polymeric membranes: how porosity, thickness and roughness affect the heterogeneous nucleation kinetics
Advancing the chemical engineering fundamentals
Interfacial & Colloidal Phenomena - III (T2-6c)
Keywords: protein crystallization, heterogeneous nucleation, microporous hydrophobic membranes
Interest for protein crystallization is today relevant in medical, pharmaceutical, agricultural and chemical industries. The innovative use of polymeric membranes as solid substrate promoting heterogeneous nucleation of proteins has been proposed by our group few years ago [1]. The experimental evidences of shorter induction times, accelerated nucleation rates and excellent diffractometric quality, obtained for various proteins (lysozyme, trypsin, catalase etc.), make this technique attractive [2] and simultaneously increase the need for a better understanding of the role of the membrane in the early stage of nuclei formation.
This work aims to investigate, both theoretically and experimentally, the effect of the morphological parameters of the membrane structure (porosity, thickness, roughness) on the crystallization kinetics of proteins, so creating the premise for a micro-scale design and a better engineering of the crystallization process.
The rate of solvent extraction through a microporous membrane, whose critical influence on the crystallization kinetics is confirmed by the linear dependence existing between the logarithm of the nucleation rate and the second power of the supersaturation, has been measured under different conditions (pH, precipitant concentration, temperature) and related to membrane porosity, tortuosity, pore size and thickness, under the regime of Knudsen-limited diffusion.
The Gibbs energy barrier to the formation of critical nuclei on a polymeric membrane, calculated as difference between the energy gained upon formation of a bulk phase and the energy required to form new surface area, was evaluated by solving the appropriate energy balance including the positive contributions of interfacial free energies of the nucleus-liquid and nucleus-substrate interfaces, and the negative contribution for the liquid-substrate interface displaced.
The Young equation - strictly valid for smooth, planar and homogeneous surfaces - has been modified in order to take into account the effect of porosity or roughness on the observed contact angle.
Model predictions - based on the contact angle data of protein solutions, porosity data from SEM and gas permeability tests, and AFM measurement of surface roughness -, significantly diverge from results of Volmer theory (valid under the same hypothesis of Young equation) already at porosity > 0.1 and for surfaces moderately hydrophobic (contact angle: 90-110°).
A substantial agreement with the nucleation rates measured for lysozyme and catalase on different polymeric supports (LiCl or poly-vinylpyrrolidone modified poly-vinylidene fluoride membranes, Teflon, modified poly-etheretherketone membranes) has been verified.
Modifications of the shape and size of crystals grown on different membranes, in most cases resulting in the elongation of the longitudinal axis, have been also observed and discussed.
[1] E. Curcio, G. Di Profio, E. Drioli, J. of Crystal Growth 2003, 247, 166
[2] G. Di Profio, E. Curcio, E. Drioli, J. Struct. Biol. 2005, 150, 41
Presented Thursday 20, 11:00 to 11:20, in session Interfacial & Colloidal Phenomena - III (T2-6c).
