(5s) Fundamental and Applied Investigations in Protein and Pharmaceutical Crystallization

My research is geared toward acquiring fundamental understanding about the governing processes of nano particle formation and developing innovative methods and techniques to apply this knowledge in solving problems of great interest to the industry and academia. To that end, my current research focus is on studying the particulate processes in protein and pharmaceutical systems. The insights obtained through my studies will assist not only the practice of industrial crystallization, but also the crystallization of proteins for structure determination, and rational drug design methodology. A few specific issues that I will address in this poster are:

1. Influencing Polymorph Selectivity by Regulation of Diffusive Mixing in Microfluidic Devices

Rapid polymorph screening techniques that reduce the time, effort and material consumed are an essential requirement in pharmaceutical industry. Microfluidic technology offers an effective approach in developing innovative methods and techniques to address this issue. Here, a microfluidic mixer design that facilitates polymorph screening is discussed. This design effects anti-solvent crystallization utilizing the fact that mixing at the micro-scale is by diffusion only (no turbulence). Also, the rate of mixing and the composition of the liquid phase can be varied by changing the flow rates and concentrations, thus influencing the polymorph selectivity. The results of our experiments on some model systems are presented and the applicability of the concept towards the development of a high throughput microfluidic device for polymorph screening is discussed.

2. Understanding Critical Supersaturation in Solution Crystallization

A significant interest exists in industry and academia towards approaches that facilitate rapid determination of useful crystallization kinetic data. Towards that end, we developed an evaporation-based crystallization platform. Earlier studies using this platform revealed the existence of a ?critical supersaturation' in the crystallization of a wide range of model compounds [1]. It was observed that this critical supersaturation is strongly correlated to the solubility of the compound.

A theoretical analysis of this observation using the classical nucleation theory is presented. Since the rate of evaporation of solvent is very slow, we consider the droplet to be in a stationary nucleation regime at all points of time and examine the evolution of the ?quasi-equilibrium cluster size distribution' in the droplet [2]. The critical supersaturation can then be explained as the minimum driving force required for the formation of ?critical nuclei' that have a maximum chance of survival in a given volume of solution. Preliminary analysis suggests that the classical nucleation theory qualitatively explains the experimental data [3] and captures the significant trends. The meaning of the ?critical supersaturation' in light of this analysis is explored and the implications toward solution crystallization are discussed.

3. Identifying Key Solution Parameters in Protein Crystal Nucleation

Crystallization of biological molecules is the first step (and usually a rate limiting step) in structure determination through X-ray diffraction. Formation of stable crystals of proteins and protein/nucleic acid complexes from solution is dominated by the kinetics of nucleation of these clusters. Given the sparse amount of kinetic data available, the nucleation rates of a model protein system, tetragonal hen egg-white lysozyme (HEWL), were measured at various solution conditions by the method of initial rates [4]. It was observed that, for HEWL / NaCl system, equal solubility conditions produce equal nucleation kinetics at a given initial protein concentration, even when the solution conditions such as pH, ionic strength and temperature are different [5]. This observation, if shown to be valid for all protein systems, could have far reaching consequences, as it will significantly narrow the spectrum of parameters to be explored to identify optimal nucleation and crystallization conditions.

References:

[1] Guangwen He, Venkateswarlu Bhamidi, Reginald B. H. Tan, Paul J. A. Kenis and Charles F. Zukoski, Cryst. Growth Des., 2006 (in press)

[2] Kashchiev, D., ?Nucleation: Basic Theory with Applications?, 2000, Butterworth-Heinemann, Oxford.

[3] Venkateswarlu Bhamidi, Guangwen He, Paul J. A. Kenis and Charles F. Zukoski, ?Critical Supersaturation in Solution Crystallization as a Function of Equilibrium Cluster Size Distribution?. (manuscript in preparation)

[4] Bhamidi, V., S. Varanasi, and Schall, C. A., Cryst. Growth Des., 2 (2002), 395-400.

[5] Bhamidi, V., S. Varanasi, and Schall, C. A., Langmuir, 21 (2005), 9044-9050