Particle assisted protein crystal nucleation as a protein purification platform for pharmaceutical manufacturing

Abstract

Slow protein crystal nucleation is a major barrier to using crystallization as a separation and purification strategy in protein drug manufacturing. This work investigates the use of functionalized nanoparticles in solution to act as in situ templates for initiating crystal nucleation. We used lysozyme, a protein with well-characterized crystallization conditions, to evaluate the nucleation rates of crystals grown on the functionalized particles. The nucleation rates were measured using an emulsion based technique that involves imaging protein-containing drops at regular intervals for several hours and then using an image recognition algorithm to count the number of crystals that formed in the drops. On a microfluidic chip, a supersaturated solution of lysozyme is mixed with a stream containing precipitants and functionalized nanoparticles. The functional groups used include bio-conjugates, which bind covalently with biomolecules, and are commonly used in diagnostic imaging and targeted drug delivery. The nucleation kinetics of lysozyme were compared in cases with and without added nanoparticles, both bare and functionalized. This work suggests that nanoparticles functionalized with bio-conjugates such as n-hydroxysuccinimide and maleimide may enhance crystal nucleation.