Spray freezing into liquid nitrogen for highly stable protein nanostructured microparticles.

The objectives of this study were to produce nanostructured protein microparticles with the spray freezing into liquid (SFL) cryogenic process and to demonstrate a smaller degree of protein denaturation and aggregation than observed in spray freeze drying (SFD). Nanostructured microparticles were formed by atomization of an aqueous buffer solution containing bovine serum albumin (BSA) with and without excipients beneath the surface of a cryogenic liquid. Lyophilization was used to sublime the water in the frozen particles. The resulting BSA dry powder was characterized by size exclusion chromatography, Fourier-transform infrared spectroscopy, scanning electron microscopy (SEM), light scattering, and specific surface area analysis. SEM revealed highly porous microparticle with features smaller than 500 nm. The specific surface area of the BSA microparticles ranged from 19.2 to 97.7 m(2)/g as a function of the total protein and excipient content in the aqueous feed solution. SFL produced less denaturation and aggregation of protein monomer than SFD, despite the extremely high surface areas in both processes. The intense atomization and ultra-rapid freezing in the SFL process lead to nanostructured BSA microparticles with high surface areas. Protein denaturation and aggregation are reduced in SFL relative to SFD. The more rapid freezing in SFL lowers the time for proteins to aggregate or diffuse to water-air and water-ice interfaces where they may be denatured.