Supercritical fluid processing of proteins I: Lysozyme precipitation from organic solution

The solution enhanced dispersion by supercritical fluid (SEDS) process was used to evaluate the effect of the processing variables on the biological a nd physicochemical characteristics of lysozyme protein particles produced f rom an organic solution of dimethylsulfoxide (DMSO) using an experimental d esign procedure. The processing variables were temperature, pressure, solut ion concentration and the flow-rates of supercritical carbon dioxide and a protein solution. Solutions of hen egg lysozyme (0.5-1%, w/v) in DMSO were dispersed using supercritical carbon dioxide as the antisolvent, and partic les precipitated in a particle formation vessel. The morphology, particle s ize and size distribution and biological activity of the protein were deter mined. The precipitates were also examined with high sensitivity differenti al scanning calorimetry (HSDSC) and high-performance cation-exchange chroma tography. The amount of residual DMSO was determined using headspace gas ch romatography. Particle size measurements showed the precipitates to be aggl omerates with primary particles of size 1-5 mu m, containing <20 ppm of res idual solvent. The activity of the precipitates varied between 44 and 100% depending on the experimental conditions. The similarity of HSDSC data for unprocessed and processed samples indicated that the SEDS process does not cause major denaturation of lysozyme when prepared from DMSO solutions. By optimising of working conditions, the SEDS process can produce micron-sized particles of lysozyme with minimal loss of biological activity. (C) 2000 E lsevier Science BN. All rights reserved.