Purpose: The aim of this study was to establish the high pressure homogenization of proteins in non-aqueous suspension as an alternative method for classical micronization strategies and to investigate the effect of high pressure on protein stability and bioactivity.
Methods: The influence of drug loading, homogenization pressure and cycles on particle size reduction was investigated by experimental design using a Box Behnken matrix with insulin as a model compound. Particle size measurements were performed by laser light scattering. Protein stability was investigated by HPLC and HPLC-MS analysis and the bioactivity of insulin was tested in a chondrocyte proliferation assay. For investigations into the effect of temperature on protein stability, insulin was micronized in molten lipid at 75 degrees C in one cycle at 1,000 bar.
Results: Within one homogenization cycle at 1,500 bar, the particle size of insulin could be reduced from 15.8 to 7.3 microm, six cycles resulted in a particle size of 3.7 microm d(0.5) (50% of the particles are smaller than the indicated value). Evaluation of the response surface diagram revealed that the homogenization pressure had the highest impact on micronization efficiency, followed by the number of homogenization cycles. Protein stability was maintained during the micronization process as well as bioactivity. Micronization at elevated temperature (75 degrees C) had no effect on protein stability.
Conclusion: High pressure homogenization of protein suspensions can be used as an alternative method for the micronization of proteins without affecting protein stability or bioactivity.