Micronization of insulin by high pressure homogenization.
OBJECTIVE
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.


CONCLUSIONS
High pressure homogenization of protein suspensions can be used as an alternative method for the micronization of proteins without affecting protein stability or bioactivity.
Maschke
Angelika
A
Department of Pharmaceutical Technology, University of Regensburg, 93040 Regensburg, Germany.
Calí
Nadia
N
Appel
Bernhard
B
Kiermaier
Josef
J
Blunk
Torsten
T
Göpferich
Achim
A
D000465:Algorithms; D002851:Chromatography, High Pressure Liquid; D004339:Drug Compounding / Q000379:methods; D004355:Drug Stability; D005079:Excipients; D007004:Hypoglycemic Agents / Q000008:administration & dosage*; D007328:Insulin / Q000008:administration & dosage*; D013058:Mass Spectrometry; D008855:Microscopy, Electron, Scanning; D010316:Particle Size; D011208:Powders; D011312:Pressure; D013696:Temperature; D014280:Triglycerides
D005079:Excipients; D007004:Hypoglycemic Agents; D007328:Insulin; D011208:Powders; D014280:Triglycerides; C433530:miglyol 810