Effects of Formulation Development Methods on the stability of Model Protein Pharmaceuticals Embedded in Solid Lipid Matrices.

This study was conducted to investigate the influence of formulation development methods on the stability (secondary structure, aggregation and biological activity) of protein drugs embedded in lipid matrices. Catalase, horseradish peroxidase and α-chymotrypsin were employed as model proteins while, Precirol® AT05 (glyceryl palmitostearate) was used as lipid matrix. Protein loaded lipid matrices were prepared using melting and mixing and wet granulation methods. Attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy, size exclusion chromatography (SEC) and biological activity analyses were performed. ATR FT-IR analysis indicated significant interference of the lipid with the protein amide-I band which was eliminated using spectral subtraction. Wet granulation method induced more changes in protein secondary structure compared to melting and mixing method. SEC analysis gave evidence of protein aggregation for catalase upon adopting the wet granulation method. The biological activity of catalase was found to reduce significantly than other two proteins upon using wet granulation method which might be ascribed to both secondary structure alterations and the formation of aggregates. Horseradish peroxidase and α-chymotrypsin did not form any soluble aggregates. In conclusion, melting and mixing method emerged as a better incorporation method compared to wet granulation because of better stability shown by the formulated proteins.