Protein Structure in Solid Lipid Matrices Intended for Potential Dosage Forms
Statement of the problem. Protein drugs are important therapeutic agents and lipids can be used to prepare protein dosage forms. However, proteins may degrade during the formulation processing leading to changes in conformation and the loss of biological activity. Objective. The objective of this study was to understand the influence of inherent conformational stability of proteins on their stability in solid lipid matrices prepared using two different procedures namely melting and mixing and wet granulation. Materials and methods. Bovine serum albumin (BSA), catalase (CT), horseradish peroxidase (HRP), α-chymotrypsin (CTP) and lysozyme (LZ) having a rank order of inherent conformational stability were used, while, Precirol® AT05 (glycerol palmitostearate, melting point 58°C) was employed as the lipid matrix. Each protein was heated alone as a solid and in solution at 70°C and was incorporated into Precirol® AT05. Protein-loaded lipid matrices were also incubated in simulated gastric fluids for two hours. ATR spectroscopy, enzyme activity assay and size exclusion chromatography (SEC) were performed. Results and discussion. Precirol® ATO5 interference with BSA amide-I band was subtracted up to 90% w/w lipid content and the method was applied on the other model proteins to analyse secondary structure. With an exception of catalase, heat-exposure of protein solids caused minimum changes in the secondary structure and enzyme activity, whilst the solution form induced more secondary structure changes and resulted in a statistically significant loss of enzyme activity. The loss of enzyme activity in solution was found to depend on protein and suggested a rank order of thermal stability with LZ=HRP > CTP > CT which was in accordance with the rank order of their inherent conformational stability. ATR spectroscopy predicted the loss of enzyme activity in the solution state. ATR analysis of proteins embedded in lipid implants showed changes in secondary structures. In vitro release studies revealed different release profiles for proteins after 48 hours dissolution run and suggested a rank order of release with BSA > HRP > LZ > CT > CTP. SEC analysis exhibited aggregation for BSA and catalase. Results of enzyme activity showed variations with CT 49.29 ± 5.86%, HRP 72.99 ± 11.94 %, CTP 83.58 ± 7.08% and LZ 82.14 ± 15.67%. However, enzyme activity was not statistically different from the controls and loss was in alignment with the rank order of protein reported stability. Incubation of BSA, CT and CTP lipid matrices in simulated gastric fluids caused changes in the secondary structure, loss of enzyme activity and aggregation for BSA and catalase. However, increasing the lipid: protein mass ratio reduced these changes and thereby improved the protein stability. Conclusions. Protein-loaded lipid matrices could be prepared using melting and mixing and wet granulation procedures. ATR spectroscopy could analyse protein secondary structure in lipid matrices provided lipid interference is minimized. Upon thermal stress, proteins as solutions are more vulnerable to stability changes than as solids. The rank order of protein inherent conformational stability has an alignment with protein stability changes in solutions, in solid lipid matrices and upon exposure to simulated gastric fluids.
Advisor: Medlicott, Natalie; Jorgensen, Lene
Degree Name: Doctor of Philosophy
Degree Discipline: School of Pharmacy
Publisher: University of Otago
Keywords: Protein; Solid lipid; ATR Spectroscopy; Enzyme activity analysis; BSA; Catalase; Horseradish peroxidase; Aplha chymotrypsin; Lysozyme; Glycerol Palmitostearate; Size exclusion chromatography
Research Type: Thesis