Mechanism of formation of a novel self-assembled protein microsphere system
Date of Completion
Health Sciences, Pharmacy
The development of proteins into pharmaceutical products is limited by protein denaturation during formulation and processing. The objective of this research was to systematically investigate the effect of changes in the protein solution environment [e.g. pH, temperature, concentration (protein and polymer) and polymer molecular weight] to determine the mechanism of formation of a novel self-assembled protein microsphere system. ^ Soluble complexes were observed in mixtures containing native bovine serum albumin (BSA), poly[ethylene glycol] (PEG) and poly[vinylpyrrolidone] (PVP) at 25°C in acetate buffer (pH 5). Changes in the circular dichroism, fluorescence, surface tension and dynamic light scattering (DLS) data of protein/polymer mixtures occurred at 55°C indicating protein conformational changes. Therefore, microspheres formed by temperature modulated self-assembly and the major mechanism of microsphere formation appears to be similar to simple coacervation involving protein denaturation and aggregation. ^ Freshly prepared microspheres were relatively spherical; had rough outer surfaces; uniform, porous cores; were uncharged and tended to aggregate. The microspheres were composed of protein and polymer with uniform distribution of the protein throughout each particle. Less than 30% of the protein was released over a seven day study period and most of the protein secondary structure was lost. ^ Soluble complexes of native BSA and the polymer, either PEG or PVP, at high concentrations and molecular weights were observed at 25°C in acetate buffer (pH 5). An aggregation exotherm, which overlapped with an unfolding endotherm, was observed with differential scanning calorimetry (DSC). The area of the aggregation peak decreased with increase in polymer concentration and this peak was not observed at high polymer molecular weights and concentrations. Proteintpolymer interactions appear to be related to absence of the aggregation peak determined by DLS and DSC, respectively. BSA precipitation with the polymers was dependent on the temperature, polymer, concentration, and molecular weight. ^ Various preparatory conditions such as pH, temperature, concentration (protein and polymer) and polymer molecular weight were determined to influence microsphere formation and protein denaturation. Elevated temperatures were found to be the most denaturing condition, Microsphere formation can be induced at 25°C with high PEG concentrations in combination with PVP in acetate buffer at pH 5. ^
Radwick, Allison Elizabeth, "Mechanism of formation of a novel self-assembled protein microsphere system" (1999). Doctoral Dissertations. AAI9946753.