Abstract

The goal of protein therapeutic formulation research is to create effective solutions in which the protein activity is maintained in solution and is stable throughout the shelf life.
Stabilisation of protein in dry and solution forms under certain conditions like temperature, pH has been a challenge in protein therapeutic drug production.
Investigating therapeutic protein physiochemical properties and aggregation are critical for development of formulations for enhancement of the drug efficacy and safety. Challenges in the analysis are usually compounded in the formulation with various excipients, small volumes and high concentrations.
The aim of this research work is to show the effects of beta cyclodextrin and pluronic F-127 on the stability of protein on lysozyme, insulin and antibody (Immunoglobulin), as well as their effects of concentration, and other solution conditions. The drying (spray-dried (SD); electrosprayed (ESD)) effect on lysozyme formulations with or without excipients was also investigated. High sensitivity differential scanning calorimetry (HSDSC), Differential scanning calorimetry (DSC) and other characterization techniques such as Dynamic light scattering (DLS), Thermogravimetry analysis (TGA), High performance liquid chromatography (HPLC), Scanning electron microscope (SEM) and Ultraviolet-Visible (UV-Vis) spectrophotometry were applied to assess the thermal stability and aggregation of these proteins including and excluding excipients. Beta cyclodextrin and pluronic F-127 demonstrated several promising qualities on lysozyme with the drying technique applied. Addition of beta cyclodextrin to spray dried lysozyme improved and maintained the biological activity.
Furthermore, the presence of beta cyclodextrin and pluronic F-127 maintained the denaturation temperature of spray dried lysozyme. A combination of both excipients with the drying technique exhibited a form of stability by maintaining the enzymatic activity of lysozyme. Electrospray technique in the presence and absence of excipients improved lysozyme stability.
The thermogram obtained for Insulin in phosphate buffered saline thermal denaturation was shifted to an increased Tm temperature of 81.2°C in the presence of pluronic F-127 at 10°C scan rate compared to unprocessed insulin at 78.44°C. In the same instance, beta cyclodextrin was not effective on the thermal stability of the insulin, rather, a reduced effect at Tm of 74.06°C and 71.45°C respectively was observed at both scan rates (10°C and 60°C). A change in the conformation of insulin affected the transition peak’s shape, sharpness, and position. Pluronic F-127 was able to enhance or preserve the helical structure of insulin by producing a stabilizing effect. No aggregation was observed for 1:1%w/v insulin, pluronic F-127 dissolved in phosphate buffered saline. Beta cyclodextrin and pluronic F-127 combination could cause the increase in the activity and stability of lysozyme using the correct formulation and technique following results obtained from electrosprayed technique. The thermogram of the antibody (IgG) showed a transition followed by a sharp aggregation peak. The thermograms showed different reactions to pH, as the effect of pH on thermal stability was investigated at pH 5.5 and 7.4 from the aggregation investigation. Increase in pH (7.4) did not show a difference in the denaturation temperature when compared to pH 5.5. Both excipients appeared to improved protein stability in their unique ways.