Abstract

The formulation, therapeutic delivery and prediction of the stability of proteins are very challenging due to their varied chemical and physical properties. A number of approaches to stabilise protein formulations, such as protein encapsulation and characterisation, using physical and chromatographic methods, were evaluated in an attempt to overcome these challenges. The aim of this project was to evaluate the impact of common formulation variables (pH, strength and composition of buffers and excipients) on liquid formulations of lysozyme and trypsin. The use of a Quality by Design (QbD) approach was adopted in liquid and nanocapsule formulations with the application of mathematical models to obtain optimised formulations in order to tailor the desired attributes.
Protein formulations were prepared according to a mathematical design of experiments by changing the pH, type of buffer and concentration, and nature of excipients. Each formulation was characterised by Differential Scanning Calorimetry (DSC) and enzymatic assay. Subsequently, each factor was optimised, and optimised formulations were prepared. These new formulations were characterised, and their stabilities investigated using the ‘Size Exclusion Chromatography Method’, which was developed and validated as a stability indicating assay for encapsulated lysozyme, deoxyribonuclease I (DNase I), and trypsin. Hydrophilic liquid chromatography methods were applied to measure the excipients’ stability during the shelf lives of the formulations.
Polymeric nanocapsules were prepared by double emulsion methods (solid/oil/water and water/oil/water), based on the QbD experiments. Critical quality attributes were determined in order to achieve the quality target product profile. The formulations were developed by using Poly (DL-lactide-co-caprolactone) copolymers in two different molar ratios (86:14 and 40:60) for lactide and Ɛ-caprolactone blocks, respectively. The nanocapsules’ spherical morphology and size were investigated by Transmission Electron Microscope and Dynamic Light Scattering (DLS), respectively. In addition, protein entrapment efficiency was determined. The proteins’ release profiles, in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), were assessed. The application of QbD principles reduced the length and cost of development and provided optimum protein formulations and promising results within a short time.
The formulations of lysozyme, at pH (4-5) and trypsin, at pH 3, retained their biological activity and conformational stability as illustrated, by having the highest transition temperature values. The phosphate buffer had the most stabilising effect on formulations and trehalose maintained the proteins’ integrity and biological activity. Using DSC and DLS to predict long term stability produced promising results. The proteins’ encapsulation efficiencies were significantly (p<0.05) affected by the copolymers’ compositions. Moreover, the drug release profile in SIF over 24 hours was affected by copolymer ratios, with 64% drug release in total. The release in SGF was 8%, suggesting protection of orally-delivered proteins from degradation by gastric enzymes. Adding trehalose and the encapsulation of solid proteins helped proteins to retain up to 97.4% of the original biomolecule activity.