STABILISING HEMOCYANINS: STEPS TOWARDS FORMULATION OF NEW PROTEIN PHARMACEUTICALS

Abstract

Haliotis Rubra hemocyanin is a promising antiviral candidate for treating Herpes Simples Viruses, however this protein is not stable and forms irreversible aggregates, destroying any therapeutic activity. In this study we have analysed the stability of this protein, developing diagnostic techniques for measuring its stability, and have created a stable formulation which maintains its native structure even after long term storage. The extreme size of hemocyanins requires specialist techniques to study as they do not readily diffuse into electrophoresis gels and tend to aggregate easily. Photon Correlation Spectroscopy and Mass Spectroscopy identified a structure of 44nm and 7.39MDa. Making this one of the largest proteins ever studied. High resolution DSC was applied to study denaturation, with a denaturation temperature of 76.3oC observed. This was entirely irreversible, necessitating the Sanchez-Ruiz model for investigation. Denaturation activation energy was 528 ± 63 kJ mol-1, aggregation also occurred simultaneously at 76oC. The hemocyanin stability was improved through formulation, with the effect of buffers, salts and sugars investigated. The highest stability formulation we discovered, through combining all available adjuvants consisted of 50mM HEPES at pH 7.4 with 100 mM NaCl and 100 mM sucrose solution to stabilise 1 mg mL-1 Haliotis Rubra hemocyanin. This final formulation had a TD of 78.9oC and an Ea of 726 kJ mol-1, a 38% increase in stability. This sample was then stored for nine months, with the sample retaining its thermal stability and oligomeric state. As we have formulated the protein into a solution with FDA approved adjuvants and proven long term stability, the hemocyanin sample is now suitable for therapeutic testing. Creating a stable hemocyanin formulation will allow for viable drug testing and will show the true efficacy of native Haliotis Rubra hemocyanin.