Ultrasound induced nucleation for controlled crystal formation within complex solute and active pharmaceutical ingredient systems.

Aims: This study aims to understand the parameters and methods to achieve successful freeze drying of biological material where activity is retained. Background: Bioscience is expanding the opportunity for discovery and selection of active therapeutic molecules. Large numbers of protein molecules have been made available as potential cures or treatments to a wide variety of diseases. Every protein is unstable in the aqueous environment to varying degrees, with most requiring storage to prevent damage before they are used in a research laboratory or as a drug therapy, vaccine or diagnostic test. Freezing is the most common method of reducing degradation to a level suitable for daily laboratory use. However, the Pharmaceutical industry has two real barriers to commercialisation of frozen biological products. The cost of products that are stored and supplied within the 'cold chain' is far greater than those that can be effectively stored at room temperature. Also, although the frozen state is protective over the medium term (1 year) its ability to protect for periods greater than two years is limited. For pharmaceutical products with defined limits of activity and purity it does not take a large amount of degradation to result in the rejection of a product batch. To overcome the issues associated with cold storage, Pharmaceutical companies have sought alternative storage methods. Drying to the powdered state is particularly appealing as materials are more stable and can be transported at room temperature. Transfer to the powder state is not without problems and although the final powder state may be free of degradation, evaporative drying and other simple forms of water removal can result in high levels of degradation. Freeze drying is the industry's preferred method for obtaining dry state materials, although involving large capital investment, infrastructure is in place worldwide. Freeze drying is not a new technology but the recent trend of biologic drugs brings far greater challenges than those associated with small molecules. Freeze drying involves three main stages (freezing, primary drying and secondary drying). Freezing of the material defines the powder's final structure and morphology. Ice is first nucleated and then allowed to grow in size. Primary drying removes the frozen ice crystals using the process of sublimation, the pressures involved in this step allow the solid ice to be removed as vapour, importantly without transition through the liquid state. Scientific rationale: It is well known that agitation induces freezing in super cooled liquids. Ultrasound is one method of causing controlled agitation and has previously been used in freeze drying of small molecules. However, it is not widely used as existing systems work well for small molecules. Systems for freeze drying biologics are not well established and existing methods give low activity recovery (Zheng & Sun, Trends in Food Science & Technology 17 (2006) 16-23). Freezing using ultrasound induced nucleation develops small ice crystal formations so it is expected to prevent damage of proteins and cells which in turn allows the freeze drying process to be completed with retention of higher activity than conventional freezing alone. Pilot work in Dr Ingham's lab has shown its potential with enzymes systems (Aspariginase, B-Galactosidase) and it is expected that this can be improved and extrapolated to cell based systems. Objectives of the study: 1, To better understand the freezing process for freeze drying, we aim to alter the nucleation of ice, particularly targeting high concentration protein solutions (Aspariginase, B-Galactosidase) and complex systems including bacteria (E. coli HBIOI) and mammalian cells (caco-2). 2, Optimise freeze drying parameters for a range of model materials including proteins, bacterial and mammalian cells and collagen scaffolds 3, Apply new parameters to pharmaceutically useful biological systems