Freeze drying, also commonly known as lyophilisation, is a widely used manufacturing method used in the final stages of production of many biopharmaceuticals, especially those whose solution stability is poor. It is a physical drying process in which an aqueous based solution containing the active protein (as well as other stabilisers) is frozen, and then the frozen water is sublimed and desorbed away from the solution under vacuum, leaving a glassy solid cake of the active product behind. Biopharmaceutical solutions themselves can have short shelf lives, primarily due to protein degradation in solution, whereas freeze dried formulations can provide improved stability and long term storage life. Despite the importance of this area, there is a dearth of academic research and expertise in the UK on freeze drying, especially of biopharmaceuticals. It is also commonly accepted that freeze drying is non-optimised, with consequently poor product control and thus a limited understanding of how to optimise both product and process with regard to product quality and economic and energy-efficient processing. These problems result in a significant environmental and cost burden, including increased times of product to market. In this study the effects of various aspects of formulation and the freeze-drying process on biopharmaceutical materials in terms of their structure, function, activity, and other aspects of product quality, including the level of residual water and its distribution within the freeze-dried material, will be investigated. While a number of publications exist that describe studies carried out by a number of workers in the field, such publications have tended to limit themselves to describing individual studies on single products, or single-variable studies for a small number of 'model' products - often with the risk of over-extrapolating the significance and meaning of the data produced. Other studies have concentrated on the mathematical modelling of issues such as heat and mass transfer during the freeze-drying process - usually for ice alone, or a single model solution - which may bear no relation to what would happen for a real product being processed. The opportunity here is to undertake a multi-variable, multi-product study in the field of lyophilisation therefore exists, to help bridge the knowledge gap that currently persists, which is relevant to the biopharmaceutical, vaccine, bio-products and diagnostics industries. Specifically in this project, the relationship between a number of key input and output variables needs to be identified and understood for real products. Input variables would typically comprise: formulation excipients, total solute concentration, pH, solution-state stability, container design and dimensions, fill depth, cooling rate, initial freezing temperature, presence or absence of an annealing step, primary drying (sublimation) temperature and pressure. Output variables currently studied include: product appearance (including surface anomalies and visible heterogeneity), residual water content, activity/potency, molecular integrity, surface area, porosity, crystal/polymorphic form of active ingredient and/or excipients, thermal properties (such as glass transition temperature, crystallisation events, relaxation), stability, degradation product levels, process economics and scale-up issues. Additionally, there are a number of in-process variables that would be studied (eg local temperatures, vial concentrations, rates of water loss from vials etc) in order to further the understanding of the product during the freezing, sublimation and desorption processes as well as examining the final lyophilised product. In conclusion it is expected that this project will inform on the rational and optimised design of industrial biopharmaceuticals which use freeze drying manufacturing operations.

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