Lyophilization of proteins - an in-situ study on structural changes and molecular interactions

More and more therapeutic proteins are getting into market due to the rapid development of biotechnology. Most of these protein macromolecules are inherantly unstable and it is important to stabilise them to preserve sufficient shelf life for storage and transport. These macro functional molecules have several levels of structure, referred to as primary, secondary, tertiary and quaternary structure, and their structure is directly linked to their functions. Stablising their structure often preserves their functions. This is usually done by adding various stablisers (excipients) and processing them into a stable status - a process referred as formulation. Almost all structural changes involve interactions with water, and hence the primary approach to elongate shelf life is to reduce water availability and activity by preferably removing water completely and transferring the aqueous formulation into a dry powder, and the latter can be easily stored and transported at ambient temperature. As many proteins are sensitive to elevated temperature, freeze-drying has been the process of choice to accomplish acceptable shelf life for protein formulations. Freeze-drying or 'lyophilization' is a drying process where the solvent, usually water, is first frozen and then removed by sublimation at low temperature and low pressure (typically 6.6Pa to 66Pa). However, freeze drying can disrupt the stabilizing forces and allowing the molecule to unfold, denature and aggregate. Hence stable formulation and process design is required to avoid biological activity loss and possible immunogenic effect of the structurally and/or chemically altered protein molecules. Time and material consuming 'trial-and-error' approaches adding excipients that have the potential to stabilize, e.g. saccharides, and/or varying the freeze-drying conditions are usually used to address stability issues. However, neither the mechanisms of stabilisation nor the relative contributions of each individual stress during freeze-drying are completely understood. The knowledge and understanding of when the degradation of proteins occur during lyophilization would be beneficial in designing more stable formulations and optimizing the freezing-drying process from a product quality perspective. A technical challenge has been how to identify the protein molecular structures and characterise their interactions with excipients during each stage of freeze drying. In this project we will adopt two advanced imaging techniques, (i) focal plane array (FPA)- Fourier Transform Infrared Spectroscopy (FTIR), which provides protein structural information, and (ii) multiphton microscopy (MPM), which extends the study into three dimensional for the in situ real-time study of freeze drying at well defined protocols provided by a cryostage. The data on interactions between proteins and stabilizing excipients as well as on the protein secondary structure, at micron and nanometre scales, during cooling, freezing and drying will be obtained, which will lead to better formulation. The results will be validated with industrial lyophilisation process and we will develop practical guidance on protein formulation.

The aim of the project is to identify and characterise protein secondary structure changes in each step of lyophilization and link these to functional loss of proteins, which leads to guided approach to achieve better formulation. Experimental studies will be performed at well defined freezing and drying conditions using a commercial lyophilisation cryostage with exemplar proteins and chosen excipients. Focal Plane Array - Fourier Transform Infrared Spectroscopy (FPA-FTIR) and 3-dimensional spectral Multi-Photon Microscopy (MPM) are used in parallel for the in situ and in real time study on the structural changes of protein molecules and intermolecular interactions in protein formulations during each unit operation of the freeze drying process. FPA-FTIR characterises protein second structures and their changes in real time by analysis of the conformation sensitive Amid I band located between 1600 and 1700 1/cm. FPA technology permits the simultaneously measurements of 64x64 i.e. 4096 spectra in seconds. All 4096 spectra are spectroscopically evaluated, which means that the analysis can range from single-band intensity plots to mathematical approaches such as cluster analysis. MPM can reveal protein-protein interactions, protein aggregations, freeze concentration, the porous structure of the 'cake' and possible structural changes within three dimensional domain. Linking these spectral data at micro and nano scales to the outcome in an industrial freeze dryer will be made and validated. Attempts will be made to predict formulation outcome using heuristic and mechanistic modelling. The results will guide the selection of excipients and freeze drying protocols based on structural information of the proteins.