Title : Effects of Oscillatory Flow on the Nucleation and Crystallisation of Insulin

Protein crystallisation has immense potential to be used for separation and formulation applications on an industrial scale. Crystallisation under static condition is often limited by the diffusion of molecules from the bulk solution to the growing crystal surface. This results in a lower overall yield of the product and longer crystallisation periods. Hence, protein crystallisation under flow conditions has attracted attention with the capability to improve the convective protein transport, reducing the crystallisation time and improving yield. In this study, we investigate the effects of flow, specifically intermittent oscillatory flow on protein (insulin) crystallisation. It was observed that the nucleation and crystal yield is very much influenced by the flow conditions and herein we present some key observations in insulin crystallisation conducted under intermittent oscillatory flow. For oscillatory flow velocities ranging from 6 to 16 mm/min at a frequency of 1 cycle/min, a considerable increase in nucleation has been observed with an increase in flow rate resulting in the formation of large number of crystals in tubes with flow compared to the stationary tubes. Also 50% yield for flow crystallisation was achieved in contrast to only 24% yield for the stationary growth over the 48 hour period. The flow condition was thus found to affect the number and size of crystals. In addition, the intermittent flow pattern utilised in this study helps to separately understand the influence of flow on nucleation and crystal growth. The flow strategy proposed herein could potentially be utilised to optimise crystallisation processes for proteins so as to be used in downstream separation and formulation of products. Abstract Protein crystallisation has immense potential to be used for separation and formulation applications on an industrial scale. Crystallisation under static condition is often limited by the diffusion of molecules from the bulk solution to the growing crystal surface. This results in a lower overall yield of the product and longer crystallisation periods. Hence, protein crystallisation under flow conditions has attracted attention with the capability to improve the convective protein transport, reducing the crystallisation time and improving yield. In this study, we investigate the effects of flow, specifically intermittent oscillatory flow on protein (insulin) crystallisation. It was observed that the nucleation and crystal yield is very much influenced by the flow conditions and herein we present some key observations in insulin crystallisation conducted under intermittent oscillatory flow. For oscillatory flow velocities ranging from 6 to 16 mm/min at a frequency of 1 cycle/min, a considerable increase in nucleation has been observed with an increase in flow rate resulting in the formation of large number of crystals in tubes with flow compared to the stationary tubes. Also 50% yield for flow crystallisation was achieved in contrast to only 24% yield for the stationary growth over the 48 hour period. The flow condition was thus found to affect the number and size of crystals. In addition, the intermittent flow pattern utilised in this study helps to separately understand the influence of flow on nucleation and crystal growth. The flow strategy proposed herein could potentially be utilised to optimise crystallisation processes for proteins so as to be used in downstream separation and formulation of products.