Insulin crystallization: The route from hanging-drop vapour diffusion to controlled crystallization in droplet microfluidics

Although half a century has passed since the mapping of the insulin structure, this globular protein is still currently being investigated. Crystalline drug formulations might replace the subcutaneous injections for diabetes treatment. However, these crystalline forms require carefully tailored properties to sustain controlled release (e.g. longer storage lifetimes and higher purity). Among others, crystal size polydispersity might reduce the speed of insulin release. Here, two distinct crystallization techniques are applied to study insulin crystallization, hanging-drop vapour diffusion and microbatch (droplet microfluidics), and an operating regime map is derived. Rhombohedral insulin crystals with variable size are produced inside the microdroplets for a broad range of insulin concentrations, while aggregate and/or agglomerate formation is observed during the hanging-drop experiments at low protein concentrations. These differences are likely explained by the way acetone evaporation is controlled in each technique. In fact, circular dichroism spectra reveal differences on insulin's secondary structure at variable acetone concentration, including the reduction of α-helical content, with no visible β-sheet-rich structures or random coils. Finally, contrary to what has been reported for other proteins, no temperature effect on insulin crystal size is observed within a range from 10 °C to 20 °C. This work highlights the formation of insulin crystals in confined microdroplets towards a better control over the insulin crystal size.