Protein crystallisation using the gas bubble as a soft template

Protein crystallisation is a potential method for protein therapeutics purification since it has lower production cost and results in products with higher purities and increased stability during drug formulation, storage, and delivery. However, protein crystallisation is difficult due to the protein’s structural complexity and requirement for biological activity. Recent research has shown that using gas bubbles as soft templates can improve the crystallisation process. The purpose of this thesis is to systematically study protein crystallisation induced by gas bubbles, using the lysozyme as the model protein and NaCl as the precipitant. The study is divided into three different scales: µL-scale in the hanging drop set-up, mL-scale using a microfluidic device, and 100 mL-scale in a stirred tank batch crystallizer.

The hanging-drop experiments revealed that nucleation could occur earlier with the addition of an air bubble and the induction time reduced with the increased lysozyme or NaCl concentrations. In experiments with an air bubble, crystals were observed attached to the bubble surface. The crystal with a curved facet would appear to have nucleated and grown on the air bubble surface. The number density of protein crystals near the bubble surface was up to 50% higher than the one in the droplet without the bubble, showing that the gas-liquid interface could act as a heterogeneous nucleation site for protein crystallisation. For the studied experimental conditions, the mass yield was also found to increase up to 80% more by adding air bubbles into the droplet.

In 1 mL scale experiments, a microfluidic device was used to mix the four different gas bubbles with the same starting bubble size into the crystallisation solution. The protein was found to act as a surfactant, stabilising the gas bubble in the crystallisation solution by lowering the surface tension of the bubbles and adding a mass transfer resistance at the interface. The change in size of the gas bubbles was found to decrease with increasing protein concentrations and over time, resulting in an arrangement of small and large bubbles. This was likely due to the Ostwald ripening caused by the Laplace pressure difference among bubbles. When bubbles were injected into the solution, the crystals tended to form on the surface of the gas bubbles, likely due to the strong interaction between the crystals and the bubble surfaces. The protein crystallisation at the gas-liquid interface was hypothesised to be a combination effect of adsorption and heterogeneous nucleation, reducing the free energy required for nucleation and promoting the nucleation of proteins. The population density of lysozyme crystals increased with an increasing order of solubility for four types of gases: carbon dioxide > oxygen > nitrogen > helium. Among the four different microbubbles examined, the carbon dioxide bubbles were found to be more likely to form crystals on its bubble surfaces, with over 90% of lysozyme crystals formed on the bubble surface. The difference in the crystal formation among different gas types could be the result of their variations in polarisability and zeta potential.

The crystallisation in a 100 mL stirred tank crystalliser applied both in-situ Process Analytical Technologies (PATs) and off-line measurements to monitor the experiments with and without gas. Using in-situ crystal number count is more accurate in detecting nucleation than off-line microscopic observation and concentration measurements. The induction time was found to decrease with increasing protein concentration for the same NaCl concentration, agreeing with the results in the smaller-scale experiments. The introduction of gas bubbles was found to significantly reduce the induction time for lysozyme crystallisation, achieving up to 92% reduction from the one without gas bubbles. This difference in induction time was caused by both the heterogeneous nucleation mechanism, which was previously discovered in smaller-scale experiments, and the “self-seeding” mechanism by the continuous gas flow. Three stages of crystallisation were discovered in protein crystallisation in the batch crystalliser for all experiments: slow nucleation stage, rapid crystallisation stage, and slow growth and breakages stage. The crystal yield was found to increase with the higher lysozyme or NaCl concentrations within the same experiment time. The crystal yield in the experiments with gas bubbles was consistently higher than the one without gas bubbles due to the enhanced nucleation by the gas bubbles upon achieving equilibrium. Overall, the results in the 100 mL study were in line with the findings from µL and mL experiments, showing a decrease in the induction time and an increase in crystal yield with the introduction of gas bubbles. The results provide a foundation for understanding the impact of gas bubbles on protein crystallisation and for selecting suitable conditions for industrial production.