(347g) Investigation of a Silica Nanoparticle-Aided Protein Crystallisation System Via Model Parametrisation and Process Performance Comparison

Recent work on template-induced biomolecule crystallisation has highlighted the potential of ‘template’ surfaces to be used as heterogeneous seeds to improve nucleation kinetics, interacting with the solute via functional group matching, topography or epitaxial relationships amongst other effects to guide the macromolecule into forming a stable crystalline nucleus (Artusio & Pisano, 2018). Silica nanoparticles (NPs) have been shown to be widely-applicable nucleation promoters, but their effectiveness in scaled-up macromolecule systems remains uncertain due to lack of extensive testing (Verma et al., 2023; Weichsel et al., 2017).

In-silico high-fidelity models can guide in-vitro experiments, be used for process optimisation and control, and have already been shown to aid scale-up efforts to drive process intensification (Szilágyi et al., 2022; Casas-Orozco et al., 2023; Mitchell et al., 2023). The models however require parametrisation against in-vitro measurements. Parameter estimation of templated systems has generally been limited to low-volume experiments, and there are conflicting hypotheses of how the templates affect kinetic parameters (Thakore, Sood & Bansal, 2020; Verma et al., 2022). Further investigation is needed to elucidate how the templates affect scaled-up crystallisation and to explore their usefulness for process optimisation.

In this work high-fidelity models of the isothermal antisolvent batch crystallisation of lysozyme in a 40 ml volume vessel are presented. Lysozyme was firstly crystallised in a template-free solution, and secondly in the presence of irregular hydroxyl-functionalised porous silica templates. Lysozyme concentration was measured with a Nanodrop UV/Vis following filtration to remove crystal fines. The final CSD was measured after filtration and drying of the solution. The first two models (referred to as UNS and T-FREE) were parametrised against unseeded and templated crystallisation measurements. Since templates are thought to only affect nucleation kinetics, a third model (T-FIX) was parametrised with fixed growth kinetic parameters from UNS to investigate how the templates affect nucleation parameters and overall kinetics.

A lower pre-exponential constant and surface energy are estimated for T-FREE and T-FIX, while the ‘free’ model had a higher pre-exponential empirical growth constant. The templated models T-FREE and T-FIX have a ‘flatter’ nucleation rate trajectory across the relevant range of supersaturations observed in-vitro compared to the unseeded model. In-silico simulations of the template system show that low-intermediate initial concentrations, the silica NP-seeded system can generate a higher crystal mass and enable low-S nucleation.

In conclusion, three crystallisation models were parametrised against medium scale experiments of unseeded and templated lysozyme in an antisolvent batch crystalliser. Parametric comparison and crystallisation simulations show in this case the templates do not indiscriminately hasten nucleation kinetics for any supersaturation. Rather, the parameterised model suggests template-induced nucleation has a lower dependence on supersaturation compared to unseeded systems. To support kinetic parameter comparison across the two systems, the differences in deposited crystal mass over the course of a batch provide another source of information to assess silica NPs as a template, and inform how it might be used for process optimisation and intensification.

Acknowledgments

This work was supported by the Engineering and Physical Sciences Research Council (EPSRC) for the Imperial College London Doctoral Training Partnership (DTP) and by AstraZeneca UK Ltd through a CASE studentship award.

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