Crystalline form of proteins has great advantages for different industries such as pharmaceuticals, which also has a high demand of specific crystals size with narrow distribution. This is challenging due to the inherent difficulty of having reproducible, controllable, and profitable processes. The easiest way to control crystalline polydispersity is by directing the crystallization process. Nucleation, as the first stage of the process, predetermines the final number of crystals and control their size but difficult to control due to its stochastic nature. Therefore, overcoming the nucleation step seems the simple way to tune the final product. Seeding in a metastable solution seems the simple way to do it.

Hydrogels have demonstrated their ability to produce higher quality crystals, influencing nucleation and growth, while providing a non-convection medium. Thus, using the gelled batch method as a means of crystallization seems as a good option due to its simplicity and its ability to be scalable.

Still gelling a crystallizable protein metastable solution introduce a new set of variables to be adjusted in order to: properly compartment the space (gel homogeneity, etc.), control the influence of the gel of the crystallization and seed stability, etc. In this work we present the preliminary results of our strategy to control the homogeneity and final size of the lysozyme crystals by means of agarose batch seeding. Our preliminary results clearly show that there is a direct correlation between the initial size of the seeds and the final size of the crystals, maintaining a narrow size distribution.^1–4

References:

(1) Nanev, C. N. Crystal Size Distribution Resulting from the Time Dependence of Crystal Nucleation.Cryst.Res.Technol.2018,53(5),1700248. https://doi.org/10.1002/crat.201700248.

(2) Pu, S.; Hadinoto, K. Continuous Crystallization as a Downstream Processing Step of Pharmaceutical Proteins: A Review. Chem. Eng. Res. Des. 2020, 160, 89–104. https://doi.org/10.1016/j.cherd.2020.05.004.

(3) Gavira, J. A. Current Trends in Protein Crystallization. Arch. Biochem. Biophys. 2016, 602, 3–11. https://doi.org/10.1016/j.abb.2015.12.010.

(4) Contreras-Montoya, R.; Arredondo-Amador, M.; Escolano-Casado, G.; Mañas-Torres, M. C.; González, M.; Conejero-Muriel, M.; Bhatia, V.; Díaz-Mochón, J. J.; Martínez-Augustin, O.; Sánchez de Medina, F.; Lopez-Lopez, M. T.; Conejero-Lara, F.; Gavira, J. A.; Álvarez De Cienfuegos, L. PAGE PENDING_Insulin Crystals Grown in Short-Peptide Supramolecular Hydrogels Show Enhanced Thermal Stability and Slower Release Profile. ACS Appl. Mater. Interfaces 2021. https://doi.org/10.1021/acsami.1c00639.