Enhancing the crystallisation of insulin using amino acids as soft-templates to control nucleation

Abstract

Amino acids have been widely used in protein formulations to increase the protein’s stability. In this study, amino acids have been introduced as soft-templates to control the nucleation of proteins....

Studying the impact of the pre-exponential factor on templated nucleation

“…16 Over the past few decades, the 'state-of-the-art' approaches responsible for studying heterogeneous nucleation have been used for the non-specic adsorption of molecules on the heterosurface, 18 specic interactions such as functional group matching, 19 or selective interactions such as lattice matching. 20 The materials that have successfully served the function of the heterosurface for heterogeneous nucleation are pharmaceutical excipients, 14,[21][22][23] silanised glass substrates, 13,24,25 amino acids, 26 self-assembled monolayers, 27 bio-compatible 28 and synthetic polymers, 29,30 and porous substrates. 31 Protein crystallization has also been achieved with chemically modied surfaces.…”

Unraveling the Impact of pH on the Crystallization of Pharmaceutical Proteins: A Case Study of Human Insulin

“…Quite often, the pH of the final crystallization cocktail is different from the pH of the stock protein solution because the addition of additives, precipitants, or additional solvents impacts the pH. 11,20 However, the overall determination of the pH of the crystallization cocktail is crucial, as it modulates the net surface charge on the protein's surface and therefore dictates the strength of the Coulombic forces between the protein molecules. 6 In addition, the pH also governs the strength of electrostatic interactions between the protein and solvent molecules as well as additives and ions present in the solution.…”

“…25,26 Additionally, investigation of the nucleation and growth rate of the basic protein lysozyme (pI = 10.7) or the acidic protein insulin (pI = 5.3) has revealed that a shift toward a more acidic (for lysozyme) or basic (for insulin) solution pH enhances the nucleation and growth rate. 7,11,20,27,28 Recently, it has been shown that insulin crystallization can be enhanced significantly with the addition of the basic amino acid arginine. 20 The deprotonation of arginine leads to a pH shift toward a more basic pH, thereby, significantly enhancing the crystallization of insulin.…”

“…7,11,20,27,28 Recently, it has been shown that insulin crystallization can be enhanced significantly with the addition of the basic amino acid arginine. 20 The deprotonation of arginine leads to a pH shift toward a more basic pH, thereby, significantly enhancing the crystallization of insulin.…”

Protein crystallisation facilitated by silica particles to compensate for the adverse impact from protein impurities

“…To address this concern, recent studies showed heterogeneous nucleants were able to provide a better control of the nucleation process and thus had the potential to facilitate rational design of selective protein crystallisation for isolation and purification steps. 23,24 A wide range of such nucleants with designed chemical and physical properties have been developed, for instance, porous silicon 25 , nanowrinkle substrates 26,27 , mesoporous MOFs 28 , magnetic particles 29 , DNA origami 30 , amino acids 31 , etc. In most of the cases, the focus has been on using the templates to increase the possibility of crystallisation from protein solution with high purity.…”