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

Abstract

One of the most crucial parameters in protein crystallization is pH, as it governs the protein's electrostatic interactions. However, the fundamental role of pH on crystallization still remains unknown. Here, we systematically investigated the crystallization of human insulin (isoelectric point 5.3) at various pHs between 6.0 and 6.7 at different supersaturation ratios, up to 20.9. Our results demonstrate that the pH has an opposing effect on solubility and nucleation rate as a shift in pH toward a more basic milieu increases the solubility by 5-fold while the onset of nucleation was accelerated by a maximum of 8.6-fold. To shed light on this opposing effect, we evaluated the protein-protein interactions as a function of pH by measuring the second virial coefficient and hydrodynamic radius and showed that a change in pH of less than one unit has no significant impact on the protein-protein interactions. As it is widely understood that the increase in protein solubility as a function of pH is due to the increase in the repulsive electrostatic interactions, we have demonstrated that the increase in insulin solubility and decrease in the onset of nucleation are independent of the protein-protein interactions. We hypothesize that it is the electrostatic interactions between both ions and solvent molecules and the protein residues that are governing the crystallization of human insulin. The findings of this study will be of crucial importance for the design of novel crystallization pathways.

Figure 1

Desupersaturation curve for insulin in the presence of different zinc salts with concentrations of 3.5 mM at pH 6.7 and 24.0 °C.

Figure 2

Insulin desupersaturation curves at different pH with an initial insulin concentration of 3 mg·mL^–1 (S_t=0 = 20.9 at pH 6.0 to S_t=0 = 4.2 at pH 6.7) and a ZnSO₄ concentration of 3.5 mM at 24.0 °C.

Figure 3

Insulin solubility as a function of pH with the addition of ZnSO₄ at 24.0 °C.

Figure 4

Desupersaturation curve (left) and obtained crystal yield (right) for insulin crystallization at various supersaturation ratios and pH at 24.0 °C. The average pH values are 6.0, 6.2, 6.5, and 6.7.

Figure 5

Induction time of insulin crystallization as a function of the initial supersaturation ratio at constant pH (top) and as a function of H⁺ concentration in solution at a constant initial supersaturation ratio (bottom) at 24.0 °C.

Figure 6

Hydrodynamic radius and diffusion coefficient of insulin at different solution pHs as a function of concentration at 24.0 °C.

Figure 7

Insulin crystals with the addition of ZnCl₂ (left), ZnAc (middle), or ZnSO₄ (right) at 24.0 °C. Images were taken after 90.0 h. Scale bar is for all images.

Figure 8

Intensity of scattered light (Kc/R_θ) as a function of insulin concentration for different solution pH at 24.0 °C. An average molecular weight of 32.7 kDa for the insulin hexamer was found.

Figure 9

Estimated parameter A and B of the classical nucleation equation (eq 5) for homogeneous nucleation as a function of pH at 24.0 °C. For discussion, see text and derivation in Supporting Information.