Human insulin polymorphism upon ligand binding and pH variation: the case of 4-ethylresorcinol

Abstract

This study focuses on the effects of the organic ligand 4-ethylresorcinol on the crystal structure of human insulin using powder X-ray crystallography. For this purpose, systematic crystallization experiments have been conducted in the presence of the organic ligand and zinc ions within the pH range 4.50-8.20, while observing crystallization behaviour around the isoelectric point of insulin. Highthroughput crystal screening was performed using a laboratory X-ray diffraction system. The most representative samples were selected for synchrotron X-ray diffraction measurements, which took place at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS). Four different crystalline polymorphs have been identified. Among these, two new phases with monoclinic symmetry have been found, which are targets for the future development of microcrystalline insulin drugs.

Applications of X-ray Powder Diffraction in Protein Crystallography and Drug Screening

“…Despite significant efforts devoted to the structural characterization of HI and its complexes with different ligands, there are still novel crystalline phases to be discovered complementary to the rich diagram of phase transitions including the C222 1 and C2 polymorphs identified a few years ago [120,122], and two previously unknown monoclinic formulations, P2 1(α) & P2 1(γ) , reported by our team [30][31][32].…”

“…An important feature of crystalline matter for pharmaceutical industries towards drug development is polymorphism, meaning the ability of a molecule or compound to exist in one or more molecular as well as crystalline phases [98]. Variation in the crystallization conditions like solvent polarity, initial macromolecular concentration, and precipitant agents may result in different crystal and/or molecular polymorphs [30][31][32]99].…”

“…T stands for an extended, R f for a "frayed" intermediate and R for a helical conformation, while the subscript is indicative of the number of monomers that exhibit the aforementioned arrangement [120]. Phenolic or non-phenolic organic molecules that can act as ligands have been used in HI co-crystallization experiments, resulting in a diverse assortment of polymorphs [30][31][32]98,[120][121][122].…”

“…To date, our research has been focused on the polymorph identification using the XRPD method for HI in the absence and presence of organic ligands and phenolic derivatives in pH variation. Ligands such as phenol and resorcinol derivatives which led to the formation of more than one monoclinic symmetry polymorphs are of particular interest [31,32] (Figure 11). The previously referenced P2 1(γ) crystal polymorph (a = 87.0749 7 Table 2, while in pH: 6.7 to 8.6 and 6.2 to 8.1, the complexes adopt the rhombohedral R3 symmetry, with R 6 and T 3 R 3 f HI conformation accordingly [26,31].…”

“…Owing to the simplicity of the XRPD data collection process and the sensitivity of the method, since each polymorph reveals a unique pattern, the technique has become a robust tool for thorough examination of a wide range of microcrystalline precipitates [23,25]. Screening of crystalline polymorphism [30][31][32] is a very important step in the structural characterization of molecules of pharmaceutical interest as different crystalline polymorphs are often associated with modified physicochemical properties and/or biological activity. A pharmaceutical composition may consist of more than one component, each of which is an active pharmaceutical ingredient [33].…”