High-throughput macromolecular polymorph screening via an NMR and X-ray powder diffraction synergistic approach: the case of human insulin co-crystallized with resorcinol derivatives

Abstract

Regular injections of insulin provide life-saving benefits to millions of diabetics. Apart from native insulin and insulin analogue formulations, microcrystalline insulin suspensions are also commercially available. The onset of action of the currently available basal insulins relies on the slow dissociation of insulin hexamers in the subcutaneous space due to the strong binding of small organic ligands. With the aim of identifying insulin–ligand complexes with enhanced pharmacokinetic and pharmacodynamic profiles, the binding affinity of two resorcinol-based molecules (4-chlororesorcinol and 4-bromoresorcinol) and the structural characteristics of insulin upon co-crystallization with them were investigated in the present study. `In solution' measurements were performed via saturation transfer difference (STD) NMR. Co-crystallization upon pH variation resulted in the production of polycrystalline precipitates, whose structural characteristics (i.e. unit-cell symmetry and dimension) were assessed. In both cases, different polymorphs (four and three, respectively) of monoclinic symmetry (P21 and C2 space groups) were identified via X-ray powder diffraction. The results demonstrate the efficiency of a new approach that combines spectroscopy and diffraction techniques and provides an innovative alternative for high-throughput examination of insulin and other therapeutic proteins.

Small-Angle X-ray Scattering as a Powerful Tool for Phase and Crystallinity Assessment of Monoclonal Antibody Crystallites in Support of Batch Crystallization

“…Several methods have nevertheless successfully been used for the detection and physical characterization of protein crystals, including secondary order nonlinear imaging of chiral crystals (SONICC), , solid-state nuclear magnetic resonance (ssNMR), − Raman, and Infrared (IR). , High-resolution diffraction techniques, generally requiring the use of synchrotron radiation, also play an important role in the characterization of mAb single crystals. These include high-resolution SCXRD, XRPD, ,− and electron diffraction (ED) . They can provide precise unit cell parameters of the mAb crystal lattice, their molecular conformation, water environment, and a direct assessment of crystallinity based on the quality of the resulting diffractions; for example, peak width, intensity, and positions from XRPD.…”

“…This is predominantly due to their weak diffraction patterns and the tendency of these crystals to exhibit broad signals caused by strains in their crystal lattice. Several methods have nevertheless successfully been used for the detection and physical characterization of protein crystals, including secondary order nonlinear imaging of chiral crystals (SONICC), , solid-state nuclear magnetic resonance (ssNMR), − Raman, and Infrared (IR). , High-resolution diffraction techniques, generally requiring the use of synchrotron radiation, also play an important role in the characterization of mAb single crystals. These include high-resolution SCXRD, XRPD, ,− and electron diffraction (ED) .…”