Analysis of an industrial production suspension ofBacillus lentussubtilisin crystals by powder diffraction: a powerful quality-control tool

Abstract

A microcrystalline suspension of Bacillus lentus subtilisin (Savinase) produced during industrial large-scale production was analysed by X-ray powder diffraction (XRPD) and X-ray single-crystal diffraction (MX). XRPD established that the bulk microcrystal sample representative of the entire production suspension corresponded to space group P212121, with unit-cell parameters a = 47.65, b = 62.43, c = 75.74 Å, equivalent to those for a known orthorhombic crystal form (PDB entry 1ndq). MX using synchrotron beamlines at the Diamond Light Source with beam dimensions of 20 × 20 µm was subsequently used to study the largest crystals present in the suspension, with diffraction data being collected from two single crystals (∼20 × 20 × 60 µm) to resolutions of 1.40 and 1.57 Å, respectively. Both structures also belonged to space group P2(1)2(1)2(1), but were quite distinct from the dominant form identified by XRPD, with unit-cell parameters a = 53.04, b = 57.55, c = 71.37 Å and a = 52.72, b = 57.13, c = 65.86 Å, respectively, and refined to R = 10.8% and Rfree = 15.5% and to R = 14.1% and Rfree = 18.0%, respectively. They are also different from any of the forms previously reported in the PDB. A controlled crystallization experiment with a highly purified Savinase sample allowed the growth of single crystals of the form identified by XRPD; their structure was solved and refined to a resolution of 1.17 Å with an R of 9.2% and an Rfree of 11.8%. Thus, there are at least three polymorphs present in the production suspension, albeit with the 1ndq-like microcrystals predominating. It is shown how the two techniques can provide invaluable and complementary information for such a production suspension and it is proposed that XRPD provides an excellent quality-control tool for such suspensions.

Rietveld Refinement for Macromolecular Powder Diffraction

“…On the contrary, powder diffraction has long been employed for characterizing crystalline, nanocrystalline, and amorphous powders of pharmaceutical importance, and due to the development of extensive databases, materials can be analyzed efficiently and in a high-throughput manner by their diffraction pattern. , Preliminary structural information, such as phase identification and characterization of unit-cell parameters, is also readily available from macromolecular powder samples. ,,,− ,,,,,,− Microcrystalline formulations provide enhanced bioavailability as well as increased stability and unique dissolution characteristics compared to amorphous or “in solution” counterparts. Although there are numerous attempts − in the literature toward the development of macromolecular microcrystalline formulations, the only successful large-scale example remains that of human insulin.…”

Conformational heterogeneity of Savinase from NMR, HDX-MS and X-ray diffraction analysis

“…Molecular replacement was performed using the published 1.4 Å resolution crystal structure of wild-type Savinase (PDB accession code: 4CFY) as the search model (Frankaer et al, 2014). Refinement was performed by PHENIX (Liebschner et al, 2019), to establish preliminary single-conformer structures for the RT and cryogenic crystals.…”

“…The substrate specificity is low but hydrophobic residues are favoured at positions P1 and P4 of the substrate (Schechter & Berger, 1967). In the structure of Savinase, two structural metal binding sites are found to be occupied by Na + and Ca 2+ (Frankaer et al, 2014).…”

Insulin polymorphism induced by two polyphenols: new crystal forms and advances in macromolecular powder diffraction

“…X-ray powder diffraction (XRPD) permits the analysis of microcrystals as an ensemble. XRPD has already been successfully employed for the structural characterization of proteins (Von Dreele et al, 2000;Basso et al, 2005;Margiolaki et al, 2007Margiolaki et al, , 2013Margiolaki & Wright, 2008;Karavassili et al, 2017) and peptides (Inouye & Kirschner, 2006;Fujii et al, 2011;Fili et al, 2019), resulting in medium-resolution models ($2.5-5 Å ), as well as for crystal and polymorph screening (Margiolaki et al, 2005;Norrman et al, 2006;Hartmann et al, 2010;Hunter et al, 2011;Karavassili et al, 2012;Frankaer et al, 2014;Das et al, 2015;Valmas et al, 2015Valmas et al, , 2018Fili et al, 2015Fili et al, , 2016Trampari et al, 2018;Spiliopoulou et al, 2020). The method is ideal for the identification (Collings et al, 2010), refinement and quantification of multiple protein polymorphs coexisting in the same sample (Basso et al, 2005).…”