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

Abstract

A microcrystalline suspension ofBacillus lentussubtilisin (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 groupP212121, with unit-cell parametersa= 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 groupP212121, but were quite distinct from the dominant form identified by XRPD, with unit-cell parametersa= 53.04,b = 57.55,c= 71.37 Å anda= 52.72,b= 57.13,c= 65.86 Å, respectively, and refined toR= 10.8% andRfree= 15.5% and toR= 14.1% andRfree= 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 anRof 9.2% and anRfreeof 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.