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Analysis of an industrial production suspension of Bacillus lentus subtilisin crystals by powder diffraction: A powerful quality-control tool

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Author(s)

  • C.G. Frankaer
  • O.V. Moroz
  • J.P. Turkenburg
  • S.I. Aspmo
  • M. Thymark
  • E.P. Friis
  • K. Stahl
  • J.E. Nielsen
  • K.S. Wilson
  • P. Harris

Department/unit(s)

Publication details

JournalActa Crystallographica Section D: Biological Crystallography
DatePublished - Apr 2014
Issue number4
Volume70
Number of pages9
Pages (from-to)1115-1123
Original languageEnglish

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 P212121, 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 R free = 15.5% and to R = 14.1% and R free = 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 R free 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.

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