title: Mapping the solid-state properties of crystalline lysozyme during pharmaceutical unit-operations.
abstract: Bulk crystallisation of protein therapeutic molecules towards their controlled drug delivery is of interest to the biopharmaceutical industry. The complexity of biotherapeutic molecules is likely to lead to complex material properties of crystals in the solid state and to complex transitions. This complexity is explored using batch crystallised lysozyme as a model. The effects of drying and milling on the solid-state transformations of lysozyme crystals were monitored using differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), FT-Raman, and enzymatic assay. XRPD was used to characterise crystallinity and these data supported those of crystalline lysozyme which gave a distinctive DSC thermogram. The apparent denaturation temperature (Tm) of the amorphous lysozyme was ∼201 °C, while the Tm of the crystalline form was ∼187 °C. Raman spectra supported a more α-helix rich structure of crystalline lysozyme. This structure is consistent with reduced cooperative unit sizes compared to the amorphous lysozyme and is consistent with a reduction in the Tm of the crystalline form. Evidence was obtained that milling also induced denaturation in the solid-state, with the denatured lysozyme showing no thermal transition. The denaturation of the crystalline lysozyme occurred mainly through its amorphous form. Interestingly, the mechanical denaturation of lysozyme did not affect its biological activity on dissolution. Lysozyme crystals on drying did not become amorphous, while milling-time played a crucial role in the crystalline-amorphous-denatured transformations of lysozyme crystals. DSC is shown to be a key tool to monitor quantitatively these transformations.
authors.affiliation: Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK; Faculty of Pharmacy, University of Damascus, Damascus, Syria; School of Pharmacy, University of Bradford, Bradford BD7 1DP, UK. Electronic address: mam71@bath.ac.uk.
authors.forename: Mohammad Amin
authors.initials: MA
authors.lastname: Mohammad
authors.affiliation: School of Pharmacy, University of Bradford, Bradford BD7 1DP, UK. Electronic address: i.m.grimsey@bradford.ac.uk.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Grimsey
authors.affiliation: School of Pharmacy, University of Bradford, Bradford BD7 1DP, UK. Electronic address: r.t.forbes@bradford.ac.uk.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Forbes
mesh_terms: D000818:Animals; D002151:Calorimetry; D002152:Calorimetry, Differential Scanning; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D002645:Chickens; D003460:Crystallization; D003890:Desiccation; D004529:Egg White; D006863:Hydrogen-Ion Concentration; D008837:Micrococcus; D009113:Muramidase / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D013696:Temperature; D013818:Thermogravimetry; D014867:Water / Q000737:chemistry; D014961:X-Ray Diffraction
chemical_list: D014867:Water; D009113:Muramidase
keywords: Crystalline-amorphous-denatured transformations; Differential scanning calorimetry; FT-Raman; Lysozyme crystals; Milling; X-ray powder diffraction