Effect of mechanical denaturation on surface free energy of protein powders.

Globular proteins are important both as therapeutic agents and excipients. However, their fragile native conformations can be denatured during pharmaceutical processing, which leads to modification of the surface energy of their powders and hence their performance. Lyophilized powders of hen egg-white lysozyme and ß-galactosidase from Aspergillus oryzae were used as models to study the effects of mechanical denaturation on the surface energies of basic and acidic protein powders, respectively. Their mechanical denaturation upon milling was confirmed by the absence of their thermal unfolding transition phases and by the changes in their secondary and tertiary structures. Inverse gas chromatography detected differences between both unprocessed protein powders and the changes induced by their mechanical denaturation. The surfaces of the acidic and basic protein powders were relatively basic, however the surface acidity of ß-galactosidase was higher than that of lysozyme. Also, the surface of ß-galactosidase powder had a higher dispersive energy compared to lysozyme. The mechanical denaturation decreased the dispersive energy and the basicity of the surfaces of both protein powders. The amino acid composition and molecular conformation of the proteins explained the surface energy data measured by inverse gas chromatography. The biological activity of mechanically denatured protein powders can either be reversible (lysozyme) or irreversible (ß-galactosidase) upon hydration. Our surface data can be exploited to understand and predict the performance of protein powders within pharmaceutical dosage forms.

Equation to Line the Borders of the Folding-Unfolding Transition Diagram of Lysozyme.

It is important for the formulators of biopharmaceuticals to predict the folding-unfolding transition of proteins. This enables them to process proteins under predetermined conditions, without denaturation. Depending on the apparent denaturation temperature (Tm) of lysozyme, we have derived an equation describing its folding-unfolding transition diagram. According to the water content and temperature, this diagram was divided into three different areas, namely, the area of the water-folded lysozyme phase, the area of the water-folded lysozyme phase and the bulk water phase, and the area of the denatured lysozyme phase. The water content controlled the appearance and intensity of the Raman band at ∼1787 cm(-1) when lysozyme powders were thermally denatured at temperatures higher than Tm.

Mapping the solid-state properties of crystalline lysozyme during pharmaceutical unit-operations.

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.

The impact of low-level inorganic impurities on key physicochemical properties of paracetamol.

Trace inorganic impurities in active pharmaceutical ingredients (APIs) while having limited toxicological significance might affect the down stream processing properties of those substances. The level of impurities in paracetamol batches was quantified and mapped using inductively coupled polarization mass spectrometry (ICP-MS) and scanning electron microscopy coupled with energy dispersive X-ray microanalysis (SEM-EDX). The physical form of samples was assessed using X-ray powder diffraction (XRPD) and characterised thermally using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Surface properties were evaluated using inverse gas chromatography (IGC) and moisture sorption. Size distribution was measured using an aerosizer with dry powder dispersion. Physical analysis confirmed that the batches were of the same physical form and particle size distribution was shown to be similar. The SEM-EDX analysis however revealed the presence of aluminium on the surface of particles. This was supported by ICP-MS analysis, which showed different levels of aluminium between batches ranging from 0.1 to 5.6 ppm. IGC indicated that the batches with the highest aluminium content had the highest dispersive free energy. Differences in levels of inorganic impurities typically not considered significant in drug substance specifications correspond with differences in physical properties of APIs, with potential downstream consequences for processing and finished product performance.

Quantitative analysis of mannitol polymorphs. FT-Raman spectroscopy.

Mannitol is a polymorphic excipient which is usually used in pharmaceutical products as the beta form, although other polymorphs (alpha and delta) are common contaminants. Binary mixtures containing beta and delta mannitol were prepared to quantify the concentration of the beta form using FT-Raman spectroscopy. Spectral regions characteristic of each form were selected and peak intensity ratios of beta peaks to delta peaks were calculated. Using these ratios, a correlation curve was established which was then validated by analysing further samples of known composition. The results indicate that levels down to 2% beta could be quantified using this novel, non-destructive approach. Potential errors associated with quantitative studies using FT-Raman spectroscopy were also researched. The principal source of variability arose from inhomogeneities on mixing of the samples; a significant reduction of these errors was observed by reducing and controlling the particle size range. The results show that FT-Raman spectroscopy can be used to rapidly and accurately quantitate polymorphic mixtures.

Quantitative analysis of mannitol polymorphs. X-ray powder diffractometry--exploring preferred orientation effects.

Mannitol is a polymorphic parmaceutical excipient, which commonly exists in three forms: alpha, beta and delta. Each polymorph has a needle-like morphology, which can give preferred orientation effects when analysed by X-ray powder diffractometry (XRPD) thus providing difficulties for quantitative XRPD assessments. The occurrence of preferred orientation may be demonstrated by sample rotation and the consequent effects on X-ray data can be minimised by reducing the particle size. Using two particle size ranges (<125 and 125-500 microm), binary mixtures of beta and delta mannitol were prepared and the delta component was quantified. Samples were assayed in either a static or rotating sampling accessory. Rotation and reducing the particle size range to <125 microm halved the limits of detection and quantitation to 1 and 3.6%, respectively. Numerous potential sources of assay errors were investigated; sample packing and mixing errors contributed the greatest source of variation. However, the rotation of samples for both particle size ranges reduced the majority of assay errors examined. This study shows that coupling sample rotation with a particle size reduction minimises preferred orientation effects on assay accuracy, allowing discrimination of two very similar polymorphs at around the 1% level.

Analysis of the surface energy of pharmaceutical powders by inverse gas chromatography.

The behavior of pharmaceutical solids, during either processing or use, can be noticeably affected by the surface energetics of the constituent particles. Several techniques exist to measure the surface energy, for example, sessile drop, and dynamic contact angle measurements. Inverse gas chromatography (IGC) is an alternative technique where the powder surface is characterized by the retention behavior of minute quantities of well-characterized vapors that are injected into a column containing the material of interest. Recently published articles using IGC on pharmaceutical powders have ranged from linking surface energetic data with triboelectric charging to studying the effect of surface moisture on surface energetics. Molecular modelling has also recently been used to explore the links between IGC data and the structural and chemical factors that influence surface properties, thereby achieving predictive knowledge regarding powder behavior during processing. In this minireview, the reported applications of IGC in the analysis of pharmaceutical powders are summarized and the major findings highlighted.