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

Structural transitions of monoolein bicontinuous cubic phase induced by inclusion of protein lysozyme solutions

Inclusion of protein lysozyme molecules in lipidic monoolein cubic phase induces a transition from a $\rm Pn\bar{3}m$ structure to $\rm Im\bar{3}m$ one. Small-angle X-ray scattering (SAXS) method with high intensity synchrotron radiation enabled us to follow closely the transition depending on the conditions of lysozyme solutions. We showed that concentrated lysozyme solutions induced the appearance of the $\rm Im\bar{3}m$ structure coexisting with the $\rm Pn\bar{3}m$ structure. From the relation between the lattice parameters of these two structures it was shown that they were related by the Bonnet transformation of underlying triply periodic minimal surfaces. We found that the transition also occurred at lower lysozyme concentration when NaCl induced attraction between lysozyme molecules. The origin of the transition was considered as a frustration in the cubic phase where lysozyme molecules were highly confined. A simple estimation of the frustration was given, which took into account of the translational entropy of lysozyme molecules. At the highest concentration of lysozyme and NaCl the $\rm Im\bar{3}m$ structure was found to disappear and left only the $\rm Pn\bar{3}m$ structure. This was probably either due to the crystallization or phase separation of lysozyme solutions ongoing microscopically, which absorbed lysozyme molecules from channels of the cubic phase and thus removed the frustration.Comment: 8 pages, 5 figure

Light scattering and phase behavior of Lysozyme-PEG mixtures

Measurements of liquid-liquid phase transition temperatures (cloud points) of mixtures of a protein (lysozyme) and a polymer, poly(ethylene glycol) (PEG) show that the addition of low molecular weight PEG stabilizes the mixture whereas high molecular weight PEG was destabilizing. We demonstrate that this behavior is inconsistent with an entropic depletion interaction between lysozyme and PEG and suggest that an energetic attraction between lysozyme and PEG is responsible. In order to independently characterize the lysozyme/PEG interactions, light scattering experiments on the same mixtures were performed to measure second and third virial coefficients. These measurements indicate that PEG induces repulsion between lysozyme molecules, contrary to the depletion prediction. Furthermore, it is shown that third virial terms must be included in the mixture's free energy in order to qualitatively capture our cloud point and light scattering data. The light scattering results were consistent with the cloud point measurements and indicate that attractions do exist between lysozyme and PEG.Comment: 5 pages, 2 figures, 1 tabl

Release of proteins via ion exchange from albumin-heparin microspheres

Albumin-heparin and albumin microspheres were prepared as ion exchange gels for the controlled release of positively charged polypeptides and proteins. The adsorption isotherms of chicken egg and human lysozyme, as model proteins, on microspheres were obtained. An adsorption isotherm of chicken egg lysozyme on albumin-heparin microspheres was linear until saturation was abruptly reached,\ud \ud The adsorption isotherms of human lysozyme at low and high ionic strength were typical of adsorption isotherms of proteins on ion exchange gels. The adsorption of human lysozyme on albumin-heparin and albumin microspheres fit the Freundlich equation suggesting heterogeneous binding sites. This was consistent with the proposed multivalent, electrostatic interactions between human lysozyme and negatively charged microspheres. Scatchard plots of the adsorption processes of human lysozyme on albumin-heparin and albumin microspheres suggested negative cooperativity, while positive cooperativity was observed for chicken egg lysozyme adsorption on albumin-heparin microspheres.\ud \ud Human lysozyme loading of albumin-heparin microspheres was 3 times higher than with albumin microspheres, with long term release occurring via an ion exchange mechanism. Apparent diffusion coefficients of 2.1 × 10-1 and 3.9 × 10-11cm2/sec were obtained for the release of human lysozyme from albumin-heparin and albumin microspheres, respectively. The release was found to be independent of diffusion, since the rate determining step was likely an adsorption/desorption processes. An apparent diffusion coefficient of 4.1 × 10-12 cm2/sec was determined for the release of chicken egg lysozyme from albumin-heparin microspheres.\ud \ud Low release of the lysozymes from albumin-heparin microspheres was observed in deionized water, consistent with the proposed ion exchange release mechanism. Overall, albumin-heparin microspheres demonstrated enhanced ion exchange characteristics over albumin microspheres

Anti-bacterial activity of lysozyme in pitching yeast and effect of lysozyme on yeast fermentation

Lysozyme has antibacterial activity against Gram-positive bacteria and has no activity against yeast. As such, lysozyme can be used for the specific inhibition of beer spoilage bacteria. After all, the most frequently identified beer spoilage bacteria are lactic acid bacteria and brewers’ yeast is the culture used for the fermentation. Hen egg white lysozyme (300 mg/L) is tested for the antibacterial activity against four described Gram-positive beer spoilage bacteria in industrial pitching yeast and in industrial beer with refermentation in the bottle. The influence of industrial pitching yeast treated with lysozyme on the fermentation performance is also studied

The potential use of hen egg white lysozyme as an antimicrobial agent in foods : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Food Technology at Massey University

The potential use of lysozyme as an antimicrobial agent in foods was investigated in model food systems(brainheart infusion broth) using factorial designed experiments and in mussel and cottage cheese food systems. Optical density or absorbance was used as a tool to monitor the growth response of Listeria monocytogenes and C. tyrobutyricum in brain heart infusion broth under the combined influence of pH(5.5, 6.5), lysozyme (0.2mg/ml, 3mg/ml) and different chelating agents(ethylene diaminetetraacetic acid(EDTA), glycine, gluco delta lactone(GDL), citric acid, sodium phosphate dibasic(SPDB) and sodium hexametaphosphate(SHMP)(10mM, 25mM). Using 2 3 full factorial design experiments, the yield of the organisms (expressed as the area under the curve of a plot of change in optical density at 600nm vs time) was taken as the quantitative response variable for each treatment. These yield values were then used for (a) statistical analysis to determine which of the single or interactive factors tested significantly reduced the yield, (b) formulation of a mathematical regression equation which could be used to predict microbial growth within the limits of the factors studied. Diagnostic plots were constructed to evaluate further how well the statistical model fit the observed yield values. Plots of residuals versus predicted yield values appeared to suggest that a transformation of the response would improve the fit of the models. No other serious reservations were suggested by the diagnostic plots. Goodness of fit of the models was also evaluated by the R-squared values. Significant two-way and three-way interactions between lysozyme, pH and EDTA, GDL, citric acid and glycine were exhibited. Response surface methodology(RSM) was used to (a) characterize the response of L. monocytogenes to variation in treatment combinations and (b) show non-linearity of models(or interaction of factors). Generally yield was minimal in treatment where pH was low, with high lysozyme and chelator. Based on equal molar concentrations, the antimicrobial activity of the different chelating agents was in the order EDTA > GDL > citric acid > glycine > adipic acid > SHMP > SPDB. The same ranking was true for the degree to which each chelating agent enhanced lysozyme activity. Based on broth culture studies, the chelating agents EDTA, GDL, glycine, citric acid and adipic acid were demonstrated to have potential for use as antimicrobial agents in combination with lysozyme in food systems. Results of a 2 5 factorial design indicated that the 5 factors, lysozyme, GDL, pH, inoculum level and temperature were important in the inhibition of L. monocytogenes. Results of the broth culture studies gave a good reflection of the survival of L. monocytogenes in the food system. The variable combinations interacted to decrease the growth of L. monocytogenes and extended the lag phase duration. However C. tyrobutyricum was more tolerant to the different treatment combinations other than EDTA. A study of protein interference demonstrated that the antimicrobial activity of the lysozyme-GDL preservation system was not inhibited by the presence of proteins. The food system study demonstrated that the lysozyme-GDL treatment combination has potential for use as a preservative in refrigerated low pH ready-to-eat foods. The susceptibility of L. monocytogenes to lysozyme-GDL treatment in both broth culture and food systems increased as the temperature was reduced(25C-5C) and as the pH decreased(pH6.5-pH5.5). Food system studies demonstrated that modified atmosphere packaging(96.58%N2 , 2.09%O2 and 1.34% CO2) has no influence of the growth of L. monocytogenes. The susceptibility of L. monocytogenes to lysozyme-GDL was a stable characteristic, remaining unchanged during the entire study. Attempts to select for greater lysozyme-GDL resistance by testing populations grown from lysozyme-GDL survivors isolated at the end of the food system study was unsuccessful. There was no evidence that L. monocytogenes was resistant to the lysozyme-GDL treatment

Concentration polarization phenomena during dead-end ultrafiltration of protein mixtures. The influence of solute-solute interactions

The flux decline behaviour of some charged proteins and of binary mixtures of charged solutes during unstirred dead-end ultrafiltration has been studied. The mixtures consisted of the proteins bovine serum albumin, (BSA), α-lactalbumin and/or lysozyme. Of special interest were α-lactalbumin and lysozyme because these proteins are physico-chemically identical, except for the sign of their charge at the conditions used (pH = 7.4, I=0.125 N and T=20°C). The ultrafiltration properties were studied using the boundary layer resistance model. Ultrafiltration of single protein solutions of α-lactalbumin and of lysozyme showed identical characteristics. The fouling behaviour during ultrafiltration of binary mixtures of the three components appeared to be dependent on both the charge of the solutes and the (unequal) dimensions of the solutes. A mixture of oppositely charged proteins (i.e., BSA/lysozyme or α-lactalbumin/lysozyme) sometimes showed a considerable increase of the resistance of the concentrated layer near the membrane, depending on the mixing ratio of the two proteins. When equally charged (i.e., BSA/α-lactalbumin) proteins are ultrafiltered, a small decrease of the resistance could be observed, again depending on the mixing ratio of the proteins. The charge of the proteins, especially opposite charges, appeared to influence the flux behaviour more than the slightly denser packing of the solutes (as a result of unequal dimensions) would account for