Robert T. Forbes

Introduction: Protein aggregation and its reversibility over time has been measured for a number of protein systems by Taylor dispersion analysis (TDA). This technique has many advantages over the conventional techniques currently employed; including its fast analysis times, large concentration range, and nanolitre sample sizes. Here the results obtained by TDA are compared to those obtained from dynamic light scattering (DLS). Materials and Methods: The ActiPix D100 employs UV area imaging and Taylor dispersion analysis (TDA) for determining diffusion coefficients and hydrodynamic radii of proteins in solution. The detector monitors broadening of a band of a therapeutic protein injected into a stream of buffer solution being driven through a fused-silica capillary. The band is imaged at two windows, the first on entry to and the second on exit from a loop in the capillary. The hydrodynamic radius follows from the measured differences between peak times and variances at the two windows. This technique was used to analyse various unstressed and stressed BSA samples over periods of time. The results were compared to those obtained from DLS and thermal methods. Results and Discussion: Hydrodynamic radius results obtained using TDA were in good correlation to those obtained using DLS. TDA and DLS both indicated that over time some aggregates were resolubilised and that the aggregate level had decreased, however DLS was less sensitive than TDA to these subtle changes. TDA was also shown to be more advantageous for measuring aggregation given it is not as susceptible to large particulates affecting the results as was the case with the DLS. Conclusions: Results for these samples indicate good correlation between the techniques for investigating protein aggregation behaviour. The ability to quantify accurately aggregate levels is being explored. TDA showed notable advantages over DLS when looking at solutions containing large particulates.

Aqueous solutions of hen egg lysozyme (3% w/v) were dispersed and precipitated by a homogenous mixture of supercritical carbon dioxide–ethanol using the Solution Enhanced Dispersion by Supercritical fluid (SEDS) process. The effects of different working conditions, such as temperature, pressure and the flow rates of the solution and ethanol, on the particle-formation process were studied. The morphology, particle size and size distribution and biological activity of the protein were determined. The precipitates were examined with high-sensitivity differential scanning calorimetry (HSDSC) and high-performance cation-exchange chromatography. Particle size measurements showed the precipitates to be aggregates with primary particles of size 1–5 μm. The similarity of HSDSC data for unprocessed and processed samples indicated that the different physical forces that stabilise the native form of lysozyme are unchanged after SEDS processing. From FT-Raman spectroscopic studies secondary structural changes were observed in certain SEDS-produced lysozyme, with most processed samples displaying a slightly more disordered secondary structure than the unprocessed sample. However, SEDS samples produced at 200 bar and 40 °C exhibited negligible disturbance. Thus the SEDS process utilising aqueous solution was able to bring about size reduction of lysozyme with minimal loss of biological activity.