Experimental Work

Bovine serum albumin (BSA), Tris buffer salt and potassium nitrate (both ACS reagent grade) were purchased from Sigma-Aldrich Co, Dorset, UK. All materials were used for analysis without any further purification. A triple filtered (filter pore size 0.22 µm) 10 mM TRIS buffer (pH 7.2) was used to prepare four sets of 4 mg/mL BSA solution.

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3.3.2 Method – Sample Preparation

Control sample – Stock solutions of 4 mg/mL BSA solution were prepared in a triple filtered 10 mM

TRIS buffer (pH 7.2) directly in a regular glass cuvette to prevent particulate contamination. The cuvette was immediately capped and sealed with parafilm. The cuvette was gently rotated manually until all the BSA particulates were dissolved into the filtered medium. The control samples were incubated at 4 °C for 20 minutes to ensure maximum dissolution of all protein solids before particle size analysis. To ensure no variation in concentrations, UV-vis absorbance at 280 nm was recorded and the concentration determined from the calibration plot presented in Figure 2-14. The recorded results were in agreement with each other (data not shown).

Stirring - Stock solution of 4 mg/mL BSA was prepared in a 50 mL beaker and covered with parafilm.

The solution was mixed using low shear magnetic stirring at a speed of 130 RPM (0.6 RCF). The sample was agitated continuously for up to 60 minutes in intervals of 10 minutes. The agitated samples were incubated at 4 °C for 20 minutes to ensure maximum dissolution of all protein solids before particle size analysis.

Shearing through needle – 4 mg/mL solutions of BSA were prepared by adding directly the solvent

and solute into regular glass cuvettes and sealed with a parafilm. The solutions were manually agitated via repeated inversion for up to 60 seconds and allowed to settle for further 60 seconds. The samples were subjected to high shear stress via the passage through a hypodermic needle (attached to a 10 mL syringe) with an internal diameter of 0.04 cm and length of 9 cm. The total shear produced in the syringe and needle was estimated as has been reported earlier (Charm and Wong, 1970). The amount of shear stress on protein molecule can be expressed as the product of shear rate (s) and residence time (t) as presented in Eqn. 3-1.

𝑺𝒉𝒆𝒂𝒓 𝒔𝒕𝒓𝒆𝒔𝒔 = 𝒔 × 𝒕 = 𝟖/𝟑 × 𝒍 × 𝒓 Eqn. 3-1

where l is the length (mm) and r is the radius (mm) of the capillary tube. In this work, the relationship between the number of sample solution expulsions from the syringe and shear stress is as presented in Table 3-1 and henceforth all data will be presented shear stress.

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Table 3-1 Theoretical estimates of shear stress experienced by BSA solution agitated using syringe. Number of Ejections from syringe Ejection Cycle (double ejection) Calculated shear stress (Pa)

5 10 4.8 10 20 9.6 20 40 19.2 30 60 28.8 40 80 38.4 50 100 48.0

To ensure no variation in concentrations, UV-vis absorbance at 280 nm was recorded and the concentration determined from the calibration plot presented in Figure 2-14. The recorded results were in agreement with each other (data not shown).

Mechanical Shaking - 4 mg/mL solutions of BSA was prepared directly in a regular glass cuvettes,

capped immediately and sealed with parafilm. Samples were placed in a bath sonicator with a frequency of 20 kHz, and the power output set to deliver an average value of 30W. Ice was added to the water in the bath to maintain the temperature below 20 °C. Each cycle consisted of 120 seconds sonication followed by 60 seconds incubation. The agitated samples were further equilibrated for 20 minutes at 4 °C before particle size analysis. To ensure no variation in concentrations, UV-vis absorbance at 280 nm was recorded and the concentration determined from the calibration plot presented in Figure 2-14. The recorded results were in agreement with each other (data not shown).

3.3.3 Data acquisition and analysis

Particle size analysis of protein macromolecules (control and agitated) were analysed with dynamic

light scattering (DLS) (Zetasizer Omni, Brookhaven). The measurements were carried out at an angle of 173 ° (back scattering), which is advantageous compared to 90 ° angle measurement as a large area (proportion of the sample in cuvette) is analysed. Continuous sampling was taking for up to 120 seconds to ensure a true representation of the sample at a temperature of 25 °C. All measurements were repeated at least five (5) consecutive times.

The data was initially analysed using the non-negatively constrained least squares (NNLS) (Tscharnuter, 2000) and further fitted with multi modal distribution. In principle, when different distribution of sizes is present, the measured effective diameter (or Z-average) is a mean average diameter weighted by the intensity of light scattered by all the distributions. This is represented mathematically as Eqn. 3-2.

116 | P a g e 𝟏 𝐃𝐳 = ∑ 𝐟_𝐢𝐃_𝐢𝟔_{𝐏(𝛉)𝐃} 𝐢 −𝟏 ∑ 𝐟𝐢𝐃𝐢𝟔𝐏(𝛉) Eqn. 3-2

Where Dz is the z-average (effective diameter), fi is the number of particles, Di is the diameter and P (θ) is the scattering angle of the particle.

However, in the case where more than one (1) distribution band originating from more than one (1) particle size population, the effective diameter takes into account all the distributions. Therefore, in this work, the data presented will be the weight average diameter of individual populations since this approach considers directly the distribution of interest. Similarly, the polydispersity index, which is a measure of the broadness of the distribution also takes into account all the distribution broadness. Hence, the broadness of distribution will also be estimated as the FWHM as described in Chapter 2. This may be used as a quantitative tool for relative analysis of the effects of agitation in time (low shear and mechanical vibrations) or shear intensity (high shear).

Turbidimetry measurements (UV-vis 360 nm) were carried out using Evolution 60S (Thermo Fisher

Scientific) to complement the observations made from particle size analysis. Samples were measured at 25 °C following the method discussed in Chapter 2. For non-agitated BSA solution, the samples were measured (directly without any agitation) in intervals of 10 minutes for up to 120 minutes and in each instance incubated at 4 °C. For agitated BSA solution, the cuvette was gently rotated manually to avoid bigger sized particles settling at the bottom of the cuvette. The apparent absorbance of the samples was measured using regular plastic cuvette with 1 cm path length in triplicates and used for further analysis.

Thioflavin T Assay - Fluorescence spectroscopy was used to detect the presence of protein aggregates

in the agitated solution. BSA sample solutions were measured using LS55 spectrofluorometer (Perkin- Elmer, UK) connected to a water bath to maintain the sample temperature. Aliquots of 2 mL individual 4 mg/mL BSA sample solutions were placed into a regular glass cuvette with 1 cm path length and 20 µL of a 1 mM of Thioflavin T solution added to the solution. The solution was equilibrated at 25 °C for five minutes before samples were measured. The solution was excited at 445 nm (2.5 nm excitation slit) and an emission spectra acquired between the wavelengths of 455 nm to 550 nm (5 nm emission slit).

The data presented from each measurement was a cumulated average from three repeated (n=1x3) analysis of the same sample to reduce signal to noise ratio. Measurements were carried out in

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triplicates and the averages used for analysis (n=3x3). As described in earlier sections, the intensity and positional shift of the local maxima for each data were analysed for all types of agitation/stress.