Purpose: To formulate and stabilize some examples of proteins using lysozyme as the model of protein with excipients.
• To prepare dried formulations using spraydrying and electrospraying technique with and without excipients using betacyclodextrin and pluronic F-127 .
• To characterize the protein using techniques like Differential Scanning Calorimetry for thermal stability analysis and UV-Vis- spectrophotometry for enzymatic analysis.
Methods: PREPARATION OF SPRAY DRIED LYSOZYME
Various concentrations of 1% lysozyme solution 1g of lysozyme in 100ml of distilled water) with the addition of some excipients (betacyclodextrin, pluronic) was prepared and spray dried using a Mini Buchi Spray Dryer B-290 model of spray drier. The prepared concentrations of feed solution were passed from a pipe through the atomising nozzle of 0.5mmdiameter to the drying chamber via a peristaltic feed pump at a flow rate of 20ml/min, dried with an inlet temperature of 130±3 oC, outlet temperature of 73±oC. Cooling was circulated through the jacket around the nozzle for protein degradation minimisation. Spraydried powder was collected in the cyclone separator and stored in a vial in a desiccator and stored in the fridge
PREPARATION OF ELECTRO SPRAYED LYSOZYME
Various concentrations of 1% lysozyme solution with various excipients; dissolved in 80% ethanol and 20% water (80:20) was homogenised by a magnetic stirring and loaded into the needle with a programmable syringe pump. The lysozyme solution in the axial compartment was slightly pressurised to achieve stable spraying. A positive high voltage was used to maintain the voltage rage of 8-18kv. A collecting slide was placed on a ground rotating drum controlled by a stepping motor.
DIFFERENTIAL SCANNING CALORIMETRY
A 2-4mg of solid sample of various formulations were analysed with a DSC Q1000 UK thermal analysis instrument made in England. Each formulation was sealed in a DSC aluminium pan with lids and loaded into the cells. An empty pan with lid was used a reference, flowrate of nitrogen at 50ml/min, equilibrated at 10.00°C. Ramp 0.001°C to 300.00°C.
ENZYMATIC ANALYSIS LYSOZYME
This biological activity is obtained with an established enzymatic potency assay. The activity of lysozyme was assayed by hydrolysing a bacterial suspension (20mg%) of Micrococcus lysodeikticus in phosphate buffer. A
concentration of 20g in 90ml of 0.0067M, at a pH of 6.24 and 10ml of 1% NaCl was prepared. A 0.01% (w/v) of each sample with 0.01g of various prepared formulation dissolved in 25ml of buffer. The biological activity was determined with 0.5ml of each prepared protein added to 2.5ml of the bacterial suspension and a decrease in the absorbance with M501 single beam UV-Vis spectrophotometer at a wavelength of 450nm. The time of activity was measured at (0, 1, 2, 3) minutes. The result of the activity was determined with the application of Shugar 1952 equation3.
Activity (units/mg) =∆450nm/min/ 0.001 x mg enzyme present in the mixture.
Results: DSC was used to analyse the protein denaturation and the effect of the drying technique chosen on the stability of protein with the solid sample lysozyme. The results for the unprocessed lysozyme and spray dried lysozyme showed little or no denaturation however, electro sprayed lysozyme has shown slight increase in temperature making it more stable compared to spray dried and unprocessed lysozyme as shown in Figure 1
High denaturation temperature for electro spray dried lysozyme with excipient (betacyclodextrin and pluronic)
have shown some stability but subject to confirmation with other techniques. Spray dried lysozyme with excipients showed no peak as shown in Figure 2.
The biological activity of all prepared formulation are shown in Figure 3 at three (3) different time interval 60 seconds, 120 seconds and 180 seconds. At 60 seconds, spray dried formulation showed 100% activity and decreased its activity by 10% after 120 seconds while electro sprayed formulation showed 90% activity at 60 seconds and increasing by 10% at 120 seconds. Also, all
formulations with excipient have shown 90-100% activity confirming the stabilizing effect of the excipients except spray dried lysozyme with pluronic that has shown less stability with 60% at 60 seconds and 30% at 120seconds
Conclusion: It is possible to state that a combination of betacyclodextrin and pluronic could increase the activity and stability of protein using the right technique and formulation as electrospray has shown in the data.
Although spray dried lysozyme with excipients has shown some certain activities in the biological activity and has not shown a sign of denaturation, it is important to note that the protein can still be active but a certain level or part of the structure might have been affected.
Excipients for protein characterization seem to look promising with some positive and negative outcomes
Amal Elkordy– Reader, University of Sunderland, Sunderland, England