Browsing by Subject "trehaloosi"
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(2010)The objective of the research was to study the effects of different polymers, sugars, and cell handlings on the viabilities of ARPE-19 and ARPE-19-SEAP-2-neo cells after freeze-drying. Also the residual moisture content of the freeze-dried samples and the amount of intracellular sugar after incubation in trehalose medium were measured. The mixtures used in the study contained different polymers (e.g. polyvinylpyrrolidone, alginate, polyethylene glycol) and sugars (sucrose, trehalose and mannitol). Some cells were incubated or heated in trehalose medium before freeze-drying in order to increase the amount of intracellular sugar. The aim of the heating was also to increase the heat shock protein formation in the cells. The samples were mainly freeze-dried in 24 well plates. The same freeze-drying parameters were used in all freeze-drying runs. The freeze-drying cycle lasted 38.5 hours (freezing 2.5 h, primary drying 32 h, and secondary drying 4 h). In the freezing stage the samples were froze to -40 °C and the freezing rate was 1 °C/minute. In the primary drying stage the shelf temperature was mainly -35 °C and the pressure was 150 mTorr. The viabilities of the samples after freeze-drying were determined by measuring the fluorescence after 3 and 6 hours from addition of the Alamar Blue indicator dye. The residual moisture contents were measured by thermogravimeter. According to the results, mixture containing glycerol (9%) and PEG 10 000 (18%) was the best lyoprotectant in the study (70% viability after 3 hours). However, the viability decreased significantly (24% viability) in the measurement after 6 hours. Similar viability decrease was observed among all lyoprotectant mixtures used in the study. Extracellular sugars rarely had positive effect on the viability results. The 12 days incubation in 150 mM trehalose medium before freeze-drying affected positively to the post freeze-drying viability. Shorter incubation time or heating did not induce the same effect. Intracellular sugar measurements revealed, that the amount of intracellular trehalose was multiple after 12 days of incubation in 150 mM trehalose medium compared to the cells that were not incubated. The residual moisture contents of the samples varied between 0-7%. The residual moisture content of the sample containing glycerol 9% and PEG 10 000 18% was 1,5%.
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(2010)Immunoglobulin G is very unstable and that is why it is very challenging to formulate and process it. Because of the unstability, IgG is vulnerable to changes in pH, heat and mechanical stress. Exposure to these stresses makes IgG aggregate more easily and lose its biological activity. To restore stability, IgG is formulated to a solid product from which it can be regenerated. With TFF (Tangential Flow Filtration) IgG can be purified from other components. The filtration is based on a half-permeable membrane which permeates the other components except for the IgG. The filtration pressure is the force which keeps the liquid flowing. It is important to control this pressure, too high or too low pressure will damage the IgG. IgG can also be protected with polysorbate which is a surfactant going to the protein/liquid interface and therefore stabilizing IgG. IgG does not stay stable in liquid very long so it has to be lyophilized to improve its process- and storage stability. Lyophilization is a long and energy consuming process. Optimisation of the process is therefore essential to save time and resources. First IgG is freezed to produce ice. Primary drying is the second step, sublimation will change ice to water vapor. Secondary drying is based on water desorption, that way residual water is removed from the lyophilizate. The drying process is carried out altering shelf temperature on which the samples are placed. Chamber pressure is also an important factor in IgG stabilisation. These factors have their impact on IgG stability. Also adding disaccharide, trehalose, in the formulation increases the stability of IgG. The purpose of this work was to optimise both the filtration and lyophilisation process so that IgG would remain as stable as possible. During preliminary testing the results showed that magnetic stirring prior to filtration will damage the IgG, showing aggregation and less biological activity. Aggregation was measured with DLS and biological activity with ELISA. Changes in the secondary structure after lyophilisation were measured with CD. The actual filtration tests were carried out using three different filtration pressures and two different polysorbate 20 concentrations, and water. The results showed that IgG is most stable in 1,25 bar filtration pressure and 0,01 % polysorbate concentration. There was less aggregation and more biological activity. The filtration tests proved to be challenging because there were several parameters that were difficult to control. The same challenge was faced when analysing the results. Lyophilisation tests were carried out using three different pressures during primary drying and three different heating rates during secondary drying. The analysis methods were the same as during filtration tests. In addition, the IgG secondary structure changes were under investigation. The lyophilisation tests showed that trehalose clearly protects the IgG. Visually lyophilised samples which contained trehalose were mechanically more stable than those which did not contain trehalose. The analysis showed that the pressure of 60 mTorr and low heating rate (5 °C/h) resulted in better stability of IgG, aggregation was lower and biological activity higher. During lyophilisation no changes in the secondary structure was seen in CD. This was possibly due to lack of sensitivity of the analysis method.
Now showing items 1-2 of 2