stress and slow freeze thaw cycles including accelerated stability studies via incubation at 50 u00b0 c for one to two weeks at several time points e g t 0 t 7 t 14 the stability of the molecules was examined with and without stress using the following tools right angle light scatter rals intrinsic and extrinsic fluorescence if ef in conjunction with analytical methods like high performance size exclusion chromatography hp sec rals is employed to detect and monitor the subtle changes in the associative behavior of the molecule which can result in aggregation and or precipitation rals monitors macroscopic changes as a soluble molecule transitions to insoluble aggregates the if assay measures stress induced conformational changes in proteins as observed by changes in the tryptophan environment ef utilizes an external non covalent polarity sensitive fluorescent probe to examine a protein 39 s apparent exposure of hydrophobic clefts and to monitor possible changes in this parameter as a function of various environmental stresses and conditions after reading initial rals or turbidity approximately 400 u02dc u03bcl of sample was frozen slowly in an eppendorf tube by placing it in a u2212 80 u00b0 c freezer after freezing was complete minimum of four hours all samples were thawed on the bench top room temperature the process was repeated for a total of 5 cycles.