Effects of Tween 80 and Sucrose on Acute Short-Term Stability and Long-Term Storage at −20 C of a Recombinant Hemoglobin

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Abstract
The addition of low levels of surfactant polyoxyethylene 20 sorbitan monooleate, Tween 80, to recombinant hemoglobin in phosphate-buffered saline minimized the level of protein aggregation during acute freeze-thaw studies. Addition of sucrose alone to the phosphate-buffered saline formulation, up to 0.5 M, provided minimal protection against freeze-thaw induced aggregation. In contrast to the acute stability studies, long-term storage at -20 degrees C induced aggregation and methemoglobin formation in those formulations containing only Tween 80 in phosphate-buffered saline. Addition of sucrose between 0.1 and 0.5 M to the formulation prevented formation of aggregates and severely arrested methemoglobin formation during the long-term -20 degrees C storage. Specific binding of Tween 80 to the hemoglobin was not observed using 16-doxyl stearic acid partitioning techniques with electron paramagnetic resonance. Minor structural changes to the protein secondary structure during freezing in the absence and presence of Tween 80 were observed with Fourier transform infrared spectroscopy. The alterations were partially prevented by addition of the sucrose. It is likely that the Tween 80 severely reduced protein aggregation during the acute stability studies by preventing the hemoglobin from reaching the air-liquid interface or the liquid-surface interfaces. The reduction in methemoglobin formation and aggregation observed during long-term storage can be accounted for on the premise that the sucrose reduced localized unfolding of the protein in a manner similar to the preferential exclusion theory (Arakawa, T.; and Timasheff, S. N. 1982, Biochemistry 1982, 21, 6536-6544). These studies demonstrate that acute formulation screening studies, albeit useful, may not necessarily predict protein stability during long-term storage.