Stability of ribonuclease A in solution and the freeze-dried state

The solution stability of bovine pancreatic ribonuclease A (RNase) in phosphate buffer (pH 4.0, 6.4, and 10.0) at 45 degrees C decreased with increasing pH. Soluble aggregates were formed at each pH and corresponded qualitatively to the loss of enzymatic activity in the samples. Freeze drying of RNase resulted in no immediate loss of enzymatic activity in both the presence and absence of phosphate buffer salts. Freeze-dried RNase stored at 45 degrees C lost enzymatic activity and formed nondissociable aggregates at rates described by the following rank order of formulation contents: distilled water less than pH 6.4 phosphate buffer, less than pH 4.0 phosphate buffer less than pH 10.0 phosphate buffer. The amount of residual moisture remaining in the freeze-dried cakes was directly related to the rate of enzymatic activity loss and aggregate formation. The degradation rate was also directly related to the concentration of phosphate buffer salts added to the freeze-dried formulation.