The Adverse Effect of Glycation of Human Serum Albumin on its Preservative Activity in the Freeze-Drying and Accelerated Degradation of Alkaline Phosphatase

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Abstract
The effects of a glycated protein additive on the stability of freeze-dried biological standards were studied using alkaline phosphatase as a model. Alkaline phosphatase was formulated with artificially glycated albumin, freeze-dried, sealed into glass ampoules and subjected to accelerated degradation studies at temperatures from —20 to 56°C. Alkaline phosphatase, freeze-dried without an additive in neutral buffer, lost over 95% of its activity, but when freeze-dried with human serum albumin it retained approximately 70% of the initial activity. Both deliberately glycated and native albumin protected approximately 70% of the initial activity on freeze-drying and this protection was maintained during storage of the freeze-dried product at temperatures of 20°C or below for up to 16 weeks. At 37°C or above, alkaline phosphatase activity was lost in a time- and temperature-related manner with changes appearing in the SDS-PAGE gels and FPLC chromatograms but, with the artificially glycated albumin formulations, this loss of activity and the changes in the gels and chromatograms happened earlier and at lower temperatures. Formulations with trehalose at 1% w/v and 15% w/v, but without albumin, preserved some 40% of alkaline phosphatase activity following freeze-drying. Further, approximately half of that activity was maintained after 16 weeks' storage at all temperatures up to 56°C by the 15% trehalose without albumin. This study indicates that, in the formulation of freeze-dried biological standards, if albumin is to be used, the initial degree of glycation should be kept to a minimum and combinations of albumin and reducing sugar should be avoided. Trehalose 15% provides an acceptable alternative where exposure to high temperature is likely to occur.