Physical stability of a recombinant alpha 1-antitrypsin injection.

Physical stability of a recombinant alpha 1-antitrypsin (rAAT) injection solution, namely, loss of rAAT to particulate formation was studied. The rAAT injection solution (1.0 mg/ml, pH 7.5, triple buffer of phosphate, Tris, and glycine, 0.075 M each) was filtered through a 0.2-micron filter and packaged in individual vials with rubber stoppers and aluminum seals. The vials were stored at 90, 80, 60, 45, 35, and 25 degrees C, and samples taken at predetermined time intervals for each storage condition. Each sample was then filtered through a 0.2-micron filter to remove particulates. The filtrate was then assayed for total protein by the biuret-phenol method. A typical first-order loss of rAAT was observed. Data were fitted to a first-order kinetics, and the shelf life (defined as t90, the time for the product to reach 90% remaining) was calculated from the resultant rate constant. The shelf lives were plotted against reciprocals of storage temperatures (a modified Arrhenius plot). A linear regression line (r = 0.9831) was drawn through the data points and extrapolated to 4 degrees C. At 4 degrees C, the predicted shelf life of the rAAT solution is 5.1 months. In real life, a batch of the rAAT solution showed that 61% of the rAAT remained in the filtrate after 18 months of storage at 4 degrees C. The observed stability compares fairly well with the predicted value of 69% for the same duration of storage at 4 degrees C. From the slope of the linear regression line, the activation energy for the particulate formation of the rAAT was calculated to be 19 kcal. This value is comparable to the activation energy of 20 kcal for ovalbumin denaturation reaction reported by Simpson and Kauzmann.