PROTEIN NEBULIZATION .1. STABILITY OF LACTATE-DEHYDROGENASE AND RECOMBINANT GRANULOCYTE-COLONY-STIMULATING FACTOR TO AIR-JET NEBULIZATION

The effect of air-jet nebulization on the stability of two proteins was examined. Lactate dehydrogenase (LDH) and recombinant human granulocyte colony stimulating factor (rhG-CSF) were chosen as the test proteins. The enzymatic activity of LDH can be readily monitored using a colorimetric assay. rhG-CSF was also studied as it has potential to be delivered to the systemic circulation via the lung. Aerosolization of LDH using a three-jet Collison nebulizer resulted in a time-dependent loss of enzymatic activity. The extent of inactivation was dependent upon the applied air pressure and the volume of fluid in the nebulizer reservoir. Inactivation at 10 and 40 psig was 12 +/- 5 (n = 6) and 18 +/- 10% (n = 6), respectively, after a period of 10 min nebulization using a 40 mi starting volume, whereas an initial volume of 10 ml resulted in 57 +/- 14% loss of activity after 10 min at 40 psig. rhG-CSF was affected in two ways by aerosolization. First, non-covalent aggregates were formed with time as observed by size exclusion chromatography. Second, a chemical change was induced and this was reflected in the formation of a higher mobility band on native gel electrophoresis. The change in mobility was thought to be due to an altered charge state of the protein, possibly through deamidation. Both the aggregated and degraded forms accounted for approx. 40% of the protein after 10 min. Polyethylene glycol (PEG) 1000 markedly reduced the deleterious effects of nebulization on the proteins. A 10 ml solution of protein containing 1% w/v PEG 1000 retained approximate to 100% of the original activity of LDH after 10 min nebulization at 40 psig while both forms of rhG-CSF degradation were held to less than 10% for the same set of operating conditions.