FACTORS AFFECTING THE IN-VITRO RELEASE OF RECOMBINANT HUMAN INTERFERON-GAMMA (RHIFN-GAMMA) FROM PLGA MICROSPHERES

A long-acting depot formulation of recombinant human interferon-gamma (rhIFN-gamma) was achieved by microencapsulation of rhIFN-gamma in pol ylactic-coglycolic acid (PLGA) microspheres by a water-in-oil-in-water technique. The release of protein was assessed with different release devices and buffer systems. The quality of the released protein was q uantitated by sodium dodecyl sulfate-size exclusion chromatography, EL ISA, and bioactivity assays. The microencapsulation process resulted i n an encapsulation efficiency of 100% and the initial release of bioac tive, native protein with no subsequent release. Further investigation suggested that the protein did not bind to the PLGA, but a constant a nd small amount of protein adsorbed to the filter device used for the release studies. The composition of the release media (pH, buffer spec ies, salt concentration, ionic strength, and type and concentration of surfactants) had a profound effect on the in vitro release rate. The effect was mainly due to the differential solubility, stability, and a ggregation of rhIFN-gamma in the various systems for protein inside th e microspheres or released into the bulk solution. The quality of the protein released from the microspheres was also affected by the buffer media upon storage at 5 degrees C, which, in turn, affected the quant ification of released protein. The bicinchoninic acid method typically used to quantitate protein release underestimated protein release bec ause of aggregation. Protein released after several days was less acti ve than the starting material and had lost activity as a result of the inherent instability of rhIFN-gamma at 37 degrees C. The release devi ce, buffer species, pH, and excipients must be assessed in release stu dies of proteins from polymer matrices because the protein stability a nd release is dependent on these variables. These studies also indicat ed that rhIFN-gamma was encapsulated and released from PLGA in a bioac tive form, but its stability at 37 degrees C, which was greatly affect ed by the release conditions, limits the duration of release of native , bioactive protein to 7 days or less.