Microencapsulation of rh-erythropoietin, using biodegradable poly(D,L-lactide-co-glycolide): Protein stability and the effects of stabilizing excipients

Parenteral delivery systems allowing controlled drug release over one month are of particular interest for proteins and peptides. We investigated the microencapsulation of recombinant human erythropoietin (EPO), a stimulating factor of red blood cell production, into poly(D,L-lactide-co-glycolide) (PLG) microspheres, using a water-in-oil-in-water (W/O/W) double emulsion technique. The integrity and stability of EPO during microencapsulation and storage was characterized. Effects of various excipients on in vitro release properties and formation of EPO aggregates were investigated. The formation of EPO aggregates in the W/O/W double emulsion technique was mainly influenced by the first homogenizing step, when preparing the water-in-oil (W/O) emulsion, whereas the subsequent processing steps, including drying, proved to be noncritical. A rotor/stator homogenizer generated ca. 5%, covalently bound EPO aggregates, ultrasonication and vortexing slightly increased aggregate-formation as demonstrated by size-exclusion chromatography and native-polyacrylamide gel electrophoresis (PAGE). Using excipients, such as hydroxypropyl-beta-cyclodextrin, L-arginine, or bovine serum albumin (BSA), a distinct reduction of the formation of EPO aggregates could be achieved. The discontinuous in vitro release behavior from PLG microspheres was not significantly modified by these additives, influencing predominantly the initial drug release phase. During the in vitro release, an accumulation of EPO aggregates in the residual microparticles was detected, which could not be suppressed by excipients. An accelerated stability test demonstrated no change in drug content, release behavior and aggregate profile over 56 days at -20, 8 degrees C or room temperature. (C) 1997 Elsevier Science B.V.