Poly(ethylene glycol) as stabilizer and emulsifying agent: a novel stabilization approach preventing aggregation and inactivation of proteins upon encapsulation in bioerodible polyester microspheres

Protein aggregation and inactivation are major problems associated with the encapsulation of pharmaceutical proteins in biodegradable microspheres. The objectives of this study were to identify the causes of aggregation and inactivation of two model enzymes upon solid-in-oil-in-water (s/o/w) encapsulation in poly(lactic-co-glycolic) acid (PLGA) microspheres in order to rationally develop approaches assuring their stability. S/o/w encapsulation of gamma-chymotrypsin in PLGA microspheres caused aggregation of ca. 30% and halved its specific activity. Co-lyophilization with poly(ethylene glycol) (PEG) substantially reduced the loss in enzyme activity but 8% of the protein still aggregated during encapsulation. Model studies performed under conditions relevant to the encapsulation procedure allowed pinpointing the cause of gamma-chymotrypsin instability, which was mainly the formation of the oil-in-water emulsion. To prevent aggregation in this encapsulation step, the most commonly used emulsifying agent polyvinyl alcohol (PVA) was replaced by PEG because it is known to reduce protein aggregation at interfaces. The use of PEG as the emulsifying agent in the aqueous and organic phase prevented gamma-chymotrypsin inactivation and aggregation during encapsulation. The stabilization approach also worked for the model protein horseradish peroxidase and thus is of a general nature. (C) 2002 Elsevier Science B.V. All rights reserved.