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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.

Development of alternative procedures to the commonly employed water-in-oil-in-water technique to encapsulate proteins in polymers is needed due to protein stability issues. Herein the model protein gamma-chymotrypsin has been encapsulated in poly(D,L-lactic-co-glycolic)acid (PLGA) microspheres using the solid-in-oil-in-water (s/o/w) encapsulation technique. The model protein was chosen because it has a measurable biological activity and its unfolding is irreversible. The latter make the protein an excellent sensor for unfolding events in the encapsulation procedure. While lyophilization did not cause any irreversible aggregation or loss in activity, encapsulation of the lyophilized enzyme by the s/o/w technique proved detrimental to its integrity. Specifically, 34% of the encapsulated protein was aggregated and the specific activity of enzyme released within 24 h was reduced to ca. 50% of that prior to encapsulation. FTIR spectra demonstrated substantial encapsulation-induced perturbations of the secondary structure of gamma-chymotrypsin. To achieve stabilization of gamma-chymotrypsin during encapsulation, excipients were employed during the initial lyophilization process. When gamma-chymotrypsin was co-lyophilized with poly(ethylene glycol) (PEG) the formation of non-covalent aggregates inside the microspheres decreased significantly to 8%. FTIR data showed that PEG prevented encapsulation-induced structural perturbations. In contrast, the amount of aggregates remained high (34%) when gamma-chymotrypsin was co-lyophilized with trehalose. No additional non-soluble aggregates were formed during 1 week of in vitro release. Furthermore, the amount of non-soluble aggregates in the microspheres after encapsulation correlated with the amount of non-released protein. Therefore in vitro release did not cause aggregation. Similar results were found with respect to the retention of the specific enzyme activity where PEG afforded excellent stability.