OBJECTIVE A previous study from our group has shown that in the acidic microclimate of poly(lactide-co-glycolide) (PLGA) implants, encapsulated BSA forms insoluble noncovalent aggregates and is hydrolyzed during in vitro release. Incorporation of Mg(OH)2 strongly inhibits these mechanisms of instability and facilitates continuous protein release. The purpose of this study was to determine the protein stabilization mechanism in the presence of basic additives. METHODS BSA, as a model protein, was encapsulated in PLGA millicylinders by a solvent extrusion method. The release of BSA from the PLGA millicylinders with and without basic additives (Mg(OH)2, Ca(OH)2, ZnCO3 and Ca3(PO4)2) in a physiological buffer was carried out at 37 degrees C and quantified by a modified Bradford assay. The insoluble aggregates extracted from the polymer with acetone were reconstituted in a denaturing (6 M urea) or denaturing/reducing solvent (6 M urea/10 mM DTT) to determine the type of aggregation. RESULTS Aggregation of encapsulated BSA was inhibited with increasing amount of base co-encapsulated in the polymer, irrespective of the type of base used. The pH drop in the release medium and extent of acid-catalyzed PLGA degradation were both inhibited in the presence of base. The resultant effect was also reflected in an increase in water uptake and porosity of the devices. The inhibition and mechanism of BSA aggregation was correlated with the basicity of the additive. For Ca(OH)2, at 3% loading, covalent BSA aggregation due to thioldisulfide interchange was observed (indicative of ionization of albumin's free thiol at high pH), whereas at 3% ZnCO3 or Ca3(PO4)2, a higher percentage of non-covalent aggregates was observed compared to Mg(OH)2. Decreasing the loading of BSA at constant Mg(OH)2 content caused an increase in BSA aggregation. CONCLUSIONS The mechanism by which Mg(OH)2 stabilizes encapsulated BSA in PLGA implants is through neutralizing the acidic microclimate pH in the polymer. The successful neutralization afforded by the basic additives requires a percolating network of pores connecting both base and protein. The microclimate pH inside PLGA implants can be controlled by selecting the type of basic salt, which suggests a potential approach to optimize the stability of encapsulated pharmaceuticals in PLGA including therapeutic proteins.