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The goal of this study was to investigate the entrapment of 3 different model proteins (tetanus toxoid, lysozyme, and insulin) into poly(D,L-lactic acid) and poly(D,L-lactic-co-glycolic acid) nanoparticles and to address process-related stability issues. For that purpose, a modified nanoprecipitation method as well as 2 emulsion-based encapsulation techniques (ie, a solid-in oil-in water (s/o/w) and a double emulsion (w1/o/w2) method) were used. The main modification of nanoprecipitation involved the use of a wide range of miscible organic solvents such as dimethylsulfoxide and ethanol instead of the common acetone and water. The results obtained showed that tetanus toxoid and lysozyme were efficiently incorporated by the double emulsion procedure when ethyl acetate was used as solvent (>80% entrapment efficiency), whereas it was necessary to use methylene chloride to achieve high insulin entrapment efficiencies. The use of the s/o/w method or the formation of a more hydrophobic protein-surfactant ion pair did not improve protein loading. The nanoprecipitation method led to a homogenous population of small nanoparticles (with size ranging from ≈130 to 560 nm) and in some cases also improved experimental drug loadings, especially for lysozyme (entrapment efficiency >90%). With respect to drug content determination, a simple and quick matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method provided results very close to those obtained by reverse phase-high-performance liquid chromatography. With respect to protein stability, the duration and intensity of sonication were not a concern for tetanus toxoid, which retained more than 95% of its antigenicity after treatment for 1 minute. Only a high methylene chloride:water ratio was shown to slightly decrease toxoid antigenicity. Finally, no more than 3.3% of A21 desamido insulin and only traces of covalent insulin dimer were detected in nanoparticles. In conclusion, both the double emulsion and nanoprecipitation methods allowed efficient protein encapsulation. MALDI-TOF MS allowed accurate drug content determination. The manufacturing processes evaluated did not damage the primary structure of insulin.