Homogeneous PLA/insulin solutions containing different amounts of 350, 750 or 1900 Da PEG (0-75 wt.% PEG) were processed by semi-continuous compressed CO2 anti-solvent precipitation to fabricate protein-loaded polymeric nano-particles. Proper operative conditions (temperature, pressure, CO2 flow rate and washing time) yielded more than 70% product recovery. Scanning electron microscopy, transmission electron microscopy and light scattering demonstrated that spherical, smooth surfaced particles with size below 1 microm could be obtained. X-ray diffraction analysis showed that the gas anti-solvent process modifies the polylactide crystalline state. PEG concentration and molecular weight were found to affect both optimal operative conditions and morphological and biopharmaceutical properties of the final product. Insulin loading yield dropped from 95% to 65% by increasing the 1900 Da PEG content from 0 to 75 wt.% or the PEG molecular weight from 350 to 1900 Da. The release rate increased significantly as the PEG content in the formulation increases. After 3-month incubation the drug released raised from 10% to 100% by increasing the 1900 Da PEG content from 23 to 7 wt.%. Formulations containing the same 350, 750 or 1900 Da PEG amount (67 wt.% PEG) displayed similar release profiles. Insulin release was found to take place by diffusion mechanism, despite the observation of matrix degradation.