Measuring the heterogeneity of protein loading in PLG microspheres using flow cytometry

Poly (DL-lactide co-glycolide) (PLG) microspheres with mean sizes up to 1 microm containing Fluorescein Isothiocyanate labelled Bovine Serum Albumin (FITC-BSA) were prepared by the water-in oil-in water (w/o/w) emulsion solvent evaporation technique. Protein loading and loading efficiency determined by the BCA total protein assay increased with microsphere size as measured by laser diffractometry. Protein loaded microspheres were analysed using flow cytometry (FC) to provide fast and reproducible measurements of the size and protein loading of individual microspheres within a sample thereby quantifying in detail the batch heterogeneity. The FC analysis demonstrated that as the size of individual microspheres within a batch increased, so the protein loading tended to increase. For example, the protein loading of microspheres increased from 2.7 to 8.9 wt.% as the size of microspheres increased from 0.42 to 1.45 microm, respectively. Measurements taken during a subsequent protein release experiment indicated that smaller microspheres within a sample released their protein more quickly than larger sizes. Flow cytometry has been shown to provide detailed information, at the level of individual microspheres, about the heterogeneity in size and protein loading of a microsphere sample and could thus lead to improvement of the release characteristics of microsphere-based delivery systems for biopharmaceuticals.