Interaction of Recombinant Human Bone Morphogenetic Protein-2 with Poly (d,lLactide-co-glycolide) Microspheres

Abstract

The combination of recombinant human bone morphogenetic protein-2 (rhBMP-2) with poly(d,l lactide-co-glycolide) (PLGA) porous microspheres provided for “sustained release” of the protein from the microspheres. Soaking 50:50 PLGA microspheres in a buffered rhBMP-2 solution for a sufficient period of time to permit equilibrium binding enabled quantification of “free” and “bound” protein. “Free” protein is defined as protein present within the porous matrix of the microspheres, whereas “bound” refers to protein adsorbed to PLGA surfaces. Kinetics of the rhBMP-2-microsphere association revealed that equilibrium was attained within 8 hr for two buffer systems (arginine/histidine, pH 6.50; and glutamic acid/sodium glutamate, pH 4.50). Increasing the concentration of the rhBMP-2 stock solution used for the interaction studies from 0.025 to 1.0 mg/ml increased the amount of rhBMP-2 adsorbed and the concentration of free rhBMP-2. Beyond a 1.0 mg/mL concentration, only free rhBMP-2 levels increased. Linearized Langmuir treatment of the adsorption data yielded values corresponding to monolayer coverage of the microspheres (C_m) and the equilibrium adsorption constant (K) of 0.17 μg/cm² and 7.57 ml/mg, respectively. Studies performed to determine the effect of ionic strength revealed that increasing NaCl and buffer concentration decreased the amount of protein adsorbed. rhBMP-2 release studies, conducted in an isotonic phosphate buffered saline, pH 7.4 vehicle, revealed that free rhBMP-2 was released during an initial period of 72–96 hr. Following this period, there was no discernible release of rhBMP-2 from the microspheres for up to 7 days, suggesting that the bound protein would remain at a defect site and release slowly upon erosion of the polymer. Mass balances performed by using an extraction buffer of high ionic strength confirmed this prediction.