Rational improvement of protein stability upon encapsulation in biodegradable microspheres

Sustained-release of pharmaceutical proteins from biodegradable polymers offers new opportunities in the treatment and prevention of diseases. However, the most significant obstacle that has emerged is the instability of the protein during encapsulation and release. Therefore, successful controlled release of proteins has been a daunting task and, until recently, only a small number of therapeutic proteins have been slowly and completely released in a native state from biodegradable polymers.; To overcome these limitations, the s/o/w encapsulation technique was explored as an alternative method to obtain protein-loaded microspheres. The main problem hampering the use of this technique is the low encapsulation efficiency for proteins (<20%). In this study, the selection of critical encapsulation parameters allowed to increase the encapsulation efficiency of BSA from 7% to >90%. However, encapsulation of BSA showed that s/o/w encapsulation procedure caused some structural perturbations and aggregation. Structural perturbations and aggregation of BSA were minimized throughout the encapsulation procedure when two conditions were fulfilled: BSA powder was obtained by co-lyophilization with trehalose and loss of trehalose in the organic solvent/water emulsion step was avoided.; Model studies performed under conditions relevant to the encapsulation procedure allowed to pinpoint the cause of protein instability. The deleterious stress responsible for the mechanism of instability was mainly the formation of the organic solvent/water emulsion. Stabilization strategies were focused on overcoming the interface-induced denaturation by avoiding the protein adsorption to the interface. Amongst them was the addition of an amphiphilic additive, PEG, able to compete with the protein by the organic solvent/water interface. After PEG addition, the inactivation of protein was completely eliminated and the aggregation was largely prevented. To completely prevent aggregation upon encapsulation a novel stabilization approach was developed by using PEG as emulsifying agent during the microspheres preparation. Both, the generality of the organic solvent/water interface-induced protein instability (BSA, gamma-chymotrypsin and horseradish peroxidase) and the stabilization approach using PEG were demonstrated. Lastly, the covalent modification of proteins with PEG showed to be another attractive stabilizing approach upon encapsulation. The importance of optimizing the degree of PEG modification necessary to protect the protein during the encapsulation process was revealed.