Effect Of Formulation Process Of Sustained-release PLGA Microspheres On The Protein Activity And Immunogenicity

The polymer-based microsphere dosage forms successfully applied in sustained-release delivery of peptide drugs become no longer feasible for protein drugs due to the fragile higher structure of these macromolecules and adverse immune response induced by their structural changes. A technology breakthrough relies on improved formulation strategies by which structural denaturing and antibody developing of proteins can be avoided. In the present study, erythropoietin (EPO) is selected as a typical model to examine how formulation parameters help to improve efficacy and safety of protein drugs which easily denature and induce immunogenicity.EPO is a protein hormone of consisting 165 amino acids and 30400 Da in molecular weight which is well used for treatment of patients with anemia due to kidney diseases, cancer, chemotherapy, or HIV infection. EPO tops the global sales of recombinant protein drugs and easily denatured in formulation process of sustained-release microspheres. Since the present treatment with EPO requiring frequent injection for years or even a life-time will lead to a serious patient compliance problem, sustained-release delivery is highly demanded in improving its administration. However, aggregation and denaturing of EPO caused by the formulation processes may lead to severe immunogenicity such as the PRCA (pure red cell aplasia) associated with the EPO injection, turning EPO from an anemia curer to an anemia causer.To avoid exposing EPO to water-oil and water-air interfaces, factors known to denature proteins, a unique approach involving pre-loading the protein into solvent-resistant polysaccharide glassy particles via a freezing-induced phase separation and non-water phase microencapsulation (Solid-in-oil-in-hydrophilic oil or S/O/hO) was proposed by our laboratory. The present study is aimed to examine how these new approaches improve protein stability during each formulation step using SEC-HPLC, ELISA, and UT-7 cells proliferation as well as BALB/C mice as a model.The experiment results showed that the polysaccharide glassy particles prepared by the said method are spherical, smooth and 1~5μm in diameter. SEC-HPLC assay confirmed no aggregation up to this step of formulation. The activity retention was about 90%.These glassy particles were microencapsulated into PLGA microspheres using S/O/hO and solid-in-oil-in-oil (S/O/O), as compared with more conventional water-in-oil-in-water (W/O/W) method. The encapsulation efficiency of EPO for all these microspheres was about 75%. EPO recovered from microspheres by W/O/W and S/O/O showed irreversible aggregates, while aggregated EPO from microspheres by S/O/hO method were reversible. Anti-EPO antibodies assay by ELISA indicated significantly reduced antibodies development in the mice given microspheres by S/O/hO, as compared with those given microspheres by W/O/W and S/O/O. In terms of UT-7 cells proliferation, sera from mice given W/O/W showed significant suppressing effect than those from mice given S/O/O and S/O/hO microspheres.The SEC-HPLC, ELISA and UT-7 cells proliferation assays provided consistent evidence that microencapsulation by S/O/hO method may offer better EPO protection in formulation process of sustained-release dosage forms.