One of the most challenging tasks in the development of protein pharmaceuticals is to deal with the physical and the chemical instabilities of the protein, which invariably lead to loss of biological activity. In order to avoid these problems, it is necessary to design vehicles that will minimize the degradation, maximize in vivo activity, and provide controlled release of encapsulated proteins. Polymers have been well-studied as matrices for drug delivery systems in the pharmaceutical industry, and encapsulating proteins into polymer microparticles is one of the most commonly used methods for parenteral delivery. It is well known that the mechanisms affecting protein stability are protein-specific; therefore, it is necessary to elucidate principles to rationally select polymer formulations to stabilize the specific protein of interest. Preservation of protein structure is important in order to generate effective immune responses when recombinant or purified proteins are used in vaccines.
Yersinia pestis is the etiological agent of bubonic and pneumonic plagues to which an estimated 200 million human deaths are attributable. In recent years, plague vaccine design, like that for many other diseases, has focused on the use of recombinant proteins such as F1 and V, which are found on the surface of the Y. pestis bacterium. The design of biodegradable polymeric delivery systems based on polyanhydrides that would provide for improved structural integrity of Y. pestis antigens is the main goal of this study.
Accordingly, the full length Y. pestis fusion protein (F1-V) or a recombinant Y. pestis fusion protein (F1B2T1-V10) were encapsulated and released from microparticles based on 1,6-bis(p-carboxyphenoxy)hexane (CPH) and sebacic acid (SA) copolymers and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and CPH copolymers fabricated by cryogenic atomization. An enzyme-linked immunosorbent assay was used to measure changes in the antigenicity of the released proteins. The recombinant F1B2T1-V10 was unstable upon release from the hydrophobic CPH:SA microparticles, but maintained its structure and antigenicity in the amphiphilic CPTEG:CPH system. The full length F1-V was stably released from both CPH:SA and CPTEG:CPH microparticles. In order to determine the effect of the anhydride monomers on the protein structure, changes in the primary, secondary, and tertiary structure, as well as the antigenicity of both Y. pestis antigens were measured after incubation in the presence of saturated solutions of SA, CPH, and CPTEG anhydride monomers. The results indicated that the amphiphilic environment provided by the CPTEG monomer was important to preserve the structure and antigenicity of both proteins. These studies offer an approach by which a thorough understanding of the mechanisms governing antigenic instability can be elucidated in order to optimize the in vivo performance of biodegradable delivery devices as protein carriers and/or vaccine adjuvants.