Effect of Particle Synthesis Method on Protein Release and Stability

Polyanhydride particle-based vaccines overcome several limitations of current vaccines owing to their ability to encapsulate different types of antigenic payloads, provide tunable release kinetics of payloads, and induce protective immunity against multiple respiratory infections. In this work, two particle synthesis methods were compared by analyzing the structure and antigenicity of released proteins from particles made by these methods. Flash nanoprecipitation is a lab-scale method to synthesize protein-loaded particles. Spray drying is a scalable method that allows for the production of protein-loaded particles. The polyanhydride copolymer used for both synthesis methods was composed of a 20:80 M ratio of 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane and 1,6-bis(p-carboxyphenoxy)hexane. The three proteins used in this work are bovine serum albumin (a model, globular protein), and SARS-CoV-2 spike and bovine RSV post-F protein (both clinically relevant proteins). The release kinetics of the encapsulated proteins were studied, and the structure of the released proteins was analyzed using gel electrophoresis and fluorescence spectroscopy. The antigenicity of the released spike and post-F was estimated based on the binding of positive mouse sera from previously inoculated mice. Our results indicate that both flash nanoprecipitation and spray drying resulted in particle formulations that provided a burst release of protein followed by a sustained period of release. The primary and tertiary structures and the antigenicity of the released proteins were maintained consistently across both methods. Altogether, these studies indicate that spray drying can be used to generate particles that stabilize encapsulated antigens and for at-scale particle synthesis in the future.