This application seeks to develop a long-acting depot formulation for antibodies, using the inverse Flash NanoPrecipitation (iFNP) platform being commercialized by Optimeos Life Sciences. Microparticle depot formulations have been tested for decades to provide sustained release. The therapeutic is traditionally entrapped in a water-insoluble polymer matrix, with release proceeding as the polymer degrades. The traditional microparticle structure has significant limitations, including low therapeutic content and poor therapeutic stability. Consequently, there are currently no marketed microparticle depots for proteins. By contrast, the microparticles produced by Optimeos are formed by aggregating nanoparticles together to produce mechanically strong nanocomposite microparticles. The nanoparticles are produced by iFNP, a scalable and continuous process for encapsulating water-soluble compounds. The nanoparticle structure permits much higher loadings and forms a protective shell that will limit antibody instability during extended release. The iFNP technology has been extensively studied for peptide delivery, with demonstrated therapeutic weight content in the depot up to 10 times higher than currently possible with existing methods, and controlled release profiles ranging from 3 weeks to more than 3 months. The proposed research will extend the iFNP sustained release technology from peptides to proteins. Three Tumor Necrosis Factor alpha (TNFα) antibody formats will be evaluated to determine the scope of applicability of the technology. These constructs – a VHH single domain nanobody, a Fab fragment, and an IgG antibody – are of increasing complexity. This proposed study will enable the translation of the iFNP technology to more complex biologics by addressing the key process risks – chemical and structural instability of the encapsulated antibody during processing and release – through three aims: 1) Aim 1: Identify VHH microparticle formulations, produced using iFNP, with 20-40 wt% functional VHH. 2) Aim 2: Generate sustained release of active VHH over 1 and 3 months from microparticles, with weekly stability assessments indicating released VHH is > 90% native and functional. 3) Aim 3: Apply Aim 1 and Aim 2 findings to the encapsulation of Fab and IgG antibodies, producing sustained release over 1 and 3 months with released antibody > 90% native and functional. The performance of iFNP will be evaluated using VHH antibodies as an initial model because they are rapidly cleared following injection. Formulation design will build on the rules derived for peptide delivery using the iFNP process, produced under an STTR grant between Princeton University and Optimeos. Key stability measurements (ELISA, SEC, mass spectrometry) will be conducted via a collaboration with Integral Molecular. These results will be generalizable to other antibodies, allowing us to expand into the treatment of other diseases rapidly. Crucially, sustained delivery of prote...