(408f) Low-Viscosity Highly Concentrated Dispersions of Stable Protein Nanoclusters for Subcutaneous Injection

Stabilizing proteins at high concentration is of broad interest in drug delivery, for treatment of cancer and many other diseases. Proteins have a tendency to undergo irreversible aggregation, gelation or precipitation at high concentrations due to unfolding caused by specific short ranged forces. Herein, we create highly concentrated antibody dispersions (up to 260 mg/ml) comprising of dense equilibrium nanoclusters of protein [monoclonal antibody (mAb) 1B7, polyclonal sheep Immunoglobulin G (IgG) and bovine serum albumin (BSA)] molecules, which upon dilution in vitro or administration in vivo, remain conformationally stable and biologically active. The nanoclusters are formed by adding trehalose as a cosolute which strengthens the short-ranged attraction between protein molecules. The protein cluster diameter was reversibly tuned from 50 to 300 nm by balancing short-ranged attraction against long–ranged electrostatic repulsion of weakly charged protein at a pH near the isoelectric point (pI).  The high protein volume fraction within the cluster stabilizes the protein conformation through a self-crowding mechanism and the primarily repulsive electrostatic interactions between the clusters keep them colloidally stable, preventing gelation. Upon dilution of the dispersion in vitro, the clusters rapidly dissociated into fully active protein monomers as shown with biophysical analysis (SEC, dynamic light scattering (DLS), circular dichroism (CD) and SDS-PAGE) and sensitive biological assays.  In vivo subcutaneous injection into mice results in indistinguishable pharmacokinetics versus a standard antibody solution.  This tunablility of multi-scale interactions , specifically inter-monomer short range attraction and inter-cluster long range repulsion, to produce nanoclusters is not protein-specific. Therefore this nanocluster concept can be applied to a wide range of therapeutic proteins without the need to engineer modified proteins through changing the amino acid sequence. Stable protein dispersions with low viscosities may potentially enable patient self-administration by subcutaneous injection of antibody therapeutics being discovered and developed.