Highly concentrated formulations of stable protein (often >200 mg/ml) with low viscosities are needed for subcutaneous delivery to treat a variety of diseases. Herein, we demonstrate that proteins can be assembled into colloidally stable dispersions of distinct dense nanoclusters using depletion interactions from trehalose. The nanoclusters, formed by either concentrating a protein solution or diluting lyophilized protein powder, exhibit equilibrium hydrodynamic diameters without undergoing gelation at very high protein concentrations (up to 320 mg/ml). Despite very different formation pathways, the diameters of the nanoclusters are the same in both cases and depend only on the final composition. Nanocluster diameter appears to be governed by a balance of strong short ranged attraction and relatively weak longer ranged replusion as described by an equilibrium model. The nanocluster diameter was unchanged after two weeks at room temperature, or after frozen storage. Upon dilution into buffer, the nanoclusters dissociate into active monomeric protein.

Reversible protein nanoclusters with tunable sizes

245th National Meeting (2013)

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