Protein stability at high concentrations (200-400 mg/ml) is of broad interest in science and human health. In cellular environments, a variety of macromolecules crowd proteins into in a compact state, stabilizing folded proteins, as described theoretically and observed experimentally.  However, short-ranged attractive and other interactions between closely spaced proteins and model colloids often produce irreversible aggregation and precipitation. In vitro, these undesirable events dominate, thus novel concepts are needed to mimic nature successfully to stabilize proteins at high concentration. We have created concentrated antibody protein dispersions, in the form of dense 80-400 nm clusters of protein molecules, which are conformationally stabilized by crowding and retain therapeutic activity in vivo.  The size of the relatively large, equilibrium nanoclusters is tuned by addition of macromolecular crowding molecules, which provides strong short-range attractions to balance weak longer-range electrostatic repulsions between individual protein molecules near the isoelectric point.  Whereas the locally concentrated environment within nanoclusters stabilizes the native state of protein molecules through self-crowding, weak interactions between nanoclusters result in colloidally-stable, translucent dispersions with low viscosities.  Not only is the protein native structure maintained within the nanocluster, but upon dilution, the clusters reversibly dissociate into native monomeric protein molecules with high biological activity.  The viscosities of antibody nanocluster dispersions are sufficiently low to allow subcutaneous injection to mice at typical therapeutic antibody dosages. Administration of dispersions resulted in high bioavailability and biological activity, similar to a sub-cutaneous injection of an antibody solution at the same dosage. Comparison of in vitro and in vivo data suggests the antibody remains stable due to crowding effects as it is transferred from the dispersion buffer to similarly-crowded physiological compartments. Dispersions of protein nanoclusters may enable patient self-administation of biological pharmaceuticals and provide insight into protein stability in vivo.

Stabilization of proteins at high concentrations by crowding

243rd National Meeting (2012)

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