Concentrated Dispersions of Equilibrium Protein Nanoclusters That Reversibly Dissociate into Active Monomers

Abstract

Stabilizing proteins at high concentration is of broad interest in drug delivery, for treatment of cancer and many other diseases. Herein, we create highly concentrated antibody dispersions (up to 260 mg/mL) comprising dense equilibrium nanoclusters of protein (monoclonal antibody 1B7, polyclonal sheep immunoglobulin G, and bovine serum albumin) molecules which, upon dilution in vitro or administration in vivo, remain conformationally stable and biologically active. The extremely concentrated environment within the nanoclusters (∼700 mg/mL) provides conformational stability to the protein through a novel self-crowding mechanism, as shown by computer simulation, while the primarily repulsive nanocluster interactions result in colloidally stable, transparent dispersions. 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. This behavior is described semiquantitatively with a free energy model which includes the fractal dimension of the clusters. Upon dilution of the dispersion in vitro, the clusters rapidly dissociated into fully active protein monomers as shown with biophysical analysis (SEC, DLS, CD, and SDS-PAGE) and sensitive biological assays. Since the concept of forming nanoclusters by tuning colloid interactions is shown to be general, it is likely applicable to a variety of biological therapeutics, mitigating the need to engineer protein stability through amino acid modification. In vivo subcutaneous injection into mice results in indistinguishable pharmacokinetics versus a standard antibody solution. Stable protein dispersions with low viscosities may potentially enable patient self-administration by subcutaneous injection of antibody therapeutics being discovered and developed.

Generic, phenomenological, on-the-fly renormalized repulsion model for self-limited organization of terminal supraparticle assemblies

“…In the weak screening limit, this is relevant because the effective repulsion between two NPs from different SPs incorporates the many-body effects from constituent NPs in each SP. In the strong screening limit and with sufficiently big counterions (16) or with stabilizing polymers (27,29), the accumulation of the counterion layers (or adsorbed polymeric stabilizers) causes the increase in A inter ij as the SPs grow in size. Using the argument as above, we propose that the repulsion strength between the NPs in the same SP, A intra ij , also depends on the relative positions of the NPs to the SP center of mass.…”

“…Because van der Waals forces decay much faster than electrostatic forces and are less sensitive to screening effects (27), the total van der Waals attraction increases with the SP radius (26). Using the same renormalization rationale as above, we observe that the rate of change in e inter ij ∼ V…”

“…An enormous body of theoretical studies that followed has been successful in characterizing the thermodynamic stability of finite-sized aggregates in various systems such as polyelectrolytes (5,7,11,(15)(16)(17)(18)(19), colloidal suspensions (20-24), and block copolymer and protein solutions (4,8,(25)(26)(27)(28)(29)(30). The optimal size of the assembled clusters is often attributed to the balance of short-ranged attraction and longer-ranged repulsion between the primary building blocks.…”

Structures and functions in the crowded nucleus: new biophysical insights

“…Simulations show that model particles form clusters in crowded conditions (Figure 2A) [24]. Cluster formation is observed quite commonly in colloidal systems [25], and the concentration of protein in clusters formed in a crowded solution may reach up to ∼700 mg/ml [26] (Figure 2B). The formation of compartments can also be regarded as phase separation, where entropic attractions in a mixture of macromolecules result in expulsion of one component as a separate phase [27].…”

Wrap-and-Strip Technology of Protein–Polyelectrolyte Complex for Biomedical Application

“…Such crystalline-state protein is pristine without impurities or aggregates, but the crystallization itself is time-consuming and the preparation of sufficient quantities of protein is difficult. A similar mechanism has been proposed using the nanoparticle dispersion of the protein-cluster state [46]. Johnston et al, have demonstrated that 1B7 antibody in a high-concentration trehalose solution forms a kind of protein colloid with a diameter of 50-300 nm via short-range hydrophobic interactions.…”