Concentrated Dispersions of Equilibrium Protein Nanoclusters That Reversibly Dissociate into Active Monomers
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- 作者：Keith P. Johnston ; Jennifer A. Maynard ; Thomas M. Truskett ; Ameya U. Borwankar ; Maria A. Miller ; Brian K. Wilson ; Aileen K. Dinin ; Tarik A. Khan ; Kevin J. Kaczorowski
- 刊名：ACS Nano
- 出版年：2012
- 出版时间：February 28, 2012
- 年：2012
- 卷：6
- 期：2
- 页码：1357-1369
- 全文大小：714K
- 年卷期：v.6,no.2(February 28, 2012)
- ISSN：1936-086X
文摘
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.