Our advanced formulation engineering approach uses plasticizers to suppress high frequency vibrational motions that protein side chains typically undergo while encased in glassy sugar matrices. These plasticizers act to fill the so-called 'free volume' that exist between the glassy sugar matrix and the surface of proteins, thereby linking the side chains of the proteins directly to the structurally rigid glassy scaffold. The net effect is the dampening of the protein's high frequency local vibrations, which minimizes its' molecular mobility and consequently promotes long term stability.
Background
The removal of water followed by the addition of pharmaceutical excipients that act as stabilizers, particularly those that form amorphous glassy matrices, is a general paradigm that is used to stabilize complex macromolecules such as proteins. However, the molecular spatial arrangements and interactions of the protein side chains with the host excipient matrix are not well understood. Recently, the application of small-angle neutron scattering to pharmaceutical solids have revealed molecular details of how proteins are encased in these excipient matrices and how such interactions can be further optimized1,2. These studies suggested that under sub-optimal formulation conditions, high frequency molecular vibrations (at picosecond timescale), which the stabilizers themselves undergo, may transmit locally to the encased proteins, thereby causing instability, denaturation and loss of activity. It was discovered that such native molecular motions could be dynamically suppressed with the addition of small molecular weight, FDA approved plasticizers such as glycerol, DMSO, sorbitol, etc. that conceptually act as molecular shock absorbers to dampen the impact of these molecular vibrations on proteins, and thereby help maintain better structural retention during storage. When judiciously added (at levels revealed by neutron scattering measurements), these plasticizer stabilizers enhanced the stability 10 to 100 times over that afforded by sugar-based stabilizers alone (Fig.1). This formulation stabilization technology represents a new paradigm in pharmaceutical stabilization that is applicable to a wide variety of drying processes (freeze drying, spray drying, etc.) and products including protein, peptides, nucleic acids, and larger entities such as viruses and bacterial products.
Figure 1. Stabilization of horse radish peroxidase (HRP) enzyme using plasticizer DMSO. More than 10-fold improvement in the storage stability of HRP was observed with the addition of the plasticizer DMSO over trehalose glass alone. Data from Cicerone, M. et al. US Patent Application Pub. No.: US2004/0014164 A1
1Cicerone MT, Tellington A, Trost L, Sokolov A. Substantially improved stability of biological agents in dried form. BioProc. Int. 2003. 1:36–47.
2Cicerone MT and Soles CL. Fast dynamics and Stabilization of Proteins: Binary glasses of Trehalose and Glycerol. Biophysical J. 2004. 86:3836-45.
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