Physical Instability of Proteins96

Chemical alterations are not the only source of protein instability. A protein is a large, globular polymer that exists in some specific forms of secondary, tertiary, and quaternary structure. A protein is not a fixed, rigid structure. The molecule is in dynamic motion, and the structure samples an array of three-dimensional space. During this motion, non-covalent intramolecular bonds can break, reform, and break again, but the overall shape remains centered around an energy minimum that represents the most likely (and pharmacologically active) conformer of the molecule. Any major change in the conformation can abolish the activity of the protein. Small drug molecules do not demonstrate this problem. A globular protein normally folds so that the hydropho-bic groups are directed to the inside and the hydrophilic groups are directed to the outside. This arrangement facilitates the water solubility of the protein. If the normal protein unfolds, it can refold to yield changes in hydrogen bonding, charge, and hydrophobic effects. The protein loses its globular structure, and the hydrophobic groups can be reposi-tioned to the outside. The unfolded protein can subsequently undergo further physical interactions. The loss of the globular structure of a protein is referred to as denaturation.

Denaturation is, by far, the most widely studied aspect of protein instability. In the process, the three-dimensional folding of the native molecule is disrupted at the tertiary and, possibly, the secondary structure level. When a protein denatures, physical structure rather than chemical composition changes. The normally globular protein unfolds, exposing hydrophobic residues and abolishing the native three-dimensional structure. Factors that affect the denaturation of proteins are temperature, pH, ionic strength of the medium, inclusion of organic solutes (urea, guanidine salts, acetamide, and for-mamide), and the presence of organic solvents such as alcohols or acetone. Denaturation can be reversible or irreversible. If the denatured protein can regain its native form when the denaturant is removed by dialysis, reversible denat-uration will occur. Denatured proteins are generally insoluble in water, lack biological activity, and become susceptible to enzymatic hydrolysis. The air-water interface presents a hy-drophobic surface that can facilitate protein denaturation. Interfaces like these are commonly encountered in drug delivery devices and intravenous (IV) bags.

Surface adsorption of proteins is characterized by adhesion of the protein to surfaces, such as the walls of the containers

Figure 4.11 • A. Protein decomposition reactions. B. ^-Elimination. B

of the dosage form and drug delivery devices, ampuls, and IV tubing. Proteins can adhere to glass, plastics, rubber, polyethylene, and polyvinylchloride. This phenomenon is referred to as flocculation. The internal surfaces of IV delivery pumps and IV delivery bags pose particular problems of this kind. Flocculated proteins cannot be dosed properly.

Aggregation results when protein molecules, in aqueous solution, self-associate to form dimers, trimers, tetramers, hexamers, and large macromolecular aggregates. Self-association depends on the pH of the medium as well as solvent composition, ionic strength, and dielectric properties. Moderate amounts of denaturants (below the concentration that would cause denaturation) may also cause protein aggregation. Partially unfolded intermediates have a tendency to aggregate. Concentrated protein solutions, such as an im-munoglobulin for injection, may aggregate with storage time on the shelf. The presence of particulates in the preparation is the pharmacist's clue that the antibody solution is defective.

Precipitation usually occurs along with denaturation. Detailed investigations have been conducted with insulin, which forms a finely divided precipitate on the walls of an infusion device or its dosage form container. It is believed that insulin undergoes denaturation at the air-water interface, facilitating the precipitation process. The concentration of zinc ion, pH, and the presence of adjuvants such as protamine also affect the precipitation reaction of insulin.

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