Reconstitution of lyophilized therapeutic protein: Effects of formulation, processing techniques and surfaces.

摘要

To study the use of polysorbate 20 in a reconstitution solution on the affect of the aggregation of lyophilized recombinant human interferon-γ (rhIFN-γ), we lyophilized four formulations separately in two buffer systems. After reconstitution with a 0.03% polysorbate 20 solution, aggregation levels in all formulations were either reduced or similar to those found after reconstitution with water. Dissolution of the lyophilized powders was measured by monitoring the oxidation of Fe(CN)6−4 ion at the surface of a platinum rotating disk electrode. The presence of 0.03% polysorbate 20 in the reconstitution solution slowed the dissolution rates of the buffer and sucrose formulations, allowing more time for the protein to refold while decreasing the maximum concentration of the protein at the dissolution interface, thus reducing the total amount of aggregation.; rhIFN-γ was lyophilized or spray-lyophilized in trehalose formulations. Powder surfaces were characterized using electron spectroscopy for chemical analysis (ESCA), time-of-flight secondary ion mass spectroscopy (ToF SIMS), X-ray diffraction and specific surface area measurements. ESCA indicated that the surface coverage by rhIFN-γ was greatest for the spray-lyophilized powder formulated without surfactant, and the surface coverage was decreased by the addition of 0.12% polysorbate 20. ToF SIMS showed that polysorbate 20 is on the surface of the lyophilized powders in place of the protein. Significant losses of soluble protein were observed after atomization, but the losses were decreased in the 0.12% polysorbate 20 formulation. The primary loss of soluble rhIFN-γ occurred at the air/liquid interface.; rhIFN-γ was lyophilized or spray-lyophilized in sucrose and hydroxyethyl starch (HES) formulations. Ice/liquid interfacial areas were manipulated via the freezing method as well as through the addition of an annealing step during lyophilization; rhIFN-γ adsorption was modified by the addition of nonionic surfactants. Adsorption of rhIFN-γ to ice/liquid interfaces alone was not responsible for aggregation; drying was necessary to cause aggregation in lyophilized sucrose formulations. Annealing decreased the excess free volume of the glassy state, resulting in samples with more native-like protein structure in the dried state and increased recovery upon reconstitution. In addition, annealing decreased the primary drying time, slowed the reconstitution step, and decreased air/liquid interface formation upon reconstitution.