The criteria for dried-protein stability include minimum lyophilization-induced unfolding, with proteins native in the dried
solid; a powder with Tg higher than the desired storage temperature; low residual moisture (<1%) in the cake; and formulation
conditions (such as pH) that inhibit chemical degradation reactions unaffected by glass transition (such as oxidation). The
goal is to design the fastest and most robust (acceptable quality even with variations in operating parameters) processing
cycle: one that consumes the least amount of energy, does not compromise product quality, and produces a mechanically strong,
rapidly dissoluble cake. The cycle must be controlled for reproducibility and rapid correction of any problems that develop.
The challenges. Early lyophilized products were found to be inactive after rehydration, but no one knew where the damage had occurred. Infrared
spectroscopy showed that proteins were unfolded in the dried solids. Stabilizers were added to prevent unfolding. Proteins
need protection to maintain native structure during lyophilization. Sucrose can protect them during both freezing and drying.
Until recently, assessing the necessary additives was impossible until after rehydration. However, Fourier-transform infrared
(FTIR) spectroscopy allows the study of protein structures in any state, enabling scientists to discover that all unprotected
proteins, except granulocyte colony-stimulating factor, unfold during lyophilization. They either regain their native conformation
during rehydration (reversible unfolding) or they aggregate. To prevent aggregation, scientists prevent unfolding by using
a stabilizer to retain the native structure in the dried product. Proteins that do refold are also protected by stabilizers
from unfolding, which is a critical parameter for successful lyophilization.
Workers load a large-scale lyophilizer.
John Carpenter of the University of Colorado is a recognized expert in protein lyophilization. In their chapter in Biotechnology and BioPharmaceutical Manufacturing, Processing, and Preservation, Carpenter and Byeong Chang, founder of Integrity Biosolution, noted that formulators should watch for contaminants in their
excipients. For example, mannitol may crystallize during short-term transient heating. "Think about the temperature of warehouses
in tropical countries," they cautioned. "Amorphous mannitol can crystallize on exposure to >45°C."
A New Stability Method for Vaccines