Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-specific stabilization. I. Enzyme activity and calorimetric studies.

Stabilization of labile proteins during lyophilization requires protection of the protein against both freezing and dehydration stresses. Solutions of 1-10% (wt/vol) polyethylene glycol (PEG) fully protected both lactate dehydrogenase and phosphofructokinase during freezing and thawing, but did not stabilize the proteins during freeze-drying. Thus, with this lyophilization system a second compound could be tested for its capacity to stabilize dried proteins, independent of its ability to provide cryopreservation. In the presence of low concentrations of glucose or trehalose (which alone provided minimal protection) and 1% PEG (wt/vol), almost full enzyme activity was recovered after freeze-drying and rehydration. Differential scanning calorimetry indicated that the PEG was crystalline and the sugars were amorphous in the dried samples. Experiments with lactose and mannitol demonstrated that if these compounds also crystallized during freeze-drying, protein stabilization was reduced or abolished. PEG stabilizes the proteins during freezing, due to preferential exclusion of PEG from the protein's surface. The sugars protect the proteins during dehydration by hydrogen bonding to the dried protein, thus serving as water substitutes. This report provides the first example of stabilization of proteins during lyophilization through separate, specific treatments of the fundamentally different stresses of freezing and dehydration.