Stabilizers in the freeze-drying of proteins: Mechanism of stabilization

The mechanisms of stabilization by sugars during freeze drying and storage are still controversial. Since the mechanism of stabilization serves as a guideline for the formulation strategy in the pharmaceutical industry, the objective of this study is to develop a series of experiments to differentiate between the mechanisms that have been proposed and to see if yet another mechanism (unknown) is required. First, the multiple glass transitions in frozen sucrose solutions are investigated using modulated differential scanning calorimeter (DSC). If the low temperature transition, Tg′, is a glass transition, and drying is carried out below Tg′, a thermodynamic stabilization mechanism during drying presents some conceptual problems. That is, the time for establishment of equilibrium between folded and unfolded states is not likely long on the time scale of drying. Our data show that: (1) Annealing causes the thermal event denoted Tg′ to disappear; (2) The area of the endotherm at Tg′ in the non-reversing heat flow curve increases with annealing time, which indicates that the transition at Tg′ must also be a glass transition, corresponding to the newly created maximally freeze concentrated sucrose phase. ^ Second, the relationships among protein stability, mobility and protein structure was studied in an effort to clarify the mechanism of protein stabilization in the solid state. Protein degradation in various formulations was quantified using size exclusion (SEC) and ion-exchange chromatography (IEX). Fourier Transform Infrared (FTIR) spectroscopy was used to characterize protein secondary structure in various formulations. The rate of enthalpy relaxation, d(ΔHr)/dt, directly measured by the thermal activity monitor (TAM) [12], was analyzed using the derivative version of the modified stretched exponential function (MSE) equation to evaluate τβ, an indication of the molecular mobility in various formulations. It was found that the protein stability can be improved by the addition of sugars. The storage stability of proteins correlated best with the degree of retention of native structure of protein during freeze drying. The glass dynamics, as measured by τ, could not explain the mechanism of stabilization. Addition of a small amount of sorbitol to sucrose based formulations produced a “more native” structure, smaller relaxation time and provided improved stability. However, it is possible that the β-relaxation rather than the α-relaxation (i.e. the τ we measured) is critical to the stability. Therefore, more work is still needed in this area.