Method development and analysis of the water content of the maximally freeze concentrated solution suitable for protein lyophilisation

During freeze-drying of a liquid formulation, a freeze-concentrate is formed in the first phase, the freezing step.Understanding the composition of the maximally freeze concentrated solution can help to judge the process stability of biopharmaceuticals during lyophilisation. Our objective was to develop a suitable method to determine the water content of the maximally freeze concentrated solution using differential scanning calorimetry (DSC). Three different methods were compared: (i) the intercept of the glass transition temperature of the maximally freeze concentrated solution Tg′ and the melting temperature Tm for a concentration series, (ii) the linear regression of the melting enthalpy starting from the onset of Tg′ until the end of the melting event for a concentration series, and (iii) a one-point determination of the amount of unfrozen water. While Method 1 is accurate but requires the analysis of a high number of samples, Method 3 requires only one single sample, with a loss of accuracy. Method 2 works best taking sample preparation and accuracy into account. Various systems containing sugar (sucrose, trehalose) and other excipients (histidine buffer, phosphate buffer, sodium chloride, arginine hydrochloride, arginine citrate) were evaluated with different antibody concentrations to evaluate the composition of the maximally freeze concentrated solution. The freeze concentrates exhibited a water content of 20–30%, slightly dependent on the excipients, but independent of the antibody concentration. The methodology we developed is broadly applicable for the analysis of the composition of maximally freeze concentrated solutions and can help to elucidate protein stability during lyophilisation.