Acidity changes in glycine and L-histidine buffers, mannitol, and their mixtures after freezing and lyophilization

Freezing and lyophilization are commonly used methods for stabilizing pharmaceutical and biochemical formulations. However, these processes can introduce a variety of freezing-induced stresses that may lead not to stabilization, but rather to the destabilization of active molecules. One of the most significant of these stresses is freezing-induced acidity change, which has been shown to cause protein aggregation, loss of structural integrity, and increased chemical reactivity. While buffers are routinely used in liquid formulations to minimize pH fluctuations, several studies have demonstrated that certain buffers not only fail to maintain pH during freezing but may actively contribute to acidity shifts. In this study, we investigate the effects of cooling rate, initial pH, mannitol concentration, and lyophilization on acidity in glycine and L-histidine buffer systems in the solid state (both frozen and lyophilized) using UV-VIS spectroscopy and differential scanning calorimetry. Our results indicate that the freezing of amino acid buffers causes a slight increase in pH (basification); however, changes in acidity are not solely the consequence of freezing as they also occur during lyophilization. Notably, in L-histidine with mannitol at pH 7, lyophilization induces acidification of up to 4 units - opposite to the direction observed during freezing. Furthermore, we explore the correlation between vitrification of the freeze-concentrated solution and freezing-induced acidity changes, as quantified using the Hammett acidity function (H_2-). These findings may inform the rational design of more robust stabilization strategies.