Investigation of Freeze/Thaw-Related Quality Attributes of a Liquid Biopharmaceutical Formulation: The Role of Saccharide Excipients

Abstract

Saccharides, including sucrose, trehalose, mannitol, and sorbitol, are commonly employed as stabilizers, cryoprotectants, and/or tonicity adjusters in protein formulations. During the thawing of a protein-containing formulated bulk drug substance conducted prior to a drug product (DP) filling operation, a white, crystalline precipitate was observed. In addition, upon thawing, vial breakage was observed for filled DP that had been previously frozen at -40 °C. To investigate the causes of both phenomena, the freeze/thaw behavior of the formulation components was studied. Multiple physical characterization techniques, including differential scanning calorimetry (DSC), electrical resistance measurements, thermomechanical analysis (TMA), and powder X-ray diffraction (PXRD), were utilized to characterize the formulations. The PXRD pattern of precipitate collected from thawed bulk was consistent with that of a mannitol control. An exothermic transition observed by DSC, a sharp increase in electrical resistance detected via resistivity measurements, and the onset of volumetric expansion of the frozen matrix evident in the TMA curve offer evidence that the frozen mannitol solution undergoes transitions at or near the vial breakage temperature (-22 to -23 °C) observed during warming. In addition, osmolality measurements taken from fractionated aliquots of frozen samples indicated that non-uniform concentration gradients contributed to precipitation of mannitol observed at larger scales. Small-scale laboratory experiments (i.e., 10-125 mL) failed to adequately predict behavior at larger scale (i.e., in 1 L and 2 L bottles). Upon linking the detrimental behavior to the freeze/thaw properties of the tonicity adjustor, mannitol, alternative saccharide excipients, including sorbitol, sucrose, and trehalose, were evaluated at isotonic concentrations over a temperature range of -80 to 25 °C using physical-chemical techniques and visual observation. Neither precipitation nor vial breakage was observed for the alternate saccharides. Recommendations for saccharide selection are provided based on storage conditions and scale considerations for liquid biopharmaceutical formulations. Saccharides, including sucrose, trehalose, mannitol, and sorbitol, are commonly employed as stabilizers, cryoprotectants and/or tonicity adjusters in protein formulations. During thawing of formulated bulk drug substance, a white, crystalline precipitate was observed. In addition, upon thawing, vial breakage was observed for filled drug product that had been previously frozen at -40 °C. To investigate the causes of both phenomena, multiple physical characterization techniques were utilized to characterize the formulations. The powder X-ray diffraction pattern of precipitate collected from thawed bulk was consistent with that of a mannitol control. Upon linking the detrimental behavior to the freeze/thaw properties of the tonicity adjustor, mannitol, alternative saccharide excipients, including sorbitol, sucrose, and trehalose, were evaluated at isotonic concentrations over a temperature range of -80 to 25 °C using physico-chemical techniques and visual observation. Neither precipitation nor vial breakage was observed for the alternate saccharides. Recommendations for saccharide selection are given based on storage conditions and scale considerations for liquid biopharmaceutical formulations.