Zakharov Boris, Fisyuk Alexander, Fitch Andy, Watier Yves, Kostyuchenko Anastasia, Varshney Dushyant, Sztucki Michael, Boldyreva Elena, Shalaev Evgenyi
Institute of Solid State Chemistry and Mechanochemistry SB RAS, ul. Kutateladze, 18, Novosibirsk 630128, Russian Federation; Novosibirsk State University, ul. Pirogova, 2, Novosibirsk 630090, Russian Federation.
Department of Organic Chemistry, Omsk F. M. Dostoevsky State University, Mira Avenue 55A, Omsk 644077, Russian Federation; Laboratory of New Organic Materials, Omsk State Technical University, Mira Avenue 11, Omsk 644050, Russian Federation.
J Pharm Sci. 2016 Jul;105(7):2129-38. doi: 10.1016/j.xphs.2016.04.020. Epub 2016 Jun 7.
Ice formation and recrystallization is a key phenomenon in freezing and freeze-drying of pharmaceuticals and biopharmaceuticals. In this investigation, high-resolution synchrotron X-ray diffraction is used to quantify the extent of disorder of ice crystals in binary aqueous solutions of a cryoprotectant (sorbitol) and a protein, bovine serum albumin. Ice crystals in more dilute (10 wt%) solutions have lower level of microstrain and larger crystal domain size than these in more concentrated (40 wt%) solutions. Warming the sorbitol-water mixtures from 100 to 228 K resulted in partial ice melting, with simultaneous reduction in the microstrain and increase in crystallite size, that is, recrystallization. In contrast to sorbitol solutions, ice crystals in the BSA solutions preserved both the microstrain and smaller crystallite size on partial melting, demonstrating that BSA inhibits ice recrystallization. The results are consistent with BSA partitioning into quasi-liquid layer on ice crystals but not with a direct protein-ice interaction and protein sorption on ice surface. The study shows for the first time that a common (i.e., not-antifreeze) protein can have a major impact on ice recrystallization and also presents synchrotron X-ray diffraction as a unique tool for quantification of crystallinity and disorder in frozen aqueous systems.
冰的形成和重结晶是药物和生物制药冷冻及冻干过程中的关键现象。在本研究中，高分辨率同步加速器X射线衍射被用于量化二元水溶液中冰晶的无序程度，该二元水溶液包含一种冷冻保护剂（山梨醇）和一种蛋白质——牛血清白蛋白。较稀（10 wt%）溶液中的冰晶比更浓（40 wt%）溶液中的冰晶具有更低的微观应变水平和更大的晶畴尺寸。将山梨醇 - 水混合物从100 K加热至228 K会导致部分冰融化，同时微观应变降低，微晶尺寸增加，即发生重结晶。与山梨醇溶液不同，BSA溶液中的冰晶在部分融化时保持了微观应变和较小的微晶尺寸，这表明BSA抑制了冰的重结晶。这些结果与BSA分配到冰晶的准液态层一致，但与蛋白质 - 冰的直接相互作用以及蛋白质在冰表面的吸附不一致。该研究首次表明一种常见的（即非抗冻）蛋白质会对冰的重结晶产生重大影响，并且还展示了同步加速器X射线衍射作为量化冷冻水体系中结晶度和无序度的独特工具。
