Ice Recrystallization in a Solution of a Cryoprotector and Its Inhibition by a Protein: Synchrotron X-Ray Diffraction Study.
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.
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.
Boris
B
Zakharov
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.
Alexander
A
Fisyuk
ESRF-The European Synchrotron Radiation Facility, 71 av des Martyrs, Grenoble 38043, France.
Andy
A
Fitch
ESRF-The European Synchrotron Radiation Facility, 71 av des Martyrs, Grenoble 38043, France.
Yves
Y
Watier
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.
Anastasia
A
Kostyuchenko
Novartis Vaccines and Diagnostics, Holly Springs, North Carolina 27540.
Dushyant
D
Varshney
ESRF-The European Synchrotron Radiation Facility, 71 av des Martyrs, Grenoble 38043, France.
Michael
M
Sztucki
Institute of Solid State Chemistry and Mechanochemistry SB RAS, ul. Kutateladze, 18, Novosibirsk 630128, Russian Federation.
Elena
E
Boldyreva
Pharmaceutical R&D, Allergan Inc., Irvine, California 92612. Electronic address: shalaev_evgenyi@allergan.com.
Evgenyi
E
Shalaev
D002152:Calorimetry, Differential Scanning; D003451:Cryoprotective Agents / Q000737:chemistry*; D003460:Crystallization; D005612:Freeze Drying; D005615:Freezing; D010316:Particle Size; D019999:Pharmaceutical Solutions; D011506:Proteins / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013012:Sorbitol / Q000737:chemistry; D017356:Synchrotrons; D014961:X-Ray Diffraction
D003451:Cryoprotective Agents; D019999:Pharmaceutical Solutions; D011506:Proteins; D012710:Serum Albumin, Bovine; D013012:Sorbitol
X-ray powder diffractometry; calorimetry (DSC); crystal defects; freeze drying/lyophilization; phase transition; proteins