Freeze-thaw of pharmaceutical solutions: counter-intuitive finding of an increase in mechanical stress between Tg” and Tg’ in frozen sucrose/water mixtures

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Freeze-thaw of pharmaceutical solutions: counter-intuitive finding of an increase in mechanical stress between Tg” and Tg’ in frozen sucrose/water mixtures. / Kostyuchenko, Anastasia; Zakharov, Boris; Fisyuk, Alexander et al.

In: Journal of Pharmaceutical Sciences, Vol. 114, No. 7, 103800, 07.2025.

Research output: Contribution to journal › Article › peer-review

BibTeX

@article{811f6b6518d24a55985f6bcec5982c85,

title = "Freeze-thaw of pharmaceutical solutions: counter-intuitive finding of an increase in mechanical stress between Tg” and Tg{\textquoteright} in frozen sucrose/water mixtures",

abstract = "High-resolution synchrotron X-ray powder diffraction has been utilized to detect mechanical stresses in frozen solutions, via analysis of the profiles of the diffraction peaks of ice. Increase in the width of the peaks (peaks broadening) reflects disruption of the crystal lattice, with contributions including a decrease in the crystallite size and an increase in microstrain. Frozen sucrose solutions (5 and 10 % w/v) were frozen at 100 K and then heated and annealed at -45 °C (228 K) and -15 °C (258 K), i.e. between the two apparent glass transition events in the freeze-concentrated solutions (Tg” and Tg{\textquoteright}), and above the Tg{\textquoteright}, respectively. A decrease in microstrain and an increase in ice crystallite size were observed during annealing at -15 °C (above the Tg{\textquoteright}), which is consistent with Ostwald ripening of ice crystals. Unexpectedly, and for the first time, an opposite trend was observed during annealing at the lower temperature of -45 °C, between Tg” and Tg{\textquoteright}. To the best of our knowledge, this is the first report of an increased strain in the crystalline ice domains and of simultaneous size reduction during annealing of frozen aqueous solutions. Considering that the interaction with ice crystals may result in protein destabilization, the size and microstrain of ice crystallites can serve as markers of the freeze/thaw stresses on biopharmaceuticals. A practical implication is that a prolonged hold between Tg” and Tg{\textquoteright} may increase stress imposed on protein molecules, i.e. a lower temperature is not always better for preserving biopolymers when freezing their aqueous solutions.",

keywords = "Aqueous solutions, Freezing, Ice crystals, Microstrain, Size of crystalline domains, Synchrotron X-ray diffraction",

author = "Anastasia Kostyuchenko and Boris Zakharov and Alexander Fisyuk and Bakul Bhatnagar and Yurii Seryotkin and Elena Boldyreva and Evgenyi Shalaev",

note = "The study was supported by ESRF proposals LS-2601 and LS-2742. BZ and EB acknowledge support from Ministry of Science and Higher Education of the Russian Federation (BZ - governmental order for SRF SKIF Boreskov Institute of Catalysis FWUR-2024-0042, EB - Program of the Strategic Academic Leadership \u201CPriority-2030\u2033). We also thank Mashikoane Mogodi and Andy Fitch for expert assistance in the experiments, and Andy Fitch for helpful discussions and theoretical X-ray diffraction pattern for Ih. We thank anonymous reviewers for thoughtful and valuable comments and suggestions, which helped us to improve the manuscript.",

year = "2025",

month = jul,

doi = "10.1016/j.xphs.2025.103800",

language = "English",

volume = "114",

journal = "Journal of Pharmaceutical Sciences",

issn = "0022-3549",

publisher = "Elsevier Science Publishing Company, Inc.",

number = "7",

}

RIS

TY - JOUR

T1 - Freeze-thaw of pharmaceutical solutions: counter-intuitive finding of an increase in mechanical stress between Tg” and Tg’ in frozen sucrose/water mixtures

AU - Kostyuchenko, Anastasia

AU - Zakharov, Boris

AU - Fisyuk, Alexander

AU - Bhatnagar, Bakul

AU - Seryotkin, Yurii

AU - Boldyreva, Elena

AU - Shalaev, Evgenyi

N1 - The study was supported by ESRF proposals LS-2601 and LS-2742. BZ and EB acknowledge support from Ministry of Science and Higher Education of the Russian Federation (BZ - governmental order for SRF SKIF Boreskov Institute of Catalysis FWUR-2024-0042, EB - Program of the Strategic Academic Leadership \u201CPriority-2030\u2033). We also thank Mashikoane Mogodi and Andy Fitch for expert assistance in the experiments, and Andy Fitch for helpful discussions and theoretical X-ray diffraction pattern for Ih. We thank anonymous reviewers for thoughtful and valuable comments and suggestions, which helped us to improve the manuscript.

PY - 2025/7

Y1 - 2025/7

N2 - High-resolution synchrotron X-ray powder diffraction has been utilized to detect mechanical stresses in frozen solutions, via analysis of the profiles of the diffraction peaks of ice. Increase in the width of the peaks (peaks broadening) reflects disruption of the crystal lattice, with contributions including a decrease in the crystallite size and an increase in microstrain. Frozen sucrose solutions (5 and 10 % w/v) were frozen at 100 K and then heated and annealed at -45 °C (228 K) and -15 °C (258 K), i.e. between the two apparent glass transition events in the freeze-concentrated solutions (Tg” and Tg’), and above the Tg’, respectively. A decrease in microstrain and an increase in ice crystallite size were observed during annealing at -15 °C (above the Tg’), which is consistent with Ostwald ripening of ice crystals. Unexpectedly, and for the first time, an opposite trend was observed during annealing at the lower temperature of -45 °C, between Tg” and Tg’. To the best of our knowledge, this is the first report of an increased strain in the crystalline ice domains and of simultaneous size reduction during annealing of frozen aqueous solutions. Considering that the interaction with ice crystals may result in protein destabilization, the size and microstrain of ice crystallites can serve as markers of the freeze/thaw stresses on biopharmaceuticals. A practical implication is that a prolonged hold between Tg” and Tg’ may increase stress imposed on protein molecules, i.e. a lower temperature is not always better for preserving biopolymers when freezing their aqueous solutions.

AB - High-resolution synchrotron X-ray powder diffraction has been utilized to detect mechanical stresses in frozen solutions, via analysis of the profiles of the diffraction peaks of ice. Increase in the width of the peaks (peaks broadening) reflects disruption of the crystal lattice, with contributions including a decrease in the crystallite size and an increase in microstrain. Frozen sucrose solutions (5 and 10 % w/v) were frozen at 100 K and then heated and annealed at -45 °C (228 K) and -15 °C (258 K), i.e. between the two apparent glass transition events in the freeze-concentrated solutions (Tg” and Tg’), and above the Tg’, respectively. A decrease in microstrain and an increase in ice crystallite size were observed during annealing at -15 °C (above the Tg’), which is consistent with Ostwald ripening of ice crystals. Unexpectedly, and for the first time, an opposite trend was observed during annealing at the lower temperature of -45 °C, between Tg” and Tg’. To the best of our knowledge, this is the first report of an increased strain in the crystalline ice domains and of simultaneous size reduction during annealing of frozen aqueous solutions. Considering that the interaction with ice crystals may result in protein destabilization, the size and microstrain of ice crystallites can serve as markers of the freeze/thaw stresses on biopharmaceuticals. A practical implication is that a prolonged hold between Tg” and Tg’ may increase stress imposed on protein molecules, i.e. a lower temperature is not always better for preserving biopolymers when freezing their aqueous solutions.

KW - Aqueous solutions

KW - Freezing

KW - Ice crystals

KW - Microstrain

KW - Size of crystalline domains

KW - Synchrotron X-ray diffraction

UR - https://www.mendeley.com/catalogue/b6e3af61-f8f5-3008-bc14-6cdba4ba63c6/

UR - https://pubmed.ncbi.nlm.nih.gov/40280484/

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-105004986582&origin=inward&txGid=9b4fb6ec58be39253a1e202d26e12518

U2 - 10.1016/j.xphs.2025.103800

DO - 10.1016/j.xphs.2025.103800

M3 - Article

C2 - 40280484

VL - 114

JO - Journal of Pharmaceutical Sciences

JF - Journal of Pharmaceutical Sciences

SN - 0022-3549

IS - 7

M1 - 103800

ER -

ID: 66565039