The high viscosity encountered during freezing in glycerol solutions: Effects on cryopreservation

Morris, G.J.; Goodrich, Martha; Acton, Elizabeth; Fonseca, Fernanda

doi:10.1016/j.cryobiol.2006.01.003

Cited by 90 publications

(71 citation statements)

References 20 publications

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“…The shift in T g2 Ј following annealing suggests that the process of ice formation during cooling is limited by high viscosity, even at the lowest rate of cooling examined (5°C min Ϫ1 ). The effects of the cooling rate on the process of ice formation in glycerol solutions and the ultrastructure of the freezeconcentrated matrix are consistent with previous work carried out on the ternary system of H 2 O-NaCl-glycerol (28).…”

Section: Discussionsupporting

confidence: 79%

“…In the binary system of glycerol-water, the measured viscosity exceeded 1,000 cPa at Ϫ40°C, while the viscosity of a ternary system, glycerol-waterNaCl, exceeded 100,000 cPa at Ϫ55°C. Because of high viscosity, the diffusion process becomes limited, and the amount of ice formed becomes dependent on the rate of cooling (28). In the current study, the measured osmolality of the solution (10% [wt/wt] glycerol plus growth medium) in which the cells were suspended was 1,600 mosM, which is very similar (1,549 mosM) to that of the ternary system of water-glycerol (10% [wt/wt])-NaCl (0.15 M).…”

Section: Discussionmentioning

confidence: 99%

“…However, no direct evidence of intracellular ice formation in cryopreserved bacteria has been presented. An alternative mechanism has been proposed in which cell damage occurring after rapid cooling rates is caused by an osmotic imbalance encountered during thawing, not by the formation of intracellular ice (28).…”

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confidence: 99%

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Stabilization of Frozen Lactobacillus delbrueckii subsp. bulgaricus in Glycerol Suspensions: Freezing Kinetics and Storage Temperature Effects

Fonseca

Marin

Morris

2006

Appl Environ Microbiol

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The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at ؊196°C and ؊20°C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (؊196°C and ؊80°C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at ؊20°C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.Concentrates of lactic acid bacteria (LAB) are widely used as starters for manufacturing cheese, fermented milk, meat, vegetables, and bread products (19). Freezing and storage at low temperatures (ϽϪ40°C) are commonly applied to preserve the viability of concentrates while maintaining their technological properties upon thawing (acidification activity, production of aroma compounds, and contribution to product texture). However, bacterial resistance to freezing and to frozen storage depends on the strain, culture conditions before freezing, harvesting, formulation (type and concentration of cryoprotectant), freezing conditions, and final storage temperature (9).Freezing is a critical step in the production of LAB concentrates, as it affects both the viability and acidification activity upon thawing (10, 32). However, the mechanisms of freezing damage to bacteria are not well understood. Two damage mechanisms dependent on the cooling rate have usually been proposed. At low freezing rates, cellular damage is principally caused by osmotic stress to cells (so-called "solution effects") (24). The formation of extracellular ice induces high solute concentrations in the extracellular medium, exposing the cells to an increase in ionic concentration, changes in pH, etc., for extended periods of time. At very high cooling rates, it has been assumed that damage is due to the formation of int...

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Section: Discussionsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 99%

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Stabilization of Frozen Lactobacillus delbrueckii subsp. bulgaricus in Glycerol Suspensions: Freezing Kinetics and Storage Temperature Effects

Fonseca

Marin

Morris

2006

Appl Environ Microbiol

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“…S1), the amount of ice crystal formation will be much less than that computed from the equilibrium-phase diagram of the two-component glycerol-water mixture at atmospheric pressure (45). If the system were at thermal equilibrium during cooling, ∼80% of the water would be ice crystal as predicted by the phase diagram, thereby concentrating the protein in a supercooled water-glycerol mixture by a factor of approximately three.…”

Section: Discussionmentioning

confidence: 96%

Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron–electron resonance

Lerch

Yang²,

Brooks³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The dominance of a single native state for most proteins under ambient conditions belies the functional importance of higherenergy conformational states (excited states), which often are too sparsely populated to allow spectroscopic investigation. Application of high hydrostatic pressure increases the population of excited states for study, but structural characterization is not trivial because of the multiplicity of states in the ensemble and rapid (microsecond to millisecond) exchange between them. Sitedirected spin labeling in combination with double electron-electron resonance (DEER) provides long-range (20-80 Å) distance distributions with angstrom-level resolution and thus is ideally suited to resolve conformational heterogeneity in an excited state populated under high pressure. DEER currently is performed at cryogenic temperatures. Therefore, a method was developed for rapidly freezing spin-labeled proteins under pressure to kinetically trap the highpressure conformational ensemble for subsequent DEER data collection at atmospheric pressure. The methodology was evaluated using seven doubly-labeled mutants of myoglobin designed to monitor selected interhelical distances. For holomyoglobin, the distance distributions are narrow and relatively insensitive to pressure. In apomyoglobin, on the other hand, the distributions reveal a striking conformational heterogeneity involving specific helices in the pressure range of 0-3 kbar, where a molten globule state is formed. The data directly reveal the amplitude of helical fluctuations, information unique to the DEER method that complements previous rate determinations. Comparison of the distance distributions for pressure-and pH-populated molten globules shows them to be remarkably similar despite a lower helical content in the latter.EPR | dipolar spectroscopy | compressibility A lthough a well-defined native state is dominant for most proteins under physiological conditions, excited states involving large (multiangstrom) conformational changes relative to the native state have been shown to play critical roles in biological function (1-3). Even for low-lying excited states with relative energies on the order of a few kilocalories (1 kcal = 4.184 kJ) per mole, the equilibrium population is too small to be characterized by most spectroscopic methods, but such states can be reversibly populated by hydrostatic pressure (4-9). It is generally agreed that the mechanism for population of excited states by pressure is hydration of cavities in the excited state relative to the ground state (10-12), but other mechanisms are possible (13). The pressures required to populate low-lying excited states are typically a few kilobars, corresponding to perturbation energies of a few kilocalories per mole. At these low energies, pressure should not strongly remodel the energy landscape itself but simply shift the relative population of preexisting conformers (14). Thus, pressure should be a powerful perturbation technique for studying functional excited states at equilibrium (15)....

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“…However, there are practical limitations that can preclude its applicability in preparing pharmaceutically relevant, highly concentrated solutions for use in HDX-MS experiments, such as the introduction of unwanted protein stresses, changes in reconstitution time, variations in viscosity, etc. [12][13][14][15][16][17]. Nevertheless, many of these challenges may be solved using alternative lyophilization strategies and protocols, but the success of this approach will depend on the protein and its specific formulation.…”

Section: Introductionmentioning

confidence: 99%

Conformational Analysis of Proteins in Highly Concentrated Solutions by Dialysis-Coupled Hydrogen/Deuterium Exchange Mass Spectrometry

Houde

Nazari

Bou-Assaf

et al. 2016

J. Am. Soc. Mass Spectrom.

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Abstract. When highly concentrated, an antibody solution can exhibit unusual behaviors, which can lead to unwanted properties, such as increased levels of protein aggregation and unusually high viscosity. Molecular modeling, along with many indirect biophysical measurements, has suggested that the cause for these phenomena can be due to short range electrostatic and/or hydrophobic protein-protein interactions. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for investigating protein conformation, dynamics, and interactions. However, Btraditional^continuous dilution labeling HDX-MS experiments have limited utility for the direct analysis of solutions with high concentrations of protein. Here, we present a dialysis-based HDX-MS (di-HDX-MS) method as an alternative HDX-MS labeling format, which takes advantage of passive dialysis rather than the classic dilution workflow. We applied this approach to a highly concentrated antibody solution without dilution or significant sample manipulation, prior to analysis. Such a method could pave the way for a deeper understanding of the unusual behavior of proteins at high concentrations, which is highly relevant for development of biopharmaceuticals in industry.

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The high viscosity encountered during freezing in glycerol solutions: Effects on cryopreservation

Cited by 90 publications

References 20 publications

Stabilization of Frozen Lactobacillus delbrueckii subsp. bulgaricus in Glycerol Suspensions: Freezing Kinetics and Storage Temperature Effects

Stabilization of Frozen Lactobacillus delbrueckii subsp. bulgaricus in Glycerol Suspensions: Freezing Kinetics and Storage Temperature Effects

Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron–electron resonance

Conformational Analysis of Proteins in Highly Concentrated Solutions by Dialysis-Coupled Hydrogen/Deuterium Exchange Mass Spectrometry

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