Compositions and methods for stabilizing a biological material or biologically-derived material

Abstract

Embodiments described herein generally relate to compositions and methods for stabilizing a biological material or biologically-derived material. Embodiments described herein also generally relate to sugar glasses and glass forming sugars. Compositions of the present disclosure may be characterized as stabilizing a biological material or biologically-derived material of interest present in the composition in a dry state at a temperature of ambient temperature or higher. In an embodiment is provided a composition that includes a biological material of interest or a biologically-derived material of interest; and an additive or additive mixture comprising: a sucrose compound comprising monomeric sucrose, a polysucrose, or a combination thereof; and optionally glycerol. In another embodiment is provided a composition that includes a biological material or biologically-derived material of interest; and an additive mixture comprising: a disaccharide; and a polyol that is different from the disaccharide.

Description

TITLE: Compositions and Methods for Stabilizing a Biological Material or Biologically-Derived MaterialInventor: Thomas C. BoothbyGOVERNMENT RIGHTS

[0001] The invention was made with United States government support under Grant No.2213983 awarded by the National Science Foundation. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims benefit of and priority to U.S. Provisional Patent Application No. 63 / 569,685, filed on March 25, 2024, and U.S. Provisional Patent Application No. 63 / 569,690, filed on March 25, 2024, each of which is incorporated herein by reference in their entireties.FIELD

[0003] Embodiments described herein generally relate to compositions and methods for stabilizing a biological material or biologically-derived material. Embodiments described herein also generally relate to sugar glasses and glass forming sugars.BACKGROUND

[0004] A number of organisms, spread across every biological kingdom, are capable of surviving near-complete water loss through a process known as anhydrobiosis (Greek for “life without water”). As organisms dry, they face a number of physical and chemical changes to their cellular environment. As water is lost, cellular constituents are concentrated, molecular crowding increases, pH and ionic concentrations change, and osmotic pressure increases. These physiochemical changes lead to detrimental perturbations such as protein unfolding, aggregation, and membrane leakage. Drying is not an all-or- nothing process and these changes as well as the perturbation they induce, occur along a continuum, with some perturbations occurring earlier as an organism is dehydrating while others manifest later, once more substantial amounts of water have been lost. How organisms survive desiccation is one of the enduring mysteries of organismal physiology. Historically, anhydrobiosis has been thought to be mediated, at least in part, through the concentration of cellular constituents until these constituents solidify into a vitrified material (a glass). In this hypothesis, known as the ‘vitrification hypothesis,’ glasses slow physicaland biochemical change, making them natural promoters of desiccation tolerance. Within the anhydrobiosis field, vitrification is considered a necessary process for desiccation tolerance.

[0005] However, a major shortcoming of the vitrification hypothesis is the observation that essentially every biological, or sufficiently heterogeneous, system will vitrify when dried, regardless of whether it is desiccation-tolerant or desiccation-sensitive. This observation implies that while vitrification is necessary, it is not sufficient for desiccation tolerance and that there must be some property, or properties, that distinguishes a protective from a non-protective vitrified state. The properties distinguishing a desiccation-protective glass from a non-protective glass are not currently fully understood. Previous studies have identified that small additions of glycerol changes the enzyme-protective capacity of trehalose. However, the material properties of these mixtures and how they correspond with changes in the level of protection have not been investigated.

[0006] There is a need for new and improved understanding of sugar glasses. There is also a need for new and improved understanding of how material properties correspond with changes in the level of protection. There is also a need for new compositions and methods for stabilizing a biological material or a biologically-derived material.SUMMARY

[0007] Embodiments described herein generally relate to compositions and methods for stabilizing a biological material or biologically-derived material. Embodiments described herein also generally relate to sugar glasses. The inventor found that a sucrose monomer or a polysucrose, with or without glycerol, may be used to stabilize and / or protect a biological material. The effect of the polymer size of sucrose on glassy properties associated with protection in the vitrified state was investigated. Using different-sized sucrose compounds — monomeric sucrose and two different-sized sucrose polymers, — the inventor found that each of the glassy properties investigated including — enzyme protection, water content, glass transition temperature, and glass former fragility — were affected by the size of the sucrose compounds. However, only one vitrified property, glass transition temperature, correlated with protection during drying under the conditions tested. The results indicated that the size of vitrifying protectants may have a profound effect on glassy properties as well as on how these properties correlate with protection in the dry state.Beyond desiccation tolerance, these findings provide insights for the development of new technologies for the stabilization of biological material in the dry state.

[0008] Embodiments described herein also generally relate to glass forming sugars. The inventor found that the enzyme-protective capacities of different glass forming sugars correlate with distinct material properties. The inventor also found that material properties of dried anhydrobiotic organisms may differ dramatically when examined in desiccation- tolerant life stages and desiccation-intolerant life stages. The inventor also found that organismal desiccation tolerance is concomitant with changes in glassy properties including increased glass transition temperature and reduced glass former fragility.

[0009] In an embodiment, a composition is provided. The composition includes an exogenous disaccharide. The composition further includes an intrinsically disordered protein (IDP). The composition further includes a biological material of interest and / or a biologically-derived material of interest, the biological material of interest and biologically- derived material of interest different from the IDP. The composition has a water content that is about 15 wt% or less based on a total wt% of the composition, the total wt% of the composition equal to 100 wt%.

[0010] In another embodiment, a composition is provided. The composition includes a biological material of interest and / or a biologically-derived material of interest. The composition further includes an additive or additive mixture comprising: a sucrose compound comprising monomeric sucrose, a polysucrose having a molecular weight that is in a range from about 10 kDa to about 800 kDa, or a combination thereof; and optionally glycerol. The composition is characterized as stabilizing the biological material of interest or biologically-derived material of interest of the composition in a dry state at a temperature of 20°C or higher.

[0011] In another embodiment, a composition is provided. The composition includes a biological material of interest and / or a biologically-derived material of interest. The composition further includes an additive mixture comprising: a disaccharide; and a polyol that is different from the disaccharide. The composition is characterized as stabilizing the biological material of interest or biologically-derived material of interest of the composition in a dry state at a temperature of 20°C or higher.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] So that the manner in which the above recited features of the present disclosure may be understood in detail, a more particular description of the disclosure, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only exemplary embodiments and are therefore not to be considered limiting of its scope, and may admit to other equally effective embodiments.

[0013] FIG. 1 shows a correlation plot of water content versus Lactate dehydrogenase (LDH) enzyme protection for all sugar-glycerol mixtures (sugar is sucrose, polysucrose 70, or polysucrose 400) indicating that water content does not correlate with the enzyme- protective capacity of any sucrose-glycerol glasses while water content strongly correlates with glass transition temperature (Tg).

[0014] FIGS. 2, 3, and 4 show non-limiting data indicating that monomeric sucrose strongly correlates with the Tg. FIG. 2 is a correlation plot of glass transition onset (Tg onset) values versus LDH protection for all sucrose-glycerol mixtures. FIG. 3 is a correlation plot of glass transition midpoint (Tg midpoint) values versus LDH protection for all sucrose-glycerol mixtures. FIG. 4 is a correlation plot of glass transition endset (Tg endset) values versus LDH protection for all sucrose-glycerol mixtures. Sucroses tested included monomeric sucrose, polysucrose 70, and polysucrose 400.

[0015] FIG. 5 is a correlation plot of glass former fragility (m-index) versus LDH protection for all sucrose-glycerol mixtures, indicating that glass forming fragility does not correlate with LDH enzyme protection in all three types of sucrose-glycerol mixtures. Sucroses tested included monomeric sucrose, polysucrose 70, and polysucrose 400.

[0016] FIG. 6 is a correlation plot of water content versus LDH protection for all disaccharide-glycerol mixtures, indicating that water content correlates with the enzyme- protective capacity of maltose-glycerol but not sucrose- or trehalose-glycerol glasses. Error bars (shown as hatching) represent 95% confidence interval (CI).

[0017] FIG. 7 is a correlation plot of glass transition midpoint versus LDH protection for all disaccharide-glycerol mixtures, indicating that anti-plasticization of maltose-glycerol glasses and sucrose-glycerol glasses, but not trehalose-glycerol glasses, correlate with enzyme protection. Error bars (shown as hatching) represent 95% confidence interval (CI).

[0018] FIG. 8 is a correlation plot of glass former fragility (m-index) versus LDH protection for all disaccharide-glycerol mixtures, indicating that reduced glass forming fragility of maltose-glycerol glasses and trehalose-glycerol glasses, but not sucrose-glycerol glasses, correlates with enzyme protection. Error bars (shown as hatching) represent 95% confidence interval (CI).

[0019] FIGS. 9 and 10 show non-limiting data indicating that in vivo glass transition temperature is increased, and glass former fragility is reduced in desiccation-tolerant versus sensitive life stages of diverse anhydrobiotic organisms. FIG. 9 includes bar charts of glass transition offset values for selected desiccation-tolerant organisms organized by organism and life stage. FIG. 10 includes bar charts of glass former fragility (m-index) values for selected desiccation-tolerant organisms organized by organism and life stage. Statistics calculated using T-test: p-value << 0.05 (*), pairwise relationships not shown are not significant.

[0020] FIG. 11 is a correlation plot of Texp - Tg versus LDH protection for maltose-, sucrose-, and trehalose-glycerol mixtures, indicating that the difference between glass transition temperature and the temperature at which the enzyme protection assay was performed was significant for maltose-glycerol mixtures. Texp is the temperature at which the enzyme (LDH) protection assay was performed. Error bars (shown as hatching) represent 95% confidence interval (CI).

[0021] FIG. 12 shows data for the evaluation of LDH enzyme fold protection in trehalose using different drying conditions. Error bars represent l*standard error (SE).

[0022] FIG. 13 shows data for the evaluation of citrate synthase (CS) enzyme fold protection in trehalose using different drying conditions. Error bars represent 1*SE.

[0023] FIG. 14 shows data for the evaluation of the fold protection of DNA in trehalose using different drying conditions. DNA integrity was measured using the DNA Integrity Number (DIN). Error bars represent 1*SE.

[0024] FIG. 15 shows data for the evaluation of the fold protection of RNA in trehalose using different drying conditions. RNA integrity was measured using the RNA Integrity Number (RIN). Error bars represent 1*SE.

[0025] FIG. 16 shows data for the correlation between fold protection of LDH enzyme in trehalose and the glassy properties in hotplate drying at 30°C.

[0026] FIG. 17 shows data for the correlation between fold protection of LDH enzyme in trehalose and the glassy properties in hotplate drying at 37°C.

[0027] FIG. 18 shows data for the correlation between fold protection of LDH enzyme in trehalose and the glassy properties in humidified drying at 30°C.

[0028] FIG. 19 shows data for the correlation between fold protection of LDH enzyme in trehalose and the glassy properties in humidified drying at 37°C.

[0029] FIG. 20 shows data for the correlation between fold protection of LDH enzyme in trehalose and the glassy properties in humidified drying at 44°C.

[0030] FIG. 21 shows non-limiting data for LDH enzyme protection in various formulations and concentrations of the following: Tris alone, trehalose alone, CAHS D alone, or CAHS D with trehalose. Statistics calculated using a one-way ANOVA and Tukey post-hoc test: p-value < 0.05 (*) p-value < 0.01 (**), p-value < 0.001 (***), and error bars represent 1*SE. Tris is Tris(hydroxymethyl)aminomethane hydrochloride.

[0031] FIG. 22 shows non-limiting data for long-term preservation of LDH enzyme activity in different formulations and concentrations of the following: Tris alone, trehalose alone, CAHS D alone, or CAHS D with trehalose. Statistics calculated using a one-way ANOVA and Tukey post-hoc test: p-value < 0.05 (*) p-value < 0.01 (**), p-value < 0.001 (***), and error bars represent 1*SE.

[0032] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. It is contemplated that elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation.DETAILED DESCRIPTION

[0033] Embodiments described herein generally relate to compositions and methods for stabilizing a biological material or biologically-derived material. The compositions may be utilized to stabilize a biological material of interest (or biologically-derived material of interest) present in the composition in a dry state and / or at temperatures of, for example, ambient temperature or higher. As used herein, a “composition” may include component(s) of the composition, reaction product(s) of two or more components of the composition, a remainder balance of remaining starting component(s), or combinations thereof.

[0034] The use of headings is for purposes of convenience only and does not limit the scope of the present disclosure. Embodiments described herein may be combined with other embodiments.

[0035] Embodiments described herein generally relate to compositions that may be utilized to stabilize a biological material of interest present in the composition in a dry state and / or at temperatures of, for example, ambient temperature or higher.

[0036] Compositions described herein may include a biological material of interest, an additive (or an additive mixture), an optional intrinsically disordered protein (IDP), and optionally water. These compositions may be characterized as protecting and / or stabilizing the biological material of interest present in the composition in a dry state at a temperature of ambient temperature or higher (e.g., about ambient temperature to less than about 100°C, such as a temperature of room temperature or higher). Ambient temperature refers to 20°C.

[0037] The term “dry state”, when referring to a composition described herein, refers to a composition that has about 15 wt% or less water based on the total wt% of the composition. In some examples, a composition in a dry state has an amount of water in a range from about 0 wt% or more, about 15 wt% or less, or combinations thereof, such as from greater than 0 wt% to about 15 wt%, from about 2 wt% to about 12 wt%, from about 5 wt% to about 15 wt%, from about 6 wt% to about 14 wt%, such as from about 7 wt% to about 12 wt%, such as from about 8 wt% to about 10 wt%, or from about 10 wt% to about 14 wt% based on a total wt% of the composition. Any of the foregoing numbers may be used singly to describe an open-ended range or in combination to describe a close-ended range.

[0038] The biological material of interest is different from the IDP. The biological material of interest may be in its natural state, in a modified state, derived from a living organism, and / or synthesized. The biological material of interest may include a peptide, a polypeptide, a protein, an enzyme, an antibody, a globular protein, a hormone, a natural product, a derivative thereof, a component thereof, or combinations thereof, among others. The biological material of interest may include a biologically-derived material of interest. A biologically-derived material of interest may be derived from a peptide, a polypeptide, a protein, an enzyme, an antibody, a globular protein, a hormone, a natural product, a derivative thereof, a component thereof, or combinations thereof, among others. The biological material of interest and the biologically-derived material of interest is not an IDPand is not derived from an IDP. The biological material of interest or the biologically- derived material of interest may be a pharmaceutical.

[0039] Any suitable optional IDP may be utilized such as a wild-type CAHS D protein or a mutant CAHS D protein. Unlike typical well-folded proteins, IDPs are characterized by a lack of defined tertiary structure, and instead exist as an ensemble of dynamic, interconverting conformations.

[0040] The additive or additive mixture is exogenous to the other components present in the composition. For example, the additive or additive mixture is exogenous to the biological material of interest (or biologically-derived material of interest) and to the IDP. A total wt% of the additive or additive mixture is based on the total amount of the components present in the additive or additive mixture and is equal to 100 wt%.

[0041] The additive or additive mixture may include one or more of the following:

[0042] (i) a disaccharide, a polysucrose (polymeric sucrose), or a combination thereof; and / or

[0043] (ii) a polyol that is different from the disaccharide and the polysucrose.

[0044] Any suitable disaccharide may be utilized. Suitable disaccharides may include sucrose, trehalose, maltose, lactose, or combinations thereof, such as sucrose, trehalose, or combinations thereof. More than one disaccharide may be used.

[0045] Any suitable polysucrose, interchangeably referred to herein as polymeric sucrose, may be utilized. The polysucrose may have a molecular weight that is from about 10 kDa to about 800 kDa, such as from about 70 kDa to about 400 kDa. More than one polysucrose may be used.

[0046] Any suitable polyol, that is different from the disaccharide and the polysucrose, may be utilized. Suitable polyols may include, for example, glycerol, ethylene glycol, propylene glycol, methyl glycol trimethylene glycol, neopentyl glycol, diethylene glycol, triethylene glycol, polyethylene glycol, polypropylene glycol, or combinations thereof, such as glycerol. More than one polyol, that is different from the di saccharide and the polysucrose, may be used

[0047] The additive or additive mixture may include:

[0048] (i) an amount of the disaccharide, a polysucrose, or combination thereof that may be in a range from about 85 wt% to about 100 wt%, such as from about 86 wt% to about 99wt%, such as from about 87 wt% to about 98 wt%, such as from about 88 wt% to about 97 wt%, such as from about 89 wt% to about 96 wt%, such as from about 90 wt% to about 95 wt%, such as from about 91 wt% to about 94 wt%, such as from about 92 wt% to about 93 wt% based on a total wt% of the additive or additive mixture. Any of the foregoing numbers may be used singly to describe an open-ended range or in combination to describe a close- ended range; and

[0049] (ii) an amount of the polyol, that is different from the disaccharide and the polysucrose, that may be in a range from about 0 wt% to about 15 wt%, or from greater than 0 wt% to about 15 wt%, such as from about 1 wt% to about 14 wt%, such as from about 2 wt% to about 13 wt%, such as from about 3 wt% to about 12 wt%, such as from about 4 wt% to about 11 wt%, such as from about 5 wt% to about 10 wt%, such as from about 6 wt% to about 9 wt%, such as from about 7 wt% to about 8 wt% based on a total wt% of the additive or additive mixture. Any of the foregoing numbers may be used singly to describe an open-ended range or in combination to describe a close-ended range.

[0050] The additive or additive mixture may be glassy and without crystallinity upon drying or desiccating (indicating that the additive mixture vitrifies).

[0051] Compositions described herein may further include water. For example, the composition may include 15 wt% or less of water based on a total wt% of the composition. The composition has an amount of water that may be from about 0 wt% or more, about 15 wt% or less, or combinations thereof, such as from greater than 0 wt% to about 15 wt%, or from about 2 wt% to about 12 wt%, or from about 5 wt% to about 15 wt%, or from about 6 wt% to about 14 wt%, or from about 7 wt% to about 12 wt%, or from about 8 wt% to about 10 wt%, or from about 10 wt% to about 14 wt%, or from about 5 wt% to about 12 wt% based on a total wt% of the composition. Any of the foregoing numbers may be used singly to describe an open-ended range or in combination to describe a close-ended range.

[0052] A total wt% of the composition may be based on the amount of the biological and / or biologically-derived material of interest (e.g., a polypeptide, protein, polypeptidederived material, protein-derived material, etc.), plus the amount of the additive (or additive mixture), plus the amount of the optional IDP, plus the amount of the optional water. The total wt% of the composition is 100 wt%.

[0053] As described above, the composition may be utilized to stabilize the biological material of interest of the composition at temperatures of, for example, ambient temperature or higher. Such temperatures at which the composition may be utilized to stabilize the biological material of interest may include a temperature in a range from about 20°C or more, 100°C or less, or combinations thereof, such as from about 20°C to about 50°C, such as from about 25°C to about 40°C, such as from about 30°C to about 35°C or from about 35°C to about 40°C. Any of the foregoing numbers may be used singly to describe an open- ended range or in combination to describe a close-ended range.

[0054] In some embodiments, which may be combined with other embodiments, a composition may include a molar ratio of the IDP to the exogenous disaccharide in the composition that is about 1 : 1,000 or more, such as 1 :5000 or more, such as 1 : 10,000 or more, or in a range from about 1 : 10,000 to about 1 : 100,000, such as from about 1 :20,000 to about 1 :90,000, such as from about 1 :30,000 to about 1 :80,000, such as from about 1 :40,000 to about 1 :70,000, such as from about 1 :50,000 to about 1 :60,000. Any of the foregoing numbers may be used singly to describe an open-ended range or in combination to describe a close-ended range. The composition may include other components described herein.

[0055] In some embodiments, which may be combined with other embodiments, a composition may include a molar ratio of the biological material (and / or biologically- derived material) of interest to the additive or additive mixture that may be in a range from about 1 :0.1 to about 1 : 100 (biological material: additive), such as from about 1 :0.5 to about 1 :50, such as from about 1 :2.5 to about 1 :25, such as from about 1 :5 to about 1 : 15, such as about 1 : 10. Any of the foregoing numbers may be used singly to describe an open-ended range or in combination to describe a close-ended range. The composition may include other components described herein.

[0056] For example, a composition may include a molar ratio of component A:component B, where component A refers to a biological material of interest and / or biologically-derived material of interest, and component B refers to the additive or additive mixture. For example, a composition may include a LDFLsugar-glycerol molar ratio of about 1 : 10.

[0057] Embodiments of the present disclosure also generally relate to methods of forming the compositions, methods of stabilizing a biological material of interest and / orbiologically-derived material of interest, and to methods of forming a stabilized biological material of interest and / or a stabilized biologically-derived material of interest. Embodiments described herein may be used to stabilize the least one biological material of interest (and / or biologically-derived material of interest) present in the composition at temperatures of, for example, ambient temperature or higher.

[0058] In at least one embodiment, one or all of these methods may generally include introducing or contacting the biological material of interest and / or biologically-derived material of interest with the additive (or additive mixture) and the optional IDP to form the composition comprising the biological material of interest and / or biologically-derived material of interest, the additive (or additive mixture), and the optional IDP, thereby, for example, stabilizing the biological material of interest and / or biologically-derived material of interest.

[0059] Introducing or contacting may be performed under conditions that include suitable temperatures, pressures, and rates of introduction of the biological material of interest and / or biologically-derived material of interest with the additive (or additive mixture) and the optional IDP. The conditions may also include mixing the components of the composition by any suitable mixing process.

[0060] Prior to forming the composition, the components (e.g., the additive(s), the biological material of interest and / or biologically-derived material of interest, and the optional IDP) may be present in an aqueous material such as water or an aqueous buffer. The aqueous buffer may be any suitable aqueous buffer such as Tris or PBS. The components may be mixed.

[0061] The method may further include removing at least a portion of the liquid content of the composition. The liquid content may include the aqueous material. Removing at least a portion of the liquid content may include drying, or at least partially drying, the composition that includes the biological material of interest and / or biologically-derived material of interest, the additive (or additive mixture), and the optional IDP. Any suitable method of drying may be utilized such as, for example, air drying, evaporating (e.g., with a hot plate), dehydrating, desiccating, vacuum desiccating, vacuum drying, spray drying, freeze drying, spray-freeze drying, lyophilizing, foam drying, or combinations thereof, among other suitable methods.

[0062] The drying or at least partially drying provides the composition in the dry state, e.g., having a water content in a range from 0 wt% to about 15 wt% as described above.

[0063] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the present disclosure, and are not intended to limit the scope of embodiments of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used but some experimental errors and deviations should be accounted for.ExamplesI. The Effect of Sucrose Polymer Size on Glass Transition Temperature, Glass Former Fragility, and Water Retention during Drying

[0064] Properties of vitrified systems that may promote protection have been investigated with disaccharides. Glassy properties — such as glass former fragility, glass transition temperature, and water content — may be associated with protection. Protective sugar-glasses made up of similarly sized components, but with distinct chemical properties, modulate distinct properties that correlate with protection. However, what has not been investigated is the effect of protectant size on glassy properties, and how these properties correlate with protection.

[0065] Water is essential for all active life processes. Despite this, there are a number of organisms that survive prolonged desiccation. The vitrification hypothesis posits that such organisms survive desiccation by forming non-crystalline amorphous (vitrified) solids, often through the accumulation of protective disaccharides. In line with this theory, vitrification has been shown to be essential for desiccation tolerance in many organisms that survive extreme drying. However, not all vitrified materials are protective and certain physiochemical properties correlate with the protection in the glassy state. Furthermore, physiochemical properties that correlate with protection may vary depending on the chemical nature of similarly sized protectants. While the chemistry of protectants has been probed in relation to the protective properties they induce when vitrified, the effect of protectant size on glassy properties and protection during drying has not been investigated. Herein, the inventor studied the effect of the size of the sucrose compounds (monomeric sucrose and two-different sized polysucroses) on glassy properties associated with protection in the vitrified state.

[0066] Specifically monomer sucrose, and the polymers polysucrose 70 and polysucrose 400 (70 and 400 refer to the molecular weight of the polymers in kDa) were investigated. Using these three different-sized sucrose compounds, it was found that each of the glassy properties investigated — including enzyme protection, water content, glass transition temperature, and glass former fragility — were affected by the size of the sucrose compound. However, only one vitrified property, glass transition temperature, was found to correlate with protection during drying. This correlation was heavily dependent on the size of the sucrose compounds. Increased glass transition midpoint temperature was found to correlate positively with protection conferred by monomeric sucrose (p-value = 0.009, R2= 0.840), whereas the correlation was found to be bi-phasic for polysucrose 70, and the correlation was found to have an inverse relationship for polysucrose 400 (p-value = 0.120, R2= 0.490). The results indicated that the size of vitrifying protectants may have a profound effect on glassy properties as well as on how these properties correlate with protection in the dry state. Beyond desiccation tolerance, these findings provide insights for the development of new technologies for the stabilization of a biological material or a biologically-derived material in the dry state.

[0067] The vitrification hypothesis is one of the major hypotheses with regard to how desiccation-tolerant organisms survive extreme drying. This hypothesis posits that mediators of anhydrobiosis, such as trehalose and sucrose, work through the induction of highly viscous states which ultimately form non-crystalline, amorphous glass-like (vitrified) solids. The viscosity induced by these vitrifying mediators is thought to slow detrimental processes, such as protein unfolding and aggregation as well as membrane fusion. Ultimately, the formation of non-crystalline solids is thought to be protective in that it limits crystallization, which if left unchecked has been shown to damage intracellular components and cellular superstructure. Through empirical studies, vitrification has been established as an essential mechanism for desiccation tolerance in a number of systems. However, while vitrification is considered essential, it is not sufficient for desiccation tolerance. This is illustrated by the simple logic that any sufficiently heterogeneous system, such as a typical non-desiccation tolerant cell, will vitrify when dried, but not all cells are anhydrobiotic. This implies that there is some property or properties that distinguishes protective from non- protective glasses.

[0068] Previous work has identified several properties of vitrified systems that are linked to their protective capacity during desiccation. The first of these is the glass transition temperature (Tg), the temperature at which a vitrified material transitions from a glass-like state to a more rubbery phase. Additionally, other studies have demonstrated that increasing the Tg (anti-plasticization) of many dry systems has a stabilizing effect, whereas decreasing Tg (plasticization) of a vitrified system in many situations has a negative impact on protection.

[0069] Glass former fragility, which was classically described by Angell as log viscosity as a function of the temperature as it approaches Tg, may more intuitively be thought of as how a material increases in viscosity as its temperature approaches the glass transition. With regard to desiccation, a strong glass former would increase in viscosity as water is lost and continue to do so to the point of vitrification. Conversely, a fragile glass former would not increase in viscosity dramatically in the initial stages of drying, but rather would increase in viscosity only close to the point at which it vitrifies. Glass former fragility has been mostly studied in the context of cryogenic vitrification not dry vitrification.

[0070] Water content is an additional property of a vitrified material that has been considered with regard to glassy properties that could influence protection. Water within a vitrified system has been shown to be protective in and of itself, providing hydrogen bonds that may stabilize proteins and maintain membrane organization. Water within a vitrified system may also influence other glassy properties, and is typically considered as a plasticizer of vitrified systems, with the addition of water causing a decrease in Tg. Contrary to that, water has also been shown to have anti-plasticizing effects on some vitreous systems. Thus, while water may act to stabilize biological materials as well as a plasticizer / anti-plasticizer of vitreous systems, its direct correlation with protection in the glassy state remains mysterious.

[0071] Glassy properties of sugars vary from sugar to sugar. What has not been investigated is the effect of protectant size on glassy property(ies) — glass former fragility, glass transition temperature, and water content — and how these glassy property(ies) correlate with protection.

[0072] Here, this question was investigated using a series of mixtures that included distinct sucrose compounds (monomeric sucrose, polysucrose 70, and polysucrose 400) andvarying glycerol content. Sucrose, a di saccharide made of glucose and fructose connected through the C1-C2 glycosidic linkage, is a naturally occurring sugar found in many desiccation-tolerant plants, and its contributions during vitrification are believed to be the reasons for cell survival during desiccation. Because of its protective properties, sucrose is used in the food and pharmaceutical industry to stabilize food and drugs in the dry state. Polysucroses are sucrose polymers formed by a highly branched structure by copolymerization of sucrose with epichlorohydrin. In contrast to sucrose monomers, polysucroses are not found as naturally occurring excipients. Instead, polysucroses are used in the biological field for purposes such as separation technology and to study glomerular physiology. Polysucrose 70 and polysucrose 400 were selected to provide an extended range of size difference relative to sucrose and one another. Polysucrose 400 is a commonly available polymer size of sucrose. Polysucrose 70 was selected as an intermediate size between the monomer and polysucrose 400. The nomenclature polysucrose 70 and polysucrose 400 is used to indicate molecular weights of 70 kDa and 400 kDa respectively.

[0073] It was found that the size of the sucrose species (sucrose compounds) affected each glassy property investigated herein. However, only Tg correlated with protection under the conditions tested. Interestingly, the correlation between Tg and protection was found to vary between samples comprising different-sized sucrose compounds. Tg and protection was found to have a positive correlation for mixtures formulated with monomeric sucrose, but this correlation was found to be negative for mixtures comprising polysucrose 400 and found to be bi-phasic for samples comprising polysucrose 70.

[0074] These results provide insights into how size affects the properties and protection conferred by glassy systems.LA. Materials, Experiments, and MethodsI.A.l. Disaccharide-glycerol Mixtures

[0075] Three species of sucrose were commercially obtained: D-sucrose (342 g / mol) from Sigma-Aldrich (S0389-500G), Ficoll 70 (polysucrose 70; 70 kDa) and Ficoll 400 (polysucrose 400; 400 kDa) from Sigma-Aldrich (SC-257529A and 26873-85-8 respectively). Glycerol was sourced from Biobasic (GB0232).

[0076] To prepare mixtures, a 50 mL stock solution of 0.25 mM polysucrose 400 was made. Polysucrose 70 and monomeric sucrose solutions were made in reference to thepolysucrose 400 solution such that each solution contained the same number of sucrose monomers.

[0077] From these stock solutions, a series of solutions were prepared via the addition of glycerol. Sucrose content was varied from 100.0% to 87.5% by adding glycerol in weight by weight ratio (sugar 100.0%, sugar 97.5% + glycerol 2.5%, sugar 95.0% + glycerol 5.0%, sugar 92.5% + glycerol, 7.5% sugar 90.0% + glycerol 10.0%, and sugar 87.5% + glycerol 12.5%), where sugar refers to the sucrose compounds. Table 1-1 shows carbohydrate content (wt%) and molarity (mol / L) in each mixture containing one of three sucrose species (sucrose compounds): monomeric sucrose, polysucrose 70, and polysucrose 400, and increasing amounts of glycerol in weight by weight ratio.Table I- 1

[0078] These mixtures (samples) shown in Table 1-1 were then desiccated, as described below, prior to the investigations to provide desiccated mixture (samples).I. A.2. Sample Desiccation

[0079] 2 mL from each aqueous sample was dispensed into individual plastic weigh boats and desiccated using a speedvac (Savant SpeedVac SCI 10 with a Thermo OFP400 vacuum pump) for 16 hours. Each experiment was performed in triplicate. After the 16-hour desiccation, known weights of dried samples were loaded to pre-massed differential scanning calorimeter (DSC) aluminum hermetic pans (TA 900793.901) and aluminum hermetic lids (901684.901), while known weights of dried samples were loaded to pre-tared thermogravimetric analyzer (TGA) platinum crucibles (TA 957207.904). Tg values and the glass former fragility (m-index) values were derived from DSC thermograms (data not shown) and the percentage of remaining water content was calculated using TGA (data not shown).I.A.3. Calculation of glass transition temperature

[0080] Dried samples (3-4 mg) were placed in pre-weighed DSC pans and loaded in the DSC machine. The glassy state of the sugars (the monomeric sucrose and the two polysucroses) were obtained experimentally via quench-cooling. The sample was heated up from 30°C to 200°C at a rate of 10°C / min and then cooled to 0°C immediately. Subsequently, the sample was heated again by raising the temperature to 250°C at a rate of 10°C / min. Tg for each of the three sugar species was obtained as the midpoint of the onset and offset (endset) temperatures using Trios software. Average midpoint values were taken from the triplicates from each type of sucrose-glycerol mixtures.I.A.4. Calculation of water content in vitrified samples

[0081] Samples were run on a TGA (TGA5500 instrument) in 100 pL platinum crucibles (TA 952018.906). Crucibles were tared prior to each run and prior to loading the samples. Then the analysis was performed to determine a material’s thermal stability and its fraction of volatile components by monitoring the weight changes along with the temperature. The dried samples (10-11 mg) were placed in TGA plates and loaded in theTGA machine. The program was started at 30°C and then heated to 250°C at 10°C / min rate. Water loss from each sample of three species of sugars (the monomeric sucrose and the two polysucroses) was calculated in order to determine the remaining percentage of water content in the dried samples. Thermograms were used to calculate mass differences that occur after ~100°C but before the thermal denaturation at ~180°C. The Trios software “Smart Analysis” tool was used to identify the inflection point between mass loss events occurring from starting masses of samples and the mass of samples at the plateau. The average values were taken from the triplicates from each species of sucrose-glycerol mixtures.I. A.5. Lactate dehydrogenase (LDH) enzyme protection assay

[0082] LDH assays were performed according to the methodology described in Boothby TC et al., “Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation,” Mol Cell, 2017, 65: 975-984.e5.

[0083] The stock solutions of 25 mM Tris HC1 (pH 7.0), 100 mM sodium phosphate (pH 6.0), 2 mM pyruvate, and 10 mM NADH were prepared in bulk and stored at 4°C prior to the experiment. L-Lactate Dehydrogenase (LDH) was received from Sigma (SKU #10127230001). Prior to assay, LDH was diluted to a working concentration of 1 g / L using a pre-prepared stock solution of 25 mM Tris HC1 (pH 7.0). Experimental sugar-glycerol mixtures were formulated with LDH at a 1 : 10 (LDH: sugar-glycerol) ratio. For all samples, 90 pL from each sample was added with 10 pL of LDH into a 1.5 mL microcentrifuge tube and a total volume of 100 pL was split in half as a control and an experimental set. The experimental set was kept in the vacuum desiccator and the control set was kept in the refrigerator (4°C) for 16 hours. Experiment was performed with triplicates from each sample for each of the three different species of sucrose. After vacuum desiccation, control and experimental excipient mixtures were rehydrated using molecular grade water and brought to 250 pL total volume. A nanodrop instrument using quartz cuvettes was first blanked against the 980 pL of 100 mM sodium phosphate and 2 mM pyruvate solution. The nanodrop instrument was used to take absorbance readings every two seconds at the 340 nm wavelength (A340) for 60 seconds. Absorbance reading was taken by 10 pL of rehydrated control mixture combined with 10 pL of NADH in 980 pL of 100 mM sodium phosphate and 2 mM pyruvate solution. Then the absorbance reading was taken for the experimentalmixture using the same procedure as the controls. A ratio of experimental over control, multiplied by one hundred, will produce the percent protection of the experimental mixture. Likewise percent protection was calculated for each mixture of three species of sucrose. The average protection percentages were taken from the triplicates from each type of sucroseglycerol mixtures.I. A.6. Fragility (m-index) calculation

[0084] Trios software (TA Instruments TRIOS version #5.0.0.44608) was used to perform analysis of onset and endset points in order to calculate the glass former fragility (m-index). Calculations of the glass m-index were performed based on equations 10 and 14 as described in Crowley KJ et al., “The use of thermal methods for predicting glass-former fragility,” Thermochim Acta, 2001, 380: 79-93.

[0085] On a thermogram with a completed heating ramp to 250°C, the degradation peak, melt peak, and glass transition were identified. The Trios software built-in onset and endset analysis was used to determine the glass transition onset and endset and m-index was calculated using Crowley equation 10 where m is the alternative fragility parameter, AErgis the activation enthalpy of structural relaxation at Tg, R is the gas constant, Tg is the experimental glass transition temperature onset, and equation 14 where AE, / is the activation enthalpy for viscosity, R is the gas constant, Tg is the experimental glass transition onset temperature, Tg"'7is the experimental glass transition endset temperature, and constant is an empirical constant of 5. A mean of each set of replicates was obtained.I. A. 7. Statistical methods

[0086] One-Way ANOVA was utilized to compare the means in independent groups of sucrose-glycerol and polysucrose-glycerol mixtures in order to determine whether there is statistical difference between one or more mixtures. The Tukey post-hoc test was used to assess the significance of differences between pairs of individual group means. Rstudio (R 4.2.2) and the package ‘ggpubr’ was used to calculate the One-Way ANOVA, Tukey post- hoc test, p-value, and R2results. A p-value of less than 0.05 being one level of statistical significance (*), less than 0.01 being two levels of statistical significance (**), and less than 0.001 being three levels of statistical significance (***). All error values in bar graphs represent one standard error (SE) and 95% confidence interval (CI) levels in correlation graphs. Regression analysis was performed using the simple linear model for monomericsucrose and polysucrose 400 correlations. Polysucrose 70 produced a bi-phasic correlation, and for such relationships fitting data is less exacting and accurate, and therefore such data was not fitted.LB. Non-limiting ResultsI.B.1. Disaccharide-glycerol mixtures had varying levels of protection during desiccation

[0087] To begin to address the question of how protectant size influences glassy properties and protection, a series of mixtures were generated, each utilizing one of three sucrose species: monomeric sucrose, polysucrose 70, and polysucrose 400, and increasing amounts of glycerol (0.0, 2.5, 5.0, 7.5, 10.0, and 12.5% glycerol in weight by weight ratio) (Table 1-1). Samples were prepared such that the total number of sucrose monomers in each sample was 5 / I 03moles in 50 mL. Next assessed was the ability of each of these 18 mixtures to preserve the function of the labile enzyme lactate dehydrogenase (LDH) during desiccation. Previous reports have shown that LDH is sensitive to desiccation and that drying and rehydration of this enzyme results in loss in functionality. As demonstrated herein, LDH functionality may be preserved by co-incubation with protective vitrifying agents, including sucrose, prior to drying.

[0088] Pure monomeric sucrose provided 54.243% protection to LDH. A decrease in protection in monomeric sucrose mixtures was found with the addition of 7.5% glycerol, though this was not significant, and with each step-wise addition beyond that, significant decreases in protection were found relative to pure monomer sucrose. Pure polysucrose 70, which provides the same number of sucrose monomers (5* l 03moles / 50 mL) as the pure sucrose monomer sample, provided 79.883% protection to LDH during drying. For mixtures containing polysucrose 70, protection did not decrease with additions of glycerol up to 7.5%, after which there was a dramatic dropoff in protection. It was noted that the poly sucrose 70 biphasic result was different from the sucrose and polysucrose 400 linear results. As a result, these assays were repeated multiple times (3 technical replicates and 3 biological replicates) on different days. In all cases, the same results were obtained. Thus, it was concluded that the result with polysucrose 70 was surprising but real. For pure polysucrose 400, which provides the same number of sucrose molecules as the pure monomer sucrose and the other polysucrose sample, provided 59.037% protection to LDH subjected to desiccation. Mixtures containing polysucrose 400 differed further from mixtures containing monomericsucrose or polysucrose 70, in that protection was not affected by the addition of glycerol at any levels used in this study. Apart from that, there was an increase of protection with addition of 10.0% and 12.5% glycerol to the mixture, but this was not statistically significant relative to pure poly sucrose 400.

[0089] These data demonstrate that the size of the sucrose species affects its protective capacity, with pure poly sucrose 70 providing more protection to LDH than mixtures of pure monomeric sucrose or polysucrose 400 that include the same number of sucrose monomers. Furthermore, these results indicate additions of glycerol affect the protective capacity of different sized sucrose species in distinct ways.I.B.2. Water content varied between glasses that include different sucrose species, but did not correlate with the protective capacity

[0090] After determining that the protective capacity of sucrose in different polymerized states differed during drying, the inventor examined whether there are any glassy properties of these sugars that correlate with their ability to provide protection from desiccation. To investigate the correlation between properties of vitrified systems comprising differently- sized sucrose species and the protection, first assessed was whether or not the water content could account for these differences.

[0091] In the field of desiccation tolerance, it has been suggested that desiccation- tolerant organisms may be able to retain more water than their desiccation-sensitive organisms. A higher amount of water could help increase the proportion of proteome, membranes, and other vital cellular components that remain solvated within the system. Conversely, retaining too much water in a dry / drying state might also be deleterious, by allowing for reduced viscosity and increased alpha-relaxations and / or beta-relaxations. Thus, desiccation-tolerant organisms likely need to precisely tune the amount of water retained within their cells to maintain viability. Therefore, water content could have an impact on in vitro stabilization of biological material in a dry state.

[0092] To assess whether there are differences in water retention in vitrified systems comprising the different sucrose species and whether or not these differences correlate with differences in protection, each of the mixtures were tested using thermogravimetric analysis (TGA). TGA produced thermograms with two distinct areas of sample mass loss, the first centered around 100°C and the second around 200°C (data not shown). These mass lossescorresponded to the evaporation of water from the samples and the degradation of the sample at high temperature, respectively. Water contents of the 18 dry mixtures ranged from 3.141% to 12.172% by weight.

[0093] Pure dry monomeric sucrose (100% sucrose) contained 5.636% retained water by weight, while pure dry polysucrose 70 (100% polysucrose 70) and pure dry polysucrose 400 (100% polysucrose 400) showed significantly higher water content (12.172% and 7.585% by weight, respectively). The water content of the dried sucrose monomer-glycerol mixtures did not change significantly with the addition of glycerol into the mixtures. For polysucrose 70, water content significantly decreases upon the addition of 12.5% glycerol. Polysucrose 400 displayed a non-monotonic response to the glycerol addition, with an initial decrease, followed by an increase, a decrease, and then a further increase.

[0094] After determining the distinct water retentive behaviors in glasses comprising different sucrose species, the relationship between water content and LDH enzyme protection was assessed. For monomeric sucrose mixtures, a negative (albeit statistically insignificant) relationship between protection and water content was found as shown in FIG. 1. This differed from the series of mixtures that included polysucrose 70, which showed a statistically significant bi-phasic relationship between water retention and protection, with samples at low average water contents (7.940%) being the least protective and samples with the most retained water (10.979%) conferring the most protection (data not shown). Finally, mixtures containing polysucrose 400 differed further, displaying a slightly positive, but statistically insignificant, trend between retained water and protection.

[0095] T aken together, the data demonstrates that water was retained in vitrified sucrose samples differently depending on the polymer state of sucrose. However, the data indicates that retained water was not a strong predictor of protection for vitrified sucrose at any size of the sucrose species tested.I.B.3. Glass transition temperature correlated with the protective capacity of sucrose species-glycerol mixtures in distinct ways

[0096] After determining that water retention is a poor indicator of the protective capacity of sucrose species-glycerol glasses, the Tg was investigated to determine whether it might be a good predictor. Tg is the temperature at which a glassy material will begin to transition into a more rubbery phase. Even small amounts of an additive, such as glycerol,may lead to changes in the Tg of a vitrifying material, which may correlate with changes to protective capacity.

[0097] The inventor investigated whether the Tg differs between sucrose speciesglycerol mixtures comprising the different-sized sucrose species and whether Tg is a good predictor of protection conferred by sucrose species. To this end, DSC was performed on the dried sucrose species-glycerol mixtures to obtain Tg onset, midpoint, and endset values (data not shown).

[0098] The Tg of pure dry sucrose (100% sucrose) was found to be significantly lower than that of pure dry poly sucrose 70 (100% poly sucrose 70) and pure dry poly sucrose 400 (100% polysucrose 400). For example, the midpoint Tg for the pure dry monomeric sucrose was measured to be 73.310°C, while the Tg of the pure dry polysucrose 70 and the pure dry polysucrose 400 were 126.603°C and 130.205°C, respectively (data not shown). The addition of glycerol to all three sucrose species tested was determined to have a similar plasticizing effect, however the range of observed plasticization varied (data not shown). For example, addition of glycerol was found to decrease the Tg of monomeric sucrose from 73.310°C (pure) to 40.907°C (87.5% sucrose), a range of 32.403°C. This same range for the dry sample to the 87.5% polysucrose 70 and 87.5% polysucrose 400 was determined to be 64.183°C and 69.282°C, respectively (data not shown).

[0099] Next evaluated was the relationship between protective capacity and the Tg onset, the Tg midpoint, and the Tg endset for each sample / mixture. Results are shown in FIGS. 2, 3, and 4. Regardless of whether onset, midpoint, or endset was considered, the same trends were found. For monomeric sucrose, a statistically significant, strong positive correlation between protection and Tg was found. This result was in stark contrast to polysucrose 400, which showed a statistically insignificant negative correlation between protection and Tg. For polysucrose 70, a statistically significant bi-phasic relationship was found between protection and Tg, where mixtures with a Tg average onset lower than 60.010°C, a Tg average midpoint lower than 69.771 °C, and a Tg average endset lower than 79.532°C provided essentially no protection, while the highest levels of protection were found for mixtures with a Tg average onset higher than 97.052°C, a Tg average midpoint higher than 104.942°C, and a Tg average endset higher than 112.834°C (data not shown).

[0100] Taken together, these results indicate that the size of the sucrose species influences the Tg of the mixtures, with monomeric sucrose transitioning at a much lower temperature than either polysucrose 70 or polysucrose 400. Furthermore, while all differently-sized species of sucrose were found to plasticize with the addition of glycerol, monomeric sucrose plasticized over a much smaller range than either of the polysucroses. Finally, and most strikingly, correlations were found between Tg and protection. However, protection provided by sucrose in different polymerized states (monomeric form or polysucroses) correlated with Tg in dramatically distinct ways (e.g., positive versus negative correlations).I.B.4. Glass former fragility of sucrose-glycerol mixtures did not correlate with enzyme protection

[0101] Because changes to Tg may influence the glass former fragility (m-index) of a material, the inventor examined whether glass former fragility also differed between the samples as well as whether there is a correlation between glass former fragility and protection during drying for mixtures comprising different-sized sucrose species. For pure samples lacking glycerol, an increase in glass former fragility was found as the size of sucrose species increased (data not shown). For each of the three different sucrose species, a decrease in glass former fragility with the addition of 2.5% glycerol was found, which was statistically insignificant for monomeric sucrose, but statistically significant for both sucrose polymers (data not shown). With the rest of the additions of glycerol (up to 12.5%), there were mild decreases in glass former fragility found for the polysucrose 400 mixtures, but these were slight and almost always statistically insignificant. Both the mixtures containing monomeric sucrose or polysucrose 70, however, showed non-monotonic behavior regardless of size of the sucrose species (data not shown).

[0102] Next assessed was the relationship between the glass former fragility of the additive mixtures and protection of LDH. This correlative analysis produced only statistically insignificant trends as shown in FIG. 5, indicating that glass former fragility is not a strong predictor of protection. Polysucrose 70, which still showed a statistically significant bi-phasic relationship between glass former fragility and protection, with samples with low average m-index (42.142) being the least protective and samples with higher average m-index value (56.126) conferring the most protection (data not shown).

[0103] Taken together these results suggest that glass former fragility is not a mechanistic driver of desiccation protection conferred by sucrose, since the glass former fragility of pure monomeric sucrose is significantly lower than that of pure polysucrose 70 and 400, but monomeric sucrose does not offer more protection than polysucrose 70 or 400. Furthermore, the addition of glycerol decreased glass former fragility in all cases, but again, there was no strong trend between this decrease in fragility and augmented protection.I.C. Non-limiting Discussion

[0104] Anhydrobiotic organisms survive during near complete water loss through different physiological and behavioral adaptations against the extreme environmental conditions. The vitrification hypothesis, a central hypothesis in the desiccation tolerance field, posits that the accumulation of sugars and other mediators of desiccation tolerance induce highly viscous states which ultimately form into amorphous solids during dehydration in order to protect the cells via the reduction of molecular motion and relaxations. While vitrification is considered to be essential for living systems to survive during desiccation, it is not sufficient for survival as virtually all sufficiently heterogeneous systems that are not desiccation tolerant will vitrify. A mixture that is not sufficiently heterogeneous may undergo crystallization rather than become vitrified. Crystallinity within a vitrified material may influence its protective capacity.

[0105] Much effort has been spent in attempting to identify properties that distinguish protective from non-protective vitrifying systems. Chief among these properties are Tg, glass former fragility, and water content. While at the organismal level, both an increase in Tg and a decrease in glass former fragility are found in desiccation-tolerant life stages versus desiccation-sensitive life stages of anhydrobiotic organisms. However, in vitro disaccharides of similar size, but with distinct chemical properties confer protection which correlates with either Tg or glass former fragility. However, when considering how the physics and material properties of a vitrified system are augmented, it is important to consider not just the chemical properties of the system’s constituents, but also their size.

[0106] Herein, a series of vitrifying mixtures that included three different sizes of sucrose species (monomeric, polysucrose 70, and polysucrose 400) and glycerol was tested. With regard to enzyme protection, water content, Tg, and glass former fragility, it was determined that the size of the sucrose species has an influence on all three of theseproperties. However, only Tg correlated with protection in the monomeric sucrose system under the conditions tested. In addition, it was found that, while the correlation between the Tg of mixtures that included monomeric sucrose had a positive correlation with protection, this relationship was bi-phasic for polysucrose 70, and there was an inverse or negative correlation between Tg and protection conferred by mixtures containing polysucrose 400.

[0107] Water plays critical roles in all active life processes as well as stabilizing diverse biological materials of interest (e.g., biological macromolecules). The proper water content for stabilizing different biological macromolecules remains to be determined, but one could envision that retaining too little water would be detrimental in that this would limit the number of stabilizing hydrogen bonds that could be made with a protein or other cellular materials. However, there might also be a penalty for retaining too much water, where excess residual water could be insufficient to fully stabilize a protein, while still allowing for some degree of molecular motion (alpha- and beta-relaxations) which could promote protein unfolding and / or aggregation.

[0108] While the water content did not correlate with protection for any of the three sucrose species, the total amount of retained water did vary, with polysucrose 70 retaining the most water, followed by polysucrose 400, and finally monomeric sucrose. The results therefore indicate that the size of the sucrose species influences water retention, but do not support the notion that water content is a property strongly predictive of sucrose-mediated protection during drying. These results do not rule out the possibility that water content influenced by species of sucrose-size might correlate with protection for other vitrifying mediators of desiccation tolerance.

[0109] Beyond stabilizing biological macromolecules, water may act as a plasticizing agent for many vitrified systems. However, as described herein, it was found that water levels are positively correlated with the Tg of poly sucrose 70 mixtures, but not with the Tg of mixtures comprising monomeric sucrose or polysucrose 400 (data not shown). This indicates an anti-plasticization effect of water on the polysucrose 70 system, which is rarely observed.

[0110] Interestingly, there were correlations between Tg and LDH protection for the sucrose-glycerol mixtures. A surprising result here is that the correlations were different between each size species of sucrose, with the Tg of monomeric sucrose correlatingpositively with protection, being bi-phasic for polysucrose 70, and being negatively correlated for polysucrose 400.[OHl] In general, an increase in cross-linking decreases the mobility of the polymer and the free volume leading to an increase in Tg. However, it was found that increasing the Tg of a glassy system comprising sucrose monomers enhances protection, while increasing the Tg of a vitrified system comprising larger polymers (e.g., polysucrose 400) decreased protection. This result may be explained by the higher water content in polysucrose 70 and polysucrose 400 compared to monomeric sucrose.

[0112] Previous work established that there is no correlation between glass former fragility and protections conferred by monomeric sucrose. However, the effect of polymer size on glass former fragility and the correlation of glass former fragility and protection for sucrose species had not been investigated until now. The results described herein show the differences in glass former fragility between samples of pure monomeric sucrose, polysucrose 70, and polysucrose 400. However, there were no clear and significant trends with regard to protection and glass former fragility among any of the series of mixtures.

[0113] Overall, the study described herein provides insights into the influence of size of the sucrose species on protection, water retention, Tg, and glass former fragility. The size of the sucrose species influenced all of these properties, but in terms of importance in predicting protection during drying, only Tg was found to be significant for the samples containing the sucrose monomer. The fact that the type of correlation (e.g., positive vs. negative correlation) that Tg and protection displayed for mixtures comprising differentsized sucrose species indicates that the size of the sucrose species influences glassy properties and that at least some of these properties confer protection.

[0114] These results provide insights into how organisms may help to stabilize their cells and sensitive cellular components during desiccation. Beyond desiccation, understanding how vitrification properties may be tuned to enhance protection has implications in the biomedical and pharmaceutical field for storage of sensitive biologies without the need for the cold-chain.II. Water Content Transition Temperature, and Fragility Influence Protection and Anhydrobiotic Capacity

[0115] Water is essential for metabolism and all life processes. Despite this, many organisms distributed across the kingdoms of life survive near-complete desiccation or anhydrobiosis. Increased intracellular viscosity, leading to the formation of a vitrified state is necessary, but not sufficient, for survival while dry. Exactly what properties of a vitrified system make it desiccation-tolerant or desiccation-sensitive are unknown.

[0116] The inventor analyzed eighteen (18) different in vitro vitrified systems — comprising one of three protective disaccharides (trehalose, sucrose, or maltose) with or without glycerol (as an example polyol) — and quantified their enzyme-protective capacity and their material properties in a dry state. Protection conferred by mixtures containing maltose correlated strongly with increased water content, increased Tg, and reduced glass former fragility, while the protection of glasses formed with sucrose correlates with increased Tg and the protection conferred by trehalose glasses correlated with reduced glass former fragility. Thus, in vitro, different vitrified sugars confer protection through distinct material properties. Next examined was the material properties of a dry desiccation-tolerant and desiccation-intolerant life stage from three different organisms. The dried desiccation- tolerant life stage of all organisms was determined to have an increased Tg and reduced glass former fragility relative to its dried desiccation intolerant life stage. These results suggest that, in nature, organismal desiccation tolerance relies on a combination of various material properties. The study presented herein advances the understanding of how protective and non-protective glasses differ in terms of material properties that promote anhydrobiosis. This knowledge presents avenues to develop novel stabilization technologies for pharmaceuticals that currently rely on the cold-chain.

[0117] As organisms dry, they face a number of physical and chemical changes to their cellular environment. As water is lost, cellular constituents are concentrated, molecular crowding increases, pH and ionic concentrations change, and osmotic pressure increases. These physiochemical changes lead to detrimental perturbations such as protein unfolding, aggregation, and membrane leakage. Drying is not an all-or-nothing process and these changes as well as the perturbation they induce, occur along a continuum, with some perturbations occurring earlier as an organism is dehydrating while others manifest later, once more substantial amounts of water have been lost. How organisms survive desiccation is one of the enduring mysteries of organismal physiology.

[0118] Historically, anhydrobiosis has been thought to be mediated, at least in part, through the concentration of cellular constituents until these constituents solidify into a vitrified material (a glass). In this hypothesis, known as the ‘vitrification hypothesis,’ glasses slow physical and biochemical change, making them natural promoters of desiccation tolerance. Within the anhydrobiosis field, vitrification is considered a necessary process for desiccation tolerance.

[0119] However, a major shortcoming of the vitrification hypothesis is the observation that essentially every biological, or sufficiently heterogeneous, system will vitrify when dried, regardless of whether it is desiccation-tolerant or -sensitive. This observation implies that while vitrification is necessary, it is not sufficient for desiccation tolerance and that there must be some property, or properties, that distinguishes a protective from a non- protective vitrified state. The properties distinguishing a desiccation-protective glass from a non-protective glass are not currently fully understood.

[0120] Previous studies have identified that small additions of glycerol changes the enzyme-protective capacity of trehalose. However, the material properties of these mixtures and how they correspond with changes in the level of protection have not been investigated. To address this gap in knowledge, the hypothesis that the enzyme-protective capacity of disaccharide-glycerol mixtures during desiccation correlates with their material properties was tested. These properties include water content, Tg, and glass former fragility.

[0121] Water content is a property of vitrified materials that has been implicated in survival during extreme desiccation. Water content refers to the weight percent of water in a desiccated sample. The water content can be measured by taking the starting mass of a desiccated sample and dividing by the mass of the sample after heating to a temperature sufficient to evaporate residual water. While hydrated, water molecules within a cell are able to solvate and then stabilize sensitive intracellular components. By retaining more water, it has been proposed that a vitrified material could prevent damage to sensitive intracellular components by maintaining hydration shells around them. Additionally, residual water is implicated in several other proposed mechanisms of desiccation tolerance such as water entrapment, preferential exclusion, and the anchorage hypothesis.

[0122] Tg is the temperature at which a vitrified solid transitions from a glassy to a rubbery state. Increases or decreases to the Tg of a vitrified material occur through theinclusion of an additive. Increasing the Tg of a vitrified material may increase the shelf-life of sensitive proteins in a dry state. At the organismal level, it has been demonstrated that many anhydrobiotic organisms survive heating up to, but not beyond, their Tg. This suggests that anhydrobiotic organisms rely on being in a vitrified state and the production of small molecules which increase Tg may be an effective strategy for increasing desiccation tolerance, or at least for increasing thermal tolerance while desiccated.

[0123] Finally, glass former fragility is a property of vitrified materials that may promote desiccation tolerance. This property distinguishes strong glass forming materials, whose viscosity increases steadily well before the liquid-to-solid transition, from fragile glass forming materials, whose viscosity increases slowly at first but then rises abruptly at the onset of vitrification. It should be noted that in this context, glass fragility / strength does not refer to the brittleness of a vitrified material, but rather to how the viscosity of the material changes as it approaches the point of vitrification. It is hypothesized that strong glass forming materials confer more protection during desiccation than their more fragile counterparts. This hypothesis relies on the logic that a fragile glass forming material will not produce a sufficiently viscous state to slow down or prevent perturbation such as protein unfolding and aggregation until it is too late. Conversely, a strong glass forming material will increase in viscosity and provide protection along the continuum of drying.

[0124] To empirically test which, if any, of these three material properties correlate with desiccation tolerance, the inventor first used a panel of simple reductive systems, each comprising two mediators of desiccation tolerance - (1) a disaccharide including maltose, sucrose, or trehalose and (2) glycerol as an example polyol different from the disaccharide. By comparing the measured material properties (water content, Tg, and glass former fragility) of the disaccharide-glycerol glasses with their in vitro enzyme-protective capacities, the inventor found that there was not a strict pattern in terms of the correlation of material properties to protection that all disaccharides follow (data not shown). Instead, it appeared that each disaccharide may have a particular material property that is best correlated with its enzyme-protective capacity.

[0125] This analysis was extended into three organismal systems, each of which has a desiccation-tolerant and desiccation-intolerant life stage(s). The inventor found that, while the enzyme-protective capacity of the in vitro systems tended to correlate with a singlematerial property, the in vivo systems showed both increased Tg and reduced glass former fragility are hallmarks of desiccation tolerance. These results suggest that anhydrobiosis may modulate multiple material properties to promote organismal desiccation tolerance.

[0126] The extension of the mechanistic understanding of the principles underlying desiccation-tolerance provide insights into how organisms can cope with changing, often extreme, environments. The inventor’s findings also help to address the decades-long paradox that some glasses are protective, and others not, by providing insights into material properties that promote the protective capacity of different vitrifying mixtures. The inventor’s work described herein, identifies increased glass transition temperature and glass former fragility as major considerations in the development of technologies for the dry preservation of pharmaceuticals and the engineering of crops that are better able to withstand climate change and extreme weather.ILA. Materials, Experiments, and MethodsII. A.1. Disaccharide-glycerol mixtures

[0127] D-Maltose monohydrate was sourced from Caisson Labs (M004-500GM). D- Sucrose was sourced from Sigma-Aldrich (S0389-500G). D-Trehalose dihydrate was sourced from VWR (VWRB3599-1KG). Glycerol was sourced from Biobasic (GB0232). Mixtures of each disaccharide and glycerol were made in 25 mM Tris at pH 7.0. Individual masses of each component were formulated (weight by weight) to additively produce mixtures of 10 g / L.

[0128] Stock solutions of the disaccharide (D-maltose, D-sucrose, and D-trehalose) were prepared. From these stock solutions, a series of solutions were prepared via the addition of glycerol. Disaccharide content was varied from 100.0% to 87.5% by adding glycerol in weight by weight ratio (disaccharide 100.0%, disaccharide 97.5% + glycerol 2.5%, disaccharide 95.0%+glycerol 5.0%, disaccharide 92.5% + glycerol 7.5%, disaccharide 90.0% + glycerol 10.0%, and disaccharide 87.5% + glycerol 12.5%). Table II- 1 shows disaccharide content (%) and molarity (mol / L) in each mixture containing one of three sucrose species: monomeric sucrose, polysucrose 70, and polysucrose 400, and increasing amounts of glycerol in weight by weight ratio.Table 11-1

[0129] These mixtures (samples) shown in Table II- 1 were then desiccated, as described below, prior to the investigations to provide desiccated mixture (samples).II. A.2. Sample Desiccation

[0130] Samples were desiccated using a speedvac (Savant SpeedVac SCI 10 with a Thermo OFP400 vacuum pump) for 16 hours. Prior to desiccation, 1 mL aliquot samples were dispensed into individual plastic weigh-boats (for aqueous samples) and at least 200 mg of organism samples were similarly loaded into individual plastic weigh-boats. The greater surface area of the weigh-boat, as opposed to Eppendorf tubes, allowed for even desiccation of the entire sample, which reduced noise on the DSC. After the 16-hour desiccation, DSC samples were transferred to pre-massed pairs of DSC aluminum hermeticpan and aluminum hermetic lids (TA 900793.901 and 901684.901, respectively) while TGA samples were transferred to pre-tared platinum crucibles (TA 957207.904), and XRD samples were kept in the desiccation weigh-boats. DSC sample masses were determined after the sample was sealed within the pan and lid.II. A.3. Single crystal X-ray diffractometry

[0131] Powder diffraction patterns for the samples were measured at 20°C on a Bruker SMART APEX II CCD area detector system equipped with a graphite monochromator and a Mo K fine-focus sealed tube operated at 1.2 kW power. The dried samples were rolled into a ball of approximate diameter 0.5 mm, mounted on a goniometer head using a glass fiber, and centered using the APEX2 software. The detector was placed at 6.12 cm during the data collection. Four frames of data were collected at four different sets of angles with a scan width of 0.2° and an exposure time of 3 min per frame. The frames were integrated using the APEX2 program. The measured powder diffraction images were integrated, and the data were plotted in the 5 to 50° 29. All analysis was performed using the APEX3 Software Suite V2017.3-0, Bruker AXS Inc.: Madison, WI, 2017.II. A.4. Lactate dehydrogenase (LDH) enzyme protection assay

[0132] LDH assays were performed using a combination of methodologies described in K. Goyal et al., “LEA proteins prevent protein aggregation due to water stress,” Biochem. J, 388 (2005) 151-157; and T.C. Boothby et al., Mol. Cell, 65 (2017) 975-984.e5.

[0133] Briefly, stock solutions of 25 mM Tris HC1 (pH 7.0), 100 mM sodium phosphate (pH 6.0), and 2 mM pyruvate prepared in bulk and stored at room temperature prior to assay. In addition, 10 mM NADH was also prepared prior to the assay and then stored at 4°C. L- Lactate Dehydrogenase (LDH) was sourced from Sigma (SKU #10127230001) and is supplied in ammonium sulfate at a pH of approximately 7. Prior to assay, LDH was diluted to a working concentration of 1 g / L. Experimental disaccharide-glycerol mixtures were formulated with LDH at a 1: 10 (LDH:disaccharide-glycerol) ratio. Enough solution was prepared for three test excipient replicates and three control replicates each with a total volume of 50 pL. Each experimental and control mixture were aliquoted into a 1.5 mL microcentrifuge tube. The test mixtures were then vacuum desiccated for 16 hours with controls kept refrigerated at 4°C. After vacuum desiccation, control and test excipient mixtures were brought to 250 pL total volume. Absorbance readings at 340 nm wavelengthwere taken every two seconds for 60 seconds with a quartz cuvette on a Thermo Scientific NanoDrop One (Thermo Scientific 840274200) spectrophotometer. A 100 mM sodium phosphate and 2mM pyruvate solution was used as a blank. For control samples and experimental samples, 10 pL of control mixture or 10 pL of experimental sample mixture were combined with 10 pL of NADH and 980 pL of the lOOmM sodium phosphate, 2mM pyruvate solution and then were measured.

[0134] This same procedure was used to measure the A340 of the experimental and control mixtures. A340 was plotted as a function of time and the slope of the linear portion of this plot calculated. A ratio of experimental over control slope, multiplied by one hundred, was taken to produce the percent protection of the experimental mixture.II. A.5. Thermogravimetric analysis

[0135] Samples were run on a TGA (TGA5500) instrument in 100 pL platinum crucibles (TA 952018.906). Crucibles were tared prior to each run and prior to sample loading. Crucibles were loaded with between 5 mg and 10 mg of sample mixture. Each sample was heated from 30°C to 220°C at a 10°C per minute ramp.

[0136] Determination of water loss was conducted using Trios software (TA Instruments, TRIOS version #5.0.0.44608). Thermograms were used to calculate starting masses of samples and the mass of samples at the plateau that occurs after ~100°C but before the thermal denaturation at ~200°C. The Trios software “Smart Analysis” tool was used to identify the inflection point between these two mass loss events.II. A.6. Differential scanning calorimetry

[0137] Samples were run on a TA DSC2500 instrument with Trios software (TA Instruments TRIOS version #5.0.0.44608). Analysis of DSC output was performed using Trios software. The heating run included loading at ambient temperatures of 20°C, then cooling at a rate of 13.2°C per minute ramp to -10°C where the temperature was equilibrated at -10°C ,and then heating using a 10°C per minute ramp to 220°C.II.A. 7. Fragility (m-index) calculation

[0138] Trios software (TA Instruments, TRIOS version #5.0.0.44608) was used to perform analysis of the DSC data. Calculations of glass former fragility (m-index) were performed based on equations 10 and 14 proposed by K.J. Crowley et al., “The use of thermal methods for predicting glass-former fragility,” Thermochim. Acta, 380 (2001) 79-93. On a thermogram with a completed heating ramp to 220°C, the degradation peak, melt peak, and glass transition were identified. The Trios software built-in Onset and Endset analysis was used to determine the glass transition onset and glass transition offset (endset). Offset and endset, when used with respect to Tg, are used interchangeably herein.

[0139] The software-identified glass transition onset and offset was used to calculate the m-index using Crowley’s equation 10 (reproduced as Eq. 1, below) where m is the alternative fragility parameter, AErgis the activation enthalpy of structural relaxation at Tg, R is the gas constant, Tg is the experimental glass transition temperature onset, and equation 14 (reproduced as Eq. 2, below) where AE^ is the activation enthalpy for viscosity, R is the gas constant, Tg is the experimental glass transition onset temperature, Tg"'7is the experimental glass transition offset temperature, and constant is an empirical constant of 5. A mean of each set of replicates was obtained.II.A.8. Selected organisms for in vivo assays

[0140] Artemia franciscana. Caenorhabditis elegans, and Saccharomyces cerevisiae were all selected based on availability of access to both desiccation-sensitive and desiccation-tolerant life stages.

[0141] Artemia franciscana adults (#1) and cysts (#11) were acquired from Northeast Brine Shrimp, LLC. A. franciscana adults were separated from culture media by tube-top filtration using a pluriSelect 200 pm pluriStrainer (#43-50200-03) prior to desiccation.

[0142] Caenorhabditis elegans non-dauered daf-2 strains were cultured in S Medium at 16°C and fed with E. coli OP50 as described in J. A. Lewis, J.T. Fleming, “Basic culture methods,” Methods Cell Biol., 48 (1995) 3-29. Dauered pre-conditioned daf-2 strain worms were cultured identically to non-dauered worms but when the initial aliquot of E. coli OP50 were consumed an additional five days of incubation at 25°C was given to starve the culture and induce dauer arrest. Dauer worms were then placed on non-spotted agar plates in a 25°C and 95% relative humidity atmosphere for 3 additional days to accomplish pre-conditioning. Both non-dauer and pre-conditioned dauer C. elegans sample sets were separated from media and residual OP50 food stock by tube-top filtration using a pluriSelect 200 pm pluriStrainer (#43-50200-03) prior to desiccation.

[0143] Saccharomyces cerevisiae strain BY4742 was cultured aerobically in YPD media at 30°C (as described in YPD media, Cold Spring Harb. Protoc., (2010) db.recl2315). After nine hours, samples were taken representing the logarithmic growth stage yeast. The remaining culture was allowed to grow for an additional five days to ensure confluent growth in the stationary phase. Both logarithmic and stationary phase samples were pelleted by centrifugation, media was decanted, and a water wash of cell pellets was performed prior to desiccation.II.A.9. Statistical methods

[0144] One-way ANOVA and Tukey post-hoc test was used for all pairwise comparisons. A p-value of less than 0.05 being one level of statistical significance (*), less than 0.01 being two levels of statistical significance (**), and less than 0.001 being three levels of statistical significance (***). All error values represent 95% confidence intervals.

[0145] Student’s T-Test was used to compare statistical differences between desiccation-tolerant and desiccation-sensitive life stages. A p-value of less than 0.05 being one level of statistical significance (*). All error values represent 95% confidence intervals. II. A.10. Resources TableII.B. Non-limiting ResultsII.B.1. Disaccharide-glycerol mixtures vitrified when dried

[0146] To address which material properties correlate with enzyme-protective capacity in a vitreous state, 18 different glass forming mixtures comprising one of three disaccharides (maltose, sucrose, or trehalose) and varying amounts of glycerol were generated. Material properties of disaccharide-glycerol mixtures and how they correlate with changes in the level of protection have not been investigated. Furthermore, it is not known to what extent additions of glycerol will influence the material and enzyme-protective properties of other disaccharides or how these two properties are linked.

[0147] Maltose is a reducing disaccharide that includes two glucose molecules joined by an u( l ^4) bond. Sucrose is a non-reducing disaccharide formed by the glycosidic linkage between Cl of a glucose molecule to the C2 of a fructose molecule. Trehalose is a non-reducing disaccharide formed through the (1-1) glycosidic linkage of two glucose molecules.

[0148] Mixtures containing 100%, 97.5%, 95%, 92.5%, 90%, and 87.5% of a single disaccharide (maltose, sucrose, or trehalose), combined with glycerol (weight % by weight % with glycerol) were created (Table II-l). Mixtures were dried overnight in a vacuum desiccator for 16 hours to produce glasses.

[0149] To ensure that these mixtures vitrified, rather than crystallized, each sample’s powder diffraction pattern was determined by powder X-ray diffraction (XRD) using Mo Ka radiation. The integrated plots of the 18 vitrifying samples were determined to contain a nearly identical broad absorption, and did not reveal any sharp peaks (data not shown). The absence of sharp peaks indicated that the samples were glassy and without crystallinity upon drying or desiccating, therefore indicating that the additive mixture vitrifies.

[0150] As a comparison, powder diffraction data was also measured for a desiccated sample of D-(+)-glucose, which is known to crystallize when dried. In contrast to the XRD patterns measured for the disaccharide-glycerol samples described herein, the diffraction pattern for the comparative glucose exhibited a large number of distinct, closely spaced peaks due to the crystalline nature of this sample (data not shown).

[0151] These results indicate that maltose, sucrose, or trehalose by themselves or in conjunction with varying amount of glycerol vitrified when dried under the drying regime used here (see Methods, above).II.B.2. Disaccharide-glycerol mixtures had varying levels of enzyme -protection during desiccation

[0152] To address the question of which property(s) of a vitrified solid correlate with enzyme-protective capacity during desiccation, the ability of the 18 disaccharide-glycerol mixtures to protect the enzyme lactate dehydrogenase (LDH) during desiccation was assessed (data not shown). LDH is sensitive to desiccation, and drying and rehydration of this enzyme results in -95-99% loss in functionality. By taking the ratio of the enzymatic activity of rehydrated versus control LDH, it was found that the protection of desiccatedLDH varied significantly between different disaccharide-glycerol mixtures (data not shown).

[0153] For the maltose-glycerol mixtures, the 100% maltose sample was determined to confer the highest level of protection, while the 87.5% maltose sample was determined to confer lowest level of protection. No significant difference in protection was found until the percentage of maltose in mixtures reached 92.5%, after which point the protection steadily decreased (data not shown).

[0154] For the sucrose-glycerol mixtures, and similar to the maltose-glycerol mixtures, the 100% sucrose sample was determined to confer the highest level of protection, while the 87.5% sucrose sample was determined to confer lowest level of protection. However, unlike the maltose samples, the sucrose samples rapidly lost enzyme-protective capacity with statistically significant decreases in protection being found upon the first (2.5%) addition of glycerol (data not shown).

[0155] The trehalose-glycerol mixtures were determined to differ from both the maltose and the sucrose mixtures in that the 97.5% trehalose sample was determined to confer the highest level of protection (data not shown). Additionally, while mixing of maltose or sucrose with glycerol resulted in decreases in protection, additions of glycerol to trehalose were determined to have a nonmonotonic relationship with protection (data not shown).

[0156] These results demonstrate that different disaccharide and glycerol mixtures provided varying levels of protection to LDH during desiccation and rehydration, with trehalose responding in a non-monotonic fashion, maltose decreasing in enzyme-protective capacity in a linear fashion, and protection conferred by sucrose decreasing exponentially as a function of glycerol content.II.B.3. Water content correlated with the enzyme-protective capacity of maltose-glycerol, but not trehalose-glycerol or sucrose-glycerol, mixtures

[0157] To assess which properties of a vitrified system correlate with protection, first assessed was whether or not the water content could account for these differences. To assess whether the differences in protection found for the vitrified samples corresponded to the amount of water they retain, each of the mixtures were tested using thermogravimetric analysis (TGA) (data not shown). The water contents of the 18 dry mixtures ranged from 10.52% to 0.81%. The pure dry trehalose sample (100% trehalose) contained about 8%water content. The pure dry sucrose sample (100% sucrose) and the pure dry maltose sample (100% maltose) were determined to contain approximately the same water content (5-6%), which is less water than the pure dry trehalose sample. For the maltose-glycerol mixtures, it was determined that the amount of retained water decreased with each addition of glycerol. In contrast, it was determined that water content did not vary significantly in any sucroseglycerol mixture. It was also found that the trehalose-glycerol mixtures only demonstrated statistically significant decreases in water content after the addition of 5% glycerol (data not shown).

[0158] After determining the distinct water retentive behaviors in dry disaccharideglycerol mixtures, the relationship between water content and protection was investigated. For each disaccharide, a positive trend between enzyme-protection and increasing water content was determined. However, the correlation between these properties varied significantly between the di saccharides. This correlative analysis produced an R2value of 0.96 (p-value = 0.00052) for maltose-containing samples, an R2value of 0.13 (p-value = 0.48) for sucrose-containing samples, and R2value of 0.28 (p-value = 0.28) for trehalose- containing samples (FIG. 6, some data not shown). For maltose-containing samples / mixtures, these results indicate that the amount of retained water is a good indicator of the enzyme-protective capacity in the dry state. However, the amount of water in mixtures made of dry trehalose or sucrose and glycerol was determined to be a poor indicator of enzyme-protective capacity (FIG. 6).

[0159] Precise determinations of water content using TGA may be complicated by an overlap between the offset of water evaporation and the onset of deterioration of a material. The inventor found that there was no such overlap for maltose-containing samples and sucrose-containing samples. However, in some cases, trehalose-containing samples showed an overlap requiring more refined methods of analysis. For example, when measuring the water content of some trehalose-glycerol mixtures, the step transitions calculated were numerous and ambiguously bordered the step transitions attributed to deterioration of a sample (data not shown). In these cases, the derivative weight loss was utilized to differentiate between water loss and sample deterioration. This may result in less accurate determinations of water content for very complex thermograms. However, the behavior of most samples, especially the maltose- and sucrose-glycerol mixtures, were much morestraightforward and allowed for simple differentiation of the water-loss and deterioration processes.II.B.4. An increase in the Tg correlated with enzyme protection conferred by maltoseglycerol and to a lesser extent sucrose-glycerol and trehalose-glycerol mixtures in the dry state

[0160] After determining that water retention was a poor indicator of the enzyme- protective capacity of trehalose-glycerol glasses and sucrose-glycerol glasses, the Tg was investigated to determine whether it might be a property that correlates with the stabilizing effects of these sugars. Even small amounts of an additive (such as glycerol) may lead to a decreased or increased Tg of a vitrifying material.

[0161] Here, the inventor assessed whether different additions of glycerol to the disaccharides would serve to increase or decrease the Tg, and whether or not these changes in Tg correlate with the enzyme-protective capacity of the mixtures.

[0162] The Tg onset and Tg offset (endset), the temperatures that the material starts and stops undergoing a change from a glassy state to a rubbery state, respectively, were assessed using DSC, and the Tg midpoint was calculated by taking the mean of the Tg onset and Tg offset. It was determined that, when considering the Tg midpoint, even small additions of glycerol (starting at 2.5%) act to decrease the Tg for trehalose, while decreasing the Tg of maltose began at 7.5% glycerol and a decrease in sucrose was only found with an even larger (12.5%) addition of glycerol (data not shown). For all disaccharide-glycerol mixtures, the Tg midpoints were determined to positively correlate with enzyme protective capacity, but only the maltose-glycerol mixtures demonstrated a significant correlation (p = 0.0063) as shown in FIG.7.

[0163] However, when considering the Tg onset or Tg offset for each set of disaccharide-glycerol mixtures, different behaviors were determined. For example, when observing changes to the Tg onset for the maltose-glycerol mixtures, even after the addition of 12.5% glycerol there was no significant change (data not shown). As opposed to maltose, sucrose was determined to have a significant decrease in Tg onset after the addition of 12.5% glycerol. Finally, the trehalose-glycerol mixtures showed statistically significant changes in the Tg onset beginning with the first addition of glycerol. The trehalose-glycerol mixtures also showed a statistically significant decrease after the addition of 12.5% glycerol. Whenobserving the Tg offset for each of the disaccharide-glycerol mixtures, it was determined that the maltose-glycerol and sucrose-glycerol mixtures demonstrated similar behavior to the Tg midpoint observations. Specifically, starting at 5% for maltose and 12.5% for sucrose, glycerol acted to decrease the Tg offset. On the other hand, trehalose did not show a statistically significant decrease in the Tg offset until after the addition of 10% glycerol.

[0164] Next was evaluated the relationship between enzyme-protective capacity and the Tg onset, Tg offset, and Tg midpoint for each mixture (FIG. 7, some data not shown). This correlative analysis produced positive correlations with R2values ranging from 0.87 to 0.81 for maltose, positive correlations with R2values ranging from 0.38 to 0.1 for sucrose, and positive correlations for trehalose the R2values ranged from 0.36 to 0.1. It was determined that the enzyme-protective capacity of the maltose-glycerol mixtures were influenced significantly by variations in their Tg and that the enzyme-protective capacity of the sucrose-glycerol mixtures and the trehalose-glycerol mixtures were moderately influenced by variations in their Tg.

[0165] The relationship between the enzyme-protective effect of the sugar mixtures and the difference between the temperature at which LDH is protected (Texp = 22°C) and their Tg was examined. It was reasoned that a mixture with a Tg close to Texp protection might be lowered due to the mixture undergoing a relaxation. Consistent with this reasoning, and for all disaccharide-glycerol mixtures, enzyme-protection and Texp - Tg had a negative correlation as shown in FIG. 11. However, only the maltose mixtures approached, but were not, a significant correlation between enzyme-protection and Texp - Tg. These results indicate that at the temperature used in this study, relaxation of dry sugar mixtures due to a similarity in Texp and Tg were not a significant influence on enzyme-protection. Texp - Tg refers to Texp minus Tg. ATg value = Texp - Tg.

[0166] Overall the results demonstrated that, in general, protection positively correlates with increasing Tg. However, this correlation was clearly stronger for some sugar-glasses (e.g., those containing maltose) compared to others (e.g., those containing sucrose).II.B.5. Vitrified maltose-glycerol and vitrified trehalose-glycerol mixtures demonstrated a reduction in glass former fragility that is correlated with enzyme-protection

[0167] Next, the glass former fragility (m-index) of the disaccharide-glycerol mixtures was empirically determined (data not shown). Thermal methods may be used to explore therelationship between the width of a glass transition and the activation enthalpy for viscosity, and the activation enthalpy for viscosity may be assumed to be equivalent to the activation enthalpy of structural relaxation at Tg. Utilizing this method and assumption, the glass former fragility (m-index) was calculated from DSC thermogram outputs (data not shown) as described in the fragility (m-index) calculation section.

[0168] The fragility of glass forming solutions comprising pure disaccharides was determined to be the lowest for maltose, followed by sucrose, and finally trehalose (data not shown). For maltose samples, the glass former fragility was determined to steadily increase with each addition of glycerol, but did not vary significantly (data not shown). However, for both sucrose and trehalose, small additions of glycerol (up to 5% and 2.5%, respectively) was determined to decrease glass former fragility (data not shown). However, larger additions of glycerol to both trehalose and sucrose was determined to increase glass former fragility (data not shown). For sucrose, variations in glass former fragility were not significant. On the other hand, after the addition of 10% glycerol, it was determined that trehalose saw significant increases in glass former fragility (data not shown).

[0169] Next, the relationship between the fragility of the glass forming mixtures and protection of LDH was investigated. For maltose and trehalose, the trend between enzymeprotection and glass former fragility was determined to be negative. In contrast, for sucrose, trend between enzyme-protection and glass former fragility was determined to be slightly positive. The strength of these trends varied dramatically between the sugars, as this correlative analysis produced an R2value of 0.64 for maltose, an R2value of 0.032 for sucrose, and an R2value of 0.77 for trehalose (FIG. 8; some data not shown). These results indicate that for maltose and trehalose, the determined variation in glass former fragility may explain some of the enzyme-protective capacity of the desiccated mixtures (FIG. 8).

[0170] It was found that the glass forming fragility of 100% sucrose was potentially behaving differently from mixtures containing glycerol additions. It was supposed that this datapoint might be “dragging” the relationship between the glass former fragility of the glass forming mixtures and protection of LDH upward into a positive correlation. A correlation was calculated excluding the pure (100%) disaccharide samples from the mixtures (97.5- 87.5%). This exclusion of the pure sucrose sample greatly improved the correlation value of the sucrose-glycerol mixtures, from an R2value of 0.032 to an R2value of 0.41 (data notshown). Interestingly, the correlation for the trehalose-glycerol mixtures was also improved, by excluding the 100% trehalose sample, from an R2value of 0.77 to an R2value of 0.83. However, the correlation for the maltose-glycerol mixtures was made worse by excluding the 100% disaccharide sample, from an R2value of 0.64 to an R2value of 0.57. These results suggest that, while pure disaccharides may behave in substantively different ways than disaccharide-glycerol mixtures, this is not always the case.II.B.6. Increased Tg and decreased glass former fragility may be characteristics of anhydrobiotic life-stages

[0171] The inventor next investigated whether the correlation between material properties — such as minimal glass former fragility or anti-plasticization, and enzymeprotection that were found in vitro — would carry over to organismal systems. Here, reduced glass former fragility and anti-plasticization were considered, but not water retention, because water may influence glass-like properties. This influence was found in the present samples. For example, for the maltose-glycerol mixtures, a very strong correlation (R2= 0.989) between water content and increased Tg was determined (data not shown).

[0172] The Tg and m-indexes of three desiccation-tolerant organisms, when in a desiccation-sensitive life stage or a desiccation-tolerant life stage, were measured. The three desiccation-tolerant organisms selected for this study were: the brine shrimp Artemia franciscana. the nematode worm Caenorhabditis elegans, and yeast Saccharomyces cerevisiae. Each of the selected organisms accumulates protective disaccharides and polyols during drying.

[0173] or A. franciscana, there was a significant increase in Tg offset between the adult (desiccation-sensitive life stage) and cyst (desiccation-tolerant life stage) as shown in FIG.9. For S. cerevisiae, a statistical increase between the exponential phase (desiccationsensitive life stage) and stationary phase (desiccation-tolerant life stage) when measuring Tg offset was found. For C. elegans, complex thermograms containing more than one obvious glass transition were found. Specifically, two glass transitions on the thermograms of non-dauer worms and three glass transitions on the thermograms of dauer pre-conditioned worms were found. The two glass transitions from non-dauer worms were paired with the first two glass transitions from the dauer pre-conditioned worms based on similarities in glass transition temperature. The trend of desiccation-tolerant life stages possessing asignificantly higher Tg offset in comparison to the desiccation-sensitive life stage continued across all three ranges of potential glass transitions for the C. elegans non-dauer and dauer pre-conditioned worms (FIG. 9). Thus, for all three of the desiccation-tolerant organisms tested, a significantly increased Tg in the anhydrobiotic state was found. These results indicate that increased Tg may be a characteristic of some desiccation-tolerant organismal systems.

[0174] Next, the inventor evaluated the three selected organisms’ life stages for changes in m-index (glass former fragility). Results are shown in FIG. 10. For A. franciscana, a significant decrease between the adult (desiccation-sensitive life stage) and cyst (desiccation-tolerant life stage) were found when measuring m-index. For S. cerevisiae, a significant decrease between the exponential phase (desiccation-sensitive life stage) and stationary phase (desiccation-tolerant life stage) was also found when measuring m-index. For C. elegans, a significant decrease in glass former fragility was found between the non- dauer (desiccation-sensitive life stage) and dauer pre-conditioned (desiccation-tolerant life stage) across all three glass transitions.

[0175] Taken together, these results indicate that (a) in vitro mixtures of protectants vary widely in what properties correlate with protection in the vitrified state, and (b) in vivo both increased Tg and decreased glass former fragility (m-index) are good indicators of survival in the dry state.ILC. Non-limiting Discussion

[0176] Since its conception, the vitrification hypothesis has provided a compelling possible explanation as to how anhydrobiotic organisms preserve their cells and cellular components during desiccation. However, while vitrification is considered necessary for desiccation tolerance it is not sufficient. This implies that there is some property of a glassy material that makes it more, or less, protective.

[0177] As described herein, the inventor quantified the enzyme-protective capacity and material properties of 18 different vitrified systems, each comprising one of three different disaccharides (maltose, sucrose, or trehalose) formulated with varying amounts of glycerol (0-12.5%). It was found that both enzyme-protective capacity and material properties of disaccharide-glycerol mixtures were modulated differently depending on the disaccharide used. Consistent with this, water retention (maltose), increased Tg (sucrose), and reducedglass fragility (trehalose) each correlated best with protection for a particular disaccharide. Interestingly, reduced glass former fragility and increased glass transition (antiplasticization) was found in desiccation-tolerant life stages of diverse organisms. Thus, individual protective properties found for reductive enzyme systems appear to be used in combination in vivo.II.C.l. Water retention, mechanisms of protection involving water, and water ’s effects on glass transition temperature and glass former fragility

[0178] The results presented herein demonstrated that different disaccharide-glycerol mixtures contain different quantities of water. Furthermore, the relationship between water content and protection also varied between disaccharide-glycerol mixtures. Maltoseglycerol mixtures displayed a significant positive correlation between water content and enzyme protection, while the sucrose-glycerol and trehalose-glycerol mixtures showed only mildly positive correlations (R2=0.13 and 0.28, respectively).

[0179] The water content of a glassy material could affect enzyme-protection through several mechanisms. Loss of water during dehydration may lead to a loss of important, stabilizing hydrogen bonds, which help to maintain protein folding. While theories, such as the water replacement hypothesis, which propose mechanisms by which loss of this hydrogen bond network can be dealt with, others such as the water entrapment, preferential exclusion / hydration hypothesis, or anchorage hypothesis offer up mechanisms by which residual water can be utilized to provide protection even at low levels.

[0180] The water entrapment hypothesis posits that a protectant which has a strong affinity for water but is preferentially excluded from client molecules could help to coordinate small amounts of residual water into proximity with desiccation-sensitive material (e.g., proteins, membrane, etc.). The effect would be to entrap and increase the local concentration of water around these desiccation-sensitive molecules which could help to maintain the hydrogen bond network needed for integrity. Conversely, the preferential exclusion hypothesis posits that a protectant which preferentially interacts with itself, to the exclusion of both water and client molecules, could in effect act as a space filling molecule. In this capacity, the protectant would reduce the overall accessible volume within the cell increasing the effective concentration of water and forcing water molecules into proximity with desiccation-sensitive molecules. Finally, the anchorage hypothesis posits that clientmolecules interact with the water-protectant matrix, and this interaction reduces the likelihood of protein unfolded since unfolding would have to lead to a recording of the water-protectant matrix.

[0181] Beyond water serving directly in the stabilization of biomolecules via the formation of a hydrogen bond network, water may also serve as a plasticizing agent of biological and hydrophilic materials. This means that increasing water content in a vitrified material typically leads to a decrease in Tg, which is also considered to reduce protection. However, while this is generally true, there have been reports of bona fide anti-plasticization effects of water. As described herein, the inventor found that the water content of maltose-, sucrose-, and trehalose-glycerol glasses have varying degrees of a strong plasticizing effect on the glass transition. The only negative correlation between water content and decreased Tg was found for the sucrose-glycerol mixtures (R2= 0.44; data not shown). For trehaloseglycerol mixtures, there was essentially no correlation between water content and decreased Tg (R2= 0.088; data not shown). Finally, in maltose-glycerol mixtures, rather than finding that water correlated with decreased Tg, a strong correlation was surprisingly found between water content and increased Tg (R2= 0.94; data not shown). This again shows how each of the disaccharides, when in a desiccated disaccharide-glycerol mixture, may display disparate changes in material properties.

[0182] When instead considering the relationship between water content and glass former fragility, the inventor again found differing results by the disaccharide-glycerol mixture used. Here, it was found that the desiccated maltose-glycerol mixtures had a strong negatively correlated relationship (R2= 0.74), that the desiccated sucrose-glycerol mixtures had a strong positively correlated relationship (R2= 0.88), and that the desiccated trehaloseglycerol mixtures had a weak positively correlated relationship (R2= 0.29) (data not shown).

[0183] These results might lead one to believe that, at the water contents examined here (< 11%), water retention might be a potential predictor of enzyme protection capacity. However, water content also appeared to influence other material properties of the vitrified system in a non-stereotyped fashion. For example, increasing water content in maltose- glycerol mixtures strongly correlated with reduced glass former fragility, while in sucrose- and trehalose-glycerol mixtures increasing water content increased glass former fragility.Thus, it may be concluded that water content itself is not a good predictor of desiccation tolerance nor of other properties of a vitrified system.II. C.2. The enzyme-protective capacity of sucrose-glycerol mixtures was most influenced by increases and decreases in Tg

[0184] Tg is the temperature at which a hard glassy material will begin to transition into a rubbery solid. The relationship of increases or decreases in Tg with protection varied between different disaccharide-glycerol mixtures but was determined to be most important for the sucrose-glycerol mixtures. While it was determined that only maltose-glycerol mixtures had a strong relationship between increased Tg and protection (FIG. 7), in the sucrose-glycerol mixtures it was determined that addition of glycerol resulted in an increase in the Tg when added up to 5%, but then further addition of glycerol caused a significant decrease in the Tg. Furthermore, the influence of glycerol on the Tg of sucrose-glycerol mixtures correlated weakly with protection but was the highest correlation with respect to enzyme-protection (FIG. 6, FIG. 7, and FIG. 8). For the trehalose-glycerol mixtures, the following was found: at first a significant decrease coinciding with the first addition of glycerol (2.5%) and then no significant variation in the glass transition midpoint until the addition of significantly more (12.5%) glycerol where it decreases the Tg, and the small changes in Tg that were found show no correlation with protection.

[0185] Thus, while increases in Tg were predictive of the protection conferred by maltose-glycerol mixtures, and to a lesser extent sucrose-glycerol, this predictive capacity did not extend to all vitrified systems.II C.3. The difference between the experimental temperature of enzyme protection and Tg did not correlate with protection

[0186] The closer a glassy protectant is to its Tg, the more molecular motion should be introduced, which could result in a loss of protective capacity. However, it was determined that the difference in the experimental temperature at which the LDH assays were conducted and the ATg of a protective glass (Texp - Tg) did not correlate significantly with enzyme- protective capacity (FIG. 11). This does not mean that, as a protective glass approaches its Tg, it does not lose enzyme-protective capacity, because it is possible that the experimental temperatures used here were still sufficiently lower than Tg to negatively impact enzymeprotection.II.C.4. The relationship between Tg and protection varied dramatically depending on whether onset, midpoint, or offset glass transition temperatures are considered

[0187] When referring to the glass transition or the material properties based on the glass transition, the standard measure is to use the glass transition midpoint. This value is inherently influenced by both the glass transition onset and offset temperatures (data not shown). The glass transition offset temperature is representative of the point at which the “glassy” nature of a vitreous system is finally overcome. In contrast, the glass transition onset is only representative of the start of the transition of a “glassy” state to a rubber-like solid. In evaluating the relationship between Tg and protection, a dramatic variation in this correlation was found depending on whether the onset, midpoint, or offset glass transition temperature was used (data not shown).II. C.5. Disaccharide-glycerol mixtures with similar concentrations produced fragilities of differing glass former fragility (m-index)

[0188] The fragility of different glass former mixtures varied by disaccharide. Only maltose and trehalose showed evidence of a relationship between glass former fragility and enzyme protection capacity. Interestingly, when comparing different disaccharide-glycerol systems that provide similar enzyme-protective capacity, those mixtures did not necessarily produce glasses with similar glass former fragility (m-index) measurements. For example, when comparing the 97.5% maltose, 97.5% sucrose, and 92.5% trehalose disaccharide- glycerol mixtures that provide approximately 30% enzyme protection during desiccation (30.21%, 30.26, and 36.72% respectively), those mixtures produced a glass former fragility (m-index) of 39.15, 74.01, and 151.81 respectively. In addition, when comparing the most enzyme-protective mixtures for each disaccharide-glycerol mixture, 100% maltose, 100% sucrose, and 97.5% trehalose, only trehalose produced the lowest glass former fragility (m- index) measurement. These results indicate that each disaccharide-glycerol system produced glass former fragility patterns that are only comparable within that system and not between different disaccharide-glycerol systems. Again, just as with water retention and shifts in the Tg, it was found that the addition of glycerol induced different degrees of glass former fragility and that this property is a good indicator of protection for some sugar glasses, but not for others.II.C.6. Changes in Tg and glass former fragility are Characteristics of organismal desiccation tolerance

[0189] The results from the in vitro experiments, while novel on their own, beg the question of whether these findings apply to whole desiccation-tolerant organisms. In particular, among the organisms studied, A. franciscana, C. elegans, and S. cerevisiae, trehalose is a major carbohydrate which is accumulated during desiccation and functionally thought to drive desiccation tolerance. In A. franciscana up to 15% of the dry weight of the cysts is attributed to trehalose accumulation. In C. elegans dauer larva that undergo preconditioning for four days will have accumulated up to approximately 375 pg of trehalose per mg of total protein. Finally, in S. cerevisiae stationary phase yeast can accumulate up to 15% of the total dry cell mass as trehalose. Of course, these organisms also accumulate a number of other disaccharides, diverse metabolites and proteins during drying, all of which may combine to contribute to glassy properties.

[0190] Examining these three different organisms in both a desiccation-tolerant and desiccation-sensitive life stage, it was found that both increased Tg and reduced m-index (glass former fragility) were characteristics of successful anhydrobiosis. Specifically, when considering changes in Tg, it was found that for each organism, A. franciscana, C. elegans, and S. cerevisiae, there was a statistically significant increase in Tg when comparing the desiccation-sensitive life stage to the desiccation-tolerant life stage. When considering the impact of glass former fragility, it was found that for each organism, A. franciscana, C. elegans, and S. cerevisiae, there was a statistically significant decrease in glass former fragility when comparing the desiccation-tolerant life stage to the desiccation-sensitive life stage.

[0191] The changes in Tg and glass former fragility appear to be protective during desiccation. It is uncertain what may be occurring to enable changes in the Tg and the glass former fragility to be protective during desiccation. A glass with a decreased Tg is one with relatively few and / or weak bonds, which do not effectively slow down molecular motions leading to the destabilization and / or aggregation of embedded client molecules over time. In contrast, a glass with an increased Tg is one with increased and / or stronger bonds leading to reduction in molecular motion and an increase in stability of a client molecule over time. This may indicate that inducing a super viscous (glassy) state is stabilizing because to unfolda protein would need to displace the embedding media. In this light, increased Tg would be protective as materials will become more fluid as the temperature they are stored at approach Tg.

[0192] While not wishing to be bound by any theory, when a glass forming mixture forms a weak glass, water is lost, and the weak glass forming ability of the mixture does not become viscous soon enough to prevent drying induced damage. Specifically, a weak glass former material will only begin gaining sufficient viscosity to confer protection after significant water loss. Conversely, a strong glass forming mixture starts to gain viscosity much earlier in the desiccation process compared to weaker glass forming materials. This steady increase in viscosity, may allow for the slowing of detrimental perturbations that manifest early during drying. One such perturbation may include the unfolding and aggregation of proteins, while another may include the fusion of membranes. Furthermore, previous studies have shown that volume loss during drying is associated with loss of cellular viability. Fragile glass formers, owing to their general lack of viscosity, might allow for more rapid cell shrinkage during drying. In contrast, strong glass formers might provide resistance to cell shrinkage via the induction of a super-viscous state early during the drying process. This model implies to some degree that it is the drying process, in addition to being in a dry state, that must be protected against.

[0193] When considering the organisms investigated within this study, A. franciscana, C. elegans, and S. cerevisiae, each organism produces a variety of metabolites that include proteins and carbohydrates which are necessary for surviving desiccation. However, as seen in other desiccation-tolerant organisms, such as rotifers, plants, and cyanobacteria, an array of disaccharides may be used instead of trehalose. While all three organisms studied here express only trehalose, the inventor has demonstrated that the use of externally added disaccharide, such as sucrose or maltose, can improve an organisms’ ability to survive desiccation. For instance, the inventor has demonstrated that uptake of maltose by way of media was able to provide desiccation protection.

[0194] While previous studies have examined the glass former fragility of seeds in relationship to their desiccation tolerance, to the inventor’s knowledge this is the first study examining changes in Tg and glass former fragility in animal and fungal systems. These comparative organismal studies show a stark contrast to the in vitro data presented herein,in that rather than a single material property correlating with protection, it appears that in living anhydrobiotic systems both increased Tg and reduced glass former fragility are generally increased. This may be due to the nature of the in vitro systems being simple, or less complex, in their interactions while the in vivo studies are by their nature much more complex, both in their material makeup and interactions. These results hint at the fact that living systems likely make use of multiple mediators of desiccation tolerance to produce protective glasses.

[0195] The study provided herein advances the understanding of what properties of a vitrified system promote desiccation tolerance and the phenomenon of anhydrobiosis both in vitro and in vivo. A deeper understanding of natural desiccation tolerance promises to provide avenues for pursuing real world applications such as biobanking of seeds and tissues, stabilization of pharmaceuticals, and the generation of stress tolerant crops.Ill, Molecular Stability Across Drying Methods: Evaluating Conditions for Room- Temperature Preservation of Biological MaterialsIII.A. Impact of Drying Methods on Molecular Stability: Fold Protection with Trehalose Versus No Protectants

[0196] LDH enzyme fold protection was evaluated with trehalose compared to no protectants. Results are shown in FIG. 12. In order to eliminate the effect of Tris and assess the effectiveness of trehalose, the fold protection was calculated by comparing LDH protection in the presence of trehalose to LDH protection in Tris buffer (no protectant). Among the tested drying conditions, a drying condition using the hotplate method at 37°C and 20% humidity was determined to yield the highest fold protection for LDH (-164) during a 30-minute drying period.

[0197] Citrate synthase (CS) fold protection was evaluated with trehalose compared to no protectants. Results are shown in FIG. 13. In order to eliminate the effect of Tris and assess the effectiveness of trehalose, the fold protection was calculated by comparing CS protection in the presence of trehalose to CS protection in Tris buffer (no protectant). Among the tested drying conditions, a drying condition using the hotplate method at 44°C and 50% humidity was determined to yield the highest fold protection for CS (-33) during a 30- minute drying period.

[0198] Next, how the drying conditions influenced the protection of other client molecules — such as DNA and RNA — in this study were examined. The results for DNA are shown in FIG. 14 and the results for RNA are shown in FIG. 15.

[0199] FIG. 14 which illustrates the fold protection of DNA in trehalose compared to Tris (no protectant). Interestingly, under all tested conditions, the DIN score remained close to 10 (resulting in a fold change in DIN of about 1.0), regardless of the methods or the presence of trehalose or Tris. Therefore, no differences were found in the fold protection of DNA between trehalose and Tris. This result indicates that, despite variations in drying conditions affecting glassy properties, DNA stability remains unaffected.

[0200] FIG. 15 illustrates the fold protection of RNA in trehalose compared to Tris. Interestingly, under all tested conditions, the RIN score remained close to 10 (resulting in a fold change in DIN of about 1.0), regardless of the methods or the presence of trehalose or Tris. Therefore, no differences were found in the fold protection of RNA between trehalose and Tris. This result indicates that, despite variations in drying conditions affecting glassy properties, RNA stability remains unaffected.III.B. Correlation Analysis of Glassy Properties and LDH Enzyme Protection under Varying Conditions

[0201] Here, the inventor examined which glassy properties may contribute to the observed fold protection. First, the inventor evaluated whether there was a link between LDH enzyme protection and glassy properties in hotplate drying at 30°C. Results are shown in FIG. 16. A strong correlation between the Tg midpoint and LDH fold protection was found, suggesting that a higher Tg may enhance LDH stability in trehalose. LDH enzyme protection versus glass former fragility (m-index) and LDH enzyme protection versus water content were also plotted (data not shown). Overall, the data showed that higher glass transition temperatures (Tg) in trehalose-based systems (trehalose-vitrified systems) may improve LDH enzyme stability, but no correlation was found between LDH enzyme protection and water content or glass former fragility.

[0202] Next, links between LDH enzyme protection and glassy properties in hotplate drying at 37°C were evaluated. Results are shown in FIG. 17. With hotplate drying at 37°C, a strong correlation between the Tg midpoint and LDH fold protection was found, suggesting that a higher Tg may enhance LDH stability in trehalose. LDH enzymeprotection versus glass former fragility (m-index) and LDH enzyme protection versus water content were also plotted (data not shown). Overall, as with hotplate drying at 30°C, the data for hotplate drying at 37°C showed that higher Tg values in trehalose-based systems (trehalose-vitrified systems) may improve LDH enzyme stability, but no correlation was found between LDH enzyme protection and water content or glass former fragility.

[0203] Links between LDH enzyme protection and glassy properties in hotplate drying at 44°C were also evaluated (data not shown). No correlation was found between LDH protection and any of the glassy properties tested (Tg, glass former fragility (m-index), or water content). This result suggested that temperature alone may inactivate LDH.

[0204] The effect of humidity was also evaluated. Links between LDH enzyme protection and glassy properties in humidified drying at 30°C, humidified drying at 37°C, and humidified drying at 44°C were evaluated. Results are shown in FIG. 18, FIG. 19, and FIG. 20. With humidified drying at 30°C (FIG. 18), humidified drying at 37°C (FIG. 19), and humidified drying at 44°C (FIG. 20), it was determined that the water content negatively correlates with LDH stability.III.C. LDH Protection Levels of CAHS D and Trehalose

[0205] LDH enzyme fold protection was evaluated with CAHS D, trehalose, or both. For these experiments, Tris, CAHS D, trehalose, or CAHS D and trehalose in different ratios were mixed with LDH and then dried at 37°C with 20% humidity using the hotplate drying method as these conditions provided the highest LDH enzyme fold protection. Results are shown in FIG. 21, indicating synergistic LDH protection when using trehalose with CAHS D.

[0206] Specifically, trehalose, itself, provided 60% protection to LDH after drying and dehydration. The sample with 0.003 mM CAHS D alone gave 4% protection. When 0.003 mM CAHS D was combined with trehalose at a 1 : 100,000 molar ratio, the observed protection (-63%) was found to be similar to the expected combined effect (-64%).

[0207] The sample with 0.03 mM CAHS D alone gave 45% protection. When 0.03 mM CAHS D was combined with trehalose to give a 1 : 10000 mixture (CAHS D:trehalose), it provided protection that closely matched the expected value. However, it was difficult to determine whether this total protection was additive or synergistic, as the expected result was already near 100%. To further investigate this, the inventor tested 0.015 mM CAHS Dat a 1 :20,000 ratio of CAHS D:trehalose. The observed effect was 94.84% for LDH enzyme protection, exceeding the combined individual protection levels (68.20%), showing the synergistic effect.III.D. Impact of Long-Term Preservation on LDH Protective Capacity in Different Formulations.

[0208] Next evaluated was the LDH protection capacity in various formulations over 8 weeks of time to determine what happens to the integrity over time in the dry state. For these experiments, LDH activity was measured after drying and storage with Tris buffer, 0.015 mM CAHS D, 300 mM trehalose, and the combination of 300 mM trehalose with 0.015 mM CAHS D. Results are shown in FIG. 22, indicating synergistic LDH protection when using trehalose with CAHS D. As shown, Tris alone (control) and 0.015 mM CAHS D alone failed to protect LDH. Even on the first day, LDH enzyme activity significantly declined after drying (at day 0, LDH protection was less than 10% when using 0.015 CAHS D). In contrast, the combination of 0.015 mM CAHS D and 300 mM trehalose showed synergistic results for LDH protection. While both 300 mM trehalose alone and the combination of 300 mM trehalose with 0.015 mM CAHS D showed decreased protection over time, the synergistic effect of the combination was consistently found at each time point tested.Embodiments Listing

[0209] The present disclosure provides, among others, the following embodiments, each of which can be considered as optionally including any alternate embodiments:

[0210] Embodiment Al. A composition, comprising: an exogenous disaccharide; an intrinsically disordered protein (IDP); and a biological material of interest and / or biologically-derived material of interest, the biological material of interest and biologically-derived material of interest different from the IDP, the composition having a water content that is about 15 wt% or less based on a total wt% of the composition (such as from 0 wt% to about 15 wt%, such as from about 1 wt% to about 14 wt%, such as from about 5 wt% to about 12 wt%), the total wt% of the composition equal to 100 wt%,the composition optionally characterized as stabilizing the biological material of interest or biologically-derived material of interest of the composition in a dry state at a temperature of about 20°C or higher (such as from about 20°C to less than 100°C).

[0211] Embodiment A2. The composition of Embodiment Al, wherein the composition comprises a molar ratio of the IDP to the exogenous disaccharide is about 1 : 1,000 or more.

[0212] Embodiment A3. The composition of any one of Embodiments A1-A2, wherein the composition comprises a molar ratio of the IDP to the exogenous disaccharide is in a range from about 1 : 10,000 to about 1 : 100,000, such as from about 1 :20,000 to about 1 :90,000, such as from about 1 :30,000 to about 1 :80,000, such as from about 1 :40,000 to about 1 :70,000, such as from about 1 :50,000 to about 1 :60,000.

[0213] Embodiment A4. The composition of any one of Embodiments A1-A3, wherein the exogenous disaccharide comprises trehalose, sucrose, or combinations thereof.

[0214] Embodiment A5. The composition of any one of Embodiments A1-A4, wherein the IDP comprises CAHS D.

[0215] Embodiment A6. A composition, comprising: a biological material of interest and / or biologically-derived material of interest; and an additive or additive mixture comprising: a sucrose compound comprising monomeric sucrose, a polysucrose having a molecular weight that is in a range from about 10 kDa to about 800 kDa, or a combination thereof; and optionally glycerol, wherein the composition is characterized as stabilizing the biological material of interest and / or biologically-derived material of interest of the composition in a dry state at a temperature of about 20°C or higher (such as from about 20°C to less than 100°C).

[0216] Embodiment A7. The composition of Embodiment A6, wherein the molecular weight of the poly sucrose is in a range from about 70 kDa to about 400 kDa.

[0217] Embodiment A8. The composition of any one of Embodiments A6-A7, wherein the additive mixture comprises: from about 85 wt% to 100 wt% of the sucrose compound based on a total wt% of the additive or additive mixture, the total wt% of the additive or additive mixture is 100wt% (such as from about 86 wt% to about 99 wt%, such as from about 87 wt% to about 98 wt%, such as from about 88 wt% to about 97 wt%, such as from about 89 wt% to about 96 wt%, such as from about 90 wt% to about 95 wt%, such as from about 91 wt% to about 94 wt%, such as from about 92 wt% to about 93 wt%); and from 0 wt% to about 15 wt% of the optional glycerol based on a total wt% of the additive or additive mixture (such as from about 1 wt% to about 14 wt%, such as from about 2 wt% to about 13 wt%, such as from about 3 wt% to about 12 wt%, such as from about 4 wt% to about 11 wt%, such as from about 5 wt% to about 10 wt%, such as from about 6 wt% to about 9 wt%, such as from about 7 wt% to about 8 wt%).

[0218] Embodiment A9. The composition of any one of Embodiments A6-A8, wherein the additive or additive mixture comprises the polysucrose.

[0219] Embodiment A10. The composition of Embodiment A9, wherein the additive or additive mixture is characterized as having: a Tg (onset) in a range from about 43°C to about 131°C, such as from about 50°C to about 125°C, such as from about 60°C to about 115°C, such as from about 70°C to about 105°C, such as from about 80°C to about 95°C; a Tg (midpoint) in a range from about 50°C to about 136°C, such as from about 60°C to about 125°C, such as from about 70°C to about 115°C, such as from about 80°C to about 105°C, such as from about 90°C to about 95°C; a Tg (endset) in a range from about 60°C to about 140°C, such as from about 70°C to about 130°C, such as from about 80°C to about 120°C, such as from about 90°C to about 110°C, such as from about 95°C to about 105°C; or combinations thereof, wherein Tg onset < Tg midpoint < Tg endset.

[0220] Embodiment Al 1. The composition of any one of Embodiments A6-A8, wherein the additive or additive mixture comprises the monomeric sucrose.

[0221] Embodiment A12. The composition of Embodiment Al l, wherein the additive or additive mixture is characterized as having: a Tg (onset) in a range from about 27°C to about 73 °C, such as from about 30°C to about 70°C, such as from about 35°C to about 65°C, such as from about 40°C to about 60°C, such as from about 45°C to about 55°C;a Tg (midpoint) in a range from about 35°C to about 79°C, such as from about 40°C to about 75°C, such as from about 45°C to about 70°C, such as from about 50°C to about 65°C, such as from about 55°C to about 60°C; a Tg (endset) in a range from about 42°C to about 85°C, such as from about 45°C to about 80°C, such as from about 50°C to about 75°C, such as from about 55°C to about 70°C, such as from about 60°C to about 65°C; or combinations thereof, wherein Tg onset < Tg midpoint < Tg endset.

[0222] Embodiment A13. The composition of any one of Embodiments A6-A12, wherein the additive or additive mixture is characterized as glassy without crystallinity.

[0223] Embodiment A14. A composition, comprising: a biological material of interest and / or biologically-derived material of interest; an additive mixture comprising: a disaccharide; and a polyol that is different from the disaccharide, wherein the composition is characterized as stabilizing the biological material of interest and / or biologically-derived material of interest of the composition in a dry state at a temperature of about 20°C or higher (such as from about 20°C to less than 100°C).

[0224] Embodiment A15. The composition of Embodiment A14, wherein the polyol comprises glycerol, ethylene glycol, propylene glycol, methyl glycol trimethylene glycol, neopentyl glycol, diethylene glycol, triethylene glycol, polyethylene glycol, polypropylene glycol, or combinations thereof.

[0225] Embodiment A16. The composition of any one of Embodiments A14-A15, wherein the disaccharide comprises trehalose, sucrose, maltose, lactose, or combinations thereof.

[0226] Embodiment A17. The composition of any one of Embodiments A14-A16, wherein the additive mixture comprises: from about 85 wt% to less than 100 wt% of the disaccharide based on a total wt% of the additive mixture, the total wt% of the additive mixture is 100 wt% (such as from about 86 wt% to about 99 wt%, such as from about 87 wt% to about 98 wt%, such as from about 88 wt% to about 97 wt%, such as from about 89 wt% to about 96 wt%, such as fromabout 90 wt% to about 95 wt%, such as from about 91 wt% to about 94 wt%, such as from about 92 wt% to about 93 wt%); and from greater than 0 wt% to about 15 wt% of the polyol based on a total wt% of the additive mixture (such as from about 1 wt% to about 14 wt%, such as from about 2 wt% to about 13 wt%, such as from about 3 wt% to about 12 wt%, such as from about 4 wt% to about 11 wt%, such as from about 5 wt% to about 10 wt%, such as from about 6 wt% to about 9 wt%, such as from about 7 wt% to about 8 wt%).

[0227] Embodiment A18. The composition of any one of Embodiments A14-A17, wherein the additive mixture is characterized as having: a Tg (onset) in a range from about 22°C to about 76°C, such as from about 25°C to about 70°C, such as from about 30°C to about 65°C, such as from about 35°C to about 60°C such as from about 40°C to about 55°C, such as from about 45°C to about 50°C; a Tg (midpoint) in a range from about 23°C to about 83°C, such as from about 25°C to about 80°C, such as from about 30°C to about 75°C, such as from about 35°C to about 70°C, such as from about 40°C to about 65°C, such as from about 45°C to about 60°C, such as from about 50°C to about 55°C; a Tg (endset) in a range from about 24°C to about 95°C, such as from about 25°C to about 90°C, such as from about 30°C to about 85°C, such as from about 35°C to about 80°C, such as from about 40°C to about 75°C, such as from about 45°C to about 70°C, such as from about 50°C to about 65°C, such as from about 65°C to about 60°C; or combinations thereof, wherein Tg onset < Tg midpoint < Tg endset.

[0228] Embodiment A19. The composition of any one of Embodiments A14-A18, wherein the additive mixture is characterized as having: a glass former fragility (m-index) in a range from about 20 to about 375, such as from about 50 to about 350, such as from about 100 to about 300, such as from about 150 to about 250, such as from about 150 to about 200, or from about 200 to about 250; a ATg value (value of Texp - Tg) in a range from about -18 to about -61, such as from about -20 to about -60, such as from about -25 to about -55, such as from about -30 to about -50, such as from about -35 to about -45, such as from about -35 to about -40, or from about -40 to about -45; or a combination thereof.

[0229] Embodiment A20. The composition of any one of Embodiments A14-A19, wherein the additive mixture is characterized as glassy without crystallinity.

[0230] Embodiment A21. A method of forming the composition of any one of Embodiments A1-A20, the method comprising: contacting components (e.g., biological and / or biologically-derived material of interest, additive or additive mixture, optional IDP, optional water, etc.) to form a mixture; and drying, or at least partially drying, the mixture to form the composition.

[0231] Embodiment Bl. A composition for stabilizing a biological material of interest, comprising: a biological material of interest; and an additive or additive mixture comprising: a polymeric sucrose having a molecular weight that is from about 10 kDa to about 400 kDa; and optionally glycerol, wherein the composition is characterized as stabilizing the biological material of interest of the composition in a dry state at a temperature of ambient temperature or higher.

[0232] Embodiment B2. The composition of Embodiment Bl, further comprising 15 wt% or less of water based on a total wt% of the composition, the total wt% of the composition not to exceed 100 wt%.

[0233] Embodiment B3. The composition of any one of Embodiments B1-B2, wherein the additive mixture comprises: from about 85 wt% to 100 wt% of the polymeric sucrose, based on a total wt% of the polymeric sucrose and the glycerol, the total wt% of the polymeric sucrose and the glycerol not to exceed 100 wt%; and from 0 wt% to about 15 wt% of the glycerol, based on a total wt% of the polymeric sucrose and the glycerol.

[0234] Embodiment B4. The composition of any one of Embodiments B1-B3, wherein the additive mixture has: a Tg (onset) that is from about 43°C to about 131°C; a Tg (midpoint) that is from about 50°C to about 136°C; a Tg (endset) that is from about 60°C to about 140°C; or combinations thereof.

[0235] Embodiment B5. The composition of any one of Embodiments B1-B4, wherein the additive mixture is glassy and without crystallinity.

[0236] Embodiment B6. A composition for stabilizing a biological material of interest, comprising: a biological material of interest; and an additive mixture comprising: from greater than about 85 wt% to less than 100 wt% of a sucrose monomer, based on a total wt% of the sucrose monomer and the glycerol, the total wt% of the sucrose monomer and the glycerol not to exceed 100 wt%; and from greater than 0 wt% to less than about 15 wt% of glycerol, based on the total wt% of the sucrose monomer and the glycerol, wherein the composition is characterized as stabilizing the biological material of interest of the composition in a dry state at a temperature of ambient temperature or higher.

[0237] Embodiment B7. The composition of Embodiment B6, further comprising 15 wt% or less of water based on a total wt% of the composition, the total wt% of the composition not to exceed 100 wt%.

[0238] Embodiment B8. The composition of any one of Embodiments B6-B7, wherein the additive mixture has: a Tg (onset) that is from about 27°C to about 73°C; a Tg (midpoint) that is from about 35°C to about 79°C; a Tg (endset) that is from about 42°C to about 85°C; or combinations thereof.

[0239] Embodiment B9. The composition of any one of Embodiments B6-B8, wherein the additive mixture is glassy and without crystallinity.

[0240] Embodiment B10. A method for stabilizing a biological material of interest, the method comprising: introducing a biological material of interest with an additive (or additive mixture) to form a composition, wherein the formed composition is characterized as stabilizing the biological material of interest of the formed composition in a dry state at a temperature of ambient temperature or higher; and optionally drying or desiccating the formed composition.

[0241] Embodiment Bl l. The method of Embodiment B10, wherein the formed composition is the composition of any one of Embodiments Bl -B10.

[0242] Embodiment Cl. A composition for stabilizing a biological material of interest, comprising: a biological material of interest; an additive mixture, comprising: a disaccharide; and a polyol that is different from the disaccharide, wherein the composition is characterized as stabilizing the biological material of interest of the composition in a dry state at a temperature of ambient temperature or higher.

[0243] Embodiment C2. The composition of Embodiment Cl, wherein the polyol comprises glycerol, ethylene glycol, propylene glycol, methyl glycol trimethylene glycol, neopentyl glycol, diethylene glycol, triethylene glycol, polyethylene glycol, polypropylene glycol, or combinations thereof.

[0244] Embodiment C3. The composition of any one of Embodiments C1-C2, wherein the disaccharide comprises trehalose, sucrose, maltose, lactose, or combinations thereof.

[0245] Embodiment C4. The composition of any one of Embodiments C1-C3, further comprising 15 wt% or less of water based on a total wt% of the composition, the total wt% of the composition not to exceed 100 wt%.

[0246] Embodiment C5. The composition of any one of Embodiments C1-C4, wherein the additive mixture comprises: from about 85 wt% to 100 wt% of the disaccharide, based on a total wt% of the disaccharide and the polyol, the total wt% of the disaccharide and the polyol not to exceed 100 wt%; and from 0 wt% to about 15 wt% of the polyol, based on a total wt% of the disaccharide and the polyol.

[0247] Embodiment C6. The composition of any one of Embodiments C1-C5, wherein the additive mixture has: a Tg (onset) that is from about 22°C to about 76°C; a Tg (midpoint) that is from about 23°C to about 83°C; a Tg (endset) that is from about 24°C to about 95°C; or combinations thereof.

[0248] Embodiment C7. The composition of any one of Embodiments C1-C6, wherein the additive mixture has: a glass former fragility (m-index) that is from about 20 to about 375;a ATg value (ATg = Texp - Tg) that is from about -18 to about -61; or combinations thereof.

[0249] Embodiment C8. The composition of any one of Embodiments C1-C7, wherein the additive mixture is glassy and without crystallinity.

[0250] Embodiment C9. A method for stabilizing a biological material of interest, the method comprising: introducing a biological material of interest with an additive mixture to form a composition, wherein the formed composition is characterized as stabilizing the biological material of interest of the formed composition in a dry state at a temperature of ambient temperature or higher; and optionally drying or desiccating the formed mixture.

[0251] Embodiment CIO. The method of Embodiment C9, wherein the formed composition is the composition of any one of Embodiments C1-C8.

[0252] As is apparent from the foregoing general description and the specific aspects, while forms of the aspects have been illustrated and described, various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, it is not intended that the present disclosure be limited thereby. Likewise, the term “comprising” is considered synonymous with the term “including.” Likewise whenever a composition, process operation, process operations, an element or a group of elements is preceded with the transitional phrase “comprising,” it is understood that we also contemplate the same composition or group of elements with transitional phrases “consisting essentially of,” “consisting of,” “selected from the group of consisting of,” or “Is” preceding the recitation of the composition, process operation, process operations, element, or elements and vice versa, such as the terms “comprising,” “consisting essentially of,” “consisting of’ also include the product of the combinations of elements listed after the term.

[0253] For purposes of this present disclosure, and unless otherwise specified, all numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and consider experimental error and variations that would be expected by a person having ordinary skill in the art. For the sake of brevity, only certain ranges are explicitly disclosed herein. However, ranges from anylower limit may be combined with any upper limit to recite a range not explicitly recited, as well as, ranges from any lower limit may be combined with any other lower limit to recite a range not explicitly recited, in the same way, ranges from any upper limit may be combined with any other upper limit to recite a range not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the subranges 1 to 4, 1.5 to 4.5, 1 to 2, among other subranges. As another example, the recitation of the numerical ranges 1 to 5, such as 2 to 4, includes the subranges 1 to 4 and 2 to 5, among other subranges. Additionally, within a range includes every point or individual value between its end points even though not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the numbers 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, among other numbers. Thus, every point or individual value may serve as its own lower or upper limit combined with any other point or individual value or any other lower or upper limit, to recite a range not explicitly recited.

[0254] As used herein, the indefinite article “a” or “an” shall mean “at least one” unless specified to the contrary or the context clearly indicates otherwise. For example, aspects comprising “a disaccharide” includes aspects comprising one, two, or more disaccharides, unless specified to the contrary or the context clearly indicates only one polymeric sucrose is included.

[0255] While the foregoing is directed to aspects of the present disclosure, other and further aspects of the disclosure may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

ClaimsWhat is claimed is:

1. A composition, comprising: an exogenous disaccharide; an intrinsically disordered protein (IDP); and a biological material of interest or a biologically-derived material of interest, the biological material of interest and biologically-derived material of interest different from the IDP, the composition having a water content that is about 15 wt% or less based on a total wt% of the composition, the total wt% of the composition equal to 100 wt%.

2. The composition of claim 1, wherein the composition comprises a molar ratio of the IDP to the exogenous disaccharide is about 1 :1,000 or more.

3. The composition of claim 1, wherein the composition comprises a molar ratio of the IDP to the exogenous disaccharide is in a range from about 1 : 10,000 to about 1 : 100,000.

4. The composition of claim 1, wherein the exogenous disaccharide comprises trehalose, sucrose, or combinations thereof.

5. The composition of claim 1, wherein the IDP comprises CAHS D.

6. A composition, comprising: a biological material of interest or a biologically-derived material of interest; and an additive or additive mixture comprising: a sucrose compound comprising monomeric sucrose, a polysucrose having a molecular weight that is in a range from about 10 kDa to about 800 kDa, or a combination thereof; and optionally glycerol,wherein the composition is characterized as stabilizing the biological material of interest or biologically-derived material of interest of the composition in a dry state at a temperature of 20°C or higher.

7. The composition of claim 6, wherein the molecular weight of the polysucrose is in a range from about 70 kDa to about 400 kDa.

8. The composition of claim 6, wherein the additive mixture comprises: from about 85 wt% to 100 wt% of the sucrose compound based on a total wt% of the additive or additive mixture, the total wt% of the additive or additive mixture is 100 wt%; and from 0 wt% to about 15 wt% of the optional glycerol based on the total wt% of the additive or additive mixture.

9. The composition of claim 6, wherein the additive or additive mixture comprises the polysucrose.

10. The composition of claim 9, wherein the additive or additive mixture is characterized as having: a Tg (onset) in a range from about 43 °C to about 131°C; a Tg (midpoint) in a range from about 50°C to about 136°C; a Tg (endset) in a range from about 60°C to about 140°C; or combinations thereof.

11. The composition of claim 6, wherein the additive or additive mixture comprises the monomeric sucrose.

12. The composition of claim 11, wherein the additive or additive mixture is characterized as having: a Tg (onset) in a range from about 27°C to about 73 °C; a Tg (midpoint) in a range from about 35°C to about 79°C;a Tg (endset) in a range from about 42°C to about 85°C; or combinations thereof.

13. The composition of claim 6, wherein the additive or additive mixture is characterized as glassy without crystallinity.

14. A composition, comprising: a biological material of interest or a biologically-derived material of interest; an additive mixture comprising: a disaccharide; and a polyol that is different from the disaccharide, wherein the composition is characterized as stabilizing the biological material of interest or biologically-derived material of interest of the composition in a dry state at a temperature of 20°C or higher.

15. The composition of claim 14, wherein the polyol comprises glycerol, ethylene glycol, propylene glycol, methyl glycol trimethylene glycol, neopentyl glycol, diethylene glycol, triethylene glycol, polyethylene glycol, polypropylene glycol, or combinations thereof.

16. The composition of claim 14, wherein the disaccharide comprises trehalose, sucrose, maltose, lactose, or combinations thereof.

17. The composition of claim 14, wherein the additive mixture comprises: from about 85 wt% to less than 100 wt% of the disaccharide based on a total wt% of the additive mixture, the total wt% of the additive mixture is 100 wt%; and from greater than 0 wt% to about 15 wt% of the polyol based on the total wt% of the additive mixture.

18. The composition of claim 14, wherein the additive mixture is characterized as having:a Tg (onset) in a range from about 22°C to about 76°C; a Tg (midpoint) in a range from about 23°C to about 83°C; a Tg (endset) in a range from about 24°C to about 95°C; or combinations thereof.

19. The composition of claim 14, wherein the additive mixture is characterized as having: a glass former fragility (m-index) that is in a range from about 20 to about 375; a ATg value (ATg = Texp - Tg) that is in a range from about -18 to about -61; or a combination thereof.

20. The composition of claim 14, wherein the additive mixture is characterized as glassy without crystallinity.

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