Methods of desiccating a sample and use thereof

Abstract

The disclosure relates to methods of desiccating of a sample comprising removing water from the sample at a temperature of 0 °C or above, and after the removing, freeze-drying of the sample.

Description

METHODS OF DESICCATING A SAMPLE AND USE THEREOF BACKGROUND

[0001] The disclosure relates to methods of desiccating of a sample comprising removing water from the sample at a temperature of 0 °C or above, and after the removing, freeze-drying of the sample. Desiccation is an important technique used to preserve biological materials such as cells, bacteria, viruses, fungi, DNA, and RNA.SUMMARY

[0002] The disclosure relates to methods of desiccating of a sample comprising removing water from the sample at a temperature of 0 °C or above, and after the removing, freeze-drying of the sample.BRIEF DESCRIPTION OF THE DRAWINGS

[0003] Figure 1 depicts exemplary steps of a desiccation method of the disclosure.

[0004] Figure 2 depicts alternative preservation form factors.

[0005] Figure 3 depicts a hybrid electrospinning process with microbiological samples embedded in a matrix.

[0006] Figure 4 depicts a conceptual diagram showing different thermodynamic routes for preservation of a biological material.DETAILED DESCRIPTION

[0007] In one aspect, the methods of the present disclosure comprise a method of desiccating a sample as described herein. The term “desiccating” or “desiccate” as used herein refers to the removal of water from a sample by the methods of the present disclosure. In some embodiments, the methods of the present disclosure comprise removing water from the sample. In some embodiments, the removing is performed at a temperature of about 0 °C or above, for example, about 1 °C or above, 2 °C or above, 3 °C or above, 4 °C or above, 5 °C or above, 10 °C or above, 15 °C or above, 20 °C or above, 25 °C or above, 30 °C or above, 35 °C or above, or 40 °C or above. In some embodiments, the removing is performed at a temperature of about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 °C. In some embodiments, the removing is performed at a temperature of from about 0 to 40 °C, from about 0to 35 °C, from about 0 to 30 °C, from about 0 to 25 °C, from about 0 to 20 °C, from about 0 to 15 °C, from about 0 to 10 °C, from about 0 to 5 °C, from about 0 to 4 °C, from about 5 to 25 °C, from about 5 to 20 °C, from about 5 to 15 °C, or from about 5 to 10 °C. In some embodiments, the removing is performed at room temperature (e.g., from about 18 to 25 °C, from about 19 to 25 °C, from about 20 to 25 °C, from about 21 to 25 °C, from about 22 to 25 °C, from about 18 to 24 °C, from about 18 to 23 °C, from about 18 to 22 °C, or from about 20 to 22 °C, and / or about 18, 19, 20, 21, 22, 23, 24, or 25 °C). In some embodiments, the removing is performed at physiological temperature (e.g., about 37 °C). In some embodiments, the removing is performed at refrigeration temperature (e.g., from about 1 to 5 °C, or about 1, 2, 3, 4, or 5 °C). In some embodiments, the removing is performed above the freezing temperature of water (e.g., above 0 °C).

[0008] In some embodiments, the removing comprises removing at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more of water from the sample. In some embodiments, the removing comprises removing from about 5 to 99%, from about 10 to 99%, from about 15 to 99%, from about 20 to 99%, from about 25 to 99%, from about 30 to 99%, from about 35 to 99%, from about 40 to 99%, from about 45 to 99%, from about 50 to 99%, from about 55 to 99%, from about 60 to 99%, from about 65 to 99%, from about 70 to 99%, from about 75 to 99%, from about 80 to 99%, from about 85 to 99%, from about 90 to 99%, from about 95 to 99% of water from the sample. In some embodiments, the removing comprises removing more than about 99% of water from the sample. In some embodiments, the removing comprises removing from about 95% to 99% of water from the sample.

[0009] In some embodiments, the removing comprises removing water from the sample so that a water content of the sample is below about 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 g water / g dry weight, or less. In some embodiments, the removing comprises removing water from the sample so that a water content of the sample is from about 0.1 to 1 g water / g dry weight, from about 0.1 to 0.9 g water / g dry weight, from about 0.1 to 0.8 g water / g dry weight, from about 0.1 to 0.7 g water / g dry weight, from about 0.1 to 0.6 g water / g dry weight, from about 0.1 to 0.5 g water / g dry weight, from about 0.1 to 0.4 g water / g dry weight, from about 0.1 to 0.3 g water / g dry weight, from about 0.2 to 1 g water / g dry weight, from about 0.2 to 0.9 g water / g dry weight,from about 0.2 to 0.8 g water / g dry weight, from about 0.2 to 0.7 g water / g dry weight, from about 0.3 to 0.8 g water / g dry weight, from about 0.4 to 0.9 g water / g dry weight, or from about 0.5 to 1 g water / g dry weight. In some embodiments, the removing comprises removing water from the sample so that a water content of the sample is below about 1 g water / g dry weight. In some embodiments, the removing comprises removing water from the sample so that a water content of the sample is from about 0.1 g water / g dry weight to about 0.6 g water / g dry weight. In some embodiments, the removing comprises removing water from the sample so that a water content of the sample is about 0.1 water / g dry weight. In some embodiments, the removing comprises removing water from the sample so that a water content of the sample is about 0.6 g water / g dry weight.

[0010] In some embodiments, the removing comprises removing water at a rate of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 g water / g dry weight / hr. In some embodiments, the removing comprises removing water a rate of from about 0.1 to 2 g water / g dry weight / hr, from about 0.5 to 2 g water / g dry weight / hr, from about 1 to 2 g water / g dry weight / hr, from about 1.1 to 2 g water / g dry weight / hr, from about 1.3 to 2 g water / g dry weight / hr, from about 1.4 to 2 g water / g dry weight / hr, from about 1.5 to 2 g water / g dry weight / hr, from about 1 to 1.9 g water / g dry weight / hr, from about 1 to 1.8 g water / g dry weight / hr, from about 1 to 1.7 g water / g dry weight / hr, from about 1 to 1.6 g water / g dry weight / hr, or from about 1 to 1.5 g water / g dry weight / hr. In some embodiments, the removing comprises removing water at a rate from about 1 g water / g dry weight / hr to about 2 g water / g dry weight / hr. In some embodiments, the removing comprises removing water at a rate of about 1 g water / g dry weight / hr. In some embodiments, the removing comprises removing water at a rate of about 2 g water / g dry weight / hr.

[0011] In some embodiments, the removing comprises at least one selected from the group consisting of spin drying, dip coating, spray drying, electrospinning and directional deposition. In some embodiments, the removing comprises at least two selected from the group consisting of spin drying, dip coating, spray drying, electrospinning and directional deposition. In some embodiments, the removing comprises at least three selected from the group consisting of spin drying, dip coating, spray drying, electrospinning and directional deposition. In some embodiments, the removing comprises and / or consists of spin drying, dip coating, spray drying,electrospinning and directional deposition. In some embodiments, the removing comprises spin drying. In some embodiments, the removing comprises dip coating. In some embodiments, the removing comprises spray drying. In some embodiments, the removing comprises electrospinning. In some embodiments, the removing comprises directional deposition. In some embodiments, the removing comprises drying using surface tension of the sample. In some embodiments, the removing comprises extrusion or deposition of the sample. In some embodiments, the removing comprises extrusion and deposition of the sample. In some embodiments, the removing comprises extrusion and / or deposition of the sample. In some embodiments, the removing comprises extrusion of the sample. In some embodiments, the removing comprises deposition of the sample. In some embodiments, the removing comprises at least one selected from the group consisting of direct extrusion, casting thin film by spreading the sample, electrospinning, and droplet deposition. In some embodiments, the removing comprises at least two selected from the group consisting of direct extrusion, casting thin film by spreading the sample, electrospinning, and droplet deposition. In some embodiments, the removing comprises at least three selected from the group consisting of direct extrusion, casting thin film by spreading the sample, electrospinning, and droplet deposition. In some embodiments, the removing comprises and / or consists of direct extrusion, casting thin film by spreading the sample, electrospinning, and droplet deposition. In some embodiments, the removing comprises direct extrusion. In some embodiments, the removing comprises casting thin film by spreading the sample. In some embodiments, the removing comprises electrospinning. In some embodiments, the removing comprises droplet deposition. In some embodiments, the method excludes generating a layer from the sample. In some embodiments, the method includes generating a layer from the sample.

[0012] In another aspect, the methods of the present disclosure comprise, after the removing, freeze-drying of the sample. In some embodiments, the freeze-drying comprises lowering a temperature. In some embodiments, the freeze-drying comprises lowering a temperature to below about 0, -5, -10, -15, -20, -25, -30, -35 °C, or less. In some embodiments, the freeze-drying comprises lowering a temperature to about 0, -5, -10, -15, -20, -25, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, -40 °C, or less. In some embodiments, the freeze-drying comprises lowering a temperature to below about 0 °C. In some embodiments, the freeze-drying comprises lowering a temperature of about -34 °C. In some embodiments, the freeze-dryingcomprises lowering a temperature to from about 0 to -40 °C, from about -5 to -40 °C, from about -10 to -40 °C, from about -15 to -40 °C, from about -20 to -40 °C, from about -25 to -40 °C, or from about -30 to -40 °C.

[0013] In some embodiments, the freeze-drying comprises lowering a temperature at a rate of from about 5 °C to -100 °C in 10 minutes to 200 minutes, from about 5 °C to -100 °C in 20 minutes to 120 minutes, from about 5 °C to -50 °C in 10 minutes to 200 minutes, from about 5 °C to -50 °C in 20 minutes to 120 minutes, from about 4 °C to -50 °C in 20 minutes to 120 minutes, from about 3 °C to -50 °C in 20 minutes to 120 minutes, from about 2 °C to -50 °C in 20 minutes to 120 minutes, from about 1 °C to -50 °C in 20 minutes to 120 minutes, from about 0 °C to -50 °C in 20 minutes to 120 minutes, from about 3 °C to -90 °C in 20 minutes to 120 minutes, from about 3 °C to -80 °C in 20 minutes to 120 minutes, from about 3 °C to -70 °C in 20 minutes to 120 minutes, from about 3 °C to -60 °C in 20 minutes to 120 minutes, from about 3 °C to -55 °C in 20 minutes to 120 minutes, from about 3 °C to -45 °C in 20 minutes to 120 minutes, from about 3 °C to -40 °C in 20 minutes to 120 minutes, from about 3 °C to -50 °C in 25 minutes to 120 minutes, from about 3 °C to -50 °C in 30 minutes to 120 minutes, from about 3 °C to -50 °C in 40 minutes to 120 minutes, from about 3 °C to -50 °C in 50 minutes to 120 minutes, from about 3 °C to -50 °C in 60 minutes to 120 minutes, from about 3 °C to -50 °C in 70 minutes to 120 minutes, from about 3 °C to -50 °C in 80 minutes to 120 minutes, from about 3 °C to -50 °C in 90 minutes to 120 minutes, from about 3 °C to -50 °C in 100 minutes to 120 minutes, from about 3 °C to -50 °C in 20 minutes to 110 minutes, from about 3 °C to -50 °C in 20 minutes to 100 minutes, from about 3 °C to -50 °C in 20 minutes to 90 minutes, from about 3 °C to -50 °C in 20 minutes to 80 minutes, from about 3 °C to -50 °C in 20 minutes to 70 minutes, from about 3 °C to -50 °C in 20 minutes to 60 minutes, from about 3 °C to -50 °C in 20 minutes to 50 minutes, from about 3 °C to -50 °C in 20 minutes to 40 minutes, or from about 3 °C to -50 °C in 20 minutes to 30 minutes. In some embodiments, the freeze-drying comprises lowering a temperature at a rate of about 3 °C to -50 °C in 20 minutes, 3 °C to -50 °C in 30 minutes, 3 °C to -50 °C in 40 minutes, 3 °C to -50 °C in 50 minutes, 3 °C to -50 °C in 60 minutes, 3 °C to -50 °C in 70 minutes, 3 °C to -50 °C in 80 minutes, 3 °C to -50 °C in 90 minutes, 3 °C to -50 °C in 100 minutes, 3 °C to -50 °C in 110 minutes, or 3 °C to -50 °C in 120 minutes. In some embodiments, the freeze-drying comprises lowering a temperature at a rate of from about 3 °C to -50 °C in 20 minutes to 120 minutes.

[0014] In some embodiments, the freeze-drying comprises lowering a pressure. In some embodiments, the freeze-drying comprises lowering a pressure to below about 1, 0.5, 0.1, 0.001 atm, or less. In some embodiments, the freeze-drying comprises lowering a pressure to below about 1 Torr, 0.1 Torr, 90 mTorr, 80 mTorr, 70 mTorr, 60 mTorr, 50 mTorr, 40 mTorr, 30 mTorr, 20 mTorr, 10 mTorr, 5 mTorr, 1 mTorr, 0.5 mTorr, 0.1 mTorr, or less. In some embodiments, the freeze-drying comprises lowering a pressure to about 1 Torr, 0.1 Torr, 90 mTorr, 80 mTorr, 70 mTorr, 60 mTorr, 50 mTorr, 40 mTorr, 30 mTorr, 20 mTorr, 10 mTorr, 5 mTorr, 1 mTorr, 0.5 mTorr, or 0.1 mTorr. In some embodiments, freeze-drying comprises lowering a pressure to below 50 mTorr. In some embodiments, freeze-drying comprises lowering a pressure to about 50 mTorr. In some embodiments, freeze-drying comprises lowering a pressure to from about 1 Torr to 0.1 mTorr, from about 0.1 Torr to 0.1 mTorr, from about 90 mTorr to 0.1 mTorr, from about 80 mTorr to 0.1 mTorr, from about 70 mTorr to 0.1 mTorr, from about 60 mTorr to 0.1 mTorr, from about 50 mTorr to 0.1 mTorr, from about 90 mTorr to 10 mTorr, from about 90 mTorr to 20 mTorr, from about 90 mTorr to 30 mTorr, from about 90 mTorr to 40 mTorr, from about 80 mTorr to 10 mTorr, from about 70 mTorr to 10 mTorr, from about 60 mTorr to 10 mTorr, or from about 50 mTorr to 10 mTorr.

[0015] In some embodiments, the freeze-drying comprises removing oxygen from environment of the sample. In some embodiments, the removing comprises removing at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more of oxygen from the sample. In some embodiments, the removing comprises removing from about 5 to 99%, from about 10 to 99%, from about 15 to 99%, from about 20 to 99%, from about 25 to 99%, from about 30 to 99%, from about 35 to 99%, from about 40 to 99%, from about 45 to 99%, from about 50 to 99%, from about 55 to 99%, from about 60 to 99%, from about 65 to 99%, from about 70 to 99%, from about 75 to 99%, from about 80 to 99%, from about 85 to 99%, from about 90 to 99%, from about 95 to 99% of oxygen from the sample. In some embodiments, the freeze-drying comprises removing at least about 99% of oxygen from the sample.

[0016] In some embodiments, the method excludes freezing the sample prior to the freeze-drying. In some embodiments, the method excludes lowering a temperature to below the freezing temperature of water (i.e., 0 °C) prior to the freeze-drying of the sample. In someembodiments, the method excludes lowering a temperature to the freezing temperature of water (i.e., 0 °C) or below prior to the freeze-drying of the sample. In some embodiments, the method excludes lowering a temperature to about 0, -1, -10, -100 °C, or below prior to the freeze-drying of the sample. In some embodiments, the method excludes lowering a temperature to 0 °C or below prior to the freeze-drying of the sample. In some embodiments, the method excludes lowering a temperature to below 0 °C prior to the freeze-drying of the sample.

[0017] In some embodiments, the method excludes freezing the sample after the freeze-drying of the sample. In some embodiments, the method excludes lowering a temperature to 0 °C or below after the freeze-drying of the sample.

[0018] In some embodiments, the method includes freezing the sample prior to the freeze-drying of the sample. In some embodiments, the method includes lowering a temperature to 0 °C or below prior to the freeze-drying of the sample.

[0019] In some embodiments, the method includes freezing the sample after the freeze-drying of the sample. In some embodiments, the method includes lowering a temperature to 0 °C or below after the freeze-drying of the sample.

[0020] In one aspect, the methods of the present disclosure further comprise storing the sample. As used herein, the term “storage” or “storing” as it relates to the sample is not particularly limited to any specific period of time. For example, storing the sample can be over a period of time including 1 second, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, 10 years, etc. In some embodiments, storing comprises storing the sample at a room temperature. In some embodiments, the room temperature comprises a temperature from about 18 to 25 °C, from about 19 to 25 °C, from about 20 to 25 °C, from about 21 to 25 °C, from about 22 to 25 °C, from about 18 to 24 °C, from about 18 to 23 °C, from about 18 to 22 °C, or from about 20 to 22 °C. In some embodiments, the room temperature comprises a temperature of about 18, 19, 20, 21, 22, 23, 24, or 25 °C. In some embodiments, storing comprises storing the sample at a refrigeration temperature (e.g., from about 1 to 5 °C, or about 1, 2, 3, 4, or 5 °C). In some embodiments, storing comprises storing the sample at or below the freezing temperature of water (e.g., <0 °C, or about 0, -10, -20, -30, -50, -80, -100, or -200 °C). In some embodiments, the method excludes storing the sample.

[0021] In one aspect, the methods of the present disclosure further comprise preserving thesample. As used herein, definition of the term “preserve” or “preserving” as it relates to the sample is understood by one of skill in the art. In some embodiments, preserving relates to maintaining the activity, integrity, and / or viability of a biological material (e.g., a cell, bacteria, a virus, a fungus, DNA, and RNA). In some embodiments, preserving comprises preserving the sample at a room temperature. In some embodiments, the room temperature comprises a temperature of from about 18 to 25 °C, from about 19 to 25 °C, from about 20 to 25 °C, from about 21 to 25 °C, from about 22 to 25 °C, from about 18 to 24 °C, from about 18 to 23 °C, from about 18 to 22 °C, or from about 20 to 22 °C. In some embodiments, the room temperature comprises a temperature of about 18, 19, 20, 21, 22, 23, 24, or 25 °C. In some embodiments, preserving comprises preserving the sample at a refrigeration temperature (e.g., from about 1 to 5 °C, or about 1, 2, 3, 4, or 5 °C). In some embodiments, preserving comprises preserving the sample at or below the freezing temperature of water (e.g., <0 °C, or about 0, -10, -20, -30, -50, -80, -100, or -200 °C). In some embodiments, the method excludes preserving the sample.

[0022] In one aspect, the methods of the present disclosure comprise a sample comprising a lyoprotective agent. In some embodiments, the sample comprises the lyoprotective agent at an amount of about 100 mmol, 200 mmol, 300 mmol, 400 mmol, 500 mmol, 600 mmol, 700 mmol, 800 mmol, 900 mmol, 1 mol, 2 mol, 3 mol, 4 mol, 5 mol, or more. In some embodiments, the sample comprises the lyoprotective agent at an amount of from about 100 mmol to 5 mol, from about 200 mmol to 5 mol, from about 300 mmol to 5 mol, from about 400 mmol to 5 mol, from about 500 mmol to 5 mol, from about 600 mmol to 5 mol, from about 500 mmol to 4 mol, from about 500 mmol to 3 mol, from about 500 mmol to 2 mol, from about 500 mmol to 1 mol, from about 500 mmol to 900 mmol, from about 500 mmol to 800 mmol, from about 500 mmol to 700 mmol, from about 400 mmol to 800 mmol, or from about 600 mmol to 1 mol. In some embodiments, the sample comprises the lyoprotective agent at an amount of about 600 mmol. In some embodiments, the sample comprises the lyoprotective agent at an amount of about 600 mmol or more.

[0023] In some embodiments, the sample comprises the lyoprotective agent at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 17, 18, 19, 20% (w / v), or more. In some embodiments, the sample comprises the lyoprotective agent at an amount of from about 1 to 20% (w / v), from about 1 to 19% (w / v), from about 1 to 18% (w / v), from about 1 to 17% (w / v), fromabout 1 to 16% (w / v), from about 1 to 15% (w / v), from about 1 to 14% (w / v), from about 1 to 13% (w / v), from about 1 to 12% (w / v), from about 1 to 11% (w / v), from about 1 to 10% (w / v), from about 2 to 20% (w / v), from about 3 to 20% (w / v), from about 4 to 20% (w / v), from about 5 to 20% (w / v), from about 6 to 20% (w / v), from about 7 to 20% (w / v), from about 8 to 20% (w / v), from about 9 to 20% (w / v), from about 10 to 20% (w / v), or from about 5 to 15% (w / v). In some embodiments, the sample comprises the lyoprotective agent at a concentration of about 10% (w / v) or more. In some embodiments, the sample comprises the lyoprotective agent at a concentration of about 10% (w / v).

[0024] Any suitable lyoprotective agent can be used in accordance with the methods of the present disclosure. In some embodiments, the lyoprotective agent is a monosaccharide, a disaccharide, a sugar, a sugar alcohol, a sugar acid, an alcohol, a polyol, an amino acid, a salt of an amino acid, a choline, or any derivatives thereof. In some embodiments, the lyoprotective agent comprises at least one selected from the group consisting of trehalose, sorbitol, mannitol, glycerol, sodium glutamate, choline, lactobionate, and betine. In some embodiments, the lyoprotective agent comprises at least two selected from the group consisting of trehalose, sorbitol, mannitol, glycerol, sodium glutamate, choline, lactobionate, and betine. In some embodiments, the lyoprotective agent comprises at least three selected from the group consisting of trehalose, sorbitol, mannitol, glycerol, sodium glutamate, choline, lactobionate, and betine. In some embodiments, the lyoprotective agent comprises at least four selected from the group consisting of trehalose, sorbitol, mannitol, glycerol, sodium glutamate, choline, lactobionate, and betine. In some embodiments, the lyoprotective agent comprises at least five selected from the group consisting of trehalose, sorbitol, mannitol, glycerol, sodium glutamate, choline, lactobionate, and betine. In some embodiments, the lyoprotective agent comprises at least six selected from the group consisting of trehalose, sorbitol, mannitol, glycerol, sodium glutamate, choline, lactobionate, and betine. In some embodiments, the lyoprotective agent comprises and / or consists of trehalose, sorbitol, mannitol, glycerol, sodium glutamate, choline, lactobionate, and betine. In some embodiments, the lyoprotective agent comprises trehalose. In some embodiments, the lyoprotective agent comprises sorbitol. In some embodiments, the lyoprotective agent comprises mannitol. In some embodiments, the lyoprotective agent comprises glycerol. In some embodiments, the lyoprotective agent comprises sodium glutamate. In some embodiments, the lyoprotective agent comprises choline. In some embodiments, thelyoprotective agent comprises lactobionate. Tn some embodiments, the lyoprotective agent comprises betine.

[0025] In some embodiments, the sample excludes trehalose. In some embodiments, the sample includes trehalose.

[0026] In one aspect, the methods of the present disclosure comprise a sample comprising sodium glutamate. In some embodiments, the sample excludes sodium glutamate. In some embodiments, the sample comprises sodium glutamate at a concentration of from about 0.1 to 5% (w / v), from about 0.1 to 1.9% (w / v), from about 0.1 to 1.8% (w / v), from about 0.1 to 1.7% (w / v), from about 0.1 to 1.6% (w / v), from about 0.1 to 1.5% (w / v), from about 0.1 to 1.4% (w / v), from about 0.1 to 1.3% (w / v), from about 0.1 to 1.2% (w / v), from about 0.1 to 1.1% (w / v), from about 0.1 to 1% (w / v), from about 0.2 to 2% (w / v), from about 0.3 to 2% (w / v), from about 0.4 to 2% (w / v), from about 0.5 to 2% (w / v), from about 0.6 to 2% (w / v), from about 0.7 to 2% (w / v), from about 0.8 to 2% (w / v), from about 0.9 to 2% (w / v), from about 1 to 2% (w / v), from about 0.5 to 1.5% (w / v), from about 1 to 5% (w / v), from about 0.5 to 5% (w / v), from about 0.1 to 4% (w / v), from about 0.1 to 3% (w / v), or from about 0.1 to 2% (w / v). In some embodiments, the sample comprises sodium glutamate at a concentration of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, or 5% (w / v). In some embodiments, the sample comprises sodium glutamate at a concentration of from about 0.1% (w / v) to 5% (w / v). In some embodiments, the sample comprises sodium glutamate at a concentration of from about 0.5% (w / v) to 1.5% (w / v). In some embodiments, the sample comprises sodium glutamate at a concentration of about 1% (w / v).

[0027] In one aspect, the methods of the present disclosure comprise a sample comprising sugar. In some embodiments, the sample excludes sugar. In some embodiments, the sample comprises sugar at a concentration of from about 0.01 to 5% (w / v), from about 0.1 to 1.5% (w / v), from about 0.5 to 1.5% (w / v), from about 0.1 to 0.9% (w / v), from about 0.1 to 0.8% (w / v), from about 0.1 to 0.7% (w / v), from about 0.1 to 0.6% (w / v), from about 0.1 to 0.5% (w / v), from about 0.2 to 1.0% (w / v), from about 0.3 to 1.0% (w / v), from about 0.4 to 1.0% (w / v), from about 0.5 to 1.0% (w / v), from about 0.01 to 0.05% (w / v), from about 0.01 to 0.1% (w / v), from about 0.01 to 0.5% (w / v), from about 0.01 to 1% (w / v), or from about 0.01 to 1.5% (w / v). In some embodiments, the sample comprises sugar at a concentration of about 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35,0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5% (w / v). In some embodiments, the sample comprises sugar at a concentration of from about 0.1% (w / v) to 1.0% (w / v). In some embodiments, the sample comprises sugar at a concentration of about 0.5% (w / v). In some embodiments, the sample comprises sugar at a concentration of about 0.25% (w / v).

[0028] Any suitable sugar can be used in accordance with the methods of the present disclosure. In some embodiments, the sugar comprises a sugar alcohol, a sugar acid, a carbohydrate, a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide, or any derivatives thereof. In some embodiments, the sugar comprises dextrose, fructose, galactose, glucose, lactose, trehalose, maltose, sucrose, erythritol, maltitol, sorbitol, mannitol, xylitol, lactitol, isomalt, N-acetylglucosamine, hydrogenated starch hydrolysates, or any derivatives thereof. In some embodiments, the sugar comprises at least one selected from the group consisting of sorbitol, mannitol, and sucrose. In some embodiments, the sugar comprises at least two selected from the group consisting of sorbitol, mannitol, and sucrose. In some embodiments, the sugar comprises and / or consists of sorbitol, mannitol, and sucrose. In some embodiments, the sugar comprises and / or consists of sorbitol. In some embodiments, the sugar comprises and / or consists of mannitol. In some embodiments, the sugar comprises and / or consists of sucrose.

[0029] In some embodiments, the sugar comprises sorbitol at a concentration of about 0.25% (w / v). In some embodiments, the sugar comprises mannitol at a concentration of about 0.25% (w / v). In some embodiments, the sugar comprises sorbitol and mannitol at a concentration of about 0.5% (w / v). In some embodiments, the sugar comprises sorbitol and mannitol at a concentration of from about 0.01 to 5% (w / v). In some embodiments, the sugar comprises sorbitol and mannitol at a concentration of from about 0.1 to 1.0% (w / v). In some embodiments, the sugar comprises sorbitol at a concentration of about 0.25% (w / v) and mannitol at a concentration of about 0.25% (w / v).

[0030] In one aspect, the methods of the present disclosure comprise a sample comprising a surfactant. In some embodiments, the sample excludes a surfactant. In some embodiments, the sample comprises the surfactant at a concentration of from about 0.01 to 0.1% (v / v), from about 0.02 to 0.1% (v / v), from about 0.03 to 0.1% (v / v), from about 0.04 to 0.1% (v / v), from about 0.05 to 0.1% (v / v), from about 0.01 to 0.09% (v / v), from about 0.01 to 0.08% (v / v), from about0.01 to 0.07% (v / v), from about 0.01 to 0.06% (v / v), or from about 0.01 to 0.05% (v / v). In some embodiments, the sample comprises the surfactant at a concentration of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1% (v / v). In some embodiments, the sample comprises a surfactant at a concentration of from about 0.01% (v / v) to 0.1% (v / v). In some embodiments, the sample comprises a surfactant at a concentration of about 0.05% (v / v).

[0031] Any suitable surfactant can be used in accordance with the methods of the present disclosure. Surfactants are typically organic compounds that are amphiphilic, i.e., containing both hydrophobic groups (“tails”) and hydrophilic groups (“heads”), which render surfactants soluble in both organic solvents and water. A surfactant can be classified by the presence of formally charged groups in its head. A non-ionic surfactant has no charge groups in its head, whereas an ionic surfactant carries a net charge in its head. A zwitterionic surfactant contains a head with two oppositely charged groups. Some examples of common surfactants include: 1) anionic surfactants (based on sulfate, sulfonate or carboxylate anions) such as perfluorooctanoate (PFOA or PFO), perfluorooctanesulfonate (PFOS), sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, and other alkyl sulfate salts, sodium laureth sulfate (also known as sodium lauryl ether sulfate, or SLES), and alkyl benzene sulfonate; 2) cationic surfactants (based on quaternary ammonium cations) such as cetyl trimethylammonium bromide (CTAB) (i.e., hexadecyl trimethyl ammonium bromide) and other alkyltrimethylammonium salts, cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC), benzethonium chloride (BZT); 3) long chain fatty acids and their salts, such as caprylate, caprylic acid, heptanoate, hexanoic acid, heptanoic acid, nonanoic acid, decanoic acid, and the like; 4) zwitterionic (amphoteric) surfactants, such as dodecyl betaine, cocamidopropyl betaine, and coco ampho glycinate; and 5) nonionic surfactants, such as alkyl polyethylene oxide), alkylphenol poly(ethylene oxide), copolymers of poly(ethylene oxide) and polypropylene oxide) (commercially known as Poloxamers or Poloxamines), alkyl polyglucosides including octyl glucoside, decyl maltoside, fatty alcohols (e.g., cetyl alcohol and oleyl alcohol), cocamide MEA, cocamide DEA, polysorbates (polysorbate 20 (e.g., Tween® 20), polysorbate 80 (e.g., Tween® 80), etc ), Triton® detergents, Tyloxapol, and dodecyl dimethylamine oxide. In some embodiments, the surfactant comprises Tyloxapol and / or polysorbate 80 (e.g., Tween® 80). In some embodiments, the surfactant comprises Tyloxapol. In some embodiments, the surfactant comprises polysorbate 80 (e.g., Tween® 80).

[0032] In one aspect, the methods of the present disclosure comprise a sample including a cell. In some embodiments, the sample excludes a cell. In some embodiments, the sample includes at least one cell. In some embodiments, the sample includes more than one cell.

[0033] In some embodiments, the sample includes a human cell. In some embodiments, the sample includes an animal or non-human cell. In some embodiments, the sample includes a mammalian cell. In some embodiments, the sample excludes a mammalian cell. In some embodiments, the sample excludes an animal or non-human cell. In some embodiments, the sample excludes a mammalian cell.

[0034] Examples of animals, e.g., non-human animals, e.g., non-human mammals, include, but are not limited to, non-human primates; mammals, including commercially relevant mammals, e.g., pet and live-stock animals, such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0035] In some embodiments, the sample includes a tissue sample from a subject. In some embodiments, the sample excludes a tissue sample from a subject. In some embodiments, the sample is a tissue sample from a subject. In some embodiments, the subject is a human. In some embodiments, the subject is an animal. In some embodiments, the subject is a plant. A tissue sample from a human or animal includes tissue comprising connective tissue, epithelial tissue, muscle tissue, or nervous tissue; for example, brain tissue, spinal cord tissue, and tissue from specific regions of the central nervous system, blood, plasma, serum, urine, sputum, cerebrospinal fluid, milk, ductal fluid samples, cartilage (e g., hyaline cartilage, elastic cartilage, and fibrocartilage), bone, loose connective issue (e.g., areolar connective tissue, adipose connective tissue, and reticular connective tissue), and dense connective tissue proper (e.g., dense regular connective tissue, dense irregular connective tissue, and elastic connective tissue). A tissue sample from a plant includes tissue comprising ground tissue (e.g., epidermis and stomata), vascular tissue (e.g., parenchyma, collenchyma, and sclerenchyma), and dermal tissue (e g., xylem, and phloem).

[0036] In some embodiments, the sample includes at least one selected from the group consisting of bacteria, virus, and fungus. Examples of bacteria include, but are not limitedto, Acinetobacter baumanii, Actinobacillus sp., Actinomycetes Actinomyces sp. (suchas Actinomyces israelii and Actinomyces naeslundii), Aeromonas sp. (such as Aeromonas hydrophUa. Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonascaviae), Anaplasma phagocy tophilum, Anaplasma marginale Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis, Bacillus cereus, Bacillus subtiHs, Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroides sp. (such as Bacteroides fragilis), Bartonella sp. (such as Bartonella bacilliformis and Bartonella henselae, Bifidobacterium sp., Bordetella sp. (such as Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), Borrelia sp. (such as Borrelia recurrentis, and Borrelia burgdorferi'), Brucella sp. (such as Brucella abortus, Brucella canis, Brucella melintensis and Brucella suis), Burkholderia sp. (such as Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter sp. (such as Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacterfetus), Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as, Corynebacterium diphtheriae, Corynebacteriumjeikeum and Corynebacterium), Clostridium sp. (such as Clostridium perft ingens, Clostridium dificile, Clostridium botulinum and Clostridium tetani), Eikenella corr odens, Enterobacter sp. (such as Enterobacter aerogenes, Enterobacter agglomerans, Enterobactercloacae and Escherichia coli, including opportunistic Escherichia coli, such as enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli and uropathogenic E. coli) Enterococcus sp. (suchas Enterococcus faecalis and Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia and Ehrlichia canis), Epidermophyton floccosum, Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp. (such as Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae , Haemophilushaemolyticus and Haemophilus parahaemolyticus, Helicobacter sp. (such as Helicobacter pylori, Helicobacter cinaedi and Helicobacter fennelliae), Kingella kingii, Klebsiella sp. (such as Klebsiella pneumoniae, Klebsiella granulomatis and Klebsiellaoxy toed), Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Mannheimia hemolytica,Microsporum canis, Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp., Mycobacterium sp. (such as Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium paratuberculosis, Mycobacterium intr acellular e, Mycobacterium avium, Mycobacterium bovis, and Mycobacterium marinum), My coplasm sp. (such as Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp. (such as Nocardia aster oides. Nocardia cyriacigeorgica and Nocardia brasiliensis), Neisseria sp. (such as Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella multocida, Pityrosporum orbiculare (Malassezia furfur), Plesiomonas shigelloides. Prevotella sp., Porphyromonas sp., Prevote Ila melaninogenica, Proteus sp. (such as Proteus vulgaris and Proteus mirabilis), Providencia sp. (such as Providencia alcalifaciens, Providencia rettgeri and Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Rickettsia sp. (such as Rickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii , Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) and Rickettsia typhi), Rhodococcus sp., Serratia marcescens, Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis and Salmonella typhimurium), Serratia sp. (such as Serratia marcesans and Serratia liquifaciens), Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei), Staphylococcus sp. (such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus), Streptococcus sp. (such as Streptococcus pneumoniae (for example chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, erythromycin-resistant serotype 14 Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae , tetracycline-resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, and trimethoprim-resistant serotype 23F Streptococcus pneumoniae, chloramphenicol -resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9 V Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, or trimethoprim-resistant serotype 23F Streptococcus pneumoniae). Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes, GroupA streptococci, Streptococcus pyogenes, Group B streptococci, Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus, Streptococcus equismilis, Group D streptococci, Streptococcus bovis, Group F streptococci, and Streptococcus anginosus Group G streptococci), Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such as Treponema carateum, Treponema petenue, Treponema pallidum and Treponema endemicum, Trichophyton rubrum, T. mentagrophytes, Tropherymawhippelii, Ureaplasma urealyticum, Veillonella sp., Vibrio sp. (such as Vibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela and Vibrio furnish), Yersinia sp. (such as Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis) and Xanthomonas maltophilia.

[0037] Examples of viruses include, but are not limited to, Adeno-associated virus, Aichi virus, Australian bat lyssavirus, BK polyomavirus, Banna virus, Barmah forest virus, Bunyamwera virus, Bunyavirus La Crosse, Bunyavirus snowshoe hare, Cercopithecine herpesvirus, Chandipura virus, Chikungunya virus, Coronavirus, Cosavirus A, Cowpox virus, Coxsackievirus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dhori virus, Dugbe virus, Duvenhage virus, Eastern equine encephalitis virus, Ebolavirus, Echovirus, Encephalomyocarditis virus, Epstein-Barr virus, European bat lyssavirus, GB virus C / Hepatitis G virus, Hantaan virus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis delta virus, Horsepox virus, Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus 68, 70, Human herpesvirus 1, Human herpesvirus 2, Human herpesvirus 6, Human herpesvirus 7, Human herpesvirus 8, Human immunodeficiency virus, Human papillomavirus 1, Human papillomavirus 2, Human papillomavirus 16,18, Human parainfluenza, Human parvovirus Bl 9, Human respiratory syncytial virus, Human rhinovirus, Human SARS coronavirus, Human spumaretrovirus, Human T-lymphotropic virus, Human torovirus, Influenza A virus, Influenza B virus, Influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Junin arenavirus, KI Polyomavirus, Kunjin virus, Lagos bat virus, Lake Victoria Marburgvirus, Langat virus, Lassa virus, Lordsdale virus, Louping ill virus, Lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, MERS coronavirus, Measles virus, Mengo encephalomyocarditis virus, Merkel cell polyomavirus, Mokola virus, Molluscum contagiosum virus, Monkeypox virus, Mumps virus, Murray valley encephalitis virus, New York virus, Nipah virus, Norwalk virus, Norovirus,O’nyong-nyong virus, Orf virus, Oropouche virus, Pichinde virus, Poliovirus, Punta toro phlebovirus, Puumala virus, Rabies virus, Rift valley fever virus, Rosavirus A, Ross river virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Salivirus A, Sandfly fever Sicilian virus, Sapporo virus, Semliki forest virus, Seoul virus, Severe acute respiratory syndrome coronavirus 2, Simian foamy virus, Simian virus 5, Sindbis virus, Southampton virus, St. louis encephalitis virus, Tick-borne powassan virus, Torque teno virus, Toscana virus, Uukuniemi virus, Vaccinia virus, Varicella-zoster virus, Variola virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, WU polyomavirus, West Nile virus, Yaba monkey tumor virus, Yaba-like disease virus, Yellow fever virus, and Zika virus.

[0038] Examples of fungi include, but are not limited to, Aspergillus Blastomyces dermatitidis, Blastomyces gilchrislii, Candida auris, Coccidioides immitis, Cryptococcus neoformans.Cryptococcus gattii, Histoplasma, Rhizopus oryzae, Rhizopus microspores, Lichtheimia corymbifera, Rhizomucor pusillus, Fusarium, Scedosporium, Lomentospora, Mucormycetes, Pneumocystis jirovecii, Trichophyton, Microsporum (e.g.,M. audouinii,M. canis and AT. ferrugineum), Epidermophyton floccosum, Sporothrix brasiliensis, Talaromyces marneffei, Rhizoctonia solani, Alternaria solani, Botrytis cinerea, Sclerotinia sclerotiorum, Blumeria graminis, Colletotrichum gloeosporioides, Fusarium solani, Gibberella fujikuroi, Mycosarcoma maydis, Gibberella zeae, Monilinia fructicola, Armillaria mellea, Fusarium oxysporum, Pythium ultimum, Alternaria alternata, Sclerotium rolfsii, Verticillium albo-atrum, Verticillium dahlia, Erysiphe cichoracearum, Gymnosporangium juniperi-virginianae, Pythium aphanidermatum, Thielaviopsis basicola, Taphrina caerulescens, Venturia inaequalis, Albugo Candida, Colletotrichum graminicola, Phytophthora cactorum, Puccinia graminis, Gymnosporangium clavipes, Gymnosporangium globosum, Apiosporina morbosa, Ophiostoma ulmi, Bipolaris sorokiniana, Botryosphaeria obtuse, Claviceps purpurea, Gaeumannomyces graminis, Ustilago tritici, Macrophomina phaseolina, Erysiphe betae, Heterobasidion annosum, Nectria cinnabarina, Phoma terrestris, Puccinia recondite, Pythium irregulare, Sphaeropsis sapinea, Alternaria brassicae, Bipolaris maydis, Ganoderma lucidum, Penicillium expansum, Phytophthora cinnamomic, Cladosporium herbarum, Exserohilum turcicum, Ganoderma applanatum, Peronospora parasitica, Septoria tritici, Taphrina communis, Trametes versicolor, Fusarium culmorum, Glomerella graminicola, Pseudopeziza medicaginis, Laetiporus sulphureus, Puccinia coronate, Botryosphaeria ribis, Diplocarpon rosae, Podosphaerafuhginea. Pythium debaryanum, Sclerotinia trifoliorum, Stagonosporci nodorum, Stemphylium botryosum, Agaricus bisporus, Lentinula edodes, Pleurotus ostreatus, Pleurotus eryngii, Flammulina filiformis, Tuber melanosporum, and Psilocybe cubensis.

[0039] In some embodiments, the sample includes at least two selected from the group consisting of bacteria, virus, and fungus. In some embodiments, the sample comprises and / or consists of bacteria, virus, and fungus. In some embodiments, the sample comprises and / or consists of bacteria. In some embodiments, the sample comprises and / or consists of a virus. In some embodiments, the sample comprises and / or consists of a fungus.

[0040] In some embodiments, the sample includes an isolated nucleotide molecule.“Nucleotide” or “nucleotide molecule” used herein means either a deoxyribonucleotide or a ribonucleotide or any nucleotide analogue (e.g., DNA and RNA). “Polynucleotide” or “oligonucleotide” are used interchangeably and each means a linear polymer of nucleotide monomers. Monomers making up polynucleotides and oligonucleotides are capable of specifically binding to a natural and / or artificial polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Such monomers and their internucleosidic linkages may be naturally occurring or may be analogues thereof, e.g., naturally occurring or non-naturally occurring analogues. Polynucleotides typically range in size from a few monomeric units when they are referred to as “oligonucleotides” to several thousand monomeric units. Whenever a polynucleotide or oligonucleotide is represented by a sequence of letters (upper or lower case), such as “ATGCCTG,” it will be understood that the nucleotides are in 5’ — >3 ’ order from left to right. Usually, polynucleotides comprise the four natural nucleosides (e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxy thy mi dine for DNA or their ribose counterparts for RNA) linked by phosphodiester linkages.

[0041] In some embodiments, the sample includes an isolated amino acid molecule. An “amino acid molecule” used herein means either a single amino acid, a polypeptide, or a protein.“Protein” herein means at least two amino acids linked together by a peptide bond. Accordingly, each respective protein is defined by a sequence of amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline), which is linked by bonds. As such, the sequence of amino acids is a linear sequence of a positions having an initial N-terminal position, one or more intermediate positions, and a C-terminal position.

[0042] The term “isolate” refers to a molecule that is substantially free of its natural environment. For instance, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived.

[0043] In some embodiments, the sample comprises and / or consists of a solution. As used herein the term “solution” broadly refers to a homogeneous mixture composed of one phase, including but not limited to aqueous mixtures, nonaqueous mixtures, suspensions, emulsions, drops, ointments, sprays, and liposomes. Typically, a solution comprises a solute or solutes dissolved in a solvent or solvents. A solvent can be aqueous or nonaqueous (e.g., methanol, ethanol, 1 -propanol, 2-propanol, ethyl acetate, acetone, acetonitrile, dimethylsulfoxide, diethyl ether, ethylene glycol, glycerin, hexane, tetrahydrofuran, chloroform, etc.). It is characterized in that the properties of the mixture (such as concentration, temperature, and density) can be uniformly distributed through the volume. Aqueous solutions may comprise a buffer (e g., phosphate buffer, citric acid / Na2HPC>4, citric acid / sodium citrate, sodium acetate / acetic acid, imidazole / HCl, sodium carbonate / sodium bicarbonate, phosphate buffered saline, HEPES, MES, Bis-Tris, PIPES, MOPSO, MOPS, TES, Tris, etc.) and / or may be acidic (pH<7), basic (pH>7), or neutral (pH=7). In some embodiments, the sample comprises an aqueous solution. In some embodiments, the sample comprises a nonaqueous solution. In some embodiments, the sample comprises an aqueous solution comprising a buffer.

[0044] In some embodiments, the sample is adhered to a substrate. In some embodiments, the sample is not adhered to a substrate. “Substrate” refers to a material or group of materials having a rigid or semi-rigid surface or surfaces. In some embodiments, at least one surface of the substrate will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like. In additional embodiments, the substrate may comprise at least one planar solid phase support (e g., a glass microscope slide). According to other embodiments, the substrate(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations. In some embodiments, the substrate according to someembodiments of the present disclosure excludes beads, resins, gels, and / or microspheres. In some embodiments, the substrate is a multi-well plate (e.g., a 4-well plate, a 6-well plate, an 8-well plate, a 12-well plate, a 16-well plate, a 24-well plate, a 36-well plate, a 48-well plate, a 60-well plate, a 96-well plate, or a 384-well plate). In some embodiments, the multi-well plates comprise a polymer such as polycarbonate, polyethylene, polyester, polypropylene, polystyrene, a cycloolefin polymer, a vinyl polymer, or any blends, derivatives, or copolymers thereof.“Adhered” refers to the direct attachment of the sample to the substate, with or without an adhesive.

[0045] In one aspect, methods of the present disclosure provide a desiccated sample as described herein.

[0046] In one aspect, the use of the product or method is characterized by one or more elements disclosed in the application.

[0047] The term “and / or” used herein is defined to indicate any combination of the components. Moreover, the singular forms “a,” “an,” and “the” may further include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a sample” refers to one, more than one, or mixtures of such samples, and reference to “a method” may include reference to equivalent steps and methods known to those skilled in the art, and so forth.EXAMPLESExample 1: Methods of Desiccating a Sample

[0048] The following describes an exemplary use of fast and controlled pre-desiccation strategies in presence of lyoprotective agents to stabilize biological materials in easy-to-use form factors for cryogenic and non-cryogenic storage.

[0049] The technologies described herein are geared towards creation of novel preservation formats of stabilized biomaterials (including microbiological products) that improves current state through (1) convenient storage, (2) improvement of workflow, and (3) reduction of preprocessing time required to perform assay. It has the advantage of stabilizing the microbiological materials in a desired form factor.

[0050] An important feature is fast desiccation prior to processing for lyophilization.

[0001] The technique is used to create easy-to-use form factors for storage, stabilization, and distribution of biological materials at cryogenic and non-cryogenic temperatures. The technique involves creation of small form factor materials that allows faster desiccation, followed by freezing and sublimation drying. The flow-chart in Figure 1 depicts the different exemplary steps involved in the process. A detailed description of each of these processes is provided herein.

[0052] The process is significantly different from lyophilization and other fast desiccation techniques in general as it relies on following scientific premises:

[0053] (1) Fast desiccation while reducing the form factor.

[0054] The active reduction of the form factor allows the pre-desiccated samples to be processed in at a faster rate and this enables (i) fast processing in desired form factor, (ii) reduced exposure to biomolecular injury in non-vitrified environment and (iii) less energy intensive operation. This step can include several of the known reduced form factor processing techniques including spin drying, dip coating, spray drying, electrospinning, and directional deposition.

[0055] The process involves a variety of techniques including extrusion / deposition techniques with active biological materials (e.g., bacteria, viral samples, DNA, and RNA). The extrusion / deposition techniques that actively increases the surface area of the deposited material include direct extrusion, casting thin film by active spreading of the material, electrospinning, and droplet deposition. The increase in surface area is a critical concept that facilitates fast freezing induced drying and effective sublimation of the ice crystals.

[0056] (2) Minimization of ice-induced denaturation of biological materials through use of a systematic pre-desiccation technique before being exposed to low temperatures.

[0057] This aspect of the technique involves systematic pre-desiccation of the biological materials to prevent (or limit) formation of deleterious ice crystals having large surface areas and volume with a view to minimize ice-induced denaturation of biological materials. Formation of large ice crystals have been traditionally attributed to injury of biological material due to (i) physical damage of the biological structure and molecular assembly by advancing ice crystals, (ii) charge concentration on the tip of the advancing ice crystals capable of destabilizing thebiomolecular assembly, and (iii) localized dehydration and resulting hydrolysis of biomol ecul ar structures due to uneven nature of the localized osmotic upshock caused by rapidly advancing ice crystals in a particular direction.

[0058] Furthermore, injuries related to downstream secondary processing techniques in stabilization processing are directly influenced by the nature of the ice crystals formed. In lyophilization, smaller ice crystals can create better connected porous structure. However, using the traditional formats for lyophilization in vials, smaller ice crystals may result in requirement of use of strong negative pressure environment which often increases chances of failure and denaturation of the lyophilized material. However, use of controlled ice nucleation coupled with reduction of form factors processing induced damages to the biomolecules can be minimized. This can result in reduced secondary / downstream processing time for lyophilization, reduced chances of injury to the biomolecules in the secondary / downstream processing, and reduced energy requirement during secondary / downstream processing.

[0059] The technique uses a combination of physico-chemical strategies to achieve minimization of ice-induced denaturation of the biological material. This can be modulated by influencing size, number, and nature of the ice crystals formed (influences shape). Following strategies can be undertaken to actively modulate ice crystal properties.

[0060] Figure 2 and Figure 3 depict techniques for form factor reduction prior to lyophilization.

[0061] Figure 4 depicts a conceptual phase diagram of different preservation techniques in relation to ice formation and relative loss of water.

Claims

CLAIMS1. A method of desiccating a sample, the method comprisingremoving water from the sample at a temperature of 0 °C or above, andafter the removing, freeze-drying of the sample.

2. The method according to any one of the preceding claims, wherein the sample comprises a lyoprotective agent.

3. The method according to claim 2, wherein the sample comprises the lyoprotective agent at an amount of 600 mmol or more.

4. The method according to claim 2, wherein the sample comprises the lyoprotective agent at a concentration of about 10% (w / v) or more.

5. The method according to any one of claims 2-4, wherein the lyoprotective agent comprises at least one selected from the group consisting of trehalose, sorbitol, mannitol, glycerol, sodium glutamate, choline, lactobionate, and betine.

6. The method according to any one of claims 1-5, wherein sample excludes trehalose.

7. The method according to any of one of claims 1-6, wherein the sample comprises sodium glutamate.

8. The method according claim 7, wherein the sample comprises sodium glutamate at a concentration of from 0.1% (w / v) to 5% (w / v).

9. The method according to any one of the preceding claims, wherein the sample comprises sugar.

10. The method according to claim 9, wherein the sample comprises sugar at a concentration of from 0.01% (w / v) to 5.0% (w / v).

11. The method according to claim 9 or 10, wherein the sugar comprises at least one selected from the group consisting of sorbitol, mannitol, and sucrose.

12. The method according to any one of the preceding claims, wherein the sample comprises a surfactant.

13. The method according to claim 12, wherein the sample comprises a surfactant at aconcentration of from 0.01% (v / v) to 0.1% (v / v).

14. The method according to claim 12 or 13, wherein the surfactant comprises Tyloxapol or polysorbate 80.

15. The method according to any one of the preceding claims, wherein the removing comprises at least one selected from the group consisting of spin drying, dip coating, spray drying, electrospinning and directional deposition.

16. The method according to any one of the preceding claims, wherein the removing comprises drying using surface tension of the sample.

17. The method according to any one of the preceding claims, wherein the removing comprises extrusion or deposition of the sample.

18. The method according to any one of the preceding claims, wherein the removing comprises at least one selected from the group consisting of direct extrusion, casting thin film by spreading the sample, electrospinning, and droplet deposition.

19. The method according to any one of the preceding claims, excluding generating a layer from the sample.

20. The method according to any one of the preceding claims, wherein the removing is performed at above 0 °C.

21. The method according to any one of the preceding claims, wherein the removing is performed at 4 °C or above.

22. The method according to any one of the preceding claims, wherein the removing is performed at 10 °C or above.

23. The method according to any one of the preceding claims, wherein the removing is performed at a room temperature.

24. The method according to any one of the preceding claims, wherein the removing comprises removing at least 5% of water from the sample.

25. The method according to any one of the preceding claims, wherein the removing comprises removing at least 50% of water from the sample.

26. The method according to any one of the preceding claims, wherein the removing comprises removing at least 90% of water from the sample.

27. The method according to any one of the preceding claims, wherein the removing comprises removing at least 95% of water from the sample.

28. The method according to any one of the preceding claims, wherein the removing comprises removing at least 99% of water from the sample.

29. The method according to any one of the preceding claims, wherein the removing comprises removing water from the sample so that a water content of the sample is below 1 g water / g dry weight.

30. The method according to any one of the preceding claims, wherein the removing comprises removing water from the sample so that a water content of the sample is from 0.1 g water / dry weight to 0.6 g water / g dry weight.

31. The method according to any one of the preceding claims, wherein the removing comprises removing water at a rate from 1 g water / g dry weight / hr to 2 g water / g dry weight / hr.

32. The method according to any one of the preceding claims, excluding freezing the sample prior to the freeze-drying of the sample.

33. The method according to any one of the preceding claims, excluding lowering a temperature to 0 °C or below prior to the freeze-drying of the sample.

34. The method according to any one of the preceding claims, excluding lowering a temperature to below 0 °C prior to the freeze-drying of the sample.

35. The method according to any one of the preceding claims, wherein the freeze-drying comprises lowering a temperature to below 0 °C.

36. The method according to any one of the preceding claims, wherein the freeze-drying comprises lowering a temperature to -34 °C.

37. The method according to any one of the preceding claims, wherein the freeze-drying comprises lowering a temperature at a rate of from 3 °C to -50 °C in 20 minutes to 120 minutes.

38. The method according to any one of the preceding claims, wherein the freeze-dryingcomprises lowering a pressure.

39. The method according to any one of the preceding claims, wherein the freeze-drying comprises lowering a pressure to below 1 atm.

40. The method according to any one of the preceding claims, wherein the freeze-drying comprises lowering a pressure to below about 50 mTorr.

41. The method according to any one of the preceding claims, wherein the freeze-drying comprises removing oxygen from environment of the sample.

42. The method according to any one of the preceding claims, wherein the sample includes a cell.

43. The method according to any one of claims 1-41, wherein the sample excludes a cell.

44. The method according to any one of the preceding claims, wherein the sample excludes a mammalian cell.

45. The method according to any one of the preceding claims, wherein the sample includes at least one selected from the group consisting of bacteria, virus, and fungus.

46. The method according to any one of the preceding claims, wherein the sample includes an isolated nucleotide molecule or an isolated amino acid molecule.

47. The method according to any one of the preceding claims, wherein the sample is a tissue sample from a subject.

48. The method according to any one of the preceding claims, wherein the sample is a solution.

49. The method according to any one of the preceding claims, wherein the sample is adhered to a substrate.

50. The method according to any one of the preceding claims, wherein the sample is not adhered to a substrate.

51. The method according to any one of the preceding claims, further comprising storing the sample at a room temperature.

52. The method according to any one of the preceding claims, further comprising preservingthe sample at a room temperature.

53. A desiccated sample prepared by the method of any one of the preceding claims.

54. Use of the product or method characterized by one or more elements disclosed in the application.

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