Methods of freeze-drying a sample and use thereof

Abstract

The disclosure relates to methods of freeze-drying a sample and products prepared by the methods thereof.

Description

Attorney Docket No.124324-5002-WO METHODS OF FREEZE-DRYING A SAMPLE AND USE THEREOF BACKGROUND

[0001] The ability to preserve, transfer, and recover viable microorganisms is essential for biological research. However, loss of viability is a constant concern and problems arise in the storage of viable microorganisms, especially when stored for extended periods of time. SUMMARY

[0002] The disclosure relates to methods of freeze-drying a sample, wherein the sample comprises trehalose. In some embodiments, the disclosure provides a method of producing a molded article, comprising freeze-drying a sample according to the methods described herein in a mold. In some embodiments, the disclosure provides a product prepared by the methods described herein. In some embodiments, the disclosure provides a sample comprising trehalose for freeze-drying. BRIEF DESCRIPTION OF THE DRAWINGS

[0003] Figure 1 depicts the contribution of polyols and sodium glutamate for pellet production and retrieval. Trehalose (T) alone (10%) and trehalose along with sodium glutamate, sorbitol, mannitol, and dextrin were added to investigate pellet production and retrieval. Pellet structure was assessed for integrity and rigidity after removal.

[0004] Figure 2 depicts efficient pellet production with trehalose, sorbitol mannitol, and monosodium glutamate. Trehalose (T) alone (10%) and trehalose along with sodium glutamate, sorbitol, mannitol, and dextrin were added to investigate pellet production and retrieval.

[0005] Figure 3 depicts the efficient pellet retrieval in presence of nonionic detergent. Formulation 10% trehalose + 0.25% sorbitol + 0.25% mannitol + 1% sodium glutamate used for pellet formation alone and in presence of either Tween® 80 (0.125% to 0.1%) or tyloxapol (0.125% to 0.1%) alone.

[0006] Figure 4A-Figure 4B depict the stability determination of microbes with and without tyloxapol (0.05%). Formulation 10% trehalose + 0.25% sorbitol + 0.25% mannitol + 1% sodium glutamate with and without 0.05% tyloxapol was used for pellet formation and stability study for DB1 / 154486626.1Attorney Docket No.124324-5002-WO Bacillus spizizenii (ATCC® 6633, Figure 4A) and Candida albicans (ATCC® 10231, Figure 4B) for 2 months period.

[0007] Figure 5A-Figure 5C depicts the evaluation of the resuscitation buffer. Three organisms used for the evaluation of the lyophilization buffer for high CFU pellets. S. aureus (ATCC® 6538, Figure 5A), E. coli (ATCC® 8739, Figure 5B) and C. albicans (ATCC® 10231, Figure 5C). Seven buffers were used with each containing 1XPBS as based along with 1) 0.1% Tween® 80; 2) 0.05% Tween® 80; 3) 0.025% Tween® 80; 4) 0.1% Pluronic® acid; 5) 0.05% Pluronic® acid; 6) 0.025% Pluronic® acid; and 7) 1XPBS as control.

[0008] Figure 6A-Figure 6L depict the evaluation of the resuscitation buffer for low CFU. Six organisms used for the evaluation of the lyophilization buffer for high CFU pellets. E. coli (ATCC® 8739, Figure 6A and Figure 6G), P. aeruginosa (ATCC® 9027, Figure 6B and Figure 6H), S. aureus (ATCC® 6538, Figure 6C and Figure 6I), B. spizizenii (ATCC® 6633, Figure 6D and Figure 6J), C. albicans (ATCC® 10231, Figure 6E and Figure 6K), and A. niger (ATCC® 16404, Figure 6F and Figure 6L). The two buffers used are as follows: A) 1XPBS containing 0.1% Tween® 80 (Figure 6A-Figure 6F) and B) sodium chloride peptone (Figure 6G-Figure 6L). Pellets were resuscitated in 1 ml buffer and 75 μl of which was used for plating in triplicate in each time point. Plates were incubated at appropriate temperature for each organism. NS indicates no significant difference in viability from the starting point and * indicates significant difference in viability. An additional solution of 0.1% tyloxapol was used as well with comparable performance (data not shown due to incomplete replicates).

[0009] Figure 7A-Figure 7C depict the superior (and immediate) dissolution characteristics of the pellets (i.e. “ATCC Pellet”). Figure 7A shows an image of the dissolved pellet compared to the undissolved comparative commercially available pellet. Figure 7B shows the optical density (OD) of the pellet solution compared to the comparative commercially available pellet solution. Figure 7C shows optical density (OD) of the pellet solution compared to the comparative commercially available pellet solution.

[0010] Figure 8A-Figure 8H depict the shipping study with the pellets at 4 °C and room temperature (RT). In Figure 8A-Figure 8F, six organisms were used for the evaluation of the effects of shipping temperature on viability: S. aureus (ATCC® 6538, Figure 8A), E. coli 2 DB1 / 154486626.1Attorney Docket No.124324-5002-WO (ATCC® 8739, Figure 8B), C. albicans (ATCC® 10231, Figure 8C), P. aeruginosa (ATCC® 9027, Figure 8E), B. spizizenii (ATCC® 6633, Figure 8D), and A. niger (ATCC® 16404, Figure 8F). The pellet was resuscitated in 1 ml buffer (1XPBS + 0.1% Tween® 80) and 75 μl of which was used for plating in triplicate from no shipping (NS), shipping at 4 °C (4C), and room temperature shipping (RT). Plates were incubated at appropriate temperature for each organism. Ns indicates no significant difference in viability from the NS and * indicates significant difference in viability from NS. Visual inspection of the pellets after shipping did not show any signs of physical damage from the shipping process. Figure 8G and Figure 8H show data from temperature tracking devices shipped with each set of samples for ambient shipping temperature and refrigerated shipping temperature, respectively.

[0011] Figure 9 depicts pellet formation from a lyophilization cycle. Generally, 70-90% of pellet formation occurs. Intact pellets are stored and used for further assays. DETAILED DESCRIPTION

[0012] In one aspect, the methods of the present disclosure comprise a method of freeze-drying (i.e., lyophilizing) a sample as described herein. The terms “freeze-drying” and “lyophilizing” as used herein refer generally to a process in which water is removed under low temperature and / or pressure by the methods of the present disclosure. The term “freezing” a sample as used herein refers to removing heat from a non-solid sample to solidify the sample and / or maintaining or storing a solid sample at a temperature to preserve its solid state.

[0013] In one aspect, the methods of the present disclosure exclude freezing the sample prior to the method is performed. In some embodiments, the methods of the present disclosure exclude lowering a temperature to 0 °C or below prior to the method is performed. In some embodiments, the methods of the present disclosure exclude lowering a temperature to below 0 °C prior to the freeze-drying or freezing the sample.

[0014] In some embodiments, the freeze-drying comprises freezing the sample and drying the sample. In some embodiments, the sample comprises trehalose and a detergent.

[0015] In some embodiments, the freeze-drying or freezing comprises freeze-drying or freezing at a first temperature below about 0 °C. In some embodiments, the first temperature is below about 0 °C, for example, about 1, 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 40, DB1 / 154486626.1Attorney Docket No.124324-5002-WO 45, 50, 55, or 60 °C. In some embodiments, the first temperature is from about 60 to 0 °C, from about 50 to 0 °C, from about 40 to 0 °C, from about 60 to 10 °C, from about 50 to 10 °C, from about 40 to 10 °C, from about 60 to 20 °C, from about 50 to 20 °C, from about 40 to 20 °C, from about 40 to 30 °C, from about 50 to 30 °C, or from about 60 to 30 °C. In some embodiments, the first temperature is from about 40 to 20 °C.

[0016] In some embodiments, the freeze-drying or freezing is performed at the first temperature for more than about 100 minutes, for example, about 100, 500, 1000, 1250, 1500, 1750, 2000, 2500, or 3000 minutes. In some embodiments, the freeze-drying or freezing is performed at the first temperature for from about 100 to 3000 minutes, from about 500 to 3000 minutes, from about 1000 to 3000 minutes, from about 1500 to 3000 minutes, from about 100 to 2500 minutes, from about 100 to 2000 minutes, from about 500 to 3000 minutes, from about 500 to 2500 minutes, from about 500 to 2000 minutes, from about 500 to 1500 minutes, from about 1000 to 3000 minutes, from about 1000 to 2000 minutes, from about 1250 to 2000 minutes, from about 1250 to 1750 minutes, from about 1500 to 1750 minutes, or from about 1500 to about 2000 minutes. In some embodiments, the freeze-drying or freezing is performed at the first temperature for from about 500 to 3000 minutes.

[0017] In some embodiments, the freeze-drying or freezing further comprises increasing a temperature from the first temperature to a second temperature above the first temperature. In some embodiments, the second temperature is below about 0 °C, for example, about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 °C. In some embodiments, the second temperature is from about 60 to 0 °C, from about 50 to 0 °C, from about 40 to 0 °C, from 30 to 0 °C, from 20 to 0 °C, from about 60 to 10 °C, from about 50 to 10 °C, from about 40 to 10 °C, from 30 to 10 °C, from about 60 to 20 °C, from about 50 to 20 °C, from about 40 to 20 °C, or from about 30 to 20 °C. In some embodiments, the second temperature is from about 30 to 10 °C.

[0018] In some embodiments, the freeze-drying or freezing comprises increasing the temperature at a first rate from 0.01 to 5 °C / min, for example, about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5 °C / min. In some embodiments, the first rate is from about 0.01 to 5 °C / min, from about 0.01 to 2 °C / min, from about 0.01 to 1 °C / min, from about 0.01 to 0.5 °C / min, from about 0.01 to 0.2 °C / min, from about 0.05 to 1 °C / min, from about 0.05 to 0.5 °C / min, from DB1 / 154486626.1Attorney Docket No.124324-5002-WO about 0.05 to 0.2 °C / min, from about 0.1 to 1 °C / min, from about 0.1 to 0.5 °C / min, from about 0.1 to 0.3 °C / min, or from about 0.1 to 0.2 °C / min. In some embodiments, the first rate is from about 0.01 to 1 °C / min.

[0019] In some embodiments, the freeze-drying or freezing is performed at the second temperature for more than about 1 minute, for example, about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 minutes. In some embodiments, the freeze-drying or freezing is performed at the second temperature for from about 1 to 500 minutes, from about 50 to 500 minutes, from about 100 to 500 minutes, from about 1 to 200 minutes, from about 50 to 200 minutes, from about 100 to 200 minutes, from about 100 to 150 minutes, or from about 50 to 250 minutes. In some embodiments, the freeze-drying or freezing is performed at the second temperature for from about 50 to 250 minutes.

[0020] In some embodiments, the freeze-drying or freezing further comprises increasing a temperature from the second temperature to a third temperature above the second temperature. In some embodiments, the third temperature is below or equal to 0 °C, for example, about 50, 30, 40, 30, 20, 10, or 0 °C. In some embodiments, the third temperature is from about 50 to 0 °C, from about 40 to 0 °C, from about 30 to 0 °C, from about 20 to 0 °C, from about 10 to 0 °C, or from about 5 to 0 °C. In some embodiments, the third temperature is from 10 to 0 °C.

[0021] In some embodiments, the freeze-drying or freezing comprises increasing the temperature at a second rate from 0.01 to 5 °C / min, for example, about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5 °C / min. In some embodiments, the second rate is from about 0.01 to 5 °C / min, from about 0.01 to 2 °C / min, from about 0.01 to 1 °C / min, from about 0.01 to 0.5 °C / min, from about 0.01 to 0.2 °C / min, from about 0.05 to 1 °C / min, from about 0.05 to 0.5 °C / min, from about 0.05 to 0.2 °C / min, from about 0.1 to 1 °C / min, from about 0.1 to 0.5 °C / min, from about 0.1 to 0.3 °C / min, or from about 0.1 to 0.2 °C / min. In some embodiments, the second rate is from about 0.01 to 1 °C / min.

[0022] In some embodiments, the freeze-drying or freezing is performed at the third temperature for more than about 1 minute, for example, about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 5 DB1 / 154486626.1Attorney Docket No.124324-5002-WO 120, 130, 140, or 150 minutes. In some embodiments, the freeze-drying or freezing is performed at the third temperature for from about 1 to 150 minutes, from about 1 to 100 minutes, from about 1 to 50 minutes, from about 1 to 30 minutes, from about 10 to 100 minutes, from about 10 to 90 minutes, from about 10 to 80 minutes, from about 10 to 70 minutes, from about 10 to 60 minutes, from about 10 to 50 minutes, from about 10 to 40 minutes, or from about 10 to 30 minutes. In some embodiments, the freeze-drying or freezing is performed at the third temperature for from about 1 to 150 minutes.

[0023] In some embodiments, the freeze-drying, freezing, and / or drying is performed at a pressure of less than about 500 mTorr, for example, about 500, 400, 300, 200, 100, 50, 10, 5, or 1 mTorr. In some embodiments, the freeze-drying, freezing, and / or drying is performed at a pressure of about 1 to 500 mTorr, from about 10 to 400 mTorr, from about 10 to 300 mTorr, from about 10 to 200 mTorr, from about 10 to 100 mTorr, from about 20 to 100 mTorr, from about 30 to 100 mTorr, from about 40 to 100 mTorr, from about 50 to 100 mTorr, from about 25 to 75 mTorr, from about 25 to 50 mTorr, from about 50 to 75 mTorr, from about 10 to 90 mTorr, from about 20 to 80 mTorr, from about 30 to 70 mTorr, or from about 40 to 60 mTorr. In some embodiments, the freeze-drying, freezing, and / or drying is performed at a pressure of about 10 to 100 mTorr.

[0024] In some embodiments, the freeze-drying or drying 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 freeze-drying or drying comprises removing at least about 99% of water from the sample. The removing may comprise 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. The freeze-drying or drying may comprise removing more than about 99% of water from the sample. In some embodiments, the freeze-drying or drying comprises removing from about 95% to 99% of water from the sample. DB1 / 154486626.1Attorney Docket No.124324-5002-WO

[0025] In some embodiments, the freeze-drying or drying 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. The freeze-drying or drying may comprise 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 freeze-drying or drying 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 freeze-drying or drying 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 freeze-drying or drying comprises removing water from the sample so that a water content of the sample is about 0.1 g water / g dry weight. In some embodiments, the freeze-drying or drying comprises removing water from the sample so that a water content of the sample is about 0.6 g water / g dry weight.

[0026] In some embodiments, the freeze-drying or drying 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. The freeze-drying or drying may comprise removing water a rate 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 freeze-drying or drying 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 freeze-drying or drying comprises removing water at a rate of about 1 g water / g dry DB1 / 154486626.1Attorney Docket No.124324-5002-WO weight / hr. In some embodiments, the freeze-drying or drying comprises removing water at a rate of about 2 g water / g dry weight / hr.

[0027] In some embodiments, the removing water comprises at least one selected from the group consisting of spin drying, dip coating, spray drying, electrospinning and directional deposition. The removing water may comprise drying using surface tension of the sample. The removing water may comprise extrusion and / or deposition of the sample. The removing water may comprise extrusion of the sample. The removing water may comprise deposition of the sample. In some embodiments, the removing water 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 method includes generating a layer from the sample.

[0028] In some embodiments, the freeze-drying or drying comprises removing oxygen from environment of the sample. In some embodiments, the freeze-drying or drying 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. The freeze-drying or drying may comprise 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. The freeze-drying or drying may comprise removing at least about 99% of oxygen from the sample.

[0029] In some embodiments, the methods described herein further comprising second drying the sample. In some embodiments, the methods described herein further comprising second drying the sample at a fourth temperature higher than the freeze-dying temperature. In some embodiments, the second drying comprises increasing a temperature from the third temperature to the fourth temperature above the third temperature. In some embodiments, the fourth temperature is at least about 0 °C, for example, about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 °C. In some embodiments, the fourth temperature is from about 0 to 50 °C. In some embodiments, the fourth temperature is about 1 °C or above, 2 °C or above, 3 °C or above, 4 °C DB1 / 154486626.1Attorney Docket No.124324-5002-WO 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 fourth temperature is from about 0 to 40 °C, from about 0 to 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, from about 5 to 10 °C, from about 10 to 20 °C, from about 10 to 30 °C, from about 10 to 40 °C, or from about 10 to 50 °C. In some embodiments, the fourth temperature is 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 fourth temperature is physiological temperature (e.g., about 37 °C). In some embodiments, the fourth temperature is refrigeration temperature (e.g., from about 1 to 5 °C, or about 1, 2, 3, 4, or 5 °C). In some embodiments, the fourth temperature is above the freezing temperature of water (e.g., above 0 °C).

[0030] In some embodiments, the second drying comprises increasing the temperature at a third rate from 0.01 to 5 °C / min, for example, about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5 °C / min. In some embodiments, the third rate is from about 0.01 to 5 °C / min, from about 0.01 to 2 °C / min, from about 0.01 to 1 °C / min, from about 0.1 to 5 °C / min, from about 0.1 to 2 °C / min, from about 0.1 to 1 °C / min, from about 0.5 to 5 °C / min, from about 0.5 to 2 °C / min, or from about 0.5 to 1 °C / min. In some embodiments, the third rate is from about 0.1 to 1 °C / min.

[0031] In some embodiments, the second drying is performed at the fourth temperature for more than about 1 minute, for example, about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 minutes. In some embodiments, the second drying is performed at the fourth temperature for from about 1 to 500 minutes, from about 50 to 500 minutes, from about 100 to 500 minutes, from about 100 to 400 minutes, from about 100 to 300 minutes, from about 50 to 400 minutes, from about 50 to 300 minutes, from about 150 to about 400 minutes, from about 150 to about 450 minutes, or from about 150 to about 500 minutes. In some embodiments, the second drying is performed at the fourth temperature for from about 100 to 500 minutes. DB1 / 154486626.1Attorney Docket No.124324-5002-WO

[0032] In some embodiments, the second drying is performed at a pressure of less than about 500 mTorr, for example, about 500, 400, 300, 200, 100, 50, 10, 5, or 1 mTorr. In some embodiments, the second drying is performed at a pressure of about 1 to 500 mTorr, from about 10 to 400 mTorr, from about 10 to 300 mTorr, from about 10 to 200 mTorr, from about 10 to 100 mTorr, from about 20 to 100 mTorr, from about 30 to 100 mTorr, from about 40 to 100 mTorr, from about 50 to 100 mTorr, from about 25 to 75 mTorr, from about 25 to 50 mTorr, from about 50 to 75 mTorr, from about 10 to 90 mTorr, from about 20 to 80 mTorr, from about 30 to 70 mTorr, or from about 40 to 60 mTorr. In some embodiments, the second drying is performed at a pressure of about 10 to 100 mTorr. In some embodiments, the second drying is performed at a pressure from 1 to 100 mTorr.

[0033] In some embodiments, the second drying 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 second drying comprises removing at least about 99% of water from the sample. The second drying may comprise 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. The second drying may comprise removing more than about 99% of water from the sample. In some embodiments, the second drying comprises removing from about 95% to 99% of water from the sample.

[0034] In some embodiments, the second drying 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. The second drying may comprise 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 DB1 / 154486626.1Attorney Docket No.124324-5002-WO 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 second drying 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 second drying comprises removing water from the sample so that a water content of the sample is from about 0.1 to about 0.6 g water / g dry weight. In some embodiments, the second drying comprises removing water from the sample so that a water content of the sample is about 0.1 g water / g dry weight. In some embodiments, the second drying comprises removing water from the sample so that a water content of the sample is about 0.6 g water / g dry weight.

[0035] In some embodiments, the second drying comprises removing oxygen from environment of the sample. In some embodiments, the second drying 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. The second drying may comprise 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. The second drying may comprise removing at least about 99% of oxygen from the sample.

[0036] In some embodiments, the sample comprises at least one excipient (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more excipients). In some embodiments, the sample comprises one or more excipients (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more excipients). In some embodiments, the sample comprises two or more excipients. In some embodiments, the sample comprises three or more excipients. In some embodiments, the sample comprises four or more excipients. In some embodiments, the sample comprises five or more excipients. In some embodiments, the sample comprises six or more excipients. In some embodiments, the sample comprises seven or more excipients. In some embodiments, the sample comprises eight or more excipients. In some embodiments, the sample comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 excipients.

[0037] In some embodiments, the sample comprises the one or more excipients at a concentration of at least about 0.01% (w / v), for example, about 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, DB1 / 154486626.1Attorney Docket No.124324-5002-WO 3, 2.5, 4, 4.5, 5, 6, 7, 8, 9, or 10% (w / v). In some embodiments, the sample comprises the one or more excipients at a concentration of about 1% (w / v). In some embodiments, the sample comprises the one or more excipients at a concentration of about 1.5% (w / v). In some embodiments, the sample comprises the one or more excipients at a concentration of about 2% (w / v). In some embodiments, the sample comprises the one or more excipients at a concentration from about 0.01 to 10% (w / v), from about 0.01 to 5% (w / v), from about 0.01 to 4% (w / v), from about 0.01 to 3% (w / v), from about 0.01 to 2% (w / v), from about 0.01 to 1% (w / v), from about 0.1 to 10% (w / v), from about 0.1 to 5% (w / v), from about 0.1 to 4% (w / v), from about 0.1 to 3% (w / v), from about 0.1 to 2% (w / v), from about 0.1 to 1% (w / v), from about 0.5 to 10% (w / v), from about 0.5 to 5% (w / v), from about 0.5 to 4% (w / v), from about 0.5 to 3% (w / v), from about 0.5 to 2% (w / v), from about 0.5 to 1% (w / v), from about 1 to 10% (w / v), from about 1 to 5% (w / v), from about 1 to 4% (w / v), from about 1 to 3% (w / v), or from about 1 to 2% (w / v). In some embodiments, the sample comprises the one or more excipients at a concentration from about 0.01 to 10% (w / v). In some embodiments, the sample comprises the one or more excipients at a concentration from about 1 to 2% (w / v).

[0038] Any suitable excipients can be used in accordance with the methods of the present disclosure. The one or more excipients may comprise one or more of a monosaccharide, a disaccharide, a sugar, a sugar alcohol, a sugar acid, a carbohydrate, an alcohol, a polyol, an amino acid, a salt of an amino acid, a choline, a protein, a milk, or any derivatives thereof.

[0039] In some embodiments, the one or more excipients are lyoprotective agents. The sample may comprise 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. The sample may comprise the lyoprotective agent at an amount 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 DB1 / 154486626.1Attorney Docket No.124324-5002-WO about 600 mmol or more. 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.

[0040] In some embodiments, the sample comprises one or more sugars. Any suitable sugar can be used in accordance with the methods of the present disclosure. The one or more sugars may comprise a sugar alcohol, a sugar acid, a carbohydrate, a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide, or any derivatives thereof. The one or more sugars may comprise dextrose, dextrin, 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 one or more sugars comprises trehalose, sucrose, dextrin, sorbitol, and / or mannitol. In some embodiments, the one or more sugars comprise trehalose. In some embodiments, the one or more sugars comprise sucrose. In some embodiments, the one or more sugars comprise dextrin. In some embodiments, the one or more sugars comprise sorbitol. In some embodiments, the one or more sugars comprise mannitol.

[0041] In some embodiments, the sample comprises the one or more sugars at a concentration of at least about 1% (w / v), for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50% (w / v). In some embodiments, the sample comprises the one or more sugars at a concentration of about 10% (w / v). In some embodiments, the sample comprises the one or more sugars at a concentration of about 10.5% (w / v). In some embodiments, the sample comprises the one or more sugars at a concentration of about 11% (w / v). In some embodiments, the sample comprises the one or more sugars at a concentration from about 1 to 50% (w / v), from about 1 to 45% (w / v), from about 1 to 40% (w / v), from about 1 to 30% (w / v), from about 1 to 20% (w / v), from about 1 to 15% (w / v), from about 5 to 50% (w / v), from about 5 to 45% (w / v), from about 5 to 40% (w / v), from about 5 to 30% (w / v), from about 5 to 20% (w / v), from about 5 to 15% (w / v), from about 10 to 50% (w / v), from about 10 to 45% (w / v), from about 10 to 40% (w / v), from about 10 to 30% (w / v), from about 10 to 20% (w / v), from about 10 to 15% (w / v), or from about 10 to 11% (w / v). In some embodiments, the sample comprises the one or more sugars at a concentration from about 5 to 45% (w / v). In some DB1 / 154486626.1Attorney Docket No.124324-5002-WO embodiments, the sample comprises the one or more excipients at a concentration from about 5 to 15% (w / v).

[0042] In some embodiments, the sample comprises trehalose. In some embodiments, the sample comprises trehalose at a concentration of at least about 0.1% (w / v), for example, about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50% (w / v). In some embodiments, the sample comprises trehalose at a concentration of about 10% (w / v). In some embodiments, the sample comprises trehalose at a concentration from about 0.1 to 20% (w / v), from about 1 to 20% (w / v), from about 5 to 20% (w / v), from about 10 to 20% (w / v), from about 1 to 15% (w / v), from about 1 to 10% (w / v), from about 5 to 15% (w / v), from about 5 to 10% (w / v), from about 5 to 11% (w / v), from about 9 to 11% (w / v), or from about 9 to 10% (w / v). In some embodiments, the sample comprises trehalose at a concentration from 1 to 20% (w / v). In some embodiments, the sample comprises trehalose at a concentration from 5 to 15% (w / v). In some embodiments, the sample comprises trehalose at a concentration from 5 to 11% (w / v). In some embodiments, the sample comprises trehalose at a concentration from 9 to 11% (w / v). In some embodiments, the sample comprises trehalose at a concentration from 9 to 10% (w / v).

[0043] In some embodiments, the sample comprises sucrose. In some embodiments, the sample comprises sucrose at a concentration of at least about 0.1% (w / v), for example, about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50% (w / v). In some embodiments, the sample comprises sucrose at a concentration of about 10% (w / v). In some embodiments, the sample comprises sucrose at a concentration of about 20% (w / v). In some embodiments, the sample comprises sucrose at a concentration from about 0.1 to 20% (w / v), from about 1 to 20% (w / v), from about 5 to 20% (w / v), from about 10 to 20% (w / v), from about 1 to 15% (w / v), from about 1 to 10% (w / v), from about 5 to 15% (w / v), from about 5 to 10% (w / v), from about 5 to 11% (w / v), from about 9 to 11% (w / v), or from about 9 to 10% (w / v). In some embodiments, the sample comprises sucrose at a concentration from 1 to 20% (w / v). In some embodiments, the sample comprises sucrose at a concentration from 5 to 15% DB1 / 154486626.1Attorney Docket No.124324-5002-WO (w / v). In some embodiments, the sample comprises sucrose at a concentration from 9 to 11% (w / v).

[0044] In some embodiments, the sample comprises dextrin. In some embodiments, the sample comprises dextrin at a concentration of at least about 0.1% (w / v), for example, about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30% (w / v). In some embodiments, the sample comprises dextrin at a concentration of about 0.5% (w / v). In some embodiments, the sample comprises dextrin at a concentration of about 1% (w / v). In some embodiments, the sample comprises dextrin at a concentration of about 2% (w / v). In some embodiments, the sample comprises dextrin at a concentration of about 5% (w / v). In some embodiments, the sample comprises dextrin at a concentration from about 0.1 to 20% (w / v), from about 0.1 to 15% (w / v), from about 0.1 to 10% (w / v), from about 0.1 to 5% (w / v), from about 0.5 to 20% (w / v), from about 0.5 to 15% (w / v), from about 0.5 to 10% (w / v), or from about 0.5 to 5% (w / v). In some embodiments, the sample comprises dextrin at a concentration from 0.1 to 20% (w / v). In some embodiments, the sample comprises dextrin at a concentration from 0.1 to 10% (w / v). In some embodiments, the sample comprises dextrin at a concentration from 0.5 to 5% (w / v).

[0045] In some embodiments, the sample comprises sorbitol. In some embodiments, the sample comprises sorbitol at a concentration of at least about 0.01% (w / v), for example, about 0.01, 0.05, 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30% (w / v). In some embodiments, the sample comprises sorbitol at a concentration of about 0.25% (w / v). In some embodiments, the sample comprises sorbitol at a concentration of about 0.5% (w / v). In some embodiments, the sample comprises sorbitol at a concentration of about 1% (w / v). In some embodiments, the sample comprises sorbitol at a concentration of about 1.5% (w / v). In some embodiments, the sample comprises sorbitol at a concentration of about 2.5% (w / v). In some embodiments, the sample comprises sorbitol at a concentration from about 0.01 to 20% (w / v), from about 0.01 to 10% (w / v), from about 0.01 to 5% (w / v), from about 0.01 to 2.5% (w / v), from about 0.01 to 0.5% (w / v), from about 0.01 to 0.25% (w / v), from about 0.05 to 20% (w / v), from about 0.05 to 10% (w / v), from about 0.05 to 5% (w / v), from about 0.05 to 2.5% (w / v), from about 0.05 to 0.5% (w / v), from about 0.05 to 0.25% (w / v), from about 0.1 to 20% (w / v), from about 0.1 to 10% (w / v), from about 0.1 to 5% (w / v), from about 0.1 to 2.5% (w / v), DB1 / 154486626.1Attorney Docket No.124324-5002-WO from about 0.1 to 1% (w / v), from about 0.1 to 0.5% (w / v), from about 0.1 to 0.25% (w / v), from about 0.25 to 5% (w / v), from about 0.25 to 2.5% (w / v), from about 0.25 to 1% (w / v), or from about 0.25 to 0.5% (w / v). In some embodiments, the sample comprises sorbitol at a concentration from about 0.25 to 5% (w / v). In some embodiments, the sample comprises sorbitol at a concentration from about 0.25 to 2.5% (w / v). In some embodiments, the sample comprises sorbitol at a concentration from about 0.25 to 0.5% (w / v). In some embodiments, the sample comprises sorbitol at a concentration from about 0.05 to 5% (w / v).

[0046] In some embodiments, the sample comprises mannitol. In some embodiments, the sample comprises mannitol at a concentration of at least about 0.01% (w / v), for example, about 0.01, 0.05, 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30% (w / v). In some embodiments, the sample comprises mannitol at a concentration of about 0.25% (w / v). In some embodiments, the sample comprises mannitol at a concentration of about 0.5% (w / v). In some embodiments, the sample comprises mannitol at a concentration of about 1% (w / v). In some embodiments, the sample comprises mannitol at a concentration of about 1.5% (w / v). In some embodiments, the sample comprises mannitol at a concentration of about 2.5% (w / v). In some embodiments, the sample comprises mannitol at a concentration from about 0.01 to 20% (w / v), from about 0.01 to 10% (w / v), from about 0.01 to 5% (w / v), from about 0.01 to 2.5% (w / v), from about 0.01 to 0.5% (w / v), from about 0.01 to 0.25% (w / v), from about 0.05 to 20% (w / v), from about 0.05 to 10% (w / v), from about 0.05 to 5% (w / v), from about 0.05 to 2.5% (w / v), from about 0.05 to 0.5% (w / v), from about 0.05 to 0.25% (w / v), from about 0.1 to 20% (w / v), from about 0.1 to 10% (w / v), from about 0.1 to 5% (w / v), from about 0.1 to 2.5% (w / v), from about 0.1 to 1% (w / v), from about 0.1 to 0.5% (w / v), from about 0.1 to 0.25% (w / v), from about 0.25 to 5% (w / v), from about 0.25 to 2.5% (w / v), from about 0.25 to 1% (w / v), or from about 0.25 to 0.5% (w / v). In some embodiments, the sample comprises mannitol at a concentration from about 0.25 to 5% (w / v). In some embodiments, the sample comprises mannitol at a concentration from about 0.25 to 2.5% (w / v). In some embodiments, the sample comprises mannitol at a concentration from about 0.25 to 1% (w / v). In some embodiments, the sample comprises mannitol at a concentration from about 0.05 to 5% (w / v).

[0047] In some embodiments, the sample comprises milk. In some embodiments, the sample comprises milk at a concentration of at least about 0.1% (w / v), for example, about 0.1, 0.5, 1, 2, DB1 / 154486626.1Attorney Docket No.124324-5002-WO 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50% (w / v). In some embodiments, the sample comprises milk at a concentration of about 10% (w / v). In some embodiments, the sample comprises milk at a concentration from about 0.1 to 20% (w / v), from about 1 to 20% (w / v), from about 5 to 20% (w / v), from about 10 to 20% (w / v), from about 1 to 15% (w / v), from about 1 to 10% (w / v), from about 5 to 15% (w / v), from about 5 to 10% (w / v), from about 5 to 11% (w / v), from about 9 to 11% (w / v), or from about 9 to 10% (w / v). In some embodiments, the sample comprises milk at a concentration from 1 to 20% (w / v). In some embodiments, the sample comprises milk at a concentration from 5 to 15% (w / v). In some embodiments, the sample comprises milk at a concentration from 9 to 11% (w / v).

[0048] In some embodiments, the sample comprises bovine serum albumin (BSA). In some embodiments, the sample comprises bovine serum albumin (BSA) at a concentration of at least about 0.1% (w / v), for example, about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50% (w / v). In some embodiments, the sample comprises bovine serum albumin (BSA) at a concentration of about 10% (w / v). In some embodiments, the sample comprises bovine serum albumin (BSA) at a concentration from about 0.1 to 20% (w / v), from about 1 to 20% (w / v), from about 5 to 20% (w / v), from about 10 to 20% (w / v), from about 1 to 15% (w / v), from about 1 to 10% (w / v), from about 5 to 15% (w / v), from about 5 to 10% (w / v), from about 5 to 11% (w / v), from about 9 to 11% (w / v), or from about 9 to 10% (w / v). In some embodiments, the sample comprises bovine serum albumin (BSA) at a concentration from 1 to 20% (w / v). In some embodiments, the sample comprises bovine serum albumin (BSA) at a concentration from 5 to 15% (w / v). In some embodiments, the sample comprises bovine serum albumin (BSA) at a concentration from 9 to 11% (w / v).

[0049] In some embodiments, the sample comprises sodium glutamate. In some embodiments, the sample comprises sodium glutamate at a concentration of at least about 0.1% (w / v), for example, about 0.1, 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, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50% (w / v). In some embodiments, the sample comprises sodium glutamate at a concentration of about 0.5% (w / v). DB1 / 154486626.1Attorney Docket No.124324-5002-WO In some embodiments, the sample comprises sodium glutamate at a concentration of about 1% (w / v). In some embodiments, the sample comprises sodium glutamate at a concentration of about 2% (w / v). In some embodiments, the sample comprises sodium glutamate at a concentration of about 5% (w / v). In some embodiments, the sample comprises sodium glutamate at a concentration from about 0.1 to 50% (w / v), from about 0.1 to 40% (w / v), from about 0.1 to 30% (w / v), from about 0.1 to 20% (w / v), from about 0.1 to 15% (w / v), from about 0.1 to 10% (w / v), from about 0.1 to 5% (w / v), from about 0.1 to 2% (w / v), from about 0.1 to 1% (w / v), from about 0.5 to 50% (w / v), from about 0.5 to 40% (w / v), from about 0.5 to 30% (w / v), from about 0.5 to 20% (w / v), from about 0.5 to 15% (w / v), from about 0.5 to 10% (w / v), from about 0.5 to 5% (w / v), from about 0.5 to 1% (w / v), or from about 0.5 to 1.5% (w / v). In some embodiments, the sample comprises sodium glutamate at a concentration from about 0.5 to 1.5% (w / v). In some embodiments, the sample comprises sodium glutamate at a concentration from about 0.5 to 5% (w / v). In some embodiments, the sample comprises sodium glutamate at a concentration from about 0.1 to 10% (w / v).

[0050] In some embodiments, the sample comprises one or more of trehalose, milk, sucrose, dextrin, sorbitol, mannitol, sodium glutamate, and bovine serum albumin. In some embodiments, the one or more excipients comprise one or more of milk, sucrose, dextrin, sorbitol, mannitol, sodium glutamate, and bovine serum albumin. In some embodiments, the sample comprises trehalose, sorbitol, mannitol, and sodium glutamate.

[0051] In some embodiments, the sample comprises trehalose at a concentration from about 1 to 20% (w / v), sorbitol at a concentration from about 0.05 to 5% (w / v), mannitol at a concentration from about 0.05 to 5% (w / v), and sodium glutamate at a concentration from about 0.1 to 10% (w / v). In some embodiments, the sample comprises trehalose at a concentration from about 5 to 15% (w / v), sorbitol at a concentration from about 0.1 to 1% (w / v), mannitol at a concentration from about 0.1 to 1% (w / v), and sodium glutamate at a concentration from about 0.1 to 5% (w / v). In some embodiments, the sample comprises trehalose at a concentration from about 9 to 11% (w / v), sorbitol at a concentration from about 0.1 to 0.5% (w / v), mannitol at a concentration from about 0.1 to 0.5% (w / v), and sodium glutamate at a concentration from about 0.5 to 1.5% (w / v). In some embodiments, the sample comprises 10% (w / v) trehalose, 0.25% (w / v) sorbitol, 0.25% (w / v) mannitol, and 1% (w / v) sodium glutamate. DB1 / 154486626.1Attorney Docket No.124324-5002-WO

[0052] In some embodiments, the sample comprises 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 / Na2HPO4, 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.

[0053] In some embodiments, the sample comprises a buffer. Any suitable buffer may be used in accordance with the methods described herein. Suitable buffers include, but are not limited to, phosphate buffer, citric acid / Na2HPO4, 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, and Tris. In some embodiments, the buffer comprises phosphate buffered saline (PBS). In some embodiments, the buffer is at an acidic pH (i.e., pH<7). In some embodiments, the buffer is at a basic pH (i.e., pH>7). In some embodiments, the buffer is at a neutral pH (i.e., pH=7). In some embodiments, the buffer is at physiological pH (i.e., approximately pH 7.4).

[0054] In some embodiments, the sample comprises a detergent. In some embodiments, the sample excludes a detergent. The sample may comprise the detergent at a concentration 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 1% (v / v), from about 0.02 to 1% (v / v), from about 0.03 to 1% (v / v), from about 0.04 to 1% (v / v), from about 0.05 to 1% (v / v), from about 0.01 to 0.09% (v / v), from about 0.01 to 0.08% (v / v), from about 0.01 to 0.07% (v / v), from about 0.01 to 0.06% (v / v), from about 0.01 to 0.05% (v / v), from about 0.05 to DB1 / 154486626.1Attorney Docket No.124324-5002-WO about 1% (v / v), or from about 0.05 to about 0.5% (v / v). The sample may comprise the detergent at a concentration of about 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 % (v / v). In some embodiments, the sample comprises a detergent at a concentration from about 0.01% (v / v) to 0.1% (v / v). In some embodiments, the sample comprises a detergent at a concentration of about 0.1% (v / v).

[0055] Any suitable detergent (i.e., surfactant) can be used in accordance with the methods of the present disclosure. Detergents may comprise organic compounds that are amphiphilic, i.e., containing both hydrophobic groups (“tails”) and hydrophilic groups (“heads”), which render detergents soluble in both organic solvents and water. A detergent can be classified by the presence of formally charged groups in its head. A non-ionic detergent has no charge groups in its head, whereas an ionic detergent carries a net charge in its head. A zwitterionic detergent contains a head with two oppositely charged groups. Some examples of common detergents include: 1) anionic detergents (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 detergents (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) detergents, such as dodecyl betaine, cocamidopropyl betaine, and coco ampho glycinate; and 5) nonionic detergents, such as alkyl poly(ethylene oxide), alkylphenol poly(ethylene oxide), copolymers of poly(ethylene oxide) and poly(propylene 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 (Tween® 20, Tween® 80, etc.), Triton detergents, Tyloxapol, Pluronic® acid, and dodecyl dimethylamine oxide. In some embodiments, In some embodiments, the detergent comprises a nonionic detergent. In some embodiments, the detergent comprises Tyloxapol and / or Tween® 80. DB1 / 154486626.1Attorney Docket No.124324-5002-WO

[0056] In one aspect, the methods of the present disclosure may 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.

[0057] 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.

[0058] Animals, e.g., non-human animals, e.g., non-human mammals, may 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.

[0059] 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).

[0060] In some embodiments, the sample comprises a microorganism. In some embodiments, the microorganism is selected from the group consisting of virus, bacteria, and fungus. DB1 / 154486626.1Attorney Docket No.124324-5002-WO

[0061] In some embodiments, the sample comprises bacteria. In some embodiments, the bacteria is Gram negative. In some embodiments, the bacteria is Gram positive. Examples of bacteria include, but are not limited to, Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, Actinomyces sp. (such as Actinomyces israelii and Actinomyces naeslundii), Aeromonas sp. (such as Aeromonas hydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonas caviae), Anaplasma phagocytophilum, Anaplasma marginale Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Bacillus spizizenii, 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 Campylobacter fetus), Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as, Corynebacterium diphtheriae, Corynebacterium jeikeum and Corynebacterium), Clostridium sp. (such as Clostridium perfringens, Clostridium dificile, Clostridium botulinum and Clostridium tetani), Eikenella corrodens, Enterobacter sp. (such as Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae 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. (such as 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, Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobacter sp. (such as Helicobacter pylori, DB1 / 154486626.1Attorney Docket No.124324-5002-WO Helicobacter cinaedi and Helicobacter fennelliae), Kingella kingii, Klebsiella sp. (such as Klebsiella pneumoniae, Klebsiella granulomatis and Klebsiella oxytoca), 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 intracellulare, Mycobacterium avium, Mycobacterium bovis, and Mycobacterium marinum), Mycoplasm sp. (such as Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp. (such as Nocardia asteroides, 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., Prevotella 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- DB1 / 154486626.1Attorney Docket No.124324-5002-WO resistant serotype 9V 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, Group A 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, Tropheryma whippelii, 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 furnisii), Yersinia sp. (such as Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis) and Xanthomonas maltophilia.

[0062] In some embodiments, the sample comprises a virus. 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 B19, 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 24 DB1 / 154486626.1Attorney Docket No.124324-5002-WO 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.

[0063] In some embodiments, the sample comprises a fungus. Examples of fungi include, but are not limited to, Aspergillus, Aspergillus niger, Blastomyces dermatitidis, Blastomyces gilchristii, Candida auris, Candida albicans, 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 M. 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 25 DB1 / 154486626.1Attorney Docket No.124324-5002-WO 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, Podosphaera fuliginea, Pythium debaryanum, Sclerotinia trifoliorum, Stagonospora nodorum, Stemphylium botryosum, Agaricus bisporus, Lentinula edodes, Pleurotus ostreatus, Pleurotus eryngii, Flammulina filiformis, Tuber melanosporum, and Psilocybe cubensis.

[0064] 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, deoxythymidine for DNA or their ribose counterparts for RNA) linked by phosphodiester linkages.

[0065] 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, DB1 / 154486626.1Attorney Docket No.124324-5002-WO leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), 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.

[0066] 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.

[0067] In some embodiments, the methods of the present disclosure may 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, for example, 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. The room temperature may comprise 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. The room temperature may comprise 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.

[0068] In some embodiments, the methods of the present disclosure may further comprise preserving the sample. 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. The room temperature may comprise 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. The room temperature may comprise a temperature of DB1 / 154486626.1Attorney Docket No.124324-5002-WO 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.

[0069] In one aspect, the disclosure provides a method of producing a molded article. In some embodiments, the method of producing a molded article comprises freeze-drying a sample according to the methods described herein in a mold.

[0070] The molded article may be in any suitable form including, but not limited to, a pellet, a cylinder, a sheet, a cube, a sphere, etc. In some embodiments, the molded article is a pellet.

[0071] In some embodiments, the mold is a well. In some embodiments, the well has a diameter of 1 mm or greater, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 mm. In some embodiments, the well has a diameter from about 1 to 10 mm, from about 1 to 20 mm, or from about 5 to 10 mm.

[0072] In some embodiments, the mold comprises 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). The multi-well plates may be comprised of a polymer such as polycarbonate, polyethylene, polyester, polypropylene, polystyrene, a cycloolefin polymer, a vinyl polymer, or any blends, derivatives, or copolymers thereof. In some embodiments, the mold comprises a 96-well plate.

[0073] In one aspect, the disclosure provides a product prepared by the method described herein. In some embodiments, the product is a molded article described herein. In some embodiments, the product is a pellet described herein.

[0074] In one aspect, the use of the product or method is characterized by one or more elements disclosed in the application. In some embodiments, the sample comprising trehalose as described herein is used in the method described herein.

[0075] 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 DB1 / 154486626.1Attorney Docket No.124324-5002-WO 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. EXAMPLES Example 1: Development of fast-dissolving lyo-casted material with quantified microorganism that involves use of a modified lyophilization process. Technology Development Goal

[0076] One goal of this technology development effort was the creation of especially formatted pellets that can contain a defined range of colony forming unit (CFU) upon rehydration that remain stable for at least 6 months. Ability to create these formatted pellets using already existing industrial lyophilizers was preferred.

[0077] The technology developed for this purpose allows for industrial scale up and minimizes the use of custom ordered biomaterials (including bacteria, fungus). The following were some of the high-level defining goals / factors for this technology development effort, guided by a series of voice of customer (VOC) data: 1. Pellet format: The VOC indicated a market primed with pellets that have the approximate dimensions of the inside of a 96-well plate. 2. Pellet characteristics (solubility): One of the significant pain-points identified by the VOC data was the poor dissolution characteristics of the similar products that exist in the market. 3. Industrial manufacturability: One of the requirements of the technology development was manufacturability and ability to scale up pellet manufacturing. 4. Stability of microorganisms: One of the requirements of the technology development was the microorganisms stabilized in the pellets must be stable for a minimum of six months (180 days) and CFU numbers be within a defined range (10–100 CFU for low CFU pellets and at least 105for high CFU pellets). DB1 / 154486626.1Attorney Docket No.124324-5002-WO 5. Quantified microorganisms capable of generating specific CFU number upon resuscitation using a specially formulated resuscitation buffer: This is an important characteristic of the pellets. A dual approach of high and low CFU was undertaken.

[0078] From the technology development point of view, at least these factors were addressed using a) a unique formulation and b) a modified lyophilization technique.

[0079] Formulation and modified lyophilization techniques together play a role in making sure that pellets created were stable, recoverable, and dissolve quickly following resuscitation. Evaluation of lyophilization formulation for long term stability for model organisms

[0080] As a first step towards developing a suitable pellet, a new lyophilization buffer was developed. Parallel to the buffer formulation work, a method for the creation and recovery of the pellets was also necessary, in which a lyo-casting procedure was investigated for this purpose.

[0081] Multiple formulations were evaluated with the model organisms to ensure long term stability / viability. Two model organisms were used for this study, first organism E. coli (ATCC® 8739) and S. epidermidis (ATCC® 12228), model Gram negative and Gram positive bacteria, respectively. The principal components milk, sucrose, trehalose, and dextrin were tested in isolation. Other formulation derivatives containing the principal components with the addition of polyols (sorbitol and mannitol) and proteins were also evaluated. A commercial formulation (also referred to herein as “ATCC (#12)”) contains sucrose, bovine serum albumin (BSA), and growth media components. This stability was determined for all the formulations along with the commercial formulation. Initial screening with 10% dextrin (#4, Table 1) alone and mixture of milk and sugars (#5-7, Table 1) did not produce better stability compared to the commercial formulation for both model organisms and were excluded from further study. Stability testing was performed on the formulations marked in bold (#1-3, 8-10, and 12, Table 1). Table 1. Preparation of the freeze-drying formulations.DB1 / 154486626.1Attorney Docket No.124324-5002-WO

[0082] As shown in Table 1, dried 10% milk along with three carbohydrates (sucrose, trehalose, dextrin) were used as the major components. Seven formulations were prepared marked in bold in the second column, #1-3, 8-10 and 12 (commercial formulation). Bold marked formulations were used for the stability assay. Formulation #13 used as negative control; and in this formulation, viability dropped more than four logs within 7 days. In all the formulations, 1XPBS was used as a common buffer. All the formulations were filter sterilized (0.2 μM) after preparation except milk. All the formulation were stored at room temperature away from light. All % measurements are weight / volume unless noted otherwise.

[0083] Table 2 shows the stability of E. coli at 4 °C (top) and room temperature (bottom) in different formulations after 6 months (180 days). Right column indicates the viability ratio between day 1 and day 180 for each formulation. Sor- indicates sorbitol, Man-mannitol and SG- Monosodium glutamate. Table 2. Stability of E. coli in lyophilized formulations for 6 months.31 DB1 / 154486626.1Attorney Docket No.124324-5002-WO

[0084] Table 3 shows the detailed stability of E. coli at 4 °C and room temperature in selected formulations after 6 months (180 days). Six time points used to measure the stability (1, 7, 14, 28, 56, and 180 days). Right column indicates the viability ratio between day 1 and day 180. Composition of formulation #9, #10 and #12 are indicated in Table 1. DB1 / 154486626.1yOtilnsiya )sytilnsiya )sW-2005-423421.oNtekcoDyenrottADS +E+E+)0 0 0s5y.1E1a 1.1.2er +ut sE+yaD3E+.1E.1.D ar S 2 1 2epD8.sC°2 na707070m8e2 707020ht4et + + + tnaME E Emna+E+E+E oy0t.9 5il9.6.1oo er3.3 3(M7.5.83mibT 36 atS 7 7 6R7rD 0+0+0t+a 07060y+ + +of s SEy9E.7E4.82.ti2li sEy D5E.8E.3.sabS 2 1 8Datanoit1a n 70807 SD4 401 707070l ae + + + n + + +uME3E E a.2.1.eE6E E.0.16 1 3M.m8 6 2rof 707060707 5deDzS + + + +0+0+ilisE7Ey .0E6.5E E E6.54 sDS.36.05.4h ayap D D o7 7yn 8lae08+07+08+ n08 5+0+0+niME9E E a E E E.4.0.e 0.9 2il1 1 1M2.2.9oc. 808 8 8 8 8+0+0+0+0+0E DfSEo y2E E E E+E.82.42.21y DS.82.42.1yatDaDil 1i n 8 1ba 08080808 8a e +E+ + n +0+0+t M5E E aS.9 eE E E6.27.57M.9 26.7.71.6.23neloit ))9 e0)2no )) )6268bas1#1#ita 9 es 01 1#44alu es #o( ol( (lrd a d u e#( oeCs l #( ( 5mordeadeC1 / TmroceshesFuSabrTaCBT rAoc shesFuSabrTaCBT 1ABDAttorney Docket No.124324-5002-WO

[0085] Table 4 shows the stability of S. epidermidis at 4 °C (top) and room temperature (bottom) in different formulations after 6 months (180 days). Right column indicates the viability ratio between day 1 and day 180 days for each formulation. Table 4. Stability of S. epidermidis in lyophilized formulations for 6 months.

[0086] Table 5 shows the detailed stability of S. epidermidis at 4 °C and room temperature after in selected formulations after 6 months (180 days). Six time points used to measure the stability (1, 7, 14, 28, 56, and 180 days). Right column indicates the viability ratio between day 1 and day 180. DB1 / 154486626.1yt n) yt n)OW-2005-423421.oNtekcoDyenrottA .syha 7 2 1 s 6.7 7tD y1.9.9no8a2 n 7 8 8Dma 0+0+0 88 8 7xCi° e + 2 ns4ME1E Et.4 Tae0+0+0+9.11.1RtME0E6E2ra a .ofy2.2.3tisl7 8 7yti0 inb+0+0+lib75070603oaDittS s SEay3E.1E5 at D+ + +2.2.4 Ss SE2E E.3.0lauD y4a 1 2.61 n 808 8Dmrae +0E+0 47 8 7E+ 1 na 0 0 0ofM3.5E.6.e + + +ME6E3Ed1 7 1e.7z9.1.4ili8h08080DpS +E+E+ 8E07070oD+ + +y ls 2.1y2.32.1 SE1E Eas.4.4y1 5.5n i Dasi 7 ndia8 8 8D e0+0+0+ 7 na808 8+0 0mME E EerE+ +8.1.0.M0E Ee6 8 4.22.62.1dipe8 8 8 8.D 0SS +0 0 08080fE+ +D+ + +oy4E.4E.1.SE4E E.4.1.ya 5 1 4ya 5 1 4ti Dl1Dibnaa808 8 1+0+0+ n 8 8 8a0+0+0+teSME2E.5E2 eE2E5E217.9.7M.7.9.7 .6.25en ) )66lobit )aal9 e0n1oi)e0 8144ues #(so#(ta e 9#s #( 5dlal 1dC) us( oladC / Tmorr e heC2 morde heC)21Boc s er sa T1 r c s er sa T1 DFuSab TB A#(oFuSab TB A#(Attorney Docket No.124324-5002-WO

[0087] A sharp drop in the stability of E. coli stored in milk and the commercial formulation was found compared to sugars alone (formulations #2 and 3), milk and sugars mixed with polyols and sodium glutamate (formulations #2, 3, 8, 9 and 10) both at 4 °C storage and room temperature. The addition of polyols and sodium glutamate to sugars and milk stabilized the E. coli significantly (Table 2). Table 3 shows how the addition of polyols and sodium glutamate restored the viability for a longer period for E. coli at both the storage temperatures.

[0088] Milk and the commercial formulation were found not to be suitable at restoring the stability of model Gram positive organism S. epidermidis. In a six-month period, viability was reduced more than 10-fold compared to sugars alone and sugars mixed with polyols and sodium glutamate (Table 4 and Table 5). Loss of viability was more predominant for E. coli compared to S. epidermidis (Table 3 and Table 5).

[0089] The effects of polyols (mannitol and sorbitol) and monosodium glutamate (MSG) upon survival during storage of freeze-dried bacteria was examined. There were no significant differences in viability during freeze-drying after addition of polyol or monosodium glutamate. However, these compounds were found to increase the stability of several strains during long- term storage (Carvalho, A., et al. Lait, 83, 3, May-June 2003, 203-210). In short, the addition of sorbitol, mannitol, and MSG to either sucrose or trehalose does not change the viability at 4 °C for a 6-month period but changed significantly when stored at near room temperature (22 °C). At 25°C storage, the viability drop for sucrose and trehalose alone was 15- and 30-fold, respectively. But supplementation of sorbitol, mannitol, and MSG with sucrose and trehalose made the drop in viability to 2-fold (Table 6). This indicates the combination of sorbitol, mannitol, and MSG has the preservative property at both low and elevated temperatures for long- term storage. Table 6. Effect on viability of microbes in lyophilized formulation.36 DB1 / 154486626.1Attorney Docket No.124324-5002-WO

[0090] Table 6, therefore, indicates the effect of polyols and monosodium glutamate on the long-term viability of Gram negative and Gram-positive bacteria.

[0091] While 10% sucrose and trehalose with the addition of 2.5% sorbitol, 2.5% mannitol, and 5% sodium glutamate yield very good stability, it is not possible to retrieve intact pellets from a 96 well plate. It was also found that the presence of 1XPBS strongly inhibited pellet formation in 96 well plate.

[0092] To understand the pellet production and retrieval from 96-well plate after lyophilization, pellet production with 10% sucrose and 10% trehalose alone was performed. Trehalose efficiently formed pellets, but sucrose did not. The percentage of trehalose was varied ranging from 5 to 12% (w / v) and its ability to form the intact and sturdy pellets was evaluated. The trehalose concentration range of 5 to 11% (w / v) provided the intact pellets; however, for the best mechanically stable pellets, the range of trehalose concentration was determined to be 9 to 10 % (w / v).

[0093] Previous data showed that additives like polyols and sodium glutamate benefit long-term stability. However, the optimal concentrations for long term stability of sorbitol, mannitol, and sodium glutamate (2.5%, 2.5% and 5% respectively) with 10% trehalose did not produce retrievable pellets from 96-well plate. Calibration of these components was done in alone and in presence of 10% trehalose, to make sure the formulation makes efficient pellet and retrieval. The concentrations of individual components were optimized for best pellet production without compromising viability of the stored organisms in the pellet. Sodium glutamate 0.5-1.0% (w / v), along with 10% trehalose, provided the good structural pellet. Similarly, mannitol 0.25-1.0% (w / v) and sorbitol 0.25-0.5% (w / v) produced good pellets after lyophilization. Dextrin was also tested at 0.5% (w / v) with 10% (w / v) trehalose (Figure 1, Table 7). Table 7. Contribution of polyols and sodium glutamate for pellet production and retrieval.DB1 / 154486626.1Attorney Docket No.124324-5002-WO

[0094] Next, the contribution of three additives (mannitol, sorbitol, and sodium glutamate) with the 10% (w / v) trehalose was evaluated with respect to the recovery and structure of pellets. As a control, 10% (w / v) trehalose alone has 100% recovery from 96 well plate well (16 out of 16 as shown in column 1 and 12; Figure 2, Table 8). Addition of polyols and / or sodium glutamate impacted either the recovery or the structure of pellets. For instance, columns 2 to 4 of Table 8 have ranging concentrations of mannitol and sodium glutamate with 10% (w / v) trehalose which lead to a poor recovery of pellets. The higher concentration of sodium glutamate (1% w / v) with 0.5% (w / v) mannitol and 10% (w / v) trehalose resulted in higher recovery but reduced the quality of the pellets. Therefore, sorbitol was decided to be added to the formulation mix. Out of all combinations of mannitol, sorbitol, and sodium glutamate with 10% (w / v) trehalose, it was found that column 7 and 8 of Table 8 generated the required quality and recovery of the pellet. Based on this preliminary study, the final formulation for the pellet is 0.25% (w / v) sorbitol, 0.25% (w / v) mannitol, 1.0% (w / v) sodium glutamate, and 10% trehalose. Table 8. Efficient pellet production with trehalose, sorbitol, mannitol, and monosodium glutamate.DB1 / 154486626.1Attorney Docket No.124324-5002-WO

[0095] Based on the data above, the 10% trehalose + 0.25% sorbitol + 0.25% mannitol + 1% sodium glutamate formulation produced a structurally stable and retrievable pellet (Figure 2). The stability of E. coli and S. epidermidis with the above formulation after lyophilization and storage at 4 °C was analyzed. It was found that a ~2-fold drop in the viability was found for both organisms after 5 months of storage (147 days; Table 9). This was an indication that this formulation behaves similarly to the parental formulation. This base formulation of 10% trehalose, 0.25% sorbitol, 0.25% mannitol + 1% sodium glutamate was used for the future pellet production.

[0096] Table 9 shows 10% trehalose + 0.25% sorbitol + 0.25% Mannitol + 1% sodium glutamate formulation was used to lyophilize different concentration of E. coli (ATCC® 8739) (E1-E6) and S. epidermidis (ATCC® 12228) (S1-S6). The first column shows the stability after lyophilization (1D stability). The second column shows the stability after 147 days (147D stability). Fold change in CFU indicates the CFU ratio between 1D / 147D. Table 9. Stability of E. coli and S epidermidis in the modified formulation as a pellet.147 Days stability of S epidermis (ATCC12228) in pellet form at 4C

[0097] Although it was possible to make and retrieve pellets using the composition 10% trehalose + 0.25% sorbitol + 0.25% Mannitol + 1% sodium glutamate, the number of pellet retrieval varied from one plate to the other also within batches. The shape of the pellet also varied. To address this, a nonionic detergent was added to the formulation for efficient retrieval of the pellet after lyophilization and to reduce the variability between batches. Varying DB1 / 154486626.1Attorney Docket No.124324-5002-WO percentage of Tween® 80 and tyloxapol were used in the formulation to ensure homogeneity of pellet and efficient retrieval of pellet from the plate. It was found that the addition of either 0.025% Tween® 80 or 0.05% tyloxapol (Figure 3) to the formulation above not only increases the pellet retrieval but also increases uniformity of the pellet shape. The addition of detergent also reduced batch to batch variation. After addition of the detergents, the pellets were easily removed without harsh patting. Figure 4A-B show the stability determination of microbes with and without tyloxapol (0.05%). Lyophilization Technique

[0098] The effort to achieve efficient pellet formation and recovery were based on the following strategies without significantly changing the formulation: 1. Freezing rate of the sample: Chilling rate was tested to ensure efficient pellet formation and recovery from 96-well plate. Very low cooling rate and high cooling rate result in poor the pellet recovery. Moderate cooling was most efficient for pellet formation and recovery. 2. Primary drying time: Primary drying time has an impact on the pellet formation. Increases in primary drying time had a positive impact on the pellet recovery. 3. Pressure differential (pVG / CM): Pressure differential has been successfully used before to indicate the primary drying, which needs to be determined empirically. 4. Temperature ramp: Temperature ramp used at different profiles for primary drying stages. It appears it may not have a significant effect on the pellet recovery.

[0099] Finally, freezing rate, primary drying time, and pressure differential during primary and secondary times dictated efficient pellet formation provided other factors during lyophilization remain intact along with the formulation. Resuscitation Formulation

[0100] A resuscitation formulation is required for uniform distribution of bacteria after rehydration of pellet. DB1 / 154486626.1Attorney Docket No.124324-5002-WO

[0101] Development of a resuscitation buffer for pellets: The resuscitation buffers were designed to effectively dissolve the pellets created. Per the design requirement for reference organisms and standardized assays indicated by the compendiums from USP, the pellets should be dissolved in 1 mL of resuscitation buffer and then 0.1 mL of the solution after complete dissolution is used to create a plate to create evaluation plates which are required to produce 10- 100 CFU of organisms (in plate-based method, see https: / / www.usp.org / sites / default / files / usp / document / harmonization / gen- method / q05b_pf_ira_34_6_2008.pdf).

[0102] One of the critical design criterium for the creation of a resuscitation buffer is that, while the buffer should encourage in-situ growth of the microorganisms in the buffer allowing a working window of couple of hours (preferably >4 hrs), it should not also cause the resultant CFU numbers to decrease appreciably below the acceptable limits.

[0103] Resuscitation buffer development effort was divided into two categories – (1) for pellets with high CFU and (2) for pellets with low CFU.

[0104] A small amount of nonionic detergents were chosen to facilitate uniform distribution of the pellets without significantly changing the microbial viability during the length of the assay.

[0105] Seven different rehydration formulations were tested for high CFU pellets. Each of these formulations contain a 1X PBS base with following additives: 1) 0.1% Tween® 80; 2) 0.05% Tween® 80; 3) 0.025% Tween® 80; 4) 0.1% Pluronic® acid; 5) 0.05% Pluronic® acid; 6) 0.025% Pluronic® acid. Buffer 7) 1XPBS was used as a control. An additional solution of 0.1% tyloxapol was used as well with comparable performance (data not shown).

[0106] These resuscitation formulations were tested selectively on three of the organisms S. aureus (ATCC® 6538, Figure 5A), E. coli (ATCC® 8739, Figure 5B) and C. albicans (ATCC® 10231, Figures 5C).

[0107] Three separate resuscitation formulations were tested for low CFU pellets: a) 1XPBS + 0.1% Tween® 80; b) NaCl-peptone; and c) 1XPBS + 0.1% tyloxapol. Here resuscitation formulations were optimized based on the withholding the viability of tested organisms for period of the assay. Result indicates 1XPBS + 0.1% Tween® 80 and NaCl-peptone buffers used DB1 / 154486626.1Attorney Docket No.124324-5002-WO withheld viability for most of the microorganisms for 8 hours after resuscitation (Figure 6A- Figure 6L). Fast dissolution of the pellets created using the chosen formulation and the modified lyophilization process

[0108] The pellets successfully dissolved in the resuscitation buffer(s). Figure 7A-Figure 7C compare the dissolution pattern of the buffer compared to commercially available pellets, which demonstrates the superior (and immediate) dissolution characteristics of the pellets. A side-by- side comparison of the dissolution characteristics of the pellets were undertaken to highlight the superior solubility characteristics of the pellets created using the process described above. As the set of figures indicate, the pellet dissolved in under 1 min (buffer used: 1x PBS + tyloxapol) while the comparative commercially available pellet took at least 40 mins to dissolve (buffer used: proprietary resuscitation buffer provided with commercially available pellet). Fast dissolution indicates ability to uniformly distribute the bacteria in the resuscitation buffer which translates to superior processing uniformity of the bacterial / fungal samples. Shipping Studies

[0109] Shipping studies were performed at two different shipping temperatures (4 °C and Room Temperatures, RT) to evaluate the robustness of the CFU forming ability of the pellets as well as ability of the pellets to maintain their physical integrity during shipping. The CFU evaluation was performed using standard plate method while the physical integrity of the pellets was verified using visual inspection. The data in Figure 8A-Figure 8H indicate that the pellets retained their ability to create desired CFU ranges while visual inspection (data not shown) confirmed their ability to maintain physical integrity. Conclusion

[0110] The technology development effort summarized here was able to produce lyophilized pellets that are fast dissolving, contain a defined number of CFU, and are stable for a minimum of 6 months. This was achieved through a lyo-casting procedure and the data drive optimization of the lyophilization formulation, cycle, and pre-processing. The underlying technique can be applied to multiple organisms. DB1 / 154486626.1Attorney Docket No.124324-5002-WO Example 2: Procedure for depositing thin polymer films in a 96-well plate.

[0111] This procedure was used to coat a 96-well plate with alternative charged polymers (i.e., deposit a thin film), which changes the physical properties of the plate (e.g., contact angle of buffer to the well). This process provides easy recovery of pellets.

[0112] The following polymer solutions were prepared in DI water (resistivity – 18.2 megohm): 1) Negatively charged polymer solutions: a) Poly(acrylic acid) (PAA, Sigma – 420344-100G) final solution to 5% w / v b) Poly(4-styrenesulfonic acid) solution (PSS, Sigma – 561223-100G) final solution to 5% w / v 2) Positively charged polymer solution: a) Poly(L-lysine) (Sigma – P8920-500ML) final solution to 0.1% w / v

[0113] A conventional polystyrene 96 well-plate was recovered. Each well was filled with the negatively charged polymer (~300 μl of solution) using a multiple channel pipette. (Note: Both polymers (PAA or PSS) had same effect and, therefore, can be used as alternative to each other). The plate was covered with a lid and the plate was left untouched for 5 minutes at room temperature (22-25 °C). (Note: Leaving polymer in well for a longer time (i.e., over 5 minutes) is not preferred. Avoid overexposure of polymer, for example, exposure of over an hour may result in non-uniform thick film). The solution was discarded. The plate was washed 3 times with DI water. (Note: Three-time washing is important to achieve monodisperse polymer coating on the plate well’s surface). The plate was dried using an air gun or left open in biosafety hood for 30 minutes. Each well was filled with positively charged polymer (Poly(L- Lysine)) ~300 μl using a multiple channel pipette. The plate was covered with a lid and the plate was left untouched for 5 minutes at room temperature (22-25 °C). The solution was discarded. The plate was washed 3 times with DI water. (Note: Three-time washing is important to achieve monodisperse polymer coating on the plate well’s surface). The plate was dried using an air gun or left open in biosafety hood for 30 minutes. The above steps were repeated once more. At this stage, the plate was polymer coated and ready to use for pellet formation. Example 3: Exemplary Pellet Formulation Preparation. 43 DB1 / 154486626.1Attorney Docket No.124324-5002-WO

[0114] Pipet tips 1000 μL, 20-200 μL and 1-20 μL, sterile; pipettes 1000 μL, 200 μl and 20 μl, 200 μl channel pipets; trehalose (Pfanstiehl; # T-104-4); mannitol (TCI; # M0044); sorbitol (JT Baker; # V045-07); sodium glutamate (TCI; # G0188); poly acrylic acid PAA, Sigma – 420344- 100G; poly 4-styrenesulfonic acid solution (PSS, Sigma – 561223-100G); poly(L-lysine) (Sigma; P8920-500ML) final solution to 0.1% w / v; 96 well plate (sterile); nitrile gloves; and Eppendorf tubes, sterile.

[0115] Spectrophotometer (Biotek, 800TSI or equivalent); 30 °C / 37 °C incubators; tabletop microcentrifuge (Eppendorf 5425 or equivalent); Vortex-Genie 2 digital vortex (Scientific Industries, Inc.); lyophilizer (SP Scientific Lyostar 4.0); and Raman Microspectrometer (as needed).

[0116] The work area in the Biosafety Cabinet was cleaned with deionized (DI) water and 70% isopropanol spray. The pipettes and racks were cleaned with DI water and 70% isopropanol spray. The desired number of sterile 96-well plate were prepared in the biosafety cabinet after taking out from the packaging. The filter sterilized lyophilized formulation was prepared (25 ml for each 96-well plate). For 100 ml 25g of trehalose; 0.25g of sorbitol, mannitol and 0.5g of sodium glutamate. 25 ml of the lyophilized formulation was poured into a sterile boat and 200 μl was distributed with a channel pipet (the plate was covered with the lid).

[0117] If coating of the plate was desired, then the following procedure and / or procedure described in Example 2 was performed.

[0118] The polymer solution was dissolved in DI water: 5 g of poly acrylic acid (PAA) was dissolved in 100 ml of DI water and filter sterilized. The poly(L-lysine) final solution at 0.1% w / v was also prepared. A conventional 96 well-plate was recovered from the packaging. Each well was filled with 300 μl of 5% PAA solution using a multiple channel pipette. The plate was covered with a lid and the plate was left untouched for 5 minutes at room temperature in biosafety cabinet (avoid overexposure of polymer over an hour may result in non-uniform thick film). The solution was discarded in waste container, and the plate was tapped on a fresh paper towel multiple times. Each of the wells was filled with 300 μl of sterile water, which was discarded by tapping on the paper towel. The process was repeated three times (three-time washing is important to achieve monodisperse polymer coating on the plate well’s surface). The DB1 / 154486626.1Attorney Docket No.124324-5002-WO plate was air dried in the biosafety cabinet, and the sterility of the plate was maintained by keeping the lid partially open. The above steps were repeated with poly(L-lysine) instead of PAA. Coating was complete and the plate was ready for the following step. (Note: Lyophilization formulation can be modified while using coated plate depending on the requirement of the process.)

[0119] The plate was set in the lyophilizer for the lyophilization process. A Raman microspectrometer was used to quantify residual water by comparing water (ratio of Asymmetric component of OH stretching peak and bending peak).

[0120] The pellet formation cycle was considered valid if more than 50% pellets are intact and look alike. Pellets that appear broken and not intact were not taken into consideration (Figure 9). EMBODIMENTS Embodiment 1. A method of freeze-drying a sample, the method comprising freezing the sample; and drying the sample, wherein the sample comprises trehalose and a detergent. Embodiment 2. The method according to embodiment 1, excluding freezing the sample prior to the method is performed. Embodiment 3. The method according to embodiment 1 or 2, excluding lowering a temperature to 0 °C or below prior to the freezing the sample. Embodiment 4. The method according to any one of the preceding embodiments, wherein the freeze-drying comprises freezing at a first temperature below 0 °C. Embodiment 5. The method according to embodiment 4, wherein the first temperature is from 40 to 20 °C. Embodiment 6. The method according to embodiment 4 or 5, wherein the freezing is performed at the first temperature for from 500 to 3000 minutes. DB1 / 154486626.1Attorney Docket No.124324-5002-WO Embodiment 7. The method according to any one of embodiments 4 to 6, wherein the freezing further comprises increasing a temperature from the first temperature to a second temperature above the first temperature. Embodiment 8. The method according to embodiment 7, wherein the second temperature is from 30 to 10 °C. Embodiment 9. The method according to embodiment 7 or 8, wherein the freezing comprises increasing the temperature at a first rate from 0.01 to 1 °C / min. Embodiment 10. The method according to any one of embodiments 7 to 9, wherein the freezing is performed at the second temperature for from 50 to 250 minutes. Embodiment 11. The method according to any one of embodiments 7 to 10, wherein the freezing further comprises increasing a temperature from the second temperature to a third temperature above the second temperature. Embodiment 12. The method according to embodiment 11, wherein the third temperature is from 10 to 0 °C. Embodiment 13. The method according to embodiment 11 or 12, wherein the freezing comprises increasing the temperature at a second rate from 0.01 to 1 °C / min. Embodiment 14. The method according to any one of embodiments 11 to 13, wherein the freezing is performed at the third temperature for from 1 to 150 minutes. Embodiment 15. The method according to any one of the preceding embodiments, wherein the freezing and / or drying is performed at a pressure from 10 to 100 mTorr. Embodiment 16. The method according to any one of the preceding embodiments, wherein the drying comprises removing at least 5% of water from the sample. Embodiment 17. The method according to any one of the preceding embodiments, wherein the drying comprises removing at least 50% of water from the sample. Embodiment 18. The method according to any one of the preceding embodiments, wherein the drying comprises removing at least 90% of water from the sample. DB1 / 154486626.1Attorney Docket No.124324-5002-WO Embodiment 19. The method according to any one of the preceding embodiments, wherein the drying comprises removing at least 95% of water from the sample. Embodiment 20. The method according to any one of the preceding embodiments, wherein the drying comprises removing at least 99% of water from the sample. Embodiment 21. The method according to any one of the preceding embodiments, wherein the drying comprises removing water from the sample so that a water content of the sample is below 1 g water / g dry weight. Embodiment 22. The method according to any one of the preceding embodiments, wherein the drying comprises removing water from the sample so that a water content of the sample is from 0.1 g water / g dry weight to 0.6 g water / g dry weight. Embodiment 23. The method according to any one of the preceding embodiments, wherein the drying comprises removing oxygen from environment of the sample. Embodiment 24. The method according to embodiment 23, wherein the removing oxygen from environment of the sample comprises removing at least 5% of oxygen from the sample. Embodiment 25. The method according to embodiment 23 or 24, wherein the removing oxygen from environment of the sample comprises removing at least 50% of oxygen from the sample. Embodiment 26. The method according to any one of embodiments 23 to 25, wherein removing oxygen from environment of the sample comprises removing at least 90% of oxygen from the sample. Embodiment 27. The method according to any one of embodiments 23 to 26, wherein the removing oxygen from environment of the sample comprises removing at least 95% of oxygen from the sample. Embodiment 28. The method according to any one of embodiments 23 to 27, wherein the removing oxygen from environment of the sample comprises removing at least 99% of oxygen from the sample. DB1 / 154486626.1Attorney Docket No.124324-5002-WO Embodiment 29. The method according to any one of the preceding embodiments, further comprising second drying at a fourth temperature higher than a temperature that the freezing is performed. Embodiment 30. The method according to embodiment 29, wherein the second drying comprises increasing a temperature from the third temperature to the fourth temperature above the third temperature. Embodiment 31. The method according to embodiment 29 or 30, wherein the fourth temperature is from 0 to 50 °C. Embodiment 32. The method according to any one of embodiments 29 to 31, wherein the second drying comprises increasing the temperature at a third rate from 0.1 to 5 °C / min. Embodiment 33. The method according to any one of embodiments 29 to 32, wherein the second drying is performed at the fourth temperature for from 100 to 500 minutes. Embodiment 34. The method according to any one of embodiments 29 to 33, wherein the second drying is performed at a pressure from 1 to 100 mTorr. Embodiment 35. The method according to any one of embodiments 29 to 34, wherein the second drying comprises removing at least 5% of water from the sample. Embodiment 36. The method according to any one of embodiments 29 or 35, wherein the second drying comprises removing at least 50% of water from the sample. Embodiment 37. The method according to any one of embodiments 29 to 36, wherein the second drying comprises removing at least 90% of water from the sample. Embodiment 38. The method according to any one of embodiments 29 to 37, wherein the second drying comprises removing at least 95% of water from the sample. Embodiment 39. The method according to any one of embodiments 29 to 38, wherein the second drying comprises removing at least 99% of water from the sample. Embodiment 40. The method according to any one of embodiments 29 to 39, wherein the second drying comprises removing water from the sample so that a water content of the sample is below 1 g water / g dry weight. DB1 / 154486626.1Attorney Docket No.124324-5002-WO Embodiment 41. The method according to any one of embodiments 29 to 40, wherein the second drying comprises removing water from the sample so that a water content of the sample is from 0.1 to 0.6 g water / g dry weight. Embodiment 42. The method according to any one of embodiments 29 to 41 wherein the second drying comprises removing oxygen from environment of the sample. Embodiment 43. The method according to embodiment 42, wherein the removing oxygen from environment of the sample comprises removing at least 5% of oxygen from the sample. Embodiment 44. The method according to embodiment 42 or 43, wherein the removing oxygen from environment of the sample comprises removing at least 50% of oxygen from the sample. Embodiment 45. The method according to any one of embodiments 42 to 44, wherein removing oxygen from environment of the sample comprises removing at least 90% of oxygen from the sample. Embodiment 46. The method according to any one of embodiments 42 to 45, wherein the removing oxygen from environment of the sample comprises removing at least 95% of oxygen from the sample. Embodiment 47. The method according to any one of embodiments 42 to 46, wherein the removing oxygen from environment of the sample comprises removing at least 99% of oxygen from the sample. Embodiment 48. The method according to any one of the preceding embodiments, wherein the sample comprises one or more excipients. Embodiment 49. The method according to embodiment 48, wherein the sample comprises the one or more excipients at a concentration from 0.01 to 10% (w / v). Embodiment 50. The method according to embodiment 48 or 49, wherein the one or more excipients comprise one or more of milk, sucrose, dextrin, sorbitol, mannitol, sodium glutamate, and bovine serum albumin. DB1 / 154486626.1Attorney Docket No.124324-5002-WO Embodiment 51. The method according to any one of the preceding embodiments, wherein the sample comprises trehalose at a concentration from 1 to 20% (w / v). Embodiment 52. The method according to any one of the preceding embodiments, wherein the sample comprises trehalose at a concentration from 5 to 15% (w / v). Embodiment 53. The method according to any one of the preceding embodiments, wherein the sample comprises trehalose at a concentration from 9 to 11% (w / v). Embodiment 54. The method according to any one of the preceding embodiments, wherein the sample comprises sorbitol. Embodiment 55. The method according to embodiment 54, wherein the sample comprises sorbitol at a concentration from 0.05 to 5% (w / v). Embodiment 56. The method according to any one of the preceding embodiments, wherein the sample comprises mannitol. Embodiment 57. The method according to embodiment 56, wherein the sample comprises mannitol at a concentration from 0.05 to 5% (w / v). Embodiment 58. The method according to any one of the preceding embodiments, wherein the sample comprises sodium glutamate. Embodiment 59. The method according to embodiment 58, wherein the sample comprises sodium glutamate at a concentration from 0.1 to 10% (w / v). Embodiment 60. The method according to any one of the preceding embodiments, wherein the sample comprises 10% (w / v) trehalose, 0.25% (w / v) sorbitol, 0.25% (w / v) mannitol, and 1% (w / v) sodium glutamate. Embodiment 61. The method according to any one of the preceding embodiments, wherein the sample comprises a buffer. Embodiment 62. The method according to embodiment 61, wherein the buffer comprises phosphate buffered saline (PBS). DB1 / 154486626.1Attorney Docket No.124324-5002-WO Embodiment 63. The method according to any one of the preceding embodiments, wherein the sample comprises the detergent at a concentration from 0.01 to 1% (w / v). Embodiment 64. The method according to any one of the preceding embodiments, wherein the detergent comprises a nonionic detergent. Embodiment 65. The method according to any one of the preceding embodiments, wherein the detergent comprises Tyloxapol or Tween 80. Embodiment 66. The method according to any one of the preceding embodiments, wherein the sample comprises a microorganism. Embodiment 67. The method according to embodiment 66, wherein the microorganism is selected from the group consisting of virus, bacteria, and fungus. Embodiment 68. The method according to embodiment 68, wherein the bacteria comprise Gram negative bacteria or Gram positive bacteria. Embodiment 69. A method of producing a molded article, comprising freeze-drying a sample according to the method of any one of the preceding embodiments in a mold. Embodiment 70. The method according to embodiment 69, wherein the molded article is a pellet. Embodiment 71. The method according to embodiment 69 or 70, wherein the mold is a well having a diameter from 1 to 10 mm. Embodiment 72. A product prepared by the method according to any one of the preceding embodiments. Embodiment 73. A sample comprising trehalose used in the method according to any one of the preceding embodiments. DB1 / 154486626.1

Claims

Attorney Docket No.124324-5002-WO CLAIMS 1. A method of freeze-drying a sample, the method comprising freezing the sample; and drying the sample, wherein the sample comprises trehalose and a detergent.

2. The method according to claim 1, wherein the freeze-drying comprises freezing at a first temperature below 0 °C, optionally wherein the first temperature is from 40 to 20 °C, and optionally wherein the freezing is performed at the first temperature for from 500 to 2000 minutes.

3. The method according claim 1 or 2, wherein the freezing further comprises increasing a temperature from the first temperature to a second temperature above the first temperature, and optionally wherein the second temperature is from 30 to 10 °C, optionally wherein the freezing comprises increasing the temperature at a first rate from 0.01 to 1 °C / min, and / or optionally wherein the freezing is performed at the second temperature for from 50 to 250 minutes.

4. The method according to claim 3, wherein the freezing further comprises increasing a temperature from the second temperature to a third temperature above the second temperature, and optionally wherein the third temperature is from 10 to 0 °C, optionally wherein the freezing comprises increasing the temperature at a second rate from 0.01 to 1 °C / min, and / or optionally wherein the freezing is performed at the third temperature for from 1 to 150 minutes.

5. The method according to any one of the preceding claims, wherein the drying comprises removing at least 5% of water from the sample, and optionally wherein the drying comprises removing water from the sample so that a water content of the sample is below 1 g water / g dry weight, optionally from 0.1 g water / g dry weight to 0.6 g water / g dry weight, and / or optionally wherein the drying comprises removing oxygen from the environment of the sample. DB1 / 154486626.1Attorney Docket No.124324-5002-WO 6. The method according to any one of the preceding claims, further comprising second drying at a fourth temperature of at least 0 °C, optionally wherein the second drying comprises increasing a temperature from the third temperature to the fourth temperature above the third temperature, optionally wherein the fourth temperature is from 0 to 50 °C, optionally wherein the second drying comprises increasing the temperature at a third rate from 0.1 to 5 °C / min, optionally wherein the second drying is performed at the fourth temperature for from 100 to 500 minutes, optionally wherein the second drying is performed at a pressure from 1 to 100 mTorr, optionally wherein the second drying comprises removing oxygen from environment of the sample, optionally wherein the second drying comprises removing at least 5% of water from the sample, and / or optionally wherein the second drying comprises removing water from the sample so that a water content of the sample is below 1 g water / g dry weight, optionally from 0.1 to 0.6 g water / g dry weight.

7. The method according to any one of the preceding claims, wherein the sample comprises one or more excipients, and optionally wherein the sample comprises the one or more excipients at a concentration from 0.01 to 10% (w / v), and / or optionally wherein the one or more excipients comprise one or more of milk, sucrose, dextrin, sorbitol, mannitol, sodium glutamate, and bovine serum albumin.

8. The method according to any one of the preceding claims, wherein the sample comprises trehalose at a concentration from 1 to 20% (w / v), optionally from 5 to 15% (w / v), and optionally from 9 to 11% (w / v), and optionally wherein the sample comprises the detergent at a concentration from 0.01 to 1% (w / v), optionally wherein the detergent comprises a nonionic detergent, and / or optionally wherein the detergent comprises Tyloxapol or Tween 80.

9. The method according to any one of the preceding claims, wherein the sample comprises sorbitol, optionally at a concentration from 0.05 to 5% (w / v).

10. The method according to any one of the preceding claims, wherein the sample comprises: DB1 / 154486626.1Attorney Docket No.124324-5002-WO mannitol, optionally at a concentration from 0.05 to 5% (w / v); sodium glutamate, optionally at a concentration from 0.1 to 10% (w / v); a buffer, optionally phosphate buffered saline (PBS); and / or a microorganism, optionally wherein the microorganism is selected from the group consisting of virus, bacteria, and fungus, optionally wherein the bacteria comprise Gram negative bacteria or Gram positive bacteria.

11. A method of producing a molded article, comprising freeze-drying a sample according to the method of any one of the preceding claims in a mold.

12. A product prepared by the method according to any one of the preceding claims.

13. A sample comprising trehalose used in the method according to any one of the preceding claims. DB1 / 154486626.1

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