Methods and compositions for freezing and drying biologics

Abstract

In some aspects, the present disclosure provides methods of preparing compositions, including compositions comprising a biological sample, wherein the methods comprise a deaeration step and a filtration step. In some embodiments, the presently disclosed methods further comprise a pressure-oscillation step or freezing step. These compositions may have a lower level of dissolved oxygen or fewer nanobubbles compared to compositions prepared using conventional methods. These compositions may be useful for improving the stability of biological samples upon freezing or freeze-drying.

Description

DESCRIPTIONMETHODS AND COMPOSITIONS FOR FREEZING AND DRYING BIOLOGICSBACKGROUND

[0001] This application claims the benefit of United States Provisional Patent Application No. 63 / 693,498, filed September 11, 2024, the entirety of which is incorporated herein by reference.1. Field

[0002] The present disclosure relates generally to the field of pharmaceuticals and pharmaceutical manufacture. More particularly, it concerns methods of preparing a solution comprising a sample, such as a biologic, for freezing or drying and liquid compositions formed according to such methods.2. Description of Related Art

[0003] Freezing is a fundamental step in the production of biopharmaceuticals, playing a critical role in the storage and formulation of biological drug substances. Most biological drug substances are stored as frozen solutions, while freeze- storage has also been used for early-phase clinical formulations and various commercial drug products (Authelin et al., 2020). Manufacture of lyophilized dosage forms also includes freezing as an integral part of the manufacturing process (Pardeshi et al. , 2023). However, while frozen products in general have reduced chemical degradation (e.g., hydrolysis and oxidation) compared to their liquid counterparts, the freezing process can impart physicochemical stresses on protein-based therapeutics, which eventually leads to denaturation and / or aggregation (Authelin et al. , 2020). Protein denaturation occurs when the protein loses its native structure due to external stresses including heat, pH changes or chemical interactions, whereas protein aggregation occurs when misfolded or denatured proteins clump together usually in a nonfunctional state (Ratanji et al., 2014). Air- water interfacial stress, which is induced by various factors including, but not limited to, (1) dissolved air molecules, (2) colloidally stable nanobubbles suspended in solution before freezing, (3) stable foam in solution, or (4) microbubbles formed during freezing, has been observed to cause proteins denaturation and subsequent aggregation (Dao et al., 2022). As more and more protein-based therapeutics are approved by FDA (e.g., enzymes, proteins, monoclonal antibodies, protein drug conjugate), there is a need for sample compositions thatthat can withstand multiple freeze-thaw without compromising the quality, efficacy, or biological activity of such therapeutics, as well as methods for forming such compositions.SUMMARY OF THE DISCLOSURE

[0004] In one aspect, the present disclosure provides methods of forming a filtered deaerated composition, wherein the method comprises:A. obtaining a liquid composition;B. placing the liquid composition in a vessel and subjecting the liquid composition to a deaeration step to form a de-aerated composition; andC. subjecting the de-aerated composition to a filtration step to form said filtered de-aerated composition.

[0005] In some embodiments, the liquid composition is suitable for dissolving or suspending a biological sample. In some embodiments, the liquid composition is suitable for dissolving a biological sample. In some embodiments, the liquid composition is suitable for suspending a biological sample. In some embodiments, the liquid composition comprises a biological sample. In some embodiments, the method further comprises combining the filtered de-aerated composition with a biological sample. In some embodiments, the filtered de-aerated composition is suitable for suspending a biological sample. In some embodiments, the filtered de-aerated composition is suitable for dissolving a biological sample.

[0006] In some embodiments, the deaeration step comprises stirring the liquid composition. In some embodiments, the deaeration step comprises stirring the liquid composition with a stir bar. In some embodiments, the stir bar is a polytetrafluoroethylene (PTFE) stir bar. In some embodiments, the stirring is off-center relative to the vessel. In some embodiments, the deaeration step further comprises agitation. In some embodiments, the agitation is stirring at about 200 rpm.

[0007] In some embodiments, the deaeration step occurs at about atmospheric pressure. In other embodiments, the deaeration step occurs at a pressure lower than atmospheric pressure. In some embodiments, the deaeration step occurs at a pressure of between about 3% and about 50% atmospheric pressure. In further embodiments, the deaeration step occurs at a pressure between about 3% atmospheric pressure and about 10% atmospheric pressure. In still further embodiments, the deaeration step occurs at a pressure of about 5% atmospheric pressure.

[0008] In some embodiments, the deaeration step has a duration of about 10 minutes to about 1 hour. In further embodiments, the deaeration step has a duration of about 10 minutes to about 30 minutes. In still further embodiments, the deaeration step has a duration of about 20 minutes. In some embodiments, the deaeration step comprises stirring the liquidcomposition with a PTFE stir bar at rate of about 200 rpm at a pressure of about 5% atmospheric pressure for a time period of about 20 minutes.

[0009] In some embodiments, the de-aerated composition has less dissolved oxygen than the liquid composition. In some embodiments, the de-aerated composition has a dissolved oxygen level of about 40% or less. In further embodiments, the de-aerated composition has a dissolved oxygen level of between about 10% and about 40%. In still further embodiments, the de-aerated composition has a dissolved oxygen level of about 20%. In other embodiments, the de-aerated composition has a dissolved oxygen level of about 15%. In further embodiments, the de-aerated composition has a dissolved oxygen level of about 10%. In still further embodiments, the de-aerated composition has a dissolved oxygen level of less than about 10%.

[0010] In some embodiments, the filtered de-aerated composition has less dissolved oxygen than the liquid composition. In some embodiments, the filtered de-aerated composition has a dissolved oxygen level of about 40% or less. In further embodiments, the filtered deaerated composition has a dissolved oxygen level of between about 10% and about 40%. In still further embodiments, the filtered de-aerated composition has a dissolved oxygen level of about 20%. In other embodiments, the filtered de-aerated composition has a dissolved oxygen level of about 15%. In further embodiments, the filtered de-aerated composition has a dissolved oxygen level of about 10%. In still further embodiments, the filtered de-aerated composition has a dissolved oxygen level of less than about 10%.

[0011] In some embodiments, the filtration occurs at a pressure that is less than atmospheric pressure. In further embodiments, the filtration occurs at a pressure that is between about 0.05 atm and about 1 atm. In still further embodiments, the filtration occurs at a pressure that is between about 0.1 atm and about 0.5 atm. In even further embodiments, the filtration occurs at a pressure that is about 0. 1 atm. In other embodiments, the filtration occurs at a pressure that is about 0.5 atm.

[0012] In some embodiments, the filtration step is filtration through a filter, such as a syringe filter. In some embodiments, the syringe tip filter is a 0.2 pm PES syringe filter. In some embodiments, the filter has a pore size that is about 0.2 pm. In further embodiments, the filter has a pore size that is less than 0.2 pm.

[0013] In some embodiments, the filtered de-aerated composition has fewer nanobubbles than the de-aerated composition. In some embodiments, the de-aerated composition has at least twice as many nanobubbles as the filtered de-aerated composition. In further embodiments, the de-aerated composition has at least five times as many nanobubblesas the filtered de-aerated composition. In still further embodiments, the de-aerated composition has about ten times as many nanobubbles as the filtered de-aerated composition.

[0014] In some embodiments, the liquid composition further comprises a cryoprotective agent. In some embodiments, the method further comprises combining a cryoprotective agent with the filtered de-aerated composition. In some embodiments, the cryoprotective agent is trehalose.

[0015] In some embodiments, the method further comprises a pressure oscillation step. In some embodiments, the pressure oscillation step comprises subjecting the liquid composition, the de-aerated composition, or the filtered de-aerated composition to a plurality of cycles of a first pressure and a second pressure. In some embodiments, the pressure oscillation step comprises subjecting the liquid composition to a plurality of cycles of a first pressure and a second pressure. In some embodiments, the pressure oscillation step comprises subjecting the de-aerated composition to a plurality of cycles of a first pressure and a second pressure. In some embodiments, the pressure oscillation step comprises subjecting the filtered de-aerated composition to a plurality of cycles of a first pressure and a second pressure.

[0016] In some embodiments, the first pressure is between about 0.3 atm and about 0.05 atm. In further embodiments, the first pressure is between about 0.2 atm and about 0.05 atm. In still further embodiments, the first pressure is about 0. 1 atm. In some embodiments, the second pressure is between about 0.3 atm and about 0.7 atm. In further embodiments, the second pressure is between about 0.4 atm and about 0.6 atm. In still further embodiments, the second pressure is about 0.5 atm.

[0017] In some embodiments, the plurality of cycles is between about 50 cycles and about 100 cycles. In further embodiments, the plurality of cycles is between about 70 cycles and about 90 cycles. In still further embodiments, the plurality of cycles is about 80 cycles. In some embodiments, the pressure oscillation step has a duration of between about 5 minutes and about 30 minutes. In further embodiments, the pressure oscillation step has a duration of between about 10 minutes and about 20 minutes. In still further embodiments, the pressure oscillation step has a duration of about 15 minutes.

[0018] In some embodiments, the method further comprises freezing the filtered deaerated composition to obtain a frozen de-aerated composition. In some embodiments, the freezing comprises freeze-drying. In some embodiments, the freezing comprises thin-film freezing. In some embodiments, the freezing is at a temperature between about -20°C and about -100°C. In further embodiments, the freezing is at a temperature of about -40°C. In stillfurther embodiments, the freezing is at a temperature of about -80°C. In some embodiments, the freezing comprises shelf-freezing.

[0019] In some embodiments, the biological sample comprises a protein. In some embodiments, the biological sample comprises an antibody. In some embodiments, the biological sample comprises an enzyme. In some embodiments, the biological sample comprises a cell. In some embodiments, the biological sample comprises a eukaryotic cell. In some embodiments, the sample comprises a red blood cell. In some embodiments, the sample comprises a red blood cell, a white blood cell, a platelet, or a mixture thereof. In some embodiments, the sample comprises blood. In some embodiments, the sample comprises a T- cell, such as a chimeric antigen T-cell.

[0020] In some embodiments, a biological sample is dissolved in the liquid composition. In some embodiments, a biological sample is suspended in the liquid composition. In some embodiments, a biological sample is dissolved in the filtered de-aerated composition. In some embodiments, a biological sample is suspended in the filtered de-aerated composition.

[0021] In another aspect, the present disclosure provides liquid compositions formed according to any of the embodiments described above. In another aspect, the present disclosure provides liquid compositions formed in the course of any of the methods described above. In some embodiments, the liquid compositions disclosed herein comprise a biological sample.

[0022] In another aspect, the present disclosure provides frozen compositions formed according to any of the methods described above that comprise a freezing step. In some embodiments, the frozen compositions disclosed herein comprise a biological sample.

[0023] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0025] FIG. 1 shows a schematic diagram of sample preparation prior to freezing, illustrating untreated, deaeration, and deaeration- filtration methods.

[0026] FIG. 2 shows dissolved oxygen level of bovine IgG solution from different deaeration processes. Data are mean ± S.D. (n = 3).

[0027] FIGS. 3A-3D (FIG. 3A) Total IgG aggregate counts after freeze-thaw in 2 mg / mL samples undergoing varied freezing rates (0.05 to 100 K / s) (n = 3). (FIG. 3B) SEC chromatogram, and (FIG. 3C) CD spectra of reference sample; samples after freeze-thaw with different treatments including untreated, deaeration, and deaeration-filtration. (FIG. 3D) Heatmap of IgG aggregate in samples subjected to one and three freeze-thaw cycles using freezing at -80°C (F80 and F80x3, respectively), dropping samples directly into LN2 (LN2), submerging glass vials in LN2 (LN2-S), untreated and deaeration-filtration respectively. Particles with larger equivalent circular diameter (ECD) are represented by colder colors, the numbers represent the percentage of aggregate count in the corresponding quadrant (See Microflow Imaging in Examples section below).

[0028] FIGS. 4A-4F (FIG. 4A) Concentration of subvisible protein aggregates in samples subjected to air- water interfacial stress (1,000 rpm vortexing for 2 mins) and subsequently stored for predetermined times (white bars). Representative images of the largest aggregates from samples exposed to air-water interfacial stress and stored for (FIG. 4B) 4 hrs, (FIG. 4C) 24 hrs, as well as freeze-induced stress through (FIG. 4D) freezing at -40°C and thaw and (FIG. 4E) freezing at -80°C and thaw for three cycles. (FIG. 4F) Heatmap of protein aggregates of different ECD populations, with numbers denoting the percentage of particles in each quadrant; comparing reference sample, air-water interfacially stressed samples stored for 2 to 24 hrs at 4°C (SH2, SHI 8, SH24) or room temperature (SH24 RT).

[0029] FIG. 5 shows the SEC chromatogram of IgG solutions with different treatments before freezing.

[0030] FIGS. 6A-6D show PBS lx solutions with different treatments (FIG. 6A) Visual photos of from left to right: reference, reduced pressure then repressurization, deaeration, deaeration-filtration. (FIG. 6B) Derived count rate from DLS measurements. (FIG. 6C) Number of nanobubbles as detected by nanotracking analysis and DO level. (FIG. 6D) Size distribution of nanobubbles as detected by nanotracking analysis.

[0031] FIGS. 7 A & 7B show MFI particle count of IgG 2 mg / mL samples with different pre-freezing treatments including untreated, deaeration, and deaeration-filtration. Samples were frozen at freezing rate of (FIG. 7A) ~1 K / s, and (FIG. 7B) -100 K / s

[0032] FIG. 8 shows a schematic illustration of root cause mechanisms of freeze- induced stress.

[0033] FIG. 9 shows the effect of deaeration- filtration of freezing media on red blood cell (RBC) lysis after subjecting a sample composition prepared according to the presently disclosed methods and a control composition to thin-film freezing and thawing. Red blood cells (RBCs) (500,000,000 cells / mL) were thin-film frozen at -80°C and then thawed at 37°C before measuring RBC lysis. Experiment was done once, and hemolysis was determined in triplicate.

[0034] FIG. 10 illustrates the effect of pretreatment of red blood cells (RBCs) to pressure oscillation on their lysis when being subject to thin-film freezing (TFF) and thawing. The deaeration-filtration process (DE-F) was used as a control. The experiment was done twice, and hemolysis was determined in triplicate. Data are mean ± S.D. (n = 2).

[0035] FIG. 11 shows the effect of pressure oscillation on RBC hemolysis after thin- film freeze-drying (TFFD). The experiment was done twice, and hemolysis was determined in triplicate. Data are mean ± S.D. (n ~2).DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0036] In some aspects, the present disclosure relates to methods of preparing a composition, wherein the method comprises a deaeration step and a filtration step. In some embodiments, the composition further comprises a biological sample. In some embodiments, the method further comprises freezing the composition, including freeze-drying the composition. Ins some embodiments, the method comprises subjecting the composition to apressure-oscillation step. A composition formed according to the presently disclosed methods, in some embodiments, has lower levels of dissolved air or fewer nanobubbles than before the method. Therefore, the presently disclosed methods lower the negative effects of the presence of air in a liquid sample solution upon freezing, such as freeze-induced stress, denaturation, or increased levels of aggregation upon thawing.

[0037] In some embodiments, the present invention relates to a novel method for enhancing the cryopreservation of a biological sample, such as red blood cells (RBCs), through the application of a deaeration and filtration process prior to freezing. It is hypothesized, without being bound by theory, that the deaeration-filtration process disclosed herein addresses the deleterious effects of air-water interfacial stress on cells during the freezing process. More particularly, the presently disclosed methods, in some embodiments, reduce or mitigate airwater interfacial stress during freezing of RBCs, whose cell membranes primarily consist of amphiphilic phospholipid bilayers. The presently disclosed methods facilitate the reduction or minimization of the air-water interface, thereby reducing the exposure of the phospholipids within the cell membrane to the hydrophobic air environment. In some embodiments, biological samples of compositions formed according to the present methods experience less structural disruption upon freezing in comparison to samples of compositions prepared according to methods known in the art. Therefore, the present methods provide a benefit, in some embodiments, of improved integrity of the sample cell membrane during freezing or freeze-drying. In some embodiments, the present invention discloses methods which have the benefit of decreased cell, such as RBC, cell lysis in comparison to methods known in the art.

[0038] In some embodiments, the presently disclosed methods comprise a pressure oscillation step. In some embodiments, the pressure oscillation step of the present methods has the benefit of increasing lipid bilayer flexibility. In some embodiments, the pressure oscillation step of the present methods has a benefit of exerting a transient mechanical force. In some embodiments, the increased lipid bilayer flexibility and / or the transient mechanical force either separately or in combination may, without being bound by theory, facilitate entry of cryoprotectant into a cell, for example a red blood cell. In this way, the present disclosure provides methods that facilitate uptake of cryoprotectant into biological sample cells, such as red blood cells, thereby reducing sensitivity to stresses associated with freezing, drying, or freeze-drying, such as lysis.I. Manufacturing Methods

[0039] The denaturation and subsequent aggregation of proteins due to air-water interfacial stress are well documented in scientific literature (Griffin et al., 2024; Koepf et al., 2018). Therefore, it is reasonable to assume, without necessarily being bound by the theory, that the presence of an air-water interface during the freezing process would exert stress on protein molecules (Authelin et al., 2020; Dao et al., 2022a). The present disclosure provides methods and compositions for reducing dissolved air and nanobubbles, which in turn reduces air-water interfacial stress during the freezing process.

[0040] More particularly, the present invention discloses methods for forming a liquid sample solution comprising a sample, such as a biologic, wherein a precursor sample solution (also referenced herein as a precursor solution) is subjected to certain processes, such as deaeration, filtration, or pressure oscillation to form the liquid sample solution. In some embodiments, the liquid sample solution has a lower level of dissolved air or fewer air nanobubbles than the precursor solution. The liquid sample solution may in some embodiments be frozen to provide a frozen sample composition wherein the sample has improved stability in comparison to samples as found in or samples derived from frozen sample compositions that have been prepared according to other methods known in the art.A. Deaeration.

[0041] Deaeration as used herein refers to the removal of dissolved air from a solution. Reduction of dissolved air in a solution suppresses a source of air- water induced denaturation, which contributes to aggregation. Therefore, the deaeration step of the presently disclosed methods is useful for preparing solutions which are less likely to undergo air-water-induced denaturation and subsequent aggregation. The presently disclosed methods comprising a deaeration step are therefore also useful for reducing freeze-induced denaturation of a sample.

[0042] Any process known through routine experimentation or known in the art is contemplated for use in the present methods. In some embodiments, the present disclosure involves subjecting a liquid composition, to a deaeration step. In some embodiments, the liquid composition subjected to the deaeration step comprises a biological sample. In some embodiments, the deaeration step involves placing a liquid composition in a vacuum chamber and modulating the pressure to less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 9%, less than about 8%, less thanabout 7%, less than about 6%, less than about 5%, less than about 4%, of atmospheric pressure, about 3% of atmospheric pressure, or any range derivable therein. In some embodiments, the deaeration step is performed at less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, of atmospheric pressure, about 3% of atmospheric pressure, or any range derivable therein. In some embodiments, the deaeration step is performed at less than about 10% of atmospheric pressure. In some embodiments, the deaeration step is performed at to less than about 5 % of atmospheric pressure.

[0043] The deaeration step of the present disclosure may further comprise agitation and / or chaotic mixing of the liquid composition. For example, the liquid composition may be agitated by stirring, such as magnetic stirring.

[0044] In some embodiments, the dissolved oxygen level of the liquid composition after the deaeration step, also referenced herein as the de-aerated composition, is less than about 40%. In some embodiments, the dissolved oxygen level of the de-aerated composition is about 40%, about 39%, about 38%, about 37%, about 36%, about 35%, about 34%, about 33%, about 32%, about 31%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%, or any range derivable therein. In some embodiments, the dissolved oxygen level of the de-aerated composition is between about 20% and about 10%. In some embodiments, the dissolved oxygen level of the de-aerated composition is about 15%.

[0045] The deaeration step may proceed for a time period referenced herein as the deaeration time period. In some embodiments, the reduction of dissolved air mentioned above may be measured over the course of the deaeration time period. In some embodiments, the deaeration time period is about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours, or any range derivable therein. In some embodiments, the dearation time period is about 2 hours to about 4 hours. In some embodiments, the deaeration time period is about 3 hours. In other embodiments, the deaeration time period is less than 45 minutes. In some embodiments, the deaeration time period is about 40 minutes, about 35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes, about 10 minutes, about 5minutes, or any range derivable therein. In some embodiments, the deaeration time period is between about 10 minutes and about 30 minutes. In some embodiments, the deaeration time period is about 20 minutes. In some embodiments, the deaeration time period is less than 20 minutes.B. Filtration.

[0047] In some embodiments, the de-aerated composition comprises nanobubbles, which are buoyancy-neutral and remain in a solution after being created. The presence of nanobubbles was seen to facilitate air-water-induced denaturation and subsequent aggregation. The inventors discovered that methods comprising a filtration step have the benefit of a reduced number of nanobubbles in the liquid sample solution or the de-aerated composition, thereby avoiding or reducing undesirable side effects associated with the presence of nanobubbles. More particularly, the filtration step of the presently disclosed methods is useful for preparing solutions which are less likely to undergo air-water induced denaturation and subsequent aggregation. The presently disclosed methods comprising a filtration step are therefore also useful for reducing freeze-induced denaturation of a sample. In some embodiments, the deaerated composition is subjected to a filtration step to form what is referenced herein as a filtered de-aerated composition.

[0048] The presently disclosed methods may involve any filtration method, technique, or equipment known in the art or identified by a person of skill in the art through routine optimization. In some embodiments, the filtration step involves filtering the de-aerated composition through a syringe filter. In some embodiments, the filtration is carried out under atmospheric pressure. In some embodiments, the filtration is carried out under pressure lower than atmospheric pressure. In some embodiments, the level of dissolved oxygen in the filtered de-aerated composition is higher than the level of dissolved oxygen in the liquid composition. In some embodiments, the level of dissolved oxygen in the filtered de-aerated composition is higher than the level of dissolved oxygen in the de-aerated composition. In some embodiments, the filtered de-aerated composition has a level of dissolved oxygen that is about 40%, about 39%, about 38%, about 37%, about 36%, about 35%, about 34%, about 33%, about 32%, about 31%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about1%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%, or any range derivable therein.

[0049] As mentioned above, in some embodiments the filtration step results in a reduction in nanobubbles compared to the liquid composition. In some embodiments the filtration step results in a reduction in nanobubbles compared to the de-aerated composition. In some embodiments, the filtration step reduces the amount of nanobubbles by about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, about 10.5 fold, about 11-fold, about11.5-fold, about 12-fold, about 12.5-fold, about 13-fold, about 13.5-fold, about 14-fold, about14.5-fold, about 15-fold, about 15.5-fold, about 16-fold, about 16.5-fold, about 17-fold, about17.5-fold, about 18-fold, about 18.5-fold, about 19-fold, about 19.5-fold, about 20-fold, or any range derivable therein, over the amount of nanobubbles in the de-aerated composition. In some embodiments, the filtration step reduces the amount of nanobubbles by between about 5-fold and about 20-fold over the amount of nanobubbles in the de-aerated composition. In some embodiments, the filtration step reduces the amount of nanobubbles by about 10-fold over the amount of nanobubbles in the de-aerated composition.

[0050] In some embodiments, the filtration step is performed at a pressure of about atmospheric pressure. In some embodiments, the filtration step is performed at a pressure that is less than atmospheric pressure. In some embodiments, the filtration step is performed at about 1 atm, about 0.95 atm, about 0.90 atm, about 0.85 atm, about 0.80 atm, about 0.75 atm, about 0.70 atm, about 0.65 atm, about 0.60 atm, about 0.55 atm, about 0.50 atm, about 0.45 atm, about 0.40 atm, about 0.35 atm, about 0.30 atm, about 0.25 atm, about 0.20 atm, about 0.15 atm, about 0.10 atm, about 0.05 atm, or less than 0.05 atm, or any range derivable therein. In some embodiments, the filtration step is performed at about 0.5 atm. In some embodiments, the filtration step is performed at less than 0.5 atm, such as about 0.1 atm.

[0051] The filtration step is useful for removing nanobubbles of a variety of sizes. In some embodiments, the filtration step of the presently disclosed methods reduces the amount of nanobubbles that are larger than about 220 nm in size. In some embodiments, the filtration step of the presently disclosed methods reduces the amount of nanobubbles that are about 200 nm or less in size. In some embodiments, the filtration step of the presently disclosed methods reduces the amount of nanobubbles that are about 10 nm, about 15 nm, about 20 nm, about 25nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 155 nm, about 160 nm, about 165 nm, about 170 nm, about 175 nm, about 180 nm, about 185 nm, about 190 nm, about 195 nm, about 200 nm, or any range derivable therein. In some embodiments, the filtration step reduces the amount of nanobubbles that are about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, or any range derivable therein. As mentioned above, the filtration step may comprise passing the de-aerated composition through a syringe filter, such as a 220 nm PES membrane. The filtration step of the presently disclosed methods, surprisingly, is useful for removing nanobubbles of a size that is less than the pore size of the filter.C. Freezing

[0052] While the denaturation of proteins is a prerequisite condition for aggregation, the close proximity of denatured molecules is a subsequent requirement for the formation of aggregates. This proximity is correlated with the volume of the unfrozen fraction or inter-ice-crystal channels, which are smaller at faster freezing rates (Engstrom et al., 2008). Conversely, slower freezing rates result in the formation of larger ice crystals and consequently bigger inter-ice channels, as evidenced by SEM images (Arango, n.d.; Engstrom et al., 2008). Faster freezing rates lead to smaller inter-ice channels, causing proteins to experience greater compression. Therefore, faster freezing induces more stress than slower freezing (FIG. 7). However, it is worth noting that air bubbles are inseparable from ice, as the formation of ice also expels dissolved air molecules to form air bubbles (Shao et al., 2023). Faster freezing rates are associated with more ice formation and smaller ice channels, which also result in smaller bubbles (Dao et al., 2022a). This inseparability underscores the importance of future studies employing advanced analytical physicochemical characterization techniques to discern the effects of the ice-water and air-water interfaces, providing definitive answers regarding the potential detrimental effects of air bubbles during the freezing of proteins.

[0053] In some embodiments, the present methods further comprise a freezing step. As used herein, the term freezing includes freeze-drying. The present methods provide a filtered de-aerated composition that is suitable for freezing or freeze-drying by any method that is known in the art or devisable through routine optimization. Different methods of freezing maybe classified by the rate of cooling, also referenced herein as the rate of freezing. The filtered de-aerated compositions of the present disclosure may be frozen at different rates. In some embodiments, the rate of freezing is less than 1 K / s. In some embodiments, the rate of freezing is about 0.90 K / s, about 0.80 K / s, about 0.70 K / s, about 0.60 K / s, about 0.50 K / s, about 0.40 K / s, about 0.30 K / s, about 0.20 K / s, about 0.15 K / s, about 0.10 K / s, about 0.09 K / s, about 0.08 K / s, about 0.07 K / s, about 0.06 K / s, about 0.05 K / s, about 0.04 K / s, about 0.03 K / s, about 0.02 K / s, about 0.01 K / s, or any range derivable therein. In some embodiments, the rate of freezing is between about 0.01 K / s and 0.50 K / s. In some embodiments, the rate of freezing is between about 0.03 K / s and 0.15 K / s. In some embodiments, the rate of freezing is approximately 0.5 K / s. In some embodiments, the rate of freezing is about equal to 1 K / s. In some embodiments, the rate of freezing is about 1 K / s, about 2 K / s, about 3 K / s, about 4 K / s, about 5 K / s, about 6 K / s, about 7 K / s, about 8 K / s, about 9 K / s, about 10 K / s, or any range derivable therein. In some embodiments, the rate of freezing is greater than 1 K / s. In some embodiments, the rate of freezing is about 20 K / s, about 30 K / s, about 40 K / s, about 50 K / s, about 60 K / s, about 70 K / s, about 80 K / s, about 90 K / s, about 100 K / s, about 110 K / s, about 120 K / s, or any range derivable therein. In some embodiments, the rate of freezing is between about 80 K / s and about 120 K / s. In some embodiments, the rate of freezing is about 100 K / s. The presently disclosed methods contemplate, for example, freezing the filtered de-aerated composition by placing the liquid sample solution on the shelf of a freezer such as a freezer at -80°C, by subjecting the filtered de-aerated composition to a plurality of freeze-thaw cycles, lyophilizing the filtered deaerated composition, adding the filtered de-aerated composition dropwise into liquid nitrogen, submerging a vial containing the filtered de-aerated composition in liquid nitrogen, or thin- film freezing the liquid sample solution.

[0054] In some embodiments, the method further comprises using a surface that has been cooled to a first reduced temperature. In some embodiments, the first reduced temperature is from about 25 °C to about -120 °C, from about -20 °C to about -100 °C, from about -60 °C to about -90 °C, or from about -150 °C, -125 °C, -120 °C, -110 °C, -100 °C, -75 °C, -50 °C, -25 °C, 0 °C, to about 25 °C, or any range derivable therein. In some embodiments, the liquid sample solution is applied from a height from about 1 cm to about 250 cm, from about 2.5 cm to about 100 cm, from about 5 cm to about 50 cm, or from about 0.5 cm, 1 cm, 1.5 cm, 2 cm, 2.5 cm, 5 cm, 10 cm, 15 cm, 20 cm, 25 cm, 50 cm, 75 cm, 100 cm, 150 cm, 200 cm, 250 cm, to about 300 cm, or any range derivable therein. In some embodiments, the surface rotates at a speed. In some embodiments, the speed is from about 5 rpm to about 500 rpm, fromabout 25 rpm to about 400 rpm, from about 50 rpm to about 250 rpm, from about 50 rpm to about 150 rpm, or from about 5 rpm, 10 rpm, 15 rpm, 20 rpm, 25 rpm, 50 rpm, 75 rpm, 100 rpm, 150 rpm, 200 rpm, 250 rpm, 300 rpm, 400 rpm, to about 500 rpm, or any range derivable therein.

[0055] In some embodiments, the freezing step comprises a drying process, such as lyophilization. In some embodiments, the drying process comprises two drying cycles. In some embodiments, the first drying cycle comprises drying at a first temperature from about -120 °C to about 0 °C, from about -10 °C to about -80 °C, from about -20 °C to about -60 °C, or from about -150 °C, -125 °C, -120 °C, -110 °C, -100 °C, -90 °C, -80 °C, -70 °C, -60 °C, -50 °C, -40 °C, -30 °C, -20 °C, -10 °C, to about 0 °C, or any range derivable therein. In some embodiments, the pharmaceutical composition is dried at a first reduced pressure from about 10 mTorr to 500 mTorr, from about 25 mTorr to about 250 mTorr, from about 50 mTorr to about 150 mTorr, or from about 5 mTorr, 6 mTorr, 7 mTorr, 8 mTorr, 9 mTorr, 10 mTorr, 20 mTorr, 25 mTorr, 50 mTorr, 100 mTorr, 150 mTorr, 200 mTorr, 250 mTorr, 300 mTorr, 350 mTorr, 400 mTorr, 450 mTorr, to about 500 mTorr, or any range derivable therein.

[0056] In some embodiments, the second drying cycle comprises drying at a second temperature from about 0 °C to about 80 °C, from about 10 °C to about 60 °C, from about 20 °C to about 50 °C, or from about 0 °C, 10 °C, 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, to about 80 °C, or any range derivable the rein. In some embodiments, the second drying cycle comprises drying at a reduced pressure. In some embodiments, the pharmaceutical composition is dried at a second reduced pressure from about 10 mTorr to 500 mTorr, from about 25 mTorr to about 250 mTorr, from about 50 mTorr to about 150 mTorr, or from about 10 mTorr, 15 mTorr, 20 mTorr, 25 mTorr, 50 mTorr, 75 mTorr, 100 mTorr, 150 mTorr, 200 mTorr, 250 mTorr, 300 mTorr, 350 mTorr, 400 mTorr, 450 mTorr, to about 500 mTorr, or any range derivable therein.D. Pressure Oscillation

[0057] In some embodiments, the presently disclosed methods comprise a pressure oscillation step. In some embodiments, a pressure oscillation may comprise placing a composition upon which the pressure oscillation step is to be performed inside of a vacuum chamber. The pressure within the vacuum chamber, in some embodiments, is modulated between a first and second pressure for a series of cycles. In some embodiments, the first pressure is below 1 atm. In some embodiments, the first pressure is about 0.05 atm, about 0.10atm, about 0.15 atm, about 0.20 atm, about 0.25 atm, about 0.30 atm, about 0.35 atm, about 0.40 atm, about 0.45 atm, about 0.50 atm, 0.55 atm, about 0.60 atm, about 0.65 atm, about 0.70 atm, about 0.75 atm, about 0.80 atm, about 0.85 atm, about 0.90 atm, about 0.95 atm, or any range derivable therein. In some embodiments, the first pressure is below 0.5 atm. In some embodiments, the first pressure is from about 0.05 atm to about 0.30 atm. In some embodiments, the first pressure is from about 0.05 atm to about 0.20 atm. In some embodiments, the first pressure is about 0. 1 atm.

[0058] In some embodiments, the second pressure is below 1 atm. In some embodiments, the second pressure is about 0.05 atm, about 0.10 atm, about 0.15 atm, about0.20 atm, about 0.25 atm, about 0.30 atm, about 0.35 atm, about 0.40 atm, about 0.45 atm, about 0.50 atm, 0.55 atm, about 0.60 atm, about 0.65 atm, about 0.70 atm, about 0.75 atm, about 0.80 atm, about 0.85 atm, about 0.90 atm, about 0.95 atm, or any range derivable therein.In some embodiments, the second pressure is higher than the first pressure. In some embodiments, the second pressure is from about 0.30 atm to about 0.70 atm. In some embodiments, the second pressure is from about 0.40 atm to about 0.60 atm. In some embodiments, the second pressure is about 0.50 atm.

[0059] According to the present disclosure, the pressure oscillation step may be performed for a number of cycles. In some embodiments, the pressure oscillation step involves cycling between the first pressure and the second pressure about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 55 times, about 60 times, about 65 times, about 70 times, about 75 times, about 80 times, about 85 times, about 90 times, about 95 times, about 100 times, about 105 times, about 110 times, about 115 times, about 120 times, about 125 times, about 130 times, about 135 times, about 140 times, about 145 times, about 150 times, or any number of times derivable therein. In some embodiments, the pressure oscillation step involves cycling between the first and second pressure more than about 50 times. In some embodiments, the pressure oscillation step involves cycling between the first and second pressure between about 60 times and about 90 times. In some embodiments, the pressure oscillation step involves cycling between the first and second pressure between about 70 times and about 90 times. In some embodiments, the pressure oscillation step involves cycling between the first and second pressure about 80 times.

[0060] The pressure oscillation step of the presently disclosed methods has a finite duration. In some embodiments, the pressure oscillation step of the presently disclosed methodshas a duration of about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145 minutes, about 150 minutes, or any length derivable therein. In some embodiments, the pressure oscillation step has a duration of more than about 15 minutes. In some embodiments, the pressure oscillation step has a duration of between about 15 minutes and about 45 minutes. In some embodiments, the pressure oscillation step has a duration of between about 20 and about 40 minutes. In some embodiments, the pressure oscillation step has a duration of about 30 minutes.IL Definitions

[0061] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” As used herein “another” may mean at least a second or more.

[0062] As used herein, the terms “drug”, “pharmaceutical”, “active agent”, “therapeutic agent”, “therapeutically active agent”, or “pharmaceutical active ingredient” are used interchangeably to represent a compound which invokes a therapeutic or pharmacological effect in a human or animal and is used to treat a disease, disorder, or other condition. In some embodiments, these compounds have undergone and received regulatory approval for administration to a living creature.

[0063] The term “biological sample” as used herein refers to any naturally derived substance of natural origin. Non-limiting examples of biological samples include proteins, such as enzymes or antibodies, and cells, including mixtures of cells. Biological samples as used herein include naturally derived substances which have been purified, processed, and / or formulated.

[0064] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. As used herein “another” may mean at least a second or more.

[0065] The terms “compositions,” “pharmaceutical compositions,” “formulations,” “pharmaceutical formulations,” “preparations”, and “pharmaceutical preparations” are used synonymously and interchangeably herein.

[0066] “Treating” or treatment of a disease or condition refers to executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus, “treating” or “treatment” may include “preventing” or “prevention” of disease or undesirable condition. In addition, “treating” or “treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0067] The term “therapeutic benefit” or “therapeutically effective” as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[0068] “Subject” and “patient” refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.

[0069] As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0070] “Pharmaceutically acceptable salts” means salts of compounds disclosed herein which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike; or with organic acids such as 1,2-ethanedisulfonic acid, 2 -hydroxy ethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene- 1 -carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-l-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl (benzoic acid, oxalic acid, p-chlorobenzenesull'onic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesull'onic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, A-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0071] The term “derivative thereof” refers to any chemically modified polysaccharide, wherein at least one of the monomeric saccharide units is modified by substitution of atoms or molecular groups or bonds. In one embodiment, a derivative thereof is a salt thereof. Salts are, for example, salts with suitable mineral acids, such as hydrohalic acids, sulfuric acid or phosphoric acid, for example hydrochlorides, hydrobromides, sulfates, hydrogen sulfates or phosphates, salts with suitable carboxylic acids, such as optionally hydroxylated lower alkanoic acids, for example acetic acid, glycolic acid, propionic acid, lactic acid or pivalic acid, optionally hydroxylated and / or oxo-substituted lower alkanedicarboxylic acids, for example oxalic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, pyruvic acid, malic acid, ascorbic acid, and also with aromatic, heteroaromatic or araliphatic carboxylic acids, such as benzoic acid, nicotinic acid or mandelic acid, and salts with suitable aliphatic or aromatic sulfonic acids or N-substituted sulfamic acids, for examplemethanesulfonates, benzenesulfonates, p-toluenesull'onates or A-cyclohexylsulfamates (cyclamates).

[0072] The term “dissolution” as used herein refers to a process by which a solid substance, for example a biological sample in solid form, is dispersed in molecular form in a medium. The dissolution rate of the active ingredients of the pharmaceutical dose of the invention is defined by the amount of drug substance that goes in solution per unit time under standardized conditions of liquid / solid interface, temperature and solvent composition.

[0073] The term “solubility” is defined as the amount of a compound that can be dissolved in a solvent. In particular, the particular amount may be described using the U.S. Pharmacopeia descriptive terms. In particular, the term “very soluble” means that less than 1 part of solvent is required for 1 part of solute. The term “freely soluble” means from 1 to 10 parts of solvent is required for 1 part of solute. The term “soluble” means from 10 to 30 parts of solvent is required for 1 part of solute. The term “sparingly soluble” means from 30 to 100 parts of solvent is required for 1 part of solute. The term “slightly soluble” means from 100 to 1000 parts of solvent is required for 1 part of solute. The term “very slightly soluble” means from 1000 to 10,000 parts of solvent is required for 1 part of solute. The term “practically insoluble or insoluble” means more than 10,000 parts of solvent is required for 1 part of solute.

[0074] The term “amorphous” refers to a substantially noncrystalline solid wherein the molecules are not organized in a definite lattice pattern. Alternatively, the term “crystalline” refers to a solid wherein the molecules in the solid have a definite lattice pattern. The crystallinity of the active agent in the composition is measured by powder x-ray diffraction.

[0075] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0076] As used in this specification, the term “significant” (and any form of significant such as “significantly”) is not meant to imply statistical differences between two values but only to imply importance or the scope of difference of the parameter.

[0077] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects or experimental studies. Unless another definition is applicable, the term “about” refers to ±10% of the indicated value.

[0078] As used herein, the term “substantially free of’ or “substantially free” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of all containments, by-products, and other material is present in that composition in an amount less than 2%. The term “essentially free of’ or “essentially free” is used to represent that the composition contains less than 1% of the specific component. The term “entirely free of’ or “entirely free” contains less than 0.1 % of the specific component.

[0079] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements and parameters.

[0080] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.IV. Examples

[0081] To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are given. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit andscope of the disclosure. In no way should the following examples be read to limit or define the entire scope of the disclosure.Example 1 - Illustrative Deaeration-Filtration of Sample SolutionA. Materials and Methods

[0082] Materials. Bovine serum IgG lyophilized powder (95+% SDS-PAGE, salt- free), and sterile phosphate-buffered saline (PBS, 0.01 M, pH 7.4) were from Millipore Sigma (Burlington, MA, USA).

[0083] Sample Preparation and Deaeration. Lyophilized bovine IgG was dissolved in 0.01 M pH 7.4 PBS buffer at a concentration of 2.0 mg / mL, centrifuged at 3,000 g, and subsequently filtered through a 0.2 pm polyethersulfone (PES) syringe filter. Samples undergoing this step were referred to as untreated or reference and are equivalent to a precursor solution of the present invention (FIG. 1).

[0084] The deaeration process, or the removal of dissolved air in the protein solution before freezing, involved placing a 5 mL liquid sample in a 20 mL glass vial inside a vacuum chamber at <5% atmospheric pressure or ~5 kPa (compared to 100% atmospheric pressure at 100 kPa). Simultaneously, the sample was agitated by magnetic stirring with a Teflon-coated magnetic stir bar at 200 rpm to induce agitation and chaotic mixing. These steps were collectively referred to as deaeration (FIG. 1). After deaeration, the pressure in the chamber was gradually brought back to atmospheric pressure (100 kPa) while the samplecontaining vial remained inside. No special sealing procedure was employed for the vial. The dissolved oxygen (DO2) level in the sample after this step was approximately 15%.

[0085] Subsequently, the deaerated samples could be further filtered through a 0.2 pm PES syringe filter before ultimately being subjected to freezing. This sequence of downstream processing was referred to as deaeration-filtration (FIG. 1). The filtration was carried out under normal atmospheric pressure (100 kPa) with minimum agitation to avoid introducing air back into the system. The filtration step increases the DO2 level 5% making the DO2 level of the solution before freezing at about 20%.

[0086] The DO2 level was quantified using the Milwaukee MW600 PRO Dissolved Oxygen Meter (Rocky Mount, NC, USA). The probe was calibrated at 0% using a dry probe method, a zero-oxygen solution. Next, the probe was calibrated at 100% using a probe-onlymethod, whereby the probe was not submerged in any solution to allow oxygen to diffuse through the membrane directly. Subsequently, the probe was tested against an air-saturated sample to confirm the calibration.

[0087] The rate of air back-diffusion into the sample is negligible, as demonstrated in the art. Deaerated samples, when left undisturbed, maintained their deaerated state for up to 30 minutes, as evidenced by unchanged dissolved oxygen (DO2) levels and the consistent number of microbubbles formed during freezing (due to low air diffusivity coefficient into water at 10'5cm2 / s) (Cadogan et al., 2014).

[0088] Freezing Processes and Air-Water Interfacial Stress. The freezing procedures involved several methods that have been described previously (Dao et al., 2022a). Initially, at the slowest cooling and / or freezing rate estimated at approximately 0.05 K / s, samples were either placed on the shelf of a -80°C freezer (referred to as F80) or subjected to three freeze-thaw cycles (referred to as F80x3). At a faster freezing rate of around 1 K / s, the IgG solution was rapidly cooled by quenching it dropwise into liquid nitrogen (LN2) using an 18-gauge needle (referred to as LN2). Alternatively, the IgG solution in a glass vial was directly submerged into LN2 (referred to as LN2-S). Lastly, the samples were gradually added dropwise onto a cryogenically cooled steel drum at -100°C and then collected in a vessel filled with LN2 (referred to as thin-film freezing (TFF)), achieving a cooling rate of approximately 100 K / s.

[0089] The air-water interfacial stress test involved vortexing the reference or untreated sample at 1,000 rpm for 2 minutes to induce visible bubbles and foaming of the protein samples.

[0090] Microflow Imaging (MFI). The protein samples were characterized for subvisible aggregates using micro-flow imaging (MFI) MFI5100 (ProteinSimple, San Jose, CA) equipped with a Botl autosampler. Specifically, 0.9 mL of protein solution was analyzed at a flow rate of 0.17 mL / min with a 300 pm flow cell. Before each sample was analyzed, the flow cell was flushed multiple times with detergent and subsequently with particle-free water, which contained less than 500 particles / mL. The protein sample was stirred three times with a Botl pipette at the lowest possible mixing rate to avoid the formation of stable foam. An MFI view system suit (MVSS) and its built-in image analysis software (ProteinSimple) were used to process the data. Extrinsic particles (e.g., rubber shards, foreign dust particles) were removed from the data using the “find like particles” feature. Intrinsic particles (e.g., air microbubblesand silicon oil droplets) were removed from the data by applying a filter that counts only particles with an aspect ratio of less than 0.9 and a circularity of less than 0.9. The data were represented using heat maps that include protein particles’ aspect ratio, circularity, and equivalent circular diameter (ECD). The circularity is a parameter used to describe the shape of particles and a measure of how close the shape is to a perfect circle. Circularity is often calculated using the formula: circularity = (4n x area) / perimeter2. The heat maps were further categorized into four quadrants and analyzed for the percentage of protein aggregates in each corresponding quadrant.

[0091] Nanobubbles Measurements by Nanotraeking Analysis. Nanosight Nanoparticle Tracking Analysis (NTA) (Malvern Inc.) was utilized for the analysis. PBS samples that underwent deaeration treatment were measured without dilution. A blue laser with a wavelength of 488 nm was employed, while the camera settings were adjusted to a level of 10 with a frame rate of 25 frames per second (FPS) and a detection threshold set to 3. Prior to each measurement, the machine underwent multiple wash cycles using HPLC grade water. The measurements were performed three times, with each run lasting between 30 to 60 seconds, depending on the concentration of nanobubbles present.

[0092] Dynamic Light Scattering (DLS). The Zetasizer ZS (Malvern Analytical, UK) was used for the DLS measurements to semi-quantitatively monitor air nanobubbles. The 0.01 M PBS pH 7.4 solutions with different treatment methods described in section 2.2 were loaded into a disposable plastic cuvette. The DLS measurements were carried out at 173° scattering angles. The derived count rate, measured in kilo-count-per-second (kcps), is a normalized parameter obtained by normalizing the count rate using the Zetasizer attenuator setting. The derived count rate is a nonlinear function of particle size and the number of particles in the system. It is collected and reported as a parameter to monitor the air nanobubbles. All measurements were performed in triplicate.

[0093] Size Exclusion Chromatography. Size exclusion chromatography (SEC) was utilized to analyze the monomer and oligomer contents of the IgG sample. SEC was performed using an Agilent AdvanceBio SEC 300 A 2.7 pm column, with a mobile phase consisting of 0.15 M phosphate buffer at pH 7.0 and 0.15 M sodium chloride at 0.35 mL / min. The detector was set at 220 and 280 nm with a 5 pL injection volume. The primary monomer peak of bovine IgG was eluted at 2.0 min.

[0094] Circular Dichroism. The effect of deaeration treatments on IgG secondary structures was carried out using circular dichroism (CD) spectroscopy. CD spectra were collected using a JASCO CD180 spectrophotometer from 195-260 nm for a far-UV study of the secondary structure. Bovine IgG solutions after freeze-thaw at different deaeration treatments were prepared at concentrations of 400 g / mL. All spectra were acquired at 20°C in a 2 mM PBS at pH 7.4 using quartz cuvettes with a 1 mm light path. All recorded spectra were the result of five scans conducted at a scanning speed of 50 nm / min. A blank spectrum was acquired from the corresponding media and subtracted accordingly.B. Results

[0095] Deaeration Process. In some embodiments, the present disclosure provides methods for producing sample compositions with less dissolved air. In some embodiments, the sample compositions of the present disclosure comprise a protein, such as IgG. In some embodiments, sample compositions formed according to the presently disclosed methods have fewer air bubbles, including air nanobubbles, in comparison to sample compositions formed according to other methods known in the art. In some embodiments, use of the presently disclosed methods leads to reduced formation of such air bubbles or nanobubbles. The presently disclosed methods may, in some embodiments, be useful for reducing the amount of dissolved air or reducing the amount of air bubbles in large volumes of sample composition.

[0096] Initially, a 2 mg / mL IgG solution was loaded into 20 mL vials, which were then placed in a vacuum chamber and subjected to a reduced pressure of 5 kPa (5% of atmospheric pressure). Without any form of agitation, no visible air bubble formation was observed. The DO2 level experienced negligible change even after 30 minutes (FIG. 2). Only after approximately 3 hours, the DO2 level stabilized to around 20%. This could be attributed, without being bound by theory, to the relatively slow molecular diffusion of air molecules through the liquid to the surface due to pressure difference.

[0097] When agitation was introduced in the form of magnetic stirring with a polytetrafluoroethylene (PTFE)-coated stir bar at a modest speed of 200 rpm, air bubble formation became apparent, leading to a rapid decrease in DO2 to approximately 20% within 20 minutes. PTFE is a hydrophobic surface coating the stir bar that, without being bound by theory, promoted air molecule nucleation on it to form bubbles, which grew in size, floated, and escaped, thereby facilitating the reduction of dissolved air level. In contrast, when a glass- coated stir bar was used, much fewer bubbles were formed and thus the deaeration process wasinefficient (FIG. 2). Notably, in a completely clean beaker treated with nitric acid and using a glass stir bar positioned centrally, air bubbles were predominantly observed along certain points of the beaker's glass side walls, possibly due, without being bound by theory, to localized imperfections that rendered those areas more hydrophobic.

[0098] Chaotic mixing and collisions were also observed to promote bubble formation by facilitating random collisions between air bubble nuclei (Scardina, 2004). Compared to a rather laminar flow mixing, which occurs when the stir bar is centered in the beaker, off-centered positioning of the stir bar proved to be more efficient, as it increased the chances of collision.

[0099] In summary, deaeration with stirring using a PTFE stir bar was considered the embodiment of choice and was utilized for deaeration steps described below.

[0100] Effect of deaeration on freeze-induced IgG aggregate formation. The deaeration process was implemented prior to freezing the protein samples. Initially, it was anticipated that, without being bound by theory, deaerating the samples would result in lower dissolved air levels and therefore would lead to fewer protein aggregates formed due to reduced denaturation at the air-water interface caused by air bubbles. Surprisingly, deaeration alone (FIG. 1 ), accompanied by visible bubble formation, actually increased denaturation, resulting in approximately twice the number of protein aggregates compared to untreated samples (blue versus white bars, respectively) (FIG. 3A). The deaeration treatments did not significantly altered the monomer oligomer ratio as demonstrated in SEC chromatogram (FIG. 3B). However, FIG. 3C showed that, compared to reference sample, the CD spectra of a liquid sample solution that had been subjected to a deaeration step and a filtration step is similar, suggesting, without being bound by theory, a stable secondary structure of IgG after freezethaw (orange vs. red lines). In contrast, a slight difference was detected in the 195-210 nm region of the untreated and deaeration (green and blue lines, respectively), compared to the reference; suggesting, without being bound by theory, a slight change in secondary structure of the protein molecules.

[0101] The addition of a filtration step (FIG. 1) consistently yielded lower aggregate counts after freezing across all tested freezing rates and techniques (ranging from 0.01 to 100 K / s) (FIG. 3A; red bars). The heatmap analysis revealed lower aggregate countsrepresented by less cold color-coding, indicating, without being bound, the presence of smaller aggregates as well (FIG. 3D).

[0102] While these results from FIG. 3 are promising, demonstrating that deaeration followed by filtration (e.g., deaeration-filtration in FIG. 1) helped mitigate freeze- induced denaturation effects on proteins and subsequently reduced aggregation, they raised several critical points in the lack of understanding of the observed phenomena including (1) the apparent bubble formation during deaeration exacerbated freeze-induced stress and (2) the unclear role of the filtration step after deaeration in lowering aggregation.

[0103] Filtration Step Unlikely Removed Protein Aggregate. FIG. 4A shows that subvisible particle count of untreated sample was not statistically different from deaeration-filtration sample (-1,600 # / mL). Furthermore, FIG. 4A demonstrated that the formation of IgG aggregation by air-water interfacial stress is a time-dependent process. After being exposed to air- water interfacial stress by shaking (1,000 rpm vortex for 2 minutes), the IgG solutions were stored at 4°C for 24 hours. The results showed that particle counts increased over time, from approximately 1,500 # / mL at baseline to around 30,000 # / mL at 24 hours. The highest number of aggregates was observed after the sample was stored at room temperature for 24 hours, at -60,000 # / mL (FIG. 4A). The results suggested that IgG molecules adsorbed onto the air-water interface and denatured (Dao et al., 2022b); however, aggregation occurred only when two denatured molecules collided and grew to the point detectable by MFI. Due to the random nature of the collisions, the morphology of the aggregates appeared more round in the representative few largest aggregates (FIG. 4B, FIG. 4C).

[0104] In contrast, freeze-induced stress is time-independent, as the protein aggregate counts of freeze-thaw samples reached their nominal values after thawing and remained unchanged over time in all reported data (FIG. 3A). Furthermore, a close visual examination of the largest representative freeze-induced aggregates in FIG. 3D and FIG. 3E demonstrated that the aggregates appeared more fibrous. This result suggested, without being bound by theory, that IgG aggregation due to freezing occurred likely because proteins were compressed between ices.

[0105] Based on the representative photos, it was hypothesized, without being bound by theory, that aggregates induced by air-water interfacial stress would become more rounded over time. The aspect ratio of the entire population of protein aggregates was thenanalyzed. An aspect ratio of 1.0 indicates a perfect circle, while 0.0 indicates a line. However, although the mean aspect ratio appears to increase with time, there is no statistical difference in aspect ratio between air-water stressed samples over time (FIG. 4A, red dots). This is likely because, without being bound by theory, aspect ratio values are number based; thus, one small 10 pm particle weighs as much as one 100 pm particle (1: 1 ratio), while in terms of volume, the 100 pm particle is approximately 4,000 times larger.

[0106] Taking a different approach to gain insights into the morphology of IgG aggregates, the heatmap was divided into four quadrants, with quadrant I being the most rounded. As illustrated in FIG. 4F, the percentage of air-water interfacially stressed protein aggregates in quadrant I increased over time from 54.9% to 93.0%, indicating a collision mechanism for air-water-induced stress.

[0107] Filtration Step Did Not Alter IgG Monomer Percentage. The SEC results demonstrated that both the deaeration process and the deaeration-filtration procedure, as illustrated in FIG. 1 , did not significantly alter the percentage of protein monomers (FIG. 5). A close examination of the IgG SEC chromatogram revealed nearly identical peaks. Neither filtration nor the formation of air bubbles during the deaeration step immediately altered the IgG monomer / oligomer population ratio. Furthermore, the recovery of IgG, as measured by the total area under the curve, indicated that protein recovery was not affected within the first hour of storage at 4°C following deaeration or deaeration-filtration processes. These results suggest that the syringe filter with pores of 220 nm (0.22 pm) in size, which were much larger than the IgG's hydrodynamic size of approximately 10 nm, had an unlikely effect on protein molecules.

[0108] Filtration Reduced Existing Nanobubbles After Deaeration. The deaeration step (FIG. 1) indeed reduced dissolved air, as demonstrated in FIG. 2. However, deaeration at reduced pressure combined with agitation by a PTFE magnetic stir bar unintentionally created and introduced nanobubbles into the liquid samples. Unlike larger bubbles, which can float to the top and escape, nanobubbles are buoyancy-neutral and thus remain in the solution after being created (Snell el al. , 2020). As shown in FIG. 6A, visual observation by shining a laser through the sample revealed that after deaeration, something was introduced into the solution, illuminating the path of the laser. The microscopic objects which illuminate the laser path were unlikely dust as the samples subjected to reduced pressure state and subsequently repressurized did not showed any visible light path (FIG. 6A). Mostimportantly, the laser light path was eliminated when sample treated with deaeration was accompanied by subsequent filtration step.

[0109] Dynamic light scattering (DLS) analysis confirms the presence of nanobubbles after the deaeration step (FIG. 6B). The derived count rate, which reflects the normalized scattered light intensity, increased approximately twofold after deaeration. This suggests an increase in the number or size of nanobubbles in the system. Filtration effectively removes these nanobubbles, as evidenced by the decrease in count rate from about 60 kcps to 30 kcps, bringing it close to the initial starting value of 25 kcps. Furthermore, the constant count rate after storing the deaerated samples for 240 minutes at room temperature (FIG. 6B) indicates the neutral buoyancy of the nanobubbles.

[0110] Lastly, the quantification of nanobubbles was performed using Nanosight Nanotracking Analysis. The reference sample exhibited nearly zero nanobubbles, which aligned with expectations (FIG. 6C). When pressure reduction was applied without agitation, no evident macro-hubbling occurred. The DO2 level remained similar to the reference sample and the number of nanobubbles was negligible. However, upon introducing stirring (allowing for macro-bubbling escape), the detected nanobubble count dramatically increased to 2.06 x 108particles / mL with diameters ranging from 50 to 100 nm (FIG. 6C, FIG. 6D). As expected, the nanobubble count remained consistent even after 240 minutes’ storage at room temperature. Eventually, subsequent filtration significantly reduced the nanobubble count by roughly ten times, resulting in 4 x 107bubbles / mL, although not returning to the original level of approximately zero.

[0111] Initially, it was anticipated that the 220 nm PES membrane would only filter out nanobubbles larger than 220 nm. However, upon closer examination of the nanobubble size distribution, it appeared that bubbles smaller than 220 nm were also significantly reduced. FIG. 6D illustrates that, prior to filtration (indicated by the blue line), only a minute fraction of nanobubbles exceeded 200 nm in size. Remarkably, the filtration process significantly reduced the number of bubbles in the 50-100 nm range, which is smaller than the pore size of the membrane (red line). These results suggest, without being bound by theory, that the hydrophilic PES membrane prevented the passage of hydrophobic air nanobubbles. It aligns with literature reports that there is substantial resistance when attempting to push air through a PES membrane, with bubble points ranging from 45 to 80 psi (Wavhal and Fisher, 2002).

[0112] Deaeration-filtration works cooperatively with cryoprotectant. We conducted experiments to assess the effectiveness of deaeration-filtration treatment in conjunction with a cryoprotective agent such as trehalose. At a slower freezing rate of approximately 1 K / s, deaeration-fdtration demonstrated efficacy in the absence of trehalose. However, the presence of 0.5% trehalose is sufficient to outweigh the effects of deaerationfiltration (FIG. 7A).

[0113] As expected, deaeration, coupled with evident bubbling and nanobubbles, exacerbate the freeze-induced stress on IgG and thus significantly increased the protein aggregate count (FIG. 7A, blue bars). The cryo-protective effect of trehalose is evident as higher concentrations of trehalose correlated with greater protective effects and reduced aggregate counts

[0114] In contrast, at a higher freezing rate (TFF at 100 K / s), deaeration-filtration proves highly effective even in the presence of trehalose (FIG. 7B). This could be attributed to the heightened stress levels during rapid freezing, overwhelming the protective capabilities of trehalose. These results suggest, without being bound, a combined effect of deaerationfiltration and the use of cryo-protectant.C. Discussion

[0115] Widely acknowledged to arise from the ice-water interface, mechanisms of freeze-induced stress have been the subject of numerous studies. Scientific literature consistently highlights the correlation between faster freezing rates and increased ice-water interfacial area (Bhatnagar et al., 2007; Cao et al., 2003; liang and Nail, 1998; Schwegman et al., 2009; Strambini and Gonnelli, 2007). The present disclosure provides methods that reduce freezing stress derived at least in part from protein denaturation by air bubbles trapped between ice crystal and subsequent freeze-induced protein aggregation. The inventors provide the present methods to reduce protein sub- visible aggregates that form during freezing due to (1) denaturation of protein molecules at the air- water interface and (2) proximity of these denatured protein molecules to each other (FIG. 8).

[0116] In some embodiments, the present disclosure provides methods of reducing the amount of dissolved air in a sample solution through deaeration processes, thereby leading to a decrease in air bubble formation during freezing. In some embodiments, the sample solution comprises protein, such as IgG. While it was observed that dissolved air and associatedmicron-sized air bubbles were indeed reduced (Dao et al., 2022a), the deaeration process unintentionally introduced nanobubbles into the solution (FIG. 6C), which cannot be detected by the vertical freezing microscopy (Dao et al., 2022a). Without the filtration step, these nanobubbles, having neutral buoyancy (Snell et al., 2020), remained in the liquid sample and facilitated denaturation and subsequent aggregation (FIG. 8). Only when both sources of air- water-induced denaturation were suppressed through deaeration and filtration (dissolved air and nanobubbles, respectively) were significant reductions observed in the freeze-induced denaturation of sample, such as IgG.

[0117] As proposed in the schematic illustration shown in FIG. 8, aggregation will not occur if only one of the two conditions is met, that of denaturation and close proximity. This point is supported by a study of large-scale freezing of protein where a higher IgG concentration was observed at the bottom of the container (Hauptmann et al., 2019). However, most of the protein aggregation was detected at the top region of the container, suggesting that the close proximity of native or folded proteins alone was insufficient to induce aggregation. In contrast, the inventors discovered that while simulating air-water stress via vigorous vortexing, wherein one prerequisite condition was met (a significantly large air- water interface and denatured protein molecules), the requirement for close proximity was lacking. Protein molecules are far apart in bulk liquid solution; collisions between two denatured molecules occur randomly and require time, making this process time-dependent (FIG. 4). Within the first two hours after exposing the IgG solution to highly stressful conditions (e.g., vortexing at 1 ,000 rpm for 2 minutes), there was no significant difference in aggregate count between the stressed and reference samples. In the context of close proximity, trehalose and other cryoprotectants’ main mechanisms in stabilizing protein is via vitrification through which protein molecules are immobilized in a glassy matrix therefore preventing the aggregation process from beginning and developing (Fahy and Wowk, 2015; Jain and Roy, 2009). Therefore, cryoprotectants with high tendency of forming amorphous morphology are often preferred over crystalline ones due to cryoprotectant crystal formation further exclude and compress the protein molecules (Li et al., 2023).

[0118] In terms of translational significance, the present disclosure underscores the value of deaeration-filtration to supplement trehalose as a cryoprotectant (FIG. 7B). This supplementary effect proves particularly beneficial in the downstream processing of freeze- dried powder intended for lung delivery, where a highly porous powder is preferable. However,achieving such porosity typically involves high freezing rates (e.g., through techniques like TFF or spray freeze drying) to generate numerous small ice crystals. Concurrently, maintaining low concentrations of cryo / lyo-protectants is essential to decrease powder density for enhanced lung deposition. If, despite exhausting all formulation approaches, aggregation remains unacceptably high, deaeration- filtration stands as a viable additive upstream process.Example 2 - Effect of deaeration-filtration of freezing media on red blood cell sensitivity to freeze-thawingA. Methods and Results

[0119] A sample solution (15% (w / v) dextran (MW 40000), 2.5% (w / v) bovine serum albumin (BSA), 300 mM trehalose, 100 mM sodium chloride, in 20 mM HEPES (pH 7.1)) was prepared. The sample solution was de-aerated and filtered (i.e., ‘deaerationfiltration’) according to the procedures outlined above before it was mixed with RBCs to reach an RBC concentration of 500,000,000 cells / mL The de-aerated and filtered sample was then thin-film frozen at -80°C and stored in a -80°C frozen until further use. Thawing was achieved by incubating the frozen cell suspension in a 37°C water bath. The extent of RBC lysis was determined using a modified Sigma-Aldrich method (MAKI 15). Briefly, after thawing, 25 pL of each RBC sample was transferred into a 96-well plate in triplicate. The remaining samples were centrifuged at 600 g, and 25 pL of the supernatant was transferred to three additional wells in the 96-well plate. Then, 200 pL of the hemolysis reagent (100 mM NaOH, 2.5% v / v Triton X-100 in water) was added to each well and incubated at room temperature for 5 min. Absorbance was measured at 400 nm. The percentage of RBC hemolysis was calculated by dividing extracellular hemoglobin absorbance (i.e., in the supernatant after centrifugation) by total hemoglobin absorbance. As a control, RBCs in the same freezing media that was not subject to the deaeration- filtration process were thin-film frozen and thawed. As shown in FIG. 9, the percent of RBC hemolysis was lower when RBCs were in the media that was subject to that deaeration-filtration process than when RBCs were in the media that was not subject to the process (18% vs. 26%).Example 3 - Pressure oscillation on red blood cell lysis when subject to freezing or freeze-dryingA. Methods and Results

[0120] Red blood cell (RBC) suspensions were prepared in a freezing media solution (15% (w / v) dextran (MW 40000), 2.5% (w / v) bovine serum albumin, 300 mM trehalose, 100 mM sodium chloride, 20 mM HEPES, pH 7.1) at a concentration ofapproximately 500,000,000 cells / mL Pressure oscillation involved subjecting the cell suspension to a 15-minute, approximately 80-cycle oscillation between 0.1 and 0.5 atm. Oscillation was done in a vacuum chamber, with 5 mL of RBC suspension in a 20-mL glass vial in the chamber. As a control, the RBC suspension was left untreated before being subject to thin-film freezing. As another control, the RBCs were suspended in the freezing media solution that was subject to deaeration- filtration prepared according to the methods described above. RBC cell suspensions were frozen using thin-film freezing (TFF) at -80°C or -40°C. A controlled shelf-freezing of RBCs suspended in a freezing media that was not subject to the deaeration-filtration process was used yet as another control (4°C to -40°C at l°C / min cooling rate, 4-hour hold). Frozen RBCs were rapidly thawed at 37°C, and cell lysis was assessed using a hemolysis assay.

[0121] Results in FIG. 10 showed a reduced RBC lysis when the RBCs were subject to pressure oscillation (17.8% lysis at -80°C and 21.9% lysis at -40°C) as compared to when RBCs were not subject to pressure oscillation (31.6% lysis). Moreover, the pressure oscillation appeared more effective in reducing RBC lysis than the deaeration-filtration process (24.8% lysis).

[0122] In some embodiments, preparation of frozen sample compositions wherein the sample is a cell according to the methods disclosed herein have a favorable effect on lysis of sample cells. To study the effect of a pressure oscillation step on red blood cell (RBC) lysis when they are subject to thin-film freeze drying (TFFD), RBCs (500,000,000 cells / mL) in freezing media (which in certain embodiments may also be referenced herein as either liquid composition, de-aerated composition, or filtered de-aerated composition) were subject to pressure oscillation and thin-film frozen as mentioned above. Water was removed from the frozen samples was sublimed in a Virtis freeze-dryer. As a control, RBCs in suspension were subject to conventional freeze-drying. Dried RBC samples were reconstituted with a reconstitution media (20% w / v dextran, 20 mM HEPES, pH 5.3) in a 45 °C water bath, and RBC lysis was measured as mentioned above. The percentage of hemolysis (lysed RBCs) was determined using a modified Sigma Aldrich method (MAKI 15). After reconstitution, 50 pL of each RBC sample was transferred into a 96-well plate in triplicate. Remaining samples were centrifuged at 9600x g, and 50 pL of supernatant was transferred to three additional wells. Then, 200 pL of hemolysis reagent (100 mM NaOH, 2.5% v / v Triton X-100 in water) was added to each well and incubated at room temperature for 5 minutes. Absorbance was measuredat 400 nm. The percentage of RBC hemolysis was calculated by dividing extracellular hemoglobin absorbance by total hemoglobin absorbance. As shown in FIG. 11, subjecting RBCs in suspension to pressure oscillation before thin-film freeze-drying reduced RBC lysis by ~15% (e.g., 73% vs. 85.4% without pressure oscillation). These findings support the effectiveness of embodiments of the presently disclosed methods comprising a pressure oscillation step on enhancing RBC stability during freezing, including freeze drying such as thin-film freeze drying.* *

[0123] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.References

[0124] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.Arango, S.J., n.d. Refrigeration: Impact of ice crystal size and freezing rate [WWW Document], New Food Magazine. URL https: / / www.newfoodmagazine.com / article / 23904 / refrigeration-impact-of-ice- crystal-size-and-freezing-rate / (accessed 5.20.24).Arsiccio et al. , J. Am. Chem. Soc., 142, 5722-5730, 2020.Arsiccio et al. , J. Phys. Chem. B, 122, 11390-11399, 2018.Arsiccio and Pisano, J. Pharm. Sci., 109, 2116-2130, 2020a.Authelin et al., J. Pharm. Sci., 109, 44-61, 2020.Bhatnagar et al. , J. Phys. Chem. B, 123, 5690-5699, 2019.Bhatnagar et al.,. Pharm. Dev. Technol., 12, 505-523, 2007.Bluemel et al.,. Int. J. Pharm., X 4, 100109, 2022.Cadogan et al. ,. J. Chem. Eng. Data, 59, 519-525, 2014.Cao et al.,. Biotechnol. Bioeng., 82, 684-690, 2003.Carte,. Proc. Phys. Soc. 77, 757, 1961.Chang et al.,. J. Pharm. Sci., 85, 1325-1330, 1996.Dao et al.,. Int. J. Pharm., 628, 122306, 2022a.Dao et al.,. Mol. Pharm., 19, 2662-2675, 2022b.Engstrom et al.,. Pharm. Res., 25, 1334-1346, 2008.Fahy and Wowk,. Methods in molecular biology (Clifton, N.J.) 1257, 21-82, 2015.Griffin et al.,. J. Pharm. Sci., 2024.Hauptmann et al.,. AAPS PharmSciTech, 20, 72, 2019.Heger and Klan,. J. Photochem. Photobiol. A, 187, 2007.Jain and Roy,. Protein Sci., 18, 24-36, 2009.Jiang and Nail,. Eur. J. Pharm. Biopharm., 45, 249-257, 1998.Koepf et al. ,. Ini. J. Pharm., 537, 202-212, 2018.Li et al.,. Mol. Pharm., 20, 4587-4596, 2023.Nikolaidis et al.,. Food Chem., 232, 425-433, 2017.Pardeshi et al.,. Futur. J. Pharm. Sci., 9, 99, 2023.Ratanji et al. ,. J. Immunotoxicol., 11, 99-109, 2014.Regand and Goff,. J. Dairy Sci., 85, 2722-2732, 2002.Rodrigues et al.,. J. Pharm. Sci., 100, 1316-1329, 2011.Rosa er al.,. PLoS One, 12, e0176748, 2017.Scardina,. Virginia Tech Electronic Theses, 2004.Schwegman et al.,. J. Pharm. Sci., 98, 3239-3246, 2009.Shao et al. ,. Phys. Fluids, 35, 113319, 2023.Snell et al.,. J. Pharm. Sci., 109, 284-292, 2020.Snell et al.,. J. Pharm. Sci., 105, 3057-3063, 2016.Strambini and Gabellieri,. Biophys. J., 70, 971-976, 1996.Strambini and Gonnelli,. Biophys. J., 92, 2131-2138, 2007.Twomey et al.,. Int. J. Pharm., 487, 91-100, 2015.Twomey et al.,. J. Phys. Chem. B, 117, 7889-7897, 2013.Wavhal and Fisher,. Langmuir, 19, 2002.Webb et al.,. J. Pharm. Sci., 92, 715-729, 2003.Yang et al.,. Phys. Rev. Lett., 129, 046001, 2022.

Claims

What Is Claimed Is:

1. A method of forming a filtered de-aerated composition, wherein the method comprises:A. obtaining a liquid composition;B. placing the liquid composition in a vessel and subjecting the liquid composition to a deaeration step to form a de-aerated composition; andC. subjecting the de-aerated composition to a filtration step to form said filtered de-aerated composition.

2. The method according to claim 1 , wherein the liquid composition comprises a biological sample.

3. The method according to either claim 1 or claim 2, wherein the method further comprises combining the filtered de-aerated composition with a biological sample.

4. The method according to any one of claims 1-3, wherein the deaeration step comprises stirring the liquid composition.

5. The method according to any one of claims 1-4, wherein the deaeration step further comprises agitation.

6. The method according to any one of claims 1-5, wherein the deaeration step occurs at a pressure lower than atmospheric pressure.

7. The method according to any one of claims 1-6, wherein the deaeration step comprises stirring the liquid composition with a PTFE stir bar at rate of about 200 rpm at a pressure of about 5% atmospheric pressure for a time period of about 20 minutes.

8. The method according to any one of claims 1-7, wherein the de-aerated composition has less dissolved oxygen than the liquid composition.

9. The method according to any one of claims 1-8, wherein the filtration occurs at a pressure that is less than atmospheric pressure.

10. The method according to any one of claims 1-9, wherein the filtered de-aerated composition has fewer nanobubbles than the de-aerated composition.

11. The method according to any one of claims 1-10, wherein the liquid composition further comprises a cryoprotective agent.

12. The method according to any one of claims 1-11, wherein the method further comprises combining a cryoprotective agent with the filtered de-aerated composition.

13. The method according to any one of claims 1-12, wherein the method further comprises a pressure oscillation step.

14. The method according to claim 13, wherein the pressure oscillation step comprises subjecting the filtered de-aerated composition to a plurality of cycles of a first pressure and a second pressure.

15. The method according to any one of claims 1-14, wherein the method further comprises freezing the filtered de-aerated composition to obtain a frozen de-aerated composition.

16. The method according to any one of claims 2-15, wherein the biological sample comprises a protein.

17. The method according to any one of claims 2-16, wherein the biological sample comprises a cell.

18. The method according to any one of claims 2-17, wherein the sample comprises a red blood cell.

19. A liquid composition formed according to the method of any one of claims 1-14.

20. A frozen composition formed according to the method of any one of claims 15-18.

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