Isolated cell culture components and methods for isolating the same from liquid cell culture medium

Abstract

To provide isolated cell culture components such as, e.g., biologics and / or lipids, and to provide methods for isolating cell culture components from a liquid cell culture medium.SOLUTION: Methods of the present invention may include: contacting a dehydration composition and a liquid cell culture medium comprising a target component to form a mixture; forming an at least partially dehydrated component in the mixture; and separating the at least partially dehydrated component from the mixture, thereby providing an isolated component. In some embodiments, the isolated component comprises the at least partially dehydrated component. In some embodiments, the isolated component is present in a composition (e.g., liquid phase) separated from the at least partially dehydrated component.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Related Application Information This application claims the benefit of U.S. Provisional Patent Application No. 62 / 801,225, filed February 5, 2019, and is incorporated herein by reference in its entirety.

[0002] Field The present invention relates to isolated cell culture components, such as biologics and / or lipids, and to methods for isolating cell culture components from liquid cell culture media. [Background technology]

[0003] background Pharmaceutical companies and manufacturers are seeing significant changes in their drug development and manufacturing methods due to the significant growth and transition to more complex biologically-based therapeutics. This industry-wide growth in biologics is the primary driving force in the production of millions of units of bulk biological drug substances (DS). Temperature-controlled pharmaceutical transportation is also one of the fastest growing vertical markets in the global healthcare logistics industry. Moreover, biomanufacturing is a key component of biologics manufacturing and accounts for a significant share of the biologics market.

[0004] Many companies have separated bulk drug substance manufacturing from their finished drug manufacturing operations, which means that valuable DS must be stored and shipped around the world on demand, along with clinical trial supplies and finished drug manufacturing.

[0005] It is well known that freezing can extend the shelf life of biological materials. This practice has become a common option for preserving bulk drug substances. However, the freezing method itself, and subsequent thawing, can have detrimental effects on the structure and stability of biomolecules. As freezing occurs, proteins are exposed to a variety of factors that can increase aggregation. Ice crystals create a large surface area where proteins are prone to unfolding. Removal of water into the ice phase results in "cryoconcentration," i.e., solutes (buffer salts, excipients, and the protein itself) become more concentrated, causing pH shifts and local concentration fluctuations. Freezing and thawing rates are highly dependent on volume and container size, so they can be inherently difficult to scale in a controlled manner. A typical jacketed metal freeze-thaw container holds up to 300 L of DS and requires 18 hours for freezing. Furthermore, storage at -20°C is often not sufficiently below the glass transition temperature to confer stability. As storage and shipping temperatures decrease, costs increase dramatically.

[0006] Challenges continue to arise in biological manufacturing as tighter controls over manufacturing methods are implemented. Manufacturers are seeing much greater importance on stability and temperature requirements. When companies are required to discard or destroy units of biological drug substance, the result is a much greater loss in investment and extended delays in product development timelines. Losses are numerous across industries, with over $15 billion in product losses occurring annually in the pharmaceutical industry due to temperature excursions alone, 25% of vaccines reaching their destination in a deteriorated state due to improper transportation, 30% of discarded medicines can be attributed to logistical issues, and 20% of temperature-sensitive products are damaged during transportation due to a broken cold chain.

[0007] Lyophilization can be used to provide stability to the final formulation form. However, it typically requires too much up-front development (e.g., identification of appropriate cycling conditions) to be useful at this stage, and suffers from the freezing stresses mentioned above. In addition, it is a batch-only process, a time-consuming process (a single batch can take several days), and the capital equipment and energy costs are significant.

[0008] Spray drying has been investigated as a potential alternative processing technique for biological materials. However, it frequently suffers from problems of reduced product yield and aggregation. Many proteins are surface active and aggregate at the high energy air-water interface. The required outlet temperature can also be a problem for temperature sensitive proteins. Large amounts of excipients (e.g., about 50%-90% (w / w)) are often required to stabilize the protein, and it has been shown that these excipients can separate from the protein within the particles, reducing their effectiveness. Summary of the Invention

[0009] summary A first aspect of the invention is directed to a method of isolating a component (e.g., a biological or lipid) from a liquid cell culture medium comprising contacting a dehydrated composition (e.g., a first organic solvent) with a liquid cell culture medium containing the component to form a mixture, forming an at least partially dehydrated component in the mixture, and separating the at least partially dehydrated component from the mixture to provide an isolated component. In some embodiments, the isolated component comprises the at least partially dehydrated component. In some embodiments, the isolated component is in a composition (e.g., a liquid phase) separated from the at least partially dehydrated component.

[0010] Another aspect of the invention is directed to a composition comprising a plurality of solid amorphous particles (e.g., microparticles). In some embodiments, at least a portion of the plurality of solid amorphous particles are solid amorphous particles comprising biological material (e.g., proteins, peptides, polynucleotides, etc.).

[0011] A further aspect of the invention is directed to an isolated biological material, the isolated biological material being at least partially dehydrated.

[0012] Another aspect of the invention is directed to isolated hydrophobic components (e.g., lipids and / or steroids), which may be at least partially separated from the at least partially dehydrated components and / or water soluble components present in the liquid cell culture medium.

[0013] It should be noted that aspects of the invention described with respect to one embodiment may be incorporated in a different embodiment without being specifically described therein. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination. Applicant reserves the right to modify any originally filed claim and / or to submit any new claim accordingly, including the right to amend any originally filed claim to depend from and / or incorporate any feature of any other claim or claims not originally claimed in that manner. These and other objects and / or aspects of the invention are described in detail in the specification set forth below. Further features, advantages and details of the invention will be understood by those skilled in the art from reading the drawings and the following detailed description of the invention of the preferred embodiments, and such description is merely illustrative of the invention. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is an image of a suspension containing microvitrified lysozyme particles. [Diagram 2]2 is a graph showing predicted set times based on the Epstein-Plesset equation, assuming 50 mg / mL with f=0.3 to account for bulk addition of water. Actual set times may increase due to mixing. [Diagram 3] FIG. 3 is a graph showing the residual water content in Microglassified™ bovine gamma globulin (BGG) batches produced using four different processing schemes. [Figure 4] FIG. 4 is a graph showing representative particle size distribution for an in-line process according to some embodiments of the present invention. [Diagram 5] FIG. 5 is a graph showing the reproducibility of particle size distribution using a small scale process. [Figure 6] FIG. 6 is a graph showing the reproducibility of particle size distribution using an in-line method according to some embodiments of the present invention. [Figure 7] FIG. 7 is a graph showing milligrams of residual solvent remaining per gram of microglassified™ BGG. [Figure 8] FIG. 8 is an image of a suspension of microvitrified particles in pentanol after contacting the cell composition and the pentanol, according to some embodiments of the present invention. [Figure 9] FIG. 9 is an image of a suspension of microvitrified particles in butyl lactate after contacting the cell composition and butyl lactate, according to some embodiments of the present invention. [Figure 10] FIG. 10 is an image of a suspension of microvitrified particles in pentanol after contacting the cell composition and the pentanol by vortexing, according to some embodiments of the present invention. [Figure 11]FIG. 11 is an image of a suspension of microvitrified particles in pentanol after contacting by homogenization of a cell composition and the pentanol, according to some embodiments of the present invention. [Figure 12] FIG. 12 is an image of a suspension of microvitrified particles in pentanol after contacting a highly concentrated cell composition and the pentanol by vortexing, according to some embodiments of the present invention. [Figure 13] FIG. 13 is an image of a microfluidic device being used in microvitrification according to an embodiment of the present invention. [Figure 14] Figure 14 is an image of particles produced using the microfluidic device shown in Figure 10. Mean particle diameter of 4.8 μm. [Figure 15] FIG. 15 is a graph of mass loss over time during nitrogen drying of a protein powder. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0016] The terms used in the description of the present invention herein are for the purpose of describing particular embodiments only and are not intended to limit the present invention. When used in the detailed description of the present invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0017] It will be understood that although the terms "first", "second", etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are used only to distinguish one element from another. Thus, a "first" element discussed below can also be referred to as a "second" element without departing from the teachings of the present invention. Unless specifically indicated otherwise, the arrangement of the operations (or steps) is not limited to the order presented in the claims or drawings.

[0018] Unless otherwise defined, all terms (including scientific and technical terms) used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. It is further understood that words, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of this application and related art, and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict in terms, the present specification shall control.

[0019] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0020] Unless the context indicates otherwise, it is specifically contemplated that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein can be excluded or omitted. To illustrate, if the present specification describes that a complex comprises components A, B, and C, it is specifically contemplated that any of A, B, or C, or any combination thereof, can be omitted and discarded.

[0021] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be construed as including the recited materials or steps "and which do not materially affect one or more of the basic and novel characteristics" of the claimed invention. In re Herz, 537 F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976) (emphasis in original); see also MPEP § 2111.03. Thus, the word "consisting essentially of" as used herein should not be construed as equivalent to "comprising."

[0022] It will also be understood that, as used herein, the words "example," "exemplary," and grammatical variations thereof are intended to refer to non-limiting example and / or variant embodiments discussed herein, and are not intended to indicate a preference for one or more embodiments discussed herein over one or more other embodiments.

[0023] As used herein, the term "about" when referring to a measurable value, such as an amount or concentration, is meant to include ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% variations of the specified value, as well as the specified value. For example, "about X," where X is a measurable value, is meant to include X, as well as ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% variations of X. Ranges provided herein for measurable values ​​can encompass any other ranges and / or individual values ​​therein.

[0024] As used herein, the words "increase," "increases," "increased," "increasing," "improve," "enhance," and similar words refer to an increase in a specified parameter of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more.

[0025] As used herein, the words "reduce," "reduces," "reduced," "reduction," "inhibit," and similar terms refer to a decrease in a specified parameter of at least about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 100%.

[0026] A method for isolating a component (e.g., a biological material or lipid) from a liquid cell culture medium is provided according to an embodiment of the present invention. The component can be any compound, molecule, and / or biological material present in the liquid cell culture medium. Exemplary liquid cell culture media include, but are not limited to, fermentation broth and / or microbiological culture. As understood in the art, liquid cell culture media can include cells, cellular components (e.g., lipids, cell membranes, proteins, DNA, etc.), synthetic materials (e.g., additives for stabilizing the medium (e.g., poloxamer, e.g., Poloxamer 188 PRO)), buffers, and / or sugars, etc. For example, fermentation broth can include tryptic soy broth, bacterial yeast extract, ammonium chloride, disodium phosphate, potassium phosphate, and / or glucose. Possible types of cells can be present in the liquid cell culture medium of the present invention, including, but not limited to, bacterial cells, yeast cells, and / or mammalian cells (e.g., human cells). In some embodiments, the liquid cell culture medium comprises cells that intracellularly and / or extracellularly express a biological substance that can be isolated according to the methods of the present invention.

[0027] The method of the present invention may use the liquid cell culture medium without any modification (e.g., without adding or removing anything) to the liquid cell culture medium compared to before the method. For example, the medium may be used directly from the cell culture. In some embodiments, the method of the present invention includes processing the liquid cell culture medium, including, but not limited to, concentrating, centrifuging, washing, dialysis, and / or filtering the liquid cell culture medium. One or more processing steps may be used to concentrate the cells and / or remove substances (e.g., salts and / or cells) present in the liquid cell culture medium. The component to be isolated may determine whether cells should be concentrated or removed. For example, if the component is expressed inside the cells, the cells may be concentrated, while if the component is expressed outside the cells, the cells may be removed. In some embodiments, the method of the present invention may use a liquid cell culture medium that is diluted, for example, with water or an aqueous solution (e.g., a buffer solution). In some embodiments, the methods of the invention may use liquid cell culture media that is concentrated, such as via centrifugation and / or filtration.

[0028] In some embodiments, the liquid cell culture medium may include one or more (e.g., 1, 2, 3, 4, 5, or more) additives. Exemplary additives include, but are not limited to, stabilizers, surfactants, tonicity agents, sugars, salts, antimicrobial agents, antioxidants, reducing agents, and the like. In some embodiments, the additives are present in the liquid cell culture medium at a concentration ranging from about 0.01%, about 0.1%, or about 0.5% by weight of the liquid cell culture medium to about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight.

[0029] Exemplary surfactants include, but are not limited to, polysorbates (e.g., polysorbate 20 or 80), spans, pegylated fatty esters and ethers (e.g., Laureth-4), sucrose esters, block copolymers (e.g., poloxamers), and / or ethoxylated triglycerides (e.g., ethoxylated castor oil).

[0030] Exemplary antimicrobial agents include, but are not limited to, benzoic acid and / or benzyl alcohol. Exemplary antioxidants include, but are not limited to, butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate, tert-butylhydroquinone, and / or vitamin E. Exemplary reducing agents include, but are not limited to, glutathione and / or dithiothreitol (DTT).

[0031] In some embodiments, an emulsifying solvent may be added to the liquid cell culture medium. The emulsifying solvent may form an interface with the liquid cell culture medium and / or the aqueous composition. Exemplary emulsifying solvents include, but are not limited to, alkanes, such as alkanes having 1-20 carbon atoms (i.e., C1-C20 alkanes) or C4-C20 alkanes (e.g., propane, pentane, hexane, etc.); alcohols (e.g., C5-C14 alcohols); acetates; esters; ethers; carboxylic acids; (poly)heteroatom cyclic, acyclic, linear or branched molecules; and / or lactates, such as butyl lactate and / or ethyl lactate. In some embodiments, the solvent may have carbon, nitrogen, and / or sulfur atoms. In some embodiments, the emulsifying solvent is miscible with the solvent present in the dehydrated composition of the present invention. In some embodiments, the emulsifying solvent may be an alcohol, such as pentanol, and / or a lactate, such as butyl lactate and / or ethyl lactate.

[0032] The emulsifying solvent may be present in the liquid cell culture medium in an amount sufficient to form an emulsion (e.g., a water-in-oil emulsion). In some embodiments, the emulsifying solvent is present in the liquid cell culture medium in an amount of about 20% or more, such as about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more. In some embodiments, the addition of the emulsifying solvent to the liquid cell culture medium may at least partially dissolve the cells, cell membranes, and / or lipid formations present in the liquid cell culture medium. In some embodiments, the emulsifying solvent does not dehydrate and / or is not present in the liquid cell culture medium in a concentration sufficient to dehydrate components (e.g., biological materials) present in the medium. In some embodiments, the emulsifying solvent removes water from components (e.g., biological materials) present in the liquid cell culture medium in an amount of less than about 10%, less than about 5%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1%. In some embodiments, the emulsifying solvent assists in providing a desired particle size for the liquid cell culture medium and / or emulsion comprising at least partially dehydrated components.

[0033] In some embodiments, the emulsifying solvent and water can be added separately (e.g., one at a time or simultaneously) to the liquid cell culture medium, and / or the emulsifying solvent and water can be present in the same composition that is added to the liquid cell culture medium. Water can be present in a composition with the emulsifying solvent in an amount up to its solubility limit in the emulsifying solvent, and / or can be added to the liquid cell culture medium with the emulsifying solvent.

[0034] Components separated (e.g., isolated) from liquid cell culture medium can be hydrophilic (e.g., biological substances) or hydrophobic (e.g., lipids or steroids). In some embodiments, the methods of the invention isolate biological substances present in liquid cell culture medium. The biological substances can be present in cells in the liquid cell culture medium and / or present outside of cells in the liquid cell culture medium. In some embodiments, the biological substances are expressed intracellularly and / or expressed extracellularly. "Biological substances" as used herein refers to amino acids, peptides, proteins, enzymes or fragments thereof, antibodies or fragments thereof (e.g., heavy chains, light chains, fusion proteins, Fv, and / or Fc), etc.), and / or polynucleotides, such as oligonucleotides, DNA, and / or RNA. The biological substances can include compounds and / or molecules, such as those that can bind to the biological substances, such as sugars and / or salts. In some embodiments, the biological substances can be biological drug substances (DS) and / or can be used as therapeutic agents.

[0035] An "isolated component", e.g., an "isolated biological material" or an "isolated lipid", as used herein, refers to a component that has been at least partially separated or removed from other components present in the liquid cell culture medium in which it was present. Thus, an "isolated component" may not be in a purified form (e.g., having a purity of greater than 90% by weight of the composition in which it is present). In some embodiments, an "isolated component" (e.g., an isolated biological material) is not in a pure form and / or does not have a high purity and / or concentration of greater than about 20% by weight, greater than about 25% by weight, greater than about 30% by weight, greater than about 35% by weight, greater than about 40% by weight, greater than about 45% by weight, greater than about 50% by weight, greater than about 55% by weight, greater than about 60% by weight, greater than about 65% by weight, greater than about 70% by weight, greater than about 75% by weight, greater than about 80% by weight, greater than about 85% by weight, or greater than about 90% by weight of the composition in which it is present. "Isolate" and grammatical versions thereof, as used herein, refers to at least partially separating or removing one component from another, e.g., separating components present in a liquid cell culture medium into at least two groups or phases. In some embodiments, the methods of the invention include isolating water-soluble components (e.g., biological materials) and / or water-insoluble components (e.g., lipids) from a liquid cell culture medium.

[0036] In some embodiments, the method of the present invention includes contacting a dehydrated composition (e.g., an organic solvent) and a liquid cell culture medium containing a component (e.g., a biological material or lipid) to form a mixture, forming an at least partially dehydrated component in the mixture, and separating the at least partially dehydrated component from the mixture, thereby providing an isolated component. "At least partially dehydrated component" as used herein refers to a component having a water activity of less than 1. In some embodiments, the isolated component includes the at least partially dehydrated component (e.g., an at least partially dehydrated biological material). In some embodiments, the isolated component (e.g., a water-insoluble component, e.g., a lipid) is present in a composition separated from the at least partially dehydrated component. An isolated component may be at least partially separated from other components present in the cell culture medium, although additional components, such as host cell proteins, sugars, salts, buffers, and / or additives, may be present with the isolated component. The additional components present with the isolated components may also be referred to herein as impurities, and the impurities may be at least partially dehydrated.

[0037] The method of the present invention may include separating the solid phase from the liquid phase. In some embodiments, the isolated component is present in the solid phase. In some embodiments, the isolated component is present in the liquid phase. The impurities present in the solid phase and / or liquid phase may include water-soluble and / or water-insoluble components. In some embodiments, the impurities present in the solid phase may include hydrophilic and / or water-soluble components, which may be at least partially dehydrated. In some embodiments, the impurities present in the liquid phase may include hydrophobic and / or water-insoluble components. In some embodiments, the impurities present in the liquid phase may include hydrophilic and / or water-soluble components. The hydrophilic and / or water-soluble components may be present in the liquid phase in an amount of less than about 5% by weight, less than about 4% by weight, less than about 3% by weight, less than about 2% by weight, less than about 1% by weight, less than about 0.5% by weight, or less than about 0.1% by weight of the liquid phase.

[0038] The method of the invention can remove (e.g., dissolve or extract) water from water-soluble components that may be present in the liquid cell culture medium, and provide a solidified component. In some embodiments, the method of the invention includes removing water from a biological material present in the liquid cell culture medium and providing a solidified biological material (i.e., the biological material in a solid form). Water can also be removed from impurities (e.g., water-soluble impurities) to provide a solidified impurity. The solidified component (e.g., the solidified biological material and / or impurities) can be in any form, such as microparticles, nanoparticles, microspheres, or nanospheres. In some embodiments, the solidified component is amorphous (e.g., amorphous particles). In some embodiments, at least a portion of the solidified component is amorphous, such that the solidified component can be partially amorphous. For example, the matrix of the solidified component can be amorphous, but the molecules (e.g., salts) within the matrix can be crystalline. In some embodiments, the solidified component is separated from any liquid and / or provided in the form of a powder. The process of removing water from a component (i.e., dehydrating the component) to provide a solid is referred to herein as "microvitrification," and the solidified component may be referred to herein as a "microvitrified" component. A microvitrified component is not a precipitate. In some embodiments, the compositions and / or methods of the invention do not include (i.e., are devoid of) a phase separation agent. Although phase separation may occur during the microvitrification methods of the invention, the microvitrified component is not formed by phase separation. The microvitrified component may be redissolved in the initial composition in which it was present (e.g., liquid cell culture medium or aqueous solution) and / or in an aqueous buffer.

[0039] According to some embodiments, biological material is isolated or separated from the liquid cell culture medium according to the methods of the present invention. In some embodiments, the methods include at least partially dehydrating the biological material. The methods may simultaneously remove water from the liquid cell culture medium and preserve the biological material. Extracting water from the biological material may preserve the biological material and provide a solidified biological material (i.e., microvitrified biological material). One or more properties and / or functions of the biological material may be preserved and / or maintained with the microvitrified biological material compared to the properties and / or functions of the biological material before microvitrification and / or compared to the properties and / or functions of a control biological material that was not microvitrified.

[0040] The biological material may be present in the liquid cell culture medium at a concentration of less than about 200 mg / mL, less than about 175 mg / mL, less than about 150 mg / mL, less than about 125 mg / mL, less than about 100 mg / mL, less than about 90 mg / mL, less than about 80 mg / mL, less than about 70 mg / mL, less than about 60 mg / mL, less than about 50 mg / mL, less than about 40 mg / mL, less than about 30 mg / mL, less than about 20 mg / mL, less than about 10 mg / mL, less than about 5 mg / mL, less than about 1 mg / mL, less than about 0.1 mg / mL, less than about 0.05 mg / mL, or less than about 0.01 mg / mL. In some embodiments, the biological material is present in the liquid cell culture medium at a concentration ranging from about 0.01 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.5 mg / mL, about 1 mg / mL, or about 5 mg / mL to about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, or about 50 mg / mL. In some embodiments, the biological material is at least partially encapsulated within cells present in the liquid cell culture medium.

[0041] As used herein, "contact," "contacting," "contacted," and grammatical variations thereof refer to bringing two or more materials (e.g., compositions, compounds, solvents, etc.) together to form a mixture. The bringing of the two or more materials into contact can be performed by pouring, spraying, mixing, flowing, and / or injecting the two materials or portions thereof together. For example, the bringing can include adding a solvent to a liquid cell culture medium or adding a liquid cell culture medium to a solvent. In some embodiments, the two or more materials can be brought into contact during a membrane emulsification method and / or technique. The bringing can be performed in a device, such as a mixer, homogenizer, and / or microfluidic device.

[0042] In some embodiments, contacting includes bringing together two or more materials (e.g., compositions, compounds, solvents, etc.) in sufficient proximity such that a desired reaction can be carried out under suitable conditions (e.g., water can be removed from a component present in one of the materials). In some embodiments, the contacting step in the method of the present invention forms a mixture, and the mixture can be one phase or two or more (e.g., two, three, or more) phases. In some embodiments, the mixture is a multi-phase composition in that the composition has two or more (e.g., two, three, four, or more) phases. In some embodiments, the contacting step in the method of the present invention forms a two-phase composition and / or an emulsion. In some embodiments, the contacting step in the method of the present invention forms a suspension (e.g., one or more liquid phases containing solid particles). In some embodiments, an emulsion is formed, and the emulsion may optionally be a water-in-oil emulsion, comprising droplets having a diameter and / or minimum dimension of less than about 1000 μm, less than about 900 μm, less than about 800 μm, less than about 700 μm, less than about 600 μm, less than about 500 μm, less than about 400 μm, less than about 300 μm, less than about 200 μm, less than about 100 μm, less than about 50 μm, less than about 25 μm, less than about 10 μm, less than about 5 μm, or less than about 1 μm. In some embodiments, the mixture is a water-in-oil emulsion comprising droplets having a diameter and / or minimum dimension ranging from about 5 μm, about 10 μm, about 25 μm, about 50 μm, about 100 μm, about 200 μm, about 300 μm, about 400 μm, or about 500 μm, to about 600 μm, about 700 μm, about 800 μm, about 900 μm, or about 1000 μm. In some embodiments, the mixture is a water-in-oil emulsion comprising droplets having a diameter and / or smallest dimension ranging from about 0.1 μm, about 0.5 μm, about 1 μm, about 2 μm, about 3 μm, about 4 μm, or about 5 μm, to about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, or about 15 μm.In some embodiments, the contacting step in the methods of the invention forms a mixture (e.g., an emulsion), and a component present in one of the materials is at least partially dehydrated as a result of the contacting step and / or formation of the mixture.

[0043] The liquid cell culture medium can be contacted with a dehydration composition that at least partially dehydrates components (e.g., biological material) present in the liquid cell culture medium. When the dehydration composition and the liquid cell culture medium are contacted, the dehydration composition can be present in a concentration sufficient to at least partially dehydrate components present in the liquid cell culture medium. In some embodiments, the dehydration composition includes a solvent (e.g., an organic solvent) that removes water from the components (e.g., biological material). Exemplary organic solvents include, but are not limited to, alcohols, such as alcohols having 1 to 20 carbons (i.e., C1-C20 alcohols) or C4-C20 alcohols, ester-containing compounds (e.g., C1-C20 ester-containing compounds, such as ethyl acetate, butyl acetate, triacetin, isobutyl acetate, isopropyl acetate, methyl acetate, propyl acetate, and / or butyl lactate), acetic acid, acetone, anisole, tertbutyl methyl ether, cumene, dimethyl sulfoxide, diethyl ether, ethyl formate, formic acid, alkanes (e.g., heptane, pentane, etc.), and / or methyl ethyl ketone.

[0044] In some embodiments, the dehydrated composition comprises a C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, and / or C20 alcohol and / or a C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, and / or C20 ester-containing compound. In some embodiments, the alcohol is methanol, ethanol, propanol (e.g., 1-propanol, 2-propanol), butanol (e.g., 1-butanol, 2-butanol), 2-methyl-1-propanal, 2-methyl-2-propanol, tert-butanol, pentanol (e.g., 1-pentanol, 3-methyl-1-butanol, 2,2-dimethyl-1-propanol, cyclopentanol), hexanol (e.g., 1-hexanol), cyclohexanol, ... The dehydrated composition is selected from ethanol, heptanol (e.g., 1-heptanol), octanol (e.g., 1-octanol), nonanol (e.g., 1-nonanol), decanol (e.g., 1-decanol), 2-propen-1-ol, benzyl alcohol, phenylmethanol, diphenylmethanol, undecanol, dodecanol, propyldecanol, butadecanol, pentadecanol, hexadecanol (e.g., 1-hexadecanol), and / or triphenylmethanol. In some embodiments, the dehydrated composition does not include a C10 alcohol (e.g., 1-decanol). In some embodiments, the ester-containing compound can be formed from an acid and a C1-C20, C1-C10, C1-C8, C1-C6, or C1-C4 alcohol.In some embodiments, the dehydrated compositions include isomers of linear alcohols (e.g., 2-octanol, 3-pentanol, 4-decanol), derivatives of linear alcohols or their isomers (e.g., octyldodecanol, neopentyl alcohol), di-, tri-, or quaternary hydroxylated materials (e.g., 1,4-butanediol, glycerin), unsaturated alcohols (e.g., cyclic, olefinic, or alkynyl alcohols, such as cyclohexanol, geraniol, oleic alcohol), and / or alcohols incorporating internal and / or external heteroatoms (e.g., polyethylene glycol, polypropylene glycol, lactate, etc.).

[0045] One or more (e.g., 1, 2, 3, 4, or more) solvents may be present in the dehydrating composition of the present invention. For example, in some embodiments, at least two alcohols (e.g., 1-pentanol and 1-hexadecanol), at least two ester-containing compounds (e.g., ethyl acetate and triacetin), or at least one alcohol (e.g., 1-pentanol) and one ester-containing compound (e.g., ethyl acetate) are present in the dehydrating composition. When two or more solvents are present in the dehydrating composition, they may be present in any suitable ratio. In some embodiments, the dehydrated composition comprises two solvents present in the composition in a ratio (by volume or weight) of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, or about 1:20.

[0046] The solvent (e.g., organic solvent) present in the dehydrated composition of the present invention may have a solubility in water ranging from about 0.05%, about 1%, about 2%, or about 5% to about 10%, about 12%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% (w / w). In some embodiments, the organic solvent is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%. , about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50% (w / w) in water. The solvent (e.g., organic solvent) can have an interfacial tension with water of less than about 55 mN / m. In some embodiments, the solvent present in the dehydrated compositions of the invention has an interfacial tension with water that is less than about 55, less than about 50, less than about 45, less than about 40, less than about 35, less than about 30, less than about 25, less than about 20, less than about 15, 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, less than about 3, less than about 2, or less than about 1 mN / m. In some embodiments, the solvent present in the dehydrated compositions of the invention has an interfacial tension with water that ranges from about 1, about 2, about 3, about 4, or about 5 to about 6, about 7, about 8, about 9, or about 10. In some embodiments, the solvent present in the dehydrated compositions of the invention is miscible with the emulsifying solvent.

[0047] In some embodiments, the dehydrated compositions of the invention comprise an organic solvent (e.g., an alcohol). In some embodiments, the dehydrated compositions comprise two or more C1-C20 alcohols. In some embodiments, the dehydrated compositions of the invention comprise about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 82%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94 3% by weight, approximately 24% by weight, approximately 25% by weight, approximately 26% by weight, approximately 27% by weight, approximately 28% by weight, approximately 29% by weight, approximately 30% by weight, approximately 31% by weight, approximately 32% by weight, approximately 33% by weight, approximately 34% by weight, approximately 35% by weight, approximately 36% by weight Amount %, about 37% by weight, about 38% by weight, about 39% by weight, about 40% by weight, about 41% by weight, about 42% by weight, about 43% by weight, about 44% by weight, about 45% by weight, about 46% by weight, about 47% by weight, about 48% by weight, about 49% by weight , about 50% by weight, about 51% by weight, about 52% by weight, about 53% by weight, about 54% by weight, about 55% by weight, about 56% by weight, about 57% by weight, about 58% by weight, about 59% by weight, about 60% by weight, about 61% by weight, about 62% by weight, about 63% by weight, approximately 64% by weight, approximately 65% ​​by weight, approximately 66% by weight, approximately 67% by weight, approximately 68% by weight, approximately 69% by weight, approximately 70% by weight, approximately 71% by weight, approximately 72% by weight, approximately 73% by weight, approximately 74% by weight, approximately 75% by weight, approximately 76 %, about 77% by weight, about 78% by weight, about 79% by weight, about 80% by weight, about 81% by weight, about 82% by weight, about 83% by weight, about 84% by weight, about 85% by weight, about 86% by weight, about 87% by weight, about 88% by weight, about 89% by weight, about 90% by weight, about 91% by weight, about 92% by weight, about 93% by weight, about 94% by weight, about 95% by weight, about 96% by weight, about 97% by weight, about 98% by weight, about 99% by weight, or about 100% by weight of the organic solvent.

[0048] The dehydrated composition can include one or more (e.g., 1, 2, 3, 4, 5, or more) additives (e.g., stabilizers, surfactants, tonicity agents, salts, sugars, antimicrobial agents, antioxidants, etc.). Exemplary additives (e.g., surfactants, antimicrobial agents, antioxidants, etc.) include, but are not limited to, those described above. In some embodiments, the additive is present in the dehydrated composition at a concentration ranging from about 0.01%, about 0.1%, or about 0.5% by weight of the dehydrated composition to about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, or about 35% by weight. In some embodiments, the dehydrated composition comprises a surfactant and / or sugar. In some embodiments, the surfactant may be present in the dehydrated composition in an amount of about 30% or less by weight of the dehydrated composition, for example, about 25%, about 20%, about 15%, about 10%, about 5%, or less by weight of the dehydrated composition. In some embodiments, the antimicrobial agent may be present in the dehydrated composition in an amount of about 1% or less by weight of the dehydrated composition, for example, about 0.5%, about 0.1%, or less by weight of the dehydrated composition. In some embodiments, the antioxidant may be present in the dehydrated composition in an amount of about 2.5% or less by weight of the dehydrated composition, for example, about 2%, about 1.5%, about 1%, about 0.5%, about 0.1%, or less by weight of the dehydrated composition.

[0049] In some embodiments, the dehydration composition comprises at least one alcohol selected from benzyl alcohol, 1-propanol, 1-butanol, tert-butyl, sec-butyl, 1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, 1-nonanol, 1-decanol, 1-undecanol, 1-dodecanol, propyldecanol, butadecanol, pentadecanol, and hexadecanol, and / or at least one solvent selected from water, triacetin, benzyl alcohol, acetic acid, acetone, anisole, 2-butanol, butyl acetate, tert-butyl methyl ether, cumene, dimethyl sulfoxide, ethanol, ethyl acetate, diethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, 2-methyl-1-propanol, pentane, 2-propanol, propyl acetate, and tert-butanol. Further exemplary solvents that may be present in the dehydrated composition include, but are not limited to, alkanes, such as alkanes having 1-20 carbon atoms (i.e., C1-C20 alkanes) or C4-C20 alkanes (e.g., propane, pentane, hexane, etc.); alcohols (e.g., C5-C14 alcohols); acetates; esters; ethers; carboxylic acids; (poly)heteroatom cyclic, acyclic, linear or branched molecules; and / or lactates. In some embodiments, the dehydrated composition includes a solvent having carbon, nitrogen, and / or sulfur atoms.

[0050] The method of the invention may include contacting the liquid cell culture medium with one or more (e.g., 1, 2, 3, 4, or more) dehydrated compositions. Thus, the method of the invention may include contacting the liquid cell culture medium with one or more (e.g., 1, 2, 3, 4, or more) solvents, such as a first organic solvent, a second organic solvent, etc. When more than one dehydrated composition is used in the method of the invention, the solvents present in the compositions may be the same or different. In some embodiments, the solvent present in the dehydrated composition may be more volatile than the solvent in the composition immediately prior to dehydration and / or may be a volatile organic compound (e.g., having a boiling point ranging from about 0° C., about 50° C., or about 100° C. to about 150° C., about 200° C., or about 260° C.). In some embodiments, the dehydrated composition and the at least partially dehydrated components may be contacted one or more (e.g., 1, 2, 3, 4, 5, or more) times.

[0051] Batch or in-line methods can be used to contact the liquid cell culture medium with one or more dehydrated compositions. In some embodiments, the methods include contacting the liquid cell culture medium with a first dehydrated composition to form a mixture, and then contacting the mixture with a second dehydrated composition. In some embodiments, the methods include contacting the liquid cell culture medium with a first dehydrated composition to form a mixture, separating at least partially dehydrated components from the mixture, and contacting the at least partially dehydrated components with a second dehydrated composition.

[0052] The solvent present in the dehydrated composition may form an interface with the liquid cell culture medium and / or aqueous composition. In some embodiments, the solvent present in the dehydrated composition is not miscible with water. In some embodiments, the solvent present in the dehydrated composition first used to dehydrate the component (i.e., the first dehydrated composition or the first organic solvent) is immiscible with water and / or forms an interface with water, and the solvent is at least partially water-soluble. In some embodiments, the solvent present in the dehydrated composition used after the first dehydrated composition (e.g., the second or third dehydrated composition or the second or third organic solvent) is not miscible with water.

[0053] Contacting the dehydrating composition with the liquid cell culture medium and / or forming the mixture reduces the water content of the components (e.g., biological material) present in the mixture to at least about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 90%, about 91%, about 92%, about 93%, about %, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, About 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 109%, about 110%, about 111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, about 123%, about 124%, about 125%, about 126%, about 127%, about 128%, about 129%, about 130%, about 131%, about 132%, about 133%, about 134%, about 135%, about 136%, about 137%, about This may include a reduction of about 4%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%. In some embodiments, the water content of the component is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35% to about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% after the contacting step of the present invention.

[0054] A mixture comprising a dehydrated composition and liquid cell culture medium can have a water activity of less than about 0.99, less than about 0.98, less than about 0.95, less than about 0.9, less than about 0.85, less than about 0.8, less than about 0.75, less than about 0.7, less than about 0.65, less than about 0.6, less than about 0.55, less than about 0.5, less than about 0.45, less than about 0.4, less than about 0.35, less than about 0.3, less than about 0.25, less than about 0.2, less than about 0.15, or less than about 0.1. In some embodiments, the mixture has a water activity ranging from about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, or about 0.5 to about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, or about 0.95. In some embodiments, the mixture has a fractional water saturation of less than about 0.95, less than about 0.9, less than about 0.85, less than about 0.8, less than about 0.75, less than about 0.7, less than about 0.65, less than about 0.6, less than about 0.55, less than about 0.5, less than about 0.45, less than about 0.4, less than about 0.35, less than about 0.3, less than about 0.25, less than about 0.2, less than about 0.15, or less than about 0.1. In some embodiments, the mixture has a fractional water saturation ranging from about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, or about 0.5 to about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, or about 0.95. For example, in some embodiments, a mixture containing protein may have a fractional water saturation of about 0.95 or less. In some embodiments, a mixture containing salt may have a fractional water saturation of about 0.5 or less.

[0055] At least partially dehydrated components (e.g., biological material) may have a water content of about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 1%, or about 0% after being contacted with one or more dehydration compositions. In some embodiments, the at least partially dehydrated components (e.g., biological material) have a water content ranging from about 0%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, or about 25% to about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% after contacting with one or more dehydration compositions. In some embodiments, the at least partially dehydrated components (e.g., biological material) have a water content ranging from about 0%, about 0.5%, about 1%, about 2%, about 3%, or about 4% to about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% after contacting with one or more dehydration compositions. In some embodiments, the at least partially dehydrated component (e.g., biological material) has a water activity of about 0.986, about 0.98, about 0.95, about 0.9, about 0.85, about 0.8, about 0.75, about 0.7, about 0.65, about 0.6, about 0.55, about 0.5, about 0.45, about 0.4, about 0.35, about 0.3, about 0.25, about 0.2, about 0.15, about 0.1, about 0.05, or about 0 after being contacted with one or more dehydration compositions. In some embodiments, the at least partially dehydrated component (e.g., biological material), after contact with one or more dehydrating compositions, has a water activity ranging from about 0, about 0.05, about 0.1, about 0.15, about 0.2, about 0.25, or about 0.3 to about 0.35, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, or about 0.9.

[0056] In some embodiments, after contacting the initial dehydrated composition and the liquid cell culture medium, the at least partially dehydrated components have a water content of less than about 60%, and / or in the range of about 1%, about 5%, about 10%, about 15%, about 20% or about 25%, up to about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% or about 70%. In some embodiments, after contacting the at least partially dehydrated component and a subsequent (e.g., second) dehydrated composition, which can be the same or different from the initial dehydrated composition, the at least partially dehydrated component has a water content of less than about 45%, and / or in the range of about 0.5%, about 1%, about 5%, about 10%, about 15%, or about 20%, to about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%.

[0057] The contacting step in the methods of the invention may be carried out at a temperature ranging from about -50°C, about -20°C, about -10°C, about 0°C, about 4°C, about 10°C, about 15°C, or about 20°C to about 25°C, about 20°C, about 35°C, about 40°C, about 45°C, or about 50°C. In some embodiments, the contacting step in the methods of the invention is carried out at room temperature and / or atmospheric pressure. In some embodiments, the methods of the invention may include devices, steps, and / or compositions described in U.S. Patent No. 8,013,022, which is incorporated herein by reference in its entirety.

[0058] In some embodiments, contacting a dehydrated composition (e.g., an organic solvent) and a liquid cell culture medium and / or forming a mixture comprising the dehydrated composition and the liquid cell culture medium can form a suspension, the suspension comprising a component (e.g., a biological material) in the form of a solid suspended in a liquid (e.g., the mixture). Contacting a dehydrated composition with a liquid cell culture medium to provide a mixture and forming an at least partially dehydrated component in the mixture can occur substantially simultaneously. "Substantially simultaneously," as used herein with respect to at least partially dehydrating a component, refers to at least partially dehydrating a component immediately upon contact with the dehydrated composition or within about 1 minute of initial contact with the dehydrated composition. In some embodiments, forming an at least partially dehydrated component in the mixture is accomplished within about 100 minutes or less (e.g., about 90, about 60, about 30, about 15, about 10, about 5, or about 1 minute or less) after initial contact of the dehydrated composition and the liquid cell culture medium.

[0059] In some embodiments, at least partially dehydrated components (e.g., biological materials) having a water content of less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% can be achieved within less than about 10 minutes, less than about 9 minutes, less than about 8 minutes, less than about 7 minutes, less than about 6 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, or less than about 1 minute, or within less than about 50 seconds, less than about 40 seconds, less than about 30 seconds, less than about 20 seconds, less than about 10 seconds, less than about 5 seconds, less than about 1 second, less than about 0.5 seconds, or less than about 0.1 seconds. In some embodiments, at least partially dehydrated components (e.g., biological material) having a water content of less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% can be achieved within a range of about 0.01 seconds, about 0.05 seconds, about 0.1 seconds, or about 0.5 seconds to about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 7 seconds, about 8 seconds, about 9 seconds, or about 10 seconds.

[0060] Contacting the liquid cell culture medium with a dehydrating composition can include at least partially lysing cells present in the liquid cell culture medium and / or at least partially dissolving lipids and / or cell membranes present in the liquid cell culture medium.

[0061] According to some embodiments, contacting two different compositions (e.g., a dehydrated composition and a liquid cell culture medium, or a composition comprising a solvent and at least partially dehydrated components) includes mixing them together. Mixing can be accomplished by any method known in the art, such as vortexing, stirring, static mixing, homogenizing, extruding, pumping, pouring, adding one composition to another, spraying one composition into another, and / or spraying the compositions together to form a mixture. Mixing can form an emulsion, and / or air can be incorporated into the mixture to form an emulsion. One composition can be added to the other using slow addition, rapid addition, or total addition, and mixing can be accomplished using low shear and / or high shear (e.g., about 1-10 million / second). If the two different compositions include an aqueous composition and a hydrophobic composition (e.g., a composition comprising an organic solvent), the aqueous composition can be added to the hydrophobic composition, or vice versa. In some embodiments, the two different compositions are contacted before mixing and / or in a mixer. Exemplary mixers include, but are not limited to, high pressure homogenizers, rotor-stator homogenizers, impellers, pipeline mixers (turbulent and laminar), static mixers, in-line mixers, and / or microfluidic devices (e.g., microfluidic devices with cross junctions). In some embodiments, the mixer is in-line, such as an in-line homogenizer. In some embodiments, the mixer is a device (e.g., a microfluidic device), and the two different compositions are added or injected into the device, thereby mixing the two different compositions together. Forming at least partially dehydrated components can include mixing (e.g., homogenizing) the mixture.

[0062] In some embodiments, two different compositions forming a mixture and / or a composition comprising at least partially dehydrated components can be mixed, for example, in a batch process, in a ratio that is the volume ratio of the two different compositions. For example, for a first composition and a second composition that are contacted in a batch process to form a mixture, the volume ratio can be the volume of the first composition that is mixed to the volume of the second composition that is mixed. In some embodiments, two different compositions forming a mixture and / or a composition comprising at least partially dehydrated components can be mixed, for example, in an in-line process, in a ratio based on the feed rate of the two different compositions. For example, for a first composition and a second composition that are contacted in an in-line process to form a mixture, the ratio can be the feed rate (e.g., flow rate) of the first composition during the contacting step to the feed rate (e.g., flow rate) of the second composition during the contacting step.

[0063] In some embodiments, the mixture and / or the two different compositions forming the composition comprising at least partially dehydrated components may be mixed (e.g., homogenized) at a feed rate (e.g., flow rate) ranging from about 1 mL / min, about 5 mL / min, about 10 mL / min, about 25 mL / min, about 50 mL / min, about 75 mL / min, or about 100 mL / min to about 150 mL / min, about 250 mL / min, about 500 mL / min, about 1,000 mL / min, about 1,500 mL / min, or about 2,000 mL / min. In some embodiments, the mixture and / or the two different compositions forming the composition comprising at least partially dehydrated components can be mixed (e.g., homogenized) at a feed rate ranging from about 1 L / h, about 5 L / h, about 10 L / h, about 25 L / h, about 50 L / h, about 75 L / h, or about 100 L / h to about 150 L / h, about 250 L / h, about 500 L / h, about 1,000 L / h, about 1,500 L / h, about 2,000 L / h, about 2,500 L / h, about 3,000 L / h, about 3,500 L / h, or about 4,000 L / h. The feed rates of each composition can be the same or different.

[0064] In some embodiments, two different compositions that form a mixture and / or a composition that includes at least partially dehydrated components can be contacted and / or combined in a given feed volume ratio. The two different compositions can include an aqueous composition that provides an aqueous phase (e.g., a composition that includes liquid cell culture medium and / or water), and a solvent-containing composition that provides a solvent phase (e.g., a hydrophobic composition). In some embodiments, the two different compositions are about 0.1:100, about 0.2:100, about 0.3:100, about 0.4:100, about 0.5:100, about 0.6:100, about 0.7:100, about 0.8:100, about 0.9:100, about 1:100, about 1.5:100, about 2:100, about 2.5:100, about 3:100, about 3.5:100, about 4:100, about 4.5:100, about 5:100, about 5.5:100, about 6:100, about 6.5:100, about 7:100, The aqueous composition and the solvent-containing composition may be contacted and / or combined in a feed volume ratio (aqueous composition:solvent-containing composition) of about 7.5:100, about 8:100, about 8.5:100, about 9:100, about 9.5:100, about 10:100, about 11:100, about 12:100, about 13:100:14:100, about 15:100, about 16:100, about 17:100, about 18:100, about 19:100, about 20:100, about 21:100, about 22:100, about 23:100, about 24:100, or about 25:100. In some embodiments, the two different compositions forming the mixture and / or composition comprising at least partially dehydrated components may be contacted and / or combined in a feed volume ratio of about 0.1:100, about 0.3:100, about 0.5:100, about 0.7:100, or about 1:100, to about 2:100, about 4:100, about 6:100, about 8:100, about 10:100, about 12:100, about 14:100, about 16:100, about 18:100, about 20:100, about 22:100, or about 25:100.

[0065] In some embodiments, the emulsifying solvent (or a composition comprising same) may be contacted and / or combined with the liquid cell culture medium in a feed volume ratio (liquid cell culture medium:emulsifying solvent) of about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1:3.5:1, about 4:1, about 4.5:1, or about 5:1.

[0066] The at least partially dehydrated component can be separated from the mixture and / or liquid phase using methods known to those skilled in the art. For example, the at least partially dehydrated component can be separated from the liquid phase by filtration (e.g., pressure filtration, tangential flow filtration, rotary filtration, centrifugal filtration, disc stack filtration, etc.), cyclone separation, sedimentation (e.g., sedimentation by acoustic resonance field), evaporation, centrifugation, fluid bed drying, evaporative drying, thermal drying, freeze drying, atmospheric freeze drying, spray drying, spray freeze drying, microwave / IR drying, and / or sieving. In some embodiments, the at least partially dehydrated component can be separated from the liquid phase by washing and / or solvent exchange methods, sedimentation and / or filtration processes, and / or further dried (e.g., to remove water and / or solvent) using air, vacuum, and / or freeze drying processes, and / or any combination thereof.

[0067] In some embodiments, the method of the present invention includes washing the at least partially dehydrated components with a cleaning composition. The cleaning composition may include an organic solvent, such as an alcohol (e.g., a C1-C20 alcohol), an alkane (e.g., a C4-C20 alkane), an ester, and / or an ether. Such organic solvents include, but are not limited to, those described above, and / or the organic solvent may have a solubility in water as described above. In some embodiments, the organic solvent in the cleaning composition is miscible with water. The organic solvent in the cleaning composition may be the same or different from the organic solvent in the dehydrated composition. In some embodiments, the organic solvent present in the cleaning composition may be a Class II or Class III residual solvent as classified by the Food & Drug Administration.

[0068] In some embodiments, the organic solvent present in the cleaning composition may be more volatile than the organic solvent in the dehydration composition. In some embodiments, the organic solvent in the cleaning composition is a volatile organic compound (e.g., having a boiling point ranging from about 0°C, about 50°C, or about 100°C to about 150°C, about 200°C, or about 260°C). In some embodiments, the cleaning composition may be used to wash away and / or remove the dehydration composition, emulsification solvent, organic solvent, and / or impurities. In some embodiments, the organic solvent present in the cleaning composition has the solubility of the emulsification solvent and / or the solvent present in the dehydration composition.

[0069] According to some embodiments, the method of the invention is a one-step method in which liquid cell culture medium is contacted with a dehydration composition to provide an emulsion and isolated components (e.g., at least partially dehydrated biological material). The contacting step in the one-step method can be repeated one or more times (e.g., 1, 2, 3, 4, 5, or more times) with the same or different dehydration composition. In some embodiments, the method of the invention is a two-step method that includes a pre-emulsification step in which liquid cell culture medium is contacted with an emulsification solvent to form an emulsion (e.g., a crude emulsion), and the emulsion is subsequently contacted with a dehydration composition to provide isolated components. Each of the contacting steps in the two-step method can be repeated one or more times (e.g., 1, 2, 3, 4, 5, or more times) with the same or different emulsification solvent and / or dehydration composition. The one-step or two-step process may further comprise contacting the at least partially dehydrated component or a composition comprising same with a cleaning composition optionally comprising an organic solvent that is more volatile than one or more previous organic solvents used in the process.

[0070] The methods of the invention may contact the emulsifying solvent, dehydration composition, or cleaning composition with the liquid cell culture medium, at least partially dehydrated component, or composition comprising at least partially dehydrated component for a period of time. The contact time for each of these steps may be about 1 minute, about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, or about 60 minutes, or about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours or more. In some embodiments, the contact time can be from about 1 minute, about 5 minutes, or about 15 minutes, to about 30 minutes, about 45 minutes, or about 60 minutes, or from about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours, to about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours, or more. In some embodiments, the period during which the emulsification solvent is contacted with the liquid cell culture medium is shorter than the period during which the dehydration composition and / or cleaning composition is contacted with the at least partially dehydrated component or a composition comprising same.

[0071] The methods of the invention can include storing the isolated component, e.g., the isolated biological material or lipid. In some embodiments, the methods of the invention include purifying the isolated component. In some embodiments, the methods of the invention include hydrating the at least partially dehydrated component (e.g., the biological material) and / or purifying the at least partially dehydrated component.

[0072] In some embodiments, the method of the present invention lacks a conventional lysis technique. Exemplary conventional lysis techniques include, but are not limited to, multiple freeze / thaw cycles, sonication, high pressure homogenization, grinding, and / or permeabilization of cell membranes using detergents and / or enzymes. In some embodiments, the liquid cell culture medium is not lysed (e.g., by physical disruption and / or permeabilization) before the first step of the method of the present invention. In some embodiments, the method of the present invention lacks a lysis step carried out using a one-phase composition (i.e., a plurality of cells are present in a one-phase composition). In some embodiments, the method of the present invention includes a lysis step carried out using a two-phase composition (i.e., a plurality of cells are present in a two-phase composition, such as an emulsion and / or suspension).

[0073] In some embodiments, the liquid cell culture medium and / or composition comprising at least partially dehydrated components has a pH greater than 7, e.g., about 7.5 or more, about 8 or more, about 8.5 or more, about 9 or more, about 10 or more. In some embodiments, the liquid cell culture medium and / or composition comprising at least partially dehydrated components has a pH less than 7, e.g., about 6.5 or less, about 6 or less, about 5.5 or less, about 5 or less, about 4.5 or less, about 4 or less. In some embodiments, the liquid cell culture medium and / or composition comprising at least partially dehydrated components has a pH at least ±0.5, 1, 1.5, or 2 pH units away from the isoelectric point (pI) of the desired isolated component (e.g., target biological material). In some embodiments, the pH of the composition comprising at least partially dehydrated components in the methods of the invention varies by less than about ±2, about ±1.5, about ±1, or about ±0.5 pH units from the initial pH of the composition.

[0074] The method of the present invention may include collecting and / or recycling the composition and / or its components (e.g., solvent and / or additives). In some embodiments, the composition and / or its components may be separated from the at least partially dehydrated components and optionally further processed by methods including, but not limited to, distillation, absorption, precipitation, adsorption, filtration, dialysis, and / or the like to collect and / or recycle the composition and / or its components. In some embodiments, water and / or impurities are removed from the composition and / or its components to be collected and / or recycled. In some embodiments, separating the at least partially dehydrated components from the liquid phase provides an isolated component (e.g., lipid or steroid) in the liquid phase separated from the at least partially dehydrated components. In some embodiments, water is removed from the composition and / or its components to be collected and / or recycled, for example, by distillation. In some embodiments, additives present in the composition and / or its components to be collected and / or recycled are removed, for example, by chemical separation methods (e.g., filtration).

[0075] According to some embodiments of the present invention, a composition is provided that includes a plurality of solid particles (e.g., microparticles). The solid particles can be amorphous and / or crystalline. In some embodiments, the plurality of solid particles includes amorphous particles. In some embodiments, the plurality of solid particles can include crystalline particles, for example, when small molecules (e.g., salts) that can be crystallized are present. In some embodiments, the composition can be provided and / or obtained after separation of the solid particles from a liquid phase. The plurality of solid particles can be of uniform size or can be polydisperse. In some embodiments, at least a portion of the plurality of solid particles has a size within about ±5%, about ±10%, about ±15%, about ±20%, about ±25%, about ±50%, about ±75%, about ±100%, about ±150%, about ±200% or greater of the average particle size. In some embodiments, the composition includes discrete particles. In some embodiments, the composition comprises aggregates of the biological material in an amount of about 50%, about 40%, about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% by weight of the biological material.

[0076] In some embodiments, at least a portion of the solid particles in the composition of the present invention are solid biological material particles. In some embodiments, a composition comprising a plurality of solid particles comprises at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, or about 100% by weight of the solid biological material particles. In some embodiments, the composition comprises one or more (e.g., 1, 2, 3, 4, or more) impurities, such as water-soluble components that may be at least partially dehydrated. Impurities can be present in a composition comprising a plurality of solid particles in an amount of about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 97% or more by weight of the composition. In some embodiments, a water-insoluble component (e.g., lipid) may be present in a composition comprising a plurality of solid particles in an amount of less than about 60% by weight, less than about 55% by weight, less than about 50% by weight, less than about 45% by weight, less than about 40% by weight, less than about 35% by weight, less than about 30% by weight, less than about 25% by weight, less than about 20% by weight, less than about 15% by weight, less than about 10% by weight, or less than about 5% by weight of the composition.

[0077] The method of the present invention may preserve components present in liquid cell culture medium. The components may be preserved and / or isolated from the liquid cell culture medium at multiple points in the downstream purification of the medium, which may eliminate or reduce interruptions in the process flow (e.g., due to equipment downtime) and / or allow for increased production and / or yield of the isolated components, for example, compared to methods of freezing, lyophilizing, and / or spray drying the same components. In some embodiments, the method of the present invention achieves a yield of about 0.1 g / L, about 0.5 g / L, about 5 g / L, about 10 g / L, about 25 g / L, or about 50 g / L. In some embodiments, the method of the present invention achieves a yield ranging from about 0.1 g / L, about 1 g / L, about 5 g / L, or about 10 g / L to about 15 g / L, about 20 g / L, about 25 g / L, about 30 g / L, about 40 g / L, or about 50 g / L. In some embodiments, the methods of the invention may reduce (e.g., at least about 5% or about 95% or more) the storage and / or shipping volume and / or weight of an isolated component (e.g., microvitrified biological material). In some embodiments, the methods of the invention provide isolated components that can maintain one or more properties and / or functions and / or desired activity levels even after exposure to temperature fluctuations of ±5° C., 10° C., 15° C., 20° C., or 25° C. or more.

[0078] In some embodiments, one or more properties and / or functions of a component (e.g., biological material) may be preserved and / or maintained after formation of a solid biological material according to a method of the invention (i.e., a microvitrification method) compared to the one or more properties and / or functions of the component prior to the method and / or compared to the one or more properties and / or functions of a control component. For example, in some embodiments, an at least partially dehydrated biological material, optionally upon dissolution in a solution (e.g., an aqueous solution), may have an activity within ±60%, ±55%, ±50%, ±45%, ±40%, ±35%, ±30%, ±25%, ±20%, ±15%, ±10%, or ±5% of the activity of the biological material prior to the method of the invention and / or the activity in the absence of the method of the invention (e.g., the activity of a control component). In some embodiments, the at least partially dehydrated biological material may have an increased activity (e.g., at least about 5% or more) compared to the activity of the same biological material that is frozen, lyophilized, and / or spray-dried and optionally subsequently dissolved in a solution, optionally upon dissolution in a solution. In some embodiments, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or more of the activity of the at least partially dehydrated component of the present invention is retained compared to the activity of a control component (i.e., a component lacking the method of the present invention) and / or the component prior to the method of the present invention. In some embodiments, the method of the present invention may provide at least partially dehydrated biological material with a similar or improved fluidity compared to the fluidity of the same biological material that is frozen, lyophilized, and / or spray-dried.

[0079] In some embodiments, the structure (e.g., primary, secondary, and / or tertiary) of a component (e.g., biological material) can be preserved and / or maintained after formation of a solid biological material according to the methods of the invention. In some embodiments, at least partially dehydrated biological material having a folded structure may not unfold during the methods of the invention. In some embodiments, at least partially dehydrated biological material having a folded structure may have a difference in structure of less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, or less than about 5%.

[0080] The at least partially dehydrated components of the present invention may have an increased shelf life compared to the shelf life of the same components in the absence of the method of the present invention and / or compared to the shelf life of the same components after freezing, lyophilization, and / or spray drying. "Shelf life" as used herein refers to the length of time that an ingredient maintains a certain level or higher activity. In some embodiments, the at least partially dehydrated components of the present invention have a shelf life of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks, months, and / or years when stored at a temperature ranging from about 2°C, about 4°C, about 10°C, about 15°C, or about 20°C to about 25°C, about 20°C, about 35°C, or about 40°C, optionally at about 4°C or about 25°C±5°C. In some embodiments, the at least partially dehydrated components of the invention have a shelf life of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks, months, and / or years when stored at a temperature of about 2° C. to about 8° C. or about 5° C.±3° C. In some embodiments, the methods of the invention increase the amount of time that an isolated component can be separated from cells and / or cell culture medium without purification compared to the amount of time for the same component that has been frozen, lyophilized, and / or spray dried. For example, in some embodiments, a composition of the invention comprising a plurality of isolated, microvitrified components may survive under storage conditions (e.g., at temperatures of about 4° C., about 10° C., about 15° C., or about 20° C. to about 25° C., about 20° C., about 35° C., or about 40° C.) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks, months, and / or years without further purification, and optionally without greater than 20% loss of activity.

[0081] In some embodiments, the methods of the present invention reduce the time to achieve a given amount of isolated component compared to a different method for isolating the same amount of the component. For example, in some embodiments, the methods of the present invention can reduce the amount of time from cell culture to preservation and / or purification of biological material compared to a different method (e.g., freezing, lyophilization, and / or spray drying) for preserving and / or purifying the same biological material in the same amount. In some embodiments, the methods of the present invention can achieve the same or increased amount of isolated component in an optionally shorter time compared to a different method for isolating the component. In some embodiments, the methods of the present invention provide the biological material to be preserved more quickly, optionally in a shorter time, than a different method (e.g., freezing, lyophilization, and / or spray drying), and the preserved biological material can then be purified immediately or stored and purified as needed.

[0082] The methods of the invention may include and / or be continuous and / or in-line processes. In some embodiments, the methods of the invention may include and / or be batch processes. In some embodiments, the methods of the invention are continuous and / or in-line with cell culture processes and / or processes and / or with component (e.g., biological material) purification processes and / or processes. In some embodiments, the methods of the invention are continuous in that only a portion of the total volume (e.g., less than about 50%) of the total volume of the liquid cell culture medium according to the embodiments of the invention is undergoing one or more steps of the method at a given time.

[0083] The invention is described in more detail in the following non-limiting examples. EXAMPLES

[0084] Example 1 Dry, dense particles of lysozyme were produced using a dehydration method referred to herein as microvitrification. The particles of lysozyme were produced by adding 0.16 mL of a 100 mg / mL lysozyme solution to 0.8 mL of pentanol (used here as the emulsifying solvent) and vortexing briefly to form a coarse emulsion. The emulsion was then passed 10-20 times through an Avanti Lipids manual extruder to form a fine emulsion. This emulsion was then added to a stirred volume of pentanol (7 mL) (used here as the dehydrating composition), which immediately dehydrated the aqueous droplets, resulting in a suspension of partially dehydrated particles. The extrusion method was repeated a total of three times to dehydrate a total of 0.48 mL of aqueous solution in that volume of pentanol. The suspension is shown in FIG. 1. The particles were then collected by filtration through a 0.2 micron filter. As can be seen in FIG. 1, the use of an emulsion solvent results in a suspension of small, relatively uniform particles.

[0085] Previous experiments using microglassified lysozyme (e.g., Aniket, Gaul DA, Bitterfield DL, Su JT, Li VM, Singh I, et al. Enzyme Dehydration Using Microglassification TM Preserves the Protein's Structure and Function. J Pharm Sci. 2015 Feb;104(2):640-51) demonstrated that when compared to lysozyme that was lyophilized, both samples lost activity after one month of storage at 40° C., but there was no difference between MICROGLASSIFIED™ lysozyme and lysozyme at three months.

[0086] Example 2 The micropipette dehydration technique has been used to study the kinetics of drop dissolution or dehydration. This technique allows real-time observation of the drop dissolution, and therefore of any phase changes. This technique also allows the calculation of the final particle density or protein concentration. As a tool for measuring two-phase microsystems, the micropipette technique has shown that gas-liquid and liquid-liquid systems follow the Epstein-Plesset model of kinetics, which depends on the solubility and diffusion of the soluble components in the surrounding phase:

[0087]

number

[0088] where R = drop radius, cs = solubility of the soluble component, r = density of the soluble component, D = diffusion coefficient of the soluble component, and f = saturation of the surrounding medium. Once these parameters are known (or measured using a micropipette), the dissolution profile of any other drop in the system can be predicted. The kinetic parameters measured for a single drop can then be extrapolated to predict kinetics for any drop size in any other solvent system.

[0089] The dehydration rate of a protein solution closely follows that of a water droplet (or a salt solution droplet) and is a function of droplet size, dehydration solvent, and fractional water saturation. The influence of each of these factors has been discussed (Rickard DL, Biophys J. 2010;98(6):1075-84; Duncan PB, Langmuir. 2004 Mar 30;20(7):2567-78; Bitterfield DL, Langmuir. 2016 Dec 6;32(48):12749-59; and Su JT, J Chem Phys. 2010;132(4):44506) and can be used to predict the required residence time (and therefore flow rate) in a homogenizer or other mixer. For example, Figure 2 shows the predicted settling time in various dehydration solvents as a function of final particle size. For example, for particles in the size range of 2-20 μm, dehydration is completed within 0.1 to 10 seconds.

[0090] The resulting particles can be separated from the drying solvent and disposal or reuse of the solvent can be accomplished. The drying solvent used in the method can be recycled. For example, in the microvitrification method, a 200 L batch of microvitrified protein may require approximately 14,000 L of drying solvent (e.g., octanol). Compared to freeze-drying or spray-drying, Microglassification™ offers higher throughput and potentially easier scalability. A small 200 L fermentation batch takes 220-300 hours to process in a laboratory-scale spray dryer (feed rate of approximately 15 mL / min). A pilot-scale dryer offers only a 4-fold improvement (50 mL / min). Initial testing with a laboratory-scale in-line homogenizer using the dehydration microvitrification method reached a protein feed rate of approximately 2 mL / min. However, IKA (and other vendors) offer standard scale homogenizers that maintain similar shear rates, allowing a 2500-fold improvement in scale, or 300 L / hr. A 20,000 L fermentation batch could potentially be processed in about 60 hours through a single homogenizer unit.

[0091] Many Microvitrification™ studies have been performed using small batch homogenization methods that produce milligram quantities of microvitrified product at a time. To assess the feasibility of scale-up, the method was tested using an in-line homogenizer. Bovine gamma globulin (BGG) was used as a model protein since BGG comprises a large proportion of immunoglobulin proteins that are commonly produced for multiple therapeutic purposes. Total flow rates up to 180 mL / min were obtained, and flow rate ratios of 1:26 to 1:132 were used without adverse effects on size distribution. These high flow rates result in Microvitrification™ of several grams of protein within minutes, a significant advance in achievable scalability.

[0092] The moisture content of the microvitrified BGG was determined via Karl Fischer (KF) analysis and was found to be comparable to that of the spray-dried protein (4-9%) (Figure 3). As shown in Figure 3, the final water content in the dehydrated protein was adjustable by adjusting the manner in which the final protein was isolated.

[0093] By adjusting the processing parameters for the dehydration microvitrification method, a variety of particle size distributions can be achieved (Figure 4). In addition, good reproducibility for the particles is achieved using both the smaller batch protocol (Figure 5) and the in-line manufacturing method described above (Figure 6).

[0094] The amount of residual solvent present in the microvitrified product can be predictable and well below the limit permitted by the Food and Drug Administration for Class III residual solvents (50 mg / day). Figure 7 shows the amount of residual solvent remaining per gram of microvitrified BGG.

[0095] These experiments demonstrated the feasibility of increasing material throughput using the microvitrification method.

[0096] Example 3 The stability of microvitrified model proteins, including monoclonal antibodies and enzymes, is evaluated and the effects of processing parameters are examined. Model proteins are dehydrated either at the beginning (crude drug substance) or at the end (formulated drug substance) of downstream purification and stability is evaluated over 6 months under accelerated conditions. Preservation is evaluated at the beginning and end of downstream purification, providing two potential steps for the workflow to be paused.

[0097] Preservation of extracellular biological material Microvitrification™ is used to preserve antibodies in culture medium harvested from Chinese hamster ovary (CHO) cell lines commonly used in the commercial production of therapeutic proteins. The stability of microvitrified upstream antibodies and microvitrified formulated antibodies is determined at intermediate and accelerated conditions.

[0098] Upstream Processing Experiments - Crude Drug Substance Preservation: As a model system, a non-producing CHO cell line was grown to 5 g / L and spiked with bovine gamma globulin (BGG), a protein used for this task to reduce production costs since large amounts of material are required for residual moisture and solvent analysis.

[0099] CHO cells are cultured for 14 days in a medium similar to that used in commercial production of antibodies. The cells are separated by centrifugation and BGG or monoclonal antibodies are added to the medium to mimic the antibodies produced by the cells. An aliquot from the cell culture medium is Microvitrified™ by combining the medium with the emulsion solvent and / or dehydration solvent and / or washing solvent as described above. Micropipette experiments are used to check the dehydration rate, final protein concentration, and final drop density (total solid mass / volume). The dehydration rate is used to calculate the required residence time in the Microvitrification™ process for a given particle size. The final concentration and density set the maximum f-value (fractional water saturation) that can be used. The dehydrated and collected powders are analyzed for size / morphology using microscopy, for moisture content using Karl Fischer titration, and for residual solvent using Gas Chromatography (GC) according to USP methods. Any adjustments to the Microvitrification™ method to account for the composition of the cell media are made prior to processing.

[0100] The same CHO cell line is cultured to approximately 5 g / L as described above and spiked with mAb. Aliquots from the cell culture medium are Microvitrified™ or frozen. The dehydrated powder is rehydrated to a concentration of 5 g / L, purified, and then assayed for changes in structure using SEC and capillary electrophoresis (CE). Functionality is also assessed via binding ELISA. As a control, the mAb is also purified and analyzed from cell culture medium that is not Microvitrified™. Additional samples are Microvitrified™, aliquoted into crimped glass vials under nitrogen, and placed in accelerated stability at 25°C and 40°C for up to 6 months. At predetermined time points, samples are rehydrated, purified, and assayed as described above. At the end of the study, the frozen samples are thawed, purified, and analyzed as a freeze-thaw comparison.

[0101] Downstream Processing Experiments - Preservation of Formulated Drug Substance: The same mAb used for the upstream processing experiments is used to check the Microvitrification™ protocol for different excipient-protein ratios (e.g., sucrose:protein at weight ratios from 0:1 to 1:1) via a design of experiments (DOE) approach. Micropipette experiments are used to check the dehydration rate, final protein concentration, and final drop density (total solids mass / volume). The purified and formulated mAb solution is microvitrified by combining it with emulsion solvent and / or dehydration solvent and / or wash solvent. The collected Microvitrified™ powder samples are assayed for morphology and residual moisture. Rehydrated samples are analyzed as described above. Compositions that maintained molecular structure are subjected to accelerated stability as described above. Controls include frozen solutions stored at -20°C.

[0102] Preservation of intracellular biological material The stability of the upstream single chain antibody fragment (scFv) when Microvitrified™ from fermentation broth is compared to the stability of formulated scFv when Microvitrified™ from purified solution.

[0103] Upstream processing experiment - Preservation of crude drug substance: Unlike antibody production from mammalian cells, which export proteins extracellularly, some proteins, including fusion proteins and enzymes, can be produced intracellularly from bacterial sources and require a cell lysis step (e.g., homogenization) during harvest. Microvitrification™ is performed from the whole fermentation broth. Without wishing to be bound by any particular theory, it is expected that some of the cell debris will be removed during Microvitrification™, since lipids are soluble in the organic solvents used. If the cells are not completely lysed by the Microvitrification™ method, the sample is microvitrified™ after cell lysis and removal of cell debris.

[0104] A single chain variable fragment (scFv) that binds to beta-galactosidase is produced by E. coli. An aliquot is Microvitrified™ as described above, this time from the fermentation broth with whole cells. The fermentation broth and cells are combined with the emulsion solvent and / or dehydration solvent and / or wash solvent as described above. Micropipette experiments are used to check the dehydration rate, final protein concentration, and final droplet density (total solid mass / volume). The dehydrated powder is analyzed for size / morphology using microscopy, for moisture content using Karl Fischer titration, and for residual solvent using gas chromatography (GC) according to USP methods. Any adjustments to the Microvitrification™ method are made to account for the composition of the cell culture medium.

[0105] Aliquots from the fermentation broth are Microvitrified™ or frozen. Dehydrated powder is rehydrated to a concentration of 5 g / L, purified, and then assayed for changes in structure using SEC and capillary electrophoresis (CE). Functionality is also assessed via ELISA. As a control, the scFv is purified and analyzed without Microvitrification™. Additional samples are Microvitrified™, aliquoted into crimped glass vials under nitrogen, and placed in accelerated stability at 25°C and 40°C for up to 6 months. At predetermined time points, samples are rehydrated, purified, and assayed as described above. At the end of the study, frozen samples are thawed, purified, and analyzed as a freeze-thaw comparison.

[0106] Downstream Processing Experiments - Preservation of Formulated Drug Substance: The same scFv used in the upstream processing experiments is used to check the Microvitrification™ protocol for various excipient-protein ratios (e.g., sucrose:protein weight ratios from 0:1 to 1:1) via a design of experiments (DOE) approach. Purified scFv is used. Micropipette experiments are used to check the dehydration rate, final protein concentration, and final drop density (total solids mass / volume). The purified and formulated scFv solution is microvitrified by combining it with emulsion solvent and / or dehydration solvent and / or wash solvent. Collected Microvitrified™ powder samples are assayed for morphology and residual moisture. Rehydrated samples are analyzed as described above. Compositions that maintained molecular structure are subjected to accelerated stability as described above. Controls include frozen solutions stored at -20°C.

[0107] Example 4 A pre-emulsion step is utilized in the dehydration method to dehydrate water-soluble components present in the liquid cell culture medium. In the pre-emulsion step, an emulsification solvent is used that does not significantly dissolve water, and therefore does not dehydrate the liquid cell culture medium containing water-soluble components and / or biological materials. The liquid cell culture medium and the emulsification solvent are mixed (e.g., by vortexing, stirring, homogenizing, extruding, and / or by adding or spraying an aqueous phase into the solvent phase) to thereby form a coarse emulsion. This coarse emulsion is then combined with a sufficient volume of a dehydration solvent (which can be the same or different from the emulsification solvent) to dissolve the water.

[0108] A manual extruder containing two glass Hamilton syringes connected such that their contents pass through an extrusion chamber into the other syringe can be used. Alternatively, for in-line production, a static mixer or homogenizer can be used in place of the manual extruder for batch production.

[0109] Example 5 A cell composition was prepared by suspending Micrococcus lysodeikticus (ML) cells (lyophilized) at 50 mg / mL (dry weight) in 150 mM phosphate buffer. 49 μL of the cell composition was rapidly added dropwise to 1 mL of pentanol, and the mixture was vortexed for 30 seconds. Figure 8 shows the dehydrated particles from the cell composition suspended in the pentanol dehydration solvent.

[0110] Example 6 ML cells were suspended in water at 14.2 mg / mL (dry weight). 31 μL of the cell composition was quickly added to 700 μL of butyl lactate, and the mixture was vortexed for 30 seconds. Figure 9 shows dehydrated particles from the cell composition suspended in the butyl lactate dehydration solvent. The suspension of dehydrated particles was centrifuged, the dehydration solvent was removed by pipette, and residual solvent was removed under vacuum overnight. The resulting powder was reconstituted by adding 800 μL of water. As one skilled in the art will recognize, whole cell suspensions have a higher turbidity than lysed suspensions of the cells. The turbidity of the reconstituted cells was measured at 450 nm and compared to a dilution of the unprocessed cell suspension. The cells dehydrated in butyl lactate had 76±7% (n=3) lower turbidity than the starting cell suspension (diluted to the same concentration), demonstrating that the cells dehydrated in butyl lactate were lysed during the dehydration process.

[0111] Example 7 ML cells were suspended in water at 14.2 mg / mL (dry weight). 70 μL of the cell composition was rapidly added to 2.5 mL of pentanol and the mixture was vortexed or homogenized for 30 seconds. Figure 10 shows dehydrated particles from the vortexed cell composition and Figure 11 shows dehydrated particles from the homogenized cell composition, both suspended in the pentanol dehydration solvent. As can be seen in Figure 11, the use of a homogenizer provided particles that were smaller but had a similar morphology to those that were vortexed.

[0112] The suspension of dehydrated particles was centrifuged, the dehydration solvent was removed by pipette, and residual solvent was removed under vacuum overnight. The resulting powder was reconstituted by adding 1.9 mL of water. The cells vortexed and dehydrated in pentanol had 50±6% (n=3) less turbidity than the starting cell suspension (diluted to the same concentration).

[0113] ML cells were suspended in water at 195 mg / mL (dry weight) to approximate a concentrated slurry. 170 μL of the cell composition was quickly added to 2.5 mL of pentanol and the mixture was vortexed for 30 seconds. Figure 12 shows dehydrated particles from the cell composition suspended in the pentanol dehydration solvent. These particles have a similar morphology to the particles in Figure 11, but are larger due to the higher starting concentration.

[0114] Example 8 The impure dehydrated solvent resulting from the microvitrification process is contacted with a hygroscopic material. The hygroscopic material can be any material (e.g., solid, liquid, or gaseous material) that is insoluble in the solvent of interest. Exemplary materials include, but are not limited to, magnesium sulfate, sodium sulfate, 3A molecular sieves, superabsorbent polymers, silica, alumina, bubble columns with dry air, and the like. Once the material has spent sufficient time with the impure dehydrated solvent and subsequently removed by the methods described above, it will be a purified or semi-purified solvent.

[0115] Example 9 The impure dehydrated solvent obtained from the microvitrification process can be passed through a distillation process to yield a purified or semi-purified solvent.

[0116] Example 10 The impure dehydrated solvent resulting from the micro-vitrification process can be cooled or heated to reduce the solubility of impurities in the material, or freeze other materials. If some materials are crystallized or precipitated, they can be removed by the methods described above to yield a purified or semi-purified solvent.

[0117] Example 11 As shown in FIG. 13, a protein solution was microvitrified using a microfluidic device with a crossflow junction (14×17 μm). The device was constructed from quartz with a hydrophobic coating to allow the formation of a water-in-oil emulsion. The aqueous phase (45 mg / mL lysozyme) and dehydration medium (1-pentanol) were delivered via a pressure pump with flow rate ratios of 1:44 to 1:258 (water:dehydration solvent). The aqueous phase was fed through the central channel to form droplets that were pinched off by the dehydration medium entering from a side channel. FIG. 14 shows collected particles with an average diameter of 4.8 μm produced using a flow rate ratio of 1:46. Depending on the dehydration solvent used and the initially formed droplet size, the droplets can be dehydrated and solidified before leaving the chip or collected in an external container. This method allows the production of highly uniform particles.

[0118] Example 12 The collected particles may be further dried by one of several methods to remove residual solvent resulting in a dry powder. Figure 15 shows the mass loss over time during drying by exposure to dry nitrogen. The mass shown in Figure 15 is normalized to the final mass of the dried powder consisting of 75% bovine serum albumin and 25% other solid components. The majority of the mass lost was residual dehydration and washing solvent, which was octanol and pentanol for the particles in Figure 15. Batch 1 was 617 mg of powder dried at a flow rate of 1-1.5 L / min. The amount of solvent and moisture removed was 427 mg. Batch 2 was 831 mg of powder dried at a flow rate of 2 L / min. The amount of solvent and moisture removed was 419 mg. Batch 3 was 890 mg of powder dried at a flow rate of 1-1.8 L / min. The amount of solvent and moisture removed was 367 mg.

[0119] The above is illustrative of the present invention and should not be construed as limiting thereof. The present invention is defined by the following claims, and equivalents of the claims are encompassed therein. All publications, patent applications, patents, patent publications, and other references referred to herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

Claims

1. 1. A method for isolating a biological material from a liquid cell culture medium, comprising: contacting a first organic solvent with a liquid cell culture medium containing a biological material to form a mixture; forming an at least partially dehydrated biological material in said mixture; and Separating the at least partially dehydrated biological material from the mixture to provide isolated biological material. The method comprising:

2. 13. The method of claim 1, wherein the biological material is present in the liquid cell culture medium at a concentration of less than about 200 mg / mL, less than about 100 mg / mL, less than about 50 mg / mL, less than about 30 mg / mL, less than about 20 mg / mL, less than about 10 mg / mL, less than about 5 mg / mL, less than about 1 mg / mL, less than about 0.1 mg / mL, less than about 0.05 mg / mL, or less than about 0.01 mg / mL, and optionally the biological material is at least partially encapsulated within cells present in the liquid cell culture medium.

3. 3. The method of claim 1 or 2, wherein the method simultaneously removes water from the liquid cell culture medium and preserves the biological material.

4. 4. The method of any one of claims 1 to 3, wherein contacting the first organic solvent with the liquid cell culture medium provides a suspension comprising biological material in solid (e.g. powder) form, optionally wherein the biological material in solid form has a water content of less than about 50% or less than about 25% and / or a water activity of less than about 1.

5. 5. The method of any one of claims 1 to 4, wherein contacting the first organic solvent with the liquid cell culture medium comprises at least partially dissolving cells present in the liquid cell culture medium.

6. 6. The method of any one of claims 1 to 5, wherein contacting the first organic solvent with the liquid cell culture medium comprises at least partially dissolving lipids and / or cell membranes of cells present in the liquid cell culture medium.

7. 7. The method of any one of claims 1 to 6, wherein the mixture is a water-in-oil emulsion, optionally the water-in-oil emulsion comprising droplets having a diameter and / or smallest dimension of less than about 1000 μm or less than about 500 μm.

8. The method according to any one of claims 1 to 7, wherein the first organic solvent and / or the second organic solvent are immiscible with water and have at least partial water solubility.

9. The method of any one of claims 1 to 8, wherein the first organic solvent and / or the second organic solvent has an interfacial tension with water that is less than about 10.

10. 10. The method of any one of claims 1 to 9, wherein the mixture has a water activity of less than about 1 (e.g., less than about 0.9, less than about 0.85, less than about 0.8, less than about 0.75, or less than about 0.7).

11. 11. The method of any one of claims 1 to 10, wherein the mixture has a fractional water saturation of less than about 0.95, less than about 0.9, less than about 0.85, less than about 0.8, less than about 0.75, less than about 0.7, less than about 0.65, less than about 0.6, less than about 0.55, less than about 0.5, less than about 0.45, less than about 0.4, less than about 0.35, or less than about 0.

3.

12. 12. The method of any one of claims 1 to 11, wherein contacting the first organic solvent and the liquid cell culture medium comprises providing each in a mixer (e.g., a homogenizer).

13. 13. The method of any one of claims 1 to 12, wherein the first organic solvent and / or the second organic solvent comprises an alcohol (e.g., a C1 to C20 alcohol, a C4 to C11 alcohol, or a C4 to C9 alcohol), optionally wherein the alcohol has a solubility in water of about 2% to about 15%, and / or optionally wherein the alcohol is not a C10 alcohol.

14. The method of any one of claims 1 to 13, wherein forming an at least partially dehydrated biological material comprises homogenizing the mixture.

15. 15. The method of claim 14, wherein homogenizing the mixture is performed using an in-line homogenizer.

16. Homogenizing the mixture may be at a rate of from about 1 L / hour, about 5 L / hour, about 10 L / hour, about 25 L / hour, about 50 L / hour, about 75 L / hour, or about 100 L / hour to about 150 L / hour, about 250 L / hour, about 500 L / hour, about 1,000 L / hour, about 1,500 L / hour, about 2,000 L / hour, about 2,500 L / hour, about 3,000 L / hour, about 3,500 L / hour, or about 4,000 L / hour.

16. The method of claim 14 or 15, wherein the method is performed at a feed rate ranging from about 0.1:100, about 0.5:100, about 1:100, about 2:100 or about 5:100 to about 8:100, about 10:100, about 15:100, about 21:100 or about 25:100 (liquid cell culture medium:first organic solvent).

17. 17. The method of any one of claims 1 to 16, wherein the steps of contacting the first organic solvent with the liquid cell culture medium containing the biological material and forming the at least partially dehydrated biological material are performed substantially simultaneously.

18. 18. The method of any one of claims 1 to 17, wherein separating the at least partially dehydrated biological material from the mixture comprises filtering the mixture and / or centrifuging the mixture.

19. 19. The method of any one of claims 1 to 18, further comprising washing the at least partially dehydrated biological material with a second organic solvent, optionally wherein the second organic solvent is the same as or different from the first organic solvent.

20. 20. The method of any one of claims 1 to 19, further comprising washing the at least partially dehydrated biological material with a third organic solvent, optionally the third organic solvent being the same or different from the first organic solvent and / or the second organic solvent, and / or being a volatile organic compound (e.g., having a boiling point of from about 0°C, about 50°C or about 100°C to about 150°C, about 200°C or about 260°C).

21. 21. The method of any one of claims 1 to 20, comprising concentrating, centrifuging, and / or filtering the mixture comprising the at least partially dehydrated biological material prior to washing the at least partially dehydrated biological material with a second organic solvent.

22. 22. The method of any one of claims 1 to 21, comprising contacting the liquid cell culture medium with an emulsification solvent prior to contacting the first organic solvent with the liquid cell culture medium, optionally wherein the emulsification solvent is an alkane (e.g., a C1-C6 alkane) or an alcohol (e.g., a C5-C14 alcohol).

23. 23. The method of any one of claims 1 to 22, further comprising storing the isolated biological material.

24. 24. A method according to any one of claims 1 to 23, comprising hydrating the isolated biological material and, optionally, further purifying the isolated biological material.

25. 25. The method of any one of claims 1 to 24, further comprising treating the liquid cell culture medium prior to contacting the liquid cell culture medium with the first organic solvent, optionally wherein treating the liquid cell culture medium comprises concentrating, centrifuging, washing, dialysing, and / or filtering the liquid cell culture medium (e.g. to concentrate the cells and / or to remove substances (e.g. salts and / or cells) present in the liquid cell culture medium).

26. 26. The method of any one of claims 1 to 25, further comprising isolating hydrophilic components present in the liquid cell culture medium.

27. 27. The method of any one of claims 1 to 26, wherein contacting the somatic cell culture medium with the first organic solvent comprises at least partially lysing cells present in the liquid cell culture medium, and wherein the method does not include an additional lysis step for isolating the biological material (e.g., does not include a lysis step comprising physical disruption (e.g., multiple freeze / thaw cycles, sonication, high pressure homogenization, trituration, etc.) and / or permeabilization (e.g., permeabilization using detergents and / or enzymes)).

28. 28. The method of any one of claims 1 to 27, wherein the liquid cell culture medium is not lysed (e.g. by physical disruption and / or permeabilization) prior to the first step of the method.

29. The method according to any one of claims 1 to 28, wherein the biological substance is a protein (e.g., an intracellularly expressed protein and / or an extracellularly expressed protein) and / or a peptide (e.g., an intracellularly expressed peptide and / or an extracellularly expressed peptide).

30. The method according to any one of claims 1 to 29, wherein the biological material is a polynucleotide (e.g., an oligonucleotide, DNA, RNA, etc.).

31. 31. The method of any one of claims 1 to 30, wherein the liquid cell culture medium comprises a fermentation broth, optionally comprising cells, cellular components and / or synthetic substances.

32. 32. The method according to any one of claims 1 to 31, wherein the liquid cell culture medium comprises a microbial culture, and optionally comprises cells, cellular components and / or synthetic substances.

33. 33. The method of any one of claims 1 to 32, wherein the liquid cell culture medium comprises cells (e.g. bacterial cells and / or yeast cells) and / or cellular components (e.g. lipids, cell membranes, proteins, DNA, etc.).

34. 34. The method of any one of claims 1 to 33, wherein the liquid cell culture medium comprises cells and, optionally, a buffering agent.

35. 35. The method of any one of claims 1-34, wherein the isolated biological material is in the form of solid amorphous particles (e.g., microparticles), and optionally the isolated biologic has a water content of less than about 15% (e.g., less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 4%, or less than about 2%) and / or a water activity of less than about 0.5%.

36. 36. The method of any one of claims 1 to 35, further comprising collecting and / or recycling the first organic solvent and / or the second organic solvent, and optionally further comprising removing water and / or impurities from the collected and / or recycled first organic solvent and / or second organic solvent.

37. 1. A composition comprising a plurality of solid amorphous particles (e.g., microparticles), comprising: The composition, wherein at least a portion of said plurality of solid amorphous particles are solid amorphous particles comprising biological material.

38. 38. The composition of claim 37, comprising a water insoluble component (e.g., lipid) in an amount less than about 25% by weight of the composition.

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