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

Abstract

Described herein are isolated cell culture components such as, e.g., biologics and/or lipids, and methods for isolating cell culture components from a liquid cell culture medium. 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.

That which is claimed is: 
     
         1 . A method of isolating a biologic from a liquid cell culture medium, the method comprising:
 contacting a first organic solvent and a liquid cell culture medium comprising a biologic to form a mixture;   forming an at least partially dehydrated biologic in the mixture; and   separating the at least partially dehydrated biologic from the mixture, thereby providing an isolated biologic.   
     
     
         2 . The method of  claim 1 , wherein the biologic is present in the liquid cell culture medium at a concentration of less than about 200 mg/mL, 100 mg/mL, 50 mg/mL, 30 mg/mL, 20 mg/mL, 10 mg/mL, 5 mg/mL, 1 mg/mL, 0.1 mg/mL, 0.05 mg/mL, or 0.01 mg/mL, optionally wherein the biologic is at least partially encapsulated in a cell present in the liquid cell culture medium. 
     
     
         3 . The method of any preceding claim, wherein the method simultaneously extracts water from the liquid cell culture medium and preserves the biologic. 
     
     
         4 . The method of any preceding claim, wherein contacting the first organic solvent and the liquid cell culture medium comprises providing a suspension comprising the biologic in the form of a solid (e.g., a powder), optionally wherein the biologic in the form of a solid has a water content of less than about 50% or 25% and/or a water activity of less than about 1. 
     
     
         5 . The method of any preceding claim, wherein contacting the first organic solvent and the liquid cell culture medium comprises at least partially lysing cells present in the liquid cell culture medium. 
     
     
         6 . The method of any preceding claim, wherein contacting the first organic solvent and the liquid cell culture medium comprises at least partially dissolving lipids and/or the cell membrane of cells present in the liquid cell culture medium. 
     
     
         7 . The method of any preceding claim, wherein the mixture is a water-in-oil emulsion, optionally wherein the water-in-oil emulsion comprises a droplet with a diameter and/or smallest dimension of less than about 1000 μm or less than about 500 μm. 
     
     
         8 . The method of any preceding claim, wherein the first organic solvent and/or a second organic solvent is immiscible with water and has at least partial water solubility. 
     
     
         9 . The method of any preceding claim, wherein the first organic solvent and/or a second organic solvent has an interfacial tension with water that is less than about 10. 
     
     
         10 . The method of any preceding claim, wherein the mixture has a water activity of less than about 1 (e.g., less than about 0.9, 0.85, 0.8, 0.75, or 0.7). 
     
     
         11 . The method of any preceding claim, wherein the mixture has a fractional water saturation of less than about 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, or 0.3. 
     
     
         12 . The method of any preceding claim, wherein contacting the first organic solvent and the liquid cell culture medium comprises feeding each into a mixer (e.g., a homogenizer). 
     
     
         13 . The method of any preceding claim, wherein the first organic solvent and/or a second organic solvent comprises an alcohol (e.g., a C1-C20 alcohol, C4 to C11 alcohol, or a C4-C9 alcohol), optionally wherein the alcohol has a solubility for water of about 2% to about 15% and/or optionally wherein the alcohol is not a C10 alcohol. 
     
     
         14 . The method of any preceding claim, wherein forming the at least partially dehydrated biologic comprises homogenizing the mixture. 
     
     
         15 . The method of  claim 14 , wherein homogenizing the mixture is carried out with an in-line homogenizer. 
     
     
         16 . The method of  claim 14  or  15 , wherein homogenizing the mixture is carried out with a feed rate in a range of about 1, 5, 10, 25, 50, 75, or 100 L/hour to about 150, 250, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000 L/hour and/or with a feed volume ratio of the liquid cell culture medium to first organic solvent of about 0.1:100, 0.5:100, 1:100, 2:100, or 5:100 to about 8:100, 10:100, 15:100, 20:100, or 25:100 (liquid cell culture medium:first organic solvent). 
     
     
         17 . The method of any preceding claim, wherein the step of contacting the first organic solvent and the liquid cell culture medium and the step of forming the at least partially dehydrated biologic in the mixture are performed substantially simultaneously. 
     
     
         18 . The method of any preceding claim, wherein separating the at least partially dehydrated biologic from the mixture comprises filtering the mixture and/or centrifuging the mixture. 
     
     
         19 . The method of any preceding claim, further comprising washing the at least partially dehydrated biologic with a second organic solvent, optionally wherein the second organic solvent is the same as or different than the first organic solvent. 
     
     
         20 . The method of any preceding claim, further comprising washing the at least partially dehydrated biologic with a third organic solvent, optionally wherein the third organic solvent is the same as or different than the first and/or second organic solvent and/or is a volatile organic compound (e.g., has a boiling point from about 0, 50, or 100° C. to about 150, 200, or 260° C.). 
     
     
         21 . The method of any preceding claim, wherein, prior to washing the at least partially dehydrated biologic with the second organic solvent, the method comprises concentrating, centrifuging, and/or filtering the mixture comprising the at least partially dehydrated biologic. 
     
     
         22 . The method of any preceding claim, wherein, prior to contacting the first organic solvent and liquid cell culture medium, the method comprises contacting the liquid cell culture medium with an emulsification solvent, optionally wherein the emulsification solvent is an alkane (e.g., a C1-C6 alkane) or an alcohol (e.g., a C5-C14 alcohol). 
     
     
         23 . The method of any preceding claim, further comprising storing the isolated biologic. 
     
     
         24 . The method of any preceding claim, further comprising hydrating the isolated biologic and optionally further purifying the isolated biologic. 
     
     
         25 . The method of any preceding claim, further comprising, prior to contacting the first organic solvent and the liquid cell culture medium, processing the liquid cell culture medium, optionally wherein processing the liquid cell culture medium comprises concentrating, centrifuging, washing, dialyzing, and/or filtering the liquid cell culture medium (e.g., to concentrate the cells and/or remove substances (e.g., salts and/or cells) present in the liquid cell culture medium). 
     
     
         26 . The method of any preceding claim, further comprising isolating hydrophilic components present in the liquid cell culture medium. 
     
     
         27 . The method of any preceding claim, wherein the step of contacting the first organic solvent and the liquid cell culture medium comprises at least partially lysing cells present in the liquid cell culture medium, and wherein the method is devoid of an additional lysing step to isolate the biologic (e.g., devoid of a lysis step including physical disruption (e.g., multiple freeze/thaw cycles, sonication, high-pressure homogenization, grinding, etc.) and/or permeabilization (e.g., with detergents and/or enzymes)). 
     
     
         28 . The method of any preceding claim, wherein the liquid cell culture medium is not lysed (e.g., by physical disruption and/or permeabilization) prior to a first step of the method. 
     
     
         29 . The method of any preceding claim, wherein the biologic is a protein (e.g., an intracellularly expressed protein and/or an extracellularly expressed protein) and/or peptide (e.g., an intracellularly expressed peptide and/or an extracellularly expressed peptide). 
     
     
         30 . The method of any preceding claim, wherein the biologic is a polynucleotide (e.g., an oligonucleotide, DNA, RNA, etc.). 
     
     
         31 . The method of any preceding claim, wherein the liquid cell culture medium comprises fermentation broth that optionally comprises cells, cell components, and/or synthetic materials. 
     
     
         32 . The method of any preceding claim, wherein the liquid cell culture medium comprises a microbiological culture that optionally comprises cells, cell components, and/or synthetic materials. 
     
     
         33 . The method of any preceding claim, 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 . The method of any preceding claim, wherein the liquid cell culture medium comprises cells and optionally a buffer. 
     
     
         35 . The method of any preceding claim, wherein the isolated biologic is in the form of a solid amorphous particle (e.g., a microparticle), optionally wherein the isolated biologic has a water content of less than about 15% (e.g., less than about 12%, 10%, 8%, 6%, 4%, or 2%) and/or a water activity of less than about 0.5. 
     
     
         34 . The method of any preceding claim, further comprising collecting and/or recycling the first organic solvent and/or second organic solvent, optionally further comprising removing water and/or an impurity from the collected and/or recycled first organic solvent and/or second organic solvent. 
     
     
         35 . A composition comprising a plurality of solid amorphous particles (e.g., microparticles),
 wherein at least a portion of the plurality of solid amorphous particles are solid amorphous particles comprising a biologic (e.g., a protein, peptide, etc.).   
     
     
         36 . The composition of  claim 35 , wherein the composition comprises water insoluble components (e.g., lipids) in an amount of less than about 25% by weight of the composition.