Method for perfusion culture of mammalian cells

Abstract

Provided herein is a method of perfusion culture of mammalian cells, the method comprising: providing a vessel containing mammalian cells disposed in a first liquid medium having an osmolality of about 270 mOsm / kg to about 380 mOsm / kg; incubating the mammalian cells at about 32° C. to about 39° C. for a period of time; and continuously or periodically during the period of time, removing a first volume of the first liquid medium and adding a second volume of a second liquid medium to the first liquid medium, wherein the first and second volumes are approximately equal, and wherein the osmolality of the first and second liquid media in the vessel is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period of time.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 174,900, filed April 14, 2021; the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to biotechnological methods and the production of recombinant proteins. [Background technology]

[0003] Mammalian cells containing recombinant protein-encoding nucleic acids are often used to produce proteins of therapeutic or commercial importance. The current landscape of diverse product pipelines requires biotechnology companies to develop innovative solutions for flexible and cost-effective manufacturing of therapeutic protein drug substances. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention is based, at least in part, on the discovery that a method for perfusion culture of mammalian cells comprising: providing a vessel containing mammalian cells disposed in a first liquid medium having an osmolality of about 270 mOsm / kg to about 380 mOsm / kg; incubating the mammalian cells at about 32° C. to about 39° C. for a period of time; and continuously or periodically during the period of time removing a first volume of the first liquid medium and adding a second volume of a second liquid medium to the first liquid medium, wherein the first and second volumes are approximately equal and the osmolality of the first liquid medium and the osmolality of the second liquid medium in the vessel are maintained at about 270 mOsm / kg to about 380 mOsm / kg for the period of time, the method achieves improved growth and health of the culture as compared to other perfusion cell culture methods in which the osmolality is not maintained at about 270 mOsm / kg to about 380 mOsm / kg for the period of time. [Means for solving the problem]

[0005] Provided herein is a method of perfusion culture of mammalian cells, the method including: providing a vessel containing mammalian cells disposed in a first liquid medium having an osmolality of about 270 mOsm / kg to about 380 mOsm / kg (e.g., any subrange therein); incubating the mammalian cells at about 32°C to about 39°C (e.g., any subrange therein) for a period of time; and continuously or periodically during the period of time removing a first volume of the first liquid medium and adding a second volume of a second liquid medium to the first liquid medium, wherein the first and second volumes are approximately equal and the osmolality of the first and second liquid media in the vessel is maintained at about 270 mOsm / kg to about 380 mOsm / kg (e.g., any subrange therein) over the period of time.

[0006] In some embodiments, the first liquid medium has an osmolality of about 270 mOsm / kg to about 320 mOsm / kg. In some embodiments, the second liquid medium comprises more than 8 g / L of poloxamer. In some embodiments, the second liquid medium comprises more than 10 g / L of poloxamer. In some embodiments, the second liquid medium comprises more than 12 g / L of poloxamer. In some embodiments of any of the methods described herein, the poloxamer is Pluronic F68. In some embodiments of any of the methods described herein, the second liquid medium has a pH of about 6.8 to about 7.1. In some embodiments of any of the methods described herein, the second liquid medium comprises sodium chloride. In some embodiments of any of the methods described herein, the second liquid medium has an osmolality of about 800 mOsm / kg to about 2,500 mOsm / kg. In some embodiments of any of the methods described herein, the second liquid medium has an osmolality of about 1,200 mOsm / kg to about 1,800 mOsm / kg. In some embodiments of any of the methods described herein, the second volume of the second liquid medium added is increased over the period. In some embodiments of any of the methods described herein, the second volume of the second liquid medium added is increased over the period based on the viable cell density. In some embodiments of any of the methods described herein, maintaining the osmolality of the first and second liquid medium in the vessel includes adjusting the flow rate of the second liquid medium at some point during the period.

[0007] In some embodiments, the adjusting step includes increasing the flow rate of the second liquid medium at some point during the period.

[0008] In some embodiments of any of the methods described herein, the method includes adjusting one or both of the pH and pCO2 of the first and second liquid media in the container at some point during the period.

[0009] In some embodiments, the adjusting step includes decreasing the flow rate of the second liquid medium at some point during the period.

[0010] In some embodiments of any of the methods described herein, the method further comprises increasing the pCO2 and increasing the pH of the first and second liquid media in the container over the period of time.

[0011] In some embodiments, the pCO2 and pH of the first and second liquid media in the vessel are increased over the period based on the viable cell density.

[0012] In some embodiments of any of the methods described herein, the 60×10 6 A viable cell density of greater than 100×10 cells / mL in the first and second liquid media is achieved during that period. 6 A viable cell density of greater than 120×10 cells / mL in the first and second liquid media is achieved during that period. 6 A viable cell density of > 100 cells / mL is achieved during that period.

[0013] In some embodiments of any of the methods described herein, the method further comprises monitoring the osmolarity of the first and second liquid media in the container during the period.

[0014] In some embodiments of any of the methods described herein, during the period, the removal of the first volume of the first liquid medium and the addition of the second volume of the second liquid medium are performed simultaneously. In some embodiments of any of the methods described herein, during the period, the removal of the first volume of the first liquid medium and the addition of the second volume of the second liquid medium are performed continuously. In some embodiments of any of the methods described herein, during the period, the removal of the first volume of the first liquid medium and the addition of the second volume of the second liquid medium are performed periodically.

[0015] In some embodiments of any of the methods described herein, the method further includes periodically adding a third volume of an aqueous solution to the first and second liquid media in the containers during the period, wherein the first volume and the sum of the second and third volumes are approximately equal, and wherein the osmolarity of the first, second, and third liquid media in the containers is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period.

[0016] In some embodiments, the aqueous solution is a salt solution, hi some embodiments, the salt solution is a sodium chloride solution or a potassium chloride solution.

[0017] In some embodiments of any of the methods described herein, during the period, the removal of the first volume of the first liquid medium, the addition of the second volume of the second liquid medium, and the addition of the third volume occur simultaneously. In some embodiments of any of the methods described herein, during the period, the removal of the first volume of the first liquid medium, the addition of the second volume of the second liquid medium, and the addition of the third volume occur sequentially. In some embodiments of any of the methods described herein, during the period, the removal of the first volume of the first liquid medium, the addition of the second volume of the second liquid medium, and the addition of the third volume occur periodically.

[0018] In some embodiments of any of the methods described herein, the method further includes continuously or periodically adding a third volume of a third liquid medium to the first liquid medium, wherein the first volume and the sum of the second and third volumes are approximately equal, and wherein an osmolality of the first, second, and third liquid medium in the container is maintained at about 270 mOsm / mg to about 380 mOsm / kg over the period.

[0019] In some embodiments, the third liquid medium has an osmolality that is about 270 mOsm / kg to about 380 mOsm / kg. In some embodiments of any of the methods described herein, maintaining the osmolality of the first, second, and third liquid medium in the container comprises adjusting a flow rate of the second liquid medium during the period. In some embodiments of any of the methods described herein, the adjusting comprises increasing the flow rate of the second liquid medium at a point during the period. In some embodiments of any of the methods described herein, the adjusting comprises decreasing the flow rate of the second liquid medium at a point during the period.

[0020] In some embodiments, the third liquid medium has an osmolality of about 10 mOsm / kg to about 270 mOsm / kg. In some embodiments, the third liquid medium has an osmolality of about 50 mOsm / kg to about 200 mOsm / kg.

[0021] In some embodiments of any of the methods described herein, maintaining the osmotic pressure of the first, second, and third liquid medium in the container comprises adjusting the flow rate of the third liquid medium during the period.

[0022] In some embodiments of any of the methods described herein, the adjusting step comprises increasing the flow rate of the third liquid medium at some point during the period.

[0023] In some embodiments of any of the methods described herein, the adjusting step comprises decreasing the flow rate of the third liquid medium at some point during the period.

[0024] In some embodiments of any of the methods described herein, the method further comprises adjusting one or both of the pH and pCO2 of the first, second, and third liquid media in the container at a point during the period. In some embodiments, the method comprises increasing the pCO2 and increasing the pH of the first, second, and third liquid media in the container over the period. In some embodiments, the pCO2 and pH of the first, second, and third liquid media in the container are increased over the period based on the viable cell density.

[0025] In some embodiments of any of the methods described herein, the first, second, and third liquid media are cultured at 60×10 6 A viable cell density of greater than 100×10 cells / mL is achieved during that period. In some embodiments, the first, second, and third liquid media are at least 100×10 cells / mL. 6 A viable cell density of greater than 120×10 cells / mL in the first, second, and third liquid media is achieved during that period. 6 A viable cell density of > 100 cells / mL is achieved during that period.

[0026] In some embodiments of any of the methods described herein, the method further comprises monitoring the osmolality of the first, second, and third liquid medium in the container during the period. In some embodiments of any of the methods described herein, during the period, the removal of the first volume of the first liquid medium, the addition of the second volume of the second liquid medium, and the addition of the third volume occur simultaneously. In some embodiments of any of the methods described herein, during the period, the removal of the first volume of the first liquid medium, the addition of the second volume of the second liquid medium, and the addition of the third volume occur continuously. In some embodiments of any of the methods described herein, during the period, the removal of the first volume of the first liquid medium, the addition of the second volume of the second liquid medium, and the addition of the third volume occur periodically.

[0027] In some embodiments of any of the methods described herein, the mammalian cell is a CHO cell.

[0028] In some embodiments of any of the methods described herein, the vessel is a bioreactor. In some embodiments, the bioreactor is a perfusion bioreactor.

[0029] Also provided herein is a method of producing a recombinant protein, the method including: providing a vessel containing mammalian cells, the cells comprising a nucleic acid encoding the recombinant protein, disposed in a first liquid medium having an osmolality of about 270 mOsm / kg to about 380 mOsm / kg (e.g., any subrange therein); incubating the mammalian cells at about 32° C. to about 39° C. (e.g., any subrange therein) for a period of time; continuously or periodically removing a first volume of the first liquid medium and adding a second volume of a second liquid medium to the first liquid medium during the period of time, the first and second volumes being approximately equal and the osmolality of the first and second liquid medium in the vessel being maintained at about 270 mOsm / kg to about 380 mOsm / kg (e.g., any subrange therein); and recovering the recombinant protein from the mammalian cells or from the first liquid medium and / or the second liquid medium.

[0030] In some embodiments, the first liquid medium has an osmolality of about 270 mOsm / kg to about 320 mOsm / kg. In some embodiments, the second liquid medium comprises more than 8 g / L of poloxamer. In some embodiments, the second liquid medium comprises more than 10 g / L of poloxamer. In some embodiments, the second liquid medium comprises more than 12 g / L of poloxamer. In some embodiments of any of the methods described herein, the poloxamer is Pluronic F68. In some embodiments of any of the methods described herein, the second liquid medium has a pH of about 6.8 to about 7.1. In some embodiments of any of the methods described herein, the second liquid medium comprises sodium chloride.

[0031] In some embodiments of any of the methods described herein, the second liquid medium has an osmolality of about 800 mOsm / kg to about 2,500 mOsm / kg. In some embodiments, the second liquid medium has an osmolality of about 1,200 mOsm / kg to about 1,800 mOsm / kg. In some embodiments of any of the methods described herein, the second volume of the second liquid medium added is increased over the period. In some embodiments, the second volume of the second liquid medium added is increased over the period based on the viable cell density.

[0032] In some embodiments of any of the methods described herein, maintaining the osmolarity of the first and second liquid media in the container comprises adjusting the flow rate of the second liquid medium at a time during the period. In some embodiments, the adjusting step comprises increasing the flow rate of the second liquid medium at a time during the period. In some embodiments, the method further comprises adjusting one or both of the pH and pCO2 of the first and second liquid media in the container at a time during the period. In some embodiments, the adjusting step comprises decreasing the flow rate of the second liquid medium at a time during the period.

[0033] In some embodiments of any of the methods described herein, the method further comprises increasing the pCO2 and increasing the pH of the first and second liquid media in the container over the period of time. In some embodiments, the pCO2 and pH of the first and second liquid media in the container are increased over the period of time based on the viable cell density.

[0034] In some embodiments of any of the methods described herein, the 60×10 6 A viable cell density of greater than 100×10 cells / mL in the first and second liquid media is achieved during that period. 6 A viable cell density of greater than 120×10 cells / mL in the first and second liquid media is achieved during that period. 6A viable cell density of > 100 cells / mL is achieved during that period.

[0035] In some embodiments of any of the methods described herein, the method further comprises monitoring the osmolality of the first and second liquid media in the container during the period. In some embodiments of any of the methods described herein, the removal of the first volume of the first liquid medium and the addition of the second volume of the second liquid medium occur simultaneously during the period. In some embodiments of any of the methods described herein, the removal of the first volume of the first liquid medium and the addition of the second volume of the second liquid medium occur continuously during the period. In some embodiments of any of the methods described herein, the removal of the first volume of the first liquid medium and the addition of the second volume of the second liquid medium occur periodically over the period.

[0036] In some embodiments of any of the methods described herein, the recombinant protein is secreted into the first and / or second liquid medium. In some embodiments, the recombinant protein is recovered from the first and / or second liquid medium.

[0037] In some embodiments of any of the methods described herein, the method further comprises continuously or periodically adding a third volume of an aqueous solution to the first and second liquid media in the containers during the period, wherein the first volume and the sum of the second and third volumes are about equal, and the osmolality of the first, second, and third liquid media in the containers is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period. In some embodiments, the aqueous solution is a salt solution. In some embodiments, the salt solution is a sodium chloride solution or a potassium chloride solution.

[0038] In some embodiments of any of the methods described herein, during the period, the removal of the first volume of the first liquid medium, the addition of the second volume of the second liquid medium, and the addition of the third volume occur simultaneously. In some embodiments of any of the methods described herein, during the period, the removal of the first volume of the first liquid medium, the addition of the second volume of the second liquid medium, and the addition of the third volume occur sequentially. In some embodiments of any of the methods described herein, during the period, the removal of the first volume of the first liquid medium, the addition of the second volume of the second liquid medium, and the addition of the third volume occur periodically.

[0039] In some embodiments of any of the methods described herein, the method further includes continuously or periodically adding a third volume of a third liquid medium to the first liquid medium, wherein the first volume and the sum of the second and third volumes are approximately equal, and wherein the osmolarity of the first, second, and third liquid media in the container is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period.

[0040] In some embodiments, the third liquid medium has an osmolality that is about 270 mOsm / kg to about 380 mOsm / kg. In some embodiments of any of the methods described herein, maintaining the osmolality of the first, second, and third liquid medium in the container comprises adjusting the flow rate of the second liquid medium during the period. In some embodiments of any of the methods described herein, the adjusting comprises increasing the flow rate of the second liquid medium at a time during the period. In some embodiments of any of the methods described herein, the adjusting comprises decreasing the flow rate of the second liquid medium at a time during the period. In some embodiments, the third liquid medium has an osmolality of about 0 mOsm / kg to about 270 mOsm / kg. In some embodiments, the third liquid medium has an osmolality of about 50 mOsm / kg to about 200 mOsm / kg. In some embodiments of any of the methods described herein, maintaining the osmolarity of the first, second, and third liquid medium in the container comprises adjusting a flow rate of the third liquid medium during the period. In some embodiments of any of the methods described herein, the adjusting comprises increasing a flow rate of the third liquid medium at a time during the period. In some embodiments of any of the methods described herein, the adjusting comprises decreasing a flow rate of the third liquid medium at a time during the period. In some embodiments of any of the methods described herein, the method further comprises adjusting one or both of the pH and pCO2 of the first, second, and third liquid medium in the container at a time during the period.

[0041] In some embodiments, the method includes increasing the pCO2 and increasing the pH of the first, second, and third liquid media in the vessel over the period. In some embodiments, the pCO2 and pH of the first, second, and third liquid media in the vessel are increased over the period based on viable cell density. In some embodiments of any of the methods described herein, the pCO2 and pH of the first, second, and third liquid media are increased over the period based on viable cell density. 6A viable cell density of greater than 100×10 cells / mL is achieved during that period. In some embodiments, the first, second, and third liquid media are at least 100×10 cells / mL. 6 A viable cell density of greater than 120×10 cells / mL in the first, second, and third liquid media is achieved during that period. 6 A viable cell density of > 100 cells / mL is achieved during that period.

[0042] In some embodiments of any of the methods described herein, the method further comprises monitoring the osmolality of the first, second, and third liquid medium in the container during the period. In some embodiments of any of the methods described herein, during the period, the removal of the first volume of the first liquid medium, the addition of the second volume of the second liquid medium, and the addition of the third volume occur simultaneously. In some embodiments of any of the methods described herein, during the period, the removal of the first volume of the first liquid medium, the addition of the second volume of the second liquid medium, and the addition of the third volume occur continuously. In some embodiments of any of the methods described herein, during the period, the removal of the first volume of the first liquid medium, the addition of the second volume of the second liquid medium, and the addition of the third volume occur periodically. In some embodiments of any of the methods described herein, the recombinant protein is secreted into the first, second, and / or third liquid medium. In some embodiments, the recombinant protein is recovered from the first, second, and / or third liquid medium.

[0043] In some embodiments of any of the methods described herein, the mammalian cells are CHO cells. In some embodiments of any of the methods described herein, the vessel is a bioreactor. In some embodiments, the bioreactor is a perfusion bioreactor. In some embodiments of any of the methods described herein, the recombinant protein is an immunoglobulin, an enzyme, a growth factor, a protein fragment, or an engineered protein. In some embodiments of any of the methods described herein, the recombinant protein is recovered from the mammalian cells.

[0044] In some embodiments of any of the methods described herein, the method further comprises isolating the recombinant protein. In some embodiments, the method further comprises formulating the isolated recombinant protein.

[0045] Provided herein are recombinant proteins produced by any of the methods described herein. Also provided herein are pharmaceutical compositions comprising any of the recombinant proteins described herein, and kits comprising any of the pharmaceutical compositions described herein.

[0046] Also provided herein are methods of treating a subject, comprising administering to a subject in need thereof a therapeutically effective amount of any of the pharmaceutical compositions described herein.

[0047] As used herein, the word "a" before a noun refers to one or more of that particular noun. For example, the phrase "mammalian cells" refers to "one or more mammalian cells."

[0048] The term "mammalian cell" refers to any cell from or derived from any mammal (e.g., human, hamster, mouse, green monkey, rat, pig, cow, or rabbit). For example, the mammalian cell can be an immortalized cell. In some embodiments, the mammalian cell is a differentiated or undifferentiated cell. Non-limiting examples of mammalian cells are described herein. Additional examples of mammalian cells are known in the art.

[0049] The term "culture" or "cell culture" refers to the maintenance or growth of mammalian cells under a controlled set of physical conditions.

[0050] The terms "mammalian cell culture," "culture," or "cell culture" refer to a liquid medium containing a plurality of mammalian cells maintained or grown under a controlled set of physical conditions.

[0051] The term "liquid medium" or "liquid media" refers to a liquid containing sufficient nutrients to allow cells (e.g., mammalian cells) to grow or proliferate in vitro. For example, the liquid medium may contain one or more of the following: amino acids (e.g., the 20 amino acids), purines (hypoxanthine), pyrimidines (e.g., thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate. In some embodiments, the liquid medium may contain serum from a mammal. In some embodiments, the liquid medium does not contain serum or another extract from a mammal (defined liquid medium). In some embodiments, the liquid medium may contain trace metals, mammalian growth hormone, and / or mammalian growth factor. Another example of a liquid medium is a minimal medium (e.g., a medium containing only inorganic salts, a carbon source, and water). Non-limiting examples of liquid media are described herein. Additional examples of liquid media are known in the art and commercially available. The liquid media may contain any density of mammalian cells. For example, as used herein, a volume of liquid media removed from a bioreactor may be substantially free of mammalian cells.

[0052] The term "animal-origin component-free liquid medium" refers to a liquid medium that does not contain components derived from mammals (eg, proteins or serum).

[0053] The term "serum-free liquid medium" refers to a liquid medium that does not contain mammalian serum.

[0054] The term "serum-containing liquid medium" refers to a liquid medium that contains mammalian serum.

[0055] The term "chemically defined liquid medium" is a term of art and refers to a liquid medium in which all chemical components are known. For example, a chemically defined liquid medium does not contain fetal bovine serum, bovine serum albumin, or human serum albumin because these preparations usually contain complex mixtures of albumin and lipids.

[0056] The term "protein-free liquid medium" means a liquid medium that does not contain proteins (eg, any detectable proteins).

[0057] The term "clarified liquid medium" refers to a liquid medium obtained from a bacterial or yeast cell culture that is substantially free (e.g., at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% free) of bacterial or yeast cells.

[0058] The term "agitation" means to stir or otherwise move a portion of the liquid medium in a bioreactor. This is done, for example, to increase the dissolved O2 concentration of the liquid medium in the bioreactor. Agitation can be done using any method known in the art, for example, using a device or a propeller. Exemplary devices and methods that can be used to stir a portion of the liquid medium in a bioreactor are known in the art.

[0059] The term "immunoglobulin" refers to a polypeptide that includes an amino acid sequence of at least 15 amino acids (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids) of an immunoglobulin protein (e.g., a variable domain sequence, a framework sequence, or a constant domain sequence). The immunoglobulin can include, for example, at least 15 amino acids of a light chain immunoglobulin, and can include, for example, at least 15 amino acids of a heavy chain immunoglobulin. The immunoglobulin can be an isolated antibody (e.g., IgG, IgE, IgD, IgA, or IgM). The immunoglobulin can be an antibody fragment, for example, a Fab fragment, a F(ab')2 fragment, or a scFv fragment. The immunoglobulin can also be a bispecific or trispecific antibody, or a dimeric, trimeric, multimeric antibody, or a diabody, Affibody®, or Nanobody®. An immunoglobulin may also be an engineered protein that contains at least one immunoglobulin domain (e.g., a fusion protein). Non-limiting examples of immunoglobulins are described herein, and additional examples of immunoglobulins are known in the art. Recombinant immunoglobulins are produced using any of the methods described herein.

[0060] The term "engineered protein" refers to a polypeptide that is not naturally encoded by an endogenous nucleic acid present in an organism (e.g., a mammal). Examples of engineered proteins include enzymes (e.g., having one or more amino acid substitutions, deletions, insertions, or additions that result in increased stability and / or catalytic activity of the engineered enzyme), fusion proteins, antibodies (e.g., bivalent antibodies, trivalent antibodies, or diabodies), and antigen binding proteins that include at least one engineered scaffold sequence.

[0061] The term "secreted protein" or "secreted recombinant protein" refers to a protein (e.g., a recombinant protein) that originally contained at least one secretory signal sequence when translated in a mammalian cell, and which is at least partially secreted into the extracellular space (e.g., liquid medium) through enzymatic cleavage of the secretory signal sequence within the mammalian cell. One of skill in the art will understand that a "secreted" protein need not be completely separated from the cell to be considered a secreted protein.

[0062] The term "perfusion bioreactor" refers to a bioreactor that includes a plurality of cells (e.g., mammalian cells) in a first liquid medium, and culturing the cells present in the bioreactor includes periodically or continuously removing the first liquid medium and simultaneously or immediately adding a substantially equal volume of a second liquid medium to the bioreactor. In some examples, the volume of the first liquid medium is removed and added over an incremental period (e.g., about a 24 hour period, a period between about 1 minute and about 24 hours, or a period of more than 24 hours) to incrementally change (e.g., increase or decrease) the volume of the first liquid medium (e.g., refeed of liquid medium per day) during the culture period. The rate of medium removed and replaced per day may vary depending on the particular cells being cultured, the initial seeding density, and the cell density at a particular time point. "RV" or "reactor volume" refers to the volume of liquid medium present at the start of the culture process (e.g., the total volume of liquid medium present after seeding).

[0063] "Specific productivity" or "SP" is a term of art and as used herein refers to the mass or enzymatic activity of a recombinant therapeutic protein produced per mammalian cell per day. The SP of a recombinant therapeutic antibody is usually measured as mass / cell / day. The SP of a recombinant therapeutic enzyme is usually measured as units / cell / day or (units / mass) / cell / day.

[0064] "Volumetric productivity" or "VP" is a term of art and as used herein refers to the mass or enzymatic activity of recombinant therapeutic protein produced per volume of culture (e.g., per L of bioreactor, vessel, or tubing volume) per day. The VP of recombinant therapeutic antibodies is usually measured as mass / L / day. The VP of recombinant therapeutic enzymes is usually measured as units / L / day or mass / L / day.

[0065] The phrases "a concentration of poloxamer" and "poloxamer concentration" are used interchangeably herein. The phrases "X g / L or greater poloxamer" and "X g / L or greater concentration of poloxamer" are used interchangeably herein.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art can also be used.Materials, methods, and examples are merely illustrative and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.

[0067] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0068] [Figure 1A] FIG. 1 is a graph of viable cell density (VCD) (million cells / mL) over time for batch cell culture runs (n=2) performed at different osmolalities (mOsm / kg) in shake flasks. The average osmolalities in mOsm are shown for days 0, 4, and 7. [Figure 1B]Graph of viable cell density (VCD) (million cells / mL) over time for batch cell culture runs (n=2) performed at different osmolalities (mOsm / kg) in Ambr15 microbioreactors. Average osmolalities in mOsm are shown for days 0, 2, 5, 7, and 9. [Figure 2A] 1 is a graph of mean cell diameter (μm) versus osmolarity (mOsm / kg) on ​​day 4 in shake flasks. [Figure 2B] FIG. 1 is a graph of average cell diameter (μm) versus osmolarity (mOsm / kg) on ​​day 5 in Ambr15 microbioreactors. [Figure 3A] 1 is a graph of osmolality (mOsm / kg) over time for perfusion cell culture runs performed in a bioreactor. Run V22 ("V22") had a ratio of concentrated cell medium feed to water feed that resulted in high osmolality in the bioreactor. [Figure 3B] 1 is a graph of viable cell density (VCD) (million cells / mL) (solid line) and viable cell mean diameter (μm) (dashed line) over time for perfusion cell culture runs performed in a bioreactor. Run V22 ("V22") experienced high osmolarity, resulting in a slower growth rate and reduced viability. [Figure 3C] 1 is a graph of harvest volumetric productivity (VP) (g / L / day) (solid line) and harvest titer (g / L) (dashed line) over time for perfusion cell culture runs performed in a bioreactor. Run V22 ("V22") had high osmolality and reduced productivity. [Figure 3D] 1 is a graph of % viability (solid line) and lactate dehydrogenase (LDH) production (U / L / day) (dashed line) over time for perfusion cell culture runs performed in a bioreactor. Run V22 ("V22") had high osmolality, resulting in high LDH production indicative of poor culture health. [Figure 3E] 1 is a graph of glucose concentration (g / L) (solid line) and lactate concentration (g / L) (dashed line) over time for perfusion cell culture runs performed in a bioreactor. Run V22 ("V22") had high osmolality, leading to increased lactate production. [Figure 4A] Graph of viable cell density (VCD) (million cells / mL) (solid line) and viability (%) (dashed line) over time for perfusion cell culture runs performed in Protein 3 bioreactors. High osmolarity reduced VCD and culture viability. [Figure 4B] FIG. 1 is a graph of lactate concentration (g / L) (solid line) and lactate osmolality (mOsm / kg) (dashed line) over time in a perfusion cell culture run performed in a bioreactor. Higher osmolality led to increased lactate production. [Figure 5A] FIG. 1 is a graph of osmolality (mOsm / kg) over time for a set of perfusion cell culture bioreactors showing different osmolality profiles. [Figure 5B] Graph of viable cell density (VCD) (million cells / mL) (solid line) and viable cell mean diameter (μm) (dashed line) over time for perfusion cell culture runs performed in a bioreactor. Differences in the VCD profiles correspond to differences in culture osmolality. [Figure 6A] FIG. 13 is a graph of osmolality (mOsm / kg) versus conductivity (mS / cm) for perfusion cell culture runs performed in a bioreactor with all data used. [Figure 6B] FIG. 1 is a graph of osmolality (mOsm / kg) versus conductivity (mS / cm) for perfusion cell culture runs performed in bioreactors, with data characterized by reactor scale and phase of culture (low or high biomass). [Figure 7] FIG. 1 is a graph of conductivity (mS / cm) (solid line) and osmolality (mOsm / kg) (individual data points) over time for a perfusion cell culture run performed in a bioreactor with automated conductivity feedback control. [Figure 8A] 1 is a graph of osmolality (mOsm / kg) over time in perfusion cell culture runs performed in a 10-L bioreactor with automated conductivity feedback control. [Figure 8B]FIG. 1 is a graph of viable cell density (VCD) (million cells / mL) (solid line) and viable cell mean diameter (μm) (dashed line) over time for a perfusion cell culture run performed in a 10-L bioreactor, showing a consistent VCD profile. [Figure 9] 1 is a graph of conductivity (mS / cm) versus osmolality (mOsm / kg) of osmolality standards measured using different probe types. [Figure 10] FIG. 13 is a graph of osmolality (mOsm / kg) over time for perfusion cell culture runs performed in a 100 L bioreactor with predicted osmolality from Raman spectroscopy measurements. [Figure 11] FIG. 1 is a graph of viable cell density (VCD) (million cells / mL) versus osmolality (mOsm / kg) on ​​day 30 of a 60-day perfusion cell culture process in a 10-L bioreactor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] Provided herein is a method of perfusion culture of mammalian cells, the method comprising: providing a vessel containing mammalian cells disposed in a first liquid medium having an osmolality of about 270 mOsm / kg to about 380 mOsm / kg; incubating the mammalian cells at about 32° C. to about 39° C. for a period of time; and continuously or periodically during the period of time, removing a first volume of the first liquid medium and adding a second volume of a second liquid medium to the first liquid medium, wherein the first and second volumes are approximately equal, and wherein the osmolality of the first and second liquid media in the vessel is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period of time. Some embodiments of these methods further include continuously or periodically adding a third volume of an aqueous solution to the first and second liquid media in the container during the period, wherein the sum of the first volume, the second and third volumes is approximately equal, and the osmolarity of the first, second and third liquid media in the container is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period. Some embodiments of these methods further include continuously or periodically adding a third volume of a third liquid medium to the first liquid medium, wherein the sum of the first volume, the second and third volumes is approximately equal, and the osmolarity of the first, second and third liquid media in the container is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period. In some examples, the third liquid medium comprises sodium chloride (NaCl) or potassium chloride (KCl). In some examples, maintaining the osmolarity of the first, second, and third liquid medium in the container includes adjusting the flow rate of the third liquid medium during the period. In other examples, the osmolarity is controlled by the flow rate of the third liquid medium. In other examples, the third liquid medium can have an osmolarity of about 0 mOsm / kg to about 270 mOsm / kg. For example, if the second liquid medium has an osmolarity of more than 380 mOsm / kg, a third liquid medium having an osmolarity of about 0 mOsm / kg to about 100 mOsm / kg can be used. In some examples, maintaining the osmolarity in the first, second, and third liquid medium in the container includes adjusting the flow rate of the third liquid medium during the period.

[0070] Also provided herein is a method of producing a recombinant protein, the method comprising: providing a vessel containing mammalian cells, the cells comprising a nucleic acid encoding the recombinant protein, disposed in a first liquid medium having an osmolality of about 270 mOsm / kg to about 380 mOsm / kg; incubating the mammalian cells at about 32° C. to about 39° C. for a period of time; continuously or periodically during the period of time, removing a first volume of the first liquid medium and adding a second volume of a second liquid medium to the first liquid medium, the first and second volumes being approximately equal and wherein the osmolality of the first and second liquid medium in the vessel is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period of time; and recovering the recombinant protein from the mammalian cells or from the first liquid medium and / or the second liquid medium. Some embodiments of these methods further include continuously or periodically adding a third volume of an aqueous solution to the first and second liquid media in the container during the period, wherein the sum of the first volume, the second and third volumes is approximately equal, and the osmolarity of the first, second and third liquid media in the container is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period. Some embodiments of these methods further include continuously or periodically adding a third volume of a third liquid medium to the first liquid medium, wherein the sum of the first volume, the second and third volumes is approximately equal, and the osmolarity of the first, second and third liquid media in the container is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period. In some examples, the third liquid medium comprises sodium chloride (NaCl) or potassium chloride (KCl). In some examples, maintaining the osmolarity of the first, second, and third liquid medium in the container includes adjusting the flow rate of the third liquid medium during the period. In other examples, the third liquid medium can have an osmolarity of about 0 mOsm / kg to about 270 mOsm / kg. For example, if the second liquid medium has an osmolarity of more than 380 mOsm / kg, a third liquid medium having an osmolarity of about 0 mOsm / kg to about 100 mOsm / kg can be used.In some examples, maintaining the osmotic pressure in the first, second, and third liquid medium within the container includes adjusting the flow rate of the third liquid medium during the period.

[0071] In some embodiments, the methods provided herein comprise culturing 60×10 in the first and second liquid medium, or 60×10 in the first, second, and third liquid medium. 6 Cell cultures having viable cell densities of greater than 100000000 cells / mL can be achieved during that period. Methods for determining the viable cell density of a cell culture are well known in the art.

[0072] In some embodiments, the methods described herein provide cell cultures having a cell viability percentage that is greater than 75% (e.g., greater than 80%, greater than 85%, or greater than 90%) over a culture period of at least 5 days (e.g., at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 65 days, or at least 70 days).

[0073] Culture volume (10L-10,000L fed batch, 50-500L-2000L, 50L, 500L, 1000L, 2000L) Any of the methods described herein can include incubating, and may include incubating at a volume of between about 10 L and about 10,000 L (e.g., between about 10 L and about 8,000 L, between about 10 L and about 7,000 L, between about 10 L and about 6,000 L, between about 10 L and about 5,000 L, between about 10 L and about 4,000 L, between about 10 L and about 3,000 L, between about 10 L and about 2,500 L, between about 10 L and about 2,000 L, between about 10 L and about 1,500 L, between about 10 L and about 1,000 L, between about 10 L and about 500 L, between about 10 L and about 200 L, between about 10 L and about 100 between about 20L and about 10,000L, between about 20L and about 8,000L, between about 20L and about 7,000L, between about 20L and about 6,000L, between about 20L and about 5,000L, between about 20L and about 4,000L, between about 20L and about 3,000L, between about 20L and about 2,500L, between about 20L and about 2,000L, between about 20L and about 1,500L, between about 20L and about 1,000L, between about 20L and about 500L, between about 20L and about 200L, between about 20L and about 100L, between about 100L and about 10,000L, between about 100L and about between about 100L and about 8,000L, between about 100L and about 7,000L, between about 100L and about 6,000L, between about 100L and about 5,000L, between about 100L and about 4,000L, between about 100L and about 3,000L, between about 100L and about 2,000L, between about 100L and about 1,500L, between about 100L and about 1,000L, between about 100L and about 800L, between about 100L and about 700L, between about 100L and about 600L, between about 100L and about 500L, between about 100L and about 400L, between about 100L and about 300L, between about 100L and about 200L between about 500L and about 10,000L, between about 500L and about 8,000L, between about 500L and about 7,000L, between about 500L and about 6,000L, between about 500L and about 5,000L, between about 500L and about 4,000L, between about 500L and about 3,000L, between about 500L and about 2,000L, between about 500L and about 1,500L, between about 500L and about 1,000L, between about 500L and about 750L, between about 1,000L and about 10,000L, between about 1,000L and about 8,000L, between about 1,000L and about 7,000L,between about 1,000L and about 6,000L, between about 1,000L and about 5,000L, between about 1,000L and about 4,000L, between about 1,000L and about 3,000L, between about 1,000L and about 2,000L, between about 1,000L and about 1,500L, between about 2,000L and about 10,000L, between about 2,000L and about 8,000L, between about 2,000L and about 7,000L, between about 2,000L and about 6,000L, between about 2,000L and about Between about 5,000L, between about 2,000L and about 4,000L, between about 2,000L and about 3,000L, between about 3,000L and about 10,000L, between about 3,000L and about 8,000L, between about 3,000L and about 7,000L, between about 3,000L and about 6,000L, between about 3,000L and about 5,000L, between about 3,000L and about 4,000L, between about 4,000L and about 10,000L, between about 4,000L and about 8,0 between about 4,000L and about 7,000L, between about 4,000L and about 6,000L, between about 4,000L and about 5,000L, between about 5,000L and about 10,000L, between about 5,000L and about 8,000L, between about 5,000L and about 7,000L, between about 5,000L and about 6,000L, between about 6,000L and about 10,000L, between about 6,000L and about 8,000L, between about 6,000L and about 7,000L The method further comprises providing a vessel (e.g., any of the vessels described herein) containing mammalian cells (e.g., any of the mammalian cells described herein) in a first liquid medium having a volume (at the beginning of the period) of between about 7,000 L and about 10,000 L, between about 7,000 L and about 8,000 L, or between about 8,000 L and about 10,000 L, or 10 L, 20 L, 50 L, 100 L, 500 L, 1,000 L, or 2,000 L). The step of incubating the mammalian cells is performed under conditions that allow for the maintenance and growth of the cells.

[0074] The first liquid medium may have a stoichiometric concentration of about 270 mOsm / kg to about 320 mOsm / kg (e.g., about 270 mOsm / kg to about 315 mOsm / kg, about 270 mOsm / kg to about 310 mOsm / kg, about 270 mOsm / kg to about 305 mOsm / kg, about 270 mOsm / kg to about 300 mOsm / kg, about 270 mOsm / kg to about 295 mOsm / kg, about 270 mOsm / kg to about 290 mOsm / kg, about 270 mOsm / kg to about 285 mOsm / kg, about 270 mOsm / kg to about 280 mOsm / kg, about 270 mOsm / kg to about 290 mOsm / kg, from about 275 mOsm / kg, from about 275 mOsm / kg to about 320 mOsm / kg, from about 275 mOsm / kg to about 315 mOsm / kg, from about 275 mOsm / kg to about 310 mOsm / kg, from about 275 mOsm / kg to about 305 mOsm / kg, from about 275 mOsm / kg to about 300 mOsm / kg, from about 275 mOsm / kg to about 295 mOsm / kg, from about 275 mOsm / kg to about 290 mOsm / kg, from about 275 mOsm / kg to about 285 mOsm / kg, from about 275 mOsm / kg to about 280 mOsm / kg, from about 280 mOsm / kg to about 295 mOsm / kg, from about 275 mOsm / kg to about 290 mOsm / kg, from about 275 mOsm / kg to about 285 mOsm / kg, from about 275 mOsm / kg to about 280 mOsm / kg, sm / kg to about 320 mOsm / kg, about 280 mOsm / kg to about 315 mOsm / kg, about 280 mOsm / kg to about 310 mOsm / kg, about 280 mOsm / kg to about 305 mOsm / kg, about 280 mOsm / kg to about 300 mOsm / kg, about 280 mOsm / kg to about 295 mOsm / kg, about 280 mOsm / kg to about 290 mOsm / kg, about 280 mOsm / kg to about 285 mOsm / kg, about 285 mOsm / kg to about 320 mOsm / kg, about 285 mOsm / kg to about 315 mOsm / kg, about 28 5 mOsm / kg to about 310 mOsm / kg, about 285 mOsm / kg to about 305 mOsm / kg, about 285 mOsm / kg to about 300 mOsm / kg, about 285 mOsm / kg to about 295 mOsm / kg, about 285 mOsm / kg to about 290 mOsm / kg, about 290 mOsm / kg to about 320 mOsm / kg, about 290 mOsm / kg to about 315 mOsm / kg, about 290 mOsm / kg to about 310 mOsm / kg, about 290 mOsm / kg to about 305 mOsm / kg, about 290 mOsm / kg to about 300 mOsm / kg,About 290 mOsm / kg to about 295 mOsm / kg, about 295 mOsm / kg to about 320 mOsm / kg, about 295 mOsm / kg to about 315 mOsm / kg, about 295 mOsm / kg to about 310 mOsm / kg, about 295 mOsm / kg to about 305 mOsm / kg, about 295 mOsm / kg to about 300 mOsm / kg, about 300 mOsm / kg to about 320 mOsm / kg, about 300 mOsm / kg to about 315 ... The first liquid medium may have an osmolality of about 300 mOsm / kg to about 305 mOsm / kg, about 305 mOsm / kg to about 320 mOsm / kg, about 305 mOsm / kg to about 315 mOsm / kg, about 305 mOsm / kg to about 310 mOsm / kg, about 310 mOsm / kg to about 320 mOsm / kg, about 310 mOsm / kg to about 315 mOsm / kg, or about 315 mOsm / kg to about 320 mOsm / kg. Non-limiting embodiments and examples of the first liquid medium are described herein.

[0075] In some embodiments of any of the methods described herein, the second volume of the second liquid medium (e.g., any of the exemplary second liquid medium described herein) (and optionally the third liquid medium) added to the first liquid medium (e.g., any of the exemplary first liquid medium described herein) is increased by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 45%, at least 50%, at least 75%, at least 80%, at least 90%, at least 100%, at least 100%, at least 150%, at least 10 ... %, at least 100%, at least 200 at least 300%, at least 400%, or at least 500%, or from about 10% to about 500%, from about 10% to about 400%, from about 10% to about 300%, from about 10% to about 200%, from about 10% to about 100%, from about 10% to about 50%, from about 10% to about 20%, from about 20% to about 500, from about 20% to about 400%, from about 20% to about 300%, from about 20% to about 200%, from about 20% to about 100%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, from about 30% to about 500%, from about 30% to about 400%, about 30% to about 300%, about 30% to about 200%, about 30% to about 100%, about 30% to about 50%, about 30% to about 40%, about 40% to about 500%, about 40% to about 400%, about 40% to about 300%, about 40% to about 200%, about 40% to about 100%, about 40% to about 50%, about 50% to about 500%, about 50% to about 400%, about 50% to about 300%, about 50% to about 200%, about 50% to about 100%, about 50% to about 60%, about 60% to about 500%, about 60% to about 400%, about 60% to about 300%, about 60% to about 200% , about 60% to about 100%, about 60% to about 80%, about 60% to about 70%, about 70% to about 500%, about 70% to about 400%, about 70% to about 300%, about 70% to about 200%, about 70% to about 100%, about 70% to about 80%, about 80% to about 500%, about 80% to about 400%, about 80% to about 300%, about 80% to about 200%, about 80% to about 100%, about 80% to about 90%, about 90% to about 500%, about 90% to about 400%, about 90% to about 300%, about 90% to about 200%, about 90% to about 100%, about 100% to about 500%,about 100% to about 400%, about 100% to about 300%, about 100% to about 200%, about 100% to about 150%, about 200% to about 500%, about 200% to about 400%, about 200% to about 300%, about 200% to about 250%, about 300% to about 500%, about 300% to about 400%, about 400% to about 500%, or about 450% to about 500%).

[0076] In some embodiments of any of the methods described herein, the second volume of the second liquid medium (e.g., any of the exemplary second liquid medium described herein) that is added to the first liquid medium (e.g., any of the first liquid medium described herein) (and optionally to the third liquid medium) is increased by at least 10% (e.g., 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 80%, at least 90%, at least 100%, at least 100%, at least 15 ... an increase of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 200%, at least 220%, at least 240%, at least 260%, at least 280%, at least 300%, at least 320%, at least 340%, at least 360%, at least 380%, at least 400%, at least 420%, at least 440%, at least 460%, at least 480%, or at least 500%, or from about 10% to about 500% (or, for example, any subrange of this range described herein).

[0077] In some embodiments of any of the methods described herein, the flow rate of the second liquid medium (and optionally the third liquid medium) added to the first liquid medium is at least 10% (e.g., 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 90%, at least 100%, at least 15 ... at least 85%, at least 90%, at least 95%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 200%, at least 220%, at least 240%, at least 260%, at least 280%, or at least 300%, or from about 10% to about 300%, from about 10% to about 250%, from about 10% to about 200%, from about 10% to about 150%, from about 10% to about 100%, from about 10% to about 90%, from about 10% to about 80%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about from about 50%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 20%, from about 20% to about 300%, from about 20% to about 250%, from about 20% to about 200%, from about 20% to about 150%, from about 20% to about 100%, from about 20% to about 90%, from about 20% to about 80%, from about 20% to about 70%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, from about 30% to about 300%, from about 30% to about 250%, from about 30% to about 200%, from about 30% to about 150%, from about 30% to about 100%, from about 30% to about 90%, 0% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 300%, about 40% to about 250%, about 40% to about 200%, about 40% to about 150%, about 40% to about 100%, about 40% to about 90%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 300%, about 50% to about 250%, about 50% to about 200%, about 50% to about 150%, about 50% to about 100%, about 50% to about 90%, about 50% to about 80%,about 50% to about 70%, about 50% to about 60%, about 60% to about 300%, about 60% to about 250%, about 60% to about 200%, about 60% to about 150%, about 60% to about 100%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 300%, about 70% to about 250%, about 70% to about 200%, about 70% to about 150%, about 70% to about 100%, about 70% to about 90%, about 70% to about 80%, about 80% to about 300%, about 80% to about 250%, about 80% to about 200%, about 80% to about 150%, about 80% to about 100%, about 80% to about 90%, about 90% to about 300%, about 90% to about 250%, about 90% to about 200%, about 90% to about 150%, about 90% to about 100%, about 100% to about 300%, about 100% to about 250%, about 100% to about 200%, about 100% to about 150%, about 150% to about 300%, about 150% to about 250%, about 150% to about 200%, about 200% to about 300%, about 200% to about 250%, or about 250% to about 300%).

[0078] In some embodiments of any of the methods described herein, the flow rate of the second liquid medium (and optionally the third liquid medium) added to the first liquid medium is at least 10% (e.g., at least 12%, at least 14%, at least 15%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 25%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99%, at least at least 35%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 45%, at least 46%, at least 48%, at least 50%, at least 52%, at least 54%, at least 55%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 65%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82% %, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or from about 1% to about 100%, from about 1% to about 90%, from about 1% to about 80%, from about 1% to about 70%, from about 1% to about 60%, from about 1% to about 50%, from about 1% to about 45%, from about 1% to about 40%, from about 1% to about 35%, from about 1% to about 30%, from about 1% to about 25%, from about 1% to about 20%, from about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 100%, about 5% to about 90%, about 5% to about 80%, about 5% to about 70%, about 5% to about 60%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%,about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 100%, about 15% to about 90%, about 15% to about 80%, about 15% to about 70%, about 15% to about 60%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 100%, about 25% to about 90%, about 25% to about 80%, about 25% to about 70%, about 25% to about 60%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 100%, about 35% to about 90%, about 35% to about 80%, about 35% to about 70%, about 35% to about 60%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 100%, about 40% to about 90%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 40% to about 45%, about 45% to about 100%, The concentration of the second liquid medium is reduced by about 45% to about 90%, about 45% to about 80%, about 45% to about 70%, about 45% to about 60%, about 45% to about 50%, about 50% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 100%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 100%, about 70% to about 90%, about 70% to about 80%, about 80% to about 100%, about 80% to about 90%, or about 90% to about 100%. Non-limiting embodiments and examples of the second liquid medium are described herein.

[0079] Duration (30-60 days; 7-90 days, 45 days, 60 days) In some examples of any of the methods described herein, the period of time is between about 5 days and about 100 days (e.g., between about 5 days and about 95 days, between about 5 days and about 90 days, between about 5 days and about 85 days, between about 5 days and about 80 days, between about 5 days and about 75 days, between about 5 days and about 70 days, between about 5 days and about 65 days, between about 5 days and about 60 days, between about 5 days and about 55 days, between about 5 days and about 50 days, between about 5 days and about 45 days, between about 5 days and about 40 days, between about 5 days and about 35 days, between about 5 days and about 30 days, between about between 5 days and about 25 days, between about 5 days and about 20 days, between about 5 days and about 15 days, between about 5 days and about 10 days, between about 5 days and about 7 days, between about 7 days and about 95 days, between about 7 days and about 90 days, between about 7 days and about 85 days, between about 7 days and about 80 days, between about 7 days and about 75 days, between about 7 days and about 70 days, between about 7 days and about 65 days, between about 7 days and about 60 days, between about 7 days and about 55 days, between about 7 days and about 50 days, between about 7 days and about 45 days, between about 7 days and about 40 days, between about 7 days and about 35 days, between about 7 days and about 30 days, between about 7 days and about 25 days, between about 7 days and about 20 days, between about 7 days and about 15 days, between about 7 days and about 10 days, between about 10 days and about 100 days, between about 10 days and about 95 days, between about 10 days and about 90 days, between about 10 days and about 85 days, between about 10 days and about 80 days, between about 10 days and about 75 days, between about 10 days and about 70 days, between about 10 days and about 65 days, between about 10 days and about 60 days, between about 10 days and about 55 days, between about 10 days and about 50 days, between about 10 days and about 4 between 5 days, between about 10 days and about 40 days, between about 10 days and about 35 days, between about 10 days and about 30 days, between about 10 days and about 25 days, between about 10 days and about 20 days, between about 10 days and about 15 days, between about 15 days and about 100 days, between about 15 days and about 95 days, between about 15 days and about 90 days, between about 15 days and about 85 days, between about 15 days and about 80 days, between about 15 days and about 75 days, between about 15 days and about 70 days, between about 15 days and about 65 days, between about 15 days and about 60 days, between about 15 days and about 55 days,Between about 15 days and about 50 days, Between about 15 days and about 45 days, Between about 15 days and about 40 days, Between about 15 days and about 35 days, Between about 15 days and about 30 days, Between about 15 days and about 25 days, Between about 15 days and about 20 days, Between about 30 days and about 100 days, Between about 30 days and about 90 days, Between about 30 days and about 80 days, Between about 30 days and about 70 days, Between about 30 days and about 60 days, Between about 30 days and about 50 days, Between about 30 days and about 40 days, Between about 40 days and about 100 days, Between about 40 days and about 90 days, Between about 40 days and about 80 days, Between about 40 days and about 70 days, Between about 40 days and about 60 days, About 40 days between about 50 days, between about 50 days and about 100 days, between about 50 days and about 90 days, between about 50 days and about 80 days, between about 50 days and about 70 days, between about 50 days and about 60 days, between about 60 days and about 100 days, between about 60 days and about 90 days, between about 60 days and about 80 days, between about 60 days and about 70 days, between about 70 days and about 100 days, between about 70 days and about 90 days, between about 70 days and about 80 days, between about 80 days and about 100 days, between about 80 days and about 90 days, or between about 90 days and about 100 days, or 7 days, 21 days, 28 days, 30 days, 31 days, 45 days, 60 days, or 90 days.

[0080] mammalian cells The mammalian cells cultured (e.g., perfusion cultured) in the methods provided herein can be cells that grow in suspension or adherent cells. Non-limiting examples of mammalian cells that can be cultured in any of the methods described herein include: Chinese hamster ovary (CHO) cells (e.g., CHO DG44 cells or CHO-K1s cells), Sp2.0, myeloma cells (e.g., NS / 0), B cells, hybridoma cells, T cells, human embryonic kidney (HEK) cells (e.g., HEK 293E and HEK 293F), African green monkey kidney epithelial cells (Vero) cells, NSO cells, baby hamster kidney (BHK) cells, PerC6 cells, Vero cells, HT-1080 cells, and Madin-Darby Canine (Cocker Spaniel) kidney epithelial cells (MDCK) cells. In some examples where adherent cells are cultured, the culture can also include a plurality of microcarriers (e.g., microcarriers that contain one or more pores). Additional mammalian cells that can be cultured by any of the methods described herein are known in the art.

[0081] The mammalian cell can include a recombinant nucleic acid (e.g., a nucleic acid stably integrated into the genome of the mammalian cell) encoding a recombinant protein (e.g., any of the exemplary recombinant proteins described herein). Non-limiting examples of recombinant nucleic acids encoding exemplary recombinant proteins are described below, as well as recombinant proteins that can be produced using the methods described herein. In some examples, the mammalian cells cultured in a bioreactor (e.g., any of the bioreactors described herein) are derived from a larger scale culture.

[0082] Nucleic acids encoding recombinant proteins can be introduced into mammalian cells using a variety of methods known in molecular biology and molecular genetics. Non-limiting examples include transfection (e.g., lipofection), transduction (e.g., lentiviral, adenoviral, or retroviral infection), and electroporation. In some examples, the nucleic acids encoding recombinant proteins are not stably integrated into the chromosomes of the mammalian cells (transient transfection), while in other examples, the nucleic acids are integrated. Alternatively or additionally, the nucleic acids encoding recombinant proteins can be present in a plasmid and / or in a mammalian artificial chromosome (e.g., a human artificial chromosome). Alternatively or additionally, the nucleic acids can be introduced into the cells using a viral vector (e.g., a lentiviral, retroviral, or adenoviral vector). The nucleic acids are operably linked to a promoter sequence (e.g., a strong promoter, e.g., a β-actin promoter and a CMV promoter, or an inducible promoter). The vectors containing the nucleic acids can also optionally include a selectable marker (e.g., a gene that confers hygromycin, puromycin, or neomycin resistance to mammalian cells).

[0083] In some examples, the recombinant protein is a secreted protein, released by the mammalian cell into the extracellular medium (e.g., the first and / or second liquid medium in a perfusion culture, or the first and / or feed liquid medium in a fed-batch culture). For example, a nucleic acid sequence encoding a soluble recombinant protein may include a sequence encoding a secretory signal peptide at the N- or C-terminus of the recombinant protein that is cleaved by an enzyme present in the mammalian cell and subsequently released into the extracellular medium (e.g., the first and / or second liquid medium).

[0084] Vessels (10L~10,000L fed batch, 50L~500L~2,000L, 50L, 500L, 1,000L, 2,000L) The culturing step in any of the methods described herein can be carried out using a vessel, e.g., a bioreactor (e.g., a perfusion bioreactor). The bioreactor (e.g., a perfusion bioreactor) can be between about 10 L and about 10,000 L (e.g., between about 10 L and about 8,000 L, between about 10 L and about 7,000 L, between about 10 L and about 6,000 L, between about 10 L and about 5,000 L, between about 10 L and about 4,000 L, between about 10 L and about 3,000 L, between about 10 L and about 2,500 L, between about 10 L and about 2,000 L, between about 10 L and about 1,500 L, between about 10 L and about 1,000 L, between about 10 L and about 500 L, between about 10 L and about 200 L). between about 10L and about 100L, between about 20L and about 10,000L, between about 20L and about 8,000L, between about 20L and about 7,000L, between about 20L and about 6,000L, between about 20L and about 5,000L, between about 20L and about 4,000L, between about 20L and about 3,000L, between about 20L and about 2,500L, between about 20L and about 2,000L, between about 20L and about 1,500L, between about 20L and about 1,000L, between about 20L and about 500L, between about 20L and about 200L, between about 20L and about 100L between about 100L and about 10,000L, between about 100L and about 8,000L, between about 100L and about 7,000L, between about 100L and about 6,000L, between about 100L and about 5,000L, between about 100L and about 4,000L, between about 100L and about 3,000L, between about 100L and about 2,000L, between about 100L and about 1,500L, between about 100L and about 1,000L, between about 100L and about 800L, between about 100L and about 700L, between about 100L and about 600L, between about 100L and about 500L, between about 100L and about 400L, between about 100L and about 300L, between about 100L and about 200L, between about 500L and about 10,000L, between about 500L and about 8,000L, between about 500L and about 7,000L, between about 500L and about 6,000L, between about 500L and about 5,000L, between about 500L and about 4,000L, between about 500L and about 3,000L, between about 500L and about 2,000L, between about 500L and about 1,500L, between about 500L and about 1,000L, between about 500L and about 750L, between about 1,between about 1,000L and about 10,000L, between about 1,000L and about 8,000L, between about 1,000L and about 7,000L, between about 1,000L and about 6,000L, between about 1,000L and about 5,000L, between about 1,000L and about 4,000L, between about 1,000L and about 3,000L, between about 1,000L and about 2,000L, between about 1,000L and about 1,500L, between about 2,000L and about 10,000L, between about 2,00 between about 0L and about 8,000L, between about 2,000L and about 7,000L, between about 2,000L and about 6,000L, between about 2,000L and about 5,000L, between about 2,000L and about 4,000L, between about 2,000L and about 3,000L, between about 3,000L and about 10,000L, between about 3,000L and about 8,000L, between about 3,000L and about 7,000L, between about 3,000L and about 6,000L, between about 3,000L and Between about 5,000L, between about 3,000L and about 4,000L, between about 4,000L and about 10,000L, between about 4,000L and about 8,000L, between about 4,000L and about 7,000L, between about 4,000L and about 6,000L, between about 4,000L and about 5,000L, between about 5,000L and about 10,000L, between about 5,000L and about 8,000L, between about 5,000L and about 7,000L, between about 5,000L and about 6 The internal volume (capacity) may be between about 1,000 L, between about 6,000 L and about 10,000 L, between about 6,000 L and about 8,000 L, between about 6,000 L and about 7,000 L, between about 7,000 L and about 10,000 L, between about 7,000 L and about 8,000 L, or between about 8,000 L and about 10,000 L, or about 10 L, about 20 L, about 50 L, about 100 L, about 500 L, about 1,000 L, or about 2,000 L.

[0085] Culture System Some of the methods described herein use a system (e.g., a perfusion culture system) that includes a vessel and a first liquid medium disposed within the vessel (e.g., any of the media described herein). The vessel can have an internal volume (capacity) of between about 10 L and about 25,000 L (or, for example, any subrange of this range described herein). The vessel can be made of metal (e.g., stainless steel), plastic, or glass, or any combination thereof.

[0086] The system may include an apparatus (e.g., an impeller and a motor to rotate the impeller) for agitating the liquid medium disposed within the vessel. The vessel may include one or more ports (and optionally a pump) that allow for the addition of substances (e.g., liquid medium, poloxamer-188, base or acid (to adjust pH, as appropriate)) to the liquid medium disposed within the vessel, and / or the removal of the liquid medium (e.g., clarified liquid medium or a sample of a cell culture).

[0087] The culture system may also include one or more sensors for monitoring pH, dO2, temperature, and pCO2 in the liquid medium during the culture period. In some embodiments, the culture system may include one or more sensors, including capacitance, conductivity, and / or Raman sensors. In some embodiments, the bioreactor may include one or more sensors, including capacitance, conductivity, and / or Raman sensors. In some embodiments, the filter permeation may include one or more sensors, including capacitance, conductivity, and / or Raman sensors. The culture system may include a filtration device (e.g., an instrument performing alternating tangential filtration or tangential flow filtration) that processes a volume of cell culture to provide a clarified liquid medium that is substantially free of cells. The culture system may include a heating / cooling device that allows for adjustment of the temperature of the liquid medium disposed in the vessel. Additional configurations of cell culture systems are well known in the art.

[0088] perfusion culture In some examples, perfusion culture involves removing a first volume of a first liquid medium from a vessel (e.g., a bioreactor) for a period of time and adding a second volume of a second liquid medium to the bioreactor, wherein the first volume and the second volume are approximately equal.

[0089] In other examples, the perfusion culture includes removing a first volume of a first liquid medium from a vessel (e.g., a bioreactor) and adding a second volume of a second liquid medium and a third volume of a third liquid medium to the vessel, wherein the first volume and the sum of the second and third volumes are approximately equal.

[0090] The mammalian cells are retained within the bioreactor by some cell retention device or by techniques such as cell sedimentation in settling cones.

[0091] Removal and addition of medium in perfusion culture can be performed simultaneously or sequentially, or some combination of the two. Additionally, removal and addition can be performed sequentially, e.g., between 0.1% and 400%, between about 25% and 400%, between about 25% and about 300%, between about 25% and about 200%, between about 25% and about 100%, between about 25% and about 50%, between about 50% and about 400%, between about 50% and about 100%, between about 50% and about 200%, between about 50% and about 300%, This can be done by removing and replacing between about 50% and about 400%, between about 100% and about 400%, between about 100% and about 300%, between about 100% and about 200% of the volume, or about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, or about 400% of the volume of the bioreactor.

[0092] The first volume of the first liquid medium removed and the second volume of the second liquid medium added (and optionally the third liquid medium added) are, in some examples, each kept about the same over a 24 hour period. As is known in the art, the rate (volume / unit time) of removing the first volume of the first liquid medium and the rate (volume / unit time) of adding the second volume of the second liquid medium (and optionally the rate (volume / unit time) of adding the third volume of the third liquid medium) can vary and depend on the conditions of the particular cell culture system.

[0093] The rate (volume / unit time) at which the first volume of the first liquid medium is removed and the rate (volume / unit time) at which the second volume of the second liquid medium is added (and optionally the rate (volume / unit time) at which the third volume of the third liquid medium is added) may be approximately the same or may be different.

[0094] Alternatively, the volumes removed and added may be varied by gradually increasing each over a 24-hour period. For example, the volume of the first liquid medium removed and the volume of the second liquid medium added (and optionally the volume of the third liquid medium added) within each 24-hour period may be increased over the culture period. The volume may be increased over the culture period from a volume that is between 0.5% and about 20% of the bioreactor volume. The volume may be increased over the culture period from about 25% to about 150% of the bioreactor volume or cell culture volume at the start of the culture period.

[0095] In some examples of the methods described herein, after the first 48 to 96 hours of the culture period, during each 24 hour period (within the culture period), the first volume of the first liquid medium removed and the second volume of the second liquid medium added (and optionally, the third volume of the third liquid medium added) is about 10% to about 95%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 85% to about 95%, about 60% to about 80%, or about 70% of the cell culture volume at the start of the culture period.

[0096] Those skilled in the art will understand that the first liquid medium and the second liquid medium may be the same type of medium. In other examples, the first liquid medium and the second liquid medium may be different types of liquid medium or different concentrations of liquid medium. The second liquid medium may be more concentrated with respect to one or more medium components compared to the first liquid medium and the third liquid medium.

[0097] The first volume of the first liquid medium can be removed by using any automated system. For example, alternating tangential flow filtration can be used. Alternatively, the first volume of the first liquid medium can be removed by percolation or gravity flow of the first volume of the first liquid medium through a sterile membrane with a molecular weight cutoff that excludes mammalian cells. Alternatively, the first volume of the first liquid medium can be removed by stopping or significantly reducing the rate of agitation for a period of at least 1 minute, at least 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, or 1 hour, and removing or aspirating the first volume of the first liquid medium from the top of the bioreactor.

[0098] The second volume of the second liquid medium (and optionally the third volume of the third liquid medium) can be added to the first liquid medium by a pump. The second liquid medium (and optionally the third volume of the third liquid medium) can be added to the first liquid medium by pipetting or injecting the second volume of the second liquid medium (and optionally the third volume of the third liquid medium) directly into the first liquid medium, or in an automated manner.

[0099] Some embodiments of the methods described herein include incubating the container with agitation at a temperature between about 32° C. and about 39° C. for a period of at least about 7 days; and continuously or periodically during the period, removing a first volume of a first liquid medium and adding a second volume of a second liquid medium to the first liquid medium, wherein the first and second volumes are approximately equal and wherein an osmolality of the first and second liquid media in the container is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period.

[0100] Some embodiments of the methods described herein include incubating the container with agitation at a temperature between about 32° C. and about 39° C. for a period of at least about 7 days; and continuously or periodically during that period removing a first volume of the first liquid medium and adding to the first liquid medium a second volume of the second liquid medium and a third volume of the third liquid medium, wherein the first volume and the sum of the second and third volumes are approximately equal and the osmolarity of the first, second, and third liquid media in the container is maintained at about 270 mOsm / kg to about 380 mOsm / kg over that period.

[0101] Some examples of perfusion culture include incubating a culture system containing a first liquid medium at a temperature between about 32° C. and about 39° C. with agitation for a culture period of at least about 7 days; continuously or periodically removing a first volume of the first liquid medium and adding a second volume of a second liquid medium to the first liquid medium, wherein the first and second volumes are approximately equal and wherein the osmolarity of the first and second liquid media in the vessel is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period.

[0102] Some examples of perfusion culture include incubating a culture system containing a first liquid medium at a temperature between about 32° C. and about 39° C. with agitation for a culture period of at least about 7 days; continuously or periodically removing a first volume of the first liquid medium, and adding to the first liquid medium a second volume of a second liquid medium and a third volume of a third liquid medium, wherein the first volume and the sum of the second and third volumes are approximately equal, and wherein the osmolarity of the first, second, and third liquid media in the vessel is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period.

[0103] In some embodiments, the first liquid medium has a concentration of from about 260 mOsm / kg to about 400 mOsm / kg (e.g., from about 260 mOsm / kg to about 380 mOsm / kg, from about 260 mOsm / kg to about 360 mOsm / kg, from about 260 mOsm / kg to about 350 mOsm / kg, from about 260 mOsm / kg to about 340 mOsm / kg, from about 260 mOsm / kg to about 320 mOsm / kg, from about 260 mOsm / kg to about 300 mOsm / kg, from about 280 mOsm / kg to about 400 mOsm / kg, from about 270 mOsm / kg to about 400 mOsm / kg, about 270 mOsm / kg to about 380 mOsm / kg, about 270 mOsm / kg to about 360 mOsm / kg, about 270 mOsm / kg to about 350 mOsm / kg, about 270 mOsm / kg to about 340 mOsm / kg, about 270 mOsm / kg to about 320 mOsm / kg, about 270 mOsm / kg to about 300 mOsm / kg, about 280 mOsm / kg to about 380 mOsm / kg, about 280 mOsm / kg to about 360 mOsm / kg, about 280 mOsm / kg to about 350 mOsm / kg, about 280 mOsm / kg to about 340 mOsm / kg, about 280 mOsm / kg to about 320 mOsm / kg, about 280 mOsm / kg to about 300 mOsm / kg, about 290 mOsm / kg to about 400 mOsm / kg, about 290 mOsm / kg to about 380 mOsm / kg, about 290 mOsm / kg to about 360 mOsm / kg, about 290 mOsm / kg to about 350 mOsm / kg, about 290 mOsm / kg to about 340 mOsm / kg, about 290 mOsm / kg to about 320 mOsm / kg, about 290 mOsm / kg to about 300 mOsm / kg, about 300 mOsm / kg to about 400 mOsm / kg, about 300 mOsm / kg to about 380 mOsm / kg, about 300 mOsm / kg to about 360 mOsm / kg, about 300 mOsm / kg to about 350 mOsm / kg, about 300 mOsm / kg to about 340 mOsm / kg, about 300 mOsm / kg to about 320 mOsm / kg, about 310 mOsm / kg to about 400 mOsm / kg, about 310 mOsm / kg to about 380 mOsm / kg, about 310 mOsm / kg to about 360 mOsm / kg, about 310 mOsm / kg to about 350 mOsm / kg, about 310 mOsm / kg to about 340 mOsm / kg,About 310 mOsm / kg to about 320 mOsm / kg, about 320 mOsm / kg to about 400 mOsm / kg, about 320 mOsm / kg to about 380 mOsm / kg, about 320 mOsm / kg to about 360 mOsm / kg, about 320 mOsm / kg to about 350 mOsm / kg, about 320 mOsm / kg to about 340 mOsm / kg, about 3 ...30 mOsm / kg to about 360 mOsm / kg, about 330 mOsm / kg to about 370 mOsm / kg, about 330 mOsm / kg to about 370 mOsm / kg, about 330 mOsm / kg to about 380 mOsm / kg, about 320 mOsm / kg to about 360 mOsm / kg, about 320 mOsm / kg to about 350 mOsm / kg, about 320 mOsm / kg to about 340 mOsm / kg, about 330 mOsm / kg to about 370 mOsm / kg, about 330 mOsm / kg to about 380 mOsm / kg, about 330 mOsm / kg to about 390 mOsm / kg, about 340 mOsm / kg to about 390 mOsm / kg, about 340 mOsm / kg to about 350 mOsm / kg, about 340 mO from about 330 mOsm / kg to about 400 mOsm / kg, from about 330 mOsm / kg to about 380 mOsm / kg, from about 330 mOsm / kg to about 360 mOsm / kg, from about 330 mOsm / kg to about 350 mOsm / kg, from about 330 mOsm / kg to about 340 mOsm / kg, from about 340 mOsm / kg to about 400 mOsm / kg, from about 340 mOsm / kg to about 380 mOsm / kg sm / kg, about 340 mOsm / kg to about 360 mOsm / kg, about 340 mOsm / kg to about 350 mOsm / kg, about 350 mOsm / kg to about 400 mOsm / kg, about 350 mOsm / kg to about 380 mOsm / kg, about 350 mOsm / kg to about 360 mOsm / kg, about 360 mOsm / kg to about 400 mOsm / kg, about 36 The osmolarity may be from 0 mOsm / kg to about 380 mOsm / kg, from about 370 mOsm / kg to about 400 mOsm / kg, from about 370 mOsm / kg to about 380 mOsm / kg, from about 380 mOsm / kg to about 400 mOsm / kg, from about 380 mOsm / kg to about 390 mOsm / kg, or from about 390 mOsm / kg to about 400 mOsm / kg.

[0104] In some embodiments, the first liquid medium may have an osmolality of about 260 mOsm / kg, about 270 mOsm / kg, about 280 mOsm / kg, about 290 mOsm / kg, about 300 mOsm / kg, about 310 mOsm / kg, about 320 mOsm / kg, about 330 mOsm / kg, about 340 mOsm / kg, about 350 mOsm / kg, about 360 mOsm / kg, about 380 mOsm / kg, about 390 mOsm / kg, or about 400 mOsm / kg.

[0105] In some embodiments, the second liquid medium has a pH of about 260 mOsm / kg to about 2,500 mOsm / kg (e.g., about 260 mOsm / kg to about 2,400 mOsm / kg, about 260 mOsm / kg to about 2,200 mOsm / kg, about 260 mOsm / kg to about 2,000 mOsm / kg, about 260 mOsm / kg to about 1,800 mOsm / kg, about 260 mOsm / kg to about 1,600 mOsm / kg, about 260 mOsm / kg to about 1,500 mOsm / kg, about 260 mOsm / kg to about 1,400 mOsm / kg, about 260 mOsm / kg to about 1,500 mOsm / kg, about 260 mOsm / kg to about 1,600 mOsm / kg, about 260 mOsm / kg to about 1,800 mOsm / kg, about 260 mOsm / kg to about 1,900 mOsm / kg, about 260 mOsm / kg to about 2,100 mOsm / kg, about 260 mOsm / kg to about 2,200 mOsm / kg, about 260 mOsm / kg to about 2,300 mOsm / kg, about 260 mOsm / kg to about 2,400 mOsm / kg, about 260 mOsm / kg to about 2,500 mOsm / kg, about 260 mOsm / kg to about 2,600 mOsm / kg, about 260 mOsm / kg to about 2,700 mOsm / kg, sm / kg to about 1,200 mOsm / kg, about 260 mOsm / kg to about 1,000 mOsm / kg, about 260 mOsm / kg to about 800 mOsm / kg, about 260 mOsm / kg to about 700 mOsm / kg, about 260 mOsm / kg to about 600 mOsm / kg, about 260 mOsm / kg to about 500 mOsm / kg, about 260 mOsm / kg to about 400 mOsm / kg, about 260 mOsm / kg to about 300 mOsm / kg, about 300 mOsm / kg to about 2,500 mOsm / kg, about 300 mOsm / kg to about 2,400 mOsm / kg, about 300mOsm / kg to about 2,200mOsm / kg, about 300mOsm / kg to about 2,000mOsm / kg, about 300mOsm / kg to about 1,800mOsm / kg, about 300mOsm / kg to about 1,600mOsm / kg, about 300mOsm / kg to about 1,500mOsm / kg, about 300mOsm / kg to about 1,400mOsm / kg, about 300mOsm / kg to about 1,200mOsm / kg, about 300mOsm / kg to about 1,000mOsm / kg, about 300mOsm / kg to about 800mOsm / kg, about 30 0 mOsm / kg to about 700 mOsm / kg, about 300 mOsm / kg to about 600 mOsm / kg, about 300 mOsm / kg to about 500 mOsm / kg, about 300 mOsm / kg to about 400 mOsm / kg, about 400 mOsm / kg to about 2,500 mOsm / kg, about 400 mOsm / kg to about 2,400 mOsm / kg, about 400 mOsm / kg to about 2,200 mOsm / kg, about 400 mOsm / kg to about 2,000 mOsm / kg, about 400 mOsm / kg to about 1,800 mOsm / kg, about 400 mOsm / kg to about 1,600mOsm / kg, about 400mOsm / kg to about 1,500mOsm / kg, about 400mOsm / kg to about 1,400mOsm / kg, about 400mOsm / kg to about 1,200mOsm / kg, about 400mOsm / kg to about 1,000mOsm / kg, about 400mOsm / kg to about 800mOsm / kg, about 400mOsm / kg to about 700mOsm / kg, about 400mOsm / kg to about 600mOsm / kg, about 500mOsm / kg to about 2,500mOsm / kg, about 500mOsm / kg to about 2,400mOsm / kg kg, about 500mOsm / kg to about 2,200mOsm / kg, about 500mOsm / kg to about 2,000mOsm / kg, about 500mOsm / kg to about 1,800mOsm / kg, about 500mOsm / kg to about 1,600mOsm / kg, about 500mOsm / kg to about 1,500mOsm / kg, about 500mOsm / kg to about 1,400mOsm / kg, about 500mOsm / kg to about 1,200mOsm / kg, about 500mOsm / kg to about 1,000mOsm / kg, about 500mOsm / kg to about 800mOsm / kg, 500mOsm / kg to about 700mOsm / kg, about 500mOsm / kg to about 600mOsm / kg, about 600mOsm / kg to about 2,500mOsm / kg, about 600mOsm / kg to about 2,400mOsm / kg, about 600mOsm / kg to about 2,200mOsm / kg, about 600mOsm / kg to about 2,000mOsm / kg, about 600mOsm / kg to about 1,800mOsm / kg, about 600mOsm / kg to about 1,600mOsm / kg, about 600mOsm / kg to about 1,500mOsm / kg, about 600mOsm / kg to about 1,500mOsm / kg, about mOsm / kg to about 1,400 mOsm / kg, about 600 mOsm / kg to about 1,200 mOsm / kg, about 600 mOsm / kg to about 1,000 mOsm / kg, about 600 mOsm / kg to about 800 mOsm / kg, about 600 mOsm / kg to about 700 mOsm / kg, about 800 mOsm / kg to about 2,500 mOsm / kg, about 800 mOsm / kg to about 2400 mOsm / kg, about 800 mOsm / kg to about 2200 mOsm / kg, about 800 mOsm / kg to about 2,000 mOsm / kg, about 800 mOsm / kg to about 1,800mOsm / kg, about 800mOsm / kg to about 1,600mOsm / kg, about 800mOsm / kg to about 1,500mOsm / kg, about 800mOsm / kg to about 1,400mOsm / kg, about 800mOsm / kg to about 1,200mOsm / kg, about 800mOsm / kg to about 1,000mOsm / kg, about 800mOsm / kg to about 2,400mOsm / kg, about 800mOsm / kg to about 2,200mOsm / kg, about 800mOsm / kg to about 2,000mOsm / kg, about 800mOsm / kg to about 1,800mOsm / kg, about 800mOsm / kg to about 1,600mOsm / kg, about 800mOsm / kg to about 1,500mOsm / kg, about 800mOsm / kg to about 1,400mOsm / kg, about 800mOsm / kg to about 1,200mOsm / kg, about 800mOsm / kg to about 1,000mOsm / kg, about 1,000mOsm / kg to about 2,500mOsm / kg, about 1,000mOsm / kg to about 2,400mOsm / kg, about 1,000mOsm / kg to about 2,200mOsm / kg, about 1,000mOsm / kg to about 2,000mOsm / kg m / kg, about 1,000mOsm / kg to about 1,800mOsm / kg, about 1,000mOsm / kg to about 1,600mOsm / kg, about 1,000mOsm / kg to about 1,500mOsm / kg, about 1,000mOsm / kg to about 1,400mOsm / kg, about 1,000mOsm / kg to about 1,200mOsm / kg, about 1,200mOsm / kg to about 2,500mOsm / kg, about 1,200mOsm / kg to about 2,400mOsm / kg, about 1,200mOsm / kg to about 2,200mOsm / kg, about 1,200mOsm / kg from about 1,200mOsm / kg to about 1,800mOsm / kg, from about 1,200mOsm / kg to about 1,600mOsm / kg, from about 1,200mOsm / kg to about 1,500mOsm / kg, from about 1,200mOsm / kg to about 1,400mOsm / kg, from about 1,400mOsm / kg to about 2,500mOsm / kg, from about 1,400mOsm / kg to about 2,400mOsm / kg, from about 1,400mOsm / kg to about 2,200mOsm / kg, from about 1,400mOsm / kg to about 2,000mOsm / kg,400mOsm / kg to about 1,800mOsm / kg, about 1,400mOsm / kg to about 1,600mOsm / kg, about 1,400mOsm / kg to about 1,500mOsm / kg, about 1,500mOsm / kg to about 2,500mOsm / kg, about 1,500mOsm / kg to about 2,400mOsm / kg, about 1,500mOsm / kg to about 2,200mOsm / kg g, about 1,500 mOsm / kg to about 2,000 mOsm / kg, about 1,500 mOsm / kg to about 1,800 mOsm / kg, about 1,500 mOsm / kg to about 1,600 mOsm / kg, about 1,600 mOsm / kg to about 2,500 mOsm / kg, about 1,600 mOsm / kg to about 2,400 mOsm / kg, about 1,600 mOsm / kg to about 2,200 mOsm / kg Osm / kg, about 1,600mOsm / kg to about 2,000mOsm / kg, about 1,800mOsm / kg to about 2,500mOsm / kg, about 1,800mOsm / kg to about 2,400mOsm / kg, about 1,800mOsm / kg to about 2,200mOsm / kg, about 1,800mOsm / kg to about 2,000mOsm / kg, about 2,000mOsm / kg to about 2 ,500 mOsm / kg, about 2,000 mOsm / kg to about 2,400 mOsm / kg, about 2,000 mOsm / kg to about 2,200 mOsm / kg, about 2,200 mOsm / kg to about 2,500 mOsm / kg, about 2,200 mOsm / kg to about 2,400 mOsm / kg, or about 2,400 mOsm / kg to about 2,500 mOsm / kg.

[0106] In some embodiments, the second liquid medium is at about 260 mOsm / kg, about 280 mOsm / kg, about 300 mOsm / kg, about 320 mOsm / kg, about 340 mOsm / kg, about 350 mOsm / kg, about 360 mOsm / kg, about 380 mOsm / kg, about 400 mOsm / kg, about 420 mOsm / kg, about 440 mOsm / kg, about 450 mOsm / kg, about 460 mOsm / kg, about 480 mOsm / kg, about 500 mOsm / kg, about 510 mOsm / kg, about 520 mOsm / kg, about 530 mOsm / kg, about 540 mOsm / kg, about 550 mOsm / kg, about 560 mOsm / kg, about 570 mOsm / kg, about 580 mOsm / kg, about 590 mOsm / kg, about 600 mOsm / kg, about 610 mOsm / kg, about 620 mOsm / kg, about 630 mOsm / kg, about 640 mOsm / kg, about 650 mOsm / kg, about 660 mOsm / kg, about 670 mOsm / kg, about 680 mOsm / kg, about 690 mOsm / kg, about 700 mOsm / kg, about 710 mOsm / kg, about 720 mOsm / kg, about 730 mOsm / kg, about 740 mOsm / kg, about 750 mOsm / kg, about 760 mOsm / kg, about 770 mOsm sm / kg, approx. 520mOsm / kg, approx. 540mOsm / kg, approx. 550mOsm / kg, approx. 560mOsm / kg, approx. 580mOsm / kg, approx. 600mOsm / kg, approx. 620mOsm / kg, approx. 640mOsm / kg, approximately 650mOsm / kg, approximately 660mOsm / kg, approximately 680mOsm / kg, approximately 700mOsm / kg, approximately 72mOsm / kg, approximately 740mOsm / kg, approximately 750mOsm / kg, approximately 760mOsm / kg , about 780mOsm / kg, about 800mOsm / kg, about 820mOsm / kg, about 840mOsm / kg, about 850mOsm / kg, about 860mOsm / kg, about 880mOsm / kg, about 900mOsm / kg, Approx. 920mOsm / kg, approx. 940mOsm / kg, approx. 950mOsm / kg, approx. 960mOsm / kg, approx. 980mOsm / kg, approx. 1000mOsm / kg, approx. 1,100mOsm / kg, approx. 1,200mOsm / In some embodiments, the osmolarity of the sera is about 1,300 mOsm / kg, about 1,400 mOsm / kg, about 1,500 mOsm / kg, about 1,600 mOsm / kg, about 1,700 mOsm / kg, about 1,800 mOsm / kg, about 1,900 mOsm / kg, about 2,000 mOsm / kg, about 2,100 mOsm / kg, about 2,200 mOsm / kg, about 2,300 mOsm / kg, about 2,400 mOsm / kg, or about 2,500 mOsm / kg.

[0107] Methods for determining the osmolality of liquid media are well known in the art. Osmolality is measured by freezing point depression using offline sampling. Commercially available osmometers, such as the OsmoTECH® pro multi-sample micro-osmometer, are also well known in the art. Continuous monitoring of osmolality can be performed by Raman spectroscopy. See, for example, Moretto et al., Am Pharma Rev., 14(3), 2011, and Whelan et al., Biotechnol.Prog., 28(5):1355-1362, September-October 2012.

[0108] Some embodiments of any of the methods described herein further include adding a third volume of an aqueous solution to the first and second liquid media in the container during the period, wherein the sum of the first volume, the second and the third volumes is approximately equal, and the osmolality of the first and second liquid media in the container and the aqueous solution is maintained at about 270 mOsm / kg to about 380 mOsm / kg (or, for example, any subrange of this range described herein) over the period. For example, the aqueous solution can be a salt solution, such as a sodium chloride solution and a potassium chloride solution. In some embodiments, the first liquid medium can be any of the first liquid media described herein, and the second liquid medium can be any of the second liquid media described herein.

[0109] Some embodiments of any of the methods described herein further include continuously or periodically adding a third volume of a third liquid medium to the first liquid medium, wherein the first volume and the sum of the second and third volumes are approximately equal, and the osmolarity of the first, second, and third liquid medium in the container is maintained at about 270 mOsm / kg to about 380 mOsm / kg (or, e.g., any subrange of this range) over the period. In some embodiments of these methods, the first liquid medium can be any of the exemplary first liquid mediums described herein, and the second liquid medium can be any of the exemplary second liquid mediums described herein. In some embodiments of these methods, maintaining the osmolarity in the first, second, and third liquid medium in the container includes adjusting a flow rate of the second liquid medium over the period. In some embodiments, the adjusting step includes increasing the flow rate of the second liquid medium at some point during the period. In some examples, the adjusting step includes decreasing the flow rate of the second liquid medium at some point during the period.

[0110] In some examples, the third liquid medium has a stoichiometric concentration of about 0 mOsm / kg to about 12,000 mOsm / kg (e.g., about 0 mOsm / kg to about 11,000 mOsm / kg, about 0 mOsm / kg to about 10,000 mOsm / kg, about 0 mOsm / kg to about 9,000 mOsm / kg, about 0 mOsm / kg to about 8,000 mOsm / kg, about 0 mOsm / kg to about 7,000 mOsm / kg, about 0 mOsm / kg to about 6,000 mOsm / kg, about 0 mOsm / kg to about 5,000 mOsm / kg, about 0 mOsm / kg to about 4,000 mOsm / kg). , about 0 mOsm / kg to about 3,000 mOsm / kg, about 0 mOsm / kg to about 2,000 mOsm / kg, about 0 mOsm / kg to about 1,000 mOsm / kg, about 0 mOsm / kg to about 800 mOsm / kg, about 0 mOsm / kg to about 600 mOsm / kg, about 0 mOsm / kg to about 500 mOsm / kg, about 0 mOsm / kg to about 400 mOsm / kg, about 0 mOsm / kg to about 350 mOsm / kg, about 0 mOsm / kg to about 300 mOsm / kg, about 0 mOsm / kg to about 250 mOsm / kg, about 0 mOsm / kg to about 2 00 mOsm / kg, about 0 mOsm / kg to about 150 mOsm / kg, about 0 mOsm / kg to about 100 mOsm / kg, about 0 mOsm / kg to about 80 mOsm / kg, about 0 mOsm / kg to about 60 mOsm / kg, about 0 mOsm / kg to about 40 mOsm / kg, about 0 mOsm / kg to about 20 mOsm / kg, about 0 mOsm / kg to about 10 mOsm / kg, about 0 mOsm / kg to about 5 mOsm / kg, about 0 mOsm / kg to about 1 mOsm / kg, about 1 mOsm / kg to about 12,000 mOsm / kg, about 1 mOsm / kg to about 11,0 00 mOsm / kg, about 1 mOsm / kg to about 10,000 mOsm / kg, about 1 mOsm / kg to about 9,000 mOsm / kg, about 1 mOsm / kg to about 8,000 mOsm / kg, about 1 mOsm / kg to about 7,000 mOsm / kg, about 1 mOsm / kg to about 6,000 mOsm / kg, about 1 mOsm / kg to about 5,000 mOsm / kg, about 1 mOsm / kg to about 4,000 mOsm / kg, about 1 mOsm / kg to about 3,000 mOsm / kg, about 1 mOsm / kg to about 2,000 mOsm / kg, about 1 mOsm / kg to about 1,000 mOsm / kg, about 1 mOsm / kg to about 800 mOsm / kg, about 1 mOsm / kg to about 600 mOsm / kg, about 1 mOsm / kg to about 500 mOsm / kg, about 1 mOsm / kg to about 400 mOsm / kg, about 1 mOsm / kg to about 350 mOsm / kg, about 1 mOsm / kg to about 300 mOsm / kg, about 1 mOsm / kg to about 250 mOsm / kg, about 1 mOsm / kg to about 200 mOsm / kg, about 1 mOsm / kg to about 150 mOsm / kg, about 1 mOsm / kg to about 100 mOsm / kg, about 1 mOsm / kg to about 80 mOsm / kg, about 1 mOsm / kg to about 60 mOsm / kg, about 1 mOsm / kg to about 40 mOsm / kg, about 1 mOsm / kg to about 20 mOsm / kg, about 1 mOsm / kg to about 10 mOsm / kg, about 1 mOsm / kg to about 5 mOsm / kg, about 5 mOsm / kg to about 12,000 mOsm / kg, about 5 mOsm / kg to about 11,000 mOsm / kg, about 5 mOsm / kg to about 10,000 mOsm / kg, about 5 mOsm / kg to about 9,000 mOsm / kg, From about 5mOsm / kg to about 8,000mOsm / kg, from about 5mOsm / kg to about 7,000mOsm / kg, from about 5mOsm / kg to about 6,000mOsm / kg, from about 5mOsm / kg to about 5,000mOsm / kg, from about 5mOsm / kg to about 4,000mOsm / kg, from about 5mOsm / kg to about 3,000mOsm / kg, from about 5mOsm / kg to about 2,000mOsm / kg, from about 5mOsm / kg to about 1,000mOsm / kg, from about 5mOsm / kg to about 800mOsm / kg, from about 5mOsm / kg to about 60 0 mOsm / kg, about 5 mOsm / kg to about 500 mOsm / kg, about 5 mOsm / kg to about 400 mOsm / kg, about 5 mOsm / kg to about 350 mOsm / kg, about 5 mOsm / kg to about 300 mOsm / kg, about 5 mOsm / kg to about 250 mOsm / kg, about 5 mOsm / kg to about 200 mOsm / kg, about 5 mOsm / kg to about 150 mOsm / kg, about 5 mOsm / kg to about 100 mOsm / kg, about 5 mOsm / kg to about 80 mOsm / kg, about 5 mOsm / kg to about 60 mOsm / kg, From about 5 mOsm / kg to about 40 mOsm / kg, from about 5 mOsm / kg to about 20 mOsm / kg, from about 5 mOsm / kg to about 10 mOsm / kg, from about 10 mOsm / kg to about 12,000 mOsm / kg, from about 10 mOsm / kg to about 11,000 mOsm / kg, from about 10 mOsm / kg to about 10,000 mOsm / kg, from about 10 mOsm / kg to about 9,000 mOsm / kg, from about 10 mOsm / kg to about 8,000 mOsm / kg, from about 10 mOsm / kg to about 7,000 mOsm / kg, from about 10 mOsm / kg to about 6,000 mOsm / kg, about 10mOsm / kg to about 5,000mOsm / kg, about 10mOsm / kg to about 4,000mOsm / kg, about 10mOsm / kg to about 3,000mOsm / kg, about 10mOsm / kg to about 2,000mOsm / kg, about 10mOsm / kg to about 1,000mOsm / kg, about 10mOsm / kg to about 800mOsm / kg, about 10mOsm / kg to about 600mOsm / kg, about 10mOsm / kg to about 500mOsm / kg, about 10mOsm / kg to about 400mOsm / kg, about 10mOsm / kg to about 350mOsm / kg, about 10mOsm / kg to about 5,000mOsm / kg, about 10mOsm / kg to about 6,000mOsm / kg, about 10mOsm / kg to about 7,000mOsm / kg, about 10mOsm / kg to about 8,000mOsm / kg, about 10mOsm / kg to about 9,000mOsm / kg, about 10mOsm / kg to about 10,000mOsm / kg, about 10mOsm / kg to about 10,000mOsm / kg, about 10mOsm / kg to about 20,000mOsm / kg, about 10mOsm / kg to about 30,000mOsm / kg, about 10mOsm / kg to about 40,000mOsm / kg, about 10mOsm / kg to about sm / kg, about 10 mOsm / kg to about 300 mOsm / kg, about 10 mOsm / kg to about 250 mOsm / kg, about 10 mOsm / kg to about 200 mOsm / kg, about 10 mOsm / kg to about 150 mOsm / kg, about 10 mOsm / kg to about 100 mOsm / kg, about 10 mOsm / kg to about 80 mOsm / kg, about 10 mOsm / kg to about 60 mOsm / kg, about 10 mOsm / kg to about 40 mOsm / kg, about 10 mOsm / kg to about 20 mOsm / kg, about 20 mOsm / kg to about 12,000 mOsm / kg, about 20 mOsm / kg to about 11,000 mOsm / kg, about 20 mOsm / kg to about 10,000 mOsm / kg, about 20 mOsm / kg to about 9,000 mOsm / kg, about 20 mOsm / kg to about 8,000 mOsm / kg, about 20 mOsm / kg to about 7,000 mOsm / kg, about 20 mOsm / kg to about 6,000 mOsm / kg, about 20 mOsm / kg to about 5,000 mOsm / kg, about 20 mOsm / kg to about 4,000 mOsm / kg, about 20 mOsm / kg to about 3,000 mOsm / kg, about 20 mOsm / kg to about 2,000 mOsm / kg, about 20 mOsm / kg to about 1,000 mOsm / kg, about 20 mOsm / kg to about 800 mOsm / kg, about 20 mOsm / kg to about 600 mOsm / kg, about 20 mOsm / kg to about 500 mOsm / kg, about 20 mOsm / kg to about 400 mOsm / kg, about 20 mOsm / kg to about 350 mOsm / kg, about 20 mOsm / kg to about 300 mOsm / kg, about 20 mOsm / kg to about 250 mOsm / kg, about 20 mOsm / kg to about 200 mOsm / kg, about 20 mOsm / kg to about 150 mOsm / kg sm / kg, about 20mOsm / kg to about 100mOsm / kg, about 20mOsm / kg to about 80mOsm / kg, about 20mOsm / kg to about 60mOsm / kg, about 20mOsm / kg to about 40mOsm / kg, about 40mOsm / kg to about 12,000mOsm / kg, about 40mOsm / kg to about 11,000mOsm / kg, about 40mOsm / kg to about 10,000mOsm / kg, about 40mOsm / kg to about 9,000mOsm / kg, about 40mOsm / kg to about 8,000mOsm / kg, about 40mOsm / kg to about 7,000mOsm / kg, 00mOsm / kg, about 40mOsm / kg to about 6,000mOsm / kg, about 40mOsm / kg to about 5,000mOsm / kg, about 40mOsm / kg to about 4,000mOsm / kg, about 40mOsm / kg to about 3,000mOsm / kg, about 40mOsm / kg to about 2,000mOsm / kg, about 40mOsm / kg to about 1,000mOsm / kg, about 40mOsm / kg to about 800mOsm / kg, about 40mOsm / kg to about 600mOsm / kg, about 40mOsm / kg to about 500mOsm / kg, about 40mOsm / kg to about 500mOsm / kg, about 40mOsm / kg to about 600mOsm / kg, about 40mOsm / kg to about 700mOsm / kg, about 40mOsm / kg to about 800mOsm / kg, about 40mOsm / kg to about 900mOsm / kg, about 40mOsm / kg to about 1000mOsm / kg, about 40mOsm / kg to about 15 ...2000mOsm / kg, about 40mOsm / kg to about 3000mOsm / kg, about 40mOsm / kg to about 2000mOsm / kg g to about 400 mOsm / kg, about 40 mOsm / kg to about 350 mOsm / kg, about 40 mOsm / kg to about 300 mOsm / kg, about 40 mOsm / kg to about 250 mOsm / kg, about 40 mOsm / kg to about 200 mOsm / kg, about 40 mOsm / kg to about 150 mOsm / kg, about 40 mOsm / kg to about 100 mOsm / kg, about 40 mOsm / kg to about 80 mOsm / kg, about 40 mOsm / kg to about 60 mOsm / kg, about 60 mOsm / kg to about 12,000 mOsm / kg, about 60 mOsm / kg to about 11,000mOsm / kg, about 60mOsm / kg to about 10,000mOsm / kg, about 60mOsm / kg to about 9,000mOsm / kg, about 60mOsm / kg to about 8,000mOsm / kg, about 60mOsm / kg to about 7,000mOsm / kg, about 60mOsm / kg to about 6,000mOsm / kg, about 60mOsm / kg to about 5,000mOsm / kg, about 60mOsm / kg to about about 4,000 mOsm / kg, about 60 mOsm / kg to about 3,000 mOsm / kg, about 60 mOsm / kg to about 2,000 mOsm / kg, about 60 mOsm / kg to about 1,000 mOsm / kg, about 60 mOsm / kg to about 800 mOsm / kg, about 60 mOsm / kg to about 600 mOsm / kg, about 60 mOsm / kg to about 500 mOsm / kg, about 60 mOsm / kg to about 400 mOsm / kg, about 60mOsm / kg to about 350mOsm / kg, about 60mOsm / kg to about 300mOsm / kg, about 60mOsm / kg to about 250mOsm / kg, about 60mOsm / kg to about 200mOsm / kg, about 60mOsm / kg to about 150mOsm / kg, about 60mOsm / kg to about 100mOsm / kg, about 60mOsm / kg to about 80mOsm / kg, about 80mOsm / kg to about 100mOsm / kg, about 60mOsm / kg to about 150mOsm / kg, about 60mOsm / kg to about 10 ... sm / kg to about 12,000 mOsm / kg, about 80 mOsm / kg to about 11,000 mOsm / kg, about 80 mOsm / kg to about 10,000 mOsm / kg, about 80 mOsm / kg to about 9,000 mOsm / kg, about 80 mOsm / kg to about 8,000 mOsm / kg, about 80 mOsm / kg to about 7,000 mOsm / kg, about 80 mOsm / kg to about 6,000 mOsm / kg, About 80mOsm / kg to about 5,000mOsm / kg, about 80mOsm / kg to about 4,000mOsm / kg, about 80mOsm / kg to about 3,000mOsm / kg, about 80mOsm / kg to about 2,000mOsm / kg, about 80mOsm / kg to about 1,000mOsm / kg, about 80mOsm / kg to about 800mOsm / kg, about 80mOsm / kg to about 600mOsm / kg, about 80mOsm / kg to about 500mOsm / kg, about 80mOsm / kg to about 400mOsm / kg, about 80mOsm / kg to about 350mOsm / kg g, about 80 mOsm / kg to about 300 mOsm / kg, about 80 mOsm / kg to about 250 mOsm / kg, about 80 mOsm / kg to about 200 mOsm / kg, about 80 mOsm / kg to about 150 mOsm / kg, about 80 mOsm / kg to about 100 mOsm / kg, about 100 mOsm / kg to about 12,000 mOsm / kg, about 100 mOsm / kg to about 11,000 mOsm / kg, about 100 mOsm / kg to about 10,000 mOsm / kg, about 100 mOsm / kg to about 9,000 mOsm / kg, about 100 mOsm / kg to about 8, 000mOsm / kg, about 100mOsm / kg to about 7,000mOsm / kg, about 100mOsm / kg to about 6,000mOsm / kg, about 100mOsm / kg to about 5,000mOsm / kg, about 100mOsm / kg to about 4,000mOsm / kg, about 100mOsm / kg to about 3,000mOsm / kg, about 100mOsm / kg to about 2,000mOsm / kg, about 100mOsm / kg to about 1,000mOsm / kg, about 100mOsm / kg to about 800mOsm / kg, about 100mOsm / kg to about 600mOsm / kg , about 100mOsm / kg to about 500mOsm / kg, about 100mOsm / kg to about 400mOsm / kg, about 100mOsm / kg to about 350mOsm / kg, about 100mOsm / kg to about 300mOsm / kg, about 100mOsm / kg to about 250mOsm / kg, about 100mOsm / kg to about 200mOsm / kg, about 100mOsm / kg to about 150mOsm / kg, about 150mOsm / kg to about 12,000mOsm / kg, about 150mOsm / kg to about 11,000mOsm / kg, about 150mOsm / kg to about 10,000 mOsm / kg, about 150 mOsm / kg to about 9,000 mOsm / kg, about 150 mOsm / kg to about 8,000 mOsm / kg, about 150 mOsm / kg to about 7,000 mOsm / kg, about 150 mOsm / kg to about 6,000 mOsm / kg, about 150 mOsm / kg to about 5,000 mOsm / kg, about 150 mOsm / kg to about 4,000 mOsm / kg, about 150 mOsm / kg to about 3,000 mOsm / kg, about 150 mOsm / kg to about 2,000 mOsm / kg, about 150 mOsm / kg to about 1,000 mOsm / kg, about 150mOsm / kg to about 800mOsm / kg, about 150mOsm / kg to about 600mOsm / kg, about 150mOsm / kg to about 500mOsm / kg, about 150mOsm / kg to about 400mOsm / kg, about 150mOsm / kg to about 350mOsm / kg, about 150mOsm / kg to about 300mOsm / kg, about 150mOsm / kg to about 250mOsm / kg, about 150mOsm / kg to about 200mOsm / kg, about 200mOsm / kg to about 12,000mOsm / kg, about 200mOsm / kg to about 11,0 00mOsm / kg, about 200mOsm / kg to about 10,000mOsm / kg, about 200mOsm / kg to about 9,000mOsm / kg, about 200mOsm / kg to about 8,000mOsm / kg, about 200mOsm / kg to about 7,000mOsm / kg, about 200mOsm / kg to about 6,000mOsm / kg, about 200mOsm / kg to about 5,000mOsm / kg, about 200mOsm / kg to about 4,000mOsm / kg, about 200mOsm / kg to about 3,000mOsm / kg, about 200mOsm / kg to about 2,000mOsm / kg, about 200mOsm / kg to about 1,000mOsm / kg, about 200mOsm / kg to about 800mOsm / kg, about 200mOsm / kg to about 600mOsm / kg, about 200mOsm / kg to about 500mOsm / kg, about 200mOsm / kg to about 400mOsm / kg, about 200mOsm / kg to about 350mOsm / kg, about 200mOsm / kg to about 300mOsm / kg, about 200mOsm / kg to about 250mOsm / kg, about 250mOsm / kg to about 12,000mOsm / kg, about 250mOsm / kg to about 11,000mOsm / kg, about 250mOsm / kg to about 10,000mOsm / kg, about 250mOsm / kg to about 9,000mOsm / kg, about 250mOsm / kg to about 8,000mOsm / kg, about 250mOsm / kg to about 7,000mOsm / kg, about 250mOsm / kg to about 6,000mOsm / kg, about 250mOsm / kg to about 5,000mOsm / kg, about 250mOsm / kg to about 4,000mOsm / kg, about 250mOsm / kg to about 3,000mOsm / kg, about 250mOsm / kg to about 2,00 0 mOsm / kg, about 250 mOsm / kg to about 1,000 mOsm / kg, about 250 mOsm / kg to about 800 mOsm / kg, about 250 mOsm / kg to about 600 mOsm / kg, about 250 mOsm / kg to about 500 mOsm / kg, about 250 mOsm / kg to about 400 mOsm / kg, about 250 mOsm / kg to about 350 mOsm / kg, about 250 mOsm / kg to about 300 mOsm / kg, about 300 mOsm / kg to about 12,000 mOsm / kg, about 300 mOsm / kg to about 11,000 mOsm / kg, about 300 mOsm / kg to about 10,000mOsm / kg, about 300mOsm / kg to about 9,000mOsm / kg, about 300mOsm / kg to about 8,000mOsm / kg, about 300mOsm / kg to about 7,000mOsm / kg, about 300mOsm / kg to about 6,000mOsm / kg, about 300mOsm / kg to about 5,000mOsm / kg, about 300mOsm / kg to about 4,000mOsm / kg, about 300mOsm / kg to about 3,000mOsm / kg, about 300mOsm / kg to about 2,000mOsm / kg, about 300mOsm / kg to about 5,000mOsm / kg, about 300mOsm / kg to about 6,000mOsm / kg, about 300mOsm / kg to about 7,000mOsm / kg, about 300mOsm / kg to about 8,000mOsm / kg, about 300mOsm / kg to about 9,000mOsm / kg, about 300mOsm / kg to about 10,000mOsm / kg, about 300mOsm / kg to about 11,000mOsm / kg, about 300mOsm / kg to about 12,000mOsm / kg, about 300mOsm / kg to about 13,000mOsm / kg, about 300mOsm / kg to about 14,000mOsm / kg, about 300mOsm / kg to about 15 sm / kg to about 1,000 mOsm / kg, about 300 mOsm / kg to about 800 mOsm / kg, about 300 mOsm / kg to about 600 mOsm / kg, about 300 mOsm / kg to about 500 mOsm / kg, about 300 mOsm / kg to about 400 mOsm / kg, about 300 mOsm / kg to about 350 mOsm / kg, about 350 mOsm / kg to about 12,000 mOsm / kg, about 350 mOsm / kg to about 11,000 mOsm / kg, about 350 mOsm / kg to about 10,000 mOsm / kg, about 350 mOsm / kg to about 9,000mOsm / kg, about 350mOsm / kg to about 8,000mOsm / kg, about 350mOsm / kg to about 7,000mOsm / kg, about 350mOsm / kg to about 6,000mOsm / kg, about 350mOsm / kg to about 5,000mOsm / kg, about 350mOsm / kg to about 4,000mOsm / kg, about 350mOsm / kg to about 3,000mOsm / kg, about 350mOsm / kg to about 2,000mOsm / kg, about 350mOsm / kg to about 1,000mOsm / kg, about 350mOsm / kg to about 5,000mOsm / kg, about 350mOsm / kg to about 6,000mOsm / kg, about 350mOsm / kg to about 7,000mOsm / kg, about 350mOsm / kg to about 8,000mOsm / kg, about 350mOsm / kg to about 9,000mOsm / kg, about 350mOsm / kg to about 10,000mOsm / kg, about 350mOsm / kg to about 15,000mOsm / kg, about 350mOsm / kg to about 20,000mOsm / kg, about 350mOsm / kg to about 35,000mOsm / kg, about 350mOsm / kg to about 4,000mOsm / kg, about 350mOsm / kg to about 5,000mOsm / kg, about g to about 800 mOsm / kg, about 350 mOsm / kg to about 600 mOsm / kg, about 350 mOsm / kg to about 500 mOsm / kg, about 350 mOsm / kg to about 400 mOsm / kg, about 400 mOsm / kg to about 12,000 mOsm / kg, about 400 mOsm / kg to about 11,000 mOsm / kg, about 400 mOsm / kg to about 10,000 mOsm / kg, about 400 mOsm / kg to about 9,000 mOsm / kg, about 400 mOsm / kg to about 8,000 mOsm / kg, about 400 mOsm / kg to about 10,000 mOsm / kg kg to about 7,000 mOsm / kg, about 400 mOsm / kg to about 6,000 mOsm / kg, about 400 mOsm / kg to about 5,000 mOsm / kg, about 400 mOsm / kg to about 4,000 mOsm / kg, about 400 mOsm / kg to about 3,000 mOsm / kg, about 400 mOsm / kg to about 2,000 mOsm / kg, about 400 mOsm / kg to about 1,000 mOsm / kg, about 400 mOsm / kg to about 800 mOsm / kg, about 400 mOsm / kg to about 6 ...5,000 mOsm / kg, about 400 mOsm / kg to about 6,000 mOsm / kg, about 400 mOsm mOsm / kg to about 500mOsm / kg, about 500mOsm / kg to about 12,000mOsm / kg, about 500mOsm / kg to about 11,000mOsm / kg, about 500mOsm / kg to about 10,000mOsm / kg, about 500mOsm / kg to about 9,000mOsm / kg, about 500mOsm / kg to about 8,000mOsm / kg, about 500mOsm / kg to about 7,000mOsm / kg, about 500mOsm / kg to about 6,000mOsm / kg, about 500mOsm / kg to about 5,000mOsm / kg, From about 500mOsm / kg to about 4,000mOsm / kg, from about 500mOsm / kg to about 3,000mOsm / kg, from about 500mOsm / kg to about 2,000mOsm / kg, from about 500mOsm / kg to about 1,000mOsm / kg, from about 500mOsm / kg to about 800mOsm / kg, from about 500mOsm / kg to about 600mOsm / kg, from about 600mOsm / kg to about 12,000mOsm / kg, from about 600mOsm / kg to about 11,000mOsm / kg, from about 600mOsm / kg to about 10,000mOsm / kg, from about 600mOsm / kg to about 15,000mOsm / kg, Osm / kg to about 9,000mOsm / kg, about 600mOsm / kg to about 8,000mOsm / kg, about 600mOsm / kg to about 7,000mOsm / kg, about 600mOsm / kg to about 6,000mOsm / kg, about 600mOsm / kg to about 5,000mOsm / kg, about 600mOsm / kg to about 4,000mOsm / kg, about 600mOsm / kg to about 3,000mOsm / kg, about 600mOsm / kg to about 2,000mOsm / kg, about 600mOsm / kg to about 1,000mOsm / kg, about 600mOsm / kg to about 5,000mOsm / kg, about 600mOsm / kg to about 6,000mOsm / kg, about 600mOsm / kg to about 7,000mOsm / kg, about 600mOsm / kg to about 8,000mOsm / kg, about 600mOsm / kg to about 9,000mOsm / kg, about 600mOsm / kg to about 10,000mOsm / kg, about 600mOsm / kg to about 11,000mOsm / kg, about 600mOsm / kg to about 12,000mOsm / kg, about 600mOsm / kg to about 13,000mOsm / kg, about 600mOsm / kg to about 14,000mOsm / kg, about 600mOsm / kg to about 15,0 kg to about 800mOsm / kg, about 800mOsm / kg to about 12,000mOsm / kg, about 800mOsm / kg to about 11,000mOsm / kg, about 800mOsm / kg to about 10,000mOsm / kg, about 800mOsm / kg to about 9,000mOsm / kg, about 800mOsm / kg to about 8,000mOsm / kg, about 800mOsm / kg to about 7,000mOsm / kg, about 800mOsm / kg to about 6,000mOsm / kg, about 800mOsm / kg to about 5,000mOsm / kg, about 800mOsm / kg to about to about 4,000 mOsm / kg, about 800 mOsm / kg to about 3,000 mOsm / kg, about 800 mOsm / kg to about 2,000 mOsm / kg, about 800 mOsm / kg to about 1,000 mOsm / kg, about 1,000 mOsm / kg to about 12,000 mOsm / kg, about 1,000 mOsm / kg to about 11,000 mOsm / kg, about 1,000 mOsm / kg to about 10,000 mOsm / kg, about 1,000 mOsm / kg to about 9,000 mOsm / kg, about 1,000 mOsm / kg to about 8,000 mOsm / kg, about 1,000mOsm / kg to about 7,000mOsm / kg, about 1,000mOsm / kg to about 6,000mOsm / kg, about 1,000mOsm / kg to about 5,000mOsm / kg, about 1,000mOsm / kg to about 4,000mOsm / kg, about 1,000mOsm / kg to about 3,000mOsm / kg, about 1,000mOsm / kg to about 2,000mOsm / kg, about 2,000mOsm / kg to about 12,000mOsm / kg, about 2,000mOsm / kg to about 11,000mOsm / kg, about 2,000mOsm / kg to about 10 ,000mOsm / kg, about 2,000mOsm / kg to about 9,000mOsm / kg, about 2,000mOsm / kg to about 8,000mOsm / kg, about 2,000mOsm / kg to about 7,000mOsm / kg, about 2,000mOsm / kg to about 6,000mOsm / kg, about 2,000mOsm / kg to about 5,000mOsm / kg, about 2,000mOsm / kg to about 4,000mOsm / kg, about 2,000mOsm / kg to about 3,000mOsm / kg, about 3,000mOsm / kg to about 12,000mOsm / kg, about 3,0 from about 3,000mOsm / kg to about 11,000mOsm / kg, from about 3,000mOsm / kg to about 10,000mOsm / kg, from about 3,000mOsm / kg to about 9,000mOsm / kg, from about 3,000mOsm / kg to about 8,000mOsm / kg, from about 3,000mOsm / kg to about 7,000mOsm / kg, from about 3,000mOsm / kg to about 6,000mOsm / kg, from about 3,000mOsm / kg to about 5,000mOsm / kg, from about 3,000mOsm / kg to about 4,000mOsm / kg, from about 4,000mOsm / kg to about 12, 000mOsm / kg, about 4,000mOsm / kg to about 11,000mOsm / kg, about 4,000mOsm / kg to about 10,000mOsm / kg, about 4,000mOsm / kg to about 9,000mOsm / kg, about 4,000mOsm / kg to about 8,000mOsm / kg, about 4,000mOsm / kg to about 7,000mOsm / kg, about 4,000mOsm / kg to about 6,000mOsm / kg, about 4,000mOsm / kg to about 5,000mOsm / kg, about 5,000mOsm / kg to about 12,000mOsm / kg, about 5,000mOsm / kg to about 11,000mOsm / kg, about 5,000mOsm / kg to about 10,000mOsm / kg, about 5,000mOsm / kg to about 9,000mOsm / kg, about 5,000mOsm / kg to about 8,000mOsm / kg, about 5,000mOsm / kg to about 7,000mOsm / kg, about 5,000mOsm / kg to about 5,000mOsm / kg, about 6,000mOsm / kg to about 12,000 mOsm / kg, about 6,000mOsm / kg to about 11,000mOsm / kg, about 6,000mOsm / kg to about 10,000mOsm / kg, about 6,000mOsm / kg to about 9,000mOsm / kg, about 6,000mOsm / kg to about 8,000mOsm / kg, about 6,000mOsm / kg to about 7,000mOsm / kg, about 7,000mOsm / kg to about 12,000mOsm / kg, about 7,000mOsm / kg to about 13,000mOsm / kg, about 7,000mOsm / kg to about 14,000mOsm / kg, about 7,000mOsm / kg to about 15,000mOsm / kg, about 7,000mOsm / kg to about 16,000mOsm / kg, about 7,000mOsm / kg to about 17,000mOsm / kg, about 7,000mOsm / kg to about 18,000mOsm / kg, about 7,000mOsm / kg to about 19,000mOsm / kg, about 7,000mOsm / kg to about 21,000mOsm / kg, about 7,000mOsm / kg to about 22,000mOsm / kg, about 7,000mOsm / kg to about 23,000mOsm / kg, about 7,000mOsm / kg to about 24,000mOsm / kg, about 7,000mOsm / kg to kg to about 11,000 mOsm / kg, about 7,000 mOsm / kg to about 10,000 mOsm / kg, about 7,000 mOsm / kg to about 9,000 mOsm / kg, about 7,000 mOsm / kg to about 8,000 mOsm / kg, about 8,000 mOsm / kg to about 12,000 mOsm / kg, about 8,000 mOsm / kg to about 11,000 mOsm / kg, about 8,000 mOsm / kg to about 10,000 mOsm / kg g, about 8,000 mOsm / kg to about 9,000 mOsm / kg, about 9,000 mOsm / kg to about 12,000 mOsm / kg, about 9,000 mOsm / kg to about 11,000 mOsm / kg, about 9,000 mOsm / kg to about 10,000 mOsm / kg, about 10,000 mOsm / kg to about 12,000 mOsm / kg, or about 11,000 mOsm / kg to about 12,000 mOsm / kg). In some embodiments, the third liquid medium comprises sodium chloride (NaCl) or potassium chloride (KCl).

[0111] Some embodiments further include adjusting one or both of the pH and pCO2 of the first, second, and third liquid media in the container at some point during the period. In some examples, the method includes increasing the pCO2 and increasing the pH of the first, second, and third liquid media in the container over the period. In some examples, the pCO2 and pH of the first, second, and third liquid media are measured in the container over the period.

[0112] In some embodiments of any of the methods described herein, the osmolality of the first and / or second liquid medium is controlled by one or more of the following: the ratio of the first and / or second liquid medium to an aqueous solution (e.g., water), the ratio of the first and / or second liquid medium to a concentrated osmolyte (e.g., sodium chloride), the viable cell density in the vessel (e.g., any of the vessels (e.g., bioreactors) described herein), the pH of the first and / or second liquid medium, the level of dissolved CO2 (dCO2) in the vessel (e.g., any of the vessels (e.g., bioreactors) described herein), the carbon (e.g., glucose) dosage to the vessel (e.g., any of the vessels (e.g., bioreactors) described herein).

[0113] Pluronic® F-68 (Poloxamer-188) Poloxamer-188 is a molecule known in the art. Poloxamer-188 is a non-ionic synthetic block copolymer of ethylene oxide and propylene oxide. Poloxamer-188 has an average molecular weight between 7680 and about 9510, and about 81.8% by weight (1.9% by weight) of its composition corresponds to oxyethylene. For example, poloxamer-188 has the structure shown in the following formula I, where a is 80 and b is 27. Poloxamer-188 has the CAS number 9003-11-6.

[0114] [ka]

[0115] Poloxamer-188 is commercially available from many different vendors including, for example, Sigma-Aldrich (St. Louis, MO), Mediatech, Inc. (Manassas, VA), MAST Therapeutics, Inc. (San Diego, CA), EMD Millipore (Billerica, MA), BASF (Ludwigshafen, Germany), Pluronic® F-68 Life Technologies (Carlsbad, CA), and PhytoTechnology Laboratories, LLC (Shawnee Mission, KS).

[0116] In some embodiments, the second liquid medium may include a concentration of greater than about 8 g / L (e.g., a concentration of about or greater than 9.0 g / L, a concentration of about or greater than 10.0 g / L, a concentration of about or greater than 12.0 g / L, a concentration of about or greater than 14.0 g / L, or a concentration of about or greater than 16.0 g / L).

[0117] In some embodiments, the second liquid medium can include poloxamer-188 at a concentration of between about 8 g / L and about 16 g / L (e.g., between about 8.0 g / L and about 14.0 g / L, between about 8.0 g / L and about 12.0 g / L, between about 8.0 g / L and about 10.0 g / L, between about 10.0 g / L and about 16.0 g / L, between about 10.0 g / L and about 12.0 g / L, between about 12.0 g / L and about 16.0 g / L, between about 12.0 g / L and about 14.0 g / L, between about 14.0 g / L and about 16.0 g / L, or between about 15.0 g / L and about 16.0 g / L).

[0118] In some embodiments, the third liquid medium comprises poloxamer-188 at between about 0 g / L and about 8 g / L (e.g., between about 0 g / L and about 6 g / L, about 0 g / L to about 4 g / L, about 0 g / L to about 2 g / L, about 0 g / L to about 1 g / L, between about 0.5 g / L and about 8 g / L, between about 0.5 g / L and about 6.0 g / L, between about 0.5 g / L and about 4.0 g / L, between about 0.5 g / L and about 2.0 g / L, about 1.0 g / L to about 2.0 g / L, about 2.0 g / L to about 3.0 g / L, about 3.0 g / L to about 4.0 g / L, about 3.0 g / L to about 5.0 g / L, about 3.0 g / L to about 6.0 g / L, about 3.0 g / L to about 7.0 g / L, about 3.0 g / L to about 8.0 g / L, about 3.0 g / L to about 9.0 g / L, about 3.0 g / L to about 10 ... between about 8 g / L, between about 1.0 g / L and about 6.0 g / L, between about 1.0 g / L and about 4.0 g / L, between about 2.0 g / L and about 8 g / L, between about 2.0 g / L and about 6.0 g / L, between about 2.0 g / L and about 4.0 g / L, between about 5.0 g / L and about 8 g / L, between about 5.0 g / L and about 6.0 g / L, between about 6.0 g / L and about 8 g / L, or between about 7.0 g / L and about 8 g / L.

[0119] Some embodiments of any of the methods provided herein include increasing the poloxamer-188 concentration in the culture over time. In some embodiments, the medium includes a poloxamer-188 concentration selected based on one or more factors selected from the group consisting of: gas flow rate and viable cell density in the medium. In these methods, the selected poloxamer-188 concentration in the medium can be achieved by adding poloxamer-188 to the medium prior to the culturing step and / or adding poloxamer-188 to the medium during the culturing step. The selected poloxamer-188 concentration can be any of the exemplary concentrations or ranges of poloxamer-188 described herein.

[0120] Culture medium Liquid media are known in the art. The liquid media (e.g., the first, second, and / or third liquid media) can be supplemented with mammalian serum (e.g., fetal bovine serum and bovine serum), and / or growth hormones or growth factors (e.g., insulin, transferrin, and epidermal growth factor). Alternatively or additionally, the liquid media (e.g., the first, second, and / or third liquid media) can be chemically defined, animal-origin component-free, serum-free, or serum-containing liquid media. Non-limiting examples of chemically defined, animal-origin component-free, serum-free, and serum-containing liquid media are commercially available.

[0121] The liquid medium typically contains an energy source (e.g., a carbohydrate such as glucose), essential amino acids (e.g., the basic set of 20 amino acids and cysteine), low concentrations of required vitamins and / or other organic compounds, free fatty acids, and / or trace elements. The liquid medium (e.g., the first, second, and / or third liquid medium) can be optionally supplemented with, for example, mammalian hormones or growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts and buffers (e.g., calcium, magnesium, and phosphate), nucleosides and bases (e.g., adenosine, thymidine, and hypoxanthine), protein and tissue hydrolysates, and / or any combination of these additives.

[0122] A wide variety of liquid media are known in the art that can be used to culture cells (e.g., mammalian cells) in any of the methods described herein. Media components that may also be useful in the present process include, but are not limited to, CD (chemically-defined) hydrolysates, such as CD peptones, CD polypeptides (two or more amino acids), and CD growth factors. Additional examples of liquid media and media components are known in the art.

[0123] Those skilled in the art will appreciate that the first, second and third liquid media described herein may be the same type of medium or different media.

[0124] The liquid medium obtained from the cell culture can be filtered or clarified to obtain a liquid medium that is substantially free of cells and / or viruses. Methods for filtering or clarifying liquid medium to remove cells are known in the art (e.g., 0.2 μm filtration, and filtration using an alternating tangential flow (ATF™) system). Mammalian cells can also be removed from liquid medium by using centrifugation and taking the supernatant, which is the liquid medium that is substantially free of cells, or by allowing the cells to settle to the bottom under gravity of a vessel or bioreactor that contains the liquid medium, and taking the liquid medium that is separate from the settled recombinant cells (the liquid medium that is substantially free of cells).

[0125] In some embodiments of any of the methods described herein, the first, second, and / or third liquid medium has a pH of about 6.0 to about 6.8 (e.g., any subrange therein).

[0126] In some embodiments of any of the methods described herein, the first, second and / or third liquid medium comprises sodium chloride or potassium chloride. In some embodiments of any of the methods described herein, the first, second and / or third liquid medium further comprises one or more of the following: L-arginine, L-asparagine, L-glutamine, L-histidine, hydroxy-L-proline, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-valine, choline, inositol, biotin, calcium, folic acid, niacinamide, pyridoxine, riboflavin, thiamine, vitamin B12, lipoic acid, glutathione, linoleic acid, pyruvate, HEPES, ferrous iron, citric acid, zinc, copper, selenite, ethanolamine, putrescine, spermine, potassium, phosphate, magnesium, aluminum, vanadate, molybdate, manganese, nickel, silicate, and tin.

[0127] pH(6~8, 6.5~7.5) The methods described herein are carried out at a pH of about 6.8 to about 7.1.

[0128] The first, second, and / or third liquid medium can have a pH of about 6.8 to about 7.1 (e.g., about 6.8 to about 7.0, about 6.8 to about 6.9, about 6.9 to about 7.1, about 6.9 to about 7.0, or about 7.0 to about 7.2).

[0129] In some embodiments, the first, second, and / or third liquid medium may have a pH of about 6.8, about 6.9, about 7.0, or about 7.1.

[0130] Temperature (30 o C~40 o C;37 o C) The step of incubating the mammalian cells can be carried out at a temperature of about 32°C to about 39°C (e.g., about 32°C to about 38°C, about 32°C to about 36°C, about 32°C to about 35°C, about 32°C to about 34°C, about 34°C to about 39°C, about 34°C to about 38°C, about 34°C to about 36°C, about 35°C to about 39°C, about 35°C to about 38°C, about 35°C to about 37°C, about 36°C to about 39°C, about 36°C to about 38°C, or about 37°C to about 39°C).

[0131] In some embodiments, the step of incubating the mammalian cells occurs at a temperature of about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, or about 39°C.

[0132] One of skill in the art will appreciate that the temperature can be changed at certain times during the culturing process, for example, hourly or daily. For example, the temperature can be changed or shifted (e.g., increased or decreased) about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or about 20 days or more after the cells (e.g., mammalian cells) are initially seeded into the bioreactor. For example, the temperature can be shifted upward (e.g., by up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 degrees Celsius, or a change of up to about 10 degrees Celsius). For example, the temperature can be shifted downward (e.g., by up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 °C, or by up to or about a change of 20 °C).

[0133] CO2 (Control the partial pressure of dissolved CO2 in the range of 20-200mmHg; 80-140mmHg) The incubating step can include exposing the liquid medium in the bioreactor to an atmosphere containing at most or about 15% CO2 (e.g., at most or about 14% CO2, 12% CO2, 10% CO2, 8% CO2, 6% CO2, 5% CO2, 4% CO2, 3% CO2, 2% CO2, or at most or about 1% CO2).

[0134] In some embodiments of any of the methods described herein, the method includes increasing the pCO2 of the first and second liquid media in a vessel (e.g., any of the vessels described herein (e.g., a bioreactor)) by at least 10% (e.g., at least 12%, at least 14%, at least 15%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 25%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 35%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 45%, at least 46%, at least 48%, at least 50%, at least 52%, at least 54%, at least 55%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 65%, at least 70%, at least 75 ... %, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and increasing the pH of the first and second liquid media in the vessel (e.g., any of the vessels (e.g., bioreactors) described herein) by at least 8% (e.g., at least 10%, at least 12%, at least 14%, at least 15%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 25%, at least 26%, at least 28%, at least 30%, at least 32%).

[0135] In some embodiments of any of the methods described herein, the pCO2 and pH of the first, second, and / or third liquid medium in a vessel (e.g., any of the vessels (e.g., bioreactors) described herein) are adjusted based on the viable cell density over the period (e.g., at least 12%, at least 14%, at least 15%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 25%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 35%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least an increase of 45%, at least 46%, at least 48%, at least 50%, at least 52%, at least 54%, at least 55%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 65%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0136] Recombinant Proteins Non-limiting examples of recombinant proteins that can be produced by the methods provided herein include immunoglobulins (including light and heavy chain immunoglobulins, antibodies, or antibody fragments (e.g., any of the antibody fragments described herein)), enzymes (e.g., galactosidases (e.g., α-galactosidase), Myozyme, or Cerezyme), proteins (e.g., human erythropoietin, tumor necrosis factor (TNF), or interferon α or β), or immunogenic or antigenic proteins or protein fragments (e.g., proteins for use in vaccines). The recombinant protein can be an engineered antigen-binding polypeptide that includes at least one multifunctional recombinant protein scaffold (see, e.g., the recombinant antigen-binding proteins described in Gebauer et al., Current Opin. Chem. Biol. 13:245-255, 2009; and U.S. Patent Application Publication No. 2012 / 0164066, which is incorporated herein by reference in its entirety).

[0137] Non-limiting examples of recombinant proteins that are antibodies include: panitumumab, omalizumab, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, amatucumab, and the like. Tuximab, anatumomab, anrukinzumab, apolizumab, arcitumomab, atinumab, tocilizumab, basilizimab, bectumomab, belimumab, bevacizumab, besilesomab, bezlotoxumab, biciromab, canakinumab, certolizumab, cetuximab, cixutumumab, daclizumab, denosumab, densumab, enzumab, Crizumab, edrecolomab, efalizumab, efungumab, epratuzumab, ertumaxomab, etaracizumab, figitumumab, golimumab, ibritumomab tiuxetan, igovomab, imgatuzumab, infliximab, inolimomab, ino These include tuzumab, labetuzumab, lebrikizumab, moxetumomab, natalizumab, obinutuzumab, oregovomab, palivizumab, panitumumab, pertuzumab, ranibizumab, rituximab, tocilizumab, tositumomab, tralokinumab, tucotuzumab, trastuzumab, veltuzumab, zalutumumab, and zatuximab.

[0138] Additional examples of recombinant antibodies that can be produced by the methods described herein are known in the art. Additional non-limiting examples of recombinant proteins that can be produced by the methods of the present invention include: alglucosidase alfa, laronidase, abatacept, galsulfase, lutropin alfa, antihemophilic factor, agalsidase beta, interferon beta-1a, darbepoetin alfa, tenecteplase, etanercept, coagulation factor IX, follicle stimulating hormone, interferon beta-1a, imiglucerase, dornase alfa, epoetin alfa, insulin or insulin analogs, mecaselmi. Antithrombin III, factor VIII, factor VIIa, antithrombin III, protein C, human albumin, erythropoietin, granulocyte-colony stimulating factor, granulocyte-macrophage colony stimulating factor, interleukin-11, laronidase, idursuphase, galsulfase, alpha-1-protease inhibitor, lactase, adenosine deaminase, tissue plasminogen activator, thyrotropin alpha (e.g., Thyrogen®), and alteplase. Additional examples of recombinant proteins that can be produced by the methods of the invention include acid α-glucosidase, alglucosidase alpha (e.g., Myozyme® and Lumizyme®), α-L-iduronidase (e.g., Aldurazyme®), iduronate sulfatase, heparan N-sulfatase, galactose-6-sulfatase, acid β-galactosidase, β-glucoronidase, N-acetylglucosamine-1-phosphotransferase, and β-glucoronidase. Examples of enzymes that may be used include ceramidase, α-N-acetylgalactosaminidase, acid lipase, lysosomal acid ceramidase, acid sphingomyelinase, β-glucosidase (e.g., Cerezyme® and Ceredase®), galactosylceramidase, α-galactosidase-A (e.g., Fabrazyme®), acid β-galactosidase, β-galactosidase, neuraminidase, hexosaminidase A, and hexosaminidase B.

[0139] The secreted soluble recombinant protein may be recovered from the liquid medium (e.g., one or more of the first, second, and third liquid medium) by removing or otherwise physically separating the liquid medium from the cells (e.g., mammalian cells). A variety of different methods for removing the liquid medium from the cells (e.g., mammalian cells) are known in the art, including, for example, centrifugation, filtration, pipetting, and / or aspiration. The secreted recombinant protein may then be recovered from the liquid medium and further purified using a variety of biochemical techniques, including various types of chromatography (e.g., affinity chromatography, molecular sieve chromatography, cation exchange chromatography, or anion exchange chromatography) and / or filtration (e.g., molecular weight cut-off filtration).

[0140] Recombinant protein recovery Some embodiments of the methods described herein further include recovering the recombinant protein (e.g., any of the recombinant proteins described herein) from the mammalian cells and / or one or more of the first, second, and third liquid media. In some examples, the recovering includes lysing the mammalian cells. In other examples, the recovering can include collecting the recombinant protein from the media (e.g., one or more of the first, second, and third liquid media).

[0141] Collecting, for example, about 60 × 10 6 >cells / mL, 62×10 6 cells / mL, 64×10 6 cells / mL, 66×10 6 cells / mL, 68×10 6 cells / mL, 70×10 6 cells / mL, 72×10 6 cells / mL, 74×10 6 cells / mL, 76×10 6 cells / mL, 78×10 6 cells / mL, 80×10 6 cells / mL, approximately 82×10 6Cells / mL, about 84×10 6 Cells / mL, about 86×10 6 Cells / mL, about 88×10 6 Cells / mL, about 90×10 6 Cells / mL, about 92×10 6 Cells / mL, about 94×10 6 Cells / mL, about 96×10 6 Cells / mL, about 98×10 6 Cells / mL, about 100×10 6 Cells / mL, about 102×10 6 Cells / mL, about 104×10 6 Cells / mL, about 106×10 6 Cells / mL, about 108×10 6 Cells / mL, about 110×10 6 Cells / mL, about 112×10 6 Cells / mL, about 114×10 6 Cells / mL, about 116×10 6 Cells / mL, about 118×10 6 Cells / mL, about 120×10 6 Cells / mL, about 122×10 6 Cells / mL, about 124×10 6 Cells / mL, about 126×10 6 Cells / mL, about 128×10 6 Cells / mL, about 130×10 6 Cells / mL, about 132×10 6 Cells / mL, about 134×10 6 Cells / mL, about 136×10 6 Cells / mL, about 138×10 6 Cells / mL, about 140×10 6 Cells / mL, about 142×10 6 Cells / mL, about 144×10 6 Cells / mL, about 146×10 6 Cells / mL, about 148×10 6 Cells / mL, about 150×10 6 Cells / mL, about 152×10 6 Cells / mL, about 154×10 6 Cells / mL, about 156×10 6More than cells / mL, approximately 158×10 6 More than cells / mL, approximately 160×10 6 More than cells / mL, approximately 162×10 6 More than cells / mL, approximately 164×10 6 More than cells / mL, approximately 166×10 6 More than cells / mL, approximately 168×10 6 More than cells / mL, approximately 170×10 6 More than cells / mL, approximately 172×10 6 More than cells / mL, approximately 174×10 6 More than cells / mL, approximately 176×10 6 More than cells / mL, approximately 178×10 6 More than cells / mL, approximately 180×10 6 More than cells / mL, approximately 182×10 6 More than cells / mL, approximately 184×10 6 More than cells / mL, approximately 186×10 6 More than cells / mL, approximately 188×10 6 More than cells / mL, approximately 190×10 6 More than cells / mL, approximately 192×10 6 More than cells / mL, approximately 194×10 6 More than cells / mL, approximately 196×10 6 More than cells / mL, approximately 198×10 6 >200 x 10 cells / mL, or approximately 6 This can be done after culture to achieve a viable cell density of > 1000 cells / mL.

[0142] Recombinant Protein Formulation Some embodiments of any of the methods described herein further include formulating the recombinant protein (e.g., a recombinant protein produced by any of the methods described herein, e.g., a recovered recombinant protein or a recovered and purified recombinant protein) into a pharmaceutical composition. For example, formulating can include adding a pharma- ceutically acceptable excipient to the recombinant protein (e.g., a recombinant protein produced by any of the methods described herein, e.g., a recovered recombinant protein or a recovered and purified recombinant protein). Formulating can include mixing a pharma-ceutically acceptable excipient with the recombinant protein (e.g., a recombinant protein produced by any of the methods described herein, e.g., a recovered recombinant protein or a recovered and purified recombinant protein). Examples of pharma-ceutically acceptable excipients (e.g., non-natural pharma-ceutically acceptable excipients) are well known in the art. In some embodiments, the recombinant protein (e.g., a recombinant protein produced by any of the methods described herein, e.g., a recovered recombinant protein or a recovered and purified recombinant protein) is formulated for intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular administration.

[0143] Pharmaceutical Compositions and Kits Also provided herein are pharmaceutical compositions comprising at least one of any of the recombinant proteins described herein (e.g., produced by any of the methods described herein). The pharmaceutical compositions can be formulated in any manner known in the art. The pharmaceutical compositions are formulated to be compatible with the intended route of administration (e.g., intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular administration).

[0144] In some embodiments, the pharmaceutical composition may include a pharma- ceutically acceptable carrier (e.g., phosphate buffered saline).Single or multiple administrations of the formulation may be administered, for example, depending on the dosage and frequency required and tolerated by the subject.

[0145] Also provided herein are kits comprising a pharmaceutical composition (e.g., any of the pharmaceutical compositions described herein). In some embodiments, the kit comprises instructions for carrying out any of the methods described herein. In some embodiments, the kit may comprise at least one dose (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) of any of the pharmaceutical compositions described herein.

[0146] Treatment method Provided herein is a treatment of a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions described herein comprising at least one of any of the recombinant proteins described herein (produced by any of the methods described herein).

[0147] Dosing can be performed, for example, at least once (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, or at least 12 times) per week.

[0148] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Working Example EXAMPLES

[0149] Osmotic pressure control method Methods involving perfusion culture use many liquid media, which require a great deal of expense for the preparation of each batch. Most of the liquid media are water. However, a single liquid medium cannot be used for the entire high cell density perfusion process because the osmotic pressure drops significantly due to the consumption of nutrients when the cell density is high. For example, the consumption of 12 g / L of glucose reduces the osmotic pressure by 67 mOsm / kg.

[0150] Using a single liquid medium with a typical osmolality (290 mOsm / kg to 320 mOsm / kg) results in low osmolality when cell density is high. Low osmolality conditions lead to poor cell health and reduced productivity. However, using a single high osmolality liquid medium from the beginning of cell culture (e.g., perfusion cell culture) can result in a spike in lactate production and a decline in cell culture.

[0151] The second liquid medium (4x liquid medium formulation) described herein (added to the first liquid medium) may have a relatively high osmolality (compared to the feed liquid medium for fed-batch culture) and a pH below 7 to prevent precipitation. This second liquid medium may be stable at room temperature for less than one week even at pH 6 to pH 7. At least two alternatives can be used to increase the stability of the second liquid medium: 1) removing cysteine ​​and tyrosine, or 2) using alternative forms of cysteine ​​and tyrosine (e.g., pyruvate / cysteine ​​and glycine-tyrosine, respectively).

[0152] To control osmolality in perfusion cell cultures, a concentrated second liquid medium can be used and diluted with various amounts of a third, low osmolality liquid (e.g., water). Concentrated sodium chloride (NaCl) solution can also be optionally added if increased osmolality is required. Additionally, the dissolved CO2 concentration and pH of the culture can be varied to adjust osmolality. EXAMPLES

[0153] Effect of osmolarity on cell culture performance To determine the effect of osmolality on cell culture performance (e.g., viable cell density), mammalian cells were grown in liquid media having an osmolality between about 290 mOsm / kg and about 400 mOsm / kg. Figures 1A and 1B show the viable cell density over time for mammalian cells expressing protein 1 and protein 2, respectively. The data in Figures 1A and 1B show that a delay in growth was observed when mammalian cells were grown in liquid media having an osmolality above 380 mOsm / kg.

[0154] The average cell diameters at the tested osmolalities at days 4 and 5, respectively, are shown (Figures 2A and 2B). The data in Figures 2A and 2B show that the higher the osmolality, the larger the average cell size, indicating that osmolality affects cell morphology.

[0155] Figures 3A and 3B show the osmolality and cell culture growth of two bioreactors fed with concentrated cell media (osmolality of 1320 mOsm / kg) and water, respectively. The ratio of concentrated media feed to water feed was varied during the growth phase of the culture. Vessel 22 ("V22") had a lower water to media ratio than vessel 27 ("V27"), which resulted in higher osmolality of the culture starting at day 10 (Figure 3A). This resulted in a reduced growth rate in V22 compared to V27 (Figure 3B). Higher osmolality also resulted in reduced productivity (Figure 3C) and higher lactate dehydrogenase production, indicative of reduced culture health (Figure 3D). One response to higher osmolality was increased lactate production in V22 (Figure 3E), which then continued to drive lactate even higher ("lactate spike").

[0156] 4A and 4B show that the cultures exhibited high lactate production and consequently increased osmolality. When osmolality reached above 400 mOsm / kg, lactate production increased and cell proliferation and viability decreased. EXAMPLES

[0157] Osmolarity Control Options Osmolality thresholds for perfusion cultures for safe operation ranged from 360 to 420 mOsm / kg at the upper end. Osmolality can be controlled by various means, for example: 1) by feeding concentrated cell medium and adjusting water dilution rates in conjunction with feedback control; 2) by varying process parameters that affect the underlying addition method (e.g., high pH and pCO2 are required when osmolality is high); 3) by using at least two different liquid media within a bioreactor campaign, e.g., a growth liquid medium with low osmolality, and a production liquid medium with high osmolality; 4) by adding salts, e.g., NaCl and / or KCl, at high cell densities with feedback control to increase osmolality. Process changes to control osmolality can be made manually based on offline osmolality measurements of cell-free culture permeate. Alternatively, automated feedback control can be made based on online osmolality predictions. Online osmolality can be predicted from online capacitance, online conductivity, or Raman spectroscopy measurements, along with appropriate models.

[0158] Figures 5A and 5B showed the stepwise change in osmolality of the feed medium from the growth phase to the production phase. For each bioreactor, NaCl addition was manually turned on at a given time point, and then the NaCl flow was kept constant for the duration of the experiment. The data in Figures 5A and 5B showed different VCD profiles at different osmolality despite the same capacitance set point. This shows the limitations of using a constant NaCl feed rate to control osmolality.

[0159] 6A and 6B show that the correlation between osmolality and conductivity is good at high cell densities during the harvest phase when osmolality control is required.

[0160] Conductivity was measured with an ABER probe and the average of two measurements is plotted in Figure 7. To maintain the set point, 5 M NaCl was fed to the bioreactor based on conductivity feedback control. As shown in Figure 7, control began on day 5 and salt addition was not required early in the culture period. NaCl addition began around day 10. NaCl addition based on conductivity feedback control provided consistent conductivity and osmolality throughout the recovery phase of the culture. Additionally, NaCl addition based on conductivity feedback control helps reduce osmolality excursions during process upsets compared to processes using constant NaCl addition.

[0161] The data in Figures 8A and 8B show similar viable cell density profiles at the same capacitance set point for all four experimental runs in which the automated conductivity control resulted in similar osmolarity trends.

[0162] FIG. 9 shows that the correlation between conductivity and osmolality is good using two different probe types.

[0163] FIG. 10 shows that osmolarity can be accurately predicted using Raman spectroscopy, and that Raman probes can be placed in bioreactors or in cell-free perfusion harvesters.

[0164] As shown in Figure 11, it was observed that the viable cell density in the perfusion cell culture treatments tended to reach a minimum in the middle of the treatment. This occurred because the cell size increased when the biomass concentration was controlled to maintain a constant capacitance value, as measured with a capacitance sensor in the bioreactor. In the later stages of these cultures, the cell size decreased and the viable cell density increased.

[0165] By aggregating data from multiple batches, it was found that there was a correlation between osmolality and the minimum viable cell density reached in the culture, with higher osmolality correlating with lower cell density. This finding is illustrated in the figure, where the VCD at day 30 of the 60-day treatments is plotted against the culture osmolality at day 30. The ability to control osmolality to a desired level minimized the change in VCD between treatments, improving consistency within treatments.

[0166] Other embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to illustrate, but not to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method for perfusion culture of mammalian cells, comprising: providing a container containing mammalian cells disposed in a first liquid medium having an osmolality of about 270 mOsm / kg to about 380 mOsm / kg; incubating the mammalian cells at about 32° C. to about 39° C. for a period of time; and continuously or periodically during the period, removing a first volume of the first liquid medium and adding a second volume of the second liquid medium to the first liquid medium, wherein the first and second volumes are approximately equal and an osmolality of the first and second liquid media in the container is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period.

2. 10. The method of claim 1, wherein the first liquid medium has an osmolality of about 270 mOsm / kg to about 320 mOsm / kg.

3. 2. The method of claim 1, wherein the second liquid medium comprises greater than 8 g / L of poloxamer.

4. 4. The method of claim 3, wherein the poloxamer is Pluronic F68.

5. The method according to any one of claims 1 to 4, wherein the second liquid medium comprises sodium chloride.

6. The method according to any one of claims 1 to 4, wherein the second liquid medium has an osmolality of about 800 mOsm / kg to about 2,500 mOsm / kg.

7. 10. The method of claim 1, wherein maintaining the osmotic pressure of the first and second liquid medium in the container comprises adjusting the flow rate of the second liquid medium at a point during the period.

8. 8. The method of claim 7, wherein the adjusting step comprises increasing the flow rate of the second liquid medium at some point during the period.

9. The pH and pCO of the first and second liquid media in the container ２ 9. The method of claim 8, further comprising adjusting one or both of at some point during the period.

10. 10. The method of claim 9, wherein the adjusting step comprises decreasing the flow rate of the second liquid medium at some point during the period.

11. Over that period, the pCO ２ 10. The method of claim 1, further comprising increasing the pH and increasing the

12. pCO of the first and second liquid media in the container ２ and pH is increased over the period based on viable cell density.

13. 60×10 in the first and second liquid media ６ The method of any one of claims 1 to 4, wherein a viable cell density of more than cells / mL is achieved during said period.

14. The method of any one of claims 1 to 4, further comprising monitoring the osmotic pressure of the first and second liquid media in the containers during that period.

15. 2. The method of claim 1, further comprising periodically during the period, adding a third volume of an aqueous solution to the first and second liquid media in the containers, wherein the sum of the first volume, the second and third volumes is approximately equal, and wherein an osmolality of the first, second and third liquid media in the containers is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period.

16. The method of claim 15, wherein the aqueous solution is a salt solution.

17. 2. The method of claim 1, further comprising continuously or periodically adding a third volume of a third liquid medium to the first liquid medium, wherein the first volume and the sum of the second and third volumes are approximately equal, and wherein an osmolality of the first, second, and third liquid media in the container is maintained at about 270 mOsm / mg to about 380 mOsm / kg over the period.

18. 20. The method of claim 17, wherein the third liquid medium has an osmolality that is from about 270 mOsm / kg to about 380 mOsm / kg.

19. 20. The method of claim 17 or 18, wherein maintaining the osmotic pressure of the first, second, and third liquid medium in the container comprises adjusting the flow rate of the second liquid medium during that period.

20. 20. The method of claim 17, wherein the third liquid medium has an osmolality of about 10 mOsm / kg to about 270 mOsm / kg.

21. The pH and pCO of the first, second, and third liquid media in the container ２ 20. The method of claim 17, further comprising adjusting one or both of at some point during the period.

22. Over that period, the pCO ２ and increasing the pH.

23. pCO of the first, second, and third liquid media in the container ２ and pH is increased over the period based on viable cell density.

24. 60 x 10 in the first, second, and third liquid media ６ 20. The method of claim 17, wherein a viable cell density of greater than cells / mL is achieved during that period.

25. The method according to any one of claims 1 to 4, wherein the vessel is a bioreactor.

26. 1. A method for producing a recombinant protein comprising: providing a container containing mammalian cells, the cells including a nucleic acid encoding a recombinant protein, disposed in a first liquid medium having an osmolality of about 270 mOsm / kg to about 380 mOsm / kg; incubating the mammalian cells at about 32° C. to about 39° C. for a period of time; continuously or periodically during the period, removing a first volume of the first liquid medium and adding a second volume of the second liquid medium to the first liquid medium, wherein the first and second volumes are approximately equal and an osmolality of the first and second liquid media within the container is maintained at about 270 mOsm / kg to about 380 mOsm / kg over the period; and recovering the recombinant protein from the mammalian cells or from the first liquid medium and / or the second liquid medium.

27. 27. The method of claim 26, wherein the first liquid medium has an osmolality of about 270 mOsm / kg to about 320 mOsm / kg.

28. 28. The method of claim 26 or 27, wherein the second liquid medium has an osmolality of about 800 mOsm / kg to about 2,500 mOsm / kg.

29. 27. The method of claim 26, further comprising isolating the recombinant protein.

30. 30. The method of claim 29, further comprising formulating the isolated recombinant protein.

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