Method for culturing mammalian cells

Abstract

This application provides a method for culturing mammalian cells in a liquid culture medium containing poloxamer-188 at a concentration of 1.8 g / L or higher, or the liquid culture medium containing a poloxamer-188 concentration selected based on one or more factors selected from the group consisting of: pore size, pore type, gas flow rate, viable cell density in the culture medium, and markers related to cellular stress.

Description

[0001] This invention application is a divisional application of the invention patent application filed on December 21, 2015, with application number 201580076640.3 (international application number PCT / US2015 / 067040) and entitled "Method for culturing mammalian cells".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 095,734, filed December 22, 2014, the entire contents of which are incorporated herein by reference. Technical Field

[0004] This invention relates to biotechnology and methods for manufacturing recombinant proteins. Background Technology

[0005] Mammalian cells containing nucleic acids encoding recombinant proteins are frequently used to produce proteins that are therapeutically or commercially important. In today's environment of diversified product pipelines, biotechnology companies are increasingly driven to develop innovative solutions for the highly flexible and cost-effective production of therapeutic protein drug substances. Summary of the Invention

[0006] This invention is based, at least in part, on the following discovery: mammalian cells comprising nucleic acids encoding recombinant proteins are exposed to poloxamer-188 at concentrations of 1.8 g / L or greater (e.g., greater than 2.0 g / L, greater than 2.5 g / L, greater than 3.0 g / L, greater than 3.5 g / L, greater than 4.0 g / L, greater than 4.5 g / L, greater than 5.0 g / L, greater than 5.5 g / L, greater than 6.0 g / L, greater than 6.5 g / L, or greater than 7.0 g / L). Cells survive and proliferate in the culture medium of poloxamer-188. Optimal cell growth is achieved at a specific ratio of poloxamer-188 to an antifoaming agent. High cell density is achieved by increasing the concentration of poloxamer-188 in the cell culture as a function of viable cell density. The optimal concentration of poloxamer-188 added to the cell culture to promote cell proliferation depends on one or more factors from the following group: pore size, pore type, gas flow rate, viable cell density in the culture medium, and markers related to cell stress.

[0007] This application provides a method for culturing mammalian cells, comprising: perfusing mammalian cells containing nucleic acids encoding said recombinant proteins under conditions sufficient to produce recombinant proteins, wherein the perfusing culture includes culturing in a liquid medium containing poloxamer-188 at a concentration of 1.8 g / L or higher, and wherein said poloxamer-188 is present in the medium and / or added to the medium prior to the culture step and / or added to the medium during the culture step. In some embodiments of any method described in this application, the medium contains poloxamer-188 at a concentration of 1.8 g / L or higher (e.g., 2.8 g / L or higher, 4.0 g / L or higher, 6.0 g / L or higher, 8.0 g / L or higher, or 10.0 g / L or higher).

[0008] In some embodiments of any method described in this application, the culture medium further comprises an antifoaming agent and the ratio of the antifoaming agent (g / L) to poloxamer-188 (g / L) in the culture medium is about 0.5% to about 6.0% (e.g., about 1.0% to about 5.5%, or about 1.5% to about 5.0%) in the liquid culture medium. In some embodiments of any method described in this application, the method includes adding poloxamer-188 to the culture medium prior to the culture step and / or adding poloxamer-188 to the culture medium during the culture step to provide a concentration of 1.8 g / L or higher of poloxamer-188 in the culture medium. In some embodiments of any method provided in this application, poloxamer-188 is present in the culture medium. In some embodiments of any of the methods described in this application, the method includes adding poloxamer-188 to the culture medium prior to the culture step and / or adding poloxamer-188 to the culture medium during the culture step to provide poloxamer-188 in the culture medium at a concentration of 2.8 g / L or higher (e.g., a concentration of 4.0 g / L or higher, a concentration of 6.0 g / L or higher, a concentration of 8.0 g / L or higher, or a concentration of 10.0 g / L or higher).

[0009] In some embodiments of any method described in this application, the method includes increasing the concentration of poloxamer-188 in the culture medium over time. In some embodiments of any method described in this application, increasing the concentration of poloxamer-188 in the culture medium over time is based on the viable cell density in the culture medium. In some embodiments of any method described in this application, the method includes increasing the concentration of poloxamer-188 in the culture medium when the viable cell density is approximately 35 x 10⁻⁶. 6 Cells / mL to approximately 60 x 10⁻⁶ 6 At a cell / mL concentration, the concentration of poloxamer-188 in the culture medium is increased to greater than 1.8 g / L (e.g., about 1.8 g / L to about 3.0 g / L, or about 2.3 g / L to about 3.0 g / L). In some embodiments of any method described in this application, the method includes when the viable cell density in the culture medium is about 60 x 10⁻⁶ cells / mL. 6 Cells / mL to approximately 90 x 10⁻⁶ 6 At a cell / mL concentration, the concentration of poloxamer-188 in the culture medium is increased to greater than 3.0 g / L (e.g., about 3.0 g / L to about 6.0 g / L, or about 3.6 g / L to about 6.0 g / L). In some embodiments of any method described in this application, the method includes when the viable cell density in the culture medium is greater than 90 x 10⁻⁶ cells / mL. 6 At a cell / mL concentration, the concentration of poloxamer-188 in the culture medium is increased to greater than 6.0 g / L (e.g., greater than 7.0 g / L, greater than 8.0 g / L, or greater than 10.0 g / L). Some embodiments of any method described in this application include: when the viable cell density in the culture medium is about 35 x 10⁻⁶ cells / mL... 6 Cells / mL to approximately 60 x 10⁻⁶ 6 When the cell / mL ratio is reached, increase the concentration of poloxamer-188 in the culture medium to approximately 1.8 g / L to approximately 3.0 g / L; when the viable cell density in the culture medium is approximately 60 x 10⁻⁶ cells / mL. 6 Cells / mL to approximately 90 x 10⁻⁶ 6 When the cell / mL ratio is reached, the concentration of poloxamer-188 in the culture medium is increased to approximately 3.0 g / L to approximately 6.0 g / L; and when the viable cell density in the culture medium is greater than 90 x 10⁻⁶ cells / mL, the concentration of poloxamer-188 is increased to approximately 3.0 g / L to approximately 6.0 g / L; and when the viable cell density in the culture medium is greater than 90 x 6 When the cell / mL ratio is reached, the concentration of poloxamer-188 in the culture medium is increased to greater than 6.0 g / L. Some embodiments of any method described in this application also include determining the viable cell density in the culture medium at one or more time points during the culture process.

[0010] In some embodiments of any method described in this application, the perfusion culture comprises: providing a container containing mammalian cells disposed in a first liquid culture medium; incubating the container with stirring at a temperature of about 32°C to about 40°C for at least about 7 days; and continuously or periodically removing a first volume of the first liquid culture medium and adding a second volume of the second liquid culture medium to the first liquid culture medium after the first 48 to 96 hours of the culture period, wherein the first and second volumes are approximately equal. In some embodiments of any method described in this application, the second liquid culture medium contains poloxamer-188 at a concentration higher than that in the first liquid culture medium. In some embodiments of any method described in this application, the first liquid culture medium contains poloxamer-188 at a concentration of 1.8 g / L or less, and the second liquid culture medium contains poloxamer-188 at a concentration greater than 1.8 g / L (e.g., greater than 2.8 g / L, greater than 4.0 g / L, greater than 6.0 g / L, greater than 8.0 g / L, or greater than 10.0 g / L). In some embodiments of any of the methods described in this application, the method includes adding poloxamer-188 to a second liquid culture medium before or after adding a second volume of a second liquid culture medium to a first liquid culture medium to provide poloxamer-188 in the second culture medium at a concentration of 1.8 g / L or a concentration higher than 1.8 g / L (e.g., higher than 2.8 g / L, higher than 4.0 g / L, higher than 6.0 g / L, higher than 8.0 g / L, or higher than 10.0 g / L).

[0011] Some embodiments of any method described in this application further include collecting the recombinant protein from mammalian cells, from a first liquid culture medium, or from a second liquid culture medium, or any combination thereof. Some embodiments of any method described in this application further include formulating the collected recombinant protein into a pharmaceutical composition. In some embodiments of any method described in this application, the culture achieves a depth greater than 30 x 10⁻⁶. 6 Cells / mL (e.g., greater than 50 x 10⁻⁶) 6 Cells / mL, greater than 100 x 10 6 Cells / mL, greater than 150 x 10 6 Cells / mL, or greater than 200 x 10⁻⁶ 6 After collecting the live cells (cells / mL), the cells were determined.

[0012] This application also provides a method for culturing mammalian cells, comprising: providing a culture system comprising a container, a liquid culture medium disposed within the container, and a sprayer disposed within the container, the sprayer comprising a plurality of orifices configured to dispense gas through the sprayer into the liquid culture medium; and perfusing mammalian cells containing nucleic acids encoding the recombinant protein into the culture system under conditions and gas flow rates sufficient to produce a recombinant protein in the system, wherein the concentration of poloxamer-188 contained in the culture medium is selected based on one or more factors selected from the group consisting of: pore size, pore type, gas flow rate, viable cell density in the culture medium, and markers related to cellular stress. In some embodiments of any method described in this application, the selected poloxamer-188 concentration is achieved by adding poloxamer-188 to the culture medium prior to the culture step and / or adding poloxamer-188 to the culture medium during the culture step.

[0013] In some embodiments of any method described in this application, the factor is the pore type, and the pore type is a sintered pore or a drilled pore. In some embodiments of any method described in this application, the method includes selecting a concentration of poloxamer-188 of about 2.3 g / L to about 3.3 g / L based on the pore type and pore size when the pore type is a drilled pore and the pore diameter is about 750 μm to about 1.5 mm, or selecting a concentration of poloxamer-188 of about 2.5 g / L to about 3.1 g / L based on the pore type and pore size when the pore type is a drilled pore and the pore diameter is about 900 μm to about 1.1 mm. In some embodiments of any of the methods described in this application, the method includes selecting a concentration of poloxamer-188 of about 3.3 g / L to about 4.3 g / L based on the pore type and pore size when the pore type is a borehole and the pore size is about 250 μm to about 750 μm, or selecting a concentration of poloxamer-188 of about 3.5 g / L to about 4.1 g / L based on the pore type and pore size when the pore type is a borehole and the pore size is about 400 μm to about 600 μm. In some embodiments of any method described in this application, the method includes selecting poloxamer-188 at a concentration higher than 4.3 g / L (e.g., higher than 5.0 g / L, higher than 6.0 g / L, higher than 8.0 g / L, or higher than 10.0 g / L) based on the pore type and pore size when the pore type is a borehole and the pore diameter is from 1 μm to about 250 μm (e.g., from about 1 μm to about 200 μm, from about 160 μm to about 190 μm, or from about 170 μm to about 180 μm).

[0014] In some embodiments of any method described in this application, the method includes selecting a concentration of poloxamer-188 from about 1.8 g / L to about 3.3 g / L based on the pore type and pore size when the pore type is sintered and the pore size is greater than 150 μm. In some embodiments of any method described in this application, the method includes selecting a concentration of poloxamer-188 from about 3.3 g / L to about 4.3 g / L based on the pore type and pore size when the pore type is sintered and the pore size is from about 80 μm to about 150 μm, or selecting a concentration of poloxamer-188 from about 3.5 g / L to about 4.1 g / L based on the pore type and pore size when the pore type is sintered and the pore size is from about 90 μm to about 110 μm. In some embodiments of any of the methods described in this application, the method includes selecting poloxamer-188 at a concentration higher than 4.5 g / L (e.g., higher than 5.0 g / L, higher than 6.0 g / L, higher than 8.0 g / L, or higher than 10.0 g / L) based on the pore type and pore size when the pore type is sintered pore and the pore diameter is about 1 μm to about 80 μm.

[0015] In some embodiments of any method described in this application, the perfusion culture comprises: incubating a culture system containing a first liquid culture medium under stirring for at least about 7 days at a temperature of about 32°C to about 40°C; and after the first 48 to 96 hours of the culture period, continuously or periodically removing a first volume of the first liquid culture medium and adding a second volume of second liquid culture medium to the first liquid culture medium. In some embodiments of any method described in this application, the second liquid culture medium contains poloxamer-188 at a concentration higher than that in the first liquid culture medium. In some embodiments of any method described in this application, the second culture medium contains a selected concentration of poloxamer-188.

[0016] Some embodiments of any of the methods described in this application further include collecting the recombinant protein from inside mammalian cells, from a first liquid culture medium or a second liquid culture medium, or any combination thereof. Some embodiments of any of the methods described in this application also include formulating the collected recombinant protein into a pharmaceutical composition.

[0017] In some embodiments of any method described in this application, the container is a perfusion bioreactor. In some embodiments of any method described in this application, the perfusion bioreactor has a volume of about 1.5 L to about 25,000 L. In some embodiments of any method described in this application, the culture period is greater than 10 days. In some embodiments of any method described in this application, the removal of a first volume of first liquid culture medium and the addition of a second volume of second liquid culture medium in (c) are performed continuously. In some embodiments of any method described in this application, the removal of a first volume of first liquid culture medium and the addition of a second volume of second liquid culture medium in (c) are performed periodically. In some embodiments of any method described in this application, the removal of a first volume of first liquid culture medium and the addition of a second volume of second liquid culture medium in (c) are increased over time. In some embodiments of any method described in this application, the first liquid culture medium and / or the second liquid culture medium are selected from the group consisting of: chemically defined liquid culture media, serum-free liquid culture media, serum-containing liquid culture media, liquid culture media without animal-derived components, and protein-free culture media. In some embodiments of any of the methods described in this application, during the first 48 to 96 hours of the time period, in every 24-hour period, the first volume of the first liquid culture medium removed in (c) and the second volume of the second liquid culture medium added are about 0.3 to about 10 times the volume of the first liquid culture medium or the container volume.

[0018] In some embodiments of any method described in this application, the mammalian cells are Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, NSO cells, baby hamster kidney (BHK) cells, PerC6 cells, Vero cells, or the HT-1080 cell line. In some embodiments of any method described in this application, the recombinant protein is an immunoglobulin, enzyme, growth factor, protein fragment, or engineered protein.

[0019] As used in this application, the "a" preceding a noun represents one or more specific nouns. For example, the phrase "mammal cell" means "one or more mammalian cells".

[0020] The term "mammalian cell" means any cell that is derived from or originates from any mammal (e.g., human, hamster, mouse, green monkey, rat, pig, cow, or rabbit). For example, a 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 in this application. Other examples of mammalian cells are known in the art.

[0021] The term "culture" or "cell culture" refers to the maintenance or proliferation of mammalian cells under controlled physical conditions.

[0022] The terms “mammalian cell culture,” “culture,” or “cell culture” refer to a liquid culture medium containing a variety of mammalian cells that are maintained or proliferated under controlled physical conditions.

[0023] The term "liquid culture medium" or "liquid medium" refers to a fluid containing sufficient nutrients to allow cells (e.g., mammalian cells) to grow or proliferate in vitro. For example, a liquid culture medium may contain one or more of the following: amino acids (e.g., 20 amino acids), purines (e.g., hypoxanthine), pyrimidines (e.g., thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, nicotinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate. In some embodiments, the liquid culture medium may contain serum from mammals. In some embodiments, the liquid culture medium does not contain serum from mammals or other extracts (composition-defined liquid culture medium). In some embodiments, the liquid culture medium may contain trace metals, mammalian growth hormones, and / or mammalian growth factors. Another example of a liquid culture medium is a basic culture medium (e.g., a medium containing only inorganic salts, a carbon source, and water). Non-limiting examples of liquid culture media are described in this application. Other examples of liquid culture media are known in the art and are commercially available. Liquid culture media may contain mammalian cells of any density. For example, as used in this application, a volume of liquid culture media removed from a bioreactor may be substantially free of mammalian cells.

[0024] The term "liquid culture medium without animal-derived components" means a liquid culture medium that does not contain any components derived from mammals (e.g., proteins or serum).

[0025] The term "serum-free liquid culture medium" refers to a liquid culture medium that does not contain mammalian serum.

[0026] The term "serum-containing liquid culture medium" refers to a liquid culture medium containing mammalian serum.

[0027] The term "chemically defined liquid culture medium" is a term used in the art and refers to a liquid culture medium in which all chemical components are known. For example, a chemically defined liquid culture medium does not contain fetal bovine serum, bovine serum albumin, or human serum albumin, because these preparations typically contain a complex mixture of albumin and lipids.

[0028] The term "protein-free liquid culture medium" refers to a liquid culture medium that does not contain any protein (e.g., any detectable protein).

[0029] The term "stirring" means agitating or otherwise moving a portion of a liquid culture medium in a container. This (operation) is performed to, for example, increase the dissolved O2 concentration in the liquid culture medium in a bioreactor. Stirring can be performed using any method known in the art, such as tools or a propeller. Exemplary apparatus and methods for stirring portions of liquid culture medium in a bioreactor are known in the art.

[0030] The term "immunoglobulin" refers to a polypeptide containing an amino acid sequence (e.g., a variable domain sequence, a frame sequence, or a constant domain 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. Immunoglobulins may contain, for example, at least 15 amino acids of a light chain immunoglobulin, or at least 15 amino acids of a heavy chain immunoglobulin. Immunoglobulins may be isolated antibodies (e.g., IgG, IgE, IgD, IgA, or IgM). Immunoglobulins may be subclasses of IgG (e.g., IgG1, IgG2, IgG3, or IgG4). Immunoglobulins may be antibody fragments, such as Fab fragments, F(ab')2 fragments, or scFv fragments. Immunoglobulins may also be bispecific or trispecific antibodies, or dimer, trimer, or multimeric antibodies, or bifunctional antibodies (diabody), Affibody®, or Nanobody®. Immunoglobulins can also be engineered proteins containing at least one immunoglobulin domain (e.g., fusion proteins). Non-limiting examples of immunoglobulins described herein, and further examples of immunoglobulins are known in the art. Recombinant immunoglobulins can be produced using any of the methods described herein.

[0031] The terms "protein fragment" or "peptide fragment" refer to a portion of a polypeptide sequence having a length of at least or about 4 amino acids, at least or about 5 amino acids, at least or about 6 amino acids, at least or about 7 amino acids, at least or about 8 amino acids, at least or about 9 amino acids, at least or about 10 amino acids, at least or about 11 amino acids, at least or about 12 amino acids, at least or about 13 amino acids, at least or about 14 amino acids, at least or about 15 amino acids, at least or about 16 amino acids, at least or about 17 amino acids, at least or about 18 amino acids, at least or about 19 amino acids, or at least or about 20 amino acids, or a length of more than 20 amino acids. Recombinant protein fragments can be generated using any of the methods described in this application.

[0032] The term "engineered protein" refers to a polypeptide that is not encoded by endogenous nucleic acids naturally 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, trivalent, or bifunctional antibodies), and antigen-binding proteins containing at least one recombinant scaffolding sequence. Recombinant engineered proteins can be generated using any of the methods described in this application.

[0033] The terms "secreted protein" or "secreted recombinant protein" mean a protein (e.g., a recombinant protein) that, when translated in mammalian cells, originally contains at least one secretion signal sequence and is at least partially secreted into the extracellular space (e.g., liquid culture medium) by enzymatic cleavage of the secretion signal sequence in mammalian cells. Those skilled in the art will understand that a "secreted" protein does not need to be completely separated from the cell to be considered a secreted protein.

[0034] The term "perfusion bioreactor" refers to a bioreactor containing multiple cell types (e.g., mammalian cells) in a first liquid culture medium, wherein the cells present in the bioreactor are cultured by periodically or continuously removing the first liquid culture medium and simultaneously or subsequently adding a substantially equal volume of a second liquid culture medium to the bioreactor. In some instances, the volume of the first liquid culture medium removed and the volume added change incrementally (e.g., increase or decrease) over increasing time during the culture period (e.g., approximately 24 hours, approximately 1 minute to approximately 24 hours, or longer than 24 hours) (e.g., a daily liquid culture medium replenishment rate). The portion of the culture medium removed and replaced each day may vary depending on the specific cells being cultured, the initial inoculation density, and the cell density at a particular time. "RV" or "reactor volume" refers to the volume of liquid culture medium present at the start of the culture process (e.g., the total volume of liquid culture medium present after inoculation).

[0035] The term "feed-batch bioreactor" is a term in the art and refers to a bioreactor containing multiple cell types (e.g., mammalian cells) in a first liquid culture medium, wherein the cells present in the bioreactor are cultured by periodically or continuously adding a second liquid culture medium to the first liquid culture medium without significant or substantial removal of either the first or second liquid culture medium from the cell culture. The second liquid culture medium may be the same as the first liquid culture medium. In some examples of fed-batch culture, the second liquid culture medium is a concentrated form of the first liquid culture medium. In some examples of fed-batch culture, the second liquid culture medium is added as a dry powder.

[0036] The term "nebulizer" is a term in the art and refers to a device having an inlet, a body, and a surface having one or more orifices, wherein the inlet, body, and one or more orifices are designed to allow gas to flow through the inlet, through the body, and out of the one or more orifices. When a nebulizer is used for cell culture, one or more orifices comes into contact with the cell culture, causing gas to flow out of at least one orifice and into the cell culture. Nebulizers are often described by their orifice diameter and orifice type. Non-limiting examples of nebulizers having different combinations of orifice diameter and orifice type are described in this application.

[0037] The term "aperture" is a term used in the art and refers to the diameter of a single orifice in a sprayer having a single orifice or the average diameter of two or more orifices in a sprayer having two or more orifices.

[0038] The term "marker of cellular stress" is a term in the art and refers to a biomolecule that is released into a liquid culture medium by cells under physiological stress (e.g., cells undergoing necrotic or apoptotic cell death), or is produced or present at elevated levels in cells under physiological stress (e.g., cells undergoing necrotic or apoptotic cell death). Non-limiting examples of markers of cellular stress released by cells under physiological stress include proteases (e.g., activated caspase), lactate dehydrogenase, genomic DNA (e.g., nucleosome DNA), cytochrome c, and activated PARP. Non-limiting examples of markers of cellular stress produced or present at elevated levels in cells under physiological stress include activated caspase, cytochrome c, activated PARP, and externalized phosphatidylserine.

[0039] "Specific productivity rate" or "SPR" is a term in the art and, as used herein, refers to the mass of recombinant protein or enzyme activity produced per mammalian cell per day. The SPR of recombinant therapeutic antibodies is typically measured as mass / cell / day. The SPR of recombinant therapeutic enzymes is typically measured as units / cell / day or (units / mass) / cell / day.

[0040] "Volume productivity rate" or "VPR" is a term in the art and, as used herein, refers to the mass of recombinant protein or enzyme activity produced per volume of culture (e.g., per L of bioreactor, vessel, or tube volume) per day. The VPR of recombinant therapeutic antibodies is typically measured in mass / L / day. The VPR of recombinant therapeutic enzymes is typically measured in units / L / day or (units / mass) / L / day.

[0041] The phrases “poloxamer-188 concentration of...” and “poloxamer-188 concentration” are used interchangeably in this application. The phrases “poloxamer-188 at or above X g / L” and “poloxamer-188 concentration of X g / L or higher” are used interchangeably in this application.

[0042] Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The methods and materials described in this application are used in this invention; other suitable methods and materials known in the art may also be used. The materials, methods, and examples described are merely illustrative and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned in this application are incorporated herein by reference in their entirety. In case of conflict, the specification containing the definitions shall prevail.

[0043] Other features and advantages of the invention will be apparent from the following detailed description and drawings, as well as from the claims.

[0044] Specifically, the present invention includes, but is not limited to, the following:

[0045] 1. A method for culturing mammalian cells, the method comprising:

[0046] Mammalian cells containing nucleic acids encoding the recombinant protein were perfused and cultured under conditions sufficient to produce the recombinant protein.

[0047] The perfusion culture comprises culturing in a liquid medium containing poloxamer-188 at a concentration of 1.8 g / L or higher, wherein the poloxamer-188 is present in the medium and / or added to the medium prior to the culture step and / or added to the medium during the culture step.

[0048] 2. The method of item 1, wherein the culture medium contains poloxamer-188 at a concentration of 1.8 g / L or higher.

[0049] 3. The method of item 2, wherein the culture medium contains poloxamer-188 at a concentration of 2.8 g / L or higher.

[0050] 4. The method of item 3, wherein the culture medium contains poloxamer-188 at a concentration of 4.0 g / L or higher.

[0051] 5. The method of item 4, wherein the culture medium contains poloxamer-188 at a concentration of 6.0 g / L or higher.

[0052] 6. The method of item 5, wherein the culture medium comprises poloxamer-188 at a concentration of 8.0 g / L or higher.

[0053] 7. The method of item 6, wherein the culture medium comprises poloxamer-188 at a concentration of 10.0 g / L or higher.

[0054] 8. The method of any one of items 1-7, wherein the culture medium further comprises an antifoaming agent, and the ratio of the antifoaming agent (g / L) to poloxamer-188 (g / L) in the culture medium is from about 0.5% to about 6.0% in the liquid culture medium.

[0055] 9. The method of item 8, wherein the ratio of the antifoaming agent (g / L) to poloxamer-188 (g / L) in the culture medium is from about 1.0% to about 5.5%.

[0056] 10. The method of item 9, wherein the ratio of the antifoaming agent (g / L) to poloxamer-188 (g / L) in the culture medium is from about 1.5% to about 5.0%.

[0057] 11. The method of claim 1, wherein the method comprises adding poloxamer-188 to the culture medium prior to the culture step and / or adding poloxamer-188 to the culture medium during the culture step to provide poloxamer-188 at a concentration of 1.8 g / L or higher in the culture medium.

[0058] 12. The method of item 1 or 11, wherein poloxamer-188 is present in the culture medium.

[0059] 13. The method of item 11 or 12, wherein the method comprises adding poloxamer-188 to the culture medium prior to the culture step and / or adding poloxamer-188 to the culture medium during the culture step to provide poloxamer-188 at a concentration of 2.8 g / L or higher in the culture medium.

[0060] 14. The method of item 13, wherein the method comprises adding poloxamer-188 to the culture medium prior to the culture step and / or adding poloxamer-188 to the culture medium during the culture step to provide poloxamer-188 at a concentration of 4.0 g / L or higher in the culture medium.

[0061] 15. The method of item 14, wherein the method comprises adding poloxamer-188 to the culture medium prior to the culture step and / or adding poloxamer-188 to the culture medium during the culture step to provide poloxamer-188 at a concentration of 6.0 g / L or higher in the culture medium.

[0062] 16. The method of item 15, wherein the method comprises adding poloxamer-188 to the culture medium prior to the culture step and / or adding poloxamer-188 to the culture medium during the culture step to provide poloxamer-188 at a concentration of 8.0 g / L or 8.0 g / L in the culture medium.

[0063] 17. The method of item 16, wherein the method comprises adding poloxamer-188 to the culture medium prior to the culture step and / or adding poloxamer-188 to the culture medium during the culture step to provide poloxamer-188 at a concentration of 10.0 g / L or higher in the culture medium.

[0064] 18. The method of any one of items 1-17, wherein the method comprises increasing the concentration of poloxamer-188 in the culture medium over time.

[0065] 19. The method of item 18, wherein the concentration of poloxamer-188 in the culture medium increases over time based on the density of live cells in the culture medium.

[0066] 20. The method of item 19, wherein the method includes when the viable cell density in the culture medium is about 35 x 10⁻⁶. 6 Cells / mL to approximately 60 x 10⁻⁶ 6 When the cell / mL ratio is reached, increase the concentration of poloxamer-188 in the culture medium to greater than 1.8 g / L.

[0067] 21. The method of item 20, wherein the method includes when the viable cell density in the culture medium is about 35 x 10⁻⁶. 6 Cells / mL to approximately 60 x 10⁻⁶ 6 When the cell / mL ratio is reached, increase the concentration of poloxamer-188 in the culture medium from approximately 1.8 g / L to approximately 3.0 g / L.

[0068] 22. The method of item 21, wherein the method includes, when the viable cell density in the culture medium is about 35 x 10⁻⁶ cells / day... 6 To approximately 60 x 10 6 When the cell / mL ratio is reached, increase the concentration of poloxamer-188 in the culture medium to approximately 2.3 g / L to approximately 3.0 g / L.

[0069] 23. The method of any one of items 19-22, wherein the method includes a method for a viable cell density of about 60 x 10⁻⁶ cells in the culture medium. 6 Cells / mL to approximately 90 x 10⁻⁶ 6 When the cell / mL ratio is reached, increase the concentration of poloxamer-188 in the culture medium to greater than 3.0 g / L.

[0070] 24. The method of item 23, wherein the method includes when the viable cell density in the culture medium is about 60 x 10⁻⁶. 6 Cells / mL to approximately 90 x 10⁻⁶ 6 When the cell / mL ratio is reached, increase the concentration of poloxamer-188 in the culture medium to approximately 3.0 g / L to approximately 6.0 g / L.

[0071] 25. The method of item 24, wherein the method includes, when the viable cell density in the culture medium is about 60 x 10⁻⁶. 6 Cells / mL to approximately 90 x 10⁻⁶ 6 When the cell / mL ratio is reached, increase the concentration of poloxamer-188 in the culture medium from approximately 3.6 g / L to approximately 6.0 g / L.

[0072] 26. The method of any one of items 19-25, wherein the method includes a method for when the viable cell density in the culture medium is greater than 90 x 10⁻⁶. 6 When the cell / mL ratio is reached, increase the concentration of poloxamer-188 in the culture medium to greater than 6.0 g / L.

[0073] 27. The method of item 26, wherein the method includes when the viable cell density in the culture medium is greater than 90 x 10⁻⁶. 6 When the cell / mL ratio is reached, increase the concentration of poloxamer-188 in the culture medium to greater than 7.0 g / L.

[0074] 28. The method of item 27, wherein the method includes when the viable cell density in the culture medium is greater than 90 x 10⁻⁶. 6 When the cell / mL ratio is reached, increase the concentration of poloxamer-188 in the culture medium to greater than 8.0 g / L.

[0075] 29. The method of item 28, wherein the method includes when the viable cell density in the culture medium is greater than 90 x 10⁻⁶. 6 When the cell / mL ratio is reached, increase the concentration of poloxamer-188 in the culture medium to greater than 10.0 g / L.

[0076] 30. The method of item 19, wherein the method comprises:

[0077] When the viable cell density in the culture medium is approximately 35 x 10⁻⁶ 6 To approximately 60 x 10 6 When the cell / mL ratio is reached, increase the concentration of poloxamer-188 in the culture medium to approximately 1.8 g / L to approximately 3.0 g / L;

[0078] When the viable cell density in the culture medium is approximately 60 x 10⁻⁶ 6 Cells / mL to approximately 90 x 10⁻⁶ 6 When the cell / mL ratio was reached, the concentration of poloxamer-188 in the culture medium was increased to approximately 3.0 g / L to approximately 6.0 g / L; and

[0079] When the viable cell density in the culture medium is greater than 90 x 10 6 When the cell / mL ratio is reached, increase the concentration of poloxamer-188 in the culture medium to greater than 6.0 g / L.

[0080] 31. The method of any one of items 19-30, further comprising determining the density of live cells in the culture medium at one or more time points during the culture process.

[0081] 32. The methods in items 1-31, wherein perfusion culture includes:

[0082] Provides a container containing mammalian cells configured in a first liquid culture medium;

[0083] The container was incubated with stirring at a temperature of about 32°C to about 40°C for at least about 7 days; and

[0084] During the first 48 to 96 hours of the incubation period, a first volume of first liquid culture medium is continuously or periodically removed and a second volume of second liquid culture medium is added to the first liquid culture medium, wherein the first and second volumes are approximately equal.

[0085] 33. The method of item 32, wherein the second liquid culture medium contains poloxamer-188 at a higher concentration than that in the first liquid culture medium.

[0086] 34. The method of item 32, wherein the first liquid culture medium contains poloxamer-188 at a concentration of 1.8 g / L or less, and the second liquid culture medium contains poloxamer-188 at a concentration greater than 1.8 g / L.

[0087] 35. The method of item 34, wherein the second liquid culture medium contains poloxamer-188 at a concentration higher than 2.8 g / L.

[0088] 36. The method of item 35, wherein the second liquid culture medium contains poloxamer-188 at a concentration higher than 4.0 g / L.

[0089] 37. The method of item 36, wherein the second liquid culture medium contains poloxamer-188 at a concentration higher than 6.0 g / L.

[0090] 38. The method of item 37, wherein the second liquid culture medium contains poloxamer-188 at a concentration higher than 8.0 g / L.

[0091] 39. The method of item 38, wherein the second liquid culture medium contains poloxamer-188 at a concentration higher than 10.0 g / L.

[0092] 40. The method of claim 32, wherein the method comprises adding poloxamer-188 to the second liquid culture medium before or after adding the second volume of the second liquid culture medium to the first liquid culture medium to provide poloxamer-188 at a concentration of 1.8 g / L or higher in the second culture medium.

[0093] 41. The method of item 40, wherein poloxamer-188 is added to a second liquid culture medium to provide a concentration of poloxamer-188 higher than 2.8 g / L in the second culture medium.

[0094] 42. The method of item 41, wherein poloxamer-188 is added to a second liquid culture medium to provide a concentration of poloxamer-188 higher than 4.0 g / L in the second culture medium.

[0095] 43. The method of item 42, wherein poloxamer-188 is added to a second liquid culture medium to provide a concentration of poloxamer-188 higher than 6.0 g / L in the second liquid culture medium.

[0096] 44. The method of item 43, wherein poloxamer-188 is added to a second liquid culture medium to provide a concentration of poloxamer-188 higher than 8.0 g / L in the second liquid culture medium.

[0097] 45. The method of item 44, wherein poloxamer-188 is added to a second liquid culture medium to provide a concentration of poloxamer-188 higher than 10.0 g / L in the second liquid culture medium.

[0098] 46. ​​The method of any one of items 32-45, further comprising:

[0099] The recombinant protein was collected from the mammalian cells, from the first liquid culture medium or the second liquid culture medium, or any combination thereof.

[0100] 47. The method of item 46 further includes formulating the collected recombinant protein into a pharmaceutical composition.

[0101] 48. The method of item 46, wherein the culture achieves a density greater than 30 x 10⁻⁶. 6 The live cell density per cell / mL was then determined.

[0102] 49. The method of item 48, wherein the culture achieves a size greater than 50 x 10⁻⁶. 6 The live cell density per cell / mL was then determined.

[0103] 50. The method of item 49, wherein the culture achieves a density greater than 100 x 10⁻⁶. 6 The live cell density per cell / mL was then determined.

[0104] 51. The method of item 50, wherein the culture achieves a density greater than 150 x 10⁻⁶. 6 The live cell density per cell / mL was then determined.

[0105] 52. The method of item 51, wherein the culture achieves a density greater than 200 x 10⁻⁶. 6 The live cell density per cell / mL was then determined.

[0106] 53. A method for culturing mammalian cells, the method comprising:

[0107] A culture system is provided, comprising a container, a liquid culture medium disposed within the container, and a sprayer disposed within the container, the sprayer including a plurality of orifices configured to dispense gas through the sprayer into the liquid culture medium; and

[0108] In the culture system, mammalian cells containing nucleic acids encoding recombinant proteins are perfused and cultured under conditions and gas flow rates sufficient to produce recombinant proteins in the system, wherein the concentration of poloxamer-188 contained in the culture medium is selected based on one or more factors selected from the group consisting of: pore size, pore type, gas flow rate, viable cell density in the culture medium, and markers related to cellular stress.

[0109] 54. The method of item 53, wherein the selected poloxamer-188 concentration is achieved by adding poloxamer-188 to the culture medium prior to the culture step and / or adding poloxamer-188 to the culture medium during the culture step.

[0110] 55. The method of item 53 or 54, wherein the factor is hole type and the hole type is sintered hole or drilled hole.

[0111] 56. The method of item 53, wherein the method includes selecting a concentration of poloxamer-188 of about 2.3 g / L to about 3.3 g / L based on the pore type and pore size when the pore type is a borehole and the pore diameter is about 750 μm to about 1.5 mm.

[0112] 57. The method of item 56, wherein the method includes selecting a concentration of poloxamer-188 of about 2.5 g / L to about 3.1 g / L based on the pore type and pore size when the pore type is a borehole and the pore diameter is about 900 μm to about 1.1 mm.

[0113] 58. The method of item 53, wherein the method includes selecting a concentration of poloxamer-188 of about 3.3 g / L to about 4.3 g / L based on the pore type and pore size when the pore type is a borehole and the pore diameter is about 250 μm to about 750 μm.

[0114] 59. The method of claim 58, wherein the method includes selecting a concentration of poloxamer-188 of about 3.5 g / L to about 4.1 g / L based on the pore type and pore size when the pore type is a borehole and the pore diameter is about 400 μm to about 600 μm.

[0115] 60. The method of item 53, wherein the method includes selecting poloxamer-188 at a concentration higher than 4.3 g / L based on the pore type and pore size when the pore type is a borehole and the pore diameter is about 1 μm to about 250 μm.

[0116] 61. The method of item 60, wherein the method includes selecting poloxamer-188 at a concentration higher than 4.3 g / L based on the pore type and pore size when the pore type is a borehole and the pore diameter is from about 1 μm to about 200 μm.

[0117] 62. The method of item 61, wherein the method includes selecting a concentration of poloxamer-188 higher than 4.3 g / L based on the pore type and pore size when the pore type is a borehole and the pore diameter is about 160 μm to about 190 μm.

[0118] 63. The method of item 62, wherein the method includes selecting poloxamer-188 at a concentration higher than 4.3 g / L based on the pore type and pore size when the pore type is a borehole and the pore diameter is about 170 μm to about 180 μm.

[0119] 64. The method of any one of items 60-63, wherein the method comprises selecting poloxamer-188 at a concentration higher than 5.0 g / L.

[0120] 65. The method of item 64, wherein the method includes selecting poloxamer-188 at a concentration higher than 6.0 g / L.

[0121] 66. The method of item 65, wherein the method includes selecting poloxamer-188 at a concentration higher than 8.0 g / L.

[0122] 67. The method of item 66, wherein the method includes selecting poloxamer-188 at a concentration higher than 10.0 g / L.

[0123] 68. The method of item 53, wherein the method includes selecting a concentration of poloxamer-188 from about 1.8 g / L to about 3.3 g / L based on the pore type and pore size when the pore type is sintered pore and the pore size is greater than 150 μm.

[0124] 69. The method of item 68, wherein the method includes selecting a concentration of poloxamer-188 of about 3.3 g / L to about 4.3 g / L based on the pore type and pore size when the pore type is sintered pore and the pore size is about 80 μm to about 150 μm.

[0125] 70. The method of item 69, wherein the method includes selecting a concentration of poloxamer-188 of about 3.5 g / L to about 4.1 g / L based on the pore type and pore size when the pore type is sintered pore and the pore size is about 90 μm to about 110 μm.

[0126] 71. The method of item 53, wherein the method includes selecting a concentration of poloxamer-188 higher than 4.5 g / L based on the pore type and pore size when the pore type is sintered pore and the pore size is about 1 μm to about 80 μm.

[0127] 72. The method of item 71, wherein the method comprises selecting poloxamer-188 at a concentration higher than 5.0 g / L.

[0128] 73. The method of item 72, wherein the method comprises selecting poloxamer-188 at a concentration higher than 6.0 g / L.

[0129] 74. The method of item 73, wherein the method comprises selecting poloxamer-188 at a concentration higher than 8.0 g / L.

[0130] 75. The method of item 74, wherein the method comprises selecting poloxamer-188 at a concentration higher than 10.0 g / L.

[0131] 76. The method of any one of items 53-75, wherein the perfusion culture comprises:

[0132] The culture system containing the first liquid culture medium was incubated with stirring at a temperature of about 32°C to about 40°C for at least about 7 days; and

[0133] After the first 48 to 96 hours of the incubation period, the first volume of the first liquid culture medium is removed continuously or periodically and a second volume of the second liquid culture medium is added to the first liquid culture medium.

[0134] 77. The method of item 76, wherein the second liquid culture medium contains poloxamer-188 at a concentration higher than that in the first liquid culture medium.

[0135] 78. The method of item 76 or 77, wherein the second culture medium contains a selected concentration of poloxamer-188.

[0136] 79. The method of any one of items 76-78, further comprising:

[0137] Recombinant proteins are collected from inside mammalian cells, from a first liquid culture medium or a second liquid culture medium, or any combination thereof.

[0138] 80. The method of item 79 further includes formulating the collected recombinant protein into a pharmaceutical composition.

[0139] 81. The method of item 32 or 53, wherein the container is a perfusion bioreactor.

[0140] 82. The method of item 81, wherein the perfusion bioreactor has a volume of about 1.5 L to about 25,000 L.

[0141] 83. The method of item 32 or 53, wherein the culture period is greater than 10 days.

[0142] 84. The method of item 32 or 53, wherein the removal of the first volume of the first liquid culture medium and the addition of the second volume of the second liquid culture medium in (c) are carried out continuously.

[0143] 85. The method of item 32 or 53, wherein the removal of the first volume of the first liquid culture medium and the addition of the second volume of the second liquid culture medium in (c) are performed periodically.

[0144] 86. The method of item 32 or 53, wherein the first volume of first liquid culture medium removed in (c) and the second volume of second liquid culture medium added increase over time.

[0145] 87. The method of item 32 or 53, wherein the first liquid culture medium and / or the second liquid culture medium are selected from the group consisting of: liquid culture medium with defined chemical composition, serum-free liquid culture medium, liquid culture medium containing serum, liquid culture medium without animal-derived components, and protein-free culture medium.

[0146] 88. The method of item 32 or 53, wherein, in the first 48 to 96 hours of the time period, in every 24-hour period, the first volume of first liquid culture medium removed in (c) and the second volume of second liquid culture medium added are about 0.3 to about 10 times the volume of the first liquid culture medium or the container volume.

[0147] 89. The method of any one of items 1-88, wherein the mammalian cell is Chinese hamster ovary (CHO) cell, human embryonic kidney (HEK) cell, NSO cell, young hamster kidney (BHK) cell, PerC6 cell, Vero cell, or HT-1080 cell line.

[0148] 90. The method of any one of items 1-89, wherein the recombinant protein is an immunoglobulin, an enzyme, a growth factor, a protein fragment, or an engineered protein. Attached Figure Description

[0149] Figure 1 This is a graph showing the viable cell density over time in perfusion cell culture operations performed using liquid medium without additional poloxamer-188 (at a stable concentration of 1.8 g / L poloxamer-188 throughout the culture period) (grey data) and in perfusion cell culture operations performed using liquid medium containing an increased amount of poloxamer-188 throughout the culture period (increasing from 1.8 g / L to 6.8 g / L poloxamer-188 throughout the culture period) (black data).

[0150] Figure 2 This is a graph showing the growth rate over time in perfusion cell culture operations using liquid medium containing increased amounts of poloxamer-188 (increased from 1.8 g / L to 6.8 g / L over the length of the culture period).

[0151] Figure 3This is a graph showing the percentage of viable cells in two perfusion cell culture operations performed using liquid medium without additional poloxamer-188 (with a stable concentration of 1.8 g / L poloxamer-188 during the culture period) (grey data) and a perfusion cell culture operation performed using liquid medium containing an increased amount of poloxamer-188 during the culture period (increasing from 1.8 g / L to 6.8 g / L poloxamer-188 during the culture period) (black data).

[0152] Figure 4 This is a graph showing the volumetric productivity over time in perfusion cell culture operations using liquid medium containing increased amounts of poloxamer-188 (increased from 1.8 g / L to 6.8 g / L of poloxamer-188 over the length of the culture period).

[0153] Figure 5 This is a graph showing specific productivity over time in perfusion cell culture operations using liquid medium containing increased amounts of poloxamer-188 (increased from 1.8 g / L to 6.8 g / L of poloxamer-188 over the length of the culture period).

[0154] Figure 6This is a Bis-Tris gel showing the degradation products of recombinant antibodies present in the following: clear liquid medium harvested from perfused cell cultures containing 6.8 g / L poloxamer-188 (lane 1); clear liquid medium harvested from perfused cell cultures containing 6.8 g / L poloxamer-188, incubated at room temperature for 7 days (lane 1a); clear liquid medium harvested from perfused cell cultures containing 3.95 g / L poloxamer-188 (lane 2); clear liquid medium harvested from perfused cell cultures containing 3.95 g / L poloxamer-188, incubated at room temperature for 7 days (lane 2a); and clear liquid medium harvested from perfused cell cultures containing 3.95 g / L poloxamer-188, supplemented with poloxamer-188 to reach 6.8 g / L. The concentration of poloxamer-188 was adjusted and incubated at room temperature for 9 days (lane 2b); clear liquid medium was harvested from perfused cell cultures containing 3.0 g / L poloxamer-188 (lane 3); clear liquid medium was harvested from perfused cell cultures containing 3.0 g / L poloxamer-188 and incubated at room temperature for 7 days (lane 3a); and clear liquid medium was harvested from perfused cell cultures containing 3.0 g / L poloxamer-188, supplemented with poloxamer-188 to reach a concentration of 6.8 g / L poloxamer-188 and incubated at room temperature for 9 days (lane 3b).

[0155] Figure 7 The graph shows the viable cell density over time for perfusion cell culture operations performed using a sprayer with sintered pores of 20 μm and a culture medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 5.8 g / L during the culture period) (square); for perfusion cell culture operations performed using a sprayer with drilled pores of 200 μm and a culture medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 4.8 g / L during the culture period) (triangle); and for perfusion cell culture operations performed using a sprayer with drilled pores of 500 μm and a culture medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 3.8 g / L during the culture period) (circle).

[0156] Figure 8The graph shows the specific growth rate over time for perfusion cell culture operations performed using a sprayer with sintered pores of 20 μm and a medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 5.8 g / L during the culture period) (square); for perfusion cell culture operations performed using a sprayer with pores of 200 μm and a medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 4.8 g / L during the culture period) (triangle); and for perfusion cell culture operations performed using a sprayer with pores of 500 μm and a medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 3.8 g / L during the culture period) (circle).

[0157] Figure 9 The graph shows the perfusion cell culture operations performed over the length of the culture period using a sprayer with sintered pores of 20 μm and a medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 5.8 g / L during the culture period) (square); the perfusion cell culture operations performed over the length of the culture period using a sprayer with pores of 200 μm and a medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 4.8 g / L during the culture period) (triangle); and the perfusion cell culture operations performed over the length of the culture period using a sprayer with pores of 500 μm and a medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 3.8 g / L during the culture period) (circle).

[0158] Figure 10 The graph shows the percentage of viable cells over time for perfusion cell culture operations performed using a sprayer with sintered pores of 20 μm and medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 5.8 g / L during the culture period) (square); for perfusion cell culture operations performed using a sprayer with pores of 200 μm and medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 4.8 g / L during the culture period) (triangle); and for perfusion cell culture operations performed using a sprayer with pores of 500 μm and medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 3.8 g / L) (circle).

[0159] Figure 11 The following are examples of perfusion cell culture procedures performed over a culture period: (1) using a sprayer with sintered pores of 20 μm and medium containing 1.8 g / L poloxamer-188 (hollow circle); (2) using a sprayer with sintered pores of 20 μm and medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 5.8 g / L during the culture period) (square); (3) using a sprayer with sintered pores of 200 μm and medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 4.8 g / L during the culture period) (triangle); and (4) using a sprayer with sintered pores of 500 μm. A graph showing the perfusion cell culture operation performed over a culture period of μm boreholes in a sprayer and medium containing an increased amount of poloxamer-188 (from 1.8 g / L to 3.8 g / L of poloxamer-188) (solid circles) compared to lactose dehydrogenase production over time.

[0160] Figure 12 The graph shows the volumetric productivity over time for perfusion cell culture operations using a sprayer with sintered pores of 20 μm and a medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 5.8 g / L during the culture period) (square); for perfusion cell culture operations using a sprayer with pores of 200 μm and a medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 4.8 g / L during the culture period) (triangle); and for perfusion cell culture operations using a sprayer with pores of 500 μm and a medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 3.8 g / L) (circle).

[0161] Figure 13The graph shows the aerobic glucose consumption over time for perfusion cell culture operations performed using a sprayer with sintered pores of 20 μm and a medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 5.8 g / L during the culture period) (square); for perfusion cell culture operations performed using a sprayer with drilled pores of 200 μm and a medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 4.8 g / L during the culture period) (triangle); and for perfusion cell culture operations performed using a sprayer with drilled pores of 500 μm and a medium containing an increased amount of poloxamer-188 (increasing from 1.8 g / L to 3.8 g / L during the culture period) (circle).

[0162] Figure 14 This is a graph showing the viable cell density over time for different batch shake-flask cell culture operations using CD-CHO liquid medium containing 0% defoamer-c poloxamer-188 (solid circles), 5.6% defoamer-c poloxamer-188 (hollow circles), 11.1% defoamer-c poloxamer-188 (solid squares), 27.8% defoamer-c poloxamer-188 (hollow squares), or 55.6% defoamer-c poloxamer-188 (solid triangles).

[0163] Figure 15 This is a graph showing the rate of lactose dehydrogenase production over time for different batch shake-flask cell culture operations using CD-CHO liquid medium containing 0% defoamer-c poloxamer-188 (solid circles), 5.6% defoamer-c poloxamer-188 (hollow circles), 11.1% defoamer-c poloxamer-188 (solid squares), 27.8% defoamer-c poloxamer-188 (hollow squares), or 55.6% defoamer-c poloxamer-188 (solid triangles). Detailed Implementation

[0164] This application provides a method for culturing mammalian cells, comprising: culturing mammalian cells containing nucleic acids encoding said recombinant proteins under conditions sufficient for the production of recombinant proteins, wherein the culture comprises culturing in a liquid medium containing poloxamer-188 at a concentration of 1.8 g / L or higher, wherein said poloxamer-188 is present in and / or added to the medium prior to the culture step and / or added to the medium during the culture step. A method for culturing mammalian cells is also provided, comprising: providing a culture system comprising a container, a liquid culture medium disposed within said container, and a sprayer disposed within said container, said sprayer comprising a plurality of orifices configured to dispense gas through said sprayer into said liquid culture medium; and culturing mammalian cells containing nucleic acids encoding said recombinant proteins in said culture system under conditions and gas flow rates sufficient for the production of recombinant proteins in said culture system, wherein the concentration of poloxamer-188 in said culture medium is selected based on one or more factors selected from the group consisting of: pore size, pore type, gas flow rate, viable cell density in said culture medium, and markers related to cellular stress. The exemplary and non-restrictive aspects of these methods are described below.

[0165] In some embodiments, the method provided in this application enables cell cultures having a diameter greater than 20 x 10⁻⁶. 6 Cells / mL, greater than 25 x 10 6 Cells / mL, greater than 30 x 10 6 Cells / mL, greater than 35 x 10 6 Cells / mL, greater than 40 x 10 6 Cells / mL, greater than 45 x 10 6 Cells / mL, greater than 50 x 10 6 Cells / mL, greater than 55 x 10 6 Cells / mL, greater than 60 x 10 6 Cells / mL, greater than 65 x 10 6 Cells / mL, greater than 70 x 10 6 Cells / mL, greater than 75 x 10 6 Cells / mL, greater than 80 x 10 6 Cells / mL, greater than 85 x 10 6 Cells / mL, greater than 90 x 10 6 Cells / mL, greater than 95 x 10 6 Cells / mL, greater than 100 x 10 6 Cells / mL, greater than 105 x 10 6 Cells / mL, greater than 110 x 10 6Cells / mL, greater than 115 x 10 6 Cells / mL, greater than 120 x 10 6 Cells / mL, greater than 125 x 10 6 Cells / mL, greater than 130 x 10 6 Cells / mL, greater than 135 x 10 6 Cells / mL, greater than 140 x 10 6 Cells / mL, greater than 145 x 10 6 Cells / mL, greater than 150 x 10 6 Cells / mL, greater than 155 x 10 6 Cells / mL, greater than 160 x 10 6 Cells / mL, greater than 165 x 10 6 Cells / mL, greater than 170 x 10 6 Cells / mL, greater than 175 x 10 6 Cells / mL, greater than 180 x 10 6 Cells / mL, greater than 185 x 10 6 Cells / mL, greater than 190 x 10 6 Cells / mL, greater than 195 x 10⁻⁶ 6 Cells / mL, greater than 200 x 10 6 Cells / mL, greater than 205 x 10 6 Cells / mL, greater than 210 x 10 6 Cells / mL, greater than 215 x 10 6 Cells / mL, greater than 220 x 10 6 Cells / mL, greater than 225 x 10 6 Cells / mL, greater than 230 x 10 6 Cells / mL, greater than 235 x 10 6 Cells / mL, greater than 240 x 10 6 Cells / mL, greater than 245 x 10 6 Cells / mL, or greater than 250 x 10⁻⁶ 6 Viable cell density per cell / mL. Methods for determining the viable cell density of a cell culture are known in the art.

[0166] In some embodiments, the method provided in this application provides cell cultures that have a percentage cell viability of greater than 75% (e.g., greater than 80%, greater than 85%, or greater than 90%) during 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).

[0167] In some embodiments, the method provided in this application enables cultivation that, at any point during the cultivation period, has a concentration of at least 1.5 g / L / day (e.g., at least 1.6 g / L / day, at least 1.7 g / L / day, at least 1.8 g / L / day, at least 1.9 g / L / day, at least 2.0 g / L / day, at least 2.1 g / L / day, at least 2.2 g / L / day, at least 2.3 g / L / day, at least 2.4 g / L / day, at least 2.5 g / L / day, at least 2.6 g / L / day, at least 2.7 g / L / day, at least 2.8 g / L / day, at least 2.9 g / L / day) for 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) starting at any time during the cultivation period. Volumetric productivity (VPR) of at least 3.0 g / L / d, at least 3.1 g / L / d, at least 3.2 g / L / d, at least 3.3 g / L / d, at least 3.4 g / L / d, at least 3.5 g / L / d, at least 3.6 g / L / d, at least 3.7 g / L / d, at least 3.8 g / L / d, at least 3.9 g / L / d, at least 4.0 g / L / d, at least 4.1 g / L / d, at least 4.2 g / L / d, at least 4.3 g / L / d, at least 4.4 g / L / d, or at least 4.5 g / L / d.

[0168] In some embodiments, the method provided in this application yields recombinant proteins that, upon collection, exhibit a reduction level (e.g., a reduction of about or less than 5%, a reduction of about or less than 10%, a reduction of about or less than 15%, a reduction of about or less than 20%, a reduction of about or less than 25%, a reduction of about or less than 30%, a reduction of about or less than 35%, a reduction of about or less than 40%, a reduction of about or less than 45%, a reduction of about or less than 50%, a reduction of about or less than 55%, a reduction of about or less than 60%, a reduction of about or less than 65%, a reduction of about or less than 70%, a reduction of about or less than 75%, a reduction of about or less than 80%, a reduction of about or less than 80%, a reduction of about or less than 85%, or a reduction of about or less than 90%) compared to recombinant proteins produced by similar methods excluding those cultured in a liquid medium containing 1.8 g / L or higher of poloxamer-188. The level of degradation of recombinant proteins collected from cell cultures can be detected, for example, using gel electrophoresis and / or immunoblotting.

[0169] In some embodiments, the method yields cell cultures that, starting at any time during the culture period, have a ratio of less than 60 nU / cell / day (e.g., less than 55 nU / cell / day, less than 50 nU / cell / day, less than 45 nU / cell / day, less than 50 nU / cell / day, less than 60 nU / cell / day, or less than 70) of lactate dehydrogenase production over at least about 5 (e.g., at least 10, at least 15, at least 20, at least 25, at least 25, at least 30, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or at least 70) consecutive days.

[0170] In some embodiments, the method results in cells being maintained in a steady state (stationary period) for 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, or at least 50 days), for example, at about or greater than 30 x 10⁻⁶ cells / year. 6 Cells / mL, approximately or greater than 35 x 10⁻⁶ 6 Cells / mL, approximately or greater than 40 x 10⁻⁶ 6 Cells / mL, approximately or greater than 45 x 10⁻⁶ 6 Cells / mL, approximately or greater than 50 x 10⁻⁶ 6 Cells / mL, approximately or greater than 55 x 10⁻⁶ 6 Cells / mL, approximately or greater than 60 x 10⁻⁶ 6 Cells / mL, approximately or greater than 65 x 10⁻⁶ 6 Cells / mL, approximately or greater than 70 x 10⁻⁶6 Cells / mL, approximately or greater than 75 x 10⁻⁶ 6 Cells / mL, approximately or greater than 80 x 10⁻⁶ 6 Cells / mL, approximately or greater than 85 x 10⁻⁶ 6 Cells / mL, approximately or greater than 90 x 10⁻⁶ 6 Cells / mL, approximately or greater than 95 x 10⁻⁶ 6 Cells / mL, approximately or greater than 100 x 10⁻⁶ 6 Cells / mL, approximately or greater than 10⁵ x 10⁻⁶ 6 Cells / mL, approximately or greater than 110 x 10 6 Cells / mL, approximately or greater than 115 x 10⁻⁶ 6 Cells / mL, approximately or greater than 120 x 10⁻⁶ 6 Cells / mL, approximately or greater than 125 x 10⁻⁶ 6 Cells / mL, approximately or greater than 130 x 10⁻⁶ 6 Cells / mL, approximately or greater than 135 x 10⁻⁶ 6 Cells / mL, approximately or greater than 140 x 10⁻⁶ 6 Cells / mL, approximately or greater than 145 x 10⁻⁶ 6 Cells / mL, approximately or greater than 150 x 10⁻⁶ 6 Cells / mL, approximately or greater than 155 x 10⁻⁶ 6 Cells / mL, approximately or greater than 160 x 10⁻⁶ 6 Cells / mL, approximately or greater than 165 x 10⁻⁶ 6 Cells / mL, approximately or greater than 170 x 10⁻⁶ 6 Cells / mL, approximately or greater than 175 x 10⁻⁶ 6 Cells / mL, approximately or greater than 180 x 10⁻⁶ 6 Cells / mL, approximately or greater than 185 x 10⁻⁶ 6 Cells / mL, approximately or greater than 190 x 10⁻⁶ 6 Cells / mL, approximately or greater than 195 x 10⁻⁶ 6 Cells / mL, or approximately or greater than 200 x 10⁻⁶ 6 Live cell density per cell / mL.

[0171] Culture volume

[0172] The culture step (e.g., perfusion culture step) in any of the methods may include incubating cells in a solution of 20 L to about 8,000 L (e.g., about 20 L to about 7,000 L, about 20 L to about 6,000 L, about 20 L to about 5,000 L, about 20 L to about 4,000 L, about 20 L to about 3,000 L, about 20 L to about 2,500 L, about 20 L to about 2,000 L, about 20 L to about 1,500 L, about 20 L to about 1,000 L, about 20 L to about 500 L, about 20 L to about 200 L, about 20 L to about 100 L, about 100 L to about 8,000 L, about 100 L to about 7,000 L, about 100 L to about 6,0 ...8,000 L, about 100 L to about 7,000 L, about L - approximately 5,000 L, approximately 100 L - approximately 4,000 L, approximately 100 L - approximately 3,000 L, approximately 100 L - approximately 2,000 L, approximately 100 L - approximately 1,500 L, approximately 100 L - approximately 1,000 L, approximately 100 L - approximately 800 L, approximately 100 L - approximately 700 L, approximately 100 L - approximately 600 L, approximately 100 L - approximately 500 L, approximately 100 L - approximately 400 L, approximately 100 L - approximately 300 L, approximately 100 L - approximately 200 L, approximately 500 L - approximately 8,000 L, approximately 500 L - approximately 7,000 L, approximately 500 L - approximately 6,000 L, approximately 500 L - approximately 5,000 L, approximately 500 L - approximately 4,000 L, approximately 500 L - approximately 3,000 L L, approximately 500 L - approximately 2,000 L, approximately 500 L - approximately 1,500 L, approximately 500 L - approximately 1,000 L, approximately 500 L - approximately 750 L, approximately 1,000 L - approximately 8,000 L, approximately 1,000 L - approximately 7,000 L, approximately 1,000 L - approximately 6,000 L, approximately 1,000 L - approximately 5,000 L, approximately 1,000 L - approximately 4,000 L, approximately 1,000 L - approximately 3,000 L, approximately 1,000 L - approximately 2,000 L, approximately 1,000 L - approximately 1,500 L, approximately 2,000 L - approximately 8,000 L, approximately 2,000 L - approximately 7,000 L, approximately 2,000 L - approximately 6,000 L, approximately 2,000 L - approximately 5,000 L L, approximately 2,000 L - approximately 4,000 L, approximately 2,000 L - approximately 3,000 L, approximately 3,000 L - approximately 8,000 L, approximately 3,000 L - approximately 7,000 L, approximately 3,000 L - approximately 6,000 L, approximately 3,000 L - approximately 5,000 L, approximately 3,000 L - approximately 4,000 L, approximately 4,000 L - approximately 8,000 L, approximately 4,Cell cultures in volumes of approximately 7,000 L, 4,000 L to 6,000 L, 5,000 L to 8,000 L, or approximately 7,000 L or approximately 8,000 L.

[0173] As is known in the art, the volume of a cell culture can remain substantially the same or can increase over time during the culture period (e.g., as described below).

[0174] Incubation period

[0175] The culture (e.g., perfusion culture) in any of the methods described in this application can last from about 3 days to about 100 days (e.g., about 3 days to about 95 days, about 3 days to about 90 days, about 3 days to about 85 days, about 3 days to about 80 days, about 3 days to about 75 days, about 3 days to about 70 days, about 3 days to about 65 days, about 3 days to about 60 days, about 3 days to about 55 days, about 3 days to about 50 days, about 3 days to about 45 days, about 3 days to about 40 days, about 3 days to about 35 days, about 3 days to about 30 days, about 3 days to about 25 days, about 3 days to about 20 days, about 3 days to about 15 days, about 3 days to about 10 days, about 5 days to about 100 days, about 5 days to about 95 days, about 5 days to about 90 days, about 5 days to about 85 days, about 5 days to about 80 days, about 5 days). - Approximately 75 days, approximately 5 days - approximately 70 days, approximately 5 days - approximately 65 days, approximately 5 days - approximately 60 days, approximately 5 days - approximately 55 days, approximately 5 days - approximately 50 days, approximately 5 days - approximately 45 days, approximately 5 days - approximately 40 days, approximately 5 days - approximately 35 days, approximately 5 days - approximately 30 days, approximately 5 days - approximately 25 days, approximately 5 days - approximately 20 days, approximately 5 days - approximately 15 days, approximately 5 days - approximately 10 days, approximately 10 days - approximately 100 days, approximately 10 days - approximately 95 days, approximately 10 days - approximately 90 days, approximately 10 days - approximately 85 days, approximately 10 days - approximately 80 days, approximately 10 days - approximately 75 days, approximately 10 days - approximately 70 days, approximately 10 days - approximately 65 days, approximately 10 days - approximately 60 days, approximately 10 days - approximately 55 days, approximately 10 days - approximately 50 days, approximately 10 days - approximately 45 days, approximately 10 days - Approximately 40 days, approximately 10-35 days, approximately 10-30 days, approximately 10-25 days, approximately 10-20 days, approximately 10-15 days, approximately 15-100 days, approximately 15-95 days, approximately 15-90 days, approximately 15-85 days, approximately 15-80 days, approximately 15-75 days, approximately 15-70 days Days, approximately 15-65 days, approximately 15-60 days, approximately 15-55 days, approximately 15-50 days, approximately 15-45 days, approximately 15-40 days, approximately 15-35 days, approximately 15-30 days, approximately 15-25 days, approximately 15-20 days, approximately 20-100 days, approximately 20-95 days, approximately 2 0 days - approximately 90 days, approximately 20 days - approximately 85 days, approximately 20 days - approximately 80 days, approximately 20 days - approximately 75 days, approximately 20 days - approximately 70 days, approximately 20 days - approximately 65 days, approximately 20 days - approximately 60 days, approximately 20 days - approximately 55 days, approximately 20 days - approximately 50 days, approximately 20 days - approximately 45 days, approximately 20 days - approximately 40 days, approximately 20 days - approximately 35 days, approximately 20 days - approximately 30 days, approximately 20-25 days, approximately 25-100 days, approximately 25-95 days, approximately 25-90 days, approximately 25-85 days, approximately 25-80 days, approximately 25-75 days, approximately 25-70 days, approximately 25-65 days, approximately 25-60 days, approximately 25-55 days, approximately 25-50 daysApproximately 25 days to approximately 45 days, approximately 25 days to approximately 40 days, approximately 25 days to approximately 35 days, approximately 25 days to approximately 30 days, approximately 30 days to approximately 100 days, approximately 30 days to approximately 90 days, approximately 30 days to approximately 80 days, approximately 30 days to approximately 70 days, approximately 30 days to approximately 60 days, approximately 30 days to approximately 50 days, approximately 30 days to approximately 40 days, approximately 40 days to approximately 100 days, approximately 40 days to approximately 90 days, approximately 40 days to approximately 80 days, approximately 40 days to approximately 70 days, approximately 40 days to approximately 60 days, approximately 40 days to approximately The timeframes are approximately 50 days, 50-100 days, 50-90 days, 50-80 days, 50-70 days, 50-60 days, 60-100 days, 60-90 days, 60-80 days, 60-70 days, 70-100 days, 70-90 days, 70-80 days, 80-100 days, or 90-100 days.

[0176] mammalian cells

[0177] The mammalian cells cultured (e.g., perfusion culture) in the methods provided in this application may be cells grown in suspension or adherent cells. Non-limiting examples of mammalian cells that can be cultured in any of the methods described in this application 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, young hamster kidney (BHK) cells, PerC6 cells, Vero cells, or HT-1080 cells and Madin-Darby Canine (Cocker Spaniel) kidney epithelial cells (MDCK) cells. In some embodiments of culturing adherent cells, the culture may also contain various microcarriers (e.g., microcarriers containing one or more pores). Other mammalian cells that can be cultured in any of the methods described in this application are known in the art.

[0178] The mammalian cells may contain recombinant nucleic acids encoding recombinant proteins (e.g., nucleic acids stably integrated into the genome of the mammalian cells). Non-limiting examples of recombinant nucleic acids encoding exemplary recombinant proteins are described below as recombinant proteins that can be produced using the methods described in this application. In some instances, the mammalian cells cultured in a bioreactor (e.g., any bioreactor described in this application) are derived from a larger culture.

[0179] 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., lipid transfection), transduction (e.g., lentiviral, adenoviral, or retroviral infection), and electroporation. In some cases, the nucleic acid encoding the recombinant protein is not stably integrated into the mammalian cell chromosome (transient transfection), while in other cases the nucleic acid is integrated. Alternatively or additionally, the nucleic acid encoding the recombinant protein may be present in a plasmid and / or a mammalian artificial chromosome (e.g., a human artificial chromosome). Alternatively or additionally, the nucleic acid can be introduced into the cells using a viral vector (e.g., a lentiviral, retroviral, or adenoviral vector). The nucleic acid can be operatively linked to a promoter sequence (e.g., a strong promoter, such as the β-actin promoter and the CMV promoter, or an inducible promoter). If desired, the vector containing the nucleic acid may also contain a selection marker (e.g., a gene conferring resistance to hygromycin, puromycin, or neomycin to mammalian cells).

[0180] In some cases, the recombinant protein is a secreted protein and is released from mammalian cells into an extracellular culture medium (e.g., a first and / or second liquid medium in perfusion culture or a first and / or supplemental liquid medium in fed batch culture). For example, the nucleic acid sequence encoding a soluble recombinant protein may contain a sequence encoding a secretion signal peptide at the N- or C-terminus of the recombinant protein, which is cleaved by an enzyme present in the mammalian cells and subsequently released into an extracellular culture medium (e.g., a first and / or second liquid medium).

[0181] bioreactor

[0182] The cultivation step in any of the methods described in this application may be carried out using a bioreactor (e.g., a perfusion bioreactor or a fed-batch bioreactor). Bioreactors used for the cultivation step (e.g., perfusion bioreactors or fed-batch bioreactors) may have a capacity of about 24 L to about 25,000 L (e.g., about 24 L - about 20,000 L, about 24 L - about 15,000 L, about 24 L - about 10,000 L, about 24 L - about 9,500 L, about 24 L - about 9,000 L, about 24 L - about 8,500 L, about 24 L - about 8,000 L, about 24 L - about 7,500 L, about 24 L - about 7,000 L, about 24 L - about 6,500 L, about 24 L - about 6,000 L, about 24 L - about 5,500 L, about 24 L - about 5,000 L, about 24 L - about 4,500 L, about 24 L - about 4,000 L, about 24 L - about 4,000 L, about 24 L - about 24 L). L - approximately 3,500 L, approximately 24 L - approximately 3,000 L, approximately 24 L - approximately 2,500 L, approximately 24 L - approximately 2,000 L, approximately 24 L - approximately 1,500 L, approximately 24 L - approximately 1,000 L, approximately 24 L - approximately 900 L, approximately 24 L - approximately 800 L, approximately 24 L - approximately 700 L, approximately 24 L - approximately 600 L, approximately 24 L - approximately 500 L, approximately 24 L - approximately 400 L, approximately 24 L - approximately 300 L, approximately 24 L - approximately 200 L, approximately 24 L - approximately 100 L, approximately 100 L - approximately 25,000 L, approximately 100 L - approximately 20,000 L, approximately 100 L - approximately 15,000 L, approximately 100 L - approximately 10,000 L, approximately 100 L - approximately 9,500 L L, approximately 100 L - approximately 9,000 L, approximately 100 L - approximately 8,500 L, approximately 100 L - approximately 8,000 L, approximately 100 L - approximately 7,500 L, approximately 100 L - approximately 7,000 L, approximately 100 L - approximately 6,500 L, approximately 100 L - approximately 6,000 L, approximately 100 L - approximately 5,500 L, approximately 100 L - approximately 5,000 L, approximately 100 L - approximately 4,500 L, approximately 100 L - approximately 4,000 L, approximately 100 L - approximately 3,500 L, approximately 100 L - approximately 3,000 L, approximately 100 L - approximately 2,500 L, approximately 100 L - approximately 2,000 L, approximately 100 L - approximately 1,500 L, approximately 100 L - approximately 1,000 L, approximately 100 L - approximately 900 L, approximately 100 L - approximately 800 L, approximately 100 L - approximately 700 L, approximately 100 L - approximately 600 L, approximately 100 L - approximately 500 L, approximately 100 L - approximately 400 L, approximately 100 L - approximately 300 L, approximately 100 L - approximately 200 L, approximately 200 L - approximately 25,000 L, approximately 200 L - approximately 20,000 L, approximately 200 L - approximately 15,000 L, approximately 200 L - approximately 10,000 L, approximately 200 L - approximately 9,500 L, approximately 200 L - approximately 9,000 L, approximately 200 L - approximately 8,500 L, approximately 200 L - approximately 8,000 L, approximately 200 L - approximately 7,500 L, approximately 200 L - approximately 7,000 L L, approximately 200 L - approximately 6,500 L, approximately 200 L - approximately 6,000 L, approximately 200 L - approximately 5,500 L, approximately 200 L - approximately 5,000 L, approximately 200 L - approximately 4,500 L, approximately 200 L - approximately 4,000 L, approximately 200 L - approximately 3,500 L, approximately 200 L - approximately 3,000 L, approximately 200 L - approximately 2,500 L, approximately 200 L - approximately 2,000 L, approximately 200 L - approximately 1,500 L, approximately 200 L - approximately 1,000 L, approximately 200 L - approximately 900 L, approximately 200 L - approximately 800 L, approximately 200 L - approximately 700 L, approximately 200 L - approximately 600 L, approximately 200 L - approximately 500 L, approximately 200 L - approximately 400 L, approximately 200 L - approximately 300 L, approximately 500 L - approximately 25,000 L, approximately 500 L - approximately 20,000 L, approximately 500 L - approximately 15,000 L, approximately 500 L - approximately 10,000 L, approximately 500 L - approximately 9,500 L, approximately 500 L - approximately 9,000 L, approximately 500 L - approximately 8,500 L, approximately 500 L - approximately 8,000 L, approximately 500 L - approximately 7,500 L, approximately 500 L - approximately 7,000 L, approximately 500 L - approximately 6,500 L, approximately 500 L - approximately 6,000 L, approximately 500 L - approximately 5,500 L, approximately 500 L - approximately 5,000 L, approximately 500 L - approximately 4,500 L, approximately 500 L - approximately 4,000 L, approximately 500 L - approximately 3,500 L, approximately 500 L - approximately 3,000 L, approximately 500 L - approximately 2,500 L, approximately 500 L - approximately 2,000 L, approximately 500 L - approximately 1,500 L, approximately 500 L - approximately 1,000 L, approximately 500 L - approximately 900 L, approximately 500 L - approximately 800 L, approximately 500 L - approximately 700 L, approximately 500 L - approximately 600 L, approximately 1,000 L - approximately 25,000 L, approximately 1,000 L - approximately 20,000 L, approximately 1,000 L - approximately 15,000 L, approximately 1,000 L - approximately 10,000 L, approximately 1,000 L - approximately 9,500 L, approximately 1,000 L - approximately 9,000 L, approximately 1,000 L - approximately 8,500 L, approximately 1,000 L - approximately 8,000 L, approximately 1,000 L - approximately 7,500 L, approximately 1,000 L - approximately 7,000 L, approximately 1,000 L - approximately 6,500 L, approximately 1,000 L - approximately 6,000 L, approximately 1,000 L - approximately 5,500 L, approximately 1,000 L - approximately 5,000 L, approximately 1,000 L - approximately 4,500 L, approximately 1,000 L - approximately 4,000L, approximately 1,000 L - approximately 3,500 L, approximately 1,000 L - approximately 3,000 L, approximately 1,000 L - approximately 2,500 L, approximately 1,000 L - approximately 2,000 L, approximately 1,000 L - approximately 1,500 L, approximately 2,000 L - approximately 25,000 L, approximately 2,000 L - approximately 20,000 L, approximately 2,000 L - approximately 15,000 L, approximately 2,000 L - approximately 10,000 L, approximately 2,000 L - approximately 9,500 L, approximately 2,000 L - approximately 9,000 L, approximately 2,000 L - approximately 8,500 L, approximately 2,000 L - approximately 8,000 L, approximately 2,000 L - approximately 7,500 L, approximately 2,000 L - approximately 7,000L, approximately 2,000 L - approximately 6,500 L, approximately 2,000 L - approximately 6,000 L, approximately 2,000 L - approximately 5,500 L, approximately 2,000 L - approximately 5,000 L, approximately 2,000 L - approximately 4,500 L, approximately 2,000 L - approximately 4,000 L, approximately 2,000 L - approximately 3,500 L, approximately 2,000 L - approximately 3,000 L, approximately 2,000 L - approximately 2,500 L, approximately 3,000 L - approximately 25,000 L, approximately 3,000 L - approximately 20,000 L, approximately 3,000 L - approximately 15,000 L, approximately 3,000 L - approximately 10,000 L, approximately 3,000 L - approximately 9,500 L, approximately 3,000 L - approximately 9,000L, approximately 3,000 L - approximately 8,500 L, approximately 3,000 L - approximately 8,000 L, approximately 3,000 L - approximately 7,500 L, approximately 3,000 L - approximately 7,000 L, approximately 3,000 L - approximately 6,500 L, approximately 3,000 L - approximately 6,000 L, approximately 3,000 L - approximately 5,500 L, approximately 3,000 L - approximately 5,000 L, approximately 3,000 L - approximately 4,500 L, approximately 3,000 L - approximately 4,000 L, approximately 3,000 L - approximately 3,500 L, approximately 4,000 L - approximately 9,000 L, approximately 4,000 L - approximately 25,000 L, approximately 4,000 L - approximately 20,000 L, approximately 4,000 L - approximately 15,000 L, approximately 4,000 L - approximately 10,000 L, approximately 4,000 L - approximately 9,500 L, approximately 4,000 L - approximately 9,000 L, approximately 4,000 L - approximately 8,500 L, approximately 4,000 L - approximately 8,000 L, approximately 4,000 L - approximately 7,500 L, approximately 4,000 L - approximately 7,000 L, approximately 4,000 L - approximately 6,500 L, approximately 4,000 L - approximately 6,000 L, approximately 4,000 L - approximately 5,500 L L, approximately 4,000 L - approximately 5,000 L, approximately 4,000 L - approximately 4,500 L, approximately 5,000 L - approximately 25,000 L, approximately 5,000 L - approximately 20,000 L, approximately 5,000 L - approximately 15,000 L, approximately 5,000 L - approximately 10,000 L, approximately 5,000 L - approximately 9,500 L, approximately 5,000 L - approximately 9,000 L, approximately 5,000 L - approximately 8,500 L, approximately 5,000 L - approximately 8,000 L, approximately 5,000 L - approximately 7,500 L, approximately 5,000 L - approximately 7,000 L, approximately 5,000 L - approximately 6,500 L, approximately 5,000 L - approximately 6,000 L, approximately 5,000 L - approximately 5,500 L L, approximately 6,000 L - approximately 25,000 L, approximately 6,000 L - approximately 20,000 L, approximately 6,000 L - approximately 15,000 L, approximately 6,000 L - approximately 10,000 L, approximately 6,000 L - approximately 9,500 L, approximately 6,000 L - approximately 9,000 L, approximately 6,000 L - approximately 8,500 L, approximately 6,000 L - approximately 8,000 L, approximately 6,000 L - approximately 7,500 L, approximately 6,000 L - approximately 7,000 L, approximately 6,000 L - approximately 6,500 L, 7,000 L - approximately 25,000 L, approximately 7,000 L - approximately 20,000 L, approximately 7,000 L - approximately 15,000 L, approximately 7,000 L - approximately 10,000 L, approximately 7,000 L - approximately 9,500 L, approximately 7,000 L - approximately 9,000 L, approximately 7,000 L - approximately 8,500 L, approximately 7,000 L - approximately 8,000 L, approximately 7,000 L - approximately 7,500 L, approximately 8,000 L - approximately 25,000 L, approximately 8,000 L - approximately 20,000 L, approximately 8,000 L - approximately 15,000 L, approximately 8,000 L - approximately 10,000 L, approximately 8,000 L - approximately 9,Internal volumes (capacities) of approximately 500 L, 8,000 L-9,000 L, 8,000 L-8,500 L, 9,000 L-25,000 L, 9,000 L-20,000 L, 9,000 L-15,000 L, 9,000 L-10,000 L, 9,000 L-9,500 L, 10,000 L-25,000 L, 10,000 L-20,000 L, 10,000 L-15,000 L, 15,000 L-25,000 L, or 20,000 L-25,000 L.

[0183] Cultivation System

[0184] Some methods described in this application use a culture system (e.g., a perfusion culture system) comprising a container, a liquid culture medium (e.g., any culture medium described in this application) disposed within the container, and a sprayer disposed within the container, the sprayer comprising a plurality of orifices configured to dispense gas through the sprayer into the liquid culture medium. Containers can range from approximately 24 L to approximately 20,000 L (e.g., approximately 24 L - approximately 15,000 L, approximately 24 L - approximately 10,000 L, approximately 24 L - approximately 9,500 L, approximately 24 L - approximately 9,000 L, approximately 24 L - approximately 8,500 L, approximately 24 L - approximately 8,000 L, approximately 24 L - approximately 7,500 L, approximately 24 L - approximately 7,000 L, approximately 24 L - approximately 6,500 L, approximately 24 L - approximately 6,000 L, approximately 24 L - approximately 5,500 L, approximately 24 L - approximately 5,000 L, approximately 24 L - approximately 4,500 L, approximately 24 L - approximately 4,000 L, approximately 24 L - approximately 3,500 L, approximately 24 L - approximately 3,000 L, approximately 24 L - approximately 2,500 L, approximately 24 L - approximately 20,0 ... L - approximately 2,000 L, approximately 24 L - approximately 1,500 L, approximately 24 L - approximately 1,000 L, approximately 24 L - approximately 900 L, approximately 24 L - approximately 800 L, approximately 24 L - approximately 700 L, approximately 24 L - approximately 600 L, approximately 24 L - approximately 500 L, approximately 24 L - approximately 400 L, approximately 24 L - approximately 300 L, approximately 24 L - approximately 200 L, approximately 24 L - approximately 100 L, approximately 100 L - approximately 20,000 L, approximately 100 L - approximately 15,000 L, approximately 100 L - approximately 10,000 L, approximately 100 L - approximately 9,500 L, approximately 100 L - approximately 9,000 L, approximately 100 L - approximately 8,500 L, approximately 100 L - approximately 8,000 L, approximately 100 L - approximately 7,500 L, approximately 100 L - approximately 7,000 L, approximately 100 L - approximately 6,500 L, approximately 100 L - approximately 6,000 L, approximately 100 L - approximately 5,500 L, approximately 100 L - approximately 5,000 L, approximately 100 L - approximately 4,500 L, approximately 100 L - approximately 4,000 L, approximately 100 L - approximately 3,500 L, approximately 100 L - approximately 3,000 L, approximately 100 L - approximately 2,500 L, approximately 100 L - approximately 2,000 L, approximately 100 L - approximately 1,500 L, approximately 100 L - approximately 1,000 L, approximately 100 L - approximately 900 L, approximately 100 L - approximately 800 L, approximately 100 L - approximately 700 L, approximately 100 L - approximately 600 L, approximately 100 L - approximately 500 L, approximately 100 L - approximately 400 L, approximately 100 L - approximately 300 L, approximately 100 L - approximately 200 L, approximately 200 L - approximately 20,000 L, approximately 200 L - approximately 15,000 L, approximately 200 L - approximately 10,000 L, approximately 200 L - approximately 9,500 L, approximately 200 L - approximately 9,000 L, approximately 200 L - approximately 8,500 L, approximately 200 L - approximately 8,000 L, approximately 200 L - approximately 7,500 L, approximately 200 L - approximately 7,000 L, approximately 200 L - approximately 6,500 L L, approximately 200 L - approximately 6,000 L, approximately 200 L - approximately 5,500 L, approximately 200 L - approximately 5,000 L, approximately 200 L - approximately 4,500 L, approximately 200 L - approximately 4,000 L, approximately 200 L - approximately 3,500 L, approximately 200 L - approximately 3,000 L, approximately 200 L - approximately 2,500 L, approximately 200 L - approximately 2,000 L, approximately 200 L - approximately 1,500 L, approximately 200 L - approximately 1,000 L, approximately 200 L - approximately 900 L, approximately 200 L - approximately 800 L, approximately 200 L - approximately 700 L, approximately 200 L - approximately 600 L, approximately 200 L - approximately 500 L, approximately 200 L - approximately 400 L, approximately 200 L - approximately 300 L, approximately 500 L - approximately 20,000 L, approximately 500 L - approximately 15,000 L, approximately 500 L - approximately 10,000 L, approximately 500 L - approximately 9,500 L, approximately 500 L - approximately 9,000 L, approximately 500 L - approximately 8,500 L, approximately 500 L - approximately 8,000 L, approximately 500 L - approximately 7,500 L, approximately 500 L - approximately 7,000 L, approximately 500 L - approximately 6,500 L, approximately 500 L - approximately 6,000 L, approximately 500 L - approximately 5,500 L, approximately 500 L - approximately 5,000 L, approximately 500 L - approximately 4,500 L, approximately 500 L - approximately 4,000 L, approximately 500 L - approximately 3,500 L, approximately 500 L - approximately 3,000 L, approximately 500 L - approximately 2,500 L, approximately 500 L - approximately 2,000 L, approximately 500 L - approximately 1,500 L, approximately 500 L - approximately 1,000 L, approximately 500 L - approximately 900 L, approximately 500 L - approximately 800 L, approximately 500 L - approximately 700 L, approximately 500 L - approximately 600 L, approximately 1,000 L - approximately 25,000 L, approximately 1,000 L - approximately 20,000 L, approximately 1,000 L - approximately 15,000 L, approximately 1,000 L - approximately 10,000 L, approximately 1,000 L - approximately 9,500 L, approximately 1,000 L - approximately 9,000 L, approximately 1,000 L - approximately 8,500 L, approximately 1,000 L - approximately 8,000 L, approximately 1,000 L - approximately 7,500 L, approximately 1,000 L - approximately 7,000 L, approximately 1,000 L - approximately 6,500 L, approximately 1,000 L - approximately 6,000 L, approximately 1,000 L - approximately 5,500 L, approximately 1,000 L - approximately 5,000 L, approximately 1,000 L - approximately 4,500 L, approximately 1,000 L - approximately 4,000 L, approximately 1,000 L - approximately 3,500 L, approximately 1,000 L - approximately 3,000 L L, approximately 1,000 L - approximately 2,500 L, approximately 1,000 L - approximately 2,000 L, approximately 1,000 L - approximately 1,500 L, approximately 2,000 L - approximately 20,000 L, approximately 2,000 L - approximately 15,000 L, approximately 2,000 L - approximately 10,000 L, approximately 2,000 L - approximately 9,500 L, approximately 2,000 L - approximately 9,000 L, approximately 2,000 L - approximately 8,500 L, approximately 2,000 L - approximately 8,000 L, approximately 2,000 L - approximately 7,500 L, approximately 2,000 L - approximately 7,000 L, approximately 2,000 L - approximately 6,500 L, approximately 2,000 L - approximately 6,000 L, approximately 2,000 L - approximately 5,500 L L, approximately 2,000 L - approximately 5,000 L, approximately 2,000 L - approximately 4,500 L, approximately 2,000 L - approximately 4,000 L, approximately 2,000 L - approximately 3,500 L, approximately 2,000 L - approximately 3,000 L, approximately 2,000 L - approximately 2,500 L, approximately 3,000 L - approximately 20,000 L, approximately 3,000 L - approximately 15,000 L, approximately 3,000 L - approximately 10,000 L, approximately 3,000 L - approximately 9,500 L, approximately 3,000 L - approximately 9,000 L, approximately 3,000 L - approximately 8,500 L, approximately 3,000 L - approximately 8,000 L, approximately 3,000 L - approximately 7,500 L, approximately 3,000 L - approximately 7,000 L L, approximately 3,000 L - approximately 6,500 L, approximately 3,000 L - approximately 6,000 L, approximately 3,000 L - approximately 5,500 L, approximately 3,000 L - approximately 5,000 L, approximately 3,000 L - approximately 4,500 L, approximately 3,000 L - approximately 4,000 L, approximately 3,000 L - approximately 3,500 L, approximately 4,000 L - approximately 20,000 L, approximately 4,000 L - approximately 15,000 L, approximately 4,000 L - approximately 10,000 L, approximately 4,000 L - approximately 9,500 L, approximately 4,000 L - approximately 9,000 L, approximately 4,000 L - approximately 8,500 L, approximately 4,000 L - approximately 8,000 L, approximately 4,000 L - approximately 7,500 L, approximately 4,000 L - approximately 7,000 L, approximately 4,000 L - approximately 6,500 L, approximately 4,000 L - approximately 6,000 L, approximately 4,000 L - approximately 5,500 L, approximately 4,000 L - approximately 5,000 L, approximately 4,000 L - approximately 4,500 L, approximately 5,000 L - approximately 20,000 L, approximately 5,000 L - approximately 15,000 L, approximately 5,000 L - approximately 10,000 L, approximately 5,000 L - approximately 9,500 L, approximately 5,000 L - approximately 9,000 L, approximately 5,000 L - approximately 8,500 L, approximately 5,000 L - approximately 8,000 L, approximately 5,000 L - approximately 7,500 L, approximately 5,000 L - approximately 7,000 L, approximately 5,000 L - approximately 6,500 L, approximately 5,000 L - approximately 6,000 L, approximately 5,000 L - approximately 5,500 L, approximately 6,000 L - approximately 20,000 L, approximately 6,000 L - approximately 10,000 L, approximately 6,000 L - approximately 9,500 L, approximately 6,000 L - approximately 9,000 L, approximately 6,000 L - approximately 8,500 L, approximately 6,000 L - approximately 8,000 L, approximately 6,000 L - approximately 7,500 L, approximately 6,000 L - approximately 7,000 L, approximately 6,000 L - approximately 6,500 L, approximately 7,000 L - approximately 20,000 L, approximately 7,000 L - approximately 15,000 L, approximately 7,000 L - approximately 10,000 L, approximately 7,000 L - approximately 9,500 L, approximately 7,000 L - approximately 9,000 L, approximately 7,000 L - approximately 8,500 L, approximately 7,000 L - approximately 8,000 L, approximately 7,000 L - approximately 7,500 L, approximately 8,000 L - approximately 20,000 L, approximately 8,000 L - approximately 15,000 L, approximately 8,000 L - approximately 10,000 L, approximately 8,000 L - approximately 9,500 L, approximately 8,000 L - approximately 9,000 L, approximately 8,000 L - approximately 8,500 L, approximately 9,000 L - approximately 20,000 L, approximately 9,000 L - approximately 15,000 L, approximately 9,000 L - approximately 10,000 L, approximately 9,000 L - approximately 9,500 L, approximately 10,000 L - approximately 20,000 L, approximately 10,000 L - approximately 15,000 L, or approximately 15,000 L - approximately 20,The internal volume (capacity) is 000 L. The container may be made of metal (e.g., stainless steel), plastic, or glass, or any combination thereof.

[0185] In some embodiments, one or more orifices of the sprayer may have a diameter of approximately 750 μm to approximately 1.5 mm (e.g., approximately 750 μm to approximately 1.45 mm, approximately 750 μm to approximately 1.4 mm, approximately 750 μm to approximately 1.35 mm, approximately 750 μm to approximately 1.3 mm, approximately 750 μm to approximately 1.25 mm, approximately 750 μm to approximately 1.2 mm, approximately 750 μm to approximately 1.15 mm, approximately 750 μm to approximately 1.1 mm, approximately 750 μm to approximately 1.05 mm, approximately 750 μm to approximately 1.0 mm, approximately 750 μm to approximately 950 μm, approximately 750 μm to approximately 900 μm, approximately 750 μm to approximately 850 μm, approximately 750 μm to approximately 800 μm, approximately 800 μm to approximately 1.5 mm, approximately 800 μm to approximately 1.45 mm). mm, approximately 800 μm - approximately 1.4 mm, approximately 800 μm - approximately 1.35 mm, approximately 800 μm - approximately 1.3 mm, approximately 800 μm - approximately 1.25 mm, approximately 800 μm - approximately 1.2 mm, approximately 800 μm - approximately 1.15 mm, approximately 800 μm - approximately 1.1 mm, approximately 800 μm - approximately 1.05 mm, approximately 800 μm - approximately 1.0 mm, approximately 800 μm - approximately 950 μm, approximately 800 μm - approximately 900 μm, approximately 800 μm - approximately 850 μm, approximately 850 μm - approximately 1.5 mm, approximately 850 μm - approximately 1.45 mm, approximately 850 μm - approximately 1.4 mm, approximately 850 μm - approximately 1.35 mm, approximately 850 μm - approximately 1.3 mm, approximately 850 μm - approximately 1.25 mm mm, approximately 850 μm - approximately 1.2 mm, approximately 850 μm - approximately 1.15 mm, approximately 850 μm - approximately 1.1 mm, approximately 850 μm - approximately 1.05 mm, approximately 850 μm - approximately 1.0 mm, approximately 850 μm - approximately 950 μm, approximately 850 μm - approximately 900 μm, approximately 900 μm - approximately 1.5 mm, approximately 900 μm - approximately 1.45 mm, approximately 900 μm - approximately 1.4 mm, approximately 900 μm - approximately 1.35 mm, approximately 900 μm - approximately 1.3 mm, approximately 900 μm - approximately 1.25 mm, approximately 900 μm - approximately 1.2 mm, approximately 900 μm - approximately 1.15 mm, approximately 900 μm - approximately 1.1 mm, approximately 900 μm - approximately 1.05 mm, approximately 900 μm - approximately 1.0 mm. mm, approximately 900 μm to approximately 950 μm, approximately 950 μm to approximately 1.5 mm, approximately 950 μm to approximately 1.45 mm, approximately 950 μm to approximately 1.4 mm, approximately 950 μm to approximately 1.35 mm, approximately 950 μm to approximately 1.3 mm, approximately 950 μm to approximately 1.25 mm, approximately 950 μm - approximately 1.2 mm, approximately 950 μm - approximately 1.15 mm, approximately 950 μm - approximately 1.1 mm, approximately 950 μm - approximately 1.05 mm, approximately 950 μm - approximately 1.0 mm, approximately 1.0 mm - approximately 1.5 mm, approximately 1.0 mm - approximately 1.45 mm, approximately 1.0 mm - approximately 1.4 mm, approximately 1.0 mm - approximately 1.35 mm, approximately 1.0 mm - approximately 1.3 mm, approximately 1.0 mm - approximately 1.0 mm - approximately 1.25 mm, approximately 1.0 mm - approximately 1.2 mm, approximately 1.0 mm - approximately 1.15 mm, approximately 1.0 mm - approximately 1.1 mm, approximately 1.0 mm - approximately 1.05 mm, approximately 1.05 mm - approximately 1.5 mm, approximately 1.05 mm - approximately 1.45 mm, approximately 1.05 mm - approximately 1.4 mm, approximately 1.05 mm mm - approximately 1.35 mm, approximately 1.05 mm - approximately 1.3 mm, approximately 1.05 mm - approximately 1.25 mm, approximately 1.05 mm - approximately 1.2 mm, approximately 1.05 mm - approximately 1.15 mm, approximately 1.05 mm - approximately 1.1 mm, approximately 1.1 mm - approximately 1.5 mm, approximately 1.1 mm - approximately 1.45 mm, approximately 1.1 mm - approximately 1.4 mm, approximately 1.1 mm - approximately 1.35 mm, approximately 1.1 mm - approximately 1.3 mm, approximately 1.1 mm - approximately 1.25 mm, approximately 1.1 mm - approximately 1.2 mm, approximately 1.1 mm - approximately 1.15 mm, approximately 1.15 mm - approximately 1.5 mm, approximately 1.15 mm - approximately 1.45 mm, approximately 1.15 mm - approximately 1.4 mm, approximately 1.15 mm - approximately 1.35 mm, approximately 1.15 mm - approximately 1.3 mm. mm, approximately 1.15 mm - approximately 1.25 mm, approximately 1.15 mm - approximately 1.2 mm, approximately 1.2 mm - approximately 1.5 mm, approximately 1.2 mm - approximately 1.45 mm, approximately 1.2 mm - approximately 1.4 mm, approximately 1.2 mm - approximately 1.35 mm, approximately 1.2 mm - approximately 1.3 mm, approximately 1.2 mm - approximately 1.25 mm, approximately 1.25 mm - approximately 1.5 mm, approximately 1.25 mm - approximately 1.45 mm, approximately 1.25 mm - approximately 1.4 mm, approximately 1.25 mm - approximately 1.35 mm, approximately 1.25 mm - approximately 1.3 mm, approximately 1.3 mm - approximately 1.5 mm, approximately 1.35 mm - approximately 1.5 mm, approximately 1.35 mm - approximately 1.45 mm mm, approximately 1.35 mm to approximately 1.4 mm, approximately 1.4 mm to approximately 1.5 mm, approximately 1.4 mm to approximately 1.An aperture of 45 mm (or approximately 1.45 mm to 1.5 mm).

[0186] In some embodiments, one or more orifices of the sprayer may have a diameter of about 250 μm to about 750 μm (e.g., about 250 μm to about 700 μm, about 250 μm to about 650 μm, about 250 μm to about 600 μm, about 250 μm to about 550 μm, about 250 μm to about 500 μm, about 250 μm to about 450 μm, about 250 μm to about 400 μm, about 250 μm to about 350 μm, about 250 μm to about 300 μm, about 300 μm to about 750 μm, about 300 μm to about 650 μm, about 300 μm to about 600 μm, about 300 μm to about 550 μm, about 300 μm to about 500 μm, about 300 μm to about 450 μm, about 300 μm to about 450 μm, about 300 μm to about 750 μm). μm - approximately 400 μm, approximately 300 μm - approximately 350 μm, approximately 350 μm - approximately 750 μm, approximately 350 μm - approximately 700 μm, approximately 350 μm - approximately 650 μm, approximately 350 μm - approximately 600 μm, approximately 350 μm - approximately 550 μm, approximately 350 μm - approximately 500 μm, approximately 350 μm - approximately 450 μm, approximately 350 μm - approximately 400 μm, approximately 400 μm - approximately 750 μm, approximately 400 μm - approximately 700 μm, approximately 400 μm - approximately 650 μm, approximately 400 μm - approximately 600 μm, approximately 400 μm - approximately 550 μm, approximately 400 μm - approximately 500 μm, approximately 400 μm - approximately 450 μm, approximately 450 μm - approximately 750 μm μm, approximately 450 μm - approximately 700 μm, approximately 450 μm - approximately 650 μm, approximately 450 μm - approximately 600 μm, approximately 450 μm - approximately 550 μm, approximately 450 μm - approximately 500 μm, approximately 500 μm - approximately 750 μm, approximately 500 μm - approximately 700 μm, approximately 500 μm - approximately 650 μm, approximately 500 μm - approximately 600 μm, approximately 500 μm - approximately 550 μm, approximately 550 μm - approximately 750 μm, approximately 550 μm - approximately 700 μm, approximately 550 μm - approximately 650 μm, approximately 600 μm - approximately 700 μm, approximately 600 μm - approximately 650 μm, approximately 650 μm Pore ​​sizes ranging from approximately 750 μm to approximately 650 μm to approximately 700 μm, or approximately 700 μm to approximately 750 μm.

[0187] In some embodiments, one or more orifices of the sprayer may have a diameter of about 1 μm to about 250 μm (e.g., about 1 μm to about 240 μm, about 1 μm to about 230 μm, about 1 μm to about 220 μm, about 1 μm to about 210 μm, about 1 μm to about 200 μm, about 1 μm to about 190 μm, about 1 μm to about 180 μm, about 1 μm to about 170 μm, about 1 μm to about 160 μm, about 1 μm to about 150 μm, about 1 μm to about 140 μm, about 1 μm to about 130 μm, about 1 μm to about 125 μm, about 1 μm to about 120 μm, about 1 μm to about 115 μm, about 1 μm to about 110 μm, about 1 μm to about 105 μm, about 1 μm to about 100 μm, about 1...). μm - approximately 95 μm, approximately 1 μm - approximately 90 μm, approximately 1 μm - approximately 85 μm, approximately 1 μm - approximately 80 μm, approximately 1 μm - approximately 75 μm, approximately 1 μm - approximately 70 μm, approximately 1 μm - approximately 65 μm, approximately 1 μm - approximately 60 μm, approximately 1 μm - approximately 55 μm, approximately 1 μm - approximately 50 μm, approximately 1 μm - approximately 45 μm, approximately 1 μm - approximately 40 μm, approximately 1 μm - approximately 35 μm, approximately 1 μm - approximately 30 μm, approximately 1 μm - approximately 25 μm, approximately 1 μm - approximately 20 μm, approximately 1 μm - approximately 15 μm, approximately 1 μm - approximately 10 μm, 1 μm - approximately 5 μm, 10 μm - approximately 250 μm, approximately 10 μm - approximately 240 μm, approximately 10 μm - approximately 230 μm, 10 μm - approximately 220 μm, approximately 10 μm - approximately 210 μm, approximately 10 μm - approximately 200 μm, approximately 10 μm - approximately 190 μm, 10 μm - approximately 180 μm, approximately 10 μm - approximately 170 μm, approximately 10 μm - approximately 160 μm, approximately 10 μm - approximately 150 μm, approximately 10 μm - approximately 140 μm, approximately 10 μm - approximately 130 μm, approximately 10 μm - approximately 120 μm, approximately 10 μm - approximately 110 μm, approximately 10 μm - approximately 100 μm, approximately 10 μm - approximately 90 μm, approximately 10 μm - approximately 80 μm, approximately 10 μm - approximately 70 μm, approximately 10 μm - approximately 60 μm, approximately 10 μm - approximately 50 μm, approximately 10 μm - approximately 40 μm, approximately 10 μm - approximately 30 μm μm, approximately 10 μm to approximately 20 μm, approximately 20 μm to approximately 250 μm, approximately 20 μm to approximately 240 μm, approximately 20 μm to approximately 230 μm, approximately 20 μm to approximately 220 μm, approximately 20 μm to approximately 210 μm, approximately 20 μm to approximately 200 μm, approximately 20 μm to approximately 190 μm,Approximately 20 μm - approximately 180 μm, approximately 20 μm - approximately 170 μm, approximately 20 μm - approximately 160 μm, approximately 20 μm - approximately 150 μm, approximately 20 μm - approximately 140 μm, approximately 20 μm - approximately 130 μm, approximately 20 μm - approximately 120 μm, approximately 20 μm - approximately 110 μm, approximately 20 μm - approximately 100 μm, approximately 20 μm - approximately 90 μm, approximately 20 μm - approximately 80 μm, approximately 20 μm - approximately 70 μm, approximately 20 μm - approximately 60 μm, approximately 20 μm - approximately 50 μm, approximately 20 μm - approximately 40 μm, approximately 20 μm - approximately 30 μm, approximately 30 μm - approximately 250 μm, approximately 30 μm - approximately 240 μm, approximately 30 μm - approximately 230 μm, approximately 30 μm - approximately 220 μm, approximately 30 μm - approximately 210 μm, approximately 30 μm - approximately 200 μm, approximately 30 μm - approximately 190 μm, approximately 30 μm - approximately 180 μm, approximately 30 μm - approximately 170 μm, approximately 30 μm - approximately 160 μm, approximately 30 μm - approximately 150 μm, approximately 30 μm - approximately 140 μm, approximately 30 μm - approximately 130 μm, approximately 30 μm - approximately 120 μm, approximately 30 μm - approximately 110 μm, approximately 30 μm - approximately 110 μm, approximately 30 μm - approximately 100 μm, approximately 30 μm - approximately 90 μm, approximately 30 μm - approximately 80 μm, approximately 30 μm - approximately 70 μm, approximately 30 μm - approximately 60 μm, approximately 30 μm - approximately 50 μm, approximately 30 μm - approximately 40 μm, approximately 40 μm - approximately 250 μm, approximately 40 μm - approximately 240 μm, approximately 40 μm - approximately 230 μm, approximately 40 μm - approximately 220 μm, approximately 40 μm - approximately 210 μm, approximately 40 μm - approximately 200 μm, approximately 40 μm - approximately 190 μm, approximately 40 μm - approximately 180 μm, approximately 40 μm - approximately 170 μm, approximately 40 μm - approximately 160 μm, approximately 40 μm - approximately 150 μm, approximately 40 μm - approximately 140 μm, approximately 40 μm - approximately 130 μm, approximately 40 μm - approximately 120 μm, approximately 40 μm - approximately 110 μm, approximately 40 μm - approximately 100 μm, approximately 40 μm - approximately 90 μm, approximately 40 μm - approximately 80 μm, approximately 40 μm - approximately 70 μm, approximately 40 μm - approximately 60 μm, approximately 40 μm - approximately 50 μm, approximately 50 μm - approximately 250 μm, approximately 50 μm - approximately 240 μm, approximately 50 μm - approximately 230 μm, approximately 50 μm - approximately 220 μm, approximately 50 μm - approximately 210 μm, approximately 50 μm - approximately 200 μm, approximately 50 μm - approximately 190 μm, approximately 50 μm - approximately 180 μmApproximately 50 μm - approximately 170 μm, approximately 50 μm - approximately 160 μm, approximately 50 μm - approximately 150 μm, approximately 50 μm - approximately 140 μm, approximately 50 μm - approximately 130 μm, approximately 50 μm - approximately 120 μm, approximately 50 μm - approximately 110 μm, approximately 50 μm - approximately 100 μm, approximately 50 μm - approximately 90 μm, approximately 50 μm - approximately 80 μm, approximately 50 μm - approximately 70 μm, approximately 50 μm - approximately 60 μm, approximately 60 μm - approximately 250 μm, approximately 60 μm - approximately 240 μm, approximately 60 μm - approximately 230 μm, approximately 60 μm - approximately 220 μm, approximately 60 μm - approximately 210 μm, approximately 60 μm - approximately 200 μm, approximately 60 μm - approximately 190 μm μm, approximately 60 μm-180 μm, approximately 60 μm-170 μm, approximately 60 μm-160 μm, approximately 60 μm-150 μm, approximately 60 μm-140 μm, approximately 60 μm-130 μm, approximately 60 μm-120 μm, approximately 60 μm-110 μm, approximately 60 μm-100 μm, approximately 60 μm-90 μm, approximately 60 μm-80 μm, approximately 60 μm-70 μm, approximately 70 μm-250 μm, approximately 70 μm-240 μm, approximately 70 μm-230 μm, approximately 70 μm-220 μm, approximately 70 μm-210 μm, approximately 70 μm-200 μm, approximately 70 μm-190 μm μm, approximately 70 μm-180 μm, approximately 70 μm-170 μm, approximately 70 μm-160 μm, approximately 70 μm-150 μm, approximately 70 μm-140 μm, approximately 70 μm-130 μm, approximately 70 μm-120 μm, approximately 70 μm-110 μm, approximately 70 μm-100 μm, approximately 70 μm-90 μm, approximately 70 μm-80 μm, approximately 80 μm-250 μm, approximately 80 μm-240 μm, approximately 80 μm-230 μm, approximately 80 μm-220 μm, approximately 80 μm-210 μm, approximately 80 μm-200 μm, approximately 80 μm-190 μm, approximately 80 μm-180 μm μm, approximately 80 μm-170 μm, approximately 80 μm-160 μm, approximately 80 μm-150 μm, approximately 80 μm-140 μm, approximately 80 μm-130 μm, approximately 80 μm-120 μm, approximately 80 μm-110 μm, approximately 80 μm-100 μm, approximately 80 μm-90 μm, approximately 90 μm-250 μm,Approximately 90 μm - approximately 240 μm, approximately 90 μm - approximately 230 μm, approximately 90 μm - approximately 220 μm, approximately 90 μm - approximately 210 μm, approximately 90 μm - approximately 200 μm, approximately 90 μm - approximately 190 μm, approximately 90 μm - approximately 180 μm, approximately 90 μm - approximately 170 μm, approximately 90 μm - approximately 160 μm, approximately 90 μm - approximately 150 μm, approximately 90 μm - approximately 140 μm, approximately 90 μm - approximately 130 μm, approximately 90 μm - approximately 120 μm, approximately 90 μm - approximately 110 μm, approximately 90 μm - approximately 100 μm, approximately 100 μm - approximately 250 μm, approximately 100 μm - approximately 240 μm, approximately 100 μm - approximately 230 μm, approximately 100 μm μm - approximately 220 μm, approximately 100 μm - approximately 210 μm, approximately 100 μm - approximately 200 μm, approximately 100 μm - approximately 190 μm, approximately 100 μm - approximately 180 μm, approximately 100 μm - approximately 170 μm, approximately 100 μm - approximately 160 μm, approximately 100 μm - approximately 150 μm, approximately 100 μm - approximately 140 μm, approximately 100 μm - approximately 130 μm, approximately 100 μm - approximately 120 μm, approximately 100 μm - approximately 110 μm, approximately 110 μm - approximately 250 μm, approximately 110 μm - approximately 240 μm, approximately 110 μm - approximately 230 μm, approximately 110 μm - approximately 220 μm, approximately 110 μm - approximately 210 μm, approximately 110 μm - approximately 200 μm μm, approximately 100 μm-190 μm, approximately 100 μm-180 μm, approximately 100 μm-170 μm, approximately 100 μm-160 μm, approximately 100 μm-150 μm, approximately 100 μm-140 μm, approximately 100 μm-130 μm, approximately 100 μm-120 μm, approximately 100 μm-110 μm, approximately 110 μm-250 μm, approximately 110 μm-240 μm, approximately 110 μm-230 μm, approximately 110 μm-220 μm, approximately 110 μm-210 μm, approximately 110 μm-200 μm, approximately 110 μm-190 μm, approximately 110 μm-180 μm, approximately 110 μm μm - approximately 170 μm, approximately 110 μm - approximately 160 μm, approximately 110 μm - approximately 150 μm, approximately 110 μm - approximately 140 μm, approximately 110 μm - approximately 130 μm, approximately 110 μm - approximately 120 μm, approximately 120 μm - approximately 250 μm, approximately 120 μm - approximately 240 μm, approximately 120 μm - approximately 230 μm, approximately 120 μm - approximately 220 μmApproximately 120 μm - approximately 210 μm, approximately 120 μm - approximately 200 μm, approximately 120 μm - approximately 190 μm, approximately 120 μm - approximately 180 μm, approximately 120 μm - approximately 170 μm, approximately 120 μm - approximately 160 μm, approximately 120 μm - approximately 150 μm, approximately 120 μm - approximately 140 μm, approximately 120 μm - approximately 130 μm, approximately 130 μm - approximately 250 μm, approximately 130 μm - approximately 240 μm, approximately 130 μm - approximately 230 μm, approximately 130 μm - approximately 220 μm, approximately 130 μm - approximately 210 μm, approximately 130 μm - approximately 200 μm, approximately 130 μm - approximately 190 μm, approximately 130 μm - approximately 180 μm, approximately 130 μm - approximately 170 μm, approximately 130 μm - approximately 160 μm, approximately 130 μm - approximately 150 μm, approximately 130 μm - approximately 140 μm, approximately 140 μm - approximately 250 μm, approximately 140 μm - approximately 240 μm, approximately 140 μm - approximately 230 μm, approximately 140 μm - approximately 220 μm, approximately 140 μm - approximately 210 μm, approximately 140 μm - approximately 200 μm, approximately 140 μm - approximately 190 μm, approximately 140 μm - approximately 180 μm, approximately 140 μm - approximately 170 μm, approximately 140 μm - approximately 160 μm, approximately 140 μm - approximately 150 μm, approximately 150 μm - approximately 250 μm, approximately 150 μm - approximately 240 μm, approximately 150 μm - approximately 230 μm μm, approximately 150 μm-approx. 220 μm, approximately 150 μm-approx. 210 μm, approximately 150 μm-approx. 200 μm, approximately 150 μm-approx. 190 μm, approximately 150 μm-approx. 180 μm, approximately 150 μm-approx. 170 μm, approximately 150 μm-approx. 160 μm, approximately 160 μm-approx. 250 μm, approximately 160 μm-approx. 240 μm, approximately 160 μm-approx. 230 μm, approximately 160 μm-approx. 220 μm, approximately 160 μm-approx. 210 μm, approximately 160 μm-approx. 200 μm, approximately 160 μm-approx. 190 μm, approximately 160 μm-approx. 180 μm, approximately 160 μm-approx. 170 μm, approximately 170 μm-approx. 250 μm, approximately 170 μm μm - approximately 240 μm, approximately 170 μm - approximately 230 μm, approximately 170 μm - approximately 220 μm, approximately 170 μm - approximately 210 μm, approximately 170 μm - approximately 200 μm, approximately 170 μm - approximately 190 μm, approximately 170 μm - approximately 180 μm, approximately 180 μm - approximately 250 μm, approximately 180 μm - approximately 240 μm, approximately 180 μm - approximately 230 μmApproximately 180 μm - approximately 220 μm, approximately 180 μm - approximately 210 μm, approximately 180 μm - approximately 200 μm, approximately 180 μm - approximately 190 μm, approximately 190 μm - approximately 250 μm, approximately 190 μm - approximately 240 μm, approximately 190 μm - approximately 230 μm, approximately 190 μm - approximately 220 μm, approximately 190 μm - approximately 210 μm, approximately 190 μm - approximately 200 μm, approximately 200 μm - approximately 250 μm, approximately 200 μm - approximately 240 μm, approximately 200 μm - approximately 230 μm, approximately 200 μm - approximately 220 μm, approximately 200 μm - approximately 210 μm, approximately 210 μm - approximately 250 μm, approximately 210 μm - approximately 240 μm, approximately 210 μm Pore ​​sizes ranging from approximately 230 μm to approximately 210 μm to approximately 220 μm, approximately 250 μm to approximately 220 μm, approximately 240 μm to approximately 220 μm, approximately 230 μm to approximately 230 μm, approximately 250 μm to approximately 230 μm, approximately 240 μm to approximately 230 μm, or approximately 250 μm.

[0188] One or more orifices of the atomizer may have, for example, drilled or sintered orifices. The culture system may include means for agitating the liquid culture medium disposed within the container (e.g., an impeller and a motor for rotating the impeller). The container may include one or more ports (and optionally a pump) that allow the addition of materials (e.g., liquid culture medium, poloxamer-188, base or acid (to adjust pH as needed), and / or antifoaming agents) to and / or removal of liquid culture medium (e.g., clear liquid culture medium or a sample of cell culture) disposed in the container. The culture system may also include one or more sensors for monitoring pH, dO2, temperature, and dCO2 in the liquid culture medium during the culture period. The culture system may include a filtration device (e.g., a device for alternating tangential filtration or tangential flow filtration) that processes a volume of cell culture to provide a substantially cell-free, clear liquid culture medium. The culture system may include a heating / cooling device that allows adjustment of the temperature of the liquid culture medium disposed in the container. Further features of the cell culture system are known in the art.

[0189] perfusion culture

[0190] In any of the methods described in this application, the culture step may be a perfusion culture. As is known in the art, a perfusion culture includes removing a first volume of a first liquid culture medium from a bioreactor and adding a second volume of a second liquid culture medium to the bioreactor, wherein the first volume and the second volume are approximately equal. Mammalian cells are retained in the bioreactor by some cell retention device or by some technique such as cell sedimentation in a sedimentation cone.

[0191] As is known in the art, perfusion culture differs from fed-batch culture, and the culture media and / or culture conditions used in perfusion culture often differ from those used in fed-batch culture.

[0192] In perfusion culture, the removal and addition of culture medium can be carried out simultaneously or sequentially, or some combination of both. Furthermore, removal and addition can be continuous, such as by removing and replacing 0.1% to 800%, 1%-700%, 1%-600%, 1%-500%, 1%-400%, 1%-350%, 1%-300%, 1%-250%, 1%-100%, 100%-200%, 5%-150%, 10%-50%, 15%-40%, 8%-80%, or 4%-30% of the bioreactor volume.

[0193] In some instances, the removal of a first volume of first liquid culture medium and the addition of a second volume of second liquid culture medium may be maintained at approximately the same rate every 24 hours. As is known in the art, the rates (volume / time) of removing the first volume of first liquid culture medium and the rates (volume / time) of adding the second volume of second liquid culture medium may vary and depend on the conditions of the particular cell culture system. The rates (volume / time) of removing the first volume of first liquid culture medium and the rates (volume / time) of adding the second volume of second liquid culture medium may be approximately the same or may be different.

[0194] Alternatively, the volumes removed and added can be varied by gradually increasing them over 24-hour periods. For example, the volume of a first liquid culture medium removed and a second liquid culture medium added every 24 hours can be increased during the culture period. The volumes can be increased from 0.5% to approximately 20% of the bioreactor volume during the culture period. The volumes can be increased to approximately 25% to approximately 150% of the bioreactor volume or the cell culture volume at the start of the culture period.

[0195] In some instances of the method described in this application, during the first 48 to 96 hours of the culture period, every 24 hours, the first volume of first liquid culture medium removed and the second volume of second liquid culture medium added are approximately 10% to approximately 95% of the cell culture volume at the start of the culture period, approximately 10% to approximately 20%, approximately 20% to approximately 30%, approximately 30% to approximately 40%, approximately 40% to approximately 50%, approximately 50% to approximately 60%, approximately 60% to approximately 70%, approximately 70% to approximately 80%, approximately 80% to approximately 90%, approximately 85% to approximately 95%, approximately 60% to approximately 80%, or approximately 70%.

[0196] Those skilled in the art will understand that the first liquid culture medium and the second liquid culture medium may be of the same type. In other instances, the first liquid culture medium and the second liquid culture medium may be different. Regarding one or more culture medium components, the second liquid culture medium may be more concentrated.

[0197] The first volume of the first liquid culture medium can be removed using any automated system. For example, alternating tangential flow filtration can be used. Alternatively, the first volume of the first liquid culture medium can be removed by percolating or gravity-flowing through a sterile membrane having a molecular weight cutoff for mammalian cells. Alternatively, the first volume of the first liquid culture medium can be removed by stopping or significantly reducing the stirring rate for 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 by removing or aspirating the first volume of the first liquid culture medium from the top of the bioreactor.

[0198] A second volume of the second liquid culture medium can be added to the first liquid culture medium using a pump. This can be done manually, such as by directly pipetting or injecting the second volume of the second liquid culture medium into the first liquid culture medium, or automatically.

[0199] In some embodiments of the method described in this application, the method includes (optionally providing a container containing mammalian cells configured in a first liquid culture medium) stirring and incubating the container at a temperature of about 32°C to about 40°C for at least about 7 days; and after the first 48 to 96 hours of the incubation period, continuously or periodically removing a first volume of the first liquid culture medium and adding a second volume of the second liquid culture medium to the first liquid culture medium, wherein the first and second volumes are approximately equal. In some embodiments, after about the first 48 to 96 hours of the incubation period, every 24-hour interval, the first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added is about 0.3x to about 10x the volume of the first liquid culture medium or the container.

[0200] Some examples of perfusion culture include stirring and incubating a culture system containing a first liquid culture medium at a temperature of about 32°C to about 40°C for at least about 7 days; and continuously or periodically removing a first volume of the first liquid culture medium and adding a second volume of the second liquid culture medium to the first liquid culture medium after the first 48 to 96 hours of the culture period.

[0201] Feed-based batch culture

[0202] The cultivation step in the method described in this application may include fed-batch culture. As known in the art, fed-batch culture involves incremental or continuous addition of a first liquid culture medium to an initial cell culture without substantially or significantly removing the first liquid culture medium from the cell culture. In some instances, the fed liquid culture medium is the same as the first liquid culture medium. The fed medium may be in liquid form (fed liquid culture medium) or in dry powder form. In other instances, the fed liquid culture medium is a concentrated form of the first liquid culture medium and / or added as dry powder.

[0203] The feed liquid culture medium can be added to the initial cell culture at specific or multiple time points during the culture period. For example, it can be added at the following time points after the start of the culture period: 6 hours to 7 days, approximately 6 hours to approximately 6 days, approximately 6 hours to approximately 5 days, approximately 6 hours to approximately 4 days, approximately 6 hours to approximately 3 days, approximately 6 hours to approximately 2 days, approximately 6 hours to approximately 1 day, approximately 12 hours to approximately 7 days, approximately 12 hours to approximately 6 days, approximately 12 hours to approximately 5 days, approximately 12 hours to approximately 4 days, approximately 12 hours to approximately 3 days, approximately 12 hours to approximately 2 days, approximately 1 day to approximately 7 days, approximately 1 day to approximately 6 days, approximately 1 day to approximately 5 days, approximately... Add feed liquid culture medium to the initial cell culture at time points of 1 day to approximately 4 days, approximately 1 day to approximately 3 days, approximately 1 day to approximately 2 days, approximately 2 days to approximately 7 days, approximately 2 days to approximately 6 days, approximately 2 days to approximately 5 days, approximately 2 days to approximately 4 days, approximately 2 days to approximately 3 days, approximately 3 days to approximately 7 days, approximately 3 days to approximately 6 days, approximately 3 days to approximately 5 days, approximately 3 days to approximately 4 days, approximately 4 days to approximately 7 days, approximately 4 days to approximately 6 days, approximately 4 days to approximately 5 days, approximately 5 days to approximately 7 days, or approximately 5 days to approximately 6 days.

[0204] During culture, feed liquid culture medium can be continuously added to the initial cell culture, or it can be added periodically (e.g., daily). In some instances, when feed liquid culture medium is added periodically to the initial cell culture, it can be added once, twice, three times, four times, or five times. The volume of feed liquid culture medium added to the initial cell culture or the amount of feed solid culture medium can vary (e.g., increase) during the culture period. The volume of feed liquid culture medium added to the initial cell culture at any 24-hour time during the culture period can be a portion of the bioreactor's volume. During the culture period, the volume of feed liquid culture added to the initial cell culture at any 24-hour interval can be 0.01X to about 0.3X of the bioreactor volume containing the culture (e.g., about 0.01X to about 0.28X, about 0.01X to about 0.26X, about 0.01X to about 0.24X, about 0.01X to about 0.22X, about 0.01X to about 0.20X, about 0.01X to about 0.18X, about 0.01X to about 0.16X, about 0.01X to about 0.14X, about 0.01X to about 0.12X, about...). 0.01X - approximately 0.10X, approximately 0.01X - approximately 0.08X, approximately 0.01X - approximately 0.06X, approximately 0.01X - approximately 0.04X, approximately 0.025X - approximately 0.3X, approximately 0.025X - approximately 0.28X, approximately 0.025X - approximately 0.26X, approximately 0.025X - approximately 0.24X, approximately 0.025X - approximately 0.22X, approximately 0.025X - approximately 0.20X, approximately 0.025X - approximately 0.18X, approximately 0.025X - approximately 0.16X, approximately 0.025X - approximately 0.14X, approximately 0.025X - Approximately 0.12X, approximately 0.025X - approximately 0.10X, approximately 0.025X - approximately 0.08X, approximately 0.025X - approximately 0.06X, approximately 0.025X - approximately 0.04X, approximately 0.05X - approximately 0.3X, approximately 0.05X - approximately 0.28X, approximately 0.05X - approximately 0.26X, approximately 0.05X - approximately 0.24X, approximately 0.05X - approximately 0.22X, approximately 0.05X - approximately 0.20X, approximately 0.05X - approximately 0.18X, approximately 0.05X - approximately 0.16X, approximately 0.05X - approximately 0.14X, approximately 0.05 X - approximately 0.12X, approximately 0.05X - approximately 0.10X, approximately 0.1X - approximately 0.3X, approximately 0.1X - approximately 0.28X, approximately 0.1X - approximately 0.26X, approximately 0.1X - approximately 0.24X, approximately 0.1X - approximately 0.22X, approximately 0.1X - approximately 0.20X, approximately 0.1X - approximately 0.18X, approximately 0.1X - approximately 0.16X, approximately 0.1X - approximately 0.14X, approximately 0.1X, approximately 0.15X - approximately 0.3X, approximately 0.15X - approximately 0.2X, approximately 0.2X - approximately 0.3X, or approximately 0.25X - approximately 0.3X).

[0205] In other embodiments, the volume of feed liquid culture added to the initial cell culture at any 24-hour interval during the culture period may be 0.02X to about 1.0X of the cell culture volume at the start of the culture period. (For example, approximately 0.02X - approximately 0.9X, approximately 0.02X - approximately 0.8X, approximately 0.02X - approximately 0.7X, approximately 0.02X - approximately 0.6X, approximately 0.02X - approximately 0.5X, approximately 0.02X - approximately 0.4X, approximately 0.02X - approximately 0.3X, approximately 0.02X - approximately 0.2X, approximately 0.02X - approximately 0.1X, approximately 0.05X - approximately 1.0X, approximately 0.05X - approximately 0.8X, approximately 0.05X - approximately 0.7X, approximately 0.05X - approximately 0.6X, approximately 0.05X - approximately 0.5X, approximately 0.05X - approximately 0.4X, approximately 0.05X - approximately 0.3X, approximately 0.0...) 5X - approximately 0.2X, approximately 0.05X - approximately 0.1X, approximately 0.1X - approximately 1.0X, approximately 0.1X - approximately 0.9X, approximately 0.1X - approximately 0.8X, approximately 0.1X - approximately 0.7X, approximately 0.1X - approximately 0.6X, approximately 0.1X - approximately 0.5X, approximately 0.1X - approximately 0.4X, approximately 0.1X - approximately 0.3X, approximately 0.1X - approximately 0.2X, approximately 0.2X - approximately 1.0X, approximately 0.2X - approximately 0.9X, approximately 0.2X - approximately 0.8X, approximately 0.2X - approximately 0.7X, approximately 0.2X - approximately 0.6X, approximately 0.2X - approximately 0.5X, or approximately 0.2X - approximately 0.4X).

[0206] Polosham-188

[0207] Poloxamer-188 is a known molecule in the art. Poloxamer-188 is a nonionic, synthetic block copolymer of ethylene oxide and propylene oxide. Poloxamer-188 has an average molecular weight of about 7680 to about 9510, and its composition by weight is about 81.8% ± 1.9% represented by oxyethylene. For example, Poloxamer-188 has the structure shown in Formula I, where a is 80 and b is 27. Poloxamer-188 has the CAS number 9003-11-6.

[0208]

[0209] Poloxamer-188 is commercially available from several different distributors, including, for example, Sigma-Aldrich (St. Louis, MO), Mediatech, Inc. (Manassas, VA), MASTTherapeutics, 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).

[0210] The method provided in this application includes culturing mammalian cells (e.g., mammalian cells containing nucleic acids encoding recombinant proteins) in a liquid culture medium containing a concentration of about 1.8 g / L or higher (e.g., about 1.9 g / L or higher, about 2.0 g / L or higher, about 2.1 g / L or higher, about 2.2 g / L or higher, about 2.3 g / L or higher, about 2.4 g / L or higher, about 2.5 g / L or higher, about 2.6 g / L or higher, about 2.7 g / L or higher, about 2.8 g / L or higher, and about 2.9 g / L). The concentrations are approximately 2.9 g / L or higher, approximately 3.0 g / L or higher, approximately 3.1 g / L or higher, approximately 3.2 g / L or higher, approximately 3.3 g / L or higher, approximately 3.4 g / L or higher, approximately 3.5 g / L or higher, approximately 3.6 g / L or higher, approximately 3.7 g / L or higher, approximately 3.8 g / L or higher, approximately 3.9 g / L or higher, approximately 4.0 g / L or higher, and approximately 4.1 g / L or higher. The concentrations are approximately 4.2 g / L or higher, approximately 4.3 g / L or higher, approximately 4.4 g / L or higher, approximately 4.5 g / L or higher, approximately 4.6 g / L or higher, approximately 4.7 g / L or higher, approximately 4.8 g / L or higher, approximately 4.9 g / L or higher, approximately 5.0 g / L or higher, approximately 5.1 g / L or higher, approximately 5.2 g / L or higher, approximately 5.3 g / L or higher, and approximately 5.4 g / L. The concentration is approximately 5.4 g / L or higher, or approximately 5.5 g / L or higher, or approximately 5.6 g / L or higher.7 g / L or higher concentration, approximately 5.7 g / L, approximately 5.8 g / L or higher concentration, approximately 5.9 g / L or higher concentration, approximately 6.0 g / L or higher concentration, approximately 6.1 g / L or higher concentration, approximately 6.2 g / L or higher concentration, approximately 6.3 g / L or higher concentration, approximately 6.4 g / L or higher concentration, approximately 6.5 g / L or higher concentration, approximately 6.6 g / L or higher concentration, approximately 6.7 g / L or higher concentration, approximately 6.8 g / L or higher concentration, approximately 6.9 g / L. The concentrations are approximately 6.9 g / L or higher, approximately 7.0 g / L or higher, approximately 7.1 g / L or higher, approximately 7.2 g / L or higher, approximately 7.3 g / L or higher, approximately 7.4 g / L or higher, approximately 7.5 g / L or higher, approximately 7.6 g / L or higher, approximately 7.7 g / L or higher, approximately 7.8 g / L or higher, approximately 7.9 g / L or higher, approximately 8.0 g / L or higher, and approximately 8.1 g / L or higher. The concentrations are approximately 8.2 g / L or higher, approximately 8.3 g / L or higher, approximately 8.4 g / L or higher, approximately 8.5 g / L or higher, approximately 8.6 g / L or higher, approximately 8.7 g / L or higher, approximately 8.8 g / L or higher, approximately 8.9 g / L or higher, approximately 9.0 g / L or higher, approximately 9.1 g / L or higher, approximately 9.2 g / L or higher, approximately 9.3 g / L or higher, and approximately 9.4 g / L. The concentrations are approximately 9.4 g / L or higher, approximately 9.5 g / L or higher, approximately 9.6 g / L or higher, approximately 9.7 g / L or higher, and approximately 9.8 g / L or higher.8 g / L, concentrations of approximately 9.9 g / L or higher, concentrations of approximately 10.0 g / L or higher, concentrations of approximately 10.1 g / L or higher, concentrations of approximately 10.2 g / L or higher, concentrations of approximately 10.3 g / L or higher, concentrations of approximately 10.4 g / L or higher, concentrations of approximately 10.5 g / L or higher, concentrations of approximately 10.6 g / L or higher, concentrations of approximately 10.7 g / L or higher, concentrations of approximately 10.8 g / L or higher, concentrations of approximately 10.9 g / L or higher, and concentrations of approximately 11.0 g / L. The concentrations are approximately 11.0 g / L or higher, approximately 11.1 g / L or higher, approximately 11.2 g / L or higher, approximately 11.3 g / L or higher, approximately 11.4 g / L or higher, approximately 11.5 g / L or higher, approximately 11.6 g / L or higher, approximately 11.7 g / L or higher, approximately 11.8 g / L or higher, approximately 11.9 g / L or higher, approximately 12.0 g / L or higher, and approximately 12.1 g / L or higher. The concentrations are approximately 12.2 g / L or higher, approximately 12.3 g / L or higher, approximately 12.4 g / L or higher, approximately 12.5 g / L or higher, approximately 12.6 g / L or higher, approximately 12.7 g / L or higher, approximately 12.8 g / L or higher, approximately 12.9 g / L or higher, approximately 13.0 g / L or higher, approximately 13.1 g / L or higher, approximately 13.2 g / L or higher, and approximately 13.3 g / L. The concentration is approximately 13.3 g / L or higher, approximately 13.4 g / L or higher, approximately 13.5 g / L or higher, approximately 13.6 g / L or higher, and approximately 13.7 g / L or higher concentration, approximately 13.7 g / L, approximately 13.8 g / L or higher concentration, approximately 13.9 g / L or higher concentration, approximately 14.0 g / L or higher concentration, approximately 14.1 g / L or higher concentration, approximately 14.2 g / L or higher concentration, approximately 14.3 g / L or higher concentration, approximately 14.4 g / L or higher concentration, approximately 14.5 g / L or higher concentration, approximately 14.6 g / L or higher concentration, approximately 14.7 g / L or higher concentration, approximately 14.8 g / L or higher concentration. Poloxamer-188 at concentrations of approximately 14.9 g / L or higher, or approximately 15.0 g / L or higher.

[0211] In some embodiments, the method provided in this application includes culturing mammalian cells (e.g., mammalian cells containing nucleic acids encoding recombinant proteins) in a liquid culture medium containing a concentration of about 1.8 g / L to about 16.0 g / L (e.g., about 1.8 g / L to about 15.8 g / L, about 1.8 g / L to about 15.6 g / L, about 1.8 g / L to about 15.4 g / L, about 1.8 g / L to about 15.2 g / L, about 1.8 g / L to about 15.0 g / L, about 1.8 g / L to about 14.8 g / L, about 1.8 g / L to about 14.6 g / L, about 1.8 g / L to about 14.4 g / L, about 1.8 g / L to about 14.2 g / L, about 1.8 g / L to about 14.0 g / L, about 1.8 g / L to about 13.8 g / L, about 1.8 g / L to about 16.0 g / L). g / L - approximately 13.6 g / L, approximately 1.8 g / L - approximately 13.4 g / L, approximately 1.8 g / L - approximately 13.2 g / L, approximately 1.8 g / L - approximately 13.0 g / L, approximately 1.8 g / L - approximately 12.8 g / L, approximately 1.8 g / L - approximately 12.6 g / L, approximately 1.8 g / L - approximately 12.4 g / L, approximately 1.8 g / L - approximately 12.2 g / L, approximately 1.8 g / L - approximately 12.0 g / L, approximately 1.8 g / L - approximately 11.8 g / L, approximately 1.8 g / L - approximately 11.6 g / L, approximately 1.8 g / L - approximately 11.4 g / L, approximately 1.8 g / L - approximately 11.2 g / L, approximately 1.8 g / L - approximately 11.0 g / L, approximately 1.8 g / L g / L - approximately 10.8 g / L, approximately 1.8 g / L - approximately 10.6 g / L, approximately 1.8 g / L - approximately 10.4 g / L, approximately 1.8 g / L - approximately 10.2 g / L, approximately 1.8 g / L - approximately 10.0 g / L, approximately 1.8 g / L - approximately 9.8 g / L, approximately 1.8 g / L - approximately 9.6 g / L, approximately 1.8 g / L - approximately 9.4 g / L, approximately 1.8 g / L - approximately 9.2 g / L, approximately 1.8 g / L - approximately 9.0 g / L, approximately 1.8 g / L - approximately 8.8 g / L, approximately 1.8 g / L - approximately 8.6 g / L, approximately 1.8 g / L - approximately 8.4 g / L, approximately 1.8 g / L - approximately 8.2 g / L, approximately 1.8 g / L - approximately 8.0 g / L, approximately 1.8 g / L - about 7.8 g / L, about 1.8 g / L - about 7.6 g / L, about 1.8 g / L - about 7.4 g / L, about 1.8 g / L - about 7.2 g / L, about 1.8 g / L - about 7.0 g / L, about 1.8 g / L - about 6.8 g / L, about 1.8 g / L - about 6.6 g / L, about 1.8 g / L - about 6.4 g / L, 1.8 g / L - about 6.2 g / L, approximately 1.8 g / L to approximately 6.0 g / L, approximately 1.8 g / L to approximately 5.8 g / L, approximately 1.8 g / L to approximately 5.6 g / L, approximately 1.8 g / L to approximately 5.4 g / L, approximately 1.8 g / L to approximately 5.2 g / L, approximately 1.8 g / L to approximately 5.0 g / L, approximately 1.8 g / L to approximately 4.8 g / L, approximately 1.8 g / L to approximately 4.6 g / L, approximately 1.8 g / L to approximately 4.4 g / L, approximately 1.8 g / L to approximately 4.2 g / L, approximately 1.8 g / L to approximately 4.0 g / L, approximately 1.8 g / L to approximately 3.8 g / L, approximately 1.8 g / L to approximately 3.6 g / L, approximately 1.8 g / L to approximately 3.4 g / L, approximately 1.8 g / L to approximately 3.2 g / L. g / L, approximately 1.8 g / L to approximately 3.0 g / L, approximately 1.8 g / L to approximately 2.8 g / L, approximately 2.0 g / L to approximately 16.0 g / L, approximately 2.0 g / L to approximately 15.8 g / L, approximately 2.0 g / L to approximately 15.6 g / L, approximately 2.0 g / L to approximately 15.4 g / L, approximately 2.0 g / L to approximately 15.2 g / L, approximately 2.0 g / L to approximately 15.0 g / L, approximately 2.0 g / L to approximately 14.8 g / L, approximately 2.0 g / L to approximately 14.6 g / L, approximately 2.0 g / L to approximately 14.4 g / L, approximately 2.0 g / L to approximately 14.2 g / L, approximately 2.0 g / L to approximately 14.0 g / L, approximately 2.0 g / L to approximately 13.8 g / L, approximately 2.0 g / L - approximately 13.6 g / L, approximately 2.0 g / L - approximately 13.4 g / L, approximately 2.0 g / L - approximately 13.2 g / L, approximately 2.0 g / L - approximately 13.0 g / L, approximately 2.0 g / L - approximately 12.8 g / L, approximately 2.0 g / L - approximately 12.6 g / L, approximately 2.0 g / L - approximately 12.4 g / L, approximately 2.0 g / L - approximately 12.2 g / L, approximately 2.0 g / L - approximately 12.0 g / L, approximately 2.0 g / L - approximately 11.8 g / L, approximately 2.0 g / L - approximately 11.6 g / L, approximately 2.0 g / L - approximately 11.4 g / L, approximately 2.0 g / L - approximately 11.2 g / L, approximately 2.0 g / L - approximately 11.0 g / L g / L, approx. 2.0 g / L - approx. 10.8 g / L, approx. 2.0 g / L - approx. 10.6 g / L, approx. 2.0 g / L - approx. 10.4 g / L, approx. 2.0 g / L - approx. 10.2 g / L, approx. 2.0 g / L - approx. 10.0 g / L, approx. 2.0 g / L - approx. 9.8 g / L, approx. 2.0 g / L - approx. 9.6 g / L, approx. 2.0 g / L - approx. 9.4 g / L, approx. 2.0 g / L - approx. 9.2 g / L, approx. 2.0 g / L - approx. 9.0 g / L, approx. 2.0 g / L - approx. 8.8 g / L, approximately 2.0 g / L to approximately 8.6 g / L, approximately 2.0 g / L to approximately 8.4 g / L, approximately 2.0 g / L to approximately 8.2 g / L, approximately 2.0 g / L to approximately 8.0 g / L, approximately 2.0 g / L to approximately 7.8 g / L, approximately 2.0 g / L to approximately 7.6 g / L, approximately 2.0 g / L to approximately 7.4 g / L, approximately 2.0 g / L to approximately 7.2 g / L, approximately 2.0 g / L to approximately 7.0 g / L, approximately 2.0 g / L to approximately 6.8 g / L, approximately 2.0 g / L to approximately 6.6 g / L, approximately 2.0 g / L to approximately 6.4 g / L, approximately 2.0 g / L to approximately 6.2 g / L, approximately 2.0 g / L to approximately 6.0 g / L, approximately 2.0 g / L to approximately 5.8 g / L. g / L, approximately 2.0 g / L to approximately 5.6 g / L, approximately 2.0 g / L to approximately 5.4 g / L, approximately 2.0 g / L to approximately 5.2 g / L, approximately 2.0 g / L to approximately 5.0 g / L, approximately 2.0 g / L to approximately 4.8 g / L, approximately 2.0 g / L to approximately 4.6 g / L, approximately 2.0 g / L to approximately 4.4 g / L, approximately 2.0 g / L to approximately 4.2 g / L, approximately 2.0 g / L to approximately 4.0 g / L, approximately 2.0 g / L to approximately 3.8 g / L, approximately 2.0 g / L to approximately 3.6 g / L, approximately 2.0 g / L to approximately 3.4 g / L, approximately 2.0 g / L to approximately 3.2 g / L, approximately 2.0 g / L to approximately 3.0 g / L, approximately 2.2 g / L to approximately 16.0 g / L. g / L, approximately 2.2 g / L to approximately 15.8 g / L, approximately 2.2 g / L to approximately 15.6 g / L, approximately 2.2 g / L to approximately 15.4 g / L, approximately 2.2 g / L to approximately 15.2 g / L, approximately 2.2 g / L to approximately 15.0 g / L, approximately 2.2 g / L to approximately 14.8 g / L, approximately 2.2 g / L to approximately 14.6 g / L, approximately 2.2 g / L to approximately 14.4 g / L, approximately 2.2 g / L to approximately 14.2 g / L, approximately 2.2 g / L to approximately 14.0 g / L, approximately 2.2 g / L to approximately 13.8 g / L, approximately 2.2 g / L to approximately 13.6 g / L, approximately 2.2 g / L to approximately 13.4 g / L, approximately 2.2 g / L to approximately 13.2 g / L, approximately 2.2 g / L - about 13.0 g / L, about 2.2 g / L - about 12.8 g / L, about 2.2 g / L - about 12.6 g / L, about 2.2 g / L - about 12.4 g / L, about 2.2 g / L - about 12.2 g / L, about 2.2 g / L - about 12.0 g / L, about 2.2 g / L - about 11.8 g / L, about 2.2 g / L - about 11.6 g / L, about 2.2 g / L - about 11.4 g / L, about 2.2 g / L - about 11.2 g / L, about 2.2 g / L - about 11.0 g / L, about 2.2 g / L - about 10.8 g / L, about 2.2 g / L - about 10.6 g / L, about 2.2 g / L - about 10.4 g / L, about 2.2 g / L - about 10.2 g / L, about 2.2 g / L - about 10.0 g / L, about 2.2 g / L - about 9.8 g / L, about 2.2 g / L - about 9.6 g / L, about 2.2 g / L - about 9.4 g / L, about 2.2 g / L - about 9.2 g / L, about 2.2 g / L - about 9.0 g / L, about 2.2 g / L - about 8.8 g / L, about 2.2 g / L - about 8.6 g / L, about 2.2 g / L - about 8.4 g / L, about 2.2 g / L - about 8.2 g / L. g / L, approximately 2.2 g / L to approximately 8.0 g / L, approximately 2.2 g / L to approximately 7.8 g / L, approximately 2.2 g / L to approximately 7.6 g / L, approximately 2.2 g / L to approximately 7.4 g / L, approximately 2.2 g / L to approximately 7.2 g / L, approximately 2.2 g / L to approximately 7.0 g / L, approximately 2.2 g / L to approximately 6.9 g / L, approximately 2.2 g / L to approximately 6.8 g / L, approximately 2.2 g / L to approximately 6.6 g / L, approximately 2.2 g / L to approximately 6.4 g / L, approximately 2.2 g / L to approximately 6.2 g / L, approximately 2.2 g / L to approximately 6.0 g / L, approximately 2.2 g / L to approximately 5.8 g / L, approximately 2.2 g / L to approximately 5.6 g / L, approximately 2.2 g / L to approximately 5.4 g / L. g / L, approximately 2.2 g / L to approximately 5.2 g / L, approximately 2.2 g / L to approximately 5.0 g / L, approximately 2.2 g / L to approximately 4.8 g / L, approximately 2.2 g / L to approximately 4.6 g / L, approximately 2.2 g / L to approximately 4.4 g / L, approximately 2.2 g / L to approximately 4.2 g / L, approximately 2.2 g / L to approximately 4.0 g / L, approximately 2.2 g / L to approximately 3.8 g / L, approximately 2.2 g / L to approximately 3.6 g / L, approximately 2.2 g / L to approximately 3.4 g / L, approximately 2.2 g / L to approximately 3.2 g / L, approximately 2.4 g / L to approximately 16.0 g / L, approximately 2.4 g / L to approximately 15.8 g / L, approximately 2.4 g / L to approximately 15.6 g / L, approximately 2.4 g / L to approximately 15.4 g / L. g / L, approximately 2.4 g / L to approximately 15.2 g / L, approximately 2.4 g / L to approximately 15.0 g / L, approximately 2.4 g / L to approximately 14.8 g / L, approximately 2.4 g / L to approximately 14.6 g / L, approximately 2.4 g / L to approximately 14.4 g / L, approximately 2.4 g / L to approximately 14.2 g / L, approximately 2.4 g / L to approximately 14.0 g / L, approximately 2.4 g / L to approximately 13.8 g / L, approximately 2.4 g / L to approximately 13.6 g / L, approximately 2.4 g / L to approximately 13.4 g / L, approximately 2.4 g / L to approximately 13.2 g / L, approximately 2.4 g / L to approximately 13.0 g / L, approximately 2.4 g / L to approximately 12.8 g / L, approximately 2.4 g / L to approximately 12.6 g / L, approximately 2.4 g / L to approximately 12.4 g / L, approximately 2.4 g / L to approximately 12.2 g / L, approximately 2.4 g / L to approximately 12.0 g / L, approximately 2.4 g / L to approximately 11.8 g / L, approximately 2.4 g / L to approximately 11.6 g / L, approximately 2.4 g / L to approximately 11.4 g / L, approximately 2.4 g / L to approximately 11.2 g / L, approximately 2.4 g / L to approximately 11.0 g / L, approximately 2.4 g / L to approximately 10.8 g / L, approximately 2.4 g / L to approximately 10.6 g / L. g / L. Approximately 2.4 g / L to approximately 10.4 g / L, approximately 2.4 g / L to approximately 10.2 g / L, approximately 2.4 g / L to approximately 10.0 g / L, approximately 2.4 g / L to approximately 9.8 g / L, approximately 2.4 g / L to approximately 9.6 g / L, approximately 2.4 g / L to approximately 9.4 g / L, approximately 2.4 g / L to approximately 9.2 g / L, approximately 2.4 g / L to approximately 9.0 g / L, approximately 2.4 g / L to approximately 8.8 g / L, approximately 2.4 g / L to approximately 8.6 g / L, approximately 2.4 g / L to approximately 8.4 g / L, approximately 2.4 g / L to approximately 8.2 g / L, approximately 2.4 g / L to approximately 8.0 g / L, approximately 2.4 g / L to approximately 7.8 g / L, approximately 2.4 g / L to approximately 7.6 g / L. g / L, approximately 2.4 g / L to approximately 7.4 g / L, approximately 2.4 g / L to approximately 7.2 g / L, approximately 2.4 g / L to approximately 7.0 g / L, approximately 2.4 g / L to approximately 6.9 g / L, approximately 2.4 g / L to approximately 6.8 g / L, approximately 2.4 g / L to approximately 6.6 g / L, approximately 2.4 g / L to approximately 6.4 g / L, approximately 2.4 g / L to approximately 6.2 g / L, approximately 2.4 g / L to approximately 6.0 g / L, approximately 2.4 g / L to approximately 5.8 g / L, approximately 2.4 g / L to approximately 5.6 g / L, approximately 2.4 g / L to approximately 5.4 g / L, approximately 2.4 g / L to approximately 5.2 g / L, approximately 2.4 g / L to approximately 5.0 g / L, approximately 2.4 g / L to approximately 4.8 g / L. g / L, approximately 2.4 g / L to approximately 4.6 g / L, approximately 2.4 g / L to approximately 4.4 g / L, approximately 2.4 g / L to approximately 4.2 g / L, approximately 2.4 g / L to approximately 4.0 g / L, approximately 2.4 g / L to approximately 3.8 g / L, approximately 2.4 g / L to approximately 3.6 g / L, approximately 2.4 g / L to approximately 3.4 g / L, approximately 2.6 g / L to approximately 16.0 g / L, approximately 2.6 g / L to approximately 15.8 g / L, approximately 2.6 g / L to approximately 15.6 g / L, approximately 2.6 g / L - approximately 15.4 g / L, approximately 2.6 g / L - approximately 15.2 g / L, approximately 2.6 g / L - approximately 15.0 g / L, approximately 2.6 g / L - approximately 14.8 g / L, approximately 2.6 g / L - approximately 14.6 g / L, approximately 2.6 g / L - approximately 14.4 g / L, approximately 2.6 g / L - approximately 14.2 g / L, approximately 2.6 g / L - approximately 14.0 g / L, approximately 2.6 g / L - approximately 13.8 g / L, approximately 2.6 g / L - approximately 13.6 g / L, approximately 2.6 g / L - approximately 13.4 g / L, approximately 2.6 g / L - approximately 13.2 g / L, approximately 2.6 g / L - approximately 13.0 g / L, approximately 2.6 g / L - approximately 12.8 g / L, approximately 2.6 g / L - approximately 12.6 g / L, approximately 2.6 g / L - approximately 12.4 g / L, approximately 2.6 g / L - approximately 12.2 g / L, approximately 2.6 g / L - approximately 12.0 g / L, approximately 2.6 g / L - approximately 11.8 g / L, approximately 2.6 g / L - approximately 11.6 g / L, approximately 2.6 g / L - approximately 11.4 g / L, approximately 2.6 g / L - approximately 11.2 g / L, approximately 2.6 g / L - approximately 11.0 g / L, approximately 2.6 g / L - approximately 10.8 g / L, approximately 2.6 g / L - approximately 10.6 g / L, approximately 2.6 g / L - approximately 10.4 g / L, approximately 2.6 g / L - approximately 10.2 g / L, approximately 2.6 g / L - approximately 10.0 g / L, approximately 2.6 g / L - approximately 9.8 g / L g / L, approximately 2.6 g / L to approximately 9.6 g / L, approximately 2.6 g / L to approximately 9.4 g / L, approximately 2.6 g / L to approximately 9.2 g / L, approximately 2.6 g / L to approximately 9.0 g / L, approximately 2.6 g / L to approximately 8.8 g / L, approximately 2.6 g / L to approximately 8.6 g / L, approximately 2.6 g / L to approximately 8.4 g / L, approximately 2.6 g / L to approximately 8.2 g / L, approximately 2.6 g / L to approximately 8.0 g / L, approximately 2.6 g / L to approximately 7.8 g / L, approximately 2.6 g / L to approximately 7.6 g / L, approximately 2.6 g / L to approximately 7.4 g / L, approximately 2.6 g / L to approximately 7.2 g / L, approximately 2.6 g / L to approximately 7.0 g / L, approximately 2.6 g / L - approximately 6.9 g / L, approximately 2.6 g / L - approximately 6.8 g / L, approximately 2.6 g / L - approximately 6.6 g / L, approximately 2.6 g / L - approximately 6.4 g / L, approximately 2.6 g / L - approximately 6.2 g / L, approximately 2.6 g / L - approximately 6.0 g / L, approximately 2.6 g / L - approximately 5.8 g / L, approximately 2.6 g / L - approximately 5.6 g / L, approximately 2.6 g / L - approximately 5.4 g / L, approximately 2.6 g / L - approximately 5.2 g / L, approximately 2.6 g / L - approximately 5.0 g / L, approximately 2.6 g / L - approx. 4.8 g / L, approx. 2.6 g / L - approx. 4.6 g / L, approx. 2.6 g / L - approx. 4.4 g / L, approx. 2.6 g / L - approx. 4.2 g / L, approx. 2.6 g / L - approx. 4.0 g / L, approx. 2.6 g / L - approx. 3.8 g / L, approx. 2.6 g / L - approx. 3.6 g / L, approx. 2.8 g / L - approx. 16.0 g / L, approx. 2.8 g / L - approx. 15.8 g / L, approx. 2.8 g / L - approx. 15.6 g / L, approx. 2.8 g / L - approx. 15.4 g / L, approx. 2.8 g / L - approx. 15.2 g / L, approx. 2.8 g / L - approx. 15.0 g / L, approx. 2.8 g / L - approx. 14.8 g / L, approx. 2.8 g / L - approx. 14.6 g / L g / L, approximately 2.8 g / L to approximately 14.4 g / L, approximately 2.8 g / L to approximately 14.2 g / L, approximately 2.8 g / L to approximately 14.0 g / L, approximately 2.8 g / L to approximately 13.8 g / L, approximately 2.8 g / L to approximately 13.6 g / L, approximately 2.8 g / L to approximately 13.4 g / L, approximately 2.8 g / L to approximately 13.2 g / L, approximately 2.8 g / L to approximately 13.0 g / L, approximately 2.8 g / L to approximately 12.8 g / L, approximately 2.8 g / L to approximately 12.6 g / L, approximately 2.8 g / L to approximately 12.4 g / L, approximately 2.8 g / L to approximately 12.2 g / L, approximately 2.8 g / L to approximately 12.0 g / L, approximately 2.8 g / L to approximately 11.8 g / L, approximately 2.8 g / L - approximately 11.6 g / L, approximately 2.8 g / L - approximately 11.4 g / L, approximately 2.8 g / L - approximately 11.2 g / L, approximately 2.8 g / L - approximately 11.0 g / L, approximately 2.8 g / L - approximately 10.8 g / L, approximately 2.8 g / L - approximately 10.6 g / L, approximately 2.8 g / L - approximately 10.4 g / L, approximately 2.8 g / L - approximately 10.2 g / L, approximately 2.8 g / L - approximately 10.0 g / L, approximately 2.8 g / L - approximately 9.8 g / L, approximately 2.8 g / L - approximately 9.6 g / L, approximately 2.8 g / L - approximately 9.4 g / L, approximately 2.8 g / L - approximately 9.2 g / L, approximately 2.8 g / L - approximately 9.0 g / L, approximately 2.8 g / L - approximately 8.8 g / L g / L, approximately 2.8 g / L to approximately 8.6 g / L, approximately 2.8 g / L to approximately 8.4 g / L, approximately 2.8 g / L to approximately 8.2 g / L, approximately 2.8 g / L to approximately 8.0 g / L, approximately 2.8 g / L to approximately 7.8 g / L, approximately 2.8 g / L to approximately 7.6 g / L, approximately 2.8 g / L to approximately 7.4 g / L, approximately 2.8 g / L to approximately 7.2 g / L, approximately 2.8 g / L to approximately 7.0 g / L, approximately 2.8 g / L to approximately 6.9 g / L, approximately 2.8 g / L - approx. 6.8 g / L, approx. 2.8 g / L - approx. 6.6 g / L, approx. 2.8 g / L - approx. 6.4 g / L, approx. 2.8 g / L - approx. 6.2 g / L, approx. 2.8 g / L - approx. 6.0 g / L, approx. 2.8 g / L - approx. 5.8 g / L, approx. 2.8 g / L - approx. 5.6 g / L, approx. 2.8 g / L - approx. 5.4 g / L, approx. 2.8 g / L - approx. 5.2 g / L, approx. 2.8 g / L - approx. 5.0 g / L, approx. 2.8 g / L - approx. 4.8 g / L, approx. 2.8 g / L - approx. 4.6 g / L, approx. 2.8 g / L - approx. 4.4 g / L, approx. 2.8 g / L - approx. 4.2 g / L, approx. 2.8 g / L - approx. 4.0 g / L, approx. 2.8 g / L - approximately 3.8 g / L, approximately 3.0 g / L - approximately 16.0 g / L, approximately 3.0 g / L - approximately 15.8 g / L, approximately 3.0 g / L - approximately 15.6 g / L, approximately 3.0 g / L - approximately 15.4 g / L, approximately 3.0 g / L - approximately 15.2 g / L, approximately 3.0 g / L - approximately 15.0 g / L, approximately 3.0 g / L - approximately 14.8 g / L, approximately 3.0 g / L - approximately 14.6 g / L, approximately 3.0 g / L - approximately 14.4 g / L, approximately 3.0 g / L - approximately 14.2 g / L, approximately 3.0 g / L - approximately 14.0 g / L, approximately 3.0 g / L - approximately 13.8 g / L, approximately 3.0 g / L - approximately 13.6 g / L, approximately 3.0 g / L - approximately 13.4 g / L g / L, approximately 3.0 g / L to approximately 13.2 g / L, approximately 3.0 g / L to approximately 13.0 g / L, approximately 3.0 g / L to approximately 12.8 g / L, approximately 3.0 g / L to approximately 12.6 g / L, approximately 3.0 g / L to approximately 12.4 g / L, approximately 3.0 g / L to approximately 12.2 g / L, approximately 3.0 g / L to approximately 12.0 g / L, approximately 3.0 g / L to approximately 11.8 g / L, approximately 3.0 g / L to approximately 11.6 g / L, approximately 3.0 g / L to approximately 11.4 g / L, approximately 3.0 g / L to approximately 11.2 g / L, approximately 3.0 g / L to approximately 11.0 g / L, approximately 3.0 g / L to approximately 10.8 g / L, approximately 3.0 g / L to approximately 10.6 g / L. Approximately 3.0 g / L - approximately 10.4 g / L, approximately 3.0 g / L - approximately 10.2 g / L, approximately 3.0 g / L - approximately 10.0 g / L, approximately 3.0 g / L - approximately 9.8 g / L, approximately 3.0 g / L - approximately 9.6 g / L, approximately 3.0 g / L - approximately 9.4 g / L, approximately 3.0 g / L - approximately 9.2 g / L, approximately 3.0 g / L - approximately 9.0 g / L, approximately 3.0 g / L - approximately 8.8 g / L, approximately 3.0 g / L - approximately 8.6 g / L, approximately 3.0 g / L - approx. 8.4 g / L, approx. 3.0 g / L - approx. 8.2 g / L, approx. 3.0 g / L - approx. 8.0 g / L, approx. 3.0 g / L - approx. 7.8 g / L, approx. 3.0 g / L - approx. 7.6 g / L, approx. 3.0 g / L - approx. 7.4 g / L, approx. 3.0 g / L - approx. 7.2 g / L, approx. 3.0 g / L - approx. 7.0 g / L, approx. 3.0 g / L - approx. 6.9 g / L, approx. 3.0 g / L - approx. 6.8 g / L, approx. 3.0 g / L - approx. 6.6 g / L, approx. 3.0 g / L - approx. 6.4 g / L, approx. 3.0 g / L - approx. 6.2 g / L, approx. 3.0 g / L - approx. 6.0 g / L, approx. 3.0 g / L - approx. 5.8 g / L, approx. 3.0 g / L - approximately 5.6 g / L, approximately 3.0 g / L - approximately 5.4 g / L, approximately 3.0 g / L - approximately 5.2 g / L, approximately 3.0 g / L - approximately 5.0 g / L, approximately 3.0 g / L - approximately 4.8 g / L, approximately 3.0 g / L - approximately 4.6 g / L, approximately 3.0 g / L - approximately 4.4 g / L, approximately 3.0 g / L - approximately 4.2 g / L, approximately 3.0 g / L - approximately 4.0 g / L, approximately 3.2 g / L - approximately 16.0 g / L, approximately 3.2 g / L - approximately 15.8 g / L, approximately 3.2 g / L - approximately 15.6 g / L, approximately 3.2 g / L - approximately 15.4 g / L, approximately 3.2 g / L - approximately 15.2 g / L, approximately 3.2 g / L - approximately 15.0 g / L, approximately 3.2 g / L - approximately 14.8 g / L, approximately 3.2 g / L - approximately 14.6 g / L, approximately 3.2 g / L - approximately 14.4 g / L, approximately 3.2 g / L - approximately 14.2 g / L, approximately 3.2 g / L - approximately 14.0 g / L, approximately 3.2 g / L - approximately 13.8 g / L, approximately 3.2 g / L - approximately 13.6 g / L, approximately 3.2 g / L - approximately 13.4 g / L, approximately 3.2 g / L - approximately 13.2 g / L, approximately 3.2 g / L - approximately 13.0 g / L, approximately 3.2 g / L - approximately 12.8 g / L, approximately 3.2 g / L - approximately 12.6 g / L, approximately 3.2 g / L - approximately 12.4 g / L, approximately 3.2 g / L - approximately 12.2 g / L, approximately 3.2 g / L - about 12.0 g / L, about 3.2 g / L - about 11.8 g / L, about 3.2 g / L - about 11.6 g / L, about 3.2 g / L - about 11.4 g / L, about 3.2 g / L - about 11.2 g / L, about 3.2 g / L - about 11.0 g / L, about 3.2 g / L - about 10.8 g / L, about 3.2 g / L - about 10.6 g / L, about 3.2 g / L - about 10.4 g / L, about 3.2 g / L - about 10.2 g / L, about 3.2 g / L - about 10.0 g / L, about 3.2 g / L - about 9.8 g / L, about 3.2 g / L - about 9.6 g / L, about 3.2 g / L - about 9.4 g / L, about 3.2 g / L - about 9.2 g / L, about 3.2 g / L - about 9.0 g / L, about 3.2 g / L - about 8.8 g / L, about 3.2 g / L - about 8.6 g / L, about 3.2 g / L - about 8.4 g / L, about 3.2 g / L - about 8.2 g / L, about 3.2 g / L - about 8.0 g / L, about 3.2 g / L - about 7.8 g / L, about 3.2 g / L - about 7.6 g / L, about 3.2 g / L - about 7.4 g / L, about 3.2 g / L - about 7.2 g / L, about 3.2 g / L - approximately 7.0 g / L, approximately 3.2 g / L - approximately 6.9 g / L, approximately 3.2 g / L - approximately 6.8 g / L, approximately 3.2 g / L - approximately 6.6 g / L, approximately 3.2 g / L - approximately 6.4 g / L, approximately 3.2 g / L - approximately 6.2 g / L, approximately 3.2 g / L - approximately 6.0 g / L, approximately 3.2 g / L - approximately 5.8 g / L, approximately 3.2 g / L - approximately 5.6 g / L, approximately 3.2 g / L - approximately 5.4 g / L, approximately 3.2 g / L - approximately 5.2 g / L, approximately 3.2 g / L - approximately 5.0 g / L, approximately 3.2 g / L - approximately 4.8 g / L, approximately 3.2 g / L - approximately 4.6 g / L, approximately 3.2 g / L - approximately 4.4 g / L, approximately 3.2 g / L - approximately 4.2 g / L, approximately 3.4 g / L - approximately 16.0 g / L, approximately 3.4 g / L - approximately 15.8 g / L, approximately 3.4 g / L - approximately 15.6 g / L, approximately 3.4 g / L - approximately 15.4 g / L, approximately 3.4 g / L - approximately 15.2 g / L, approximately 3.4 g / L - approximately 15.0 g / L, approximately 3.4 g / L - approximately 14.8 g / L, approximately 3.4 g / L - approximately 14.6 g / L, approximately 3.4 g / L - approximately 14.4 g / L, approximately 3.4 g / L - approximately 14.2 g / L, approximately 3.4 g / L - approximately 14.0 g / L, approximately 3.4 g / L - approximately 13.8 g / L, approximately 3.4 g / L - approximately 13.6 g / L, approximately 3.4 g / L - approximately 13.4 g / L, approximately 3.4 g / L - approximately 13.2 g / L, approximately 3.4 g / L - approximately 13.0 g / L, approximately 3.4 g / L - approximately 12.8 g / L, approximately 3.4 g / L - approximately 12.6 g / L, approximately 3.4 g / L - approximately 12.4 g / L, approximately 3.4 g / L - approximately 12.2 g / L, approximately 3.4 g / L - approximately 12.0 g / L, approximately 3.4 g / L - approximately 11.8 g / L, approximately 3.4 g / L - approximately 11.6 g / L, approximately 3.4 g / L - approximately 11.4 g / L, approximately 3.4 g / L to approximately 11.2 g / L, approximately 3.4 g / L to approximately 11.0 g / L, approximately 3.4 g / L to approximately 10.8 g / L, approximately 3.4 g / L to approximately 10.6 g / L, approximately 3.4 g / L to approximately 10.4 g / L, approximately 3.4 g / L to approximately 10.2 g / L, approximately 3.4 g / L to approximately 10.0 g / L, approximately 3.4 g / L to approximately 9.8 g / L, approximately 3.4 g / L to approximately 9.6 g / L, approximately 3.4 g / L to approximately 9.4 g / L, approximately 3.4 g / L to approximately 9.2 g / L, approximately 3.4 g / L to approximately 9.0 g / L, approximately 3.4 g / L to approximately 8.8 g / L, approximately 3.4 g / L to approximately 8.6 g / L, approximately 3.4 g / L g / L - approximately 8.4 g / L, approximately 3.4 g / L - approximately 8.2 g / L, approximately 3.4 g / L - approximately 8.0 g / L, approximately 3.4 g / L - approximately 7.8 g / L, approximately 3.4 g / L - approximately 7.6 g / L, approximately 3.4 g / L - approximately 7.4 g / L, approximately 3.4 g / L - approximately 7.2 g / L, approximately 3.4 g / L - approximately 7.0 g / L, approximately 3.4 g / L - approximately 6.9 g / L, approximately 3.4 g / L - approximately 6.8 g / L, approximately 3.4 g / L - approximately 6.6 g / L, approximately 3.4 g / L - approximately 6.4 g / L, approximately 3.4 g / L - approximately 6.2 g / L, approximately 3.4 g / L - approximately 6.0 g / L, approximately 3.4 g / L - approximately 5.8 g / L, approximately 3.4 g / L - approximately 5.6 g / L, approximately 3.4 g / L - approximately 5.4 g / L, approximately 3.4 g / L - approximately 5.2 g / L, approximately 3.4 g / L - approximately 5.0 g / L, approximately 3.4 g / L - approximately 4.8 g / L, approximately 3.4 g / L - approximately 4.6 g / L, approximately 3.4 g / L - approximately 4.4 g / L, approximately 3.6 g / L - approximately 16.0 g / L, approximately 3.6 g / L - approximately 15.8 g / L, approximately 3.6 g / L - approximately 15.6 g / L, approximately 3.6 g / L - approximately 15.4 g / L, approximately 3.6 g / L - approximately 15.2 g / L, approximately 3.6 g / L - approximately 15.0 g / L, approximately 3.6 g / L - approximately 14.6 g / L g / L, approximately 3.6 g / L to approximately 14.4 g / L, approximately 3.6 g / L to approximately 14.2 g / L, approximately 3.6 g / L to approximately 14.0 g / L, approximately 3.6 g / L to approximately 13.8 g / L, approximately 3.6 g / L to approximately 13.6 g / L, approximately 3.6 g / L to approximately 13.4 g / L, approximately 3.6 g / L to approximately 13.2 g / L, approximately 3.6 g / L to approximately 13.0 g / L, approximately 3.6 g / L to approximately 12.8 g / L, approximately 3.6 g / L to approximately 12.6 g / L, approximately 3.6 g / L - approximately 12.4 g / L, approximately 3.6 g / L - approximately 12.2 g / L, approximately 3.6 g / L - approximately 12.0 g / L, approximately 3.6 g / L - approximately 11.8 g / L, approximately 3.6 g / L - approximately 11.6 g / L, approximately 3.6 g / L - approximately 11.4 g / L, approximately 3.6 g / L - approximately 11.2 g / L, approximately 3.6 g / L - approximately 11.0 g / L, approximately 3.6 g / L - approximately 10.8 g / L, approximately 3.6 g / L - approximately 10.6 g / L, approximately 3.6 g / L - approximately 10.4 g / L, approximately 3.6 g / L - approximately 10.2 g / L, approximately 3.6 g / L - approximately 10.0 g / L, approximately 3.6 g / L - approximately 9.8 g / L, approximately 3.6 g / L - approximately 9.6 g / L, approximately 3.6 g / L - approximately 9.4 g / L, approximately 3.6 g / L - approximately 9.2 g / L, approximately 3.6 g / L - approximately 9.0 g / L, approximately 3.6 g / L - approximately 8.8 g / L, approximately 3.6 g / L - approximately 8.6 g / L, approximately 3.6 g / L - approximately 8.4 g / L, approximately 3.6 g / L - approximately 8.2 g / L, approximately 3.6 g / L - approximately 8.0 g / L, approximately 3.6 g / L - approximately 7.8 g / L, approximately 3.6 g / L - approximately 7.6 g / L, approximately 3.6 g / L - approximately 7.4 g / L, approximately 3.6 g / L - approximately 7.2 g / L, approximately 3.6 g / L - approximately 7.0 g / L, approximately 3.6 g / L - approximately 6.9 g / L g / L, approximately 3.6 g / L to approximately 6.8 g / L, approximately 3.6 g / L to approximately 6.6 g / L, approximately 3.6 g / L to approximately 6.4 g / L, approximately 3.6 g / L to approximately 6.2 g / L, approximately 3.6 g / L to approximately 6.0 g / L, approximately 3.6 g / L to approximately 5.8 g / L, approximately 3.6 g / L to approximately 5.6 g / L, approximately 3.6 g / L to approximately 5.4 g / L, approximately 3.6 g / L to approximately 5.2 g / L, approximately 3.6 g / L to approximately 5.0 g / L, approximately 3.6 g / L to approximately 4.8 g / L, approximately 3.6 g / L to approximately 4.6 g / L, approximately 3.8 g / L to approximately 16.0 g / L, approximately 3.8 g / L to approximately 15.8 g / L, approximately 3.8 g / L to approximately 15.6 g / L. g / L, approximately 3.8 g / L to approximately 15.4 g / L, approximately 3.8 g / L to approximately 15.2 g / L, approximately 3.8 g / L to approximately 15.0 g / L, approximately 3.8 g / L to approximately 14.8 g / L, approximately 3.8 g / L to approximately 14.6 g / L, approximately 3.8 g / L to approximately 14.4 g / L, approximately 3.8 g / L to approximately 14.2 g / L, approximately 3.8 g / L to approximately 14.0 g / L, approximately 3.8 g / L to approximately 13.8 g / L, approximately 3.8 g / L to approximately 13.6 g / L, approximately 3.8 g / L - approximately 13.4 g / L, approximately 3.8 g / L - approximately 13.2 g / L, approximately 3.8 g / L - approximately 13.0 g / L, approximately 3.8 g / L - approximately 12.8 g / L, approximately 3.8 g / L - approximately 12.6 g / L, approximately 3.8 g / L - approximately 12.4 g / L, approximately 3.8 g / L - approximately 12.2 g / L, approximately 3.8 g / L - approximately 12.0 g / L, approximately 3.8 g / L - approximately 11.8 g / L, approximately 3.8 g / L - approximately 11.6 g / L, approximately 3.8 g / L - approximately 11.4 g / L, approximately 3.8 g / L - approximately 11.2 g / L, approximately 3.8 g / L - approximately 11.0 g / L, approximately 3.8 g / L - approximately 10.8 g / L, approximately 3.8 g / L - approximately 10.6 g / L, approximately 3.8 g / L - approximately 10.4 g / L, approximately 3.8 g / L - approximately 10.2 g / L, approximately 3.8 g / L - approximately 10.0 g / L, approximately 3.8 g / L - approximately 9.8 g / L, approximately 3.8 g / L - approximately 9.6 g / L, approximately 3.8 g / L - approximately 9.4 g / L, approximately 3.8 g / L - approximately 9.2 g / L, approximately 3.8 g / L - approximately 9.0 g / L, approximately 3.8 g / L - approximately 8.8 g / L, approximately 3.8 g / L - approximately 8.6 g / L, approximately 3.8 g / L - approximately 8.4 g / L, approximately 3.8 g / L - approximately 8.2 g / L, approximately 3.8 g / L - approximately 8.0 g / L, approximately 3.8 g / L - approximately 7.8 g / L, approximately 3.8 g / L - approximately 7.6 g / L, approximately 3.8 g / L - approximately 7.4 g / L, approximately 3.8 g / L - approximately 7.2 g / L, approximately 3.8 g / L - approximately 7.0 g / L, approximately 3.8 g / L - approximately 6.9 g / L, approximately 3.8 g / L - approximately 6.8 g / L, approximately 3.8 g / L - approximately 6.6 g / L, approximately 3.8 g / L - approximately 6.4 g / L, approximately 3.8 g / L - approximately 6.2 g / L, approximately 3.8 g / L - approximately 6.0 g / L, approximately 3.8 g / L - approximately 5.8 g / L, approximately 3.8 g / L - approximately 5.6 g / L, approximately 3.8 g / L - approximately 5.4 g / L, approximately 3.8 g / L - approximately 5.2 g / L, approximately 3.8 g / L - approximately 5.0 g / L, approximately 3.8 g / L - approximately 4.8 g / L, approximately 4.0 g / L - approximately 16.0 g / L, approximately 4.0 g / L - approximately 15.8 g / L, approximately 4.0 g / L - approximately 15.6 g / L, approximately 4.0 g / L - approximately 15.4 g / L, approximately 4.0 g / L - approximately 15.2 g / L, approximately 4.0 g / L - approximately 15.0 g / L, approximately 4.0 g / L - approximately 14.8 g / L, approximately 4.0 g / L - approximately 14.6 g / L, approximately 4.0 g / L - approximately 14.4 g / L, approximately 4.0 g / L - approx. 14.2 g / L, approx. 4.0 g / L - approx. 14.0 g / L, approx. 4.0 g / L - approx. 13.8 g / L, approx. 4.0 g / L - approx. 13.6 g / L, approx. 4.0 g / L - approx. 13.4 g / L, approx. 4.0 g / L - approx. 12.8 g / L, approx. 4.0 g / L - approx. 12.6 g / L, approx. 4.0 g / L - approx. 12.4 g / L, approx. 4.0 g / L - approx. 12.2 g / L, approx. 4.0 g / L - approx. 12.0 g / L, approx. 4.0 g / L - approx. 11.8 g / L, approx. 4.0 g / L - approx. 11.6 g / L, approx. 4.0 g / L g / L - approximately 11.4 g / L, approximately 4.0 g / L - approximately 11.2 g / L, approximately 4.0 g / L - approximately 11.0 g / L, approximately 4.0 g / L - approximately 10.8 g / L, approximately 4.0 g / L - approximately 10.6 g / L, approximately 4.0 g / L - approximately 10.4 g / L, approximately 4.0 g / L - approximately 10.2 g / L, approximately 4.0 g / L - approximately 10.0 g / L, approximately 4.0 g / L - approximately 9.8 g / L, approximately 4.0 g / L - approximately 9.6 g / L, approximately 4.0 g / L - approximately 9.4 g / L, approximately 4.0 g / L - approximately 9.2 g / L, approximately 4.0 g / L - approximately 9.0 g / L, approximately 4.0 g / L - approximately 8.8 g / L, approximately 4.0 g / L - approximately 8.6 g / L. g / L, approximately 4.0 g / L to approximately 8.4 g / L, approximately 4.0 g / L to approximately 8.2 g / L, approximately 4.0 g / L to approximately 8.0 g / L, approximately 4.0 g / L to approximately 7.8 g / L, approximately 4.0 g / L to approximately 7.6 g / L, approximately 4.0 g / L to approximately 7.4 g / L, approximately 4.0 g / L to approximately 7.2 g / L, approximately 4.0 g / L to approximately 7.0 g / L, approximately 4.0 g / L to approximately 6.9 g / L, approximately 4.0 g / L to approximately 6.8 g / L, approximately 4.0 g / L to approximately 6.6 g / L, approximately 4.0 g / L to approximately 6.4 g / L, approximately 4.0 g / L to approximately 6.2 g / L, approximately 4.0 g / L to approximately 6.0 g / L, approximately 4.0 g / L to approximately 5.8 g / L. g / L, approximately 4.0 g / L to approximately 5.6 g / L, approximately 4.0 g / L to approximately 5.4 g / L, approximately 4.0 g / L to approximately 5.2 g / L, approximately 4.0 g / L to approximately 5.0 g / L, approximately 4.2 g / L to approximately 16.0 g / L, approximately 4.2 g / L to approximately 15.8 g / L, approximately 4.2 g / L to approximately 15.6 g / L, approximately 4.2 g / L to approximately 15.4 g / L, approximately 4.2 g / L to approximately 15.2 g / L, approximately 4.2 g / L to approximately 15.0 g / L, approximately 4.2 g / L - approximately 14.8 g / L, approximately 4.2 g / L - approximately 14.6 g / L, approximately 4.2 g / L - approximately 14.4 g / L, approximately 4.2 g / L - approximately 14.2 g / L, approximately 4.2 g / L - approximately 14.0 g / L, approximately 4.2 g / L - approximately 13.8 g / L, approximately 4.2 g / L - approximately 13.6 g / L, approximately 4.2 g / L - approximately 13.4 g / L, approximately 4.2 g / L - approximately 13.2 g / L, approximately 4.2 g / L - approximately 13.0 g / L, approximately 4.2 g / L - approximately 12.8 g / L, approximately 4.2 g / L - approximately 12.6 g / L, approximately 4.2 g / L - approximately 12.4 g / L, approximately 4.2 g / L - approximately 12.2 g / L, approximately 4.2 g / L - approximately 12.0 g / L, approximately 4.2 g / L - approximately 11.8 g / L, approximately 4.2 g / L - approximately 11.6 g / L, approximately 4.2 g / L - approximately 11.4 g / L, approximately 4.2 g / L - approximately 11.2 g / L, approximately 4.2 g / L - approximately 11.0 g / L, approximately 4.2 g / L - approximately 10.8 g / L, approximately 4.2 g / L - approximately 10.6 g / L, approximately 4.2 g / L - approximately 10.4 g / L, approximately 4.2 g / L - approximately 10.2 g / L, approximately 4.2 g / L - approximately 10.0 g / L, approximately 4.2 g / L - approximately 9.8 g / L, approximately 4.2 g / L - approximately 9.6 g / L, approximately 4.2 g / L - approximately 9.4 g / L, approximately 4.2 g / L - approximately 9.2 g / L g / L, approximately 4.2 g / L to approximately 9.0 g / L, approximately 4.2 g / L to approximately 8.8 g / L, approximately 4.2 g / L to approximately 8.6 g / L, approximately 4.2 g / L to approximately 8.4 g / L, approximately 4.2 g / L to approximately 8.2 g / L, approximately 4.2 g / L to approximately 8.0 g / L, approximately 4.2 g / L to approximately 7.8 g / L, approximately 4.2 g / L to approximately 7.6 g / L, approximately 4.2 g / L to approximately 7.4 g / L, approximately 4.2 g / L to approximately 7.2 g / L, approximately 4.2 g / L to approximately 7.0 g / L, approximately 4.2 g / L to approximately 6.9 g / L, approximately 4.2 g / L to approximately 6.8 g / L, approximately 4.2 g / L to approximately 6.6 g / L, approximately 4.2 g / L to approximately 6.4 g / L. g / L, approximately 4.2 g / L to approximately 6.2 g / L, approximately 4.2 g / L to approximately 6.0 g / L, approximately 4.2 g / L to approximately 5.8 g / L, approximately 4.2 g / L to approximately 5.6 g / L, approximately 4.2 g / L to approximately 5.4 g / L, approximately 4.2 g / L to approximately 5.2 g / L, approximately 4.4 g / L to approximately 16.0 g / L, approximately 4.4 g / L to approximately 15.8 g / L, approximately 4.4 g / L to approximately 15.6 g / L, approximately 4.4 g / L to approximately 15.4 g / L, approximately 4.4 g / L - approximately 15.2 g / L, approximately 4.4 g / L - approximately 15.0 g / L, approximately 4.4 g / L - approximately 14.8 g / L, approximately 4.4 g / L - approximately 14.6 g / L, approximately 4.4 g / L - approximately 14.4 g / L, approximately 4.4 g / L - approximately 14.2 g / L, approximately 4.4 g / L - approximately 14.0 g / L, approximately 4.4 g / L - approximately 13.8 g / L, approximately 4.4 g / L - approximately 13.6 g / L, approximately 4.4 g / L - approximately 13.4 g / L, approximately 4.4 g / L - approximately 13.2 g / L, approximately 4.4 g / L - approximately 13.0 g / L, approximately 4.4 g / L - approximately 12.8 g / L, approximately 4.4 g / L - approximately 12.6 g / L, approximately 4.4 g / L - approximately 12.4 g / L, approximately 4.4 g / L - approximately 12.2 g / L, approximately 4.4 g / L - approximately 12.0 g / L, approximately 4.4 g / L - approximately 11.8 g / L, approximately 4.4 g / L - approximately 11.6 g / L, approximately 4.4 g / L - approximately 11.4 g / L, approximately 4.4 g / L - approximately 11.2 g / L, approximately 4.4 g / L - approximately 11.0 g / L, approximately 4.4 g / L - approximately 10.8 g / L, approximately 4.4 g / L - approximately 10.6 g / L, approximately 4.4 g / L - approximately 10.4 g / L, approximately 4.4 g / L - approximately 10.2 g / L, approximately 4.4 g / L - approximately 10.0 g / L, approximately 4.4 g / L - approximately 9.8 g / L, approximately 4.4 g / L - approximately 9.6 g / L g / L, approximately 4.4 g / L to approximately 9.4 g / L, approximately 4.4 g / L to approximately 9.2 g / L, approximately 4.4 g / L to approximately 9.0 g / L, approximately 4.4 g / L to approximately 8.8 g / L, approximately 4.4 g / L to approximately 8.6 g / L, approximately 4.4 g / L to approximately 8.4 g / L, approximately 4.4 g / L to approximately 8.2 g / L, approximately 4.4 g / L to approximately 8.0 g / L, approximately 4.4 g / L to approximately 7.8 g / L, approximately 4.4 g / L to approximately 7.6 g / L, approximately 4.4 g / L to approximately 7.4 g / L, approximately 4.4 g / L to approximately 7.2 g / L, approximately 4.4 g / L to approximately 7.0 g / L, approximately 4.4 g / L to approximately 6.9 g / L, approximately 4.4 g / L to approximately 6.8 g / L. g / L, approximately 4.4 g / L to approximately 6.6 g / L, approximately 4.4 g / L to approximately 6.4 g / L, approximately 4.4 g / L to approximately 6.2 g / L, approximately 4.4 g / L to approximately 6.0 g / L, approximately 4.4 g / L to approximately 5.8 g / L, approximately 4.4 g / L to approximately 5.6 g / L, approximately 4.4 g / L to approximately 5.4 g / L, approximately 4.6 g / L to approximately 16.0 g / L, approximately 4.6 g / L to approximately 15.8 g / L, approximately 4.6 g / L to approximately 15.6 g / L, approximately 4.6 g / L - approximately 15.4 g / L, approximately 4.6 g / L - approximately 15.2 g / L, approximately 4.6 g / L - approximately 15.0 g / L, approximately 4.6 g / L - approximately 14.8 g / L, approximately 4.6 g / L - approximately 14.6 g / L, approximately 4.6 g / L - approximately 14.4 g / L, approximately 4.6 g / L - approximately 14.2 g / L, approximately 4.6 g / L - approximately 14.0 g / L, approximately 4.6 g / L - approximately 13.8 g / L, approximately 4.6 g / L - approximately 13.6 g / L, approximately 4.6 g / L - approximately 13.4 g / L, approximately 4.6 g / L - approximately 13.2 g / L, approximately 4.6 g / L - approximately 13.0 g / L, approximately 4.6 g / L - approximately 12.8 g / L, approximately 4.6 g / L - approximately 12.6 g / L, approximately 4.6 g / L - approximately 12.4 g / L, approximately 4.6 g / L - approximately 12.2 g / L, approximately 4.6 g / L - approximately 12.0 g / L, approximately 4.6 g / L - approximately 11.8 g / L, approximately 4.6 g / L - approximately 11.6 g / L, approximately 4.6 g / L - approximately 11.4 g / L, approximately 4.6 g / L - approximately 11.2 g / L, approximately 4.6 g / L - approximately 11.0 g / L, approximately 4.6 g / L - approximately 10.8 g / L, approximately 4.6 g / L - approximately 10.6 g / L, approximately 4.6 g / L - approximately 10.4 g / L, approximately 4.6 g / L - approximately 10.2 g / L, approximately 4.6 g / L - approximately 10.0 g / L, approximately 4.6 g / L - approximately 9.8 g / L g / L, approximately 4.6 g / L to approximately 9.6 g / L, approximately 4.6 g / L to approximately 9.4 g / L, approximately 4.6 g / L to approximately 9.2 g / L, approximately 4.6 g / L to approximately 9.0 g / L, approximately 4.6 g / L to approximately 8.8 g / L, approximately 4.6 g / L to approximately 8.6 g / L, approximately 4.6 g / L to approximately 8.4 g / L, approximately 4.6 g / L to approximately 8.2 g / L, approximately 4.6 g / L to approximately 8.0 g / L, approximately 4.6 g / L to approximately 7.8 g / L, approximately 4.6 g / L to approximately 7.6 g / L, approximately 4.6 g / L to approximately 7.4 g / L, approximately 4.6 g / L to approximately 7.2 g / L, approximately 4.6 g / L to approximately 7.0 g / L, approximately 4.6 g / L to approximately 6.9 g / L. g / L, approximately 4.6 g / L to approximately 6.8 g / L, approximately 4.6 g / L to approximately 6.6 g / L, approximately 4.6 g / L to approximately 6.4 g / L, approximately 4.6 g / L to approximately 6.2 g / L, approximately 4.6 g / L to approximately 6.0 g / L, approximately 4.6 g / L to approximately 5.8 g / L, approximately 4.6 g / L to approximately 5.6 g / L, approximately 4.8 g / L to approximately 16.0 g / L, approximately 4.8 g / L to approximately 15.8 g / L, approximately 4.8 g / L to approximately 15.6 g / L, approximately 4.8 g / L - approximately 15.4 g / L, approximately 4.8 g / L - approximately 15.2 g / L, approximately 4.8 g / L - approximately 15.0 g / L, approximately 4.8 g / L - approximately 14.8 g / L, approximately 4.8 g / L - approximately 14.6 g / L, approximately 4.8 g / L - approximately 14.4 g / L, approximately 4.8 g / L - approximately 14.2 g / L, approximately 4.8 g / L - approximately 14.0 g / L, approximately 4.8 g / L - approximately 13.8 g / L, approximately 4.8 g / L - approximately 13.6 g / L, approximately 4.8 g / L - approximately 13.4 g / L, approximately 4.8 g / L - approximately 13.2 g / L, approximately 4.8 g / L - approximately 13.0 g / L, approximately 4.8 g / L - approximately 12.8 g / L, approximately 4.8 g / L - approximately 12.6 g / L, approximately 4.8 g / L - approximately 12.4 g / L, approximately 4.8 g / L - approximately 12.2 g / L, approximately 4.8 g / L - approximately 12.0 g / L, approximately 4.8 g / L - approximately 11.8 g / L, approximately 4.8 g / L - approximately 11.6 g / L, approximately 4.8 g / L - approximately 11.4 g / L, approximately 4.8 g / L - approximately 11.2 g / L, approximately 4.8 g / L - approximately 11.0 g / L, approximately 4.8 g / L - approximately 10.8 g / L, approximately 4.8 g / L - approximately 10.6 g / L, approximately 4.8 g / L - approximately 10.4 g / L, approximately 4.8 g / L - approximately 10.2 g / L, approximately 4.8 g / L - approximately 10.0 g / L, approximately 4.8 g / L - approximately 9.8 g / L, approximately 4.8 g / L to approximately 9.6 g / L, approximately 4.8 g / L to approximately 9.4 g / L, approximately 4.8 g / L to approximately 9.2 g / L, approximately 4.8 g / L to approximately 9.0 g / L, approximately 4.8 g / L to approximately 8.8 g / L, approximately 4.8 g / L to approximately 8.6 g / L, approximately 4.8 g / L to approximately 8.4 g / L, approximately 4.8 g / L to approximately 8.2 g / L, approximately 4.8 g / L to approximately 8.0 g / L, approximately 4.8 g / L to approximately 7.8 g / L, approximately 4.8 g / L to approximately 7.6 g / L, approximately 4.8 g / L to approximately 7.4 g / L, approximately 4.8 g / L to approximately 7.2 g / L, approximately 4.8 g / L to approximately 7.0 g / L, approximately 4.8 g / L to approximately 6.9 g / L. g / L, approximately 4.8 g / L to approximately 6.8 g / L, approximately 4.8 g / L to approximately 6.6 g / L, approximately 4.8 g / L to approximately 6.4 g / L, approximately 4.8 g / L to approximately 6.2 g / L, approximately 4.8 g / L to approximately 6.0 g / L, approximately 4.8 g / L to approximately 5.8 g / L, approximately 5.0 g / L to approximately 16.0 g / L, approximately 5.0 g / L to approximately 15.8 g / L, approximately 5.0 g / L to approximately 15.6 g / L, approximately 5.0 g / L to approximately 15.4 g / L, approximately 5.0 g / L - approx. 15.2 g / L, approx. 5.0 g / L - approx. 15.0 g / L, approx. 5.0 g / L - approx. 14.8 g / L, approx. 5.0 g / L - approx. 14.6 g / L, approx. 5.0 g / L - approx. 14.4 g / L, approx. 5.0 g / L - approx. 14.0 g / L, approx. 5.0 g / L - approx. 13.8 g / L, approx. 5.0 g / L - approx. 13.6 g / L, approx. 5.0 g / L - approx. 13.4 g / L, approx. 5.0 g / L - approx. 13.2 g / L, approx. 5.0 g / L - approx. 13.0 g / L, approx. 5.0 g / L - approx. 12.8 g / L, approx. 5.0 g / L - approx. 12.6 g / L, approx. 5.0 g / L g / L - approximately 12.4 g / L, approximately 5.0 g / L - approximately 12.2 g / L, approximately 5.0 g / L - approximately 12.0 g / L, approximately 5.0 g / L - approximately 11.8 g / L, approximately 5.0 g / L - approximately 11.6 g / L, approximately 5.0 g / L - approximately 11.4 g / L, approximately 5.0 g / L - approximately 11.2 g / L, approximately 5.0 g / L - approximately 11.0 g / L, approximately 5.0 g / L - approximately 10.8 g / L, approximately 5.0 g / L - approximately 10.6 g / L, approximately 5.0 g / L - approximately 10.4 g / L, approximately 5.0 g / L - approximately 10.2 g / L, approximately 5.0 g / L - approximately 10.0 g / L, approximately 5.0 g / L - approximately 9.8 g / L, approximately 5.0 g / L - approximately 9.6 g / L g / L, approximately 5.0 g / L to approximately 9.4 g / L, approximately 5.0 g / L to approximately 9.2 g / L, approximately 5.0 g / L to approximately 9.0 g / L, approximately 5.0 g / L to approximately 8.8 g / L, approximately 5.0 g / L to approximately 8.6 g / L, approximately 5.0 g / L to approximately 8.4 g / L, approximately 5.0 g / L to approximately 8.2 g / L, approximately 5.0 g / L to approximately 8.0 g / L, approximately 5.0 g / L to approximately 7.8 g / L, approximately 5.0 g / L to approximately 7.6 g / L, approximately 5.0 g / L to approximately 7.4 g / L, approximately 5.0 g / L to approximately 7.2 g / L, approximately 5.0 g / L to approximately 7.0 g / L, approximately 5.0 g / L to approximately 6.9 g / L, approximately 5.0 g / L to approximately 6.8 g / L. g / L, approximately 5.0 g / L to approximately 6.6 g / L, approximately 5.0 g / L to approximately 6.4 g / L, approximately 5.0 g / L to approximately 6.2 g / L, approximately 5.0 g / L to approximately 6.0 g / L, approximately 5.1 g / L to approximately 16.0 g / L, approximately 5.1 g / L to approximately 15.8 g / L, approximately 5.1 g / L to approximately 15.6 g / L, approximately 5.1 g / L to approximately 15.4 g / L, approximately 5.1 g / L to approximately 15.2 g / L, approximately 5.1 g / L to approximately 15.0 g / L, approximately 5.1 g / L - approximately 14.8 g / L, approximately 5.1 g / L - approximately 14.6 g / L, approximately 5.1 g / L - approximately 14.4 g / L, approximately 5.1 g / L - approximately 14.2 g / L, approximately 5.1 g / L - approximately 14.0 g / L, approximately 5.1 g / L - approximately 13.8 g / L, approximately 5.1 g / L - approximately 13.6 g / L, approximately 5.1 g / L - approximately 13.4 g / L, approximately 5.1 g / L - approximately 13.2 g / L, approximately 5.1 g / L - approximately 13.0 g / L, approximately 5.1 g / L - approximately 12.8 g / L, approximately 5.1 g / L - approximately 12.6 g / L, approximately 5.1 g / L - approximately 12.4 g / L, approximately 5.1 g / L - approximately 12.2 g / L, approximately 5.1 g / L - approximately 12.0 g / L, approximately 5.1 g / L - approximately 11.8 g / L, approximately 5.1 g / L - approximately 11.6 g / L, approximately 5.1 g / L - approximately 11.4 g / L, approximately 5.1 g / L - approximately 11.2 g / L, approximately 5.1 g / L - approximately 11.0 g / L, approximately 5.1 g / L - approximately 10.8 g / L, approximately 5.1 g / L - approximately 10.6 g / L, approximately 5.1 g / L - approximately 10.4 g / L, approximately 5.1 g / L - approximately 10.2 g / L, approximately 5.1 g / L - approximately 10.0 g / L, approximately 5.1 g / L - approximately 9.8 g / L, approximately 5.1 g / L - approximately 9.6 g / L, approximately 5.1 g / L - approximately 9.4 g / L, approximately 5.1 g / L - approximately 9.2 g / L g / L, approximately 5.1 g / L to approximately 9.0 g / L, approximately 5.1 g / L to approximately 8.8 g / L, approximately 5.1 g / L to approximately 8.6 g / L, approximately 5.1 g / L to approximately 8.4 g / L, approximately 5.1 g / L to approximately 8.2 g / L, approximately 5.1 g / L to approximately 8.0 g / L, approximately 5.1 g / L to approximately 7.8 g / L, approximately 5.1 g / L to approximately 7.6 g / L, approximately 5.1 g / L to approximately 7.4 g / L, approximately 5.1 g / L to approximately 7.2 g / L, approximately 5.1 g / L to approximately 7.0 g / L, approximately 5.1 g / L to approximately 6.9 g / L, approximately 5.1 g / L to approximately 6.8 g / L, approximately 5.1 g / L to approximately 6.6 g / L, approximately 5.1 g / L to approximately 6.4 g / L. g / L, approximately 5.1 g / L to approximately 6.2 g / L, approximately 5.1 g / L to approximately 6.1 g / L, approximately 5.2 g / L to approximately 16.0 g / L, approximately 5.2 g / L to approximately 15.8 g / L, approximately 5.2 g / L to approximately 15.6 g / L, approximately 5.2 g / L to approximately 15.4 g / L, approximately 5.2 g / L to approximately 15.2 g / L, approximately 5.2 g / L to approximately 15.0 g / L, approximately 5.2 g / L to approximately 14.8 g / L, approximately 5.2 g / L to approximately 14.6 g / L, approximately 5.2 g / L - approximately 14.4 g / L, approximately 5.2 g / L - approximately 14.2 g / L, approximately 5.2 g / L - approximately 14.0 g / L, approximately 5.2 g / L - approximately 13.8 g / L, approximately 5.2 g / L - approximately 13.6 g / L, approximately 5.2 g / L - approximately 13.4 g / L, approximately 5.2 g / L - approximately 13.2 g / L, approximately 5.2 g / L - approximately 13.0 g / L, approximately 5.2 g / L - approximately 12.8 g / L, approximately 5.2 g / L - approximately 12.6 g / L, approximately 5.2 g / L - approximately 12.4 g / L, approximately 5.2 g / L - approximately 12.2 g / L, approximately 5.2 g / L - approximately 12.0 g / L, approximately 5.2 g / L - approximately 11.8 g / L, approximately 5.2 g / L - approximately 11.6 g / L, approximately 5.2 g / L - approximately 11.4 g / L, approximately 5.2 g / L - approximately 11.2 g / L, approximately 5.2 g / L - approximately 11.0 g / L, approximately 5.2 g / L - approximately 10.8 g / L, approximately 5.2 g / L - approximately 10.6 g / L, approximately 5.2 g / L - approximately 10.4 g / L, approximately 5.2 g / L - approximately 10.2 g / L, approximately 5.2 g / L - approximately 10.0 g / L, approximately 5.2 g / L - approximately 9.8 g / L, approximately 5.2 g / L - approximately 9.6 g / L, approximately 5.2 g / L - approximately 9.4 g / L, approximately 5.2 g / L - approximately 9.2 g / L, approximately 5.2 g / L - approximately 9.0 g / L, approximately 5.2 g / L - approximately 8.8 g / L g / L, approximately 5.2 g / L to approximately 8.6 g / L, approximately 5.2 g / L to approximately 8.4 g / L, approximately 5.2 g / L to approximately 8.2 g / L, approximately 5.2 g / L to approximately 8.0 g / L, approximately 5.2 g / L to approximately 7.8 g / L, approximately 5.2 g / L to approximately 7.6 g / L, approximately 5.2 g / L to approximately 7.4 g / L, approximately 5.2 g / L to approximately 7.2 g / L, approximately 5.2 g / L to approximately 7.0 g / L, approximately 5.2 g / L to approximately 6.9 g / L, approximately 5.2 g / L to approximately 6.8 g / L, approximately 5.2 g / L to approximately 6.6 g / L, approximately 5.2 g / L to approximately 6.4 g / L, approximately 5.2 g / L to approximately 6.2 g / L, approximately 5.4 g / L to approximately 16.0 g / L. g / L, approximately 5.4 g / L to approximately 15.8 g / L, approximately 5.4 g / L to approximately 15.6 g / L, approximately 5.4 g / L to approximately 15.4 g / L, approximately 5.4 g / L to approximately 15.2 g / L, approximately 5.4 g / L to approximately 15.0 g / L, approximately 5.4 g / L to approximately 14.8 g / L, approximately 5.4 g / L to approximately 14.6 g / L, approximately 5.4 g / L to approximately 14.4 g / L, approximately 5.4 g / L to approximately 14.2 g / L, approximately 5.4 g / L to approximately 14.0 g / L, approximately 5.4 g / L - approximately 13.8 g / L, approximately 5.4 g / L - approximately 13.6 g / L, approximately 5.4 g / L - approximately 13.4 g / L, approximately 5.4 g / L - approximately 13.2 g / L, approximately 5.4 g / L - approximately 13.0 g / L, approximately 5.4 g / L - approximately 12.8 g / L, approximately 5.4 g / L - approximately 12.6 g / L, approximately 5.4 g / L - approximately 12.4 g / L, approximately 5.4 g / L - approximately 12.2 g / L, approximately 5.4 g / L - approximately 12.0 g / L, approximately 5.4 g / L - approximately 11.8 g / L, approximately 5.4 g / L - approximately 11.6 g / L, approximately 5.4 g / L - approximately 11.4 g / L, approximately 5.4 g / L - approximately 11.2 g / L, approximately 5.4 g / L - approximately 11.0 g / L, approximately 5.4 g / L - approximately 10.8 g / L, approximately 5.4 g / L - approximately 10.6 g / L, approximately 5.4 g / L - approximately 10.4 g / L, approximately 5.4 g / L - approximately 10.2 g / L, approximately 5.4 g / L - approximately 10.0 g / L, approximately 5.4 g / L - approximately 9.8 g / L, approximately 5.4 g / L - approximately 9.6 g / L, approximately 5.4 g / L - approximately 9.4 g / L, approximately 5.4 g / L - approximately 9.2 g / L, approximately 5.4 g / L - approximately 9.0 g / L, approximately 5.4 g / L - approximately 8.8 g / L, approximately 5.4 g / L - approximately 8.6 g / L, approximately 5.4 g / L - approximately 8.4 g / L, approximately 5.4 g / L - approximately 8.2 g / L, approximately 5.4 g / L g / L - approximately 8.0 g / L, approximately 5.4 g / L - approximately 7.8 g / L, approximately 5.4 g / L - approximately 7.6 g / L, approximately 5.4 g / L - approximately 7.4 g / L, approximately 5.4 g / L - approximately 7.2 g / L, approximately 5.4 g / L - approximately 7.0 g / L, approximately 5.4 g / L - approximately 6.9 g / L, approximately 5.4 g / L - approximately 6.8 g / L, approximately 5.4 g / L - approximately 6.6 g / L, approximately 5.4 g / L - approximately 6.4 g / L, approximately 5.6 g / L - approximately 16.0 g / L, approximately 5.6 g / L - approximately 15.8 g / L, approximately 5.6 g / L - approximately 15.6 g / L, approximately 5.6 g / L - approximately 15.4 g / L, approximately 5.6 g / L - approximately 15.2 g / L, approximately 5.6 g / L - about 15.0 g / L, about 5.6 g / L - about 14.8 g / L, about 5.6 g / L - about 14.6 g / L, about 5.6 g / L - about 14.4 g / L, about 5.6 g / L - about 14.2 g / L, about 5.6 g / L - about 14.0 g / L, about 5.6 g / L - about 13.8 g / L, about 5.6 g / L - about 13.6 g / L, about 5.6 g / L - about 13.4 g / L, about 5.6 g / L - about 13.2 g / L, approximately 5.6 g / L to approximately 13.0 g / L, approximately 5.6 g / L to approximately 12.8 g / L, approximately 5.6 g / L to approximately 12.6 g / L, approximately 5.6 g / L to approximately 12.4 g / L, approximately 5.6 g / L to approximately 12.2 g / L, approximately 5.6 g / L to approximately 12.0 g / L, approximately 5.6 g / L to approximately 11.8 g / L, approximately 5.6 g / L to approximately 11.6 g / L, approximately 5.6 g / L to approximately 11.4 g / L, approximately 5.6 g / L to approximately 11.2 g / L, approximately 5.6 g / L to approximately 11.0 g / L, approximately 5.6 g / L to approximately 10.8 g / L, approximately 5.6 g / L to approximately 10.6 g / L, approximately 5.6 g / L to approximately 10.4 g / L. g / L, approximately 5.6 g / L to approximately 10.2 g / L, approximately 5.6 g / L to approximately 10.0 g / L, approximately 5.6 g / L to approximately 9.8 g / L, approximately 5.6 g / L to approximately 9.6 g / L, approximately 5.6 g / L to approximately 9.4 g / L, approximately 5.6 g / L to approximately 9.2 g / L, approximately 5.6 g / L to approximately 9.0 g / L, approximately 5.6 g / L to approximately 8.8 g / L, approximately 5.6 g / L to approximately 8.6 g / L, approximately 5.6 g / L to approximately 8.4 g / L, approximately 5.6 g / L to approximately 8.2 g / L, approximately 5.6 g / L to approximately 8.0 g / L, approximately 5.6 g / L to approximately 7.8 g / L, approximately 5.6 g / L to approximately 7.6 g / L, approximately 5.6 g / L to approximately 7.4 g / L. g / L, approximately 5.6 g / L to approximately 7.2 g / L, approximately 5.6 g / L to approximately 7.0 g / L, approximately 5.6 g / L to approximately 6.9 g / L, approximately 5.6 g / L to approximately 6.8 g / L, approximately 5.6 g / L to approximately 6.6 g / L, approximately 5.8 g / L to approximately 16.0 g / L, approximately 5.8 g / L to approximately 15.8 g / L, approximately 5.8 g / L to approximately 15.6 g / L, approximately 5.8 g / L to approximately 15.4 g / L, approximately 5.8 g / L to approximately 15.2 g / L, approximately 5.8 g / L to approximately 15.0 g / L, approximately 5.8 g / L to approximately 14.8 g / L, approximately 5.8 g / L to approximately 14.6 g / L, approximately 5.8 g / L to approximately 14.4 g / L. g / L, approximately 5.8 g / L to approximately 14.2 g / L, approximately 5.8 g / L to approximately 14.0 g / L, approximately 5.8 g / L to approximately 13.8 g / L, approximately 5.8 g / L to approximately 13.6 g / L, approximately 5.8 g / L to approximately 13.4 g / L, approximately 5.8 g / L to approximately 13.2 g / L, approximately 5.8 g / L to approximately 13.0 g / L, approximately 5.8 g / L to approximately 12.8 g / L, approximately 5.8 g / L to approximately 12.6 g / L, approximately 5.8 g / L to approximately 12.4 g / L, approximately 5.8 g / L - approx. 12.2 g / L, approx. 5.8 g / L - approx. 12.0 g / L, approx. 5.8 g / L - approx. 11.8 g / L, approx. 5.8 g / L - approx. 11.6 g / L, approx. 5.8 g / L - approx. 11.4 g / L, approx. 5.8 g / L - approx. 11.2 g / L, approx. 5.8 g / L - approx. 11.0 g / L, approx. 5.8 g / L - approx. 10.8 g / L, approx. 5.8 g / L - approx. 10.6 g / L, approx. 5.8 g / L - approx. 10.4 g / L, approx. 5.8 g / L - approx. 10.2 g / L, approx. 5.8 g / L - approx. 10.0 g / L, approx. 5.8 g / L - approx. 9.8 g / L, approx. 5.8 g / L - approx. 9.6 g / L, approx. 5.8 g / L - approx. 9.4 g / L g / L, approximately 5.8 g / L to approximately 9.2 g / L, approximately 5.8 g / L to approximately 9.0 g / L, approximately 5.8 g / L to approximately 8.8 g / L, approximately 5.8 g / L to approximately 8.6 g / L, approximately 5.8 g / L to approximately 8.4 g / L, approximately 5.8 g / L to approximately 8.2 g / L, approximately 5.8 g / L to approximately 8.0 g / L, approximately 5.8 g / L to approximately 7.8 g / L, approximately 5.8 g / L to approximately 7.6 g / L, approximately 5.8 g / L to approximately 7.4 g / L, approximately 5.8 g / L to approximately 7.2 g / L, approximately 5.8 g / L to approximately 7.0 g / L, approximately 5.8 g / L to approximately 6.9 g / L, approximately 5.8 g / L to approximately 6.8 g / L, approximately 6.0 g / L to approximately 16.0 g / L. g / L, approximately 6.0 g / L to approximately 15.8 g / L, approximately 6.0 g / L to approximately 15.6 g / L, approximately 6.0 g / L to approximately 15.4 g / L, approximately 6.0 g / L to approximately 15.2 g / L, approximately 6.0 g / L to approximately 15.0 g / L, approximately 6.0 g / L to approximately 14.8 g / L, approximately 6.0 g / L to approximately 14.6 g / L, approximately 6.0 g / L to approximately 14.4 g / L, approximately 6.0 g / L to approximately 14.2 g / L, approximately 6.0 g / L to approximately 14.0 g / L, approximately 6.0 g / L to approximately 13.8 g / L, approximately 6.0 g / L to approximately 13.6 g / L, approximately 6.0 g / L to approximately 13.4 g / L, approximately 6.0 g / L to approximately 13.2 g / L, approximately 6.0 g / L - about 13.0 g / L, about 6.0 g / L - about 12.8 g / L, about 6.0 g / L - about 12.6 g / L, about 6.0 g / L - about 12.4 g / L, about 6.0 g / L - about 12.2 g / L, about 6.0 g / L - about 12.0 g / L, about 6.0 g / L - about 11.8 g / L, about 6.0 g / L - about 11.6 g / L, about 6.0 g / L - about 11.4 g / L, about 6.0 g / L - about 11.2 g / L, approximately 6.0 g / L to approximately 11.0 g / L, approximately 6.0 g / L to approximately 10.8 g / L, approximately 6.0 g / L to approximately 10.6 g / L, approximately 6.0 g / L to approximately 10.4 g / L, approximately 6.0 g / L to approximately 10.2 g / L, approximately 6.0 g / L to approximately 10.0 g / L, approximately 6.0 g / L to approximately 9.8 g / L, approximately 6.0 g / L to approximately 9.6 g / L, approximately 6.0 g / L to approximately 9.4 g / L, approximately 6.0 g / L to approximately 9.2 g / L, approximately 6.0 g / L to approximately 9.0 g / L, approximately 6.0 g / L to approximately 8.8 g / L, approximately 6.0 g / L to approximately 8.6 g / L, approximately 6.0 g / L to approximately 8.4 g / L. g / L, approximately 6.0 g / L to approximately 8.2 g / L, approximately 6.0 g / L to approximately 8.0 g / L, approximately 6.0 g / L to approximately 7.8 g / L, approximately 6.0 g / L to approximately 7.6 g / L, approximately 6.0 g / L to approximately 7.4 g / L, approximately 6.0 g / L to approximately 7.2 g / L, approximately 6.0 g / L to approximately 7.0 g / L, approximately 6.2 g / L to approximately 16.0 g / L, approximately 6.2 g / L to approximately 15.8 g / L, approximately 6.2 g / L to approximately 15.6 g / L, approximately 6.2 g / L to approximately 15.4 g / L, approximately 6.2 g / L to approximately 15.2 g / L, approximately 6.2 g / L to approximately 15.0 g / L, approximately 6.2 g / L to approximately 14.8 g / L, approximately 6.2 g / L - approximately 14.6 g / L, approximately 6.2 g / L - approximately 14.4 g / L, approximately 6.2 g / L - approximately 14.2 g / L, approximately 6.2 g / L - approximately 14.0 g / L, approximately 6.2 g / L - approximately 13.8 g / L, approximately 6.2 g / L - approximately 13.6 g / L, approximately 6.2 g / L - approximately 13.4 g / L, approximately 6.2 g / L - approximately 13.2 g / L, approximately 6.2 g / L - approximately 13.0 g / L, approximately 6.2 g / L - approximately 12.8 g / L, approximately 6.2 g / L - approximately 12.6 g / L, approximately 6.2 g / L - approximately 12.4 g / L, approximately 6.2 g / L - approximately 12.2 g / L, approximately 6.2 g / L - approximately 12.0 g / L, approximately 6.2 g / L - approximately 11.8 g / L, approximately 6.2 g / L - approximately 11.6 g / L, approximately 6.2 g / L - approximately 11.4 g / L, approximately 6.2 g / L - approximately 11.2 g / L, approximately 6.2 g / L - approximately 11.0 g / L, approximately 6.2 g / L - approximately 10.8 g / L, approximately 6.2 g / L - approximately 10.6 g / L, approximately 6.2 g / L - approximately 10.4 g / L, approximately 6.2 g / L - approximately 10.2 g / L, approximately 6.2 g / L - approximately 10.0 g / L, approximately 6.2 g / L - approximately 9.8 g / L, approximately 6.2 g / L - approximately 9.6 g / L, approximately 6.2 g / L - approximately 9.4 g / L, approximately 6.2 g / L - approximately 9.2 g / L, approximately 6.2 g / L - approximately 9.0 g / L, approximately 6.2 g / L - approximately 8.8 g / L, approximately 6.2 g / L - approximately 8.6 g / L, approximately 6.2 g / L - approximately 8.4 g / L, approximately 6.2 g / L - approximately 8.2 g / L, approximately 6.2 g / L - approximately 8.0 g / L, approximately 6.2 g / L - approximately 7.8 g / L, approximately 6.2 g / L - approximately 7.6 g / L, approximately 6.2 g / L - approximately 7.4 g / L, approximately 6.2 g / L - approximately 7.2 g / L, approximately 6.4 g / L - approximately 16.0 g / L, approximately 6.4 g / L - approximately 15.8 g / L, approximately 6.4 g / L - approximately 15.6 g / L, approximately 6.4 g / L - approximately 15.4 g / L, approximately 6.4 g / L - approximately 15.2 g / L, approximately 6.4 g / L - approximately 15.0 g / L, approximately 6.4 g / L - approximately 14.8 g / L, approximately 6.4 g / L - approximately 14.6 g / L, approximately 6.4 g / L - approximately 14.4 g / L, approximately 6.4 g / L - approximately 14.2 g / L, approximately 6.4 g / L - approximately 14.0 g / L, approximately 6.4 g / L - approximately 13.8 g / L, approximately 6.4 g / L - approximately 13.6 g / L, approximately 6.4 g / L - approximately 13.4 g / L, approximately 6.4 g / L - approximately 13.2 g / L, approximately 6.4 g / L - approximately 13.0 g / L, approximately 6.4 g / L - approximately 12.8 g / L, approximately 6.4 g / L - approximately 12.6 g / L, approximately 6.4 g / L - approximately 12.4 g / L, approximately 6.4 g / L - approximately 12.2 g / L, approximately 6.4 g / L - approximately 12.0 g / L, approximately 6.4 g / L - approximately 11.8 g / L, approximately 6.4 g / L - approximately 11.6 g / L, approximately 6.4 g / L - approximately 11.4 g / L, approximately 6.4 g / L - approximately 11.2 g / L, approximately 6.4 g / L - approximately 11.0 g / L, approximately 6.4 g / L - approximately 10.8 g / L, approximately 6.4 g / L - approximately 10.6 g / L, approximately 6.4 g / L - approximately 10.4 g / L, approximately 6.4 g / L - approximately 10.2 g / L, approximately 6.4 g / L - about 10.0 g / L, about 6.4 g / L - about 9.8 g / L, about 6.4 g / L - about 9.6 g / L, about 6.4 g / L - about 9.4 g / L, about 6.4 g / L - about 9.2 g / L, about 6.4 g / L - about 9.0 g / L, about 6.4 g / L - about 8.8 g / L, about 6.4 g / L - about 8.6 g / L, about 6.4 g / L - about 8.4 g / L, about 6.4 g / L - about 8.2 g / L, about 6.4 g / L - about 8.0 g / L, about 6.4 g / L - approximately 7.8 g / L, approximately 6.4 g / L - approximately 7.6 g / L, approximately 6.4 g / L - approximately 7.4 g / L, approximately 6.6 g / L - approximately 16.0 g / L, approximately 6.6 g / L - approximately 15.8 g / L, approximately 6.6 g / L - approximately 15.6 g / L, approximately 6.6 g / L - approximately 15.4 g / L, approximately 6.6 g / L - approximately 15.2 g / L, approximately 6.6 g / L - approximately 15.0 g / L, approximately 6.6 g / L - approximately 14.8 g / L, approximately 6.6 g / L - approximately 14.6 g / L, approximately 6.6 g / L - approximately 14.4 g / L, approximately 6.6 g / L - approximately 14.2 g / L, approximately 6.6 g / L - approximately 14.0 g / L, approximately 6.6 g / L - approximately 13.8 g / L g / L, approximately 6.6 g / L to approximately 13.6 g / L, approximately 6.6 g / L to approximately 13.4 g / L, approximately 6.6 g / L to approximately 13.2 g / L, approximately 6.6 g / L to approximately 13.0 g / L, approximately 6.6 g / L to approximately 12.8 g / L, approximately 6.6 g / L to approximately 12.6 g / L, approximately 6.6 g / L to approximately 12.4 g / L, approximately 6.6 g / L to approximately 12.2 g / L, approximately 6.6 g / L to approximately 12.0 g / L, approximately 6.6 g / L to approximately 11.8 g / L, approximately 6.6 g / L to approximately 11.6 g / L, approximately 6.6 g / L to approximately 11.4 g / L, approximately 6.6 g / L to approximately 11.2 g / L, approximately 6.6 g / L to approximately 11.0 g / L, approximately 6.6 g / L - approximately 10.8 g / L, approximately 6.6 g / L - approximately 10.6 g / L, approximately 6.6 g / L - approximately 10.4 g / L, approximately 6.6 g / L - approximately 10.2 g / L, approximately 6.6 g / L - approximately 10.0 g / L, approximately 6.6 g / L - approximately 9.8 g / L, approximately 6.6 g / L - approximately 9.6 g / L, approximately 6.6 g / L - approximately 9.4 g / L, approximately 6.6 g / L - approximately 9.2 g / L, approximately 6.6 g / L - approximately 9.0 g / L, approximately 6.6 g / L - approximately 8.8 g / L, approximately 6.6 g / L - approximately 8.6 g / L, approximately 6.6 g / L - approximately 8.4 g / L, approximately 6.6 g / L - approximately 8.2 g / L, approximately 6.6 g / L - approximately 8.0 g / L, approximately 6.6 g / L - about 7.8 g / L, about 6.6 g / L - about 7.6 g / L, about 6.8 g / L - about 16.0 g / L, about 6.8 g / L - about 15.8 g / L, about 6.8 g / L - about 15.6 g / L, about 6.8 g / L - about 15.4 g / L, about 6.8 g / L - about 15.2 g / L, about 6.8 g / L - about 15.0 g / L, about 6.8 g / L - about 14.8 g / L, about 6.8 g / L - about 14.6 g / L, approximately 6.8 g / L to approximately 14.4 g / L, approximately 6.8 g / L to approximately 14.2 g / L, approximately 6.8 g / L to approximately 14.0 g / L, approximately 6.8 g / L to approximately 13.8 g / L, approximately 6.8 g / L to approximately 13.6 g / L, approximately 6.8 g / L to approximately 13.4 g / L, approximately 6.8 g / L to approximately 13.2 g / L, approximately 6.8 g / L to approximately 13.0 g / L, approximately 6.8 g / L to approximately 12.8 g / L, approximately 6.8 g / L to approximately 12.6 g / L, approximately 6.8 g / L to approximately 12.4 g / L, approximately 6.8 g / L to approximately 12.2 g / L, approximately 6.8 g / L to approximately 12.0 g / L, approximately 6.8 g / L to approximately 11.8 g / L. g / L, approximately 6.8 g / L to approximately 11.6 g / L, approximately 6.8 g / L to approximately 11.4 g / L, approximately 6.8 g / L to approximately 11.2 g / L, approximately 6.8 g / L to approximately 11.0 g / L, approximately 6.8 g / L to approximately 10.8 g / L, approximately 6.8 g / L to approximately 10.6 g / L, approximately 6.8 g / L to approximately 10.4 g / L, approximately 6.8 g / L to approximately 10.2 g / L, approximately 6.8 g / L to approximately 10.0 g / L, approximately 6.8 g / L to approximately 9.8 g / L, approximately 6.8 g / L to approximately 9.6 g / L, approximately 6.8 g / L to approximately 9.4 g / L, approximately 6.8 g / L to approximately 9.2 g / L, approximately 6.8 g / L to approximately 9.0 g / L, approximately 6.8 g / L - approximately 8.8 g / L, approximately 6.8 g / L - approximately 8.6 g / L, approximately 6.8 g / L - approximately 8.4 g / L, approximately 6.8 g / L - approximately 8.2 g / L, approximately 6.8 g / L - approximately 8.0 g / L, approximately 6.8 g / L - approximately 7.8 g / L, approximately 7.0 g / L - approximately 16.0 g / L, approximately 7.0 g / L - approximately 15.8 g / L, approximately 7.0 g / L - approximately 15.6 g / L, approximately 7.0 g / L - approximately 15.4 g / L, approximately 7.0 g / L - approximately 15.2 g / L, approximately 7.0 g / L - approximately 15.0 g / L, approximately 7.0 g / L - approximately 14.8 g / L, approximately 7.0 g / L - approximately 14.6 g / L, approximately 7.0 g / L - approximately 14.4 g / L g / L, approximately 7.0 g / L to approximately 14.2 g / L, approximately 7.0 g / L to approximately 14.0 g / L, approximately 7.0 g / L to approximately 13.8 g / L, approximately 7.0 g / L to approximately 13.6 g / L, approximately 7.0 g / L to approximately 13.4 g / L, approximately 7.0 g / L to approximately 13.2 g / L, approximately 7.0 g / L to approximately 13.0 g / L, approximately 7.0 g / L to approximately 12.8 g / L, approximately 7.0 g / L to approximately 12.6 g / L, approximately 7.0 g / L to approximately 12.4 g / L, approximately 7.0 g / L - approx. 12.2 g / L, approx. 7.0 g / L - approx. 12.0 g / L, approx. 7.0 g / L - approx. 11.8 g / L, approx. 7.0 g / L - approx. 11.6 g / L, approx. 7.0 g / L - approx. 11.4 g / L, approx. 7.0 g / L - approx. 11.2 g / L, approx. 7.0 g / L - approx. 11.0 g / L, approx. 7.0 g / L - approx. 10.8 g / L, approx. 7.0 g / L - approx. 10.6 g / L, approx. 7.0 g / L - approx. 10.4 g / L, approx. 7.0 g / L - approx. 10.2 g / L, approx. 7.0 g / L - approx. 10.0 g / L, approx. 7.0 g / L - approx. 9.8 g / L, approx. 7.0 g / L - approx. 9.6 g / L, approx. 7.0 g / L - approx. 9.4 g / L g / L, approximately 7.0 g / L to approximately 9.2 g / L, approximately 7.0 g / L to approximately 9.0 g / L, approximately 7.0 g / L to approximately 8.8 g / L, approximately 7.0 g / L to approximately 8.6 g / L, approximately 7.0 g / L to approximately 8.4 g / L, approximately 7.0 g / L to approximately 8.2 g / L, approximately 7.0 g / L to approximately 8.0 g / L, approximately 7.2 g / L to approximately 16.0 g / L, approximately 7.2 g / L to approximately 15.8 g / L, approximately 7.2 g / L to approximately 15.6 g / L, approximately 7.2 g / L to approximately 15.4 g / L, approximately 7.2 g / L to approximately 15.2 g / L, approximately 7.2 g / L to approximately 15.0 g / L, approximately 7.2 g / L to approximately 14.8 g / L, approximately 7.2 g / L - approximately 14.6 g / L, approximately 7.2 g / L - approximately 14.4 g / L, approximately 7.2 g / L - approximately 14.2 g / L, approximately 7.2 g / L - approximately 14.0 g / L, approximately 7.2 g / L - approximately 13.8 g / L, approximately 7.2 g / L - approximately 13.6 g / L, approximately 7.2 g / L - approximately 13.4 g / L, approximately 7.2 g / L - approximately 13.2 g / L, approximately 7.2 g / L - approximately 13.0 g / L, approximately 7.2 g / L - approximately 12.8 g / L, approximately 7.2 g / L - approximately 12.6 g / L, approximately 7.2 g / L - approximately 12.4 g / L, approximately 7.2 g / L - approximately 12.2 g / L, approximately 7.2 g / L - approximately 12.0 g / L, approximately 7.2 g / L - about 11.8 g / L, about 7.2 g / L - about 11.6 g / L, about 7.2 g / L - about 11.4 g / L, about 7.2 g / L - about 11.2 g / L, about 7.2 g / L - about 11.0 g / L, about 7.2 g / L - about 10.8 g / L, about 7.2 g / L - about 10.6 g / L, about 7.2 g / L - about 10.4 g / L, about 7.2 g / L - about 10.2 g / L, about 7.2 g / L - about 10.0 g / L, about 7.2 g / L - about 9.8 g / L, approximately 7.2 g / L to approximately 9.6 g / L, approximately 7.2 g / L to approximately 9.4 g / L, approximately 7.2 g / L to approximately 9.2 g / L, approximately 7.2 g / L to approximately 9.0 g / L, approximately 7.2 g / L to approximately 8.8 g / L, approximately 7.2 g / L to approximately 8.6 g / L, approximately 7.2 g / L to approximately 8.4 g / L, approximately 7.2 g / L to approximately 8.2 g / L, approximately 7.4 g / L to approximately 16.0 g / L, approximately 7.4 g / L to approximately 15.8 g / L, approximately 7.4 g / L to approximately 15.6 g / L, approximately 7.4 g / L to approximately 15.4 g / L, approximately 7.4 g / L to approximately 15.2 g / L, approximately 7.4 g / L to approximately 15.0 g / L, approximately 7.4 g / L - approximately 14.8 g / L, approximately 7.4 g / L - approximately 14.6 g / L, approximately 7.4 g / L - approximately 14.4 g / L, approximately 7.4 g / L - approximately 14.2 g / L, approximately 7.4 g / L - approximately 14.0 g / L, approximately 7.4 g / L - approximately 13.8 g / L, approximately 7.4 g / L - approximately 13.6 g / L, approximately 7.4 g / L - approximately 13.4 g / L, approximately 7.4 g / L - approximately 13.2 g / L, approximately 7.4 g / L - approximately 13.0 g / L, approximately 7.4 g / L - approximately 12.8 g / L, approximately 7.4 g / L - approximately 12.6 g / L, approximately 7.4 g / L - approximately 12.4 g / L, approximately 7.4 g / L - approximately 12.2 g / L, approximately 7.4 g / L - approximately 12.0 g / L, approximately 7.4 g / L - approximately 11.8 g / L, approximately 7.4 g / L - approximately 11.6 g / L, approximately 7.4 g / L - approximately 11.4 g / L, approximately 7.4 g / L - approximately 11.2 g / L, approximately 7.4 g / L - approximately 11.0 g / L, approximately 7.4 g / L - approximately 10.8 g / L, approximately 7.4 g / L - approximately 10.6 g / L, approximately 7.4 g / L - approximately 10.4 g / L, approximately 7.4 g / L - approximately 10.2 g / L, approximately 7.4 g / L - approximately 10.0 g / L, approximately 7.4 g / L - approximately 9.8 g / L, approximately 7.4 g / L - approximately 9.6 g / L, approximately 7.4 g / L - approximately 9.4 g / L, approximately 7.4 g / L - approximately 9.2 g / L g / L, approximately 7.4 g / L to approximately 9.0 g / L, approximately 7.4 g / L to approximately 8.8 g / L, approximately 7.4 g / L to approximately 8.6 g / L, approximately 7.4 g / L to approximately 8.4 g / L, approximately 7.6 g / L to approximately 16.0 g / L, approximately 7.6 g / L to approximately 15.8 g / L, approximately 7.6 g / L to approximately 15.6 g / L, approximately 7.6 g / L to approximately 15.4 g / L, approximately 7.6 g / L to approximately 15.2 g / L, approximately 7.6 g / L to approximately 15.0 g / L, approximately 7.6 g / L - approximately 14.8 g / L, approximately 7.6 g / L - approximately 14.6 g / L, approximately 7.6 g / L - approximately 14.4 g / L, approximately 7.6 g / L - approximately 14.2 g / L, approximately 7.6 g / L - approximately 14.0 g / L, approximately 7.6 g / L - approximately 13.8 g / L, approximately 7.6 g / L - approximately 13.6 g / L, approximately 7.6 g / L - approximately 13.4 g / L, approximately 7.6 g / L - approximately 13.2 g / L, approximately 7.6 g / L - approximately 13.0 g / L, approximately 7.6 g / L - approximately 12.8 g / L, approximately 7.6 g / L - approximately 12.6 g / L, approximately 7.6 g / L - approximately 12.4 g / L, approximately 7.6 g / L - approximately 12.2 g / L, approximately 7.6 g / L - approximately 12.0 g / L, approximately 7.6 g / L - approximately 11.8 g / L, approximately 7.6 g / L - approximately 11.6 g / L, approximately 7.6 g / L - approximately 11.4 g / L, approximately 7.6 g / L - approximately 11.2 g / L, approximately 7.6 g / L - approximately 11.0 g / L, approximately 7.6 g / L - approximately 10.8 g / L, approximately 7.6 g / L - approximately 10.6 g / L, approximately 7.6 g / L - approximately 10.4 g / L, approximately 7.6 g / L - approximately 10.2 g / L, approximately 7.6 g / L - approximately 10.0 g / L, approximately 7.6 g / L - approximately 9.8 g / L, approximately 7.6 g / L - approximately 9.6 g / L, approximately 7.6 g / L - approximately 9.4 g / L, approximately 7.6 g / L - approximately 9.2 g / L g / L, approximately 7.6 g / L to approximately 9.0 g / L, approximately 7.6 g / L to approximately 8.8 g / L, approximately 7.6 g / L to approximately 8.6 g / L, approximately 7.8 g / L to approximately 16.0 g / L, approximately 7.8 g / L to approximately 15.8 g / L, approximately 7.8 g / L to approximately 15.6 g / L, approximately 7.8 g / L to approximately 15.4 g / L, approximately 7.8 g / L to approximately 15.2 g / L, approximately 7.8 g / L to approximately 15.0 g / L, approximately 7.8 g / L to approximately 14.8 g / L, approximately 7.8 g / L to approximately 14.6 g / L, approximately 7.8 g / L to approximately 14.4 g / L, approximately 7.8 g / L to approximately 14.2 g / L, approximately 7.8 g / L to approximately 14.0 g / L, approximately 7.8 g / L - approximately 13.8 g / L, approximately 7.8 g / L - approximately 13.6 g / L, approximately 7.8 g / L - approximately 13.4 g / L, approximately 7.8 g / L - approximately 13.2 g / L, approximately 7.8 g / L - approximately 13.0 g / L, approximately 7.8 g / L - approximately 12.8 g / L, approximately 7.8 g / L - approximately 12.6 g / L, approximately 7.8 g / L - approximately 12.4 g / L, approximately 7.8 g / L - approximately 12.2 g / L, approximately 7.8 g / L - approximately 12.0 g / L, approximately 7.8 g / L - approx. 11.8 g / L, approx. 7.8 g / L - approx. 11.6 g / L, approx. 7.8 g / L - approx. 11.4 g / L, approx. 7.8 g / L - approx. 11.2 g / L, approx. 7.8 g / L - approx. 11.0 g / L, approx. 7.8 g / L - approx. 10.8 g / L, approx. 7.8 g / L - approx. 10.6 g / L, approx. 7.8 g / L - approx. 10.4 g / L, approx. 7.8 g / L - approx. 10.2 g / L, approx. 7.8 g / L - approx. 10.0 g / L, approx. 7.8 g / L - approx. 9.8 g / L, approx. 7.8 g / L - approx. 9.6 g / L, approx. 7.8 g / L - approx. 9.4 g / L, approx. 7.8 g / L - approx. 9.2 g / L, approx. 7.8 g / L - approx. 9.0 g / L g / L, approximately 7.8 g / L to approximately 8.8 g / L, approximately 8.0 g / L to approximately 16.0 g / L, approximately 8.0 g / L to approximately 15.8 g / L, approximately 8.0 g / L to approximately 15.6 g / L, approximately 8.0 g / L to approximately 15.4 g / L, approximately 8.0 g / L to approximately 15.2 g / L, approximately 8.0 g / L to approximately 15.0 g / L, approximately 8.0 g / L to approximately 14.8 g / L, approximately 8.0 g / L to approximately 14.6 g / L, approximately 8.0 g / L to approximately 14.4 g / L, approximately 8.0 g / L to approximately 14.2 g / L, approximately 8.0 g / L to approximately 14.0 g / L, approximately 8.0 g / L to approximately 13.8 g / L, approximately 8.0 g / L to approximately 13.6 g / L, approximately 8.0 g / L g / L - approximately 13.4 g / L, approximately 8.0 g / L - approximately 13.2 g / L, approximately 8.0 g / L - approximately 13.0 g / L, approximately 8.0 g / L - approximately 12.8 g / L, approximately 8.0 g / L - approximately 12.6 g / L, approximately 8.0 g / L - approximately 12.4 g / L, approximately 8.0 g / L - approximately 12.2 g / L, approximately 8.0 g / L - approximately 12.0 g / L, approximately 8.0 g / L - approximately 11.8 g / L, approximately 8.0 g / L - approximately 11.6 g / L, approximately 8.0 g / L - approximately 11.4 g / L, approximately 8.0 g / L - approximately 11.2 g / L, approximately 8.0 g / L - approximately 11.0 g / L, approximately 8.0 g / L - approximately 10.8 g / L, approximately 8.0 g / L - approximately 10.6 g / L, approximately 8.0 g / L - approximately 10.4 g / L, approximately 8.0 g / L - approximately 10.2 g / L, approximately 8.0 g / L - approximately 10.0 g / L, approximately 8.0 g / L - approximately 9.8 g / L, approximately 8.0 g / L - approximately 9.6 g / L, approximately 8.0 g / L - approximately 9.4 g / L, approximately 8.0 g / L - approximately 9.2 g / L, approximately 8.0 g / L - approximately 9.0 g / L, approximately 8.5 g / L - approximately 16.0 g / L, approximately 8.5 g / L - approximately 15 g / L.5 g / L, approximately 8.5 g / L to approximately 15.0 g / L, approximately 8.5 g / L to approximately 14.5 g / L, approximately 8.5 g / L to approximately 14.0 g / L, approximately 8.5 g / L to approximately 13.5 g / L, approximately 8.5 g / L to approximately 13.0 g / L, approximately 8.5 g / L to approximately 12.5 g / L, approximately 8.5 g / L to approximately 12.0 g / L, approximately 8.5 g / L to approximately 11.5 g / L, approximately 8.5 g / L to approximately 11.0 g / L, approximately 8.5 g / L to approximately 10.5 g / L, approximately 8.5 g / L to approximately 10.0 g / L, approximately 8.5 g / L to approximately 9.5 g / L, approximately 9.0 g / L to approximately 16.0 g / L, approximately 9.0 g / L to approximately 15.5 g / L. g / L, approximately 9.0 g / L to approximately 15.0 g / L, approximately 9.0 g / L to approximately 14.5 g / L, approximately 9.0 g / L to approximately 14.0 g / L, approximately 9.0 g / L to approximately 13.5 g / L, approximately 9.0 g / L to approximately 13.0 g / L, approximately 9.0 g / L to approximately 12.5 g / L, approximately 9.0 g / L to approximately 12.0 g / L, approximately 9.0 g / L to approximately 11.5 g / L, approximately 9.0 g / L to approximately 11.0 g / L, approximately 9.0 g / L to approximately 10.5 g / L, approximately 9.0 g / L to approximately 10.0 g / L, approximately 9.5 g / L to approximately 16.0 g / L, approximately 9.5 g / L to approximately 15.5 g / L, approximately 9.5 g / L to approximately 15.0 g / L, approximately 9.5 g / L - approximately 14.5 g / L, approximately 9.5 g / L - approximately 14.0 g / L, approximately 9.5 g / L - approximately 13.5 g / L, approximately 9.5 g / L - approximately 13.0 g / L, approximately 9.5 g / L - approximately 12.5 g / L, approximately 9.5 g / L - approximately 12.0 g / L, approximately 9.5 g / L - approximately 11.5 g / L, approximately 9.5 g / L - approximately 11.0 g / L, approximately 9.5 g / L - approximately 10.5 g / L, approximately 10.0 g / L - approximately 16.0 g / L, approximately 10.0 g / L - approximately 15.5 g / L, approximately 10.0 g / L - approximately 15.0 g / L, approximately 10.0 g / L - approximately 14.5 g / L, approximately 10.0 g / L - approximately 14.0 g / L, approximately 10.0 g / L - approximately 13.5 g / L, approximately 10.0 g / L - approximately 13.0 g / L, approximately 10.0 g / L - approximately 12.5 g / L, approximately 10.0 g / L - approximately 12.0 g / L, approximately 10.0 g / L - approximately 11.5 g / L, approximately 10.0 g / L - approximately 11.0 g / L, approximately 10.5 g / L - approximately 16.0 g / L, approximately 10.5 g / L - approximately 15.5 g / L, approximately 10.5 g / L - approximately 15.0 g / L, approximately 10.5 g / L - approximately 14.5 g / L, approximately 10.5 g / L to approximately 14.0 g / L, approximately 10.5 g / L to approximately 13.5 g / L, approximately 10.5 g / L to approximately 13.0 g / L, approximately 10.5 g / L to approximately 12.5 g / L, approximately 10.5 g / L to approximately 12.0 g / L, approximately 10.5 g / L to approximately 11.5 g / L, approximately 11.0 g / L to approximately 16.0 g / L, approximately 11.0 g / L to approximately 15.5 g / L, approximately 11.0 g / L to approximately 15.0 g / L, approximately 11.0 g / L to approximately 14.5 g / L, approximately 11.0 g / L to approximately 14.0 g / L, approximately 11.0 g / L to approximately 13.5 g / L, approximately 11.0 g / L to approximately 13.0 g / L, approximately 11.0 g / L g / L - approximately 12.5 g / L, approximately 11.0 g / L - approximately 12.0 g / L, approximately 11.5 g / L - approximately 16.0 g / L, approximately 11.5 g / L - approximately 15.5 g / L, approximately 11.5 g / L - approximately 15.0 g / L, approximately 11.5 g / L - approximately 14.5 g / L, approximately 11.5 g / L - approximately 14.0 g / L, approximately 11.5 g / L - approximately 13.5 g / L, approximately 11.5 g / L - approximately 13.0 g / L, approximately 11.5 g / L - approximately 12.5 g / L, approximately 12.0 g / L - approximately 16.0 g / L, approximately 12.0 g / L - approximately 15.5 g / L, approximately 12.0 g / L - approximately 15.0 g / L, approximately 12.0 g / L - approximately 14.5 g / L g / L, approximately 12.0 g / L to approximately 14.0 g / L, approximately 12.0 g / L to approximately 13.5 g / L, approximately 12.0 g / L to approximately 13.0 g / L, approximately 12.5 g / L to approximately 16.0 g / L, approximately 12.5 g / L to approximately 15.5 g / L, approximately 12.5 g / L to approximately 15.0 g / L, approximately 12.5 g / L to approximately 14.5 g / L, approximately 12.5 g / L to approximately 14.0 g / L, approximately 12.5 g / L to approximately 13.5 g / L, approximately 13.0 g / L to approximately 16.0 g / L, approximately 13.0 g / L to approximately 15.5 g / L, approximately 13.0 g / L to approximately 15.0 g / L, approximately 13.0 g / L to approximately 14.5 g / L, approximately 13.0 g / L. g / L - about 14.0 g / L, about 13.5 g / L - about 16.0 g / L, about 13.5 g / L - about 15.5 g / L, about 13.5 g / L - about 15.0 g / L, about 13.5 g / L - about 14.5 g / L, about 14.0 g / L - about 16.0 g / L, about 14.0 g / L - about 15.5 g / L, about 14.0 g / L - about 15.0 g / L, about 14.5 g / L - about 16.0 g / L, about 14.5 g / L - about 15.Poloxamer-188 (5 g / L, or approximately 15.0 g / L to 16.0 g / L).

[0212] In some embodiments, poloxamer-188 is present in the culture medium prior to the culture step and / or poloxamer-188 is added to the culture medium and / or added to the culture medium during the culture step. For example, the method provided in this application may further include adding poloxamer-188 prior to and / or during the culture step to provide a concentration of 1.8 g / L or higher in the culture medium (e.g., any exemplified concentration or range of poloxamer-188 described in this application). In some instances, additional poloxamer-188 is added to a liquid culture medium containing some poloxamer-188 to provide a concentration of 1.8 g / L or higher of poloxamer-188 (e.g., any exemplified concentration or range of poloxamer-188 described in this application). For example, poloxamer-188 can be added to the first liquid medium, the second liquid medium, and / or the feed liquid medium before and / or during the culture step.

[0213] In some instances, poloxamer-188 is added to the culture medium in one or more (e.g., two, three, four, or five) aliquots during the culturing step. Poloxamer-188 can be added to the culture medium continuously. In some instances, the first liquid culture medium may contain poloxamer-188, and additional poloxamer-188 may be added to the medium during the culturing period. In some embodiments, poloxamer-188 is present in the culture medium.

[0214] When the culture step includes perfusion culture, the second liquid culture medium may contain poloxamer-188 at a higher concentration than that in the first liquid culture medium. For example, the first liquid culture medium may contain poloxamer-188 at a concentration of 1.8 g / L or less, while the second liquid culture medium contains poloxamer-188 at a concentration greater than 1.8 g / L (e.g., any concentration or range of poloxamer-188 described in this application, such as concentrations greater than 1.9 g / L, greater than 2.0 g / L, greater than 2.1 g / L, greater than 2.2 g / L, greater than 2.3 g / L, greater than 2.4 g / L, greater than 2.5 g / L, greater than 2.6 g / L, greater than 2.7 g / L, greater than 2.8 g / L, greater than 2.9 g / L, greater than 3.0 g / L, greater than 3.1 g / L, greater than 3.2 g / L, greater than 3.3 g / L, greater than 3.4 g / L, greater than 3.5 g / L, greater than 3.6 g / L, and greater than 3.7 g / L). Concentrations above 3.8 g / L, 3.9 g / L, 4.0 g / L, 4.1 g / L, 4.2 g / L, 4.3 g / L, 4.4 g / L, 4.5 g / L, 4.6 g / L, 4.7 g / L, 4.8 g / L, 4.9 g / L, 5.0 g / L, 5.1 g / L, 5.2 g / L, 5.3 g / L, 5.4 g / L, 5.5 g / L, 5.6 g / L, 5.7 g / L, 5.8 g / L, 5.9 g / L, 6.0 g / L, 6.1 g / L, 6.2 g / L, 6.3 g / L, and 6.4 g / L are listed. Concentrations above 6.5 g / L, 6.6 g / L, 6.7 g / L, 6.8 g / L, 6.9 g / L, 7.0 g / L, 7.1 g / L, 7.2 g / L, 7.3 g / L, 7.4 g / L, 7.5 g / L, 7.6 g / L, 7.7 g / L, 7.8 g / L, 7.9 g / L, 8.0 g / L, 8.1 g / L, 8.2 g / L, 8.3 g / L, 8.4 g / L, 8.5 g / L, 8.6 g / L, 8.7 g / L, 8.8 g / L, and 8.5 g / L.9 g / L, concentrations higher than 9.0 g / L, concentrations higher than 9.1 g / L, concentrations higher than 9.2 g / L, concentrations higher than 9.3 g / L, concentrations higher than 9.4 g / L, concentrations higher than 9.5 g / L, concentrations higher than 9.6 g / L, concentrations higher than 9.7 g / L, concentrations higher than 9.8 g / L, concentrations higher than 9.9 g / L, concentrations higher than 10.0 g / L, concentrations higher than 10.1 g / L, concentrations higher than 10.2 g / L, concentrations higher than 10.3 g / L, concentrations higher than 10.4 g / L, concentrations higher than 10.5 g / L, concentrations higher than 10.6 g / L, concentrations higher than 10.7 g / L, concentrations higher than 10.8 g / L, concentrations higher than 10.9 g / L, concentrations higher than 11.0 g / L, concentrations higher than 11.1 g / L, concentrations higher than 11.2 g / L, concentrations higher than 11.3 g / L, concentrations higher than 11.4 g / L. Concentrations above 11.5 g / L, 11.6 g / L, 11.7 g / L, 11.8 g / L, 11.9 g / L, 12.0 g / L, 12.1 g / L, 12.2 g / L, 12.3 g / L, 12.4 g / L, 12.5 g / L, 12.6 g / L, 12.7 g / L, 12.8 g / L, 12.9 g / L, 13.0 g / L, 13.1 g / L, 13.2 g / L, 13.3 g / L, 13.4 g / L, 13.5 g / L, 13.6 g / L, 13.7 g / L, and 13.8 g / L are listed. Concentrations exceeding 13.9 g / L, 14.0 g / L, 14.1 g / L, 14.2 g / L, 14.3 g / L, 14.4 g / L, 14.5 g / L, 14.6 g / L, 14.7 g / L, 14.8 g / L, 14.9 g / L, 15.0 g / L, 15.1 g / L, 15.2 g / L, 15.3 g / L, 15.4 g / L, 15.5 g / L, 15.6 g / L, 15.7 g / L, 15.8 g / L, 15.9 g / L, or 16.0 g / L (Poloxamer-188).

[0215] When the culture step includes fed batch culture, the fed liquid culture medium may contain poloxamer-188 at a higher concentration than that in the first liquid culture medium. For example, the first liquid culture medium may include poloxamer-188 at a concentration of 1.8 g / L or less, while the feed liquid culture medium may contain concentrations higher than 1.8 g / L (e.g., higher than 2.0 g / L, higher than 2.5 g / L, higher than 3.0 g / L, higher than 3.5 g / L, higher than 4.0 g / L, higher than 4.5 g / L, higher than 5.0 g / L, higher than 5.5 g / L, higher than 6.0 g / L, higher than 6.5 g / L, higher than 7.0 g / L, higher than 7.5 g / L, higher than 8.0 g / L, higher than 8.5 g / L, higher than 9.0 g / L, higher than 9.5 g / L, higher than 10 g / L, higher than 10.5 g / L, higher than 11.0 g / L, higher than 11.5 g / L, higher than 12.0 g / L). Concentrations above 12.5 g / L, 13.0 g / L, 14.0 g / L, 15.0 g / L, 16.0 g / L, 17.0 g / L, 18.0 g / L, 19.0 g / L, 20.0 g / L, 21.0 g / L, 22.0 g / L, 23.0 g / L, 24.0 g / L g / L, concentration higher than 24.5 g / L, concentration higher than 25.0 g / L, concentration higher than 25.5 g / L, concentration higher than 26.0 g / L, concentration higher than 26.5 g / L, concentration higher than 27.0 g / L, concentration higher than 28.0 g / L, concentration higher than 28.5 g / L, concentration higher than 29.0 g / L, concentration higher than 29.5 g / L, concentration higher than 30.0 g / L, concentration higher than 30.5 g / L, concentration higher than 31.0 g / L, concentration higher than 31.5 g / L, concentration higher than 32.0 g / L, concentration higher than 32.5 g / L, concentration higher than 33.0 g / L, concentration higher than 33.5 g / L, concentration higher than 34.0 g / L, concentration higher than 34.5 g / L, concentration higher than 35.0 g / L, concentration higher than 35.5 g / L, concentration higher than 36.0 g / L, concentration higher than 36.Poloxamer-188 at concentrations above 5 g / L, above 37.0 g / L, above 37.5 g / L, above 38.0 g / L, above 38.5 g / L, above 39.0 g / L, above 39.5 g / L, or above 40.0 g / L.

[0216] Some embodiments of any of the methods provided in this application involve increasing the concentration of poloxamer-188 in the culture over time. In some embodiments, the culture medium contains a concentration of poloxamer-188 selected based on one or more factors chosen from the group consisting of: pore size, pore type, gas flow rate, viable cell density in the culture medium, and markers related to cellular stress. In these methods, the selected concentration of poloxamer-188 in the culture medium can be achieved by adding poloxamer-188 to the culture medium prior to the culture step and / or by adding poloxamer-188 to the culture medium during the culture step. The selected concentration of poloxamer-188 can be any of the exemplified concentrations or ranges of poloxamer-188 described in this application.

[0217] The concentration of poloxamer-188 is selected based on one or more factors.

[0218] Some embodiments of the method described in this application include culturing mammalian cells in a liquid culture medium containing a concentration of poloxamer-188, the concentration of poloxamer-188 being selected based on one or more factors selected from the group consisting of: pore size, pore type, gas flow rate, viable cell density in the culture medium, and markers related to cellular stress.

[0219] Different examples of hole types are known in the art. Non-limiting examples of hole types are sintered holes and drilled holes.

[0220] Various gas flow rates suitable for cell culture are known in the art. Generally, a higher gas flow rate indicates a higher poloxamer-188 concentration (compared to a culture step using a lower gas flow rate), while a lower gas flow rate indicates a lower poloxamer-188 concentration (compared to a culture step using a higher gas flow rate).

[0221] Various non-limiting viable cell densities are described in this application. Generally, a higher viable cell density indicator should be selected (compared to a medium containing a lower viable cell density) at a higher poloxamer-188 concentration, while a lower viable cell density indicator should be selected (compared to a medium containing a higher viable cell density) at a lower poloxamer-188 concentration.

[0222] Exemplary markers of cellular stress released by cells under physiological stress include proteases (e.g., activated caspase), lactate dehydrogenase, genomic DNA (e.g., nucleosome DNA), cytochrome c, and activated PARP. Non-limiting examples of markers of cellular stress produced or present at elevated levels in cells under physiological stress include activated caspase, cytochrome c, activated PARP, and externalized phosphatidylserine. An elevated level of one or more (e.g., two, three, four, five, six, or seven) of any of these markers of cellular stress compared to a control indicates that a higher concentration of poloxamer-188 should be selected. A level of one or more markers of cellular stress that is approximately the same as or lower than a control indicates that a lower concentration of poloxamer-188 should be selected. In any example described in this application, the control level may be the level in control cells that do not undergo apoptosis or are not cultured under conditions that would trigger necrosis. Methods for detecting the levels of one or more biomarkers of cellular stress are known in the art. Commercially available kits can be used to measure said one or more biomarkers of cellular stress.

[0223] In any of the embodiments described in this application, the selected concentration of poloxamer-188 is achieved by adding poloxamer-188 to the culture medium prior to the culture step and / or adding poloxamer-188 to the culture medium during the culture step. In some embodiments, the method includes drilling when the hole type is a borehole and the hole diameter is approximately 750 μm to approximately 1.5 mm (e.g., approximately 750 μm to approximately 1.45 mm, approximately 750 μm to approximately 1.40 mm, approximately 750 μm to approximately 1.35 mm, approximately 750 μm to approximately 1.30 mm, approximately 750 μm to approximately 1.25 mm, approximately 750 μm to approximately 1.20 mm, approximately 750 μm to approximately 1.15 mm, approximately 750 μm to approximately 1.10 mm, approximately 750 μm to approximately 1.05 mm, approximately 750 μm to approximately 1.00 mm, approximately 750 μm to approximately 950 μm, approximately 750 μm to approximately 900 μm, approximately 750 μm to approximately 850 μm, approximately 750 μm to approximately 800 μm, approximately 800 μm to approximately 1.5 mm ... μm - approximately 1.45 mm, approximately 800 μm - approximately 1.40 mm, approximately 800 μm - approximately 1.35 mm, approximately 800 μm - approximately 1.30 mm, approximately 800 μm - approximately 1.25 mm, approximately 800 μm - approximately 1.20 mm, approximately 800 μm - approximately 1.15 mm, approximately 800 μm - approximately 1.10 mm, approximately 800 μm - approximately 1.05 mm, approximately 800 μm - approximately 1.00 mm, approximately 800 μm - approximately 950 μm, approximately 800 μm - approximately 900 μm, approximately 800 μm - approximately 850 μm, approximately 850 μm - approximately 1.5 mm, approximately 850 μm - approximately 1.45 mm, approximately 850 μm - approximately 1.40 mm, approximately 850 μm - approximately 1.35 mm, approximately 850 μm - approximately 1.30 mm mm, approximately 850 μm - approximately 1.25 mm, approximately 850 μm - approximately 1.20 mm, approximately 850 μm - approximately 1.15 mm, approximately 850 μm - approximately 1.10 mm, approximately 850 μm - approximately 1.05 mm, approximately 850 μm - approximately 1.00 mm, approximately 850 μm - approximately 950 μm, approximately 850 μm - approximately 900 μm, approximately 900 μm - approximately 1.5 mm, approximately 900 μm - approximately 1.45 mm, approximately 900 μm - approximately 1.40 mm, approximately 900 μm - approximately 1.35 mm, approximately 900 μm - approximately 1.30 mm, approximately 900 μm - approximately 1.25 mm, approximately 900 μm - approximately 1.20 mm, approximately 900 μm - approximately 1.15 mm, approximately 900 μm - approximately 1.10 mm, approximately 900 μm - approximately 1.05 mm, approximately 900 μm - approximately 1.00mm, approximately 900 μm - approximately 950 μm, approximately 950 μm - approximately 1.5 mm, approximately 950 μm - approximately 1.45 mm, approximately 950 μm - approximately 1.40 mm, approximately 950 μm - approximately 1.35 mm, approximately 950 μm - approximately 1.30 mm, approximately 950 μm - approximately 1.25 mm, approximately 950 μm - approximately 1.20 mm, approximately 950 μm - approximately 1.15 mm, approximately 950 μm - approximately 1.10 mm, approximately 950 μm - approximately 1.05 mm, approximately 950 μm - approximately 1.00 mm, approximately 1.0 mm - approximately 1.5 mm, approximately 1.0 mm - approximately 1.45 mm, approximately 1.0 mm - approximately 1.40 mm, approximately 1.0 mm - approximately 1.35 mm, approximately 1.0 mm - approximately 1.30 mm, approximately 1.0 mm mm - approximately 1.25 mm, approximately 1.0 mm - approximately 1.20 mm, approximately 1.0 mm - approximately 1.15 mm, approximately 1.0 mm - approximately 1.10 mm, approximately 1.0 mm - approximately 1.05 mm, approximately 1.05 mm - approximately 1.5 mm, approximately 1.05 mm - approximately 1.45 mm, approximately 1.05 mm - approximately 1.40 mm, approximately 1.05 mm - approximately 1.35 mm, approximately 1.05 mm - approximately 1.30 mm, approximately 1.05 mm - approximately 1.25 mm, approximately 1.05 mm - approximately 1.20 mm, approximately 1.05 mm - approximately 1.15 mm, approximately 1.05 mm - approximately 1.10 mm, approximately 1.10 mm - approximately 1.5 mm, approximately 1.10 mm - approximately 1.45 mm, approximately 1.10 mm - approximately 1.40 mm, approximately 1.10 mm - approximately 1.35 mm, approximately 1.10 mm - approximately 1.30 mm, approximately 1.10 mm - approximately 1.25 mm, approximately 1.10 mm - approximately 1.20 mm, approximately 1.10 mm - approximately 1.15 mm, approximately 1.15 mm - approximately 1.5 mm, approximately 1.15 mm - approximately 1.45 mm, approximately 1.15 mm - approximately 1.40 mm, approximately 1.15 mm - approximately 1.35 mm, approximately 1.15 mm - approximately 1.30 mm, approximately 1.15 mm - approximately 1.25 mm, approximately 1.15 mm - approximately 1.20 mm, approximately 1.20 mm - approximately 1.5 mm, approximately 1.20 mm - approximately 1.45 mm, approximately 1.20 mm - approximately 1.40 mm, approximately 1.20 mm - approximately 1.35 mm, approximately 1.20 mm - approximately 1.30 mm, approximately 1.20 mm - approximately 1.25 mm, approximately 1.25 mm - approximately 1.5 mm mm, approximately 1.25 mm to approximately 1.45 mm, approximately 1.25 mm to approximately 1.40 mm, approximately 1.25 mm to approximately 1.35 mm, approximately 1.25 mm to approximately 1.30 mm, approximately 1.When selecting pore sizes of approximately 2.3 g / L to approximately 3.3 g / L based on pore type and diameter (e.g., approximately 2.3 g / L to approximately 3.2 g / L, approximately 2.3 g / L to approximately 3.1 g / L, approximately 2.3 g / L to approximately 3.0 g / L, approximately 2.3 g / L to approximately 2.9 g / L, approximately 2.3 g / L to approximately 2.8 g / L, approximately 2.3 g / L to approximately 2.3 g / L), 30 mm to approximately 1.5 mm, approximately 1.35 mm to approximately 1.5 mm, approximately 1.35 mm to approximately 1.45 mm, approximately 1.35 mm to approximately 1.40 mm, approximately 1.35 mm to approximately 1.40 mm, approximately 1.30 mm to approximately 1.40 mm, approximately 1.40 mm to approximately 1.40 mm, approximately 1.40 mm to approximately 1.50 mm, approximately 1.40 mm to approximately 1.45 mm, or approximately 1.45 mm to approximately 1.50 mm), the appropriate pore size is approximately 2.3 g / L to approximately 3.3 g / L (e.g., approximately 2.3 g / L to approximately 3.2 g / L, approximately 2.3 g / L to approximately 3.1 g / L, approximately 2.3 g / L to approximately 3.0 g / L, approximately 2.3 g / L to approximately 2.9 g / L, approximately 2.3 g / L to approximately 2.8 g / L, approximately 2.3 g / L to approximately 2.3 g / L). g / L - approx. 2.7 g / L, approx. 2.3 g / L - approx. 2.6 g / L, approx. 2.3 g / L - approx. 2.5 g / L, approx. 2.4 g / L - approx. 3.3 g / L, approx. 2.4 g / L - approx. 3.2 g / L, approx. 2.4 g / L - approx. 3.1 g / L, approx. 2.4 g / L - approx. 3.0 g / L, approx. 2.4 g / L - approx. 2.9 g / L, approx. 2.4 g / L - approx. 2.8 g / L, approx. 2.4 g / L - approx. 2.7 g / L, approx. 2.4 g / L - approx. 2.6 g / L, approx. 2.5 g / L - approx. 3.3 g / L, approx. 2.5 g / L - approx. 3.2 g / L, approx. 2.5 g / L - approx. 3.1 g / L, approx. 2.5 g / L - approx. 3.0 g / L, approx. 2.5 g / L g / L - approx. 2.9 g / L, approx. 2.5 g / L - approx. 2.8 g / L, approx. 2.5 g / L - approx. 2.7 g / L, approx. 2.6 g / L - approx. 3.3 g / L, approx. 2.6 g / L - approx. 3.2 g / L, approx. 2.6 g / L - approx. 3.1 g / L, approx. 2.6 g / L - approx. 3.0 g / L, approx. 2.6 g / L - approx. 2.9 g / L, approx. 2.6 g / L - approx. 2.8 g / L, approx. 2.7 g / L - approx. 3.3 g / L, approx. 2.8 g / L - approx. 3.1 g / L, approx. 2.7 g / L - approx. 3.0 g / L, approx. 2.7 g / L - approx. 2.9 g / L, approx. 2.8 g / L - approx. 3.3 g / L, approx. 2.8 g / L g / L - about 3.2 g / L, about 2.8 g / L - about 3.1 g / L, about 2.8 g / L - about 3.0 g / L, about 2.9 g / L - about 3.3 g / L, about 2.9 g / L - about 3.2 g / L, about 2.9 g / L - about 3.1 g / L, about 3.0 g / L - about 3.3 g / L, about 3.0 g / L - about 3.2 g / L, or about 3.1 g / L - about 3.A concentration of poloxamer-188 of 3 g / L.

[0224] In some embodiments, the method includes cases where the hole type is a drill hole and the hole diameter is approximately 250 μm to approximately 750 μm (e.g., approximately 250 μm to approximately 700 μm, approximately 250 μm to approximately 650 μm, approximately 250 μm to approximately 600 μm, approximately 250 μm to approximately 550 μm, approximately 250 μm to approximately 500 μm, approximately 250 μm to approximately 450 μm, approximately 250 μm to approximately 400 μm, approximately 250 μm to approximately 350 μm, approximately 250 μm to approximately 300 μm, approximately 300 μm to approximately 750 μm, approximately 300 μm to approximately 700 μm, approximately 300 μm to approximately 650 μm, approximately 300 μm to approximately 600 μm, approximately 300 μm to approximately 550 μm, approximately 300 μm to approximately 500 μm). μm, approximately 300 μm - approximately 450 μm, approximately 300 μm - approximately 400 μm, approximately 300 μm - approximately 350 μm, approximately 350 μm - approximately 750 μm, approximately 350 μm - approximately 700 μm, approximately 350 μm - approximately 650 μm, approximately 350 μm - approximately 600 μm, approximately 350 μm - approximately 550 μm, approximately 350 μm - approximately 500 μm, approximately 350 μm - approximately 450 μm, approximately 350 μm - approximately 400 μm, approximately 400 μm - approximately 750 μm, approximately 400 μm - approximately 700 μm, approximately 400 μm - approximately 650 μm, approximately 400 μm - approximately 600 μm, approximately 400 μm - approximately 550 μm, approximately 400 μm - approximately 500 μm, approximately 400 μm μm - approximately 450 μm, approximately 450 μm - approximately 750 μm, approximately 450 μm - approximately 700 μm, approximately 450 μm - approximately 650 μm, approximately 450 μm - approximately 600 μm, approximately 450 μm - approximately 550 μm, approximately 450 μm - approximately 500 μm, approximately 500 μm - approximately 750 μm, approximately 500 μm - approximately 700 μm, approximately 500 μm - approximately 650 μm, approximately 500 μm - approximately 600 μm, approximately 500 μm - approximately 700 μm, approximately 500 μm - approximately 600 μm, approximately 600 μm - approximately 750 μm, approximately 600 μm - approximately 700 μm When selecting a pore size of approximately 3.3 g / L to approximately 4.3 g / L (e.g., approximately 3.3 g / L to approximately 4.2 g / L, approximately 3.3 g / L to approximately 4.1 g / L, approximately 3.3 g / L to approximately 4.0 g / L, approximately 3 μm, approximately 600 μm to approximately 650 μm, approximately 650 μm to approximately 750 μm, approximately 650 μm to approximately 700 μm, or approximately 700 μm and 750 μm), the pore size is selected based on the pore type and pore size.3 g / L - approx. 3.9 g / L, approx. 3.3 g / L - approx. 3.8 g / L, approx. 3.3 g / L - approx. 3.7 g / L, approx. 3.3 g / L - approx. 3.6 g / L, approx. 3.3 g / L - approx. 3.5 g / L, approx. 3.4 g / L - approx. 4.3 g / L, approx. 3.4 g / L - approx. 4.2 g / L, approx. 3.4 g / L - approx. 4.1 g / L, approx. 3.4 g / L - approx. 4.0 g / L, approx. 3.4 g / L - approx. 3.9 g / L, approx. 3.4 g / L - approx. 3.8 g / L, approx. 3.4 g / L - approx. 3.7 g / L, approx. 3.4 g / L - approx. 3.6 g / L, approx. 3.5 g / L - approx. 4.3 g / L, approx. 3.5 g / L - approx. 4.2 g / L, approx. 3.5 g / L g / L - approximately 4.1 g / L, approximately 3.5 g / L - approximately 4.0 g / L, approximately 3.5 g / L - approximately 3.9 g / L, approximately 3.5 g / L - approximately 3.8 g / L, approximately 3.5 g / L - approximately 3.7 g / L, approximately 3.6 g / L - approximately 4.3 g / L, approximately 3.6 g / L - approximately 4.2 g / L, approximately 3.6 g / L - approximately 4.1 g / L, approximately 3.6 g / L - approximately 4.0 g / L, approximately 3.6 g / L - approximately 3.9 g / L, approximately 3.6 g / L - approximately 3.8 g / L, approximately 3.7 g / L - approximately 4.3 g / L, approximately 3.7 g / L - approximately 4.2 g / L, approximately 3.7 g / L - approximately 4.1 g / L, approximately 3.7 g / L - approximately 4.0 g / L, approximately 3.7 g / L Poloxamer-188 concentrations ranging from approximately 3.9 g / L, 3.8 g / L to approximately 4.3 g / L, 3.8 g / L to approximately 4.2 g / L, 3.8 g / L to approximately 4.1 g / L, 3.8 g / L to approximately 4.0 g / L, 3.9 g / L to approximately 4.3 g / L, 3.9 g / L to approximately 4.2 g / L, 3.9 g / L to approximately 4.1 g / L, 4.0 g / L to approximately 4.3 g / L, 4.0 g / L to approximately 4.2 g / L, or approximately 4.1 g / L to approximately 4.3 g / L.

[0225] The method may include, for example, when the hole type is a drill hole and the hole diameter is approximately 1 μm to approximately 250 μm (e.g., approximately 1 μm to approximately 200 μm, approximately 1 μm to approximately 150 μm, approximately 1 μm to approximately 100 μm, approximately 1 μm to approximately 50 μm, approximately 1 μm to approximately 25 μm, approximately 10 μm to approximately 250 μm, approximately 10 μm to approximately 200 μm, approximately 10 μm to approximately 150 μm, approximately 10 μm to approximately 100 μm, approximately 10 μm to approximately 50 μm, approximately 10 μm to approximately 25 μm, approximately 25 μm to approximately 250 μm, approximately 25 μm to approximately 200 μm, approximately 25 μm to approximately 150 μm, approximately 25 μm to approximately 100 μm, approximately 25 μm to approximately 50 μm, approximately 50 μm to approximately 2 ... When the pore size is selected as approximately 4.3 g / L (e.g., above approximately 4.4 g / L, above approximately 4.5 g / L, above approximately 4.6 g / L, above approximately 4.7 g / L, above approximately 4.8 g / L, above approximately 4.9 g / L, above approximately 5.0 g / L, above approximately 5.1 g / L, above approximately 5.2 g / L, above approximately 5.3 g / L), based on the pore type and pore size, the concentration is selected to be higher than approximately 4.3 g / L (e.g., above approximately 200 μm, approximately 50 μm to approximately 150 μm, approximately 50 μm to approximately 100 μm, approximately 100 μm to approximately 250 μm, approximately 100 μm to approximately 200 μm, approximately 100 μm to approximately 150 μm, approximately 150 μm to approximately 250 μm, approximately 150 μm to approximately 200 μm, or approximately 200 μm to approximately 250 μm). Concentrations above approximately 5.4 g / L, 5.5 g / L, 5.6 g / L, 5.7 g / L, 5.8 g / L, 5.9 g / L, 6.0 g / L, 6.1 g / L, 6.2 g / L, 6.3 g / L, 6.4 g / L, 6.5 g / L, 6.6 g / L, 6.7 g / L, 6.8 g / L, 6.9 g / L, 7.0 g / L, 7.1 g / L, 7.2 g / L, 7.3 g / L, 7.4 g / L, 7.5 g / L, 7.6 g / L, and 7.7 g / L. g / L, concentration higher than about 7.8 g / L, concentration higher than about 7.9 g / L, concentration higher than about 8.0 g / L, concentration higher than about 8.1 g / L, concentration higher than about 8.2 g / L, concentration higher than about 8.2 g / L, concentration higher than about 8.3 g / L, concentration higher than about 8.Poloxamer-188 concentrations exceeding approximately 4 g / L, and concentrations exceeding approximately 8.5 g / L, 8.6 g / L, 8.7 g / L, 8.8 g / L, 8.9 g / L, 9.0 g / L, 9.1 g / L, 9.2 g / L, 9.3 g / L, 9.4 g / L, 9.5 g / L, 9.6 g / L, 9.7 g / L, 9.8 g / L, 9.9 g / L, or 10.0 g / L.

[0226] In some instances, the method includes cases where the hole type is a drill hole and the hole diameter is approximately 160 μm to approximately 190 μm (e.g., approximately 160 μm to approximately 185 μm, approximately 160 μm to approximately 180 μm, approximately 160 μm to approximately 175 μm, approximately 160 μm to approximately 170 μm, approximately 160 μm to approximately 165 μm, approximately 165 μm to approximately 190 μm, approximately 165 μm to approximately 185 μm, approximately 165 μm to approximately 180 μm, approximately 165 μm to approximately 175 μm, approximately 165 μm to approximately 170 μm, approximately 170 μm to approximately 190 μm, approximately 170 μm to approximately 185 μm, approximately 170 μm to approximately 180 μm, approximately 170 μm to approximately 175 μm, approximately 175 μm to approximately 190 μm). When the pore size is approximately 175 μm to approximately 185 μm, approximately 175 μm to approximately 180 μm, approximately 180 μm to approximately 190 μm, approximately 180 μm to approximately 185 μm, or approximately 185 μm to approximately 190 μm, the concentration is selected based on the pore type and pore size for concentrations higher than 4.3 g / L (e.g., concentrations higher than approximately 4.4 g / L, concentrations higher than approximately 4.5 g / L, concentrations higher than approximately 4.6 g / L, concentrations higher than approximately 4.7 g / L, concentrations higher than approximately 4.8 g / L, concentrations higher than approximately 4.9 g / L, concentrations higher than approximately 5.0 g / L, concentrations higher than approximately 5.1 g / L, concentrations higher than approximately 5.2 g / L, concentrations higher than approximately 5.3 g / L, concentrations higher than approximately 5.4 g / L, concentrations higher than approximately 5.5 g / L, concentrations higher than approximately 5.6 g / L, concentrations higher than approximately 5.7 g / L, and concentrations higher than approximately 5.8 g / L). g / L, concentration higher than about 5.9 g / L, concentration higher than about 6.0 g / L, concentration higher than about 6.1 g / L, concentration higher than about 6.2 g / L, concentration higher than about 6.3 g / L, concentration higher than about 6.4 g / L, concentration higher than about 6.5 g / L, concentration higher than about 6.6 g / L, concentration higher than about 6.7 g / L, concentration higher than about 6.8 g / L, concentration higher than about 6.9 g / L, concentration higher than about 7.0 g / L, concentration higher than about 7.1 g / L, concentration higher than about 7.2 g / L, concentration higher than about 7.3 g / L, concentration higher than about 7.4 g / L, concentration higher than about 7.5 g / L, concentration higher than about 7.6 g / L, concentration higher than about 7.7 g / L, concentration higher than about 7.8 g / L, concentration higher than about 7.9 g / L, concentration higher than about 8.0 g / L, concentration higher than about 8.1 g / L, concentration higher than about 8.2 g / L. g / L, concentration higher than about 8.2 g / L, concentration higher than about 8.3 g / L, concentration higher than about 8.4 g / L, concentration higher than about 8.5 g / L, concentration higher than about 8.6 g / L, concentration higher than about 8.7 g / L, concentration higher than about 8.8 g / L, concentration higher than about 8.Poloxamer-188 concentrations exceeding approximately 9 g / L, and concentrations exceeding approximately 9.0 g / L, 9.1 g / L, 9.2 g / L, 9.3 g / L, 9.4 g / L, 9.5 g / L, 9.6 g / L, 9.7 g / L, 9.8 g / L, 9.9 g / L, or 10.0 g / L.

[0227] In some embodiments, the method includes cases where the pore type is a sintered pore and the pore diameter is greater than 150 μm (e.g., greater than 155 μm, greater than 160 μm, greater than 165 μm, greater than 170 μm, greater than 175 μm, greater than about 180 μm, greater than 185 μm, greater than about 190 μm, greater than about 195 μm, greater than about 200 μm, greater than about 205 μm, greater than about 210 μm, greater than about 215 μm, greater than about 220 μm, greater than about 225 μm, greater than about 230 μm, greater than about 235 μm, greater than about 240 μm, greater than about 245 μm, greater than about 250 μm, greater than about 255 μm, greater than about 260 μm, greater than about 265 μm, greater than about 270 μm, greater than about 275 μm, greater than about 280 μm, greater than about 290 μm, greater than about 300 μm). When the pore size is greater than approximately 350 μm, greater than approximately 400 μm, greater than approximately 450 μm, or greater than approximately 500 μm, the concentration is selected based on the pore type and pore size as approximately 1.8 g / L to approximately 3.3 g / L (e.g., approximately 1.8 g / L to approximately 3.2 g / L, approximately 1.8 g / L to approximately 3.1 g / L, approximately 1.8 g / L to approximately 3.0 g / L, approximately 1.8 g / L to approximately 2.9 g / L, approximately 1.8 g / L to approximately 2.8 g / L, approximately 1.8 g / L to approximately 2.7 g / L, approximately 1.8 g / L to approximately 2.6 g / L, approximately 1.8 g / L to approximately 2.5 g / L, approximately 1.8 g / L to approximately 2.4 g / L, approximately 1.8 g / L to approximately 2.3 g / L, approximately 1.8 g / L to approximately 2.2 g / L, approximately 1.8 g / L to approximately 2.2 g / L). g / L - approx. 2.1 g / L, approx. 1.8 g / L - approx. 2.0 g / L, approx. 1.9 g / L - approx. 3.3 g / L, approx. 1.9 g / L - approx. 3.2 g / L, approx. 1.9 g / L - approx. 3.1 g / L, approx. 1.9 g / L - approx. 3.0 g / L, approx. 1.9 g / L - approx. 2.9 g / L, approx. 1.9 g / L - approx. 2.8 g / L, approx. 1.9 g / L - approx. 2.7 g / L, approx. 1.9 g / L - approx. 2.6 g / L, approx. 1.9 g / L - approx. 2.5 g / L, approx. 1.9 g / L - approx. 2.4 g / L, approx. 1.9 g / L - approx. 2.3 g / L, approx. 1.9 g / L - approx. 2.2 g / L, approx. 1.9 g / L - approx. 2.1 g / L, approx. 2.0 g / L g / L - about 3.3 g / L, about 2.0 g / L - about 3.2 g / L, about 2.0 g / L - about 3.1 g / L, about 2.0 g / L - about 3.0 g / L, about 2.0 g / L - about 2.9 g / L, about 2.0 g / L - about 2.8 g / L, about 2.0 g / L - about 2.7 g / L, about 2.0 g / L - about 2.6 g / L, approximately 2.0 g / L to approximately 2.5 g / L, approximately 2.0 g / L to approximately 2.4 g / L, approximately 2.0 g / L to approximately 2.3 g / L, approximately 2.0 g / L to approximately 2.2 g / L, approximately 2.1 g / L to approximately 3.3 g / L, approximately 2.1 g / L to approximately 3.2 g / L, approximately 2.1 g / L to approximately 3.1 g / L, approximately 2.1 g / L to approximately 3.0 g / L, approximately 2.1 g / L to approximately 2.9 g / L, approximately 2.1 g / L to approximately 2.8 g / L, approximately 2.1 g / L to approximately 2.7 g / L, approximately 2.1 g / L to approximately 2.6 g / L, approximately 2.1 g / L to approximately 2.5 g / L, approximately 2.1 g / L to approximately 2.4 g / L, approximately 2.1 g / L to approximately 2.3 g / L. g / L, approximately 2.2 g / L to approximately 3.3 g / L, approximately 2.2 g / L to approximately 3.2 g / L, approximately 2.2 g / L to approximately 3.1 g / L, approximately 2.2 g / L to approximately 3.0 g / L, approximately 2.2 g / L to approximately 2.9 g / L, approximately 2.2 g / L to approximately 2.8 g / L, approximately 2.2 g / L to approximately 2.7 g / L, approximately 2.2 g / L to approximately 2.6 g / L, approximately 2.2 g / L to approximately 2.5 g / L, approximately 2.2 g / L to approximately 2.4 g / L, approximately 2.3 g / L to approximately 3.3 g / L, approximately 2.3 g / L to approximately 3.2 g / L, approximately 2.3 g / L to approximately 3.1 g / L, approximately 2.3 g / L to approximately 3.0 g / L, approximately 2.3 g / L to approximately 2.9 g / L. g / L, approximately 2.3 g / L to approximately 2.8 g / L, approximately 2.3 g / L to approximately 2.7 g / L, approximately 2.3 g / L to approximately 2.6 g / L, approximately 2.3 g / L to approximately 2.5 g / L, approximately 2.4 g / L to approximately 3.3 g / L, approximately 2.4 g / L to approximately 3.2 g / L, approximately 2.4 g / L to approximately 3.1 g / L, approximately 2.4 g / L to approximately 3.0 g / L, approximately 2.4 g / L to approximately 2.9 g / L, approximately 2.4 g / L to approximately 2.8 g / L, approximately 2.4 g / L to approximately 2.7 g / L, approximately 2.4 g / L to approximately 2.6 g / L, approximately 2.5 g / L to approximately 3.3 g / L, approximately 2.5 g / L to approximately 3.2 g / L, approximately 2.5 g / L to approximately 3.1 g / L. g / L, approximately 2.5 g / L to approximately 3.0 g / L, approximately 2.5 g / L to approximately 2.9 g / L, approximately 2.5 g / L to approximately 2.8 g / L, approximately 2.5 g / L to approximately 2.7 g / L, approximately 2.6 g / L to approximately 3.3 g / L, approximately 2.6 g / L to approximately 3.2 g / L, approximately 2.6 g / L to approximately 3.1 g / L, approximately 2.6 g / L to approximately 3.0 g / L, approximately 2.6 g / L to approximately 2.9 g / L, approximately 2.6 g / L to approximately 2.8 g / L, approximately 2.7 g / L - about 3.3 g / L, about 2.7 g / L - about 3.2 g / L, about 2.7 g / L - about 3.1 g / L, about 2.7 g / L - about 3.0 g / L, about 2.7 g / L - about 2.9 g / L, about 2.8 g / L - about 3.3 g / L, about 2.8 g / L - about 3.2 g / L, about 2.8 g / L - about 3.1 g / L, about 2.8 g / L - about 3.0 g / L, about 2.9 g / L - about 3.3 g / L, about 2.9 g / L - about 3.2 g / L, about 2.9 g / L - about 3.1 g / L, about 3.0 g / L - about 3.3 g / L, about 3.0 g / L - about 3.2 g / L, or about 3.1 g / L - about 3.3 g / L. Poloxamer-188 (g / L).

[0228] The method may include, for example, when the pore type is a sintered pore and the pore diameter is about 80 μm to about 150 μm (e.g., about 80 μm to about 145 μm, about 80 μm to about 140 μm, about 80 μm to about 135 μm, about 80 μm to about 130 μm, about 80 μm to about 125 μm, about 80 μm to about 120 μm, about 80 μm to about 115 μm, about 80 μm to about 110 μm, about 80 μm to about 105 μm, about 80 μm to about 100 μm, about 80 μm to about 95 μm, about 80 μm to about 90 μm, about 80 μm to about 85 μm, about 85 μm to about 150 μm, about 85 μm to about 145 μm, about 85 μm to about 140 μm, about 85 μm to about 140 μm, about 85 μm to about 150 μm, about 85 μm to about 145 ...50 μm, about 85 μm to about 145 μm, about 85 μm to about 150 μm, about 85 μm to about 145 μm, about 85 μm to about 150 μm, about 85 μm to about 150 μm, about 85 μm to about 150 μm, about 85 μm to about 150 μm, about 85 μm to about 150 μm, about μm - approximately 135 μm, approximately 85 μm - approximately 130 μm, approximately 85 μm - approximately 125 μm, approximately 85 μm - approximately 120 μm, approximately 85 μm - approximately 115 μm, approximately 85 μm - approximately 110 μm, approximately 85 μm - approximately 105 μm, approximately 85 μm - approximately 100 μm, approximately 85 μm - approximately 95 μm, approximately 85 μm - approximately 90 μm, approximately 90 μm - approximately 150 μm, approximately 90 μm - approximately 145 μm, approximately 90 μm - approximately 140 μm, approximately 90 μm - approximately 135 μm, approximately 90 μm - approximately 130 μm, approximately 90 μm - approximately 125 μm, approximately 90 μm - approximately 120 μm, approximately 90 μm - approximately 115 μm, approximately 90 μm - approximately 110 μm, approximately 90 μm - approximately 105 μm, approximately 90 μm - approximately 100 μm, approximately 90 μm - approximately 95 μm, approximately 95 μm - approximately 150 μm, approximately 95 μm - approximately 145 μm, approximately 95 μm - approximately 140 μm, approximately 95 μm - approximately 135 μm, approximately 95 μm - approximately 130 μm, approximately 95 μm - approximately 125 μm, approximately 95 μm - approximately 120 μm, approximately 95 μm - approximately 115 μm, approximately 95 μm - approximately 110 μm, approximately 95 μm - approximately 105 μm, approximately 95 μm - approximately 100 μm, approximately 100 μm - approximately 150 μm, approximately 100 μm - approximately 145 μm, approximately 100 μm - approximately 140 μm, approximately 100 μm - approximately 135 μm, approximately 100 μm - approximately 130 μm μm, approximately 100 μm-125 μm, approximately 100 μm-120 μm, approximately 100 μm-115 μm, approximately 100 μm-110 μm, approximately 100 μm-105 μm, approximately 105 μm-150 μm, approximately 105 μm-145 μm, approximately 105 μm-140 μm, approximately 105 μm-135 μm,Approximately 105 μm - approximately 130 μm, approximately 105 μm - approximately 125 μm, approximately 105 μm - approximately 120 μm, approximately 105 μm - approximately 115 μm, approximately 105 μm - approximately 110 μm, approximately 110 μm - approximately 150 μm, approximately 110 μm - approximately 145 μm, approximately 110 μm - approximately 140 μm, approximately 110 μm - approximately 135 μm, approximately 110 μm - approximately 130 μm, approximately 110 μm - approximately 125 μm, approximately 110 μm - approximately 120 μm, approximately 110 μm - approximately 115 μm, approximately 115 μm - approximately 150 μm, approximately 115 μm - approximately 145 μm, approximately 115 μm - approximately 140 μm, approximately 115 μm - approximately 135 μm, approximately 115 μm - approximately 130 μm, approximately 115 μm - approximately 125 μm, approximately 115 μm - approximately 120 μm, approximately 120 μm - approximately 150 μm, approximately 120 μm - approximately 145 μm, approximately 120 μm - approximately 140 μm, approximately 120 μm - approximately 135 μm, approximately 120 μm - approximately 130 μm, approximately 120 μm - approximately 125 μm, approximately 125 μm - approximately 150 μm, approximately 125 μm - approximately 145 μm, approximately 125 μm - approximately 140 μm, approximately 125 μm - approximately 135 μm, approximately 125 μm - approximately 130 μm, approximately 130 μm - approximately 150 μm, approximately 130 μm - approximately 145 μm, approximately 130 μm - approximately 140 μm, approximately 130 μm - approximately 135 μm When selecting concentrations of approximately 3.3 g / L to approximately 4.3 g / L based on pore type and pore size (e.g., approximately 3.3 g / L to approximately 4.2 g / L, approximately 3.3 g / L to approximately 4.1 g / L, approximately 3.3 g / L to approximately 4.0 g / L, approximately 3.3 g / L to approximately 3.9 g / L, approximately 3.3 g / L to approximately 3.8 g / L, approximately 3.3 g / L to approximately 3.7 g / L, approximately 3.3 g / L to approximately 3.6 g / L, approximately 3.3 g / L to approximately 3.5 g / L), the concentration range is approximately 3.3 g / L to approximately 4.5 g / L. g / L, approximately 3.4 g / L to approximately 4.3 g / L, approximately 3.4 g / L to approximately 4.2 g / L, approximately 3.4 g / L to approximately 4.1 g / L, approximately 3.4 g / L to approximately 4.0 g / L, approximately 3.4 g / L to approximately 3.9 g / L, approximately 3.4 g / L to approximately 3.8 g / L, approximately 3.4 g / L to approximately 3.7 g / L, approximately 3.4 g / L to approximately 3.6 g / L, approximately 3.5 g / L to approximately 4.3 g / L.Approximately 3.5 g / L - approximately 4.2 g / L, approximately 3.5 g / L - approximately 4.1 g / L, approximately 3.5 g / L - approximately 4.0 g / L, approximately 3.5 g / L - approximately 3.9 g / L, approximately 3.5 g / L - approximately 3.8 g / L, approximately 3.5 g / L - approximately 3.7 g / L, approximately 3.6 g / L - approximately 4.3 g / L, approximately 3.6 g / L - approximately 4.2 g / L, approximately 3.6 g / L - approximately 4.1 g / L, approximately 3.6 g / L - approximately 4.0 g / L, approximately 3.6 g / L - approximately 3.9 g / L, approximately 3.6 g / L - approximately 3.8 g / L, approximately 3.7 g / L - approximately 4.3 g / L, approximately 3.7 g / L - approximately 4.2 g / L, approximately 3.7 g / L - approximately 4.1 g / L, approximately 3.7 g / L Poloxamer-188 with concentrations of approximately 4.0 g / L, 3.7 g / L to approximately 3.9 g / L, 3.8 g / L to approximately 4.3 g / L, 3.8 g / L to approximately 4.2 g / L, 3.8 g / L to approximately 4.1 g / L, 3.8 g / L to approximately 4.0 g / L, 3.9 g / L to approximately 4.3 g / L, 3.9 g / L to approximately 4.2 g / L, 3.9 g / L to approximately 4.1 g / L, 4.0 g / L to approximately 4.3 g / L, 4.0 g / L to approximately 4.2 g / L, or 4.1 g / L to approximately 4.3 g / L.

[0229] The method may include, for example, selecting a concentration of about 4.5 g / L or a concentration higher than about 4.5 g / L (e.g., a concentration of about 4.6 g / L or a concentration greater than 4.6 g / L) based on the pore type and pore size when the pore type is a sintered pore and the pore size is about 1 μm to about 80 μm (e.g., about 1 μm to about 50 μm, about 1 μm to about 25 μm, about 1 μm to about 10 μm, about 10 μm to about 50 μm, about 10 μm to about 25 μm, about 25 μm to about 80 μm, about 25 μm to about 50 μm, or about 50 μm to about 80 μm). The concentrations are approximately 4.7 g / L or greater than 4.7 g / L, approximately 4.8 g / L or greater than 4.8 g / L, approximately 4.9 g / L or greater than 4.9 g / L, approximately 5.0 g / L or greater than 5.0 g / L, approximately 5.1 g / L or greater than 5.1 g / L, approximately 5.2 g / L or greater than 5.2 g / L, approximately 5.3 g / L or greater than 5.3 g / L, approximately 5.4 g / L or greater than 5.4 g / L, approximately 5.5 g / L or greater than 5.5 g / L, approximately 5.6 g / L or greater than 5.6 g / L, approximately 5.7 g / L or greater than 5.7 g / L, and approximately 5.8 g / L or greater than 5.8 g / L. The concentrations are approximately 6.0 g / L or greater than 6.0 g / L, approximately 6.1 g / L or greater than 6.1 g / L, approximately 6.2 g / L or greater than 6.2 g / L, approximately 6.3 g / L or greater than 6.3 g / L, approximately 6.4 g / L or greater than 6.4 g / L, approximately 6.5 g / L or greater than 6.5 g / L, approximately 6.6 g / L or greater than 6.6 g / L, approximately 6.7 g / L or greater than 6.7 g / L, approximately 6.8 g / L or greater than 6.8 g / L, approximately 6.9 g / L or greater than 6.9 g / L, approximately 7.0 g / L or greater than 7.0 g / L, and approximately 7.1 g / L or greater than 7.1 g / L. The concentration is approximately 7.2 g / L or greater than 7.2 g / L, the concentration is approximately 7.3 g / L or greater than 7.3 g / L, the concentration is approximately 7.4 g / L or greater than 7.4 g / L, the concentration is approximately 7.5 g / L or greater than 7.5 g / L, and the concentration is approximately 7.6 g / L or a concentration greater than 7.6 g / L, a concentration of approximately 7.7 g / L or greater than 7.7 g / L, a concentration of approximately 7.8 g / L or greater than 7.8 g / L, a concentration of approximately 7.9 g / L or greater than 7.9 g / L, a concentration of approximately 8.0 g / L or greater than 8.0 g / L, a concentration of approximately 8.1 g / L or greater than 8.1 g / L, a concentration of approximately 8.2 g / L or greater than 8.2 g / L, a concentration of approximately 8.3 g / L or greater than 8.3 g / L, a concentration of approximately 8.4 g / L or greater than 8.4 g / L, a concentration of approximately 8.5 g / L or greater than 8.5 g / L, a concentration of approximately 8.6 g / L or greater than 8.6 g / L, a concentration of approximately 8.7 g / L or greater than 8.7 g / L, a concentration of approximately 8.8 g / L. The concentrations are approximately 8.8 g / L or greater, approximately 8.9 g / L or greater, approximately 9.0 g / L or greater, approximately 9.1 g / L or greater, approximately 9.2 g / L or greater, approximately 9.3 g / L or greater, approximately 9.4 g / L or greater, approximately 9.5 g / L or greater, approximately 9.6 g / L or greater, approximately 9.7 g / L or greater, approximately 9.8 g / L or greater, approximately 9.9 g / L or greater, or approximately 10.0 g / L. Poloxamer-188 (at a concentration of g / L or greater than 10.0 g / L).

[0230] When the culture is a perfusion culture, the second liquid culture medium may include a selected concentration of poloxamer-188.

[0231] Based on increased poloxamer-188 concentration due to increased live cell density

[0232] Some embodiments of any method described in this application include increasing the concentration of poloxamer-188 in the culture medium over time based on the live cell density in the culture medium. For example, any method described in this application may include when the live cell density in the culture medium is about 20 x 10⁻⁶. 6 Cells / mL to approximately 60 x 10⁻⁶ 6 Cells / mL (e.g., approximately 20 x 10⁻⁶) 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 20 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6Cells / mL, approximately 20 x 10 6 Cells / mL - Approximately 54 x 10 6 Cells / mL, approximately 20 x 10 6 Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 20 x 10 6 Cells / mL - Approximately 50 x 10 6 Cells / mL, approximately 20 x 10 6 Cells / mL - Approximately 48 x 10 6 Cells / mL, approximately 20 x 10 6 Cells / mL - Approximately 46 x 10 6 cells / mL, approximately 20 x 10 6 Cells / mL - Approximately 44 x 10 6 Cells / mL, approximately 20 x 10 6 Cells / mL - Approximately 42 x 10 6 Cells / mL, approximately 20 x 10 6 Cells / mL to approximately 40 x 10 6 Cells / mL, approximately 20 x 10 6 Cells / mL - Approximately 38 x 10 6 Cells / mL, approximately 20 x 10 6 Cells / mL - Approximately 36 x 10 6 Cells / mL, approximately 20 x 10 6 Cells / mL - Approximately 34 x 10 6 Cells / mL, approximately 20 x 10 6 Cells / mL - Approximately 32 x 10 6 Cells / mL, approximately 20 x 10 6 Cells / mL - Approximately 30 x 10 6 Cells / mL, approximately 20 x 10 6 cells / mL to approximately 28 x 10 6 Cells / mL, approximately 20 x 10 6 Cells / mL - Approximately 26 x 10 6 Cells / mL, approximately 20 x 10 6 Cells / mL - Approximately 24 x 10 6 Cells / mL, approximately 20 x 10 6 Cells / mL - Approximately 22 x 10 6 Cells / mL, approximately 22 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 22 x 10 6Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 22 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6 Cells / mL, approximately 22 x 10⁻⁶ 6 Cells / mL - Approximately 54 x 10 6 Cells / mL, approximately 22 x 10 6 Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 22 x 10 6 Cells / mL - Approximately 50 x 10 6 Cells / mL, approximately 22 x 10 6 Cells / mL - Approximately 48 x 10 6 Cells / mL, approximately 22 x 10 6 Cells / mL - Approximately 46 x 10 6 Cells / mL, approximately 22 x 10 6 Cells / mL - Approximately 44 x 10 6 Cells / mL, approximately 22 x 10 6 Cells / mL - Approximately 42 x 10 6 Cells / mL, approximately 22 x 10 6 Cells / mL to approximately 40 x 10 6 Cells / mL, approximately 22 x 10 6 Cells / mL - Approximately 38 x 10 6 Cells / mL, approximately 22 x 10 6 Cells / mL - Approximately 36 x 10 6 Cells / mL, approximately 22 x 10 6 Cells / mL - Approximately 34 x 10 6 Cells / mL, approximately 22 x 10 6 Cells / mL - Approximately 32 x 10 6 Cells / mL, approximately 22 x 10 6 Cells / mL - Approximately 30 x 10 6 cells / mL, approximately 22 x 10 6 Cells / mL to approximately 28 x 10⁻⁶ 6 Cells / mL, approximately 22 x 10 6 Cells / mL - Approximately 26 x 10 6 Cells / mL, approximately 22 x 10 6 Cells / mL - Approximately 24 x 10 6 Cells / mL, approximately 24 x 10 6 Cells / mL - Approximately 60 x 10 6Cells / mL, approximately 24 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 24 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6 Cells / mL, approximately 24 x 10 6 Cells / mL - Approximately 54 x 10⁻⁶ 6 Cells / mL, approximately 24 x 10 6 Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 24 x 10 6 Cells / mL - Approximately 50 x 10 6 Cells / mL, approximately 24 x 10 6 Cells / mL - Approximately 48 x 10 6 Cells / mL, approximately 24 x 10 6 Cells / mL - Approximately 46 x 10 6 Cells / mL, approximately 24 x 10 6 Cells / mL - Approximately 44 x 10 6 Cells / mL, approximately 24 x 10 6 Cells / mL - Approximately 42 x 10 6 cells / mL, approximately 24 x 10 6 Cells / mL to approximately 40 x 10 6 Cells / mL, approximately 24 x 10 6 Cells / mL - Approximately 38 x 10 6 Cells / mL, approximately 24 x 10 6 Cells / mL - Approximately 36 x 10 6 Cells / mL, approximately 24 x 10 6 Cells / mL - Approximately 34 x 10 6 Cells / mL, approximately 24 x 10 6 Cells / mL - Approximately 32 x 10 6 Cells / mL, approximately 24 x 10 6 Cells / mL - Approximately 30 x 10 6 Cells / mL, approximately 24 x 10 6 Cells / mL to approximately 28 x 10⁻⁶ 6 Cells / mL, approximately 24 x 10 6 Cells / mL - Approximately 26 x 10 6 Cells / mL, approximately 26 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 26 x 10 6Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 26 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6 Cells / mL, approximately 26 x 10 6 Cells / mL - Approximately 54 x 10 6 Cells / mL, approximately 26 x 10 6 Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 26 x 10 6 Cells / mL - Approximately 50 x 10 6 Cells / mL, approximately 26 x 10 6 Cells / mL - Approximately 48 x 10 6 cells / mL, approximately 26 x 10 6 Cells / mL - Approximately 46 x 10 6 Cells / mL, approximately 26 x 10 6 Cells / mL - Approximately 44 x 10 6 Cells / mL, approximately 26 x 10 6 Cells / mL - Approximately 42 x 10 6 Cells / mL, approximately 26 x 10 6 Cells / mL to approximately 40 x 10 6 Cells / mL, approximately 26 x 10 6 Cells / mL - Approximately 38 x 10 6 Cells / mL, approximately 26 x 10 6 Cells / mL - Approximately 36 x 10 6 Cells / mL, approximately 26 x 10 6 Cells / mL - Approximately 34 x 10 6 Cells / mL, approximately 26 x 10 6 Cells / mL - Approximately 32 x 10 6 Cells / mL, approximately 26 x 10 6 Cells / mL - Approximately 30 x 10 6 Cells / mL, approximately 26 x 10 6 Cells / mL to approximately 28 x 10⁻⁶ 6 Cells / mL, approximately 28 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 28 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 28 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6Cells / mL, approximately 28 x 10 6 Cells / mL - Approximately 54 x 10 6 Cells / mL, approximately 28 x 10 6 Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 28 x 10 6 Cells / mL - Approximately 50 x 10 6 Cells / mL, approximately 28 x 10 6 Cells / mL - Approximately 48 x 10 6 cells / mL, approximately 28 x 10 6 Cells / mL - Approximately 46 x 10 6 Cells / mL, approximately 28 x 10 6 Cells / mL - Approximately 44 x 10 6 Cells / mL, approximately 28 x 10 6 Cells / mL - Approximately 42 x 10 6 Cells / mL, approximately 28 x 10 6 Cells / mL to approximately 40 x 10 6 Cells / mL, approximately 28 x 10 6 Cells / mL - Approximately 38 x 10 6 Cells / mL, approximately 28 x 10 6 Cells / mL - Approximately 36 x 10 6 Cells / mL, approximately 28 x 10 6 Cells / mL - Approximately 34 x 10 6 Cells / mL, approximately 28 x 10 6 Cells / mL - Approximately 32 x 10 6 Cells / mL, approximately 28 x 10 6 Cells / mL - Approximately 30 x 10 6 Cells / mL, approximately 30 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 30 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 30 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6 Cells / mL, approximately 30 x 10 6 Cells / mL - Approximately 54 x 10 6 Cells / mL, approximately 30 x 10 6 Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 30 x 10 6Cells / mL - Approximately 50 x 10 6 Cells / mL, approximately 30 x 10 6 Cells / mL - Approximately 48 x 10 6 Cells / mL, approximately 30 x 10 6 Cells / mL - Approximately 46 x 10 6 cells / mL, approximately 30 x 10 6 Cells / mL - Approximately 44 x 10 6 Cells / mL, approximately 30 x 10 6 Cells / mL - Approximately 42 x 10 6 Cells / mL, approximately 30 x 10 6 Cells / mL to approximately 40 x 10 6 Cells / mL, approximately 30 x 10 6 Cells / mL - Approximately 38 x 10 6 Cells / mL, approximately 30 x 10 6 Cells / mL - Approximately 36 x 10 6 Cells / mL, approximately 30 x 10 6 Cells / mL - Approximately 34 x 10 6 Cells / mL, approximately 30 x 10 6 Cells / mL - Approximately 32 x 10 6 Cells / mL, approximately 32 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 32 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 32 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6 Cells / mL, approximately 32 x 10 6 Cells / mL - Approximately 54 x 10 6 Cells / mL, approximately 32 x 10 6 Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 32 x 10 6 Cells / mL - Approximately 50 x 10 6 Cells / mL, approximately 32 x 10 6 Cells / mL - Approximately 48 x 10 6 Cells / mL, approximately 32 x 10 6 Cells / mL - Approximately 46 x 10 6 Cells / mL, approximately 32 x 10 6 Cells / mL - Approximately 44 x 10 6Cells / mL, approximately 32 x 10 6 Cells / mL - Approximately 42 x 10 6 Cells / mL, approximately 32 x 10 6 Cells / mL to approximately 40 x 10 6 Cells / mL, approximately 32 x 10 6 Cells / mL - Approximately 38 x 10 6 Cells / mL, approximately 32 x 10 6 Cells / mL - Approximately 36 x 10 6 Cells / mL, approximately 32 x 10 6 Cells / mL - Approximately 34 x 10 6 Cells / mL, approximately 34 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 34 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 34 x 10 6 Cells / mL to approximately 56 x 10 6 Cells / mL, approximately 34 x 10 6 Cells / mL - Approximately 54 x 10 6 Cells / mL, approximately 34 x 10 6 Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 34 x 10 6 Cells / mL - Approximately 50 x 10 6 Cells / mL, approximately 34 x 10 6 Cells / mL - Approximately 48 x 10 6 Cells / mL, approximately 34 x 10 6 Cells / mL - Approximately 46 x 10 6 Cells / mL, approximately 34 x 10 6 Cells / mL - Approximately 44 x 10 6 cells / mL, approximately 34 x 10 6 Cells / mL - Approximately 42 x 10 6 Cells / mL, approximately 34 x 10 6 Cells / mL to approximately 40 x 10 6 Cells / mL, approximately 34 x 10 6 Cells / mL - Approximately 38 x 10 6 Cells / mL, approximately 34 x 10 6 Cells / mL - Approximately 36 x 10 6 Cells / mL, approximately 36 x 10 6Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 36 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 36 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6 Cells / mL, approximately 36 x 10 6 Cells / mL - Approximately 54 x 10 6 Cells / mL, approximately 36 x 10 6 Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 36 x 10 6 Cells / mL - Approximately 50 x 10 6 Cells / mL, approximately 36 x 10 6 Cells / mL - Approximately 48 x 10 6 Cells / mL, approximately 36 x 10 6 Cells / mL - Approximately 46 x 10 6 Cells / mL, approximately 36 x 10 6 Cells / mL - Approximately 44 x 10 6 cells / mL, approximately 36 x 10 6 Cells / mL - Approximately 42 x 10 6 Cells / mL, approximately 36 x 10 6 Cells / mL to approximately 40 x 10 6 Cells / mL, approximately 36 x 10 6 Cells / mL - Approximately 38 x 10 6 Cells / mL, approximately 38 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 38 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 38 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6 Cells / mL, approximately 38 x 10 6 Cells / mL - Approximately 54 x 10⁻⁶ 6 Cells / mL, approximately 38 x 10 6 Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 38 x 10 6 Cells / mL - Approximately 50 x 10 6 Cells / mL, approximately 38 x 10 6 Cells / mL - Approximately 48 x 10 6Cells / mL, approximately 38 x 10 6 Cells / mL - Approximately 46 x 10 6 Cells / mL, approximately 38 x 10 6 Cells / mL - Approximately 44 x 10 6 Cells / mL, approximately 38 x 10 6 Cells / mL - Approximately 42 x 10 6 cells / mL, approximately 38 x 10 6 Cells / mL to approximately 40 x 10 6 Cells / mL, approximately 40 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 40 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 40 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6 Cells / mL, approximately 40 x 10 6 Cells / mL - Approximately 54 x 10 6 Cells / mL, approximately 40 x 10 6 Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 40 x 10 6 Cells / mL - Approximately 50 x 10 6 Cells / mL, approximately 40 x 10 6 Cells / mL - Approximately 48 x 10 6 Cells / mL, approximately 40 x 10 6 Cells / mL - Approximately 46 x 10 6 Cells / mL, approximately 40 x 10 6 Cells / mL - Approximately 44 x 10 6 Cells / mL, approximately 40 x 10 6 Cells / mL - Approximately 42 x 10 6 Cells / mL, approximately 42 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 42 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 42 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6 Cells / mL, approximately 42 x 10 6 Cells / mL - Approximately 54 x 10 6 Cells / mL, approximately 42 x 10⁻⁶ 6Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 42 x 10 6 Cells / mL - Approximately 50 x 10 6 Cells / mL, approximately 42 x 10 6 Cells / mL - Approximately 48 x 10 6 Cells / mL, approximately 42 x 10 6 Cells / mL - Approximately 46 x 10 6 Cells / mL, approximately 42 x 10 6 Cells / mL - Approximately 44 x 10 6 Cells / mL, approximately 44 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 44 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 44 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6 Cells / mL, approximately 44 x 10 6 Cells / mL - Approximately 54 x 10 6 Cells / mL, approximately 44 x 10 6 Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 44 x 10 6 Cells / mL - Approximately 50 x 10 6 Cells / mL, approximately 44 x 10 6 Cells / mL - Approximately 48 x 10 6 Cells / mL, approximately 44 x 10 6 Cells / mL - Approximately 46 x 10 6 cells / mL, approximately 46 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 46 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 46 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6 Cells / mL, approximately 46 x 10 6 Cells / mL - Approximately 54 x 10 6 Cells / mL, approximately 46 x 10 6 Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 46 x 10 6 Cells / mL - Approximately 50 x 10 6Cells / mL, approximately 46 x 10 6 Cells / mL - Approximately 48 x 10 6 Cells / mL, approximately 48 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 48 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 48 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6 Cells / mL, approximately 48 x 10 6 Cells / mL - Approximately 54 x 10 6 Cells / mL, approximately 48 x 10 6 Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 48 x 10 6 Cells / mL - Approximately 50 x 10 6 Cells / mL, approximately 50 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 50 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 50 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6 Cells / mL, approximately 50 x 10 6 Cells / mL - Approximately 54 x 10 6 Cells / mL, approximately 50 x 10 6 Cells / mL - Approximately 52 x 10 6 Cells / mL, approximately 52 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 52 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 52 x 10 6 Cells / mL to approximately 56 x 10⁻⁶ 6 Cells / mL, approximately 52 x 10 6 Cells / mL - Approximately 54 x 10⁻⁶ 6 Cells / mL, approximately 54 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 54 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, approximately 54 x 10 6Cells / mL to approximately 56 x 10⁻⁶ 6 Cells / mL, approximately 56 x 10 6 Cells / mL - Approximately 60 x 10 6 Cells / mL, approximately 56 x 10 6 Cells / mL - Approximately 58 x 10 6 Cells / mL, or approximately 58 x 10⁻⁶ 6 Cells / mL - Approximately 60 x 10 6When the concentration of poloxamer-188 in the culture medium is increased to greater than 1.8 g / L (e.g., about 1.8 g / L - about 3.0 g / L, about 1.8 g / L - about 2.9 g / L, about 1.8 g / L - about 2.8 g / L, about 1.8 g / L - about 2.7 g / L, about 1.8 g / L - about 2.5 g / L, about 1.8 g / L - about 2.4 g / L, about 1.8 g / L - about 2.3 g / L, about 1.8 g / L - about 2.2 g / L, about 1.8 g / L - about 2.1 g / L, about 1.8 g / L - about 2.0 g / L, about 1.9 g / L - about 3.0 g / L, about 1.9 g / L - about 2.9 g / L, about 1.9 g / L - about 2.8 g / L, about 1.9 g / L), the concentration of poloxamer-188 in the culture medium is increased to greater than 1.8 g / L (e.g., about 1.9 g / L - about 3.0 g / L, about 1.9 g / L - about 2.9 g / L, about 1.9 g / L - about 2.8 g / L, about 1.9 g / L - about 2.8 g / L). g / L - approx. 2.7 g / L, approx. 1.9 g / L - approx. 2.6 g / L, approx. 1.9 g / L - approx. 2.5 g / L, approx. 1.9 g / L - approx. 2.4 g / L, approx. 1.9 g / L - approx. 2.3 g / L, approx. 1.9 g / L - approx. 2.2 g / L, approx. 1.9 g / L - approx. 2.1 g / L, approx. 2.0 g / L - approx. 3.0 g / L, approx. 2.0 g / L - approx. 2.9 g / L, approx. 2.0 g / L - approx. 2.8 g / L, approx. 2.0 g / L - approx. 2.7 g / L, approx. 2.0 g / L - approx. 2.6 g / L, approx. 2.0 g / L - approx. 2.5 g / L, approx. 2.0 g / L - approx. 2.4 g / L, approx. 2.0 g / L - approx. 2.3 g / L, approx. 2.0 g / L g / L - about 2.2 g / L, about 2.1 g / L - about 3.0 g / L, about 2.1 g / L - about 2.9 g / L, about 2.1 g / L - about 2.8 g / L, about 2.1 g / L - about 2.7 g / L, about 2.1 g / L - about 2.6 g / L, about 2.1 g / L - about 2.5 g / L, about 2.1 g / L - about 2.4 g / L, about 2.1 g / L - about 2.3 g / L, about 2.2 g / L - about 3.0 g / L, about 2.2 g / L - about 2.9 g / L, about 2.2 g / L - about 2.8 g / L, about 2.2 g / L - about 2.7 g / L, about 2.2 g / L - about 2.6 g / L, about 2.2 g / L - about 2.5 g / L g / L, approximately 2.2 g / L to approximately 2.4 g / L, approximately 2.3 g / L to approximately 3.0 g / L, approximately 2.3 g / L to approximately 2.9 g / L, approximately 2.3 g / L to approximately 2.8 g / L, approximately 2.3 g / L to approximately 2.7 g / L, approximately 2.3 g / L to approximately 2.6 g / L, approximately 2.3 g / L to approximately 2.5 g / L, approximately 2.4 g / L to approximately 3.0 g / L, approximately 2.4 g / L to approximately 2.9 g / L, approximately 2.4 g / L to approximately 2.8 g / L, approximately 2.4 g / L - about 2.7 g / L, about 2.4 g / L - about 2.6 g / L, about 2.5 g / L - about 3.0 g / L, about 2.5 g / L - about 2.9 g / L, about 2.5 g / L - about 2.8 g / L, about 2.5 g / L - about 2.7 g / L, about 2.6 g / L - about 3.0 g / L, about 2.6 g / L - about 2.9 g / L, about 2.6 g / L - about 2.8 g / L, about 2.7 g / L - about 3.0 g / L, about 2.7 g / L - about 2.9 g / L, or about 2.8 g / L - about 3.0 g / L.

[0233] Any method described in this application may include when the live cell density in the culture medium is about 60 x 10⁻⁶. 6 Cells / mL to approximately 90 x 10⁻⁶ 6 Cells / mL (e.g., approximately 60 x 10⁻⁶) 6 Cells / mL - Approximately 88 x 10 6 Cells / mL, approximately 60 x 10 6 Cells / mL to approximately 86 x 10⁻⁶ 6 Cells / mL, approximately 60 x 10 6 Cells / mL - Approximately 84 x 10 6 Cells / mL, approximately 60 x 10 6 Cells / mL - Approximately 82 x 10⁻⁶ 6 Cells / mL, approximately 60 x 10 6 Cells / mL - Approximately 80 x 10 6 Cells / mL, approximately 60 x 10 6 Cells / mL - Approximately 78 x 10 6 Cells / mL, approximately 60 x 10 6 Cells / mL - Approximately 76 x 10 6 Cells / mL, approximately 60 x 10 6 Cells / mL - Approximately 74 x 10 6 Cells / mL, approximately 60 x 10 6 Cells / mL - Approximately 72 x 10 6 Cells / mL, approximately 60 x 10 6 Cells / mL to approximately 70 x 10⁻⁶ 6 Cells / mL, approximately 60 x 10⁻⁶ 6 Cells / mL - Approximately 68 x 10 6 Cells / mL, approximately 60 x 10 6 Cells / mL - Approximately 66 x 10 6 Cells / mL, approximately 60 x 10 6Cells / mL - Approximately 64 x 10 6 Cells / mL, approximately 60 x 10 6 Cells / mL - Approximately 62 x 10 6 Cells / mL, approximately 62 x 10 6 Cells / mL - Approximately 90 x 10⁻⁶ 6 Cells / mL, approximately 62 x 10 6 Cells / mL - Approximately 88 x 10 6 Cells / mL, approximately 62 x 10 6 Cells / mL to approximately 86 x 10⁻⁶ 6 Cells / mL, approximately 62 x 10 6 Cells / mL - Approximately 84 x 10 6 Cells / mL, approximately 62 x 10 6 Cells / mL - Approximately 82 x 10⁻⁶ 6 Cells / mL, approximately 62 x 10 6 Cells / mL - Approximately 80 x 10 6 Cells / mL, approximately 62 x 10 6 Cells / mL - Approximately 78 x 10 6 Cells / mL, approximately 62 x 10 6 Cells / mL - Approximately 76 x 10 6 Cells / mL, approximately 62 x 10 6 Cells / mL - Approximately 74 x 10 6 Cells / mL, approximately 62 x 10 6 Cells / mL - Approximately 72 x 10 6 Cells / mL, approximately 62 x 10 6 Cells / mL to approximately 70 x 10⁻⁶ 6 Cells / mL, approximately 62 x 10 6 Cells / mL - Approximately 68 x 10 6 Cells / mL, approximately 62 x 10 6 Cells / mL - Approximately 66 x 10 6 Cells / mL, approximately 62 x 10 6 Cells / mL - Approximately 64 x 10 6 Cells / mL, approximately 64 x 10 6 Cells / mL - Approximately 90 x 10⁻⁶ 6 Cells / mL, approximately 64 x 10 6 Cells / mL - Approximately 88 x 10 6 Cells / mL, approximately 64 x 10 6 Cells / mL to approximately 86 x 10⁻⁶ 6Cells / mL, approximately 64 x 10 6 Cells / mL - Approximately 84 x 10 6 Cells / mL, approximately 64 x 10 6 Cells / mL - Approximately 82 x 10⁻⁶ 6 Cells / mL, approximately 64 x 10 6 Cells / mL - Approximately 80 x 10 6 Cells / mL, approximately 64 x 10 6 Cells / mL - Approximately 78 x 10 6 Cells / mL, approximately 64 x 10 6 Cells / mL - Approximately 76 x 10 6 Cells / mL, approximately 64 x 10 6 Cells / mL - Approximately 74 x 10 6 Cells / mL, approximately 64 x 10 6 Cells / mL - Approximately 72 x 10 6 Cells / mL, approximately 64 x 10 6 Cells / mL to approximately 70 x 10⁻⁶ 6 Cells / mL, approximately 64 x 10 6 Cells / mL - Approximately 68 x 10 6 Cells / mL, approximately 64 x 10 6 Cells / mL - Approximately 66 x 10 6 Cells / mL, approximately 66 x 10 6 Cells / mL - Approximately 90 x 10⁻⁶ 6 Cells / mL, approximately 66 x 10 6 Cells / mL - Approximately 88 x 10 6 Cells / mL, approximately 66 x 10 6 Cells / mL to approximately 86 x 10⁻⁶ 6 Cells / mL, approximately 66 x 10 6 Cells / mL - Approximately 84 x 10 6 Cells / mL, approximately 66 x 10 6 Cells / mL - Approximately 82 x 10⁻⁶ 6 Cells / mL, approximately 66 x 10 6 Cells / mL - Approximately 80 x 10 6 Cells / mL, approximately 66 x 10 6 Cells / mL - Approximately 78 x 10 6 Cells / mL, approximately 66 x 10 6 Cells / mL - Approximately 76 x 10 6 Cells / mL, approximately 66 x 10 6Cells / mL - Approximately 74 x 10 6 Cells / mL, approximately 66 x 10 6 Cells / mL - Approximately 72 x 10 6 Cells / mL, approximately 66 x 10 6 Cells / mL to approximately 70 x 10⁻⁶ 6 Cells / mL, approximately 66 x 10 6 Cells / mL - Approximately 68 x 10 6 Cells / mL, approximately 68 x 10 6 Cells / mL - Approximately 90 x 10⁻⁶ 6 Cells / mL, approximately 68 x 10 6 Cells / mL - Approximately 88 x 10 6 Cells / mL, approximately 68 x 10 6 Cells / mL to approximately 86 x 10⁻⁶ 6 Cells / mL, approximately 68 x 10 6 Cells / mL - Approximately 84 x 10 6 Cells / mL, approximately 68 x 10 6 Cells / mL - Approximately 82 x 10⁻⁶ 6 Cells / mL, approximately 68 x 10 6 Cells / mL - Approximately 80 x 10 6 Cells / mL, approximately 68 x 10 6 Cells / mL - Approximately 78 x 10 6 Cells / mL, approximately 68 x 10 6 Cells / mL - Approximately 76 x 10 6 Cells / mL, approximately 68 x 10 6 Cells / mL - Approximately 74 x 10 6 Cells / mL, approximately 68 x 10 6 Cells / mL - Approximately 72 x 10 6 Cells / mL, approximately 68 x 10 6 Cells / mL to approximately 70 x 10⁻⁶ 6 Cells / mL, approximately 70 x 10⁻⁶ 6 Cells / mL - Approximately 90 x 10⁻⁶ 6 Cells / mL, approximately 70 x 10 6 Cells / mL - Approximately 88 x 10 6 Cells / mL, approximately 70 x 10 6 Cells / mL to approximately 86 x 10⁻⁶ 6 Cells / mL, approximately 70 x 10 6 Cells / mL - Approximately 84 x 10 6Cells / mL, approximately 70 x 10 6 Cells / mL - Approximately 82 x 10⁻⁶ 6 Cells / mL, approximately 70 x 10 6 Cells / mL - Approximately 80 x 10 6 Cells / mL, approximately 70 x 10 6 Cells / mL - Approximately 78 x 10 6 Cells / mL, approximately 70 x 10 6 Cells / mL - Approximately 76 x 10 6 Cells / mL, approximately 70 x 10 6 Cells / mL - Approximately 74 x 10 6 Cells / mL, approximately 70 x 10 6 Cells / mL - Approximately 72 x 10 6 Cells / mL, approximately 72 x 10 6 Cells / mL - Approximately 90 x 10⁻⁶ 6 Cells / mL, approximately 72 x 10 6 Cells / mL - Approximately 88 x 10 6 Cells / mL, approximately 72 x 10 6 Cells / mL to approximately 86 x 10⁻⁶ 6 Cells / mL, approximately 72 x 10 6 Cells / mL - Approximately 84 x 10 6 Cells / mL, approximately 72 x 10 6 Cells / mL - Approximately 82 x 10⁻⁶ 6 Cells / mL, approximately 72 x 10 6 Cells / mL - Approximately 80 x 10 6 Cells / mL, approximately 72 x 10 6 Cells / mL - Approximately 78 x 10 6 Cells / mL, approximately 72 x 10 6 Cells / mL - Approximately 76 x 10 6 Cells / mL, approximately 72 x 10 6 Cells / mL - Approximately 74 x 10 6 Cells / mL, approximately 74 x 10 6 Cells / mL - Approximately 90 x 10⁻⁶ 6 Cells / mL, approximately 74 x 10 6 Cells / mL - Approximately 88 x 10 6 Cells / mL, approximately 74 x 10 6 Cells / mL to approximately 86 x 10⁻⁶ 6 Cells / mL, approximately 74 x 10 6Cells / mL - Approximately 84 x 10 6 Cells / mL, approximately 74 x 10 6 Cells / mL - Approximately 82 x 10⁻⁶ 6 Cells / mL, approximately 74 x 10 6 Cells / mL - Approximately 80 x 10 6 Cells / mL, approximately 74 x 10 6 Cells / mL - Approximately 78 x 10 6 Cells / mL, approximately 74 x 10 6 Cells / mL - Approximately 76 x 10 6 Cells / mL, approximately 76 x 10 6 Cells / mL - Approximately 90 x 10⁻⁶ 6 Cells / mL, approximately 76 x 10 6 Cells / mL - Approximately 88 x 10 6 Cells / mL, approximately 76 x 10 6 Cells / mL to approximately 86 x 10⁻⁶ 6 Cells / mL, approximately 76 x 10⁻⁶ 6 Cells / mL - Approximately 84 x 10 6 Cells / mL, approximately 76 x 10 6 Cells / mL - Approximately 82 x 10⁻⁶ 6 Cells / mL, approximately 76 x 10 6 Cells / mL - Approximately 80 x 10 6 Cells / mL, approximately 76 x 10 6 Cells / mL - Approximately 78 x 10 6 Cells / mL, approximately 78 x 10 6 Cells / mL - Approximately 90 x 10⁻⁶ 6 Cells / mL, approximately 78 x 10 6 Cells / mL - Approximately 88 x 10 6 Cells / mL, approximately 78 x 10 6 Cells / mL to approximately 86 x 10⁻⁶ 6 Cells / mL, approximately 78 x 10 6 Cells / mL - Approximately 84 x 10 6 Cells / mL, approximately 78 x 10 6 Cells / mL - Approximately 82 x 10⁻⁶ 6 Cells / mL, approximately 78 x 10 6 Cells / mL - Approximately 80 x 10 6 Cells / mL, approximately 80 x 10 6 Cells / mL - Approximately 90 x 10⁻⁶ 6Cells / mL, approximately 80 x 10 6 Cells / mL - Approximately 88 x 10 6 Cells / mL, approximately 80 x 10 6 Cells / mL to approximately 86 x 10⁻⁶ 6 Cells / mL, approximately 80 x 10⁻⁶ 6 Cells / mL - Approximately 84 x 10 6 Cells / mL, approximately 80 x 10 6 Cells / mL - Approximately 82 x 10⁻⁶ 6 Cells / mL, approximately 82 x 10 6 Cells / mL - Approximately 90 x 10⁻⁶ 6 Cells / mL, approximatel...

Claims

1. A method for culturing mammalian cells, the method comprising: Mammalian cells containing nucleic acids encoding the recombinant protein were perfused and cultured under conditions sufficient to produce the recombinant protein. The perfusion culture comprises culturing in a liquid medium containing poloxamer-188 at a concentration of 1.8 g / L or higher, wherein the poloxamer-188 is present in the medium and / or added to the medium prior to the culture step and / or added to the medium during the culture step.

2. The method of claim 1, wherein the culture medium comprises poloxamer-188 at a concentration of 1.8 g / L or higher.

3. The method of claim 2, wherein the culture medium comprises poloxamer-188 at a concentration of 2.8 g / L or higher.

4. The method of claim 3, wherein the culture medium comprises poloxamer-188 at a concentration of 4.0 g / L or higher.

5. The method of claim 4, wherein the culture medium comprises poloxamer-188 at a concentration of 6.0 g / L or higher.

6. The method of claim 5, wherein the culture medium comprises poloxamer-188 at a concentration of 8.0 g / L or higher.

7. The method of claim 6, wherein the culture medium comprises poloxamer-188 at a concentration of 10.0 g / L or higher.

8. The method of any one of claims 1-7, wherein the culture medium further comprises an antifoaming agent, and the ratio of the antifoaming agent (g / L) to poloxamer-188 (g / L) in the culture medium is from about 0.5% to about 6.0% in the liquid culture medium.

9. The method of claim 8, wherein the ratio of the antifoaming agent (g / L) to poloxamer-188 (g / L) in the culture medium is from about 1.0% to about 5.5%.

10. The method of claim 9, wherein the ratio of the defoamer (g / L) to poloxamer-188 (g / L) in the culture medium is from about 1.5% to about 5.0%.

Images