Methods of culturing a mammalian cell
12486524 ยท 2025-12-02
Assignee
Inventors
- Christopher Hwang (Cambridge, MA, US)
- Timothy Johnson (Cambridge, MA, US)
- Jason Walther (Cambridge, MA, US)
- Cheng Cheng (Cambridge, MA, US)
- Jonathan Wang (Cambridge, MA, US)
- Neha Shah (Cambridge, MA, US)
- Seul-A Bae (Cambridge, MA, US)
Cpc classification
C12N2500/50
CHEMISTRY; METALLURGY
C12N5/0043
CHEMISTRY; METALLURGY
C07K2317/14
CHEMISTRY; METALLURGY
C07K16/00
CHEMISTRY; METALLURGY
International classification
C07K16/00
CHEMISTRY; METALLURGY
Abstract
Provided herein are methods of culturing a mammalian cell in a liquid medium including poloxamer-188 at a concentration of 1.8 g/L or at a greater concentration than 1.8 g/L more or a liquid medium that includes a poloxamer-188 concentration that is selected based on one or more factors selected from the group of: pore size, pore type, gas flow rate, viable cell density in the medium, and markers related to cell stress.
Claims
1. A method of culturing a CHO cell in a liquid culture medium to achieve a viable cell density of greater than 6010.sup.6 cells/mL, the method comprising: providing a culturing system comprising a vessel, a liquid culture medium disposed within the vessel, and a sparger disposed within the vessel comprising a plurality of pores configured to dispense gas through the sparger into the liquid culture medium; and perfusion culturing a CHO cell comprising a recombinant protein-encoding nucleic acid in the culturing system comprising the liquid culture medium comprising an amount of poloxamer-188 and antifoam under conditions and a gas flow rate sufficient to produce the recombinant protein in the system, wherein the ratio of antifoam (g/L) to poloxamer-188 (g/L) is about 1.0% to about 3.0% in the liquid culture medium, and wherein: (i) the plurality of pores are drilled pores with a pore size of 900 m to 1.1 mm and the poloxamer-188 concentration is 2.5 g/L to 3.1 g/L; (ii) the plurality of pores are drilled pores with a pore size of 400 m to 600 m and the poloxamer-188 concentration is 3.5 g/L to 4.1 g/L; (iii) the plurality of pores are drilled pores with a pore size of 1 m to 250 m and the poloxamer-188 concentration is 4.3 g/L to 8.0 g/L; or (iv) the plurality of pores are sintered pores with a pore size of 100 m and the poloxamer-188 concentration is 5.8 g/L, and wherein the method results in a viable cell density in the liquid culture medium that is greater than 6010.sup.6 cells/mL.
2. The method of claim 1, wherein the plurality of pores are drilled pores with a pore size of 900 m to 1.1 mm and the poloxamer-188 concentration is 2.5 g/L to 3.1 g/L.
3. The method of claim 1, wherein the plurality of pores are drilled pores with a pore size of 1 m to 250 m and the poloxamer-188 concentration is 4.3 g/L to 8.0 g/L.
4. The method of claim 1, wherein the plurality of pores are drilled pores with a pore size of 400 m to 600 m and the poloxamer-188 concentration is 3.5 g/L to 4.1 g/L.
5. The method of claim 1, wherein the plurality of pores are sintered pores with a pore size that is 100 m and the poloxamer-188 concentration is 5.8 g/L.
Description
DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION
(16) Provided herein are methods of culturing a mammalian cell that include culturing a mammalian cell including a recombinant protein-encoding nucleic acid under conditions sufficient to produce the recombinant protein, where the culturing includes culturing in a liquid medium including poloxamer-188 at a concentration of 1.8 g/L or at a greater concentration than 1.8 g.Math.L and where the poloxamer-188 is present in the medium and/or added to the medium prior to the culturing step and/or is added to the medium during the culturing step. Also provided are methods of culturing a mammalian cell that include providing a culture system including a vessel, a liquid medium disposed within the vessel, and a sparger disposed within the vessel including a plurality of pores configured to dispense gas through the sparger into the liquid medium, and culturing a mammalian cell including a recombinant protein-encoding nucleic acid in the culturing system under conditions and a gas flow rate sufficient to produce the recombinant protein in the system, where the medium includes a poloxmer-188 concentration that is selected based on one or more factors selected from the group of: pore size, pore type, gas flow rate, viable cell density in the medium, and markers related to cell stress. Exemplary non-limiting aspects of these methods are described below.
(17) In some embodiments, the methods provided herein can achieve a cell culture having a viable cell density of greater than 2010.sup.6 cells/mL, greater than 2510.sup.6 cells/mL, greater than 3010.sup.6 cells/mL, greater than 3510.sup.6 cells/mL, greater than 4010.sup.6 cells/mL, greater than 4510.sup.6 cells/mL, greater than 5010.sup.6 cells/mL, greater than 5510.sup.6 cells/mL, greater than 6010.sup.6 cells/mL, greater than 6510.sup.6 cells/mL, greater than 7010.sup.6 cells/mL, greater than 7510.sup.6 cells/mL, greater than 8010.sup.6 cells/mL, greater than 8510.sup.6 cells/mL, greater than 9010.sup.6 cells/mL, greater than 9510.sup.6 cells/mL, greater than 10010.sup.6 cells/mL, greater than 10510.sup.6 cells/mL, greater than 11010.sup.6 cells/mL, greater than 11510.sup.6 cells/mL, greater than 12010.sup.6 cells/mL, greater than 12510.sup.6 cells/mL, greater than 13010.sup.6 cells/mL, greater than 13510.sup.6 cells/mL, greater than 14010.sup.6 cells/mL, greater than 14510.sup.6 cells/mL, greater than 15010.sup.6 cells/mL, greater than 15510.sup.6 cells/mL, greater than 16010.sup.6 cells/mL, greater than 16510.sup.6 cells/mL, greater than 17010.sup.6 cells/mL, greater than 17510.sup.6 cells/mL, greater than 18010.sup.6 cells/mL, greater than 18510.sup.6 cells/mL, greater than 19010.sup.6 cells/mL, greater than 19510.sup.6 cells/mL, greater than 20010.sup.6 cells/mL, greater than 20510.sup.6 cells/mL, greater than 21010.sup.6 cells/mL, greater than 21510.sup.6 cells/mL, greater than 22010.sup.6 cells/mL, greater than 22510.sup.6 cells/mL, greater than 23010.sup.6 cells/mL, greater than 23510.sup.6 cells/mL, greater than 24010.sup.6 cells/mL, greater than 24510.sup.6 cells/mL, or greater than 25010.sup.6 cells/mL. Methods for determining the viable cell density of a cell culture are well known in the art.
(18) In some embodiments, the methods provided herein provide for a cell culture having a percentage cell viability that is greater than 75% (e.g., greater than 80%, greater than 85%, or greater than 90%) over a culturing 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). In some embodiments, the method provided herein can achieve a culture having a volumetric productivity rate (VPR) of at least 1.5 g/L/d (e.g., at least 1.6 g/L/d, at least 1.7 g/L/d, at least 1.8 g/L/d, at least 1.9 g/L/d, at least 2.0 g/L/d, at least 2.1 g/L/d, at least 2.2 g/L/day, at least 2.3 g/L/d, at least 2.4 g/L/d, at least 2.5 g/L/d, at least 2.6 g/L/d, at least 2.7 g/L/d, at least 2.8 g/L/d, at least 2.9 g/L/d, 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) over 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 culturing period.
(19) In some embodiments, the methods provided herein result in the production of a recombinant protein that when collected has a decreased level (e.g., a decrease of about or less than 5%, a decrease of about or less than 10%, a decrease of about or less than 15%, a decrease of about or less than 20%, a decrease of about or less than 25%, a decrease of about or less than 30%, a decrease of about or less than 35%, a decrease of about or less than 40%, a decrease of about or less than 45%, a decrease of about or less than 50%, a decrease of about or less than 55%, a decrease of about or less than 60%, a decrease of about or less than 65%, a decrease of about or less than 70%, a decrease of about or less than 75%, a decrease of about or less than 80%, a decrease of about or less than 80%, a decrease of about or less than 85%, or a decrease of about or less than 90%) of degradation (as compared to a recombinant protein produced in a similar method that does not include culturing in a liquid medium including 1.8 g/L or more poloxamer-188). The level of degradation of a recombinant protein collected from a cell culture can be detected, e.g., using gel electrophoresis and/or immunoblotting.
(20) In some embodiments, the methods result in a cell culture that has a specific lactate dehydrogenase production 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 40 nU/cell/day, less than 35 nU/cell/day, less than 30 nU/cell/day, less than 25 nU/cell/day, or less than 20 nU/cell/day) over at least about 5 (e.g., at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or at least 70) continuous days starting at any time within the culturing period.
(21) In some embodiments, the methods result in the maintenance of the cells in a steady state (stationary phase) 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), e.g., at a viable cell density about or greater than 3010.sup.6 cells/mL, about or greater than 3510.sup.6 cells/mL, about or greater than 4010.sup.6 cells/mL, about or greater than 4510.sup.6 cells/mL, about or greater than 5010.sup.6 cells/mL, about or greater than 5510.sup.6 cells/mL, about or greater than 6010.sup.6 cells/mL, about or greater than 6510.sup.6 cells/mL, about or greater than 7010.sup.6 cells/mL, about or greater than 7510.sup.6 cells/mL, about or greater than 8010.sup.6 cells/mL, about or greater than 8510.sup.6 cells/mL, about or greater than 9010.sup.6 cells/mL, about or greater than 9510.sup.6 cells/mL, about or greater than 10010.sup.6 cells/mL, about or greater than 10510.sup.6 cells/mL, about or greater than 11010.sup.6 cells/mL, about or greater than 11510.sup.6 cells/mL, about or greater than 12010.sup.6 cells/mL, about or greater than 12510.sup.6 cells/mL, about or greater than 13010.sup.6 cells/mL, about or greater than 13510.sup.6 cells/mL, about or greater than 14010.sup.6 cells/mL, about or greater than 14510.sup.6 cells/mL, about or greater than 15010.sup.6 cells/mL, about or greater than 15510.sup.6 cells/mL, about or greater than 16010.sup.6 cells/mL, about or greater than 16510.sup.6 cells/mL, about or greater than 17010.sup.6 cells/mL, about or greater than 17510.sup.6 cells/mL, about or greater than 18010.sup.6 cells/mL, about or greater than 18510.sup.6 cells/mL, about or greater than 19010.sup.6 cells/mL, about or greater than 19510.sup.6 cells/mL, or about or greater than 20010.sup.6 cell s/mL.
(22) Culture Volumes
(23) The culturing step (e.g., perfusion culturing step) in any of the methods described can include incubating a cell culture having a volume of between 20 L and about 8,000 L (e.g., between about 20 L and about 7,000 L, between about 20 L and about 6,000 L, between about 20 L an about 5,000 L, between about 20 L and about 4,000 L, between about 20 L and about 3,000 L, between about 20 L and about 2,500 L, between about 20 L and about 2,000 L, between about 20 L and about 1,500 L, between about 20 L and about 1,000 L, between about 20 L and about 500 L, between about 20 L and about 200 L, between about 20 L and about 100 L, between about 100 L and about 8,000 L, between about 100 L and about 7,000 L, between about 100 L and about 6,000 L, between about 100 L and about 5,000 L, between about 100 L and about 4,000 L, between about 100 L and about 3,000 L, between about 100 L and about 2,000 L, between about 100 L and about 1,500 L, between about 100 L and about 1,000 L, between about 100 L and about 800 L, between about 100 L and about 700 L, between about 100 L and about 600 L, between about 100 L and about 500 L, between about 100 L and about 400 L, between about 100 L and about 300 L, between about 100 L and about 200 L, between about 500 L and about 8,000 L, between about 500 L and about 7,000 L, between about 500 L and about 6,000 L, between about 500 L and about 5,000 L, between about 500 L and about 4,000 L, between about 500 L and about 3,000 L, between about 500 L and about 2,000 L, between about 500 L and about 1,500 L, between about 500 L and about 1,000 L, between about 500 L and about 750 L, between about 1,000 L and about 8,000 L, between about 1,000 L and about 7,000 L, between about 1,000 L and about 6,000 L, between about 1,000 L and about 5,000 L, between about 1,000 L and about 4,000 L, between about 1,000 L and about 3,000 L, between about 1,000 L and about 2,000 L, between about 1,000 L and about 1,500 L, between about 2,000 L and about 8,000 L, between about 2,000 L and about 7,000 L, between about 2,000 L and about 6,000 L, between about 2,000 L and about 5,000 L, between about 2,000 L and about 4,000 L, between about 2,000 L and about 3,000 L, between about 3,000 L and about 8,000 L, between about 3,000 L and about 7,000 L, between about 3,000 L and about 6,000 L, between about 3,000 L and about 5,000 L, between about 3,000 L and about 4,000 L, between about 4,000 L and about 8,000 L, between about 4,000 L and about 7,000 L, between about 4,000 L and about 6,000 L, between about 4,000 L and about 5,000 L, between about 5,000 L and about 8,000 L, between about 5,000 L and about 7,000 L, between about 5,000 L and about 6,000 L, between about 6,000 L and about 8,000 L, between about 6,000 L and about 7,000 L, or between about 7,000 L or about 8,000 L) under conditions that allow for cell maintenance and proliferation in the culture.
(24) As is known in the art, the volume of a cell culture can remain substantially the same or can increase over time during the culturing period (e.g., as described below).
(25) Culturing Period
(26) Culturing (e.g., perfusion culturing) in any of the methods described herein can be performed over a period of time of between about 3 days to about 100 days (e.g., between about 3 days and about 95 days, between about 3 days and about 90 days, between about 3 days and about 85 days, between about 3 days and about 80 days, between about 3 days and about 75 days, between about 3 days and about 70 days, between about 3 days and about 65 days, between about 3 days and about 60 days, between about 3 days and about 55 days, between about 3 days and about 50 days, between about 3 days and about 45 days, between about 3 days and about 40 days, between about 3 days and about 35 days, between about 3 days and about 30 days, between about 3 days and about 25 days, between about 3 days and about 20 days, between about 3 days and about 15 days, between about 3 days and about 10 days, between about 5 days and about 100 days, between about 5 days and about 95 days, between about 5 days and about 90 days, between about 5 days and about 85 days, between about 5 days and about 80 days, between about 5 days and about 75 days, between about 5 days and about 70 days, between about 5 days and about 65 days, between about 5 days and about 60 days, between about 5 days and about 55 days, between about 5 days and about 50 days, between about 5 days and about 45 days, between about 5 days and about 40 days, between about 5 days and about 35 days, between about 5 days and about 30 days, between about 5 days and about 25 days, between about 5 days and about 20 days, between about 5 days and about 15 days, between about 5 days and about 10 days, between about 10 days and about 100 days, between about 10 days and about 95 days, between about 10 days and about 90 days, between about 10 days and about 85 days, between about 10 days and about 80 days, between about 10 days and about 75 days, between about 10 days and about 70 days, between about 10 days and about 65 days, between about 10 days and about 60 days, between about 10 days and about 55 days, between about 10 days and about 50 days, between about 10 days and about 45 days, between about 10 days and about 40 days, between about 10 days and about 35 days, between about 10 days and about 30 days, between about 10 days and about 25 days, between about 10 days and about 20 days, between about 10 days and about 15 days, between about 15 days and about 100 days, between about 15 days and about 95 days, between about 15 days and about 90 days, between about 15 days and about 85 days, between about 15 days and about 80 days, between about 15 days and about 75 days, between about 15 days and about 70 days, between about 15 days and about 65 days, between about 15 days and about 60 days, between about 15 days and about 55 days, between about 15 days and about 50 days, between about 15 days and about 45 days, between about 15 days and about 40 days, between about 15 days and about 35 days, between about 15 days and about 30 days, between about 15 days and about 25 days, between about 15 days and about 20 days, between about 20 days and about 100 days, between about 20 days and about 95 days, between about 20 days and about 90 days, between about 20 days and about 85 days, between about 20 days and about 80 days, between about 20 days and about 75 days, between about 20 days and about 70 days, between about 20 days and about 65 days, between about 20 days and about 60 days, between about 20 days and about 55 days, between about 20 days and about 50 days, between about 20 days and about 45 days, between about 20 days and about 40 days, between about 20 days and about 35 days, between about 20 days and about 30 days, between about 20 days and about 25 days, between about 25 days and about 100 days, between about 25 days and about 95 days, between about 25 days and about 90 days, between about 25 days and about 85 days, between about 25 days and about 80 days, between about 25 days and about 75 days, between about 25 days and about 70 days, between about 25 days and about 65 days, between about 25 days and about 60 days, between about 25 days and about 55 days, between about 25 days and about 50 days, between about 25 days and about 45 days, between about 25 days and about 40 days, between about 25 days and about 35 days, between about 25 days and about 30 days, between about 30 days and about 100 days, between about 30 days and about 90 days, between about 30 days and about 80 days, between about 30 days and about 70 days, between about 30 days and about 60 days, between about 30 days and about 50 days, between about 30 days and about 40 days, between about 40 days and about 100 days, between about 40 days and about 90 days, between about 40 days and about 80 days, between about 40 days and about 70 days, between about 40 days and about 60 days, between about 40 days and about 50 days, between about 50 days and about 100 days, between about 50 days and about 90 days, between about 50 days and about 80 days, between about 50 days and about 70 days, between about 50 days and about 60 days, between about 60 days and about 100 days, between about 60 days and about 90 days, between about 60 days and about 80 days, between about 60 days and about 70 days, between about 70 days and about 100 days, between about 70 days and about 90 days, between about 70 days and about 80 days, between about 80 days and about 100 days, between about 80 days and about 90 days, or between about 90 days and about 100 days).
(27) Mammalian Cells
(28) The mammalian cell cultured (e.g., perfusion cultured) in the methods provided herein can be a cell that grows in suspension or an adherent cell. Non-limiting examples of mammalian cells that can be cultured in any of the methods described herein include: Chinese hamster ovary (CHO) cells (e.g., CHO DG44 cells or CHO-K 1 s 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, an NSO cell, a baby hamster kidney (BHK) cell, a PerC6 cell, a Vero cells, a HT-1080 cell, and Madin-Darby Canine (Cocker Spaniel) kidney epithelial cells (MDCK) cells. In some examples where an adherent cell is cultured, the culture can also contain a plurality of microcarriers (e.g., microcarriers that contain one or more pores). Additional mammalian cells that can be cultured in any of the methods described herein are known in the art.
(29) The mammalian cell can contain a recombinant nucleic acid (e.g., a nucleic acid stably integrated in the mammalian cell's genome) that encodes a recombinant protein. Non-limiting examples of recombinant nucleic acids that encode exemplary recombinant proteins are described below, as are recombinant proteins that can be produced using the methods described herein. In some instances, the mammalian cell that is cultured in a bioreactor (e.g., any of the bioreactors described herein) was derived from a larger culture.
(30) A nucleic acid encoding a recombinant protein can be introduced into a mammalian cell using a wide variety of methods known in molecular biology and molecular genetics. Non-limiting examples include transfection (e.g., lipofection), transduction (e.g., lentivirus, adenovirus, or retrovirus infection), and electroporation. In some instances, the nucleic acid that encodes a recombinant protein is not stably integrated into a chromosome of the mammalian cell (transient transfection), while in others the nucleic acid is integrated. Alternatively or in addition, the nucleic acid encoding a recombinant protein can be present in a plasmid and/or in a mammalian artificial chromosome (e.g., a human artificial chromosome). Alternatively or in addition, the nucleic acid can be introduced into the cell using a viral vector (e.g., a lentivirus, retrovirus, or adenovirus vector). The nucleic acid can be operably linked to a promoter sequence (e.g., a strong promoter, such as a (3-actin promoter and CMV promoter, or an inducible promoter). A vector containing the nucleic acid can, if desired, also contain a selectable marker (e.g., a gene that confers hygromycin, puromycin, or neomycin resistance to the mammalian cell).
(31) In some instances, the recombinant protein is a secreted protein and is released by the mammalian cell into the extracellular medium (e.g., the first and/or second liquid medium in perfusion culturing or the first liquid medium and/or feed liquid medium in feed batch culturing). For example, a nucleic acid sequence encoding a soluble recombinant protein can contain a sequence that encodes a secretion signal peptide at the N- or C-terminus of the recombinant protein, which is cleaved by an enzyme present in the mammalian cell, and subsequently released into the extracellular medium (e.g., the first and/or second liquid medium).
(32) Bioreactors
(33) The culturing step in any of the methods described herein can be performed using a bioreactor (e.g., a perfusion bioreactor or a fed batch bioreactor). A bioreactor (e.g., a perfusion bioreactor or a fed batch bioreactor) used to perform the culturing step can have an internal volume (capacity) of between about 24 L to about 25,000 L (e.g., between about 24 L and about 20,000 L, between about 24 L and about 15,000 L, between about 24 L and about 10,000 L, between about 24 L and about 9,500 L, between about 24 L and about 9,000 L, between about 24 L and about 8,500 L, between about 24 L and about 8,000 L, between about 24 L and about 7,500 L, between about 24 L and about 7,000 L, between about 24 L and about 6,500 L, between about 24 L and about 6,000 L, between about 24 L and about 5,500 L, between about 24 L and about 5,000 L, between about 24 L and about 4,500 L, between about 24 L and about 4,000 L, between about 24 L and about 3,500 L, between about 24 L and about 3,000 L, between about 24 L and about 2,500 L, between about 24 L and about 2,000 L, between about 24 L and about 1,500 L, between about 24 L and about 1,000 L, between about 24 L and about 900 L, between about 24 L and about 800 L, between about 24 L and about 700 L, between about 24 L and about 600 L, between about 24 L and about 500 L, between about 24 L and about 400 L, between about 24 L and about 300 L, between about 24 L and about 200 L, between about 24 L and about 100 L, between about 100 L and about 25,000 L, between about 100 L and about 20,000 L, between about 100 L and about 15,000, between about 100 L and about 10,000 L, between about 100 L and about 9,500 L, between about 100 L and about 9,000 L, between about 100 L and about 8,500 L, between about 100 L and about 8,000 L, between about 100 L and about 7,500 L, between about 100 L and about 7,000 L, between about 100 L and about 6,500 L, between about 100 L and about 6,000 L, between about 100 L and about 5,500 L, between about 100 L and about 5,000 L, between about 100 L and about 4,500 L, between about 100 L and about 4,000 L, between about 100 L and about 3,500 L, between about 100 L and about 3,000 L, between about 100 L and about 2,500 L, between about 100 L and about 2,000 L, between about 100 L and about 1,500 L, between about 100 L and about 1,000 L, between about 100 L and about 900 L, between about 100 L and about 800 L, between about 100 L and about 700 L, between about 100 L and about 600 L, between about 100 L and about 500 L, between about 100 L and about 400 L, between about 100 L and about 300 L, between about 100 L and about 200 L, between about 200 L and about 25,000 L, between about 200 L and about 20,000 L, between about 200 L and about 15,000 L, between about 200 L and about 10,000 L, between about 200 L and about 9,500 L, between about 200 L and about 9,000 L, between about 200 L and about 8,500 L, between about 200 L and about 8,000 L, between about 200 L and about 7,500 L, between about 200 L and about 7,000 L, between about 200 L and about 6,500 L, between about 200 L and about 6,000 L, between about 200 L and about 5,500 L, between about 200 L and about 5,000 L, between about 200 L and about 4,500 L, between about 200 L and about 4,000 L, between about 200 L and about 3,500 L, between about 200 L and about 3,000 L, between about 200 L and about 2,500 L, between about 200 L and about 2,000 L, between about 200 L and about 1,500 L, between about 200 L and about 1,000 L, between about 200 L and about 900 L, between about 200 L and about 800 L, between about 200 L and about 700 L, between about 200 L and about 600 L, between about 200 L and about 500 L, between about 200 L and about 400 L, between about 200 L and about 300 L, between about 500 L and about 25,000 L, between about 500 L and about 20,000 L, between about 500 L and about 15,000 L, between about 500 L and about 10,000 L, between about 500 L and about 9,500 L, between about 500 L and about 9,000 L, between about 500 L and about 8,500 L, between about 500 L and about 8,000 L, between about 500 L and about 7,500 L, between about 500 L and about 7,000 L, between about 500 L and about 6,500 L, between about 500 L and about 6,000 L, between about 500 L and about 5,500 L, between about 500 L and about 5,000 L, between about 500 L and about 4,500 L, between about 500 L and about 4,000 L, between about 500 L and about 3,500 L, between about 500 L and about 3,000 L, between about 500 L and about 2,500 L, between about 500 L and about 2,000 L, between about 500 L and about 1,500 L, between about 500 L and about 1,000 L, between about 500 L and about 900 L, between about 500 L and about 800 L, between about 500 L and about 700 L, between about 500 L and about 600 L, between about 1,000 L and about 25,000 L, between about 1,000 L and about 20,000 L, between about 1,000 L and about 15,000 L, between about 1,000 L and about 10,000 L, between about 1,000 L and about 9,500 L, between about 1,000 L and about 9,000 L, between about 1,000 L and about 8,500 L, between about 1,000 L and about 8,000 L, between about 1,000 L and about 7,500 L, between about 1,000 L and about 7,000 L, between about 1,000 L and about 6,500 L, between about 1,000 L and about 6,000 L, between about 1,000 L and about 5,500 L, between about 1,000 L and about 5,000 L, between about 1,000 L and about 4,500 L, between about 1,000 L and about 4,000 L, between about 1,000 L and about 3,500 L, between about 1,000 L and about 3,000 L, between about 1,000 L and about 2,500 L, between about 1,000 L and about 2,000 L, between about 1,000 L and about 1,500 L, between about 2,000 L and about 25,000 L, between about 2,000 L and about 20,000 L, between about 2,000 L and about 15,000 L, between about 2,000 L and about 10,000 L, between about 2,000 L and about 9,500 L, between about 2,000 L and about 9,000 L, between about 2,000 L and about 8,500 L, between about 2,000 L and about 8,000 L, between about 2,000 L and about 7,500 L, between about 2,000 L and about 7,000 L, between about 2,000 L and about 6,500 L, between about 2,000 L and about 6,000 L, between about 2,000 L and about 5,500 L, between about 2,000 L and about 5,000 L, between about 2,000 L and about 4,500 L, between about 2,000 L and about 4,000 L, between about 2,000 L and about 3,500 L, between about 2,000 L and about 3,000 L, between about 2,000 L and about 2,500 L, between about 3,000 L and about 25,000 L, between about 3,000 L and about 20,000 L, between about 3,000 L and about 15,000 L, between about 3,000 L and about 10,000 L, between about 3,000 L and about 9,500 L, between about 3,000 L and about 9,000 L, between about 3,000 L and about 8,500 L, between about 3,000 L and about 8,000 L, between about 3,000 L and about 7,500 L, between about 3,000 L and about 7,000 L, between about 3,000 L and about 6,500 L, between about 3,000 L and about 6,000 L, between about 3,000 L and about 5,500 L, between about 3,000 L and about 5,000 L, between about 3,000 L and about 4,500 L, between about 3,000 L and about 4,000 L, between about 3,000 L and about 3,500 L, between about 4,000 L and about 9,000 L, between about 4,000 L and about 25,000 L, between about 4,000 L and about 20,000 L, between about 4,000 L and about 15,000 L, between about 4,000 L and about 10,000 L, between about 4,000 L and about 9,500 L, between about 4,000 L and about 9,000 L, between about 4,000 L and about 8,500 L, between about 4,000 L and about 8,000 L, between about 4,000 L and about 7,500 L, between about 4,000 L and about 7,000 L, between about 4,000 L and about 6,500 L, between about 4,000 L and about 6,000 L, between about 4,000 L and about 5,500 L, between about 4,000 L and about 5,000 L, between about 4,000 L and about 4,500 L, between about 5,000 L and about 25,000 L, between about 5,000 L and about 20,000 L, between about 5,000 L and about 15,000 L, between about 5,000 L and about 10,000 L, between about 5,000 L and about 9,500 L, between about 5,000 L and about 9,000 L, between about 5,000 L and about 8,500 L, between about 5,000 L and about 8,000 L, between about 5,000 L and about 7,500 L, between about 5,000 L and about 7,000 L, between about 5,000 L and about 6,500 L, between about 5,000 L and about 6,000 L, between about 5,000 L and about 5,500 L, between about 6,000 L and about 25,000 L, between about 6,000 L and about 20,000 L, between about 6,000 L and about 15,000 L, between about 6.000 L and about 10,000 L, between about 6,000 L and about 9,500 L, between about 6,000 L and about 9,000 L, between about 6,000 L and about 8,500 L, between about 6,000 L and about 8,000 L, between about 6,000 L and about 7,500 L, between about 6,000 L and about 7,000 L, between about 6,000 L and about 6,500 L, between 7,000 L and about 25,000 L, between about 7,000 L and about 20,000 L, between about 7,000 L and about 15,000 L, between about 7,000 L and about 10,000 L, between about 7,000 L and about 9,500 L, between about 7,000 L and about 9,000 L, between about 7,000 L and about 8,500 L, between about 7,000 L and about 8,000 L, between about 7,000 L and about 7,500 L, between about 8,000 L and about 25,000 L, between about 8,000 L and about 20,000 L, between about 8,000 L and about 15,000 L, between about 8,000 L and about 10,000 L, between about 8,000 L and about 9,500 L, between about 8,000 L and about 9,000 L, between about 8,000 L and about 8,500 L, between about 9,000 L and about 25,000 L, between about 9,000 L and about 20,000 L, between about 9,000 L and about 15,000 L, between about 9,000 L and about 10,000 L, between about 9,000 L and about 9,500 L, between about 10,000 L and about 25,000 L, between about 10,000 L and about 20,000 L, between about 10,000 L and about 15,000 L, between about 15,000 L and about 25,000 L, between about 15,000 L and about 20,000 L, or between about 20,000 L and about 25,000 L).
(34) Culture Systems
(35) Some of the methods described herein use a culturing system (e.g., a perfusion culturing system) that includes a vessel, a liquid medium disposed within the vessel (e.g., any of the culture media described herein), and a sparger disposed within the vessel including a plurality of pores configured to dispense gas through the sparger into the liquid medium. The vessel can have an internal volume (capacity) of between about 24 L to about 20,000 L (e.g., between about 24 L and about 15,000 L, between about 24 L and about 10,000 L, between about 24 L and about 9,500 L, between about 24 L and about 9,000 L, between about 24 L and about 8,500 L, between about 24 L and about 8,000 L, between about 24 L and about 7,500 L, between about 24 L and about 7,000 L, between about 24 L and about 6,500 L, between about 24 L and about 6,000 L, between about 24 L and about 5,500 L, between about 24 L and about 5,000 L, between about 24 L and about 4,500 L, between about 24 L and about 4,000 L, between about 24 L and about 3,500 L, between about 24 L and about 3,000 L, between about 24 L and about 2,500 L, between about 24 L and about 2,000 L, between about 24 L and about 1,500 L, between about 24 L and about 1,000 L, between about 24 L and about 900 L, between about 24 L and about 800 L, between about 24 L and about 700 L, between about 24 L and about 600 L, between about 24 L and about 500 L, between about 24 L and about 400 L, between about 24 L and about 300 L, between about 24 L and about 200 L, between about 24 L and about 100 L, between about 100 L and about 20,000 L, between about 100 L and about 15,000 L, between about 100 L and about 10,000 L, between about 100 L and about 9,500 L, between about 100 L and about 9,000 L, between about 100 L and about 8,500 L, between about 100 L and about 8,000 L, between about 100 L and about 7,500 L, between about 100 L and about 7,000 L, between about 100 L and about 6,500
(36) L, between about 100 L and about 6,000 L, between about 100 L and about 5,500 L, between about 100 L and about 5,000 L, between about 100 L and about 4,500 L, between about 100 L and about 4,000 L, between about 100 L and about 3,500 L, between about 100 L and about 3,000 L, between about 100 L and about 2,500 L, between about 100 L and about 2,000 L, between about 100 L and about 1,500 L, between about 100 L and about 1,000 L, between about 100 L and about 900 L, between about 100 L and about 800 L, between about 100 L and about 700 L, between about 100 L and about 600 L, between about 100 L and about 500 L, between about 100 L and about 400 L, between about 100 L and about 300 L, between about 100 L and about 200 L, between about 200 L and about 20,000 L, between about 200 L and about 15,000 L, between about 200 L and about 10,000 L, between about 200 L and about 9,500 L, between about 200 L and about 9,000 L, between about 200 L and about 8,500 L, between about 200 L and about 8,000 L, between about 200 L and about 7,500 L, between about 200 L and about 7,000 L, between about 200 L and about 6,500 L, between about 200 L and about 6,000 L, between about 200 L and about 5,500 L, between about 200 L and about 5,000 L, between about 200 L and about 4,500 L, between about 200 L and about 4,000 L, between about 200 L and about 3,500 L, between about 200 L and about 3,000 L, between about 200 L and about 2,500 L, between about 200 L and about 2,000 L, between about 200 L and about 1,500 L, between about 200 L and about 1,000 L, between about 200 L and about 900 L, between about 200 L and about 800 L, between about 200 L and about 700 L, between about 200 L and about 600 L, between about 200 L and about 500 L, between about 200 L and about 400 L, between about 200 L and about 300 L, between about 500 L and about 20,000 L, between about 500 L and about 15,000 L, between about 500 L and about 10,000 L, between about 500 L and about 9,500 L, between about 500 L and about 9,000 L, between about 500 L and about 8,500 L, between about 500 L and about 8,000 L, between about 500 L and about 7,500 L, between about 500 L and about 7,000 L, between about 500 L and about 6,500 L, between about 500 L and about 6,000 L, between about 500 L and about 5,500 L, between about 500 L and about 5,000 L, between about 500 L and about 4,500 L, between about 500 L and about 4,000 L, between about 500 L and about 3,500 L, between about 500 L and about 3,000 L, between about 500 L and about 2,500 L, between about 500 L and about 2,000 L, between about 500 L and about 1,500 L, between about 500 L and about 1,000 L, between about 500 L and about 900 L, between about 500 L and about 800 L, between about 500 L and about 700 L, between about 500 L and about 600 L, between about 1,000 L and about 25,000 L, between about 1,000 L and about 20,000 L, between about 1,000 L and about 15,000 L, between about 1,000 L and about 10,000 L, between about 1,000 L and about 9,500 L, between about 1,000 L and about 9,000 L, between about 1,000 L and about 8,500 L, between about 1,000 L and about 8,000 L, between about 1,000 L and about 7,500 L, between about 1,000 L and about 7,000 L, between about 1,000 L and about 6,500 L, between about 1,000 L and about 6,000 L, between about 1,000 L and about 5,500 L, between about 1,000 L and about 5,000 L, between about 1,000 L and about 4,500 L, between about 1,000 L and about 4,000 L, between about 1,000 L and about 3,500 L, between about 1,000 L and about 3,000 L, between about 1,000 L and about 2,500 L, between about 1,000 L and about 2,000 L, between about 1,000 L and about 1,500 L, between about 2,000 L and about 20,000 L, between about 2,000 L and about 15,000 L, between about 2,000 L and about 10,000 L, between about 2,000 L and about 9,500 L, between about 2,000 L and about 9,000 L, between about 2,000 L and about 8,500 L, between about 2,000 L and about 8,000 L, between about 2,000 L and about 7,500 L, between about 2,000 L and about 7,000 L, between about 2,000 L and about 6,500 L, between about 2,000 L and about 6,000 L, between about 2,000 L and about 5,500 L, between about 2,000 L and about 5,000 L, between about 2,000 L and about 4,500 L, between about 2,000 L and about 4,000 L, between about 2,000 L and about 3,500 L, between about 2,000 L and about 3,000 L, between about 2,000 L and about 2,500 L, between about 3,000 L and about 20,000 L, between about 3,000 L and about 15,000 L, between about 3,000 L and about 10,000 L, between about 3,000 L and about 9,500 L, between about 3,000 L and about 9,000 L, between about 3,000 L and about 8,500 L, between about 3,000 L and about 8,000 L, between about 3,000 L and about 7,500 L, between about 3,000 L and about 7,000 L, between about 3,000 L and about 6,500 L, between about 3,000 L and about 6,000 L, between about 3,000 L and about 5,500 L, between about 3,000 L and about 5,000 L, between about 3,000 L and about 4,500 L, between about 3,000 L and about 4,000 L, between about 3,000 L and about 3,500 L, between about 4,000 L and about 20,000 L, between about 4,000 L and about 15,000 L, between about 4,000 L and about 10,000 L, between about 4,000 L and about 9,500 L, between about 4,000 L and about 9,000 L, between about 4,000 L and about 8,500 L, between about 4,000 L and about 8,000 L, between about 4,000 L and about 7,500 L, between about 4,000 L and about 7,000 L, between about 4,000 L and about 6,500 L, between about 4,000 L and about 6,000 L, between about 4,000 L and about 5,500 L, between about 4,000 L and about 5,000 L, between about 4,000 L and about 4,500 L, between about 5,000 L and about 20,000 L, between about 5,000 L and about 15,000 L, between about 5,000 L and about 10,000 L, between about 5,000 L and about 9,500 L, between about 5,000 L and about 9,000 L, between about 5,000 L and about 8,500 L, between about 5,000 L and about 8,000 L, between about 5,000 L and about 7,500 L, between about 5,000 L and about 7,000 L, between about 5,000 L and about 6,500 L, between about 5,000 L and about 6,000 L, between about 5,000 L and about 5,500 L, between about 6,000 L and about 20,000 L, between about 6,000 L and about 10,000 L, between about 6,000 L and about 9,500 L, between about 6,000 L and about 9,000 L, between about 6,000 L and about 8,500 L, between about 6,000 L and about 8,000 L, between about 6,000 L and about 7,500 L, between about 6,000 L and about 7,000 L, between about 6,000 L and about 6,500 L, between about 7,000 L and about 20,000 L, between about 7,000 L and about 15,000 L, between about 7,000 L and about 10,000 L, between about 7,000 L and about 9,500 L, between about 7,000 L and about 9,000 L, between about 7,000 L and about 8,500 L, between about 7,000 L and about 8,000 L, between about 7,000 L and about 7,500 L, between about 8,000 L and about 20,000 L, between about 8,000 L and about 15,000 L, between about 8,000 L and about 10,000 L, between about 8,000 L and about 9,500 L, between about 8,000 L and about 9,000 L, between about 8,000 L and about 8,500 L, between about 9,000 L and about 20,000 L, between about 9,000 L and about 15,000 L, between about 9,000 L and about 10,000 L, between about 9,000 L and about 9,500 L, between about 10,000 L and about 20,000 L, between about 10,000 L and about 15,000 L, or between about 15,000 L and about 20,000 L). The vessel can be made from metal (e.g., stainless steel), plastic, or glass, or any combination thereof.
(37) In some embodiments, the one or more pores of the sparger can have a pore size of between about 750 m and about 1.5 mm (e.g., between about 750 m and about 1.45 mm, between about 750 m and about 1.4 mm, between about 750 m and about 1.35 mm, between about 750 m and about 1.3 mm, between about 750 m and about 1.25 mm, between about 750 m and about 1.2 mm, between about 750 m and about 1.15 mm, between about 750 m and about 1.1 mm, between about 750 m and about 1.05 mm, between about 750 m and about 1.0 mm, between about 750 m and about 950 m, between about 750 m and about 900 m, between about 750 m and about 850 m, between about 750 m and about 800 m, between about 800 m and about 1.5 mm, between about 800 m and about 1.45 mm, between about 800 m and about 1.4 mm, between about 800 m and about 1.35 mm, between about 800 m and about 1.3 mm, between about 800 m and about 1.25 mm, between about 800 m and about 1.2 mm, between about 800 m and about 1.15 mm, between about 800 m and about 1.1 mm, between about 800 m and about 1.05 mm, between about 800 m and about 1.0 mm, between about 800 m and about 950 m, between about 800 m and about 900 m, between about 800 m and about 850 m, between about 850 m and about 1.5 mm, between about 850 m and about 1.45 mm, between about 850 m and about 1.4 mm, between about 850 m and about 1.35 mm, between about 850 m and about 1.3 mm, between about 850 m and about 1.25 mm, between about 850 m and about 1.2 mm, between about 850 m and about 1.15 mm, between about 850 m and about 1.1 mm, between about 850 m and about 1.05 mm, between about 850 m and about 1.0 mm, between about 850 m and about 950 m, between about 850 m and about 900 m, between about 900 m and about 1.5 mm, between about 900 m and about 1.45 mm, between about 900 m and about 1.4 mm, between about 900 m and about 1.35 mm, between about 900 m and about 1.3 mm, between about 900 m and about 1.25 mm, between about 900 m and about 1.2 mm, between about 900 m and about 1.15 mm, between about 900 m and about 1.1 mm, between about 900 m and about 1.05 mm, between about 900 m and about 1.0 mm, between about 900 m and about 950 m, between about 950 m and about 1.5 mm, between about 950 m and about 1.45 mm, between about 950 m and about 1.4 mm, between about 950 m and about 1.35 mm, between about 950 m and about 1.3 mm, between about 950 m and about 1.25 mm, between about 950 m and about 1.2 mm, between about 950 m and about 1.15 mm, between about 950 m and about 1.1 mm, between about 950 m and about 1.05 mm, between about 950 m and about 1.0 mm, between about 1.0 mm and about 1.5 mm, between about 1.0 mm and about 1.45 mm, between about 1.0 mm and about 1.4 mm, between about 1.0 mm and about 1.35 mm, between about 1.0 mm and about 1.3 mm, between about 1.0 mm and about 1.25 mm, between about 1.0 mm and about 1.2 mm, between about 1.0 mm and about 1.15 mm, between about 1.0 mm and about 1.1 mm, between about 1.0 mm and about 1.05 mm, between about 1.05 mm and about 1.5 mm, between about 1.05 mm and about 1.45 mm, between about 1.05 mm and about 1.4 mm, between about 1.05 mm and about 1.35 mm, between about 1.05 mm and about 1.3 mm, between about 1.05 mm and about 1.25 mm, between about 1.05 mm and about 1.2 mm, between about 1.05 mm and about 1.15 mm, between about 1.05 mm and about 1.1 mm, between about 1.1 mm and about 1.5 mm, between about 1.1 mm and about 1.45 mm, between about 1.1 mm and about 1.4 mm, between about 1.1 mm and about 1.35 mm, between about 1.1 mm and about 1.3 mm, between about 1.1 mm and about 1.25 mm, between about 1.1 mm and about 1.2 mm, between about 1.1 mm and about 1.15 mm, between about 1.15 mm and about 1.5 mm, between about 1.15 mm and about 1.45 mm, between about 1.15 mm and about 1.4 mm, between about 1.15 mm and about 1.35 mm, between about 1.15 mm and about 1.3 mm, between about 1.15 mm and about 1.25 mm, between about 1.15 mm and about 1.2 mm, between about 1.2 mm and about 1.5 mm, between about 1.2 mm and about 1.45 mm, between about 1.2 mm and about 1.4 mm, between about 1.2 mm and about 1.35 mm, between about 1.2 mm and about 1.3 mm, between about 1.2 mm and about 1.25 mm, between about 1.25 mm and about 1.5 mm, between about 1.25 mm and about 1.45 mm, between about 1.25 mm and about 1.4 mm, between about 1.25 mm and about 1.35 mm, between about 1.25 mm and about 1.3 mm, between about 1.3 mm and about 1.5 mm, between about 1.3 mm and about 1.45 mm, between about 1.3 mm and about 1.4 mm, between about 1.3 mm and about 1.35 mm, between about 1.35 mm and about 1.5 mm, between about 1.35 mm and about 1.45 mm, between about 1.35 mm and about 1.4 mm, between about 1.4 mm and about 1.5 mm, between about 1.4 mm and about 1.45 mm, or between about 1.45 mm and about 1.5 mm).
(38) In some embodiments, the one or more pores of the sparger can have a pore size of between about 250 m and about 750 m (e.g., between about 250 m and about 700 m, between about 250 m and about 650 m, between about 250 m and about 600 m, between about 250 m and about 550 m, between about 250 m and about 500 m, between about 250 m and about 450 m, between about 250 m and about 400 m, between about 250 m and about 350 m, between about 250 m and about 300 m, between about 300 m and about 750 m, between about 300 m and about 650 m, between about 300 m and about 600 m, between about 300 m and about 550 m, between about 300 m and about 500 m, between about 300 m and about 450 m, between about 300 m and about 400 m, between about 300 m and about 350 m, between about 350 m and about 750 m, between about 350 m and about 700 m, between about 350 m and about 650 m, between about 350 m and about 600 m, between about 350 m and about 550 m, between about 350 m and about 500 m, between about 350 m and about 450 m, between about 350 m and about 400 m, between about 400 m and about 750 m, between about 400 m and about 700 m, between about 400 m and about 650 m, between about 400 m and about 600 m, between about 400 m and about 550 m, between about 400 m and about 500 m, between about 400 m and about 450 m, between about 450 m and about 750 m, between about 450 m and about 700 m, between about 450 m and about 650 m, between about 450 m and about 600 m, between about 450 m and about 550 m, between about 450 m and about 500 m, between about 500 m and about 750 m, between about 500 m and about 700 m, between about 500 m and about 650 m, between about 500 m and about 600 m, between about 500 m and about 550 m, between about 550 m and about 750 m, between about 550 m and about 700 m, between about 550 m and about 650 m, between about 550 m and about 600 m, between about 600 m and about 750 m, between about 600 m and about 700 m, between about 600 m and about 650 m, between about 650 m and about 750 m, between about 650 m and about 700 m, or between about 700 m and about 750 m).
(39) In some embodiments, the one or more pores of the sparger can have a pore size of between about 1 m and about 250 m (e.g., between about 1 m and about 240 m, between about 1 m and about 230 m, between about 1 m and about 220 m, between about 1 m and about 210 m, between about 1 m and about 200 m, between about 1 m and about 190 m, between about 1 m and about 180 m, between about 1 m and about 170 m, between about 1 m and about 160 m, between about 1 m and about 150 m, between about 1 m and about 140 m, between about 1 m and about 130 m, between about 1 m and about 125 m, between about 1 m and about 120 m, between about 1 m and about 115 m, between about 1 m and about 110 m, between about 1 m and about 105 m, between about 1 m and about 100 m, between about 1 m and about 95 m, between about 1 m and about 90 m, between about 1 m and about 85 m, between about 1 m and about 80 m, between about 1 m and about 75 m, between about 1 m and about 70 m, between about 1 m and about 65 m, between about 1 m and about 60 m, between about 1 m and about 55 m, between about 1 m and about 50 m, between about 1 m and about 45 m, between about 1 m and about 40 m, between about 1 m and about 35 m, between about 1 m and about 30 m, between about 1 m and about 25 m, between about 1 m and about 20 m, between about 1 m and about 15 m, between about 1 m and about 10 m, between 1 m and about 5 m, between 10 m and about 250 m, between about 10 m and about 240 m, between about 10 m and about 230 m, between 10 m and about 220 m, between about 10 m and about 210 m, between about 10 m and about 200 m, between about 10 m and about 190 m, between 10 m and about 180 m, between about 10 m and about 170 m, between about 10 m and about 160 m, between about 10 m and about 150 m, between about 10 m and about 140 m, between about 10 m and about 130 m, between about 10 m and about 120 m, between about 10 m and about 110 m, between about 10 m and about 100 m, between about 10 m and about 90 m, between about 10 m and about 80 m, between about 10 m and about 70 m, between about 10 m and about 60 m, between about 10 m and about 50 m, between about 10 m and about 40 m, between about 10 m and about 30 m, between about 10 m and about 20 m, between about 20 m and about 250 m, between about 20 m and about 240 m, between about 20 m and about 230 m, between about 20 m and about 220 m, between about 20 m and about 210 m, between about 20 m and about 200 m, between about 20 m and about 190 m, between about 20 m and about 180 m, between about 20 m and about 170 m, between about 20 m and about 160 m, between about 20 m and about 150 m, between about 20 m and about 140 m, between about 20 m and about 130 m, between about 20 m and about 120 m, between about 20 m and about 110 m, between about 20 m and about 100 m, between about 20 m and about 90 m, between about 20 m and about 80 m, between about 20 m and about 70 m, between about 20 m and about 60 m, between about 20 m and about 50 m, between about 20 m and about 40 m, between about 20 m and about 30 m, between about 30 m and about 250 m, between about 30 m and about 240 m, between about 30 m and about 230 m, between about 30 m and about 220 m, between about 30 m and about 210 m, between about 30 m and about 200 m, between about 30 m and about 190 m, between about 30 m and about 180 m, between about 30 m and about 170 m, between about 30 m and about 160 m, between about 30 m and about 150 m, between about 30 m and about 140 m, between about 30 m and about 130 m, between about 30 m and about 120 m, between about 30 m and about 110 m, between about 30 m and about 110 m, between about 30 m and about 100 m, between about 30 m and about 90 m, between about 30 m and about 80 m, between about 30 m and about 70 m, between about 30 m and about 60 m, between about 30 m and about 50 m, between about 30 m and about 40 m, between about 40 m and about 250 m, between about 40 m and about 240 m, between about 40 m and about 230 m, between about 40 m and about 220 m, between about 40 m and about 210 m, between about 40 m and about 200 m, between about 40 m and about 190 m, between about 40 m and about 180 m, between about 40 m and about 170 m, between about 40 m and about 160 m, between about 40 m and about 150 m, between about 40 m and about 140 m, between about 40 m and about 130 m, between about 40 m and about 120 m, between about 40 m and about 110 m, between about 40 m and about 100 m, between about 40 m and about 90 m, between about 40 m and about 80 m, between about 40 m and about 70 m, between about 40 m and about 60 m, between about 40 m and about 50 m, between about 50 m and about 250 m, between about 50 m and about 240 m, between about 50 m and about 230 m, between about 50 m and about 220 m, between about 50 m and about 210 m, between about 50 m and about 200 m, between about 50 m and about 190 m, between about 50 m and about 180 m, between about 50 m and about 170 m, between about 50 m and about 160 m, between about 50 m and about 150 m, between about 50 m and about 140 m, between about 50 m and about 130 m, between about 50 m and about 120 m, between about 50 m and about 110 m, between about 50 m and about 100 m, between about 50 m and about 90 m, between about 50 m and about 80 m, between about 50 m and about 70 m, between about 50 m and about 60 m, between about 60 m and about 250 m, between about 60 m and about 240 m, between about 60 m and about 230 m, between about 60 m and about 220 m, between about 60 m and about 210 m, between about 60 m and about 200 m, between about 60 m and about 190 m, between about 60 m and about 180 m, between about 60 m and about 170 m, between about 60 m and about 160 m, between about 60 m and about 150 m, between about 60 m and about 140 m, between about 60 m and about 130 m, between about 60 m and about 120 m, between about 60 m and about 110 m, between about 60 m and about 100 m, between about 60 m and about 90 m, between about 60 m and about 80 m, between about 60 m and about 70 m, between about 70 m and about 250 m, between about 70 m and about 240 m, between about 70 m and about 230 m, between about 70 m and about 220 m, between about 70 m and about 210 m, between about 70 m and about 200 m, between about 70 m and about 190 m, between about 70 m and about 180 m, between about 70 m and about 170 m, between about 70 m and about 160 m, between about 70 m and about 150 m, between about 70 m and about 140 m, between about 70 m and about 130 m, between about 70 m and about 120 m, between about 70 m and about 110 m, between about 70 m and about 100 m, between about 70 m and about 90 m, between about 70 m and about 80 m, between about 80 m and about 250 m, between about 80 m and about 240 m, between about 80 m and about 230 m, between about 80 m and about 220 m, between about 80 m and about 210 m, between about 80 m and about 200 m, between about 80 m and about 190 m, between about 80 m and about 180 m, between about 80 m and about 170 m, between about 80 m and about 160 m, between about 80 m and about 150 m, between about 80 m and about 140 m, between about 80 m and about 130 m, between about 80 m and about 120 m, between about 80 m and about 110 m, between about 80 m and about 100 m, between about 80 m and about 90 m, between about 90 m and about 250 m, between about 90 m and about 240 m, between about 90 m and about 230 m, between about 90 m and about 220 m, between about 90 m and about 210 m, between about 90 m and about 200 m, between about 90 m and about 190 m, between about 90 m and about 180 m, between about 90 m and about 170 m, between about 90 m and about 160 m, between about 90 m and about 150 m, between about 90 m and about 140 m, between about 90 m and about 130 m, between about 90 m and about 120 m, between about 90 m and about 110 m, between about 90 m and about 100 m, between about 100 m and about 250 m, between about 100 m and about 240 m, between about 100 m and about 230 m, between about 100 m and about 220 m, between about 100 m and about 210 m, between about 100 m and about 200 m, between about 100 m and about 190 m, between about 100 m and about 180 m, between about 100 m and about 170 m, between about 100 m and about 160 m, between about 100 m and about 150 m, between about 100 m and about 140 m, between about 100 m and about 130 m, between about 100 m and about 120 m, between about 100 m and about 110 m, between about 110 m and about 250 m, between about 110 m and about 240 m, between about 110 m and about 230 m, between about 110 m and about 220 m, between about 110 m and about 210 m, between about 110 m and about 200 m, between about 100 m and about 190 m, between about 100 m and about 180 m, between about 100 m and about 170 m, between about 100 m and about 160 m, between about 100 m and about 150 m, between about 100 m and about 140 m, between about 100 m and about 130 m, between about 100 m and about 120 m, between about 100 m and about 110 m, between about 110 m and about 250 m, between about 110 m and about 240 m, between about 110 m and about 230 m, between about 110 m and about 220 m, between about 110 m and about 210 m, between about 110 m and about 200 m, between about 110 m and about 190 m, between about 110 m and about 180 m, between about 110 m and about 170 m, between about 110 m and about 160 m, between about 110 m and about 150 m, between about 110 m and about 140 m, between about 110 m and about 130 m, between about 110 m and about 120 m, between about 120 m and about 250 m, between about 120 m and about 240 m, between about 120 m and about 230 m, between about 120 m and about 220 m, between about 120 m and about 210 m, between about 120 m and about 200 m, between about 120 m and about 190 m, between about 120 m and about 180 m, between about 120 m and about 170 m, between about 120 m and about 160 m, between about 120 m and about 150 m, between about 120 m and about 140 m, between about 120 m and about 130 m, between about 130 m and about 250 m, between about 130 m and about 240 m, between about 130 m and about 230 m, between about 130 m and about 220 m, between about 130 m and about 210 m, between about 130 m and about 200 m, between about 130 m and about 190 m, between about 130 m and about 180 m, between about 130 m and about 170 m, between about 130 m and about 160 m, between about 130 m and about 150 m, between about 130 m and about 140 m, between about 140 m and about 250 m, between about 140 m and about 240 m, between about 140 m and about 230 m, between about 140 m and about 220 m, between about 140 m and about 210 m, between about 140 m and about 200 m, between about 140 m and about 190 m, between about 140 m and about 180 m, between about 140 m and about 170 m, between about 140 m and about 160 m, between about 140 m and about 150 m, between about 150 m and about 250 m, between about 150 m and about 240 m, between about 150 m and about 230 m, between about 150 m and about 220 m, between about 150 m and about 210 m, between about 150 m and about 200 m, between about 150 m and about 190 m, between about 150 m and about 180 m, between about 150 m and about 170 m, between about 150 m and about 160 m, between about 160 m and about 250 m, between about 160 m and about 240 m, between about 160 m and about 230 m, between about 160 m and about 220 m, between about 160 m and about 210 m, between about 160 m and about 200 m, between about 160 m and about 190 m, between about 160 m and about 180 m, between about 160 m and about 170 m, between about 170 m and about 250 m, between about 170 m and about 240 m, between about 170 m and about 230 m, between about 170 m and about 220 m, between about 170 m and about 210 m, between about 170 m and about 200 m, between about 170 m and about 190 m, between about 170 m and about 180 m, between about 180 m and about 250 m, between about 180 m and about 240 m, between about 180 m and about 230 m, between about 180 m and about 220 m, between about 180 m and about 210 m, between about 180 m and about 200 m, between about 180 m and about 190 m, between about 190 m and about 250 m, between about 190 m and about 240 m, between about 190 m and about 230 m, between about 190 m and about 220 m, between about 190 m and about 210 m, between about 190 m and about 200 m, between about 200 m and about 250 m, between about 200 m and about 240 m, between about 200 m and about 230 m, between about 200 m and about 220 m, between about 200 m and about 210 m, between about 210 m and about 250 m, between about 210 m and about 240 m, between about 210 m and about 230 m, between about 210 m and about 220 m, between about 220 m and about 250 m, between about 220 m and about 240 m, between about 220 m and about 230 m, between about 230 m and about 250 m, between about 230 m and about 240 m, or between about 240 m and about 250 m).
(40) The one or more pores of the sparger can have a pore type of, e.g., a drilled pore or a sintered pore. The culturing system can include a device for agitating the liquid medium disposed within the vessel (e.g., an impellor and a motor that rotates the impellor). The vessel can include one or more ports (and optionally pumps) that allow for the addition of a material (e.g., a liquid medium, poloxamer-188, base or acid (as needed to regulate pH), and/or antifoam) to the liquid medium disposed in the vessel and/or the removal of liquid medium (e.g., a clarified liquid medium or a sample of cell culture). The culturing system can also include one or more sensors for monitoring the pH, dO.sub.2, temperature, and dCO.sub.2 in the liquid medium during a culturing period. The culturing system can include a filtration device (e.g., a device that performs alternating tangential filtration or tangential flow filtration) that processes a volume of cell culture to provide a clarified liquid medium that is substantially free of cells. The culturing system can include a heating/cooling device that allows for regulation of the temperature of the liquid medium disposed in the vessel. Additional features of cell culture systems are well known in the art.
(41) Perfusion Culturing
(42) In any of the methods described herein, the culturing step can be perfusion culturing. As is known in the art, perfusion culturing includes removing from a bioreactor a first volume of a first liquid medium, and adding to the bioreactor a second volume of a second liquid medium, wherein the first volume and the second volume are about equal. The mammalian cells are retained in the bioreactor by some cell retention device or through techniques, such as cell settling in a settling cone.
(43) As is known in the art, perfusion culturing is different than feed batch culturing, and the culture media and/or the culturing conditions used in perfusion culturing are often different than those used in feed batch culturing.
(44) The removal and addition of media in perfusion culturing can be performed simultaneously or sequentially, or some combination of the two. Further, removal and addition can be performed continuously, such as at a rate that removes and replaces a volume of between 0.1% to 800%, between 1% and 700%, between 1% and 600%, between 1% and 500%, between 1% and 400%, between 1% and 350%, between 1% and 300%, between 1% and 250%, between 1% and 100%, between 100% and 200%, between 5% and 150%, between 10% and 50%, between 15% and 40%, between 8% and 80%, or between 4% and 30% of the volume of the bioreactor.
(45) The first volume of the first liquid medium removed and the second volume of the second liquid medium added can in some instances be held approximately the same over each 24-hour period. As is known in the art, the rate at which the first volume of the first liquid medium is removed (volume/unit of time) and the rate at which the second volume of the second liquid medium is added (volume/unit of time) can be varied and depends on the conditions of the particular cell culture system. The rate at which the first volume of the first liquid medium is removed (volume/unit of time) and the rate at which the second volume of the second liquid medium is added (volume/unit of time) can be about the same or can be different.
(46) Alternatively, the volume removed and added can change by gradually increasing over each 24-hour period. For example, the volume of the first liquid medium removed and the volume of the second liquid medium added within each 24-hour period can be increased over the culturing period. The volume can be increased over the culturing period from a volume that is between 0.5% to about 20% of the bioreactor volume. The volume can be increased over the culturing period to about 25% to about 150% of the bioreactor capacity or the volume of the cell culture at the start of the culturing period.
(47) In some examples of the methods described herein, after the first 48 to 96 hours of the culturing period, in each 24-hour period (within the culturing period), the first volume of the first liquid medium removed and the second volume of the second liquid medium added is about 10% to about 95%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 85% to about 95%, about 60% to about 80%, or about 70% of the volume of the cell culture at the start of the culturing period.
(48) Skilled practitioners will appreciate that the first liquid medium and the second liquid medium can be the same type of media. In other instances, the first liquid medium and the second liquid medium can be different. The second liquid medium may be more concentrated with respect to one or more media components.
(49) The first volume of the first liquid medium can be removed by using any automated system. For example alternating tangential flow filtration may be used. Alternatively, the first volume of the first liquid medium can be removed by seeping or gravity flow of the first volume of the first liquid medium through a sterile membrane with a molecular weight cut-off that excludes the mammalian cell. Alternatively, the first volume of the first liquid medium can be removed by stopping or significantly decreasing the rate of agitation for a period of at least 1 minute, at least 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, or 1 hour, and removing or aspirating the first volume of the first liquid medium from the top of the bioreactor.
(50) The second volume of the second liquid medium can be added to the first liquid medium by a pump. The second liquid medium can be added to the first liquid medium manually, such as by pipetting or injecting the second volume of the second liquid medium directly onto the first liquid medium or in an automated fashion.
(51) In some embodiments of the methods described herein include (optionally providing a vessel containing mammalian cells disposed in a first liquid medium and) incubating the vessel with agitation and for a culturing period of at least about 7 days at a temperature between about 32 C. to about 40 C.; and continuously or periodically after the first 48 to 96 hours of the culturing period removing a first volume of the first liquid medium and adding to the first liquid medium a second volume of a second liquid medium, where the first and second volumes are about equal. In some embodiments, after about the first 48 to 96 hours of the culturing period, in each 24-hour period, the first volume of the first liquid medium removed and the second volume of the second liquid medium added is about 0.3 to about 10 of the volume of the first liquid medium or the volume of the vessel.
(52) Some examples of perfusion culturing include incubating a culturing system containing a first liquid medium with agitation and for a culturing period of at least about 7 days at a temperature between about 32 C. to about 40 C.; and continuously or periodically after the first 48 to 96 hours of the culturing period removing a first volume of the first liquid medium and adding to the first liquid medium a second volume of a second liquid medium.
(53) Fed Batch Culturing
(54) The culturing step in the methods described herein can include fed batch culturing. As is known in the art, fed batch culturing includes the incremental or continuous addition of a feed liquid medium to an initial cell culture without substantial or significant removal of the first liquid medium from the cell culture. In some instances, the feed liquid medium is the same as the first liquid medium. The feed medium may be either in a liquid form (a feed liquid medium) or a dry powder. In other instances, the feed liquid medium is a concentrated form of the first liquid medium and/or is added as a dry powder.
(55) The feed liquid medium can be added to the initial cell culture at a specific time point or at multiple time points during the culturing period. For example, the feed liquid medium can be added to the initial cell culture at a time point that is between 6 hours and 7 days, between about 6 hours and about 6 days, between about 6 hours and about 5 days, between about 6 hours and about 4 days, between about 6 hours and about 3 days, between about 6 hours and about 2 days, between about 6 hours and about 1 day, between about 12 hours and about 7 days, between about 12 hours and about 6 days, between about 12 hours and about 5 days, between about 12 hours and about 4 days, between about 12 hours and about 3 days, between about 12 hours and about 2 days, between about 1 day and about 7 days, between about 1 day and about 6 days, between about 1 day and about 5 days, between about 1 day and about 4 days, between about 1 day and about 3 days, between about 1 day and about 2 days, between about 2 days and about 7 days, between about 2 days and about 6 days, between about 2 days and about 5 days, between about 2 days and about 4 days, between about 2 days and about 3 days, between about 3 days and about 7 days, between about 3 days and about 6 days, between about 3 days and about 5 days, between about 3 days and about 4 days, between about 4 days and about 7 days, between about 4 days and about 6 days, between about 4 days and about 5 days, between about 5 days and about 7 days, or between about 5 days and about 6 days, after the start of culturing period.
(56) The feed liquid medium can be continuously added to the initial cell culture, or can be added periodically (e.g., daily) to the initial cell culture during the culturing period. In some examples when the feed liquid medium is added periodically to the initial cell culture, the feed liquid medium can be added once, twice, three times, four times, or five times. The volume of a feed liquid medium or the amount of a feed solid medium added to the initial cell culture can change (e.g., increase) during the culturing period. The volume of a feed liquid medium added to the initial cell culture over any 24 hour period during the culturing period can be some fraction of the capacity of the bioreactor. The volume of a feed liquid medium added to the initial cell culture over any 24 hour period during the culturing period can be between 0.01 and about 0.3 (e.g., between about 0.01 and about 0.28, between about 0.01 and about 0.26, between about 0.01 and about 0.24, between about 0.01 and about 0.22, between about 0.01 and about 0.20, between about 0.01 and about 0.18, between about 0.01 and about 0.16, between about 0.01 and about 0.14, between about 0.01 and about 0.12, between about 0.01 and about 0.10, between about 0.01 and about 0.08, between about 0.01 and about 0.06, between about 0.01 and about 0.04, between about 0.025 and about 0.3, between about 0.025 and about 0.28, between about 0.025 and about 0.26, between about 0.025 and about 0.24, between about 0.025 and about 0.22, between about 0.025 and about 0.20, between about 0.025 and about 0.18, between about 0.025 and about 0.16, between about 0.025 and about 0.14, between about 0.025 and about 0.12, between about 0.025 and about 0.10, between about 0.025 and about 0.08, between about 0.025 and about 0.06, between about 0.025 and about 0.04, between about 0.05 and about 0.3, between about 0.05 and about 0.28, between about 0.05 and about 0.26, between about 0.05 and about 0.24, between about 0.05 and about 0.22, between about 0.05 and about 0.20, between about 0.05 and about 0.18, between about 0.05 and about 0.16, between about 0.05 and about 0.14, between about 0.05 and about 0.12, between about 0.05 and about 0.10, between about 0.1 and about 0.3, between about 0.1 and about 0.28, between about 0.1 and about 0.26, between about 0.1 and about 0.24, between about 0.1 and about 0.22, between about 0.1 and about 0.20, between about 0.1 and about 0.18, between about 0.1 and about 0.16 between about 0.1 and about 0.14, between about 0.1, between about 0.15 and about 0.3, between about 0.15 and about 0.2, between about 0.2 and about 0.3, or between about 0.25 and about 0.3) of the capacity of a bioreactor containing the culture.
(57) In other embodiments, the volume of a feed liquid medium added to the initial cell culture over any 24 hour period in the culturing period can be between 0.02 and about 1.0 (e.g., between about 0.02 and about 0.9, between about 0.02 and about 0.8, between about 0.02 and about 0.7, between about 0.02 and about 0.6, between about 0.02 and about 0.5, between about 0.02 and about 0.4, between about 0.02 and about 0.3, between about 0.02 and about 0.2, between about 0.02 and about 0.1, between about 0.05 and about 1.0, between about 0.05 and about 0.8, between about 0.05 and about 0.7, between about 0.05 and about 0.6, between about 0.05 and about 0.5, between about 0.05 and about 0.4, between about 0.05 and about 0.3, between about 0.05 and about 0.2, between about 0.05 and about 0.1, between about 0.1 and about 1.0, between about 0.1 and about 0.9, between about 0.1 and about 0.8, between about 0.1 and about 0.7, between about 0.1 and about 0.6, between about 0.1 and about 0.5, between about 0.1 and about 0.4, between about 0.1 and about 0.3, between about 0.1 and about 0.2, between about 0.2 and about 1.0, between about 0.2 and about 0.9, between about 0.2 and about 0.8, between about 0.2 and about 0.7, between about 0.2 and about 0.6, between about 0.2 and about 0.5, or between about 0.2 and about 0.4) of the volume of the cell culture at the start of the culturing period.
(58) Poloxamer-188
(59) Poloxamer-188 is a molecule known in the art. Poloxamer-188 is a non-ionic synthetic block copolymer of ethylene oxide and propylene oxide. Poloxamer-188 has an average molecular weight of between about 7680 to about 9510, and about 81.8%1.9% of its composition by weight is represented by oxyethylene. For example, poloxamer-188 has the structure shown in Formula I below, where a is 80 and b is 27. Poloxamer-188 has a CAS number of 9003-11-6.
(60) ##STR00001##
(61) Poloxamer-188 is commercially available from a number of different vendors including, e.g., Sigma-Aldrich (St. Louis, MO), Mediatech, Inc. (Manassas, VA), MAST Therapeutics, Inc. (San Diego, CA), EMD Millipore (Billerica, MA), BASF (Ludwigshafen, Germany), Pluronic F-68 Life Technologies (Carlsbad, CA), and PhytoTechnology Laboratories, LLC (Shawnee Mission, KS).
(62) The methods provided herein include culturing a mammalian cell (e.g., a mammalian cell including a recombinant protein-encoding nucleic acid) in a liquid medium that includes poloxamer-188 at a concentration of about 1.8 g/L or at a greater concentration than 1.8 g/L (e.g., a concentration of about 1.9 g/L or a greater concentration than 1.9 g/L, a concentration of about 2.0 g/L or a greater concentration than 2.0 g/L, a concentration of about 2.1 g/L or a greater concentration than 2.1 g/L, a concentration of about 2.2 g/L or a greater concentration than 2.2 g/L, a concentration of about 2.3 g/L or a greater concentration than 2.3 g/L, a concentration of about 2.4 g/L or a greater concentration than 2.4 g/L, a concentration of about 2.5 g/L or a greater concentration than 2.5 g/L, a concentration of about 2.6 g/L or a greater concentration than 2.6 g/L, a concentration of about 2.7 g/L or a greater concentration than 2.7 g/L, a concentration of about 2.8 g/L or a greater concentration than 2.8 g/L, a concentration of about 2.9 g/L or a greater concentration than 2.9 g/L, a concentration of about 3.0 g/L or a greater concentration than 3.0 g/L, a concentration of about 3.1 g/L or a greater concentration than 3.1 g/L, a concentration of about 3.2 g/L or a greater concentration than 3.2 g/L, a concentration of about 3.3 g/L or a greater concentration than 3.3 g/L, a concentration of about 3.4 g/L or a greater concentration than 3.4 g/L, a concentration of about 3.5 g/L or a greater concentration than 3.5 g/L, a concentration of about 3.6 g/L or a greater concentration than 3.6 g/L, a concentration of about 3.7 g/L or a greater concentration than 3.7 g/L, a concentration of about 3.8 g/L or a greater concentration than 3.8 g/L, a concentration of about 3.9 g/L or a greater concentration than 3.9 g/L, a concentration of about 4.0 g/L or a greater concentration than 4.0 g/L, a concentration of about 4.1 g/L or a greater concentration than 4.1 g/L, a concentration of about 4.2 g/L or a greater concentration than 4.2 g/L, a concentration of about 4.3 g/L or a greater concentration than 4.3 g/L, a concentration of about 4.4 g/L or a greater concentration than 4.4 g/L, a concentration of about 4.5 g/L or a greater concentration than 4.5 g/L, a concentration of about 4.6 g/L or a greater concentration than 4.6 g/L, a concentration of about 4.7 g/L or a greater concentration than 4.7 g/L, a concentration of about 4.8 g/L or a greater concentration than 4.8 g/L, a concentration of about 4.9 g/L or a greater concentration than 4.9 g/L, a concentration of about 5.0 g/L or a greater concentration than 5.0 g/L, a concentration of about 5.1 g/L or a greater concentration than 5.1 g/L, a concentration of about 5.2 g/L or a greater concentration than 5.2 g/L, a concentration of about 5.3 g/L or a greater concentration than 5.3 g/L, a concentration of about 5.4 g/L or a greater concentration than 5.4 g/L, a concentration of about 5.5 g/L or a greater concentration than 5.5 g/L, a concentration of about 5.6 g/L or a greater concentration than 5.6 g/L, a concentration of about 5.7 g/L or a greater concentration than 5.7 g/L, a concentration of about 5.8 g/L or a greater concentration than 5.8 g/L, a concentration of about 5.9 g/L or a greater concentration than 5.9 g/L, a concentration of about 6.0 g/L or a greater concentration than 6.0 g/L, a concentration of about 6.1 g/L or a greater concentration than 6.1 g/L, a concentration of about 6.2 g/L or a greater concentration than 6.2 g/L, a concentration of about 6.3 g/L or a greater concentration than 6.3 g/L, a concentration of about 6.4 g/L or a greater concentration than 6.4 g/L, a concentration of about 6.5 g/L or a greater concentration than 6.5 g/L, a concentration of about 6.6 g/L or a greater concentration than 6.6 g/L, a concentration of about 6.7 g/L or a greater concentration than 6.7 g/L, a concentration of about 6.8 g/L or a greater concentration than 6.8 g/L, a concentration of about 6.9 g/L or a greater concentration than 6.9 g/L, a concentration of about 7.0 g/L or a greater concentration than 7.0 g/L, a concentration of about 7.1 g/L or a greater concentration than 7.1 g/L, a concentration of about 7.2 g/L or a greater concentration than 7.2 g/L, a concentration of about 7.3 g/L or a greater concentration than 7.3 g/L, a concentration of about 7.4 g/L or a greater concentration than 7.4 g/L, a concentration of about 7.5 g/L or a greater concentration than 7.5 g/L, a concentration of about 7.6 g/L or a greater concentration than 7.6 g/L, a concentration of about 7.7 g/L or a greater concentration than 7.7 g/L, a concentration of about 7.8 g/L or a greater concentration than 7.8 g/L, a concentration of about 7.9 g/L or a greater concentration than 7.9 g/L, a concentration of about 8.0 g/L or a greater concentration than 8.0 g/L, a concentration of about 8.1 g/L or a greater concentration than 8.1 g/L, a concentration of about 8.2 g/L or a greater concentration than 8.2 g/L, a concentration of about 8.3 g/L or a greater concentration than 8.3 g/L, a concentration of about 8.4 g/L or a greater concentration than 8.4 g/L, a concentration of about 8.5 g/L or a greater concentration than 8.5 g/L, a concentration of about 8.6 g/L or a greater concentration than 8.6 g/L, a concentration of about 8.7 g/L or a greater concentration than 8.7 g/L, a concentration of about 8.8 g/L or a greater concentration than 8.8 g/L, a concentration of about 8.9 g/L or a greater concentration than 8.9 g/L, a concentration of about 9.0 g/L or a greater concentration than 9.0 g/L, a concentration of about 9.1 g/L or a greater concentration than 9.1 g/L, a concentration of about 9.2 g/L or a greater concentration than 9.2 g/L, a concentration of about 9.3 g/L or a greater concentration than 9.3 g/L, a concentration of about 9.4 g/L or a greater concentration than 9.4 g/L, a concentration of about 9.5 g/L or a greater concentration than 9.5 g/L, a concentration of about 9.6 g/L or a greater concentration than 9.6 g/L, a concentration of about 9.7 g/L or a greater concentration than 9.7 g/L, a concentration of about 9.8 g/L or a greater concentration than 9.8 g/L, a concentration of about 9.9 g/L or a greater concentration than 9.9 g/L, a concentration of about 10.0 g/L or a greater concentration than 10.0 g/L, a concentration of about 10.1 g/L or a greater concentration than 10.1 g/L, a concentration of about 10.2 g/L or a greater concentration than 10.2 g/L, a concentration of about 10.3 g/L or a greater concentration than 10.3 g/L, a concentration of about 10.4 g/L or a greater concentration than 10.4 g/L, a concentration of about 10.5 g/L or a greater concentration than 10.5 g/L, a concentration of about 10.6 g/L or a greater concentration than 10.6 g/L, a concentration of about 10.7 g/L or a greater concentration than 10.7 g/L, a concentration of about 10.8 g/L or a greater concentration than 10.8 g/L, a concentration of about 10.9 g/L or a greater concentration than 10.9 g/L, a concentration of about 11.0 g/L or a greater concentration than 11.0 g/L, a concentration of about 11.1 g/L or a greater concentration than 11.1 g/L, a concentration of about 11.2 g/L or a greater concentration than 11.2 g/L, a concentration of about 11.3 g/L or a greater concentration than 11.3 g/L, a concentration of about 11.4 g/L or a greater concentration than 11.4 g/L, a concentration of about 11.5 g/L or a greater concentration than 11.5 g/L, a concentration of about 11.6 g/L or a greater concentration than 11.6 g/L, a concentration of about 11.7 g/L or a greater concentration than 11.7 g/L, a concentration of about 11.8 g/L or a greater concentration than 11.8 g/L, a concentration of about 11.9 g/L or a greater concentration than 11.9 g/L, a concentration of about 12.0 g/L or a greater concentration than 12.0 g/L, a concentration of about 12.1 g/L or a greater concentration than 12.1 g/L, a concentration of about 12.2 g/L or a greater concentration than 12.2 g/L, a concentration of about 12.3 g/L or a greater concentration than 12.3 g/L, a concentration of about 12.4 g/L or a greater concentration than 12.4 g/L, a concentration of about 12.5 g/L or a greater concentration than 12.5 g/L, a concentration of about 12.6 g/L or a greater concentration than 12.6 g/L, a concentration of about 12.7 g/L or a greater concentration than 12.7 g/L, a concentration of about 12.8 g/L or a greater concentration than 12.8 g/L, a concentration of about 12.9 g/L or a greater concentration than 12.9 g/L, a concentration of about 13.0 g/L or a greater concentration than 13.0 g/L, a concentration of about 13.1 g/L or a greater concentration than 13.1 g/L, a concentration of about 13.2 g/L or a greater concentration than 13.2 g/L, a concentration of about 13.3 g/L or a greater concentration than 13.3 g/L, a concentration of about 13.4 g/L or a greater concentration than 13.4 g/L, a concentration of about 13.5 g/L or a greater concentration than 13.5 g/L, a concentration of about 13.6 g/L or a greater concentration than 13.6 g/L, a concentration of about 13.7 g/L or a greater concentration than 13.7 g/L, a concentration of about 13.8 g/L or a greater concentration than 13.8 g/L, a concentration of about 13.9 g/L or a greater concentration than 13.9 g/L, a concentration of about 14.0 g/L or a greater concentration than 14.0 g/L, a concentration of about 14.1 g/L or a greater concentration than 14.1 g/L, a concentration of about 14.2 g/L or a greater concentration than 14.2 g/L, a concentration of about 14.3 g/L or a greater concentration than 14.3 g/L, a concentration of about 14.4 g/L or a greater concentration than 14.4 g/L, a concentration of about 14.5 g/L or a greater concentration than 14.5 g/L, a concentration of about 14.6 g/L or a greater concentration than 14.6 g/L, a concentration of about 14.7 g/L or a greater concentration than 14.7 g/L, a concentration of about 14.8 g/L or a greater concentration than 14.8 g/L, a concentration of about 14.9 g/L or a greater concentration than 14.9 g/L, or a concentration of about 15.0 g/L or a greater concentration than 15.0 g/L).
(63) In some embodiments, the methods provided herein include culturing a mammalian cell (e.g., a mammalian cell including a recombinant protein-encoding nucleic acid) in a liquid medium that includes poloxamer-188 at a concentration of between about 1.8 g/L and about 16.0 g/L (e.g., between about 1.8 g/L and about 15.8 g/L, between about 1.8 g/L and about 15.6 g/L, between about 1.8 g/L and about 15.4 g/L, between about 1.8 g/L and about 15.2 g/L, between about 1.8 g/L and about 15.0 g/L, between about 1.8 g/L and about 14.8 g/L, between about 1.8 g/L and about 14.6 g/L, between about 1.8 g/L and about 14.4 g/L, between about 1.8 g/L and about 14.2 g/L, between about 1.8 g/L and about 14.0 g/L, between about 1.8 g/L and about 13.8 g/L, between about 1.8 g/L and about 13.6 g/L, between about 1.8 g/L and about 13.4 g/L, between about 1.8 g/L and about 13.2 g/L, between about 1.8 g/L and about 13.0 g/L, between about 1.8 g/L and about 12.8 g/L, between about 1.8 g/L and about 12.6 g/L, between about 1.8 g/L and about 12.4 g/L, between about 1.8 g/L and about 12.2 g/L, between about 1.8 g/L and about 12.0 g/L, between about 1.8 g/L and about 11.8 g/L, between about 1.8 g/L and about 11.6 g/L, between about 1.8 g/L and about 11.4 g/L, between about 1.8 g/L and about 11.2 g/L, between about 1.8 g/L and about 11.0 g/L, between about 1.8 g/L and about 10.8 g/L, between about 1.8 g/L and about 10.6 g/L, between about 1.8 g/L and about 10.4 g/L, between about 1.8 g/L and about 10.2 g/L, between about 1.8 g/L and about 10.0 g/L, between about 1.8 g/L and about 9.8 g/L, between about 1.8 g/L and about 9.6 g/L, between about 1.8 g/L and about 9.4 g/L, between about 1.8 g/L and about 9.2 g/L, between about 1.8 g/L and about 9.0 g/L, between about 1.8 g/L and about 8.8 g/L, between about 1.8 g/L and about 8.6 g/L, between about 1.8 g/L and about 8.4 g/L, between about 1.8 g/L and about 8.2 g/L, between about 1.8 g/L and about 8.0 g/L, between about 1.8 g/L and about 7.8 g/L, between about 1.8 g/L and about 7.6 g/L, between about 1.8 g/L and about 7.4 g/L, between about 1.8 g/L and about 7.2 g/L, between about 1.8 g/L and about 7.0 g/L, between about 1.8 g/L and about 6.8 g/L, between about 1.8 g/L and about 6.6 g/L, between about 1.8 g/L and about 6.4 g/L, between 1.8 g/L and about 6.2 g/L, between about 1.8 g/L and about 6.0 g/L, between about 1.8 g/L and about 5.8 g/L, between about 1.8 g/L and about 5.6 g/L, between about 1.8 g/L and about 5.4 g/L, between about 1.8 g/L and about 5.2 g/L, between about 1.8 g/L and about 5.0 g/L, between about 1.8 g/L and about 4.8 g/L, between about 1.8 g/L and about 4.6 g/L, between about 1.8 g/L and about 4.4 g/L, between about 1.8 g/L and about 4.2 g/L, between about 1.8 g/L and about 4.0 g/L, between about 1.8 g/L and about 3.8 g/L, between about 1.8 g/L and about 3.6 g/L, between about 1.8 g/L and about 3.4 g/L, between about 1.8 g/L and about 3.2 g/L, between about 1.8 g/L and about 3.0 g/L, between about 1.8 g/L and about 2.8 g/L, between about 2.0 g/L and about 16.0 g/L, between about 2.0 g/L and about 15.8 g/L, between about 2.0 g/L and about 15.6 g/L, between about 2.0 g/L and about 15.4 g/L, between about 2.0 g/L and about 15.2 g/L, between about 2.0 g/L and about 15.0 g/L, between about 2.0 g/L and about 14.8 g/L, between about 2.0 g/L and about 14.6 g/L, between about 2.0 g/L and about 14.4 g/L, between about 2.0 g/L and about 14.2 g/L, between about 2.0 g/L and about 14.0 g/L, between about 2.0 g/L and about 13.8 g/L, between about 2.0 g/L and about 13.6 g/L, between about 2.0 g/L and about 13.4 g/L, between about 2.0 g/L and about 13.2 g/L, between about 2.0 g/L and about 13.0 g/L, between about 2.0 g/L and about 12.8 g/L, between about 2.0 g/L and about 12.6 g/L, between about 2.0 g/L and about 12.4 g/L, between about 2.0 g/L and about 12.2 g/L, about 2.0 g/L and about 12.0 g/L, between about 2.0 g/L and about 11.8 g/L, between about 2.0 g/L and about 11.6 g/L, between about 2.0 g/L and about 11.4 g/L, between about 2.0 g/L and about 11.2 g/L, between about 2.0 g/L and about 11.0 g/L, between about 2.0 g/L and about 10.8 g/L, between about 2.0 g/L and about 10.6 g/L, between about 2.0 g/L and about 10.4 g/L, between about 2.0 g/L and about 10.2 g/L, between about 2.0 g/L and about 10.0 g/L, between about 2.0 g/L and about 9.8 g/L, between about 2.0 g/L and about 9.6 g/L, between about 2.0 g/L and about 9.4 g/L, between about 2.0 g/L and about 9.2 g/L, between about 2.0 g/L and about 9.0 g/L, between about 2.0 g/L and about 8.8 g/L, between about 2.0 g/L and about 8.6 g/L, between about 2.0 g/L and about 8.4 g/L, between about 2.0 g/L and about 8.2 g/L, between about 2.0 g/L and about 8.0 g/L, between about 2.0 g/L and about 7.8 g/L, between about 2.0 g/L and about 7.6 g/L, between about 2.0 g/L and about 7.4 g/L, between about 2.0 g/L and about 7.2 g/L, between about 2.0 g/L and about 7.0 g/L, between about 2.0 g/L and about 6.8 g/L, between about 2.0 g/L and about 6.6 g/L, between about 2.0 g/L and about 6.4 g/L, between about 2.0 g/L and about 6.2 g/L, between about 2.0 g/L and about 6.0 g/L, between about 2.0 g/L and about 5.8 g/L, between about 2.0 g/L and about 5.6 g/L, between about 2.0 g/L and about 5.4 g/L, between about 2.0 g/L and about 5.2 g/L, between about 2.0 g/L and about 5.0 g/L, between about 2.0 g/L and about 4.8 g/L, between about 2.0 g/L and about 4.6 g/L, between about 2.0 g/L and about 4.4 g/L, between about 2.0 g/L and about 4.2 g/L, between about 2.0 g/L and about 4.0 g/L, between about 2.0 g/L and about 3.8 g/L, between about 2.0 g/L and about 3.6 g/L, between about 2.0 g/L and about 3.4 g/L, between about 2.0 g/L and about 3.2 g/L, between about 2.0 g/L and about 3.0 g/L, between about 2.2 g/L and about 16.0 g/L, between about 2.2 g/L and about 15.8 g/L, between about 2.2 g/L and about 15.6 g/L, between about 2.2 g/L and about 15.4 g/L, between about 2.2 g/L and about 15.2 g/L, between about 2.2 g/L and about 15.0 g/L, between about 2.2 g/L and about 14.8 g/L, between about 2.2 g/L and about 14.6 g/L, between about 2.2 g/L and about 14.4 g/L, between about 2.2 g/L and about 14.2 g/L, between about 2.2 g/L and about 14.0 g/L, between about 2.2 g/L and about 13.8 g/L, between about 2.2 g/L and about 13.6 g/L, between about 2.2 g/L and about 13.4 g/L, between about 2.2 g/L and about 13.2 g/L, between about 2.2 g/L and about 13.0 g/L, between about 2.2 g/L and about 12.8 g/L, between about 2.2 g/L and about 12.6 g/L, between about 2.2 g/L and about 12.4 g/L, between about 2.2 g/L and about 12.2 g/L, between about 2.2 g/L and about 12.0 g/L, between about 2.2 g/L and about 11.8 g/L, between about 2.2 g/L and about 11.6 g/L, between about 2.2 g/L and about 11.4 g/L, between about 2.2 g/L and about 11.2 g/L, between about 2.2 g/L and about 11.0 g/L, between about 2.2 g/L and about 10.8 g/L, between about 2.2 g/L and about 10.6 g/L. between about 2.2 g/L and about 10.4 g/L, between about 2.2 g/L and about 10.2 g/L, between about 2.2 g/L and about 10.0 g/L, between about 2.2 g/L and about 9.8 g/L, between about 2.2 g/L and about 9.6 g/L, between about 2.2 g/L and about 9.4 g/L, between about 2.2 g/L and about 9.2 g/L, between about 2.2 g/L and about 9.0 g/L, between about 2.2 g/L and about 8.8 g/L, between about 2.2 g/L and about 8.6 g/L, between about 2.2 g/L and about 8.4 g/L, between about 2.2 g/L and about 8.2 g/L, between about 2.2 g/L and about 8.0 g/L, between about 2.2 g/L and about 7.8 g/L, between about 2.2 g/L and about 7.6 g/L, between about 2.2 g/L and about 7.4 g/L, between about 2.2 g/L and about 7.2 g/L, between about 2.2 g/L and about 7.0 g/L, between about 2.2 g/L and about 6.9 g/L, between about 2.2 g/L and about 6.8 g/L, between about 2.2 g/L and about 6.6 g/L, between about 2.2 g/L and about 6.4 g/L, between about 2.2 g/L and about 6.2 g/L, between about 2.2 g/L and about 6.0 g/L, between about 2.2 g/L and about 5.8 g/L, between about 2.2 g/L and about 5.6 g/L, between about 2.2 g/L and about 5.4 g/L, between about 2.2 g/L and about 5.2 g/L, between about 2.2 g/L and about 5.0 g/L, between about 2.2 g/L and about 4.8 g/L, between about 2.2 g/L and about 4.6 g/L, between about 2.2 g/L and about 4.4 g/L, between about 2.2 g/L and about 4.2 g/L, between about 2.2 g/L and about 4.0 g/L, between about 2.2 g/L and about 3.8 g/L, between about 2.2 g/L and about 3.6 g/L, between about 2.2 g/L and about 3.4 g/L, between about 2.2 g/L and about 3.2 g/L, between about 2.4 g/L and about 16.0 g/L, between about 2.4 g/L and about 15.8 g/L, between about 2.4 g/L and about 15.6 g/L, between about 2.4 g/L and about 15.4 g/L, between about 2.4 g/L and about 15.2 g/L, between about 2.4 g/L and about 15.0 g/L, between about 2.4 g/L and about 14.8 g/L, between about 2.4 g/L and about 14.6 g/L, between about 2.4 g/L and about 14.4 g/L, between about 2.4 g/L and about 14.2 g/L, between about 2.4 g/L and about 14.0 g/L, between about 2.4 g/L and about 13.8 g/L, between about 2.4 g/L and about 13.6 g/L, between about 2.4 g/L and about 13.4 g/L, between about 2.4 g/L and about 13.2 g/L, between about 2.4 g/L and about 13.0 g/L, between about 2.4 g/L and about 12.8 g/L, between about 2.4 g/L and about 12.6 g/L, between about 2.4 g/L and about 12.4 g/L, between about 2.4 g/L and about 12.2 g/L, between about 2.4 g/L and about 12.0 g/L, between about 2.4 g/L and about 11.8 g/L, between about 2.4 g/L and about 11.6 g/L, between about 2.4 g/L and about 11.4 g/L, between about 2.4 g/L and about 11.2 g/L, between about 2.4 g/L and about 11.0 g/L, between about 2.4 g/L and about 10.8 g/L, between about 2.4 g/L and about 10.6 g/L. between about 2.4 g/L and about 10.4 g/L, between about 2.4 g/L and about 10.2 g/L, between about 2.4 g/L and about 10.0 g/L, between about 2.4 g/L and about 9.8 g/L, between about 2.4 g/L and about 9.6 g/L, between about 2.4 g/L and about 9.4 g/L, between about 2.4 g/L and about 9.2 g/L, between about 2.4 g/L and about 9.0 g/L, between about 2.4 g/L and about 8.8 g/L, between about 2.4 g/L and about 8.6 g/L, between about 2.4 g/L and about 8.4 g/L, between about 2.4 g/L and about 8.2 g/L, between about 2.4 g/L and about 8.0 g/L, between about 2.4 g/L and about 7.8 g/L, between about 2.4 g/L and about 7.6 g/L, between about 2.4 g/L and about 7.4 g/L, between about 2.4 g/L and about 7.2 g/L, between about 2.4 g/L and about 7.0 g/L, between about 2.4 g/L and about 6.9 g/L, between about 2.4 g/L and about 6.8 g/L, between about 2.4 g/L and about 6.6 g/L, between about 2.4 g/L and about 6.4 g/L, between about 2.4 g/L and about 6.2 g/L, between about 2.4 g/L and about 6.0 g/L, between about 2.4 g/L and about 5.8 g/L, between about 2.4 g/L and about 5.6 g/L, between about 2.4 g/L and about 5.4 g/L, between about 2.4 g/L and about 5.2 g/L, between about 2.4 g/L and about 5.0 g/L, between about 2.4 g/L and about 4.8 g/L, between about 2.4 g/L and about 4.6 g/L, between about 2.4 g/L and about 4.4 g/L, between about 2.4 g/L and about 4.2 g/L, between about 2.4 g/L and about 4.0 g/L, between about 2.4 g/L and about 3.8 g/L, between about 2.4 g/L and about 3.6 g/L, between about 2.4 g/L and about 3.4 g/L, between about 2.6 g/L and about 16.0 g/L, between about 2.6 g/L and about 15.8 g/L, between about 2.6 g/L and about 15.6 g/L, between about 2.6 g/L and about 15.4 g/L, between about 2.6 g/L and about 15.2 g/L, between about 2.6 g/L and about 15.0 g/L, between about 2.6 g/L and about 14.8 g/L, between about 2.6 g/L and about 14.6 g/L, between about 2.6 g/L and about 14.4 g/L, between about 2.6 g/L and about 14.2 g/L, between about 2.6 g/L and about 14.0 g/L, between about 2.6 g/L and about 13.8 g/L, between about 2.6 g/L and about 13.6 g/L, between about 2.6 g/L and about 13.4 g/L, between about 2.6 g/L and about 13.2 g/L, between about 2.6 g/L and about 13.0 g/L, between about 2.6 g/L and about 12.8 g/L, between about 2.6 g/L and about 12.6 g/L, between about 2.6 g/L and about 12.4 g/L, between about 2.6 g/L and about 12.2 g/L, between about 2.6 g/L and about 12.0 g/L, between about 2.6 g/L and about 11.8 g/L, between about 2.6 g/L and about 11.6 g/L, between about 2.6 g/L and about 11.4 g/L, between about 2.6 g/L and about 11.2 g/L, between about 2.6 g/L and about 11.0 g/L, between about 2.6 g/L and about 10.8 g/L, between about 2.6 g/L and about 10.6 g/L. between about 2.6 g/L and about 10.4 g/L, between about 2.6 g/L and about 10.2 g/L, between about 2.6 g/L and about 10.0 g/L, between about 2.6 g/L and about 9.8 g/L, between about 2.6 g/L and about 9.6 g/L, between about 2.6 g/L and about 9.4 g/L, between about 2.6 g/L and about 9.2 g/L, between about 2.6 g/L and about 9.0 g/L, between about 2.6 g/L and about 8.8 g/L, between about 2.6 g/L and about 8.6 g/L, between about 2.6 g/L and about 8.4 g/L, between about 2.6 g/L and about 8.2 g/L, between about 2.6 g/L and about 8.0 g/L, between about 2.6 g/L and about 7.8 g/L, between about 2.6 g/L and about 7.6 g/L, between about 2.6 g/L and about 7.4 g/L, between about 2.6 g/L and about 7.2 g/L, between about 2.6 g/L and about 7.0 g/L, between about 2.6 g/L and about 6.9 g/L, between about 2.6 g/L and about 6.8 g/L, between about 2.6 g/L and about 6.6 g/L, between about 2.6 g/L and about 6.4 g/L, between about 2.6 g/L and about 6.2 g/L, between about 2.6 g/L and about 6.0 g/L, between about 2.6 g/L and about 5.8 g/L, between about 2.6 g/L and about 5.6 g/L, between about 2.6 g/L and about 5.4 g/L, between about 2.6 g/L and about 5.2 g/L, between about 2.6 g/L and about 5.0 g/L, between about 2.6 g/L and about 4.8 g/L, between about 2.6 g/L and about 4.6 g/L, between about 2.6 g/L and about 4.4 g/L, between about 2.6 g/L and about 4.2 g/L, between about 2.6 g/L and about 4.0 g/L, between about 2.6 g/L and about 3.8 g/L, between about 2.6 g/L and about 3.6 g/L, between about 2.8 g/L and about 16.0 g/L, between about 2.8 g/L and about 15.8 g/L, between about 2.8 g/L and about 15.6 g/L, between about 2.8 g/L and about 15.4 g/L, between about 2.8 g/L and about 15.2 g/L, between about 2.8 g/L and about 15.0 g/L, between about 2.8 g/L and about 14.8 g/L, between about 2.8 g/L and about 14.6 g/L, between about 2.8 g/L and about 14.4 g/L, between about 2.8 g/L and about 14.2 g/L, between about 2.8 g/L and about 14.0 g/L, between about 2.8 g/L and about 13.8 g/L, between about 2.8 g/L and about 13.6 g/L, between about 2.8 g/L and about 13.4 g/L, between about 2.8 g/L and about 13.2 g/L, between about 2.8 g/L and about 13.0 g/L, between about 2.8 g/L and about 12.8 g/L, between about 2.8 g/L and about 12.6 g/L, between about 2.8 g/L and about 12.4 g/L, between about 2.8 g/L and about 12.2 g/L, between about 2.8 g/L and about 12.0 g/L, between about 2.8 g/L and about 11.8 g/L, between about 2.8 g/L and about 11.6 g/L, between about 2.8 g/L and about 11.4 g/L, between about 2.8 g/L and about 11.2 g/L, between about 2.8 g/L and about 11.0 g/L, between about 2.8 g/L and about 10.8 g/L, between about 2.8 g/L and about 10.6 g/L. between about 2.8 g/L and about 10.4 g/L, between about 2.8 g/L and about 10.2 g/L, between about 2.8 g/L and about 10.0 g/L, between about 2.8 g/L and about 9.8 g/L, between about 2.8 g/L and about 9.6 g/L, between about 2.8 g/L and about 9.4 g/L, between about 2.8 g/L and about 9.2 g/L, between about 2.8 g/L and about 9.0 g/L, between about 2.8 g/L and about 8.8 g/L, between about 2.8 g/L and about 8.6 g/L, between about 2.8 g/L and about 8.4 g/L, between about 2.8 g/L and about 8.2 g/L, between about 2.8 g/L and about 8.0 g/L, between about 2.8 g/L and about 7.8 g/L, between about 2.8 g/L and about 7.6 g/L, between about 2.8 g/L and about 7.4 g/L, between about 2.8 g/L and about 7.2 g/L, between about 2.8 g/L and about 7.0 g/L, between about 2.8 g/L and about 6.9 g/L, between about 2.8 g/L and about 6.8 g/L, between about 2.8 g/L and about 6.6 g/L, between about 2.8 g/L and about 6.4 g/L, between about 2.8 g/L and about 6.2 g/L, between about 2.8 g/L and about 6.0 g/L, between about 2.8 g/L and about 5.8 g/L, between about 2.8 g/L and about 5.6 g/L, between about 2.8 g/L and about 5.4 g/L, between about 2.8 g/L and about 5.2 g/L, between about 2.8 g/L and about 5.0 g/L, between about 2.8 g/L and about 4.8 g/L, between about 2.8 g/L and about 4.6 g/L, between about 2.8 g/L and about 4.4 g/L, between about 2.8 g/L and about 4.2 g/L, between about 2.8 g/L and about 4.0 g/L, between about 2.8 g/L and about 3.8 g/L, between about 3.0 g/L and about 16.0 g/L, between about 3.0 g/L and about 15.8 g/L, between about 3.0 g/L and about 15.6 g/L, between about 3.0 g/L and about 15.4 g/L, between about 3.0 g/L and about 15.2 g/L, between about 3.0 g/L and about 15.0 g/L, between about 3.0 g/L and about 14.8 g/L, between about 3.0 g/L and about 14.6 g/L, between about 3.0 g/L and about 14.4 g/L, between about 3.0 g/L and about 14.2 g/L, between about 3.0 g/L and about 14.0 g/L, between about 3.0 g/L and about 13.8 g/L, between about 3.0 g/L and about 13.6 g/L, between about 3.0 g/L and about 13.4 g/L, between about 3.0 g/L and about 13.2 g/L, between about 3.0 g/L and about 13.0 g/L, between about 3.0 g/L and about 12.8 g/L, between about 3.0 g/L and about 12.6 g/L, between about 3.0 g/L and about 12.4 g/L, between about 3.0 g/L and about 12.2 g/L, between about 3.0 g/L and about 12.0 g/L, between about 3.0 g/L and about 11.8 g/L, between about 3.0 g/L and about 11.6 g/L, between about 3.0 g/L and about 11.4 g/L, between about 3.0 g/L and about 11.2 g/L, between about 3.0 g/L and about 11.0 g/L, between about 3.0 g/L and about 10.8 g/L, between about 3.0 g/L and about 10.6 g/L. between about 3.0 g/L and about 10.4 g/L, between about 3.0 g/L and about 10.2 g/L, between about 3.0 g/L and about 10.0 g/L, between about 3.0 g/L and about 9.8 g/L, between about 3.0 g/L and about 9.6 g/L, between about 3.0 g/L and about 9.4 g/L, between about 3.0 g/L and about 9.2 g/L, between about 3.0 g/L and about 9.0 g/L, between about 3.0 g/L and about 8.8 g/L, between about 3.0 g/L and about 8.6 g/L, between about 3.0 g/L and about 8.4 g/L, between about 3.0 g/L and about 8.2 g/L, between about 3.0 g/L and about 8.0 g/L, between about 3.0 g/L and about 7.8 g/L, between about 3.0 g/L and about 7.6 g/L, between about 3.0 g/L and about 7.4 g/L, between about 3.0 g/L and about 7.2 g/L, between about 3.0 g/L and about 7.0 g/L, between about 3.0 g/L and about 6.9 g/L, between about 3.0 g/L and about 6.8 g/L, between about 3.0 g/L and about 6.6 g/L, between about 3.0 g/L and about 6.4 g/L, between about 3.0 g/L and about 6.2 g/L, between about 3.0 g/L and about 6.0 g/L, between about 3.0 g/L and about 5.8 g/L, between about 3.0 g/L and about 5.6 g/L, between about 3.0 g/L and about 5.4 g/L, between about 3.0 g/L and about 5.2 g/L, between about 3.0 g/L and about 5.0 g/L, between about 3.0 g/L and about 4.8 g/L, between about 3.0 g/L and about 4.6 g/L, between about 3.0 g/L and about 4.4 g/L, between about 3.0 g/L and about 4.2 g/L, between about 3.0 g/L and about 4.0 g/L, between about 3.2 g/L and about 16.0 g/L, between about 3.2 g/L and about 15.8 g/L, between about 3.2 g/L and about 15.6 g/L, between about 3.2 g/L and about 15.4 g/L, between about 3.2 g/L and about 15.2 g/L, between about 3.2 g/L and about 15.0 g/L, between about 3.2 g/L and about 14.8 g/L, between about 3.2 g/L and about 14.6 g/L, between about 3.2 g/L and about 14.4 g/L, between about 3.2 g/L and about 14.2 g/L, between about 3.2 g/L and about 14.0 g/L, between about 3.2 g/L and about 13.8 g/L, between about 3.2 g/L and about 13.6 g/L, between about 3.2 g/L and about 13.4 g/L, between about 3.2 g/L and about 13.2 g/L, between about 3.2 g/L and about 13.0 g/L, between about 3.2 g/L and about 12.8 g/L, between about 3.2 g/L and about 12.6 g/L, between about 3.2 g/L and about 12.4 g/L, between about 3.2 g/L and about 12.2 g/L, between about 3.2 g/L and about 12.0 g/L, between about 3.2 g/L and about 11.8 g/L, between about 3.2 g/L and about 11.6 g/L, between about 3.2 g/L and about 11.4 g/L, between about 3.2 g/L and about 11.2 g/L, between about 3.2 g/L and about 11.0 g/L, between about 3.2 g/L and about 10.8 g/L, between about 3.2 g/L and about 10.6 g/L. between about 3.2 g/L and about 10.4 g/L, between about 3.2 g/L and about 10.2 g/L, between about 3.2 g/L and about 10.0 g/L, between about 3.2 g/L and about 9.8 g/L, between about 3.2 g/L and about 9.6 g/L, between about 3.2 g/L and about 9.4 g/L, between about 3.2 g/L and about 9.2 g/L, between about 3.2 g/L and about 9.0 g/L, between about 3.2 g/L and about 8.8 g/L, between about 3.2 g/L and about 8.6 g/L, between about 3.2 g/L and about 8.4 g/L, between about 3.2 g/L and about 8.2 g/L, between about 3.2 g/L and about 8.0 g/L, between about 3.2 g/L and about 7.8 g/L, between about 3.2 g/L and about 7.6 g/L, between about 3.2 g/L and about 7.4 g/L, between about 3.2 g/L and about 7.2 g/L, between about 3.2 g/L and about 7.0 g/L, between about 3.2 g/L and about 6.9 g/L, between about 3.2 g/L and about 6.8 g/L, between about 3.2 g/L and about 6.6 g/L, between about 3.2 g/L and about 6.4 g/L, between about 3.2 g/L and about 6.2 g/L, between about 3.2 g/L and about 6.0 g/L, between about 3.2 g/L and about 5.8 g/L, between about 3.2 g/L and about 5.6 g/L, between about 3.2 g/L and about 5.4 g/L, between about 3.2 g/L and about 5.2 g/L, between about 3.2 g/L and about 5.0 g/L, between about 3.2 g/L and about 4.8 g/L, between about 3.2 g/L and about 4.6 g/L, between about 3.2 g/L and about 4.4 g/L, between about 3.2 g/L and about 4.2 g/L, between about 3.4 g/L and about 16.0 g/L, between about 3.4 g/L and about 15.8 g/L, between about 3.4 g/L and about 15.6 g/L, between about 3.4 g/L and about 15.4 g/L, between about 3.4 g/L and about 15.2 g/L, between about 3.4 g/L and about 15.0 g/L, between about 3.4 g/L and about 14.8 g/L, between about 3.4 g/L and about 14.6 g/L, between about 3.4 g/L and about 14.4 g/L, between about 3.4 g/L and about 14.2 g/L, between about 3.4 g/L and about 14.0 g/L, between about 3.4 g/L and about 13.8 g/L, between about 3.4 g/L and about 13.6 g/L, between about 3.4 g/L and about 13.4 g/L, between about 3.4 g/L and about 13.2 g/L, between about 3.4 g/L and about 13.0 g/L, between about 3.4 g/L and about 12.8 g/L, between about 3.4 g/L and about 12.6 g/L, between about 3.4 g/L and about 12.4 g/L, between about 3.4 g/L and about 12.2 g/L, between about 3.4 g/L and about 12.0 g/L, between about 3.4 g/L and about 11.8 g/L, between about 3.4 g/L and about 11.6 g/L, between about 3.4 g/L and about 11.4 g/L, between about 3.4 g/L and about 11.2 g/L, between about 3.4 g/L and about 11.0 g/L, between about 3.4 g/L and about 10.8 g/L, between about 3.4 g/L and about 10.6 g/L. between about 3.4 g/L and about 10.4 g/L, between about 3.4 g/L and about 10.2 g/L, between about 3.4 g/L and about 10.0 g/L, between about 3.4 g/L and about 9.8 g/L, between about 3.4 g/L and about 9.6 g/L, between about 3.4 g/L and about 9.4 g/L, between about 3.4 g/L and about 9.2 g/L, between about 3.4 g/L and about 9.0 g/L, between about 3.4 g/L and about 8.8 g/L, between about 3.4 g/L and about 8.6 g/L, between about 3.4 g/L and about 8.4 g/L, between about 3.4 g/L and about 8.2 g/L, between about 3.4 g/L and about 8.0 g/L, between about 3.4 g/L and about 7.8 g/L, between about 3.4 g/L and about 7.6 g/L, between about 3.4 g/L and about 7.4 g/L, between about 3.4 g/L and about 7.2 g/L, between about 3.4 g/L and about 7.0 g/L, between about 3.4 g/L and about 6.9 g/L, between about 3.4 g/L and about 6.8 g/L, between about 3.4 g/L and about 6.6 g/L, between about 3.4 g/L and about 6.4 g/L, between about 3.4 g/L and about 6.2 g/L, between about 3.4 g/L and about 6.0 g/L, between about 3.4 g/L and about 5.8 g/L, between about 3.4 g/L and about 5.6 g/L, between about 3.4 g/L and about 5.4 g/L, between about 3.4 g/L and about 5.2 g/L, between about 3.4 g/L and about 5.0 g/L, between about 3.4 g/L and about 4.8 g/L, between about 3.4 g/L and about 4.6 g/L, between about 3.4 g/L and about 4.4 g/L, between about 3.6 g/L and about 16.0 g/L, between about 3.6 g/L and about 15.8 g/L, between about 3.6 g/L and about 15.6 g/L, between about 3.6 g/L and about 15.4 g/L, between about 3.6 g/L and about 15.2 g/L, between about 3.6 g/L and about 15.0 g/L, between about 3.6 g/L and about 14.8 g/L, between about 3.6 g/L and about 14.6 g/L, between about 3.6 g/L and about 14.4 g/L, between about 3.6 g/L and about 14.2 g/L, between about 3.6 g/L and about 14.0 g/L, between about 3.6 g/L and about 13.8 g/L, between about 3.6 g/L and about 13.6 g/L, between about 3.6 g/L and about 13.4 g/L, between about 3.6 g/L and about 13.2 g/L, between about 3.6 g/L and about 13.0 g/L, between about 3.6 g/L and about 12.8 g/L, between about 3.6 g/L and about 12.6 g/L, between about 3.6 g/L and about 12.4 g/L, between about 3.6 g/L and about 12.2 g/L, between about 3.6 g/L and about 12.0 g/L, between about 3.6 g/L and about 11.8 g/L, between about 3.6 g/L and about 11.6 g/L, between about 3.6 g/L and about 11.4 g/L, between about 3.6 g/L and about 11.2 g/L, between about 3.6 g/L and about 11.0 g/L, between about 3.6 g/L and about 10.8 g/L, between about 3.6 g/L and about 10.6 g/L. between about 3.6 g/L and about 10.4 g/L, between about 3.6 g/L and about 10.2 g/L, between about 3.6 g/L and about 10.0 g/L, between about 3.6 g/L and about 9.8 g/L, between about 3.6 g/L and about 9.6 g/L, between about 3.6 g/L and about 9.4 g/L, between about 3.6 g/L and about 9.2 g/L, between about 3.6 g/L and about 9.0 g/L, between about 3.6 g/L and about 8.8 g/L, between about 3.6 g/L and about 8.6 g/L, between about 3.6 g/L and about 8.4 g/L, between about 3.6 g/L and about 8.2 g/L, between about 3.6 g/L and about 8.0 g/L, between about 3.6 g/L and about 7.8 g/L, between about 3.6 g/L and about 7.6 g/L, between about 3.6 g/L and about 7.4 g/L, between about 3.6 g/L and about 7.2 g/L, between about 3.6 g/L and about 7.0 g/L, between about 3.6 g/L and about 6.9 g/L, between about 3.6 g/L and about 6.8 g/L, between about 3.6 g/L and about 6.6 g/L, between about 3.6 g/L and about 6.4 g/L, between about 3.6 g/L and about 6.2 g/L, between about 3.6 g/L and about 6.0 g/L, between about 3.6 g/L and about 5.8 g/L, between about 3.6 g/L and about 5.6 g/L, between about 3.6 g/L and about 5.4 g/L, between about 3.6 g/L and about 5.2 g/L, between about 3.6 g/L and about 5.0 g/L, between about 3.6 g/L and about 4.8 g/L, between about 3.6 g/L and about 4.6 g/L, between about 3.8 g/L and about 16.0 g/L, between about 3.8 g/L and about 15.8 g/L, between about 3.8 g/L and about 15.6 g/L, between about 3.8 g/L and about 15.4 g/L, between about 3.8 g/L and about 15.2 g/L, between about 3.8 g/L and about 15.0 g/L, between about 3.8 g/L and about 14.8 g/L, between about 3.8 g/L and about 14.6 g/L, between about 3.8 g/L and about 14.4 g/L, between about 3.8 g/L and about 14.2 g/L, between about 3.8 g/L and about 14.0 g/L, between about 3.8 g/L and about 13.8 g/L, between about 3.8 g/L and about 13.6 g/L, between about 3.8 g/L and about 13.4 g/L, between about 3.8 g/L and about 13.2 g/L, between about 3.8 g/L and about 13.0 g/L, between about 3.8 g/L and about 12.8 g/L, between about 3.8 g/L and about 12.6 g/L, between about 3.8 g/L and about 12.4 g/L, between about 3.8 g/L and about 12.2 g/L, between about 3.8 g/L and about 12.0 g/L, between about 3.8 g/L and about 11.8 g/L, between about 3.8 g/L and about 11.6 g/L, between about 3.8 g/L and about 11.4 g/L, between about 3.8 g/L and about 11.2 g/L, between about 3.8 g/L and about 11.0 g/L, between about 3.8 g/L and about 10.8 g/L, between about 3.8 g/L and about 10.6 g/L. between about 3.8 g/L and about 10.4 g/L, between about 3.8 g/L and about 10.2 g/L, between about 3.8 g/L and about 10.0 g/L, between about 3.8 g/L and about 9.8 g/L, between about 3.8 g/L and about 9.6 g/L, between about 3.8 g/L and about 9.4 g/L, between about 3.8 g/L and about 9.2 g/L, between about 3.8 g/L and about 9.0 g/L, between about 3.8 g/L and about 8.8 g/L, between about 3.8 g/L and about 8.6 g/L, between about 3.8 g/L and about 8.4 g/L, between about 3.8 g/L and about 8.2 g/L, between about 3.8 g/L and about 8.0 g/L, between about 3.8 g/L and about 7.8 g/L, between about 3.8 g/L and about 7.6 g/L, between about 3.8 g/L and about 7.4 g/L, between about 3.8 g/L and about 7.2 g/L, between about 3.8 g/L and about 7.0 g/L, between about 3.8 g/L and about 6.9 g/L, between about 3.8 g/L and about 6.8 g/L, between about 3.8 g/L and about 6.6 g/L, between about 3.8 g/L and about 6.4 g/L, between about 3.8 g/L and about 6.2 g/L, between about 3.8 g/L and about 6.0 g/L, between about 3.8 g/L and about 5.8 g/L, between about 3.8 g/L and about 5.6 g/L, between about 3.8 g/L and about 5.4 g/L, between about 3.8 g/L and about 5.2 g/L, between about 3.8 g/L and about 5.0 g/L, between about 3.8 g/L and about 4.8 g/L, between about 4.0 g/L and about 16.0 g/L, between about 4.0 g/L and about 15.8 g/L, between about 4.0 g/L and about 15.6 g/L, between about 4.0 g/L and about 15.4 g/L, between about 4.0 g/L and about 15.2 g/L, between about 4.0 g/L and about 15.0 g/L, between about 4.0 g/L and about 14.8 g/L, between about 4.0 g/L and about 14.6 g/L, between about 4.0 g/L and about 14.4 g/L, between about 4.0 g/L and about 14.2 g/L, between about 4.0 g/L and about 14.0 g/L, between about 4.0 g/L and about 13.8 g/L, between about 4.0 g/L and about 13.6 g/L, between about 4.0 g/L and about 13.4 g/L, between about 4.0 g/L and about 13.2 g/L, between about 4.0 g/L and about 13.0 g/L, between about 4.0 g/L and about 12.8 g/L, between about 4.0 g/L and about 12.6 g/L, between about 4.0 g/L and about 12.4 g/L, between about 4.0 g/L and about 12.2 g/L, between about 4.0 g/L and about 12.0 g/L, between about 4.0 g/L and about 11.8 g/L, between about 4.0 g/L and about 11.6 g/L, between about 4.0 g/L and about 11.4 g/L, between about 4.0 g/L and about 11.2 g/L, between about 4.0 g/L and about 11.0 g/L, between about 4.0 g/L and about 10.8 g/L, between about 4.0 g/L and about 10.6 g/L. between about 4.0 g/L and about 10.4 g/L, between about 4.0 g/L and about 10.2 g/L, between about 4.0 g/L and about 10.0 g/L, between about 4.0 g/L and about 9.8 g/L, between about 4.0 g/L and about 9.6 g/L, between about 4.0 g/L and about 9.4 g/L, between about 4.0 g/L and about 9.2 g/L, between about 4.0 g/L and about 9.0 g/L, between about 4.0 g/L and about 8.8 g/L, between about 4.0 g/L and about 8.6 g/L, between about 4.0 g/L and about 8.4 g/L, between about 4.0 g/L and about 8.2 g/L, between about 4.0 g/L and about 8.0 g/L, between about 4.0 g/L and about 7.8 g/L, between about 4.0 g/L and about 7.6 g/L, between about 4.0 g/L and about 7.4 g/L, between about 4.0 g/L and about 7.2 g/L, between about 4.0 g/L and about 7.0 g/L, between about 4.0 g/L and about 6.9 g/L, between about 4.0 g/L and about 6.8 g/L, between about 4.0 g/L and about 6.6 g/L, between about 4.0 g/L and about 6.4 g/L, between about 4.0 g/L and about 6.2 g/L, between about 4.0 g/L and about 6.0 g/L, between about 4.0 g/L and about 5.8 g/L, between about 4.0 g/L and about 5.6 g/L, between about 4.0 g/L and about 5.4 g/L, between about 4.0 g/L and about 5.2 g/L, between about 4.0 g/L and about 5.0 g/L, between about 4.2 g/L and about 16.0 g/L, between about 4.2 g/L and about 15.8 g/L, between about 4.2 g/L and about 15.6 g/L, between about 4.2 g/L and about 15.4 g/L, between about 4.2 g/L and about 15.2 g/L, between about 4.2 g/L and about 15.0 g/L, between about 4.2 g/L and about 14.8 g/L, between about 4.2 g/L and about 14.6 g/L, between about 4.2 g/L and about 14.4 g/L, between about 4.2 g/L and about 14.2 g/L, between about 4.2 g/L and about 14.0 g/L, between about 4.2 g/L and about 13.8 g/L, between about 4.2 g/L and about 13.6 g/L, between about 4.2 g/L and about 13.4 g/L, between about 4.2 g/L and about 13.2 g/L, between about 4.2 g/L and about 13.0 g/L, between about 4.2 g/L and about 12.8 g/L, between about 4.2 g/L and about 12.6 g/L, between about 4.2 g/L and about 12.4 g/L, between about 4.2 g/L and about 12.2 g/L, between about 4.2 g/L and about 12.0 g/L, between about 4.2 g/L and about 11.8 g/L, between about 4.2 g/L and about 11.6 g/L, between about 4.2 g/L and about 11.4 g/L, between about 4.2 g/L and about 11.2 g/L, between about 4.2 g/L and about 11.0 g/L, between about 4.2 g/L and about 10.8 g/L, between about 4.2 g/L and about 10.6 g/L. between about 4.2 g/L and about 10.4 g/L, between about 4.2 g/L and about 10.2 g/L, between about 4.2 g/L and about 10.0 g/L, between about 4.2 g/L and about 9.8 g/L, between about 4.2 g/L and about 9.6 g/L, between about 4.2 g/L and about 9.4 g/L, between about 4.2 g/L and about 9.2 g/L, between about 4.2 g/L and about 9.0 g/L, between about 4.2 g/L and about 8.8 g/L, between about 4.2 g/L and about 8.6 g/L, between about 4.2 g/L and about 8.4 g/L, between about 4.2 g/L and about 8.2 g/L, between about 4.2 g/L and about 8.0 g/L, between about 4.2 g/L and about 7.8 g/L, between about 4.2 g/L and about 7.6 g/L, between about 4.2 g/L and about 7.4 g/L, between about 4.2 g/L and about 7.2 g/L, between about 4.2 g/L and about 7.0 g/L, between about 4.2 g/L and about 6.9 g/L, between about 4.2 g/L and about 6.8 g/L, between about 4.2 g/L and about 6.6 g/L, between about 4.2 g/L and about 6.4 g/L, between about 4.2 g/L and about 6.2 g/L, between about 4.2 g/L and about 6.0 g/L, between about 4.2 g/L and about 5.8 g/L, between about 4.2 g/L and about 5.6 g/L, between about 4.2 g/L and about 5.4 g/L, between about 4.2 g/L and about 5.2 g/L, between about 4.4 g/L and about 16.0 g/L, between about 4.4 g/L and about 15.8 g/L, between about 4.4 g/L and about 15.6 g/L, between about 4.4 g/L and about 15.4 g/L, between about 4.4 g/L and about 15.2 g/L, between about 4.4 g/L and about 15.0 g/L, between about 4.4 g/L and about 14.8 g/L, between about 4.4 g/L and about 14.6 g/L, between about 4.4 g/L and about 14.4 g/L, between about 4.4 g/L and about 14.2 g/L, between about 4.4 g/L and about 14.0 g/L, between about 4.4 g/L and about 13.8 g/L, between about 4.4 g/L and about 13.6 g/L, between about 4.4 g/L and about 13.4 g/L, between about 4.4 g/L and about 13.2 g/L, between about 4.4 g/L and about 13.0 g/L, between about 4.4 g/L and about 12.8 g/L, between about 4.4 g/L and about 12.6 g/L, between about 4.4 g/L and about 12.4 g/L, between about 4.4 g/L and about 12.2 g/L, between about 4.4 g/L and about 12.0 g/L, between about 4.4 g/L and about 11.8 g/L, between about 4.4 g/L and about 11.6 g/L, between about 4.4 g/L and about 11.4 g/L, between about 4.4 g/L and about 11.2 g/L, between about 4.4 g/L and about 11.0 g/L, between about 4.4 g/L and about 10.8 g/L, between about 4.4 g/L and about 10.6 g/L. between about 4.4 g/L and about 10.4 g/L, between about 4.4 g/L and about 10.2 g/L, between about 4.4 g/L and about 10.0 g/L, between about 4.4 g/L and about 9.8 g/L, between about 4.4 g/L and about 9.6 g/L, between about 4.4 g/L and about 9.4 g/L, between about 4.4 g/L and about 9.2 g/L, between about 4.4 g/L and about 9.0 g/L, between about 4.4 g/L and about 8.8 g/L, between about 4.4 g/L and about 8.6 g/L, between about 4.4 g/L and about 8.4 g/L, between about 4.4 g/L and about 8.2 g/L, between about 4.4 g/L and about 8.0 g/L, between about 4.4 g/L and about 7.8 g/L, between about 4.4 g/L and about 7.6 g/L, between about 4.4 g/L and about 7.4 g/L, between about 4.4 g/L and about 7.2 g/L, between about 4.4 g/L and about 7.0 g/L, between about 4.4 g/L and about 6.9 g/L, between about 4.4 g/L and about 6.8 g/L, between about 4.4 g/L and about 6.6 g/L, between about 4.4 g/L and about 6.4 g/L, between about 4.4 g/L and about 6.2 g/L, between about 4.4 g/L and about 6.0 g/L, between about 4.4 g/L and about 5.8 g/L, between about 4.4 g/L and about 5.6 g/L, between about 4.4 g/L and about 5.4 g/L, between about 4.6 g/L and about 16.0 g/L, between about 4.6 g/L and about 15.8 g/L, between about 4.6 g/L and about 15.6 g/L, between about 4.6 g/L and about 15.4 g/L, between about 4.6 g/L and about 15.2 g/L, between about 4.6 g/L and about 15.0 g/L, between about 4.6 g/L and about 14.8 g/L, between about 4.6 g/L and about 14.6 g/L, between about 4.6 g/L and about 14.4 g/L, between about 4.6 g/L and about 14.2 g/L, between about 4.6 g/L and about 14.0 g/L, between about 4.6 g/L and about 13.8 g/L, between about 4.6 g/L and about 13.6 g/L, between about 4.6 g/L and about 13.4 g/L, between about 4.6 g/L and about 13.2 g/L, between about 4.6 g/L and about 13.0 g/L, between about 4.6 g/L and about 12.8 g/L, between about 4.6 g/L and about 12.6 g/L, between about 4.6 g/L and about 12.4 g/L, between about 4.6 g/L and about 12.2 g/L, between about 4.6 g/L and about 12.0 g/L, between about 4.6 g/L and about 11.8 g/L, between about 4.6 g/L and about 11.6 g/L, between about 4.6 g/L and about 11.4 g/L, between about 4.6 g/L and about 11.2 g/L, between about 4.6 g/L and about 11.0 g/L, between about 4.6 g/L and about 10.8 g/L, between about 4.6 g/L and about 10.6 g/L. between about 4.6 g/L and about 10.4 g/L, between about 4.6 g/L and about 10.2 g/L, between about 4.6 g/L and about 10.0 g/L, between about 4.6 g/L and about 9.8 g/L, between about 4.6 g/L and about 9.6 g/L, between about 4.6 g/L and about 9.4 g/L, between about 4.6 g/L and about 9.2 g/L, between about 4.6 g/L and about 9.0 g/L, between about 4.6 g/L and about 8.8 g/L, between about 4.6 g/L and about 8.6 g/L, between about 4.6 g/L and about 8.4 g/L, between about 4.6 g/L and about 8.2 g/L, between about 4.6 g/L and about 8.0 g/L, between about 4.6 g/L and about 7.8 g/L, between about 4.6 g/L and about 7.6 g/L, between about 4.6 g/L and about 7.4 g/L, between about 4.6 g/L and about 7.2 g/L, between about 4.6 g/L and about 7.0 g/L, between about 4.6 g/L and about 6.9 g/L, between about 4.6 g/L and about 6.8 g/L, between about 4.6 g/L and about 6.6 g/L, between about 4.6 g/L and about 6.4 g/L, between about 4.6 g/L and about 6.2 g/L, between about 4.6 g/L and about 6.0 g/L, between about 4.6 g/L and about 5.8 g/L, between about 4.6 g/L and about 5.6 g/L, between about 4.8 g/L and about 16.0 g/L, between about 4.8 g/L and about 15.8 g/L, between about 4.8 g/L and about 15.6 g/L, between about 4.8 g/L and about 15.4 g/L, between about 4.8 g/L and about 15.2 g/L, between about 4.8 g/L and about 15.0 g/L, between about 4.8 g/L and about 14.8 g/L, between about 4.8 g/L and about 14.6 g/L, between about 4.8 g/L and about 14.4 g/L, between about 4.8 g/L and about 14.2 g/L, between about 4.8 g/L and about 14.0 g/L, between about 4.8 g/L and about 13.8 g/L, between about 4.8 g/L and about 13.6 g/L, between about 4.8 g/L and about 13.4 g/L, between about 4.8 g/L and about 13.2 g/L, between about 4.8 g/L and about 13.0 g/L, between about 4.8 g/L and about 12.8 g/L, between about 4.8 g/L and about 12.6 g/L, between about 4.8 g/L and about 12.4 g/L, between about 4.8 g/L and about 12.2 g/L, between about 4.8 g/L and about 12.0 g/L, between about 4.8 g/L and about 11.8 g/L, between about 4.8 g/L and about 11.6 g/L, between about 4.8 g/L and about 11.4 g/L, between about 4.8 g/L and about 11.2 g/L, between about 4.8 g/L and about 11.0 g/L, between about 4.8 g/L and about 10.8 g/L, between about 4.8 g/L and about 10.6 g/L. between about 4.8 g/L and about 10.4 g/L, between about 4.8 g/L and about 10.2 g/L, between about 4.8 g/L and about 10.0 g/L, between about 4.8 g/L and about 9.8 g/L, between about 4.8 g/L and about 9.6 g/L, between about 4.8 g/L and about 9.4 g/L, between about 4.8 g/L and about 9.2 g/L, between about 4.8 g/L and about 9.0 g/L, between about 4.8 g/L and about 8.8 g/L, between about 4.8 g/L and about 8.6 g/L, between about 4.8 g/L and about 8.4 g/L, between about 4.8 g/L and about 8.2 g/L, between about 4.8 g/L and about 8.0 g/L, between about 4.8 g/L and about 7.8 g/L, between about 4.8 g/L and about 7.6 g/L, between about 4.8 g/L and about 7.4 g/L, between about 4.8 g/L and about 7.2 g/L, between about 4.8 g/L and about 7.0 g/L, between about 4.8 g/L and about 6.9 g/L, between about 4.8 g/L and about 6.8 g/L, between about 4.8 g/L and about 6.6 g/L, between about 4.8 g/L and about 6.4 g/L, between about 4.8 g/L and about 6.2 g/L, between about 4.8 g/L and about 6.0 g/L, between about 4.8 g/L and about 5.8 g/L, between about 5.0 g/L and about 16.0 g/L, between about 5.0 g/L and about 15.8 g/L, between about 5.0 g/L and about 15.6 g/L, between about 5.0 g/L and about 15.4 g/L, between about 5.0 g/L and about 15.2 g/L, between about 5.0 g/L and about 15.0 g/L, between about 5.0 g/L and about 14.8 g/L, between about 5.0 g/L and about 14.6 g/L, between about 5.0 g/L and about 14.4 g/L, between about 5.0 g/L and about 14.2 g/L, between about 5.0 g/L and about 14.0 g/L, between about 5.0 g/L and about 13.8 g/L, between about 5.0 g/L and about 13.6 g/L, between about 5.0 g/L and about 13.4 g/L, between about 5.0 g/L and about 13.2 g/L, between about 5.0 g/L and about 13.0 g/L, between about 5.0 g/L and about 12.8 g/L, between about 5.0 g/L and about 12.6 g/L, between about 5.0 g/L and about 12.4 g/L, between about 5.0 g/L and about 12.2 g/L, between about 5.0 g/L and about 12.0 g/L, between about 5.0 g/L and about 11.8 g/L, between about 5.0 g/L and about 11.6 g/L, between about 5.0 g/L and about 11.4 g/L, between about 5.0 g/L and about 11.2 g/L, between about 5.0 g/L and about 11.0 g/L, between about 5.0 g/L and about 10.8 g/L, between about 5.0 g/L and about 10.6 g/L. between about 5.0 g/L and about 10.4 g/L, between about 5.0 g/L and about 10.2 g/L, between about 5.0 g/L and about 10.0 g/L, between about 5.0 g/L and about 9.8 g/L, between about 5.0 g/L and about 9.6 g/L, between about 5.0 g/L and about 9.4 g/L, between about 5.0 g/L and about 9.2 g/L, between about 5.0 g/L and about 9.0 g/L, between about 5.0 g/L and about 8.8 g/L, between about 5.0 g/L and about 8.6 g/L, between about 5.0 g/L and about 8.4 g/L, between about 5.0 g/L and about 8.2 g/L, between about 5.0 g/L and about 8.0 g/L, between about 5.0 g/L and about 7.8 g/L, between about 5.0 g/L and about 7.6 g/L, between about 5.0 g/L and about 7.4 g/L, between about 5.0 g/L and about 7.2 g/L, between about 5.0 g/L and about 7.0 g/L, between about 5.0 g/L and about 6.9 g/L, between about 5.0 g/L and about 6.8 g/L, between about 5.0 g/L and about 6.6 g/L, between about 5.0 g/L and about 6.4 g/L, between about 5.0 g/L and about 6.2 g/L, between about 5.0 g/L and about 6.0 g/L, between about 5.1 g/L and about 16.0 g/L, between about 5.1 g/L and about 15.8 g/L, between about 5.1 g/L and about 15.6 g/L, between about 5.1 g/L and about 15.4 g/L, between about 5.1 g/L and about 15.2 g/L, between about 5.1 g/L and about 15.0 g/L, between about 5.1 g/L and about 14.8 g/L, between about 5.1 g/L and about 14.6 g/L, between about 5.1 g/L and about 14.4 g/L, between about 5.1 g/L and about 14.2 g/L, between about 5.1 g/L and about 14.0 g/L, between about 5.1 g/L and about 13.8 g/L, between about 5.1 g/L and about 13.6 g/L, between about 5.1 g/L and about 13.4 g/L, between about 5.1 g/L and about 13.2 g/L, between about 5.1 g/L and about 13.0 g/L, between about 5.1 g/L and about 12.8 g/L, between about 5.1 g/L and about 12.6 g/L, between about 5.1 g/L and about 12.4 g/L, between about 5.1 g/L and about 12.2 g/L, between about 5.1 g/L and about 12.0 g/L, between about 5.1 g/L and about 11.8 g/L, between about 5.1 g/L and about 11.6 g/L, between about 5.1 g/L and about 11.4 g/L, between about 5.1 g/L and about 11.2 g/L, between about 5.1 g/L and about 11.0 g/L, between about 5.1 g/L and about 10.8 g/L, between about 5.1 g/L and about 10.6 g/L. between about 5.1 g/L and about 10.4 g/L, between about 5.1 g/L and about 10.2 g/L, between about 5.1 g/L and about 10.0 g/L, between about 5.1 g/L and about 9.8 g/L, between about 5.1 g/L and about 9.6 g/L, between about 5.1 g/L and about 9.4 g/L, between about 5.1 g/L and about 9.2 g/L, between about 5.1 g/L and about 9.0 g/L, between about 5.1 g/L and about 8.8 g/L, between about 5.1 g/L and about 8.6 g/L, between about 5.1 g/L and about 8.4 g/L, between about 5.1 g/L and about 8.2 g/L, between about 5.1 g/L and about 8.0 g/L, between about 5.1 g/L and about 7.8 g/L, between about 5.1 g/L and about 7.6 g/L, between about 5.1 g/L and about 7.4 g/L, between about 5.1 g/L and about 7.2 g/L, between about 5.1 g/L and about 7.0 g/L, between about 5.1 g/L and about 6.9 g/L, between about 5.1 g/L and about 6.8 g/L, between about 5.1 g/L and about 6.6 g/L, between about 5.1 g/L and about 6.4 g/L, between about 5.1 g/L and about 6.2 g/L, between about 5.1 g/L and about 6.1 g/L, between about 5.2 g/L and about 16.0 g/L, between about 5.2 g/L and about 15.8 g/L, between about 5.2 g/L and about 15.6 g/L, between about 5.2 g/L and about 15.4 g/L, between about 5.2 g/L and about 15.2 g/L, between about 5.2 g/L and about 15.0 g/L, between about 5.2 g/L and about 14.8 g/L, between about 5.2 g/L and about 14.6 g/L, between about 5.2 g/L and about 14.4 g/L, between about 5.2 g/L and about 14.2 g/L, between about 5.2 g/L and about 14.0 g/L, between about 5.2 g/L and about 13.8 g/L, between about 5.2 g/L and about 13.6 g/L, between about 5.2 g/L and about 13.4 g/L, between about 5.2 g/L and about 13.2 g/L, between about 5.2 g/L and about 13.0 g/L, between about 5.2 g/L and about 12.8 g/L, between about 5.2 g/L and about 12.6 g/L, between about 5.2 g/L and about 12.4 g/L, between about 5.2 g/L and about 12.2 g/L, between about 5.2 g/L and about 12.0 g/L, between about 5.2 g/L and about 11.8 g/L, between about 5.2 g/L and about 11.6 g/L, between about 5.2 g/L and about 11.4 g/L, between about 5.2 g/L and about 11.2 g/L, between about 5.2 g/L and about 11.0 g/L, between about 5.2 g/L and about 10.8 g/L, between about 5.2 g/L and about 10.6 g/L. between about 5.2 g/L and about 10.4 g/L, between about 5.2 g/L and about 10.2 g/L, between about 5.2 g/L and about 10.0 g/L, between about 5.2 g/L and about 9.8 g/L, between about 5.2 g/L and about 9.6 g/L, between about 5.2 g/L and about 9.4 g/L, between about 5.2 g/L and about 9.2 g/L, between about 5.2 g/L and about 9.0 g/L, between about 5.2 g/L and about 8.8 g/L, between about 5.2 g/L and about 8.6 g/L, between about 5.2 g/L and about 8.4 g/L, between about 5.2 g/L and about 8.2 g/L, between about 5.2 g/L and about 8.0 g/L, between about 5.2 g/L and about 7.8 g/L, between about 5.2 g/L and about 7.6 g/L, between about 5.2 g/L and about 7.4 g/L, between about 5.2 g/L and about 7.2 g/L, between about 5.2 g/L and about 7.0 g/L, between about 5.2 g/L and about 6.9 g/L, between about 5.2 g/L and about 6.8 g/L, between about 5.2 g/L and about 6.6 g/L, between about 5.2 g/L and about 6.4 g/L, between about 5.2 g/L and about 6.2 g/L, between about 5.4 g/L and about 16.0 g/L, between about 5.4 g/L and about 15.8 g/L, between about 5.4 g/L and about 15.6 g/L, between about 5.4 g/L and about 15.4 g/L, between about 5.4 g/L and about 15.2 g/L, between about 5.4 g/L and about 15.0 g/L, between about 5.4 g/L and about 14.8 g/L, between about 5.4 g/L and about 14.6 g/L, between about 5.4 g/L and about 14.4 g/L, between about 5.4 g/L and about 14.2 g/L, between about 5.4 g/L and about 14.0 g/L, between about 5.4 g/L and about 13.8 g/L, between about 5.4 g/L and about 13.6 g/L, between about 5.4 g/L and about 13.4 g/L, between about 5.4 g/L and about 13.2 g/L, between about 5.4 g/L and about 13.0 g/L, between about 5.4 g/L and about 12.8 g/L, between about 5.4 g/L and about 12.6 g/L, between about 5.4 g/L and about 12.4 g/L, between about 5.4 g/L and about 12.2 g/L, between about 5.4 g/L and about 12.0 g/L, between about 5.4 g/L and about 11.8 g/L, between about 5.4 g/L and about 11.6 g/L, between about 5.4 g/L and about 11.4 g/L, between about 5.4 g/L and about 11.2 g/L, between about 5.4 g/L and about 11.0 g/L, between about 5.4 g/L and about 10.8 g/L, between about 5.4 g/L and about 10.6 g/L. between about 5.4 g/L and about 10.4 g/L, between about 5.4 g/L and about 10.2 g/L, between about 5.4 g/L and about 10.0 g/L, between about 5.4 g/L and about 9.8 g/L, between about 5.4 g/L and about 9.6 g/L, between about 5.4 g/L and about 9.4 g/L, between about 5.4 g/L and about 9.2 g/L, between about 5.4 g/L and about 9.0 g/L, between about 5.4 g/L and about 8.8 g/L, between about 5.4 g/L and about 8.6 g/L, between about 5.4 g/L and about 8.4 g/L, between about 5.4 g/L and about 8.2 g/L, between about 5.4 g/L and about 8.0 g/L, between about 5.4 g/L and about 7.8 g/L, between about 5.4 g/L and about 7.6 g/L, between about 5.4 g/L and about 7.4 g/L, between about 5.4 g/L and about 7.2 g/L, between about 5.4 g/L and about 7.0 g/L, between about 5.4 g/L and about 6.9 g/L, between about 5.4 g/L and about 6.8 g/L, between about 5.4 g/L and about 6.6 g/L, between about 5.4 g/L and about 6.4 g/L, between about 5.6 g/L and about 16.0 g/L, between about 5.6 g/L and about 15.8 g/L, between about 5.6 g/L and about 15.6 g/L, between about 5.6 g/L and about 15.4 g/L, between about 5.6 g/L and about 15.2 g/L, between about 5.6 g/L and about 15.0 g/L, between about 5.6 g/L and about 14.8 g/L, between about 5.6 g/L and about 14.6 g/L, between about 5.6 g/L and about 14.4 g/L, between about 5.6 g/L and about 14.2 g/L, between about 5.6 g/L and about 14.0 g/L, between about 5.6 g/L and about 13.8 g/L, between about 5.6 g/L and about 13.6 g/L, between about 5.6 g/L and about 13.4 g/L, between about 5.6 g/L and about 13.2 g/L, between about 5.6 g/L and about 13.0 g/L, between about 5.6 g/L and about 12.8 g/L, between about 5.6 g/L and about 12.6 g/L, between about 5.6 g/L and about 12.4 g/L, between about 5.6 g/L and about 12.2 g/L, between about 5.6 g/L and about 12.0 g/L, between about 5.6 g/L and about 11.8 g/L, between about 5.6 g/L and about 11.6 g/L, between about 5.6 g/L and about 11.4 g/L, between about 5.6 g/L and about 11.2 g/L, between about 5.6 g/L and about 11.0 g/L, between about 5.6 g/L and about 10.8 g/L, between about 5.6 g/L and about 10.6 g/L. between about 5.6 g/L and about 10.4 g/L, between about 5.6 g/L and about 10.2 g/L, between about 5.6 g/L and about 10.0 g/L, between about 5.6 g/L and about 9.8 g/L, between about 5.6 g/L and about 9.6 g/L, between about 5.6 g/L and about 9.4 g/L, between about 5.6 g/L and about 9.2 g/L, between about 5.6 g/L and about 9.0 g/L, between about 5.6 g/L and about 8.8 g/L, between about 5.6 g/L and about 8.6 g/L, between about 5.6 g/L and about 8.4 g/L, between about 5.6 g/L and about 8.2 g/L, between about 5.6 g/L and about 8.0 g/L, between about 5.6 g/L and about 7.8 g/L, between about 5.6 g/L and about 7.6 g/L, between about 5.6 g/L and about 7.4 g/L, between about 5.6 g/L and about 7.2 g/L, between about 5.6 g/L and about 7.0 g/L, between about 5.6 g/L and about 6.9 g/L, between about 5.6 g/L and about 6.8 g/L, between about 5.6 g/L and about 6.6 g/L, between about 5.8 g/L and about 16.0 g/L, between about 5.8 g/L and about 15.8 g/L, between about 5.8 g/L and about 15.6 g/L, between about 5.8 g/L and about 15.4 g/L, between about 5.8 g/L and about 15.2 g/L, between about 5.8 g/L and about 15.0 g/L, between about 5.8 g/L and about 14.8 g/L, between about 5.8 g/L and about 14.6 g/L, between about 5.8 g/L and about 14.4 g/L, between about 5.8 g/L and about 14.2 g/L, between about 5.8 g/L and about 14.0 g/L, between about 5.8 g/L and about 13.8 g/L, between about 5.8 g/L and about 13.6 g/L, between about 5.8 g/L and about 13.4 g/L, between about 5.8 g/L and about 13.2 g/L, between about 5.8 g/L and about 13.0 g/L, between about 5.8 g/L and about 12.8 g/L, between about 5.8 g/L and about 12.6 g/L, between about 5.8 g/L and about 12.4 g/L, between about 5.8 g/L and about 12.2 g/L, between about 5.8 g/L and about 12.0 g/L, between about 5.8 g/L and about 11.8 g/L, between about 5.8 g/L and about 11.6 g/L, between about 5.8 g/L and about 11.4 g/L, between about 5.8 g/L and about 11.2 g/L, between about 5.8 g/L and about 11.0 g/L, between about 5.8 g/L and about 10.8 g/L, between about 5.8 g/L and about 10.6 g/L. between about 5.8 g/L and about 10.4 g/L, between about 5.8 g/L and about 10.2 g/L, between about 5.8 g/L and about 10.0 g/L, between about 5.8 g/L and about 9.8 g/L, between about 5.8 g/L and about 9.6 g/L, between about 5.8 g/L and about 9.4 g/L, between about 5.8 g/L and about 9.2 g/L, between about 5.8 g/L and about 9.0 g/L, between about 5.8 g/L and about 8.8 g/L, between about 5.8 g/L and about 8.6 g/L, between about 5.8 g/L and about 8.4 g/L, between about 5.8 g/L and about 8.2 g/L, between about 5.8 g/L and about 8.0 g/L, between about 5.8 g/L and about 7.8 g/L, between about 5.8 g/L and about 7.6 g/L, between about 5.8 g/L and about 7.4 g/L, between about 5.8 g/L and about 7.2 g/L, between about 5.8 g/L and about 7.0 g/L, between about 5.8 g/L and about 6.9 g/L, between about 5.8 g/L and about 6.8 g/L, between about 6.0 g/L and about 16.0 g/L, between about 6.0 g/L and about 15.8 g/L, between about 6.0 g/L and about 15.6 g/L, between about 6.0 g/L and about 15.4 g/L, between about 6.0 g/L and about 15.2 g/L, between about 6.0 g/L and about 15.0 g/L, between about 6.0 g/L and about 14.8 g/L, between about 6.0 g/L and about 14.6 g/L, between about 6.0 g/L and about 14.4 g/L, between about 6.0 g/L and about 14.2 g/L, between about 6.0 g/L and about 14.0 g/L, between about 6.0 g/L and about 13.8 g/L, between about 6.0 g/L and about 13.6 g/L, between about 6.0 g/L and about 13.4 g/L, between about 6.0 g/L and about 13.2 g/L, between about 6.0 g/L and about 13.0 g/L, between about 6.0 g/L and about 12.8 g/L, between about 6.0 g/L and about 12.6 g/L, between about 6.0 g/L and about 12.4 g/L, between about 6.0 g/L and about 12.2 g/L, between about 6.0 g/L and about 12.0 g/L, between about 6.0 g/L and about 11.8 g/L, between about 6.0 g/L and about 11.6 g/L, between about 6.0 g/L and about 11.4 g/L, between about 6.0 g/L and about 11.2 g/L, between about 6.0 g/L and about 11.0 g/L, between about 6.0 g/L and about 10.8 g/L, between about 6.0 g/L and about 10.6 g/L. between about 6.0 g/L and about 10.4 g/L, between about 6.0 g/L and about 10.2 g/L, between about 6.0 g/L and about 10.0 g/L, between about 6.0 g/L and about 9.8 g/L, between about 6.0 g/L and about 9.6 g/L, between about 6.0 g/L and about 9.4 g/L, between about 6.0 g/L and about 9.2 g/L, between about 6.0 g/L and about 9.0 g/L, between about 6.0 g/L and about 8.8 g/L, between about 6.0 g/L and about 8.6 g/L, between about 6.0 g/L and about 8.4 g/L, between about 6.0 g/L and about 8.2 g/L, between about 6.0 g/L and about 8.0 g/L, between about 6.0 g/L and about 7.8 g/L, between about 6.0 g/L and about 7.6 g/L, between about 6.0 g/L and about 7.4 g/L, between about 6.0 g/L and about 7.2 g/L, between about 6.0 g/L and about 7.0 g/L, between about 6.2 g/L and about 16.0 g/L, between about 6.2 g/L and about 15.8 g/L, between about 6.2 g/L and about 15.6 g/L, between about 6.2 g/L and about 15.4 g/L, between about 6.2 g/L and about 15.2 g/L, between about 6.2 g/L and about 15.0 g/L, between about 6.2 g/L and about 14.8 g/L, between about 6.2 g/L and about 14.6 g/L, between about 6.2 g/L and about 14.4 g/L, between about 6.2 g/L and about 14.2 g/L, between about 6.2 g/L and about 14.0 g/L, between about 6.2 g/L and about 13.8 g/L, between about 6.2 g/L and about 13.6 g/L, between about 6.2 g/L and about 13.4 g/L, between about 6.2 g/L and about 13.2 g/L, between about 6.2 g/L and about 13.0 g/L, between about 6.2 g/L and about 12.8 g/L, between about 6.2 g/L and about 12.6 g/L, between about 6.2 g/L and about 12.4 g/L, between about 6.2 g/L and about 12.2 g/L, between about 6.2 g/L and about 12.0 g/L, between about 6.2 g/L and about 11.8 g/L, between about 6.2 g/L and about 11.6 g/L, between about 6.2 g/L and about 11.4 g/L, between about 6.2 g/L and about 11.2 g/L, between about 6.2 g/L and about 11.0 g/L, between about 6.2 g/L and about 10.8 g/L, between about 6.2 g/L and about 10.6 g/L. between about 6.2 g/L and about 10.4 g/L, between about 6.2 g/L and about 10.2 g/L, between about 6.2 g/L and about 10.0 g/L, between about 6.2 g/L and about 9.8 g/L, between about 6.2 g/L and about 9.6 g/L, between about 6.2 g/L and about 9.4 g/L, between about 6.2 g/L and about 9.2 g/L, between about 6.2 g/L and about 9.0 g/L, between about 6.2 g/L and about 8.8 g/L, between about 6.2 g/L and about 8.6 g/L, between about 6.2 g/L and about 8.4 g/L, between about 6.2 g/L and about 8.2 g/L, between about 6.2 g/L and about 8.0 g/L, between about 6.2 g/L and about 7.8 g/L, between about 6.2 g/L and about 7.6 g/L, between about 6.2 g/L and about 7.4 g/L, between about 6.2 g/L and about 7.2 g/L, between about 6.4 g/L and about 16.0 g/L, between about 6.4 g/L and about 15.8 g/L, between about 6.4 g/L and about 15.6 g/L, between about 6.4 g/L and about 15.4 g/L, between about 6.4 g/L and about 15.2 g/L, between about 6.4 g/L and about 15.0 g/L, between about 6.4 g/L and about 14.8 g/L, between about 6.4 g/L and about 14.6 g/L, between about 6.4 g/L and about 14.4 g/L, between about 6.4 g/L and about 14.2 g/L, between about 6.4 g/L and about 14.0 g/L, between about 6.4 g/L and about 13.8 g/L, between about 6.4 g/L and about 13.6 g/L, between about 6.4 g/L and about 13.4 g/L, between about 6.4 g/L and about 13.2 g/L, between about 6.4 g/L and about 13.0 g/L, between about 6.4 g/L and about 12.8 g/L, between about 6.4 g/L and about 12.6 g/L, between about 6.4 g/L and about 12.4 g/L, between about 6.4 g/L and about 12.2 g/L, between about 6.4 g/L and about 12.0 g/L, between about 6.4 g/L and about 11.8 g/L, between about 6.4 g/L and about 11.6 g/L, between about 6.4 g/L and about 11.4 g/L, between about 6.4 g/L and about 11.2 g/L, between about 6.4 g/L and about 11.0 g/L, between about 6.4 g/L and about 10.8 g/L, between about 6.4 g/L and about 10.6 g/L. between about 6.4 g/L and about 10.4 g/L, between about 6.4 g/L and about 10.2 g/L, between about 6.4 g/L and about 10.0 g/L, between about 6.4 g/L and about 9.8 g/L, between about 6.4 g/L and about 9.6 g/L, between about 6.4 g/L and about 9.4 g/L, between about 6.4 g/L and about 9.2 g/L, between about 6.4 g/L and about 9.0 g/L, between about 6.4 g/L and about 8.8 g/L, between about 6.4 g/L and about 8.6 g/L, between about 6.4 g/L and about 8.4 g/L, between about 6.4 g/L and about 8.2 g/L, between about 6.4 g/L and about 8.0 g/L, between about 6.4 g/L and about 7.8 g/L, between about 6.4 g/L and about 7.6 g/L, between about 6.4 g/L and about 7.4 g/L, between about 6.6 g/L and about 16.0 g/L, between about 6.6 g/L and about 15.8 g/L, between about 6.6 g/L and about 15.6 g/L, between about 6.6 g/L and about 15.4 g/L, between about 6.6 g/L and about 15.2 g/L, between about 6.6 g/L and about 15.0 g/L, between about 6.6 g/L and about 14.8 g/L, between about 6.6 g/L and about 14.6 g/L, between about 6.6 g/L and about 14.4 g/L, between about 6.6 g/L and about 14.2 g/L, between about 6.6 g/L and about 14.0 g/L, between about 6.6 g/L and about 13.8 g/L, between about 6.6 g/L and about 13.6 g/L, between about 6.6 g/L and about 13.4 g/L, between about 6.6 g/L and about 13.2 g/L, between about 6.6 g/L and about 13.0 g/L, between about 6.6 g/L and about 12.8 g/L, between about 6.6 g/L and about 12.6 g/L, between about 6.6 g/L and about 12.4 g/L, between about 6.6 g/L and about 12.2 g/L, between about 6.6 g/L and about 12.0 g/L, between about 6.6 g/L and about 11.8 g/L, between about 6.6 g/L and about 11.6 g/L, between about 6.6 g/L and about 11.4 g/L, between about 6.6 g/L and about 11.2 g/L, between about 6.6 g/L and about 11.0 g/L, between about 6.6 g/L and about 10.8 g/L, between about 6.6 g/L and about 10.6 g/L. between about 6.6 g/L and about 10.4 g/L, between about 6.6 g/L and about 10.2 g/L, between about 6.6 g/L and about 10.0 g/L, between about 6.6 g/L and about 9.8 g/L, between about 6.6 g/L and about 9.6 g/L, between about 6.6 g/L and about 9.4 g/L, between about 6.6 g/L and about 9.2 g/L, between about 6.6 g/L and about 9.0 g/L, between about 6.6 g/L and about 8.8 g/L, between about 6.6 g/L and about 8.6 g/L, between about 6.6 g/L and about 8.4 g/L, between about 6.6 g/L and about 8.2 g/L, between about 6.6 g/L and about 8.0 g/L, between about 6.6 g/L and about 7.8 g/L, between about 6.6 g/L and about 7.6 g/L, between about 6.8 g/L and about 16.0 g/L, between about 6.8 g/L and about 15.8 g/L, between about 6.8 g/L and about 15.6 g/L, between about 6.8 g/L and about 15.4 g/L, between about 6.8 g/L and about 15.2 g/L, between about 6.8 g/L and about 15.0 g/L, between about 6.8 g/L and about 14.8 g/L, between about 6.8 g/L and about 14.6 g/L, between about 6.8 g/L and about 14.4 g/L, between about 6.8 g/L and about 14.2 g/L, between about 6.8 g/L and about 14.0 g/L, between about 6.8 g/L and about 13.8 g/L, between about 6.8 g/L and about 13.6 g/L, between about 6.8 g/L and about 13.4 g/L, between about 6.8 g/L and about 13.2 g/L, between about 6.8 g/L and about 13.0 g/L, between about 6.8 g/L and about 12.8 g/L, between about 6.8 g/L and about 12.6 g/L, between about 6.8 g/L and about 12.4 g/L, between about 6.8 g/L and about 12.2 g/L, between about 6.8 g/L and about 12.0 g/L, between about 6.8 g/L and about 11.8 g/L, between about 6.8 g/L and about 11.6 g/L, between about 6.8 g/L and about 11.4 g/L, between about 6.8 g/L and about 11.2 g/L, between about 6.8 g/L and about 11.0 g/L, between about 6.8 g/L and about 10.8 g/L, between about 6.8 g/L and about 10.6 g/L. between about 6.8 g/L and about 10.4 g/L, between about 6.8 g/L and about 10.2 g/L, between about 6.8 g/L and about 10.0 g/L, between about 6.8 g/L and about 9.8 g/L, between about 6.8 g/L and about 9.6 g/L, between about 6.8 g/L and about 9.4 g/L, between about 6.8 g/L and about 9.2 g/L, between about 6.8 g/L and about 9.0 g/L, between about 6.8 g/L and about 8.8 g/L, between about 6.8 g/L and about 8.6 g/L, between about 6.8 g/L and about 8.4 g/L, between about 6.8 g/L and about 8.2 g/L, between about 6.8 g/L and about 8.0 g/L, between about 6.8 g/L and about 7.8 g/L, between about 7.0 g/L and about 16.0 g/L, between about 7.0 g/L and about 15.8 g/L, between about 7.0 g/L and about 15.6 g/L, between about 7.0 g/L and about 15.4 g/L, between about 7.0 g/L and about 15.2 g/L, between about 7.0 g/L and about 15.0 g/L, between about 7.0 g/L and about 14.8 g/L, between about 7.0 g/L and about 14.6 g/L, between about 7.0 g/L and about 14.4 g/L, between about 7.0 g/L and about 14.2 g/L, between about 7.0 g/L and about 14.0 g/L, between about 7.0 g/L and about 13.8 g/L, between about 7.0 g/L and about 13.6 g/L, between about 7.0 g/L and about 13.4 g/L, between about 7.0 g/L and about 13.2 g/L, between about 7.0 g/L and about 13.0 g/L, between about 7.0 g/L and about 12.8 g/L, between about 7.0 g/L and about 12.6 g/L, between about 7.0 g/L and about 12.4 g/L, between about 7.0 g/L and about 12.2 g/L, between about 7.0 g/L and about 12.0 g/L, between about 7.0 g/L and about 11.8 g/L, between about 7.0 g/L and about 11.6 g/L, between about 7.0 g/L and about 11.4 g/L, between about 7.0 g/L and about 11.2 g/L, between about 7.0 g/L and about 11.0 g/L, between about 7.0 g/L and about 10.8 g/L, between about 7.0 g/L and about 10.6 g/L. between about 7.0 g/L and about 10.4 g/L, between about 7.0 g/L and about 10.2 g/L, between about 7.0 g/L and about 10.0 g/L, between about 7.0 g/L and about 9.8 g/L, between about 7.0 g/L and about 9.6 g/L, between about 7.0 g/L and about 9.4 g/L, between about 7.0 g/L and about 9.2 g/L, between about 7.0 g/L and about 9.0 g/L, between about 7.0 g/L and about 8.8 g/L, between about 7.0 g/L and about 8.6 g/L, between about 7.0 g/L and about 8.4 g/L, between about 7.0 g/L and about 8.2 g/L, between about 7.0 g/L and about 8.0 g/L, between about 7.2 g/L and about 16.0 g/L, between about 7.2 g/L and about 15.8 g/L, between about 7.2 g/L and about 15.6 g/L, between about 7.2 g/L and about 15.4 g/L, between about 7.2 g/L and about 15.2 g/L, between about 7.2 g/L and about 15.0 g/L, between about 7.2 g/L and about 14.8 g/L, between about 7.2 g/L and about 14.6 g/L, between about 7.2 g/L and about 14.4 g/L, between about 7.2 g/L and about 14.2 g/L, between about 7.2 g/L and about 14.0 g/L, between about 7.2 g/L and about 13.8 g/L, between about 7.2 g/L and about 13.6 g/L, between about 7.2 g/L and about 13.4 g/L, between about 7.2 g/L and about 13.2 g/L, between about 7.2 g/L and about 13.0 g/L, between about 7.2 g/L and about 12.8 g/L, between about 7.2 g/L and about 12.6 g/L, between about 7.2 g/L and about 12.4 g/L, between about 7.2 g/L and about 12.2 g/L, between about 7.2 g/L and about 12.0 g/L, between about 7.2 g/L and about 11.8 g/L, between about 7.2 g/L and about 11.6 g/L, between about 7.2 g/L and about 11.4 g/L, between about 7.2 g/L and about 11.2 g/L, between about 7.2 g/L and about 11.0 g/L, between about 7.2 g/L and about 10.8 g/L, between about 7.2 g/L and about 10.6 g/L. between about 7.2 g/L and about 10.4 g/L, between about 7.2 g/L and about 10.2 g/L, between about 7.2 g/L and about 10.0 g/L, between about 7.2 g/L and about 9.8 g/L, between about 7.2 g/L and about 9.6 g/L, between about 7.2 g/L and about 9.4 g/L, between about 7.2 g/L and about 9.2 g/L, between about 7.2 g/L and about 9.0 g/L, between about 7.2 g/L and about 8.8 g/L, between about 7.2 g/L and about 8.6 g/L, between about 7.2 g/L and about 8.4 g/L, between about 7.2 g/L and about 8.2 g/L, between about 7.4 g/L and about 16.0 g/L, between about 7.4 g/L and about 15.8 g/L, between about 7.4 g/L and about 15.6 g/L, between about 7.4 g/L and about 15.4 g/L, between about 7.4 g/L and about 15.2 g/L, between about 7.4 g/L and about 15.0 g/L, between about 7.4 g/L and about 14.8 g/L, between about 7.4 g/L and about 14.6 g/L, between about 7.4 g/L and about 14.4 g/L, between about 7.4 g/L and about 14.2 g/L, between about 7.4 g/L and about 14.0 g/L, between about 7.4 g/L and about 13.8 g/L, between about 7.4 g/L and about 13.6 g/L, between about 7.4 g/L and about 13.4 g/L, between about 7.4 g/L and about 13.2 g/L, between about 7.4 g/L and about 13.0 g/L, between about 7.4 g/L and about 12.8 g/L, between about 7.4 g/L and about 12.6 g/L, between about 7.4 g/L and about 12.4 g/L, between about 7.4 g/L and about 12.2 g/L, between about 7.4 g/L and about 12.0 g/L, between about 7.4 g/L and about 11.8 g/L, between about 7.4 g/L and about 11.6 g/L, between about 7.4 g/L and about 11.4 g/L, between about 7.4 g/L and about 11.2 g/L, between about 7.4 g/L and about 11.0 g/L, between about 7.4 g/L and about 10.8 g/L, between about 7.4 g/L and about 10.6 g/L. between about 7.4 g/L and about 10.4 g/L, between about 7.4 g/L and about 10.2 g/L, between about 7.4 g/L and about 10.0 g/L, between about 7.4 g/L and about 9.8 g/L, between about 7.4 g/L and about 9.6 g/L, between about 7.4 g/L and about 9.4 g/L, between about 7.4 g/L and about 9.2 g/L, between about 7.4 g/L and about 9.0 g/L, between about 7.4 g/L and about 8.8 g/L, between about 7.4 g/L and about 8.6 g/L, between about 7.4 g/L and about 8.4 g/L, between about 7.6 g/L and about 16.0 g/L, between about 7.6 g/L and about 15.8 g/L, between about 7.6 g/L and about 15.6 g/L, between about 7.6 g/L and about 15.4 g/L, between about 7.6 g/L and about 15.2 g/L, between about 7.6 g/L and about 15.0 g/L, between about 7.6 g/L and about 14.8 g/L, between about 7.6 g/L and about 14.6 g/L, between about 7.6 g/L and about 14.4 g/L, between about 7.6 g/L and about 14.2 g/L, between about 7.6 g/L and about 14.0 g/L, between about 7.6 g/L and about 13.8 g/L, between about 7.6 g/L and about 13.6 g/L, between about 7.6 g/L and about 13.4 g/L, between about 7.6 g/L and about 13.2 g/L, between about 7.6 g/L and about 13.0 g/L, between about 7.6 g/L and about 12.8 g/L, between about 7.6 g/L and about 12.6 g/L, between about 7.6 g/L and about 12.4 g/L, between about 7.6 g/L and about 12.2 g/L, between about 7.6 g/L and about 12.0 g/L, between about 7.6 g/L and about 11.8 g/L, between about 7.6 g/L and about 11.6 g/L, between about 7.6 g/L and about 11.4 g/L, between about 7.6 g/L and about 11.2 g/L, between about 7.6 g/L and about 11.0 g/L, between about 7.6 g/L and about 10.8 g/L, between about 7.6 g/L and about 10.6 g/L. between about 7.6 g/L and about 10.4 g/L, between about 7.6 g/L and about 10.2 g/L, between about 7.6 g/L and about 10.0 g/L, between about 7.6 g/L and about 9.8 g/L, between about 7.6 g/L and about 9.6 g/L, between about 7.6 g/L and about 9.4 g/L, between about 7.6 g/L and about 9.2 g/L, between about 7.6 g/L and about 9.0 g/L, between about 7.6 g/L and about 8.8 g/L, between about 7.6 g/L and about 8.6 g/L, between about 7.8 g/L and about 16.0 g/L, between about 7.8 g/L and about 15.8 g/L, between about 7.8 g/L and about 15.6 g/L, between about 7.8 g/L and about 15.4 g/L, between about 7.8 g/L and about 15.2 g/L, between about 7.8 g/L and about 15.0 g/L, between about 7.8 g/L and about 14.8 g/L, between about 7.8 g/L and about 14.6 g/L, between about 7.8 g/L and about 14.4 g/L, between about 7.8 g/L and about 14.2 g/L, between about 7.8 g/L and about 14.0 g/L, between about 7.8 g/L and about 13.8 g/L, between about 7.8 g/L and about 13.6 g/L, between about 7.8 g/L and about 13.4 g/L, between about 7.8 g/L and about 13.2 g/L, between about 7.8 g/L and about 13.0 g/L, between about 7.8 g/L and about 12.8 g/L, between about 7.8 g/L and about 12.6 g/L, between about 7.8 g/L and about 12.4 g/L, between about 7.8 g/L and about 12.2 g/L, between about 7.8 g/L and about 12.0 g/L, between about 7.8 g/L and about 11.8 g/L, between about 7.8 g/L and about 11.6 g/L, between about 7.8 g/L and about 11.4 g/L, between about 7.8 g/L and about 11.2 g/L, between about 7.8 g/L and about 11.0 g/L, between about 7.8 g/L and about 10.8 g/L, between about 7.8 g/L and about 10.6 g/L. between about 7.8 g/L and about 10.4 g/L, between about 7.8 g/L and about 10.2 g/L, between about 7.8 g/L and about 10.0 g/L, between about 7.8 g/L and about 9.8 g/L, between about 7.8 g/L and about 9.6 g/L, between about 7.8 g/L and about 9.4 g/L, between about 7.8 g/L and about 9.2 g/L, between about 7.8 g/L and about 9.0 g/L, between about 7.8 g/L and about 8.8 g/L, between about 8.0 g/L and about 16.0 g/L, between about 8.0 g/L and about 15.8 g/L, between about 8.0 g/L and about 15.6 g/L, between about 8.0 g/L and about 15.4 g/L, between about 8.0 g/L and about 15.2 g/L, between about 8.0 g/L and about 15.0 g/L, between about 8.0 g/L and about 14.8 g/L, between about 8.0 g/L and about 14.6 g/L, between about 8.0 g/L and about 14.4 g/L, between about 8.0 g/L and about 14.2 g/L, between about 8.0 g/L and about 14.0 g/L, between about 8.0 g/L and about 13.8 g/L, between about 8.0 g/L and about 13.6 g/L, between about 8.0 g/L and about 13.4 g/L, between about 8.0 g/L and about 13.2 g/L, between about 8.0 g/L and about 13.0 g/L, between about 8.0 g/L and about 12.8 g/L, between about 8.0 g/L and about 12.6 g/L, between about 8.0 g/L and about 12.4 g/L, between about 8.0 g/L and about 12.2 g/L, between about 8.0 g/L and about 12.0 g/L, between about 8.0 g/L and about 11.8 g/L, between about 8.0 g/L and about 11.6 g/L, between about 8.0 g/L and about 11.4 g/L, between about 8.0 g/L and about 11.2 g/L, between about 8.0 g/L and about 11.0 g/L, between about 8.0 g/L and about 10.8 g/L, between about 8.0 g/L and about 10.6 g/L. between about 8.0 g/L and about 10.4 g/L, between about 8.0 g/L and about 10.2 g/L, between about 8.0 g/L and about 10.0 g/L, between about 8.0 g/L and about 9.8 g/L, between about 8.0 g/L and about 9.6 g/L, between about 8.0 g/L and about 9.4 g/L, between about 8.0 g/L and about 9.2 g/L, between about 8.0 g/L and about 9.0 g/L, between about 8.5 g/L and about 16.0 g/L, between about 8.5 g/L and about 15.5 g/L, between about 8.5 g/L and about 15.0 g/L, between about 8.5 g/L and about 14.5 g/L, between about 8.5 g/L and about 14.0 g/L, between about 8.5 g/L and about 13.5 g/L, between about 8.5 g/L and about 13.0 g/L, between about 8.5 g/L and about 12.5 g/L, between about 8.5 g/L and about 12.0 g/L, between about 8.5 g/L and about 11.5 g/L, between about 8.5 g/L and about 11.0 g/L, between about 8.5 g/L and about 10.5 g/L, between about 8.5 g/L and about 10.0 g/L, between about 8.5 g/L and about 9.5 g/L, between about 9.0 g/L and about 16.0 g/L, between about 9.0 g/L and about 15.5 g/L, between about 9.0 g/L and about 15.0 g/L, between about 9.0 g/L and about 14.5 g/L, between about 9.0 g/L and about 14.0 g/L, between about 9.0 g/L and about 13.5 g/L, between about 9.0 g/L and about 13.0 g/L, between about 9.0 g/L and about 12.5 g/L, between about 9.0 g/L and about 12.0 g/L, between about 9.0 g/L and about 11.5 g/L, between about 9.0 g/L and about 11.0 g/L, between about 9.0 g/L and about 10.5 g/L, between about 9.0 g/L and about 10.0 g/L, between about 9.5 g/L and about 16.0 g/L, between about 9.5 g/L and about 15.5 g/L, between about 9.5 g/L and about 15.0 g/L, between about 9.5 g/L and about 14.5 g/L, between about 9.5 g/L and about 14.0 g/L, between about 9.5 g/L and about 13.5 g/L, between about 9.5 g/L and about 13.0 g/L, between about 9.5 g/L and about 12.5 g/L, between about 9.5 g/L and about 12.0 g/L, between about 9.5 g/L and about 11.5 g/L, between about 9.5 g/L and about 11.0 g/L, between about 9.5 g/L and about 10.5 g/L, between about 10.0 g/L and about 16.0 g/L, between about 10.0 g/L and about 15.5 g/L, between about 10.0 g/L and about 15.0 g/L, between about 10.0 g/L and about 14.5 g/L, between about 10.0 g/L and about 14.0 g/L, between about 10.0 g/L and about 13.5 g/L, between about 10.0 g/L and about 13.0 g/L, between about 10.0 g/L and about 12.5 g/L, between about 10.0 g/L and about 12.0 g/L, between about 10.0 g/L and about 11.5 g/L, between about 10.0 g/L and about 11.0 g/L, between about 10.5 g/L and about 16.0 g/L, between about 10.5 g/L and about 15.5 g/L, between about 10.5 g/L and about 15.0 g/L, between about 10.5 g/L and about 14.5 g/L, between about 10.5 g/L and about 14.0 g/L, between about 10.5 g/L and about 13.5 g/L, between about 10.5 g/L and about 13.0 g/L, between about 10.5 g/L and about 12.5 g/L, between about 10.5 g/L and about 12.0 g/L, between about 10.5 g/L and about 11.5 g/L, between about 11.0 g/L and about 16.0 g/L, between about 11.0 g/L and about 15.5 g/L, between about 11.0 g/L and about 15.0 g/L, between about 11.0 g/L and about 14.5 g/L, between about 11.0 g/L and about 14.0 g/L, between about 11.0 g/L and about 13.5 g/L, between about 11.0 g/L and about 13.0 g/L, between about 11.0 g/L and about 12.5 g/L, between about 11.0 g/L and about 12.0 g/L, between about 11.5 g/L and about 16.0 g/L, between about 11.5 g/L and about 15.5 g/L, between about 11.5 g/L and about 15.0 g/L, between about 11.5 g/L and about 14.5 g/L, between about 11.5 g/L and about 14.0 g/L, between about 11.5 g/L and about 13.5 g/L, between about 11.5 g/L and about 13.0 g/L, between about 11.5 g/L and about 12.5 g/L, between about 12.0 g/L and about 16.0 g/L, between about 12.0 g/L and about 15.5 g/L, between about 12.0 g/L and about 15.0 g/L, between about 12.0 g/L and about 14.5 g/L, between about 12.0 g/L and about 14.0 g/L, between about 12.0 g/L and about 13.5 g/L, between about 12.0 g/L and about 13.0 g/L, between about 12.5 g/L and about 16.0 g/L, between about 12.5 g/L and about 15.5 g/L, between about 12.5 g/L and about 15.0 g/L, between about 12.5 g/L and about 14.5 g/L, between about 12.5 g/L and about 14.0 g/L, between about 12.5 g/L and about 13.5 g/L, between about 13.0 g/L and about 16.0 g/L, between about 13.0 g/L and about 15.5 g/L, between about 13.0 g/L and about 15.0 g/L, between about 13.0 g/L and about 14.5 g/L, between about 13.0 g/L and about 14.0 g/L, between about 13.5 g/L and about 16.0 g/L, between about 13.5 g/L and about 15.5 g/L, between about 13.5 g/L and about 15.0 g/L, between about 13.5 g/L and about 14.5 g/L, between about 14.0 g/L and about 16.0 g/L, between about 14.0 g/L and about 15.5 g/L, between about 14.0 g/L and about 15.0 g/L, between about 14.5 g/L and about 16.0 g/L, between about 14.5 g/L and about 15.5 g/L, or between about 15.0 g/L and about 16.0 g/L).
(64) In some embodiments, the poloxamer-188 is present in the medium and/or added to the medium prior to the culturing step and/or is added to the medium during the culturing step. For example, the methods provided herein can further include adding poloxamer-188 prior to and/or during the culturing step to provide a concentration of 1.8 g/L or more (e.g., any of the exemplary concentrations of poloxamer-188 or ranges of poloxamer-188 described herein) in the medium. In some examples, additional poloxamer-188 is added to a liquid medium including some poloxamer-188 in order to provide a concentration of 1.8 g/L or more poloxamer-188 (e.g., any of the exemplary concentrations of poloxamer-188 or ranges of poloxamer-188 described herein). For example, poloxamer-188 can be added to a first liquid medium, a second liquid medium, and/or a feed liquid medium prior to and/or during the culturing step.
(65) In some examples, the poloxamer-188 is added to a medium in one or more (e.g., two, three, four, or five) aliquots during the culturing step. The poloxamer-188 can be added to the medium in a continuous manner. In some examples, the first liquid medium can contain poloxamer-188, and additional poloxamer-188 is added to the medium during the culturing period. In some embodiments, the poloxamer-188 is present in the medium.
(66) When the culturing step includes perfusion culturing, the second liquid medium can include poloxamer-188 at a greater concentration than that in the first liquid medium. For example, the first liquid medium can include poloxamer-188 at a concentration of 1.8 g/L or at a concentration or less than 1.8 g/L, and the second liquid medium includes poloxamer-188 at a greater concentration than 1.8 g/L (e.g., any of the concentrations or ranges of poloxamer-188 described herein, e.g., a greater concentration than 1.9 g/L, a greater concentration than 2.0 g/L, a greater concentration than 2.1 g/L, a greater concentration than 2.2 g/L, a greater concentration than 2.3 g/L, a greater concentration than 2.4 g/L, a greater concentration than 2.5 g/L, a greater concentration than 2.6 g/L, a greater concentration than 2.7 g/L, a greater concentration than 2.8 g/L, a greater concentration than 2.9 g/L, a greater concentration than 3.0 g/L, a greater concentration than 3.1 g/L, a greater concentration than 3.2 g/L, a greater concentration than 3.3 g/L, a greater concentration than 3.4 g/L, a greater concentration than 3.5 g/L, a greater concentration than 3.6 g/L, a greater concentration than 3.7 g/L, a greater concentration than 3.8 g/L, a greater concentration than 3.9 g/L, a greater concentration than 4.0 g/L, a greater concentration than 4.1 g/L, a greater concentration than 4.2 g/L, a greater concentration than 4.3 g/L, a greater concentration than 4.4 g/L, a greater concentration than 4.5 g/L, a greater concentration than 4.6 g/L, a greater concentration than 4.7 g/L, a greater concentration than 4.8 g/L, a greater concentration than 4.9 g/L, a greater concentration than 5.0 g/L, a greater concentration than 5.1 g/L, a greater concentration than 5.2 g/L, a greater concentration than 5.3 g/L, a greater concentration than 5.4 g/L, a greater concentration than 5.5 g/L, a greater concentration than 5.6 g/L, a greater concentration than 5.7 g/L, a greater concentration than 5.8 g/L, a greater concentration than 5.9 g/L, a greater concentration than 6.0 g/L, a greater concentration than 6.1 g/L, a greater concentration than 6.2 g/L, a greater concentration than 6.3 g/L, a greater concentration than 6.4 g/L, a greater concentration than 6.5 g/L, a greater concentration than 6.6 g/L, a greater concentration than 6.7 g/L, a greater concentration than 6.8 g/L, a greater concentration than 6.9 g/L, a greater concentration than 7.0 g/L, a greater concentration than 7.1 g/L, a greater concentration than 7.2 g/L, a greater concentration than 7.3 g/L, a greater concentration than 7.4 g/L, a greater concentration than 7.5 g/L, a greater concentration than 7.6 g/L, a greater concentration than 7.7 g/L, a greater concentration than 7.8 g/L, a greater concentration than 7.9 g/L, a greater concentration than 8.0 g/L, a greater concentration than 8.1 g/L, a greater concentration than 8.2 g/L, a greater concentration than 8.3 g/L, a greater concentration than 8.4 g/L, a greater concentration than 8.5 g/L, a greater concentration than 8.6 g/L, a greater concentration than 8.7 g/L, a greater concentration than 8.8 g/L, a greater concentration than 8.9 g/L, a greater concentration than 9.0 g/L, a greater concentration than 9.1 g/L, a greater concentration than 9.2 g/L, a greater concentration than 9.3 g/L, a greater concentration than 9.4 g/L, a greater concentration than 9.5 g/L, a greater concentration than 9.6 g/L, a greater concentration than 9.7 g/L, a greater concentration than 9.8 g/L, a greater concentration than 9.9 g/L, a greater concentration than 10.0 g/L, a greater concentration than 10.1 g/L, a greater concentration than 10.2 g/L, a greater concentration than 10.3 g/L, a greater concentration than 10.4 g/L, a greater concentration than 10.5 g/L, a greater concentration than 10.6 g/L, a greater concentration than 10.7 g/L, a greater concentration than 10.8 g/L, a greater concentration than 10.9 g/L, a greater concentration than 11.0 g/L, a greater concentration than 11.1 g/L. a greater concentration than 11.2 g/L, a greater concentration than 11.3 g/L, a greater concentration than 11.4 g/L, a greater concentration than 11.5 g/L, a greater concentration than 11.6 g/L, a greater concentration than 11.7 g/L, a greater concentration than 11.8 g/L, a greater concentration than 11.9 g/L, a greater concentration than 12.0 g/L, a greater concentration than 12.1 g/L, a greater concentration than 12.2 g/L, a greater concentration than 12.3 g/L, a greater concentration than 12.4 g/L, a greater concentration than 12.5 g/L, a greater concentration than 12.6 g/L, a greater concentration than 12.7 g/L, a greater concentration than 12.8 g/L, a greater concentration than 12.9 g/L, a greater concentration than 13.0 g/L, a greater concentration than 13.1 g/L, a greater concentration than 13.2 g/L, a greater concentration than 13.3 g/L, a greater concentration than 13.4 g/L, a greater concentration than 13.5 g/L, a greater concentration than 13.6 g/L, a greater concentration than 13.7 g/L, a greater concentration than 13.8 g/L, a greater concentration than 13.9 g/L, a greater concentration than 14.0 g/L, a greater concentration than 14.1 g/L, a greater concentration than 14.2 g/L, a greater concentration than 14.3 g/L, a greater concentration than 14.4 g/L, a greater concentration than 14.5 g/L, a greater concentration than 14.6 g/L, a greater concentration than 14.7 g/L, a greater concentration than 14.8 g/L, a greater concentration than 14.9 g/L, a greater concentration than 15.0 g/L, a greater concentration than 15.1 g/L, a greater concentration than 15.2 g/L, a greater concentration than 15.3 g/L, a greater concentration than 15.4 g/L, a greater concentration than 15.5 g/L, a greater concentration than 15.6 g/L, a greater concentration than 15.7 g/L, a greater concentration than 15.8 g/L, a greater concentration than 15.9 g/L, or a greater concentration than 16.0 g/L poloxamer-188).
(67) When the culturing step includes feed batch culturing, the feed liquid medium can include poloxamer-188 at a greater concentration than that in the first liquid medium. For example, the first liquid medium can include poloxamer-188 at a concentration of 1.8 g/L or at a concentration of less than 1.8 g/L, and the feed liquid medium includes poloxamer-188 at a greater concentration than 1.8 g/L (e.g., a greater concentration than 2.0 g/L, a greater concentration than 2.5 g/L, a greater concentration than 3.0 g/L, a greater concentration than 3.5 g/L, a greater concentration than 4.0 g/L, a greater concentration than 4.5 g/L, a greater concentration than 5.0 g/L, a greater concentration than 5.5 g/L, a greater concentration than 6.0 g/L, a greater concentration than 6.5 g/L, a greater concentration than 7.0 g/L, a greater concentration than 7.5 g/L, a greater concentration than 8.0 g/L, a greater concentration than 8.5 g/L, a greater concentration than 9.0 g/L, a greater concentration than 9.5 g/L, a greater concentration than 10 g/L, a greater concentration than 10.5 g/L, a greater concentration than 11.0 g/L, a greater concentration than 11.5 g/L, a greater concentration than 12.0 g/L, a greater concentration than 12.5 g/L, a greater concentration than 13.0 g/L, a greater concentration than 13.5 g/L, a greater concentration than 14.0 g/L, a greater concentration than 14.5 g/L, a greater concentration than 15.0 g/L, a greater concentration than 15.5 g/L, a greater concentration than 16.0 g/L, a greater concentration than 16.5 g/L, a greater concentration than 17.0 g/L, a greater concentration than 17.5 g/L, a greater concentration than 18.0 g/L, a greater concentration than 18.5 g/L, a greater concentration than 19.0 g/L, a greater concentration than 19.5 g/L, a greater concentration than 20.0 g/L, a greater concentration than 20.5 g/L, a greater concentration than 21.0 g/L, a greater concentration than 21.5 g/L, a greater concentration than 22.0 g/L, a greater concentration than 22.5 g/L, a greater concentration than 23.0 g/L, a greater concentration than 23.5 g/L, a greater concentration than 24.0 g/L, a greater concentration than 24.5 g/L, a greater concentration than 25.0 g/L, a greater concentration than 25.5 g/L, a greater concentration than 26.0 g/L, a greater concentration than 26.5 g/L, a greater concentration than 27.0 g/L, a greater concentration than 28.0 g/L, a greater concentration than 28.5 g/L, a greater concentration than 29.0 g/L, a greater concentration than 29.5 g/L, a greater concentration than 30.0 g/L, a greater concentration than 30.5 g/L, a greater concentration than 31.0 g/L, a greater concentration than 31.5 g/L, a greater concentration than 32.0 g/L, a greater concentration than 32.5 g/L, a greater concentration than 33.0 g/L, a greater concentration than 33.5 g/L, a greater concentration than 34.0 g/L, a greater concentration than 34.5 g/L, a greater concentration than 35.0 g/L, a greater concentration than 35.5 g/L, a greater concentration than 36.0 g/L, a greater concentration than 36.5 g/L, a greater concentration than 37.0 g/L, a greater concentration than 37.5 g/L, a greater concentration than 38.0 g/L, a greater concentration than 38.5 g/L, a greater concentration than 39.0 g/L, a greater concentration than 39.5 g/L. or a greater concentration than 40.0 g/L).
(68) Some embodiments of any of the methods provided herein include increasing the poloxamer-188 concentration in the culture over time. In some embodiments, the medium includes a poloxamer-188 concentration that is selected based on one or more factors selected from the group of: pore size, pore type, gas flow rate, viable cell density in the medium, and markers related to cell stress. In these methods, the selected poloxamer-188 concentration in the medium can be achieved by adding poloxamer-188 to the medium prior to the culturing step and/or by adding poloxamer-188 to the medium during the culturing step. The selected poloxmer-188 concentration can be any of the exemplary concentrations or ranges of poloxamer-188 described herein.
(69) Selecting Concentration of Poloxamer-188 Based on One or More Factors
(70) Some embodiments of the methods described herein include culturing a mammalian cell in a liquid medium including a poloxamer-188 concentration that is selected based on one or more factors selected from the group of: pore size, pore type, gas flow rate, viable cell density in the medium, and markers related to cell stress.
(71) Different examples of pore types are known in the art. Non-limiting examples of pore types are sintered pores and drilled pores.
(72) A variety of gas flow rates suitable for cell culturing are known in the art. Generally, a higher gas flow rate indicates that a higher poloxamer-188 concentration should be selected (as compared to a culturing step using a lower gas flow rate), and a lower gas flow rate indicates that a lower poloxamer-188 concentration should be selected (as compared to a culturing step using a higher gas flow rate).
(73) A variety of non-limiting viable cell densities are described herein. Generally, a higher viable cell density indicates that a higher poloxamer-188 concentration should be selected (as compared to a medium containing a lower viable cell density), and a lower viable cell density indicates that a lower poloxamer-188 concentration should be selected (as compared to a medium containing a higher viable cell density).
(74) Exemplary markers of cell stress that are released by a cell under physiological stress include proteases (e.g., activated caspases), lactate dehydrogenase, genomic DNA (e.g., nucleosomal DNA), cytochrome c, and activated PARP. Non-limiting examples of markers related to cell stress that are produced or have elevated levels in a cell under physiological stress include activated caspases, cytochrome c, activated PARP, and externalized phosphatidylserine. Elevated levels of one or more (e.g., two, three, four, five, six, or seven) of any of these markers of cell stress as compared to a control value(s) indicate that a higher poloxamer-188 concentration should be selected. Level(s) of the one or more markers of cell stress as compared to a control value(s) that are about the same or below a control value(s) indicate lower poloxamer-188 concentration should be selected. In any of the examples describe herein, the control level can be a level in a control cell that is not undergoing apoptosis or is not cultured under conditions that would trigger necrosis. Methods for detecting the level of one or more markers of cell stress are well known in the art. Commercially available kits are available to measure the one or more markers of cell stress.
(75) In any of the embodiments described herein, the selected poloxamer-188 concentration is achieved by adding poloxamer-188 to the medium prior to the culturing step and/or by adding poloxamer-188 to the medium during the culturing step. In some embodiments, the methods include selecting based on pore type and pore size a poloxamer-188 concentration of between about 2.3 g/L and about 3.3 g/L (e.g., between about 2.3 g/L and about 3.2 g/L, between about 2.3 g/L and about 3.1 g/L, between about 2.3 g/L and about 3.0 g/L, between about 2.3 g/L and about 2.9 g/L, between about 2.3 g/L and about 2.8 g/L, between about 2.3 g/L and about 2.7 g/L, between about 2.3 g/L and about 2.6 g/L, between about 2.3 g/L and about 2.5 g/L, between about 2.4 g/L and about 3.3 g/L, between about 2.4 g/L and about 3.2 g/L, between about 2.4 g/L and about 3.1 g/L, between about 2.4 g/L and about 3.0 g/L, between about 2.4 g/L and about 2.9 g/L, between about 2.4 g/L and about 2.8 g/L, between about 2.4 g/L and about 2.7 g/L, between about 2.4 g/L and about 2.6 g/L, between about 2.5 g/L and about 3.3 g/L, between about 2.5 g/L and about 3.2 g/L, between about 2.5 g/L and about 3.1 g/L, between about 2.5 g/L and about 3.0 g/L, between about 2.5 g/L and about 2.9 g/L, between about 2.5 g/L and about 2.8 g/L, between about 2.5 g/L and about 2.7 g/L, between about 2.6 g/L and about 3.3 g/L, between about 2.6 g/L and about 3.2 g/L, between about 2.6 g/L and about 3.1 g/L, between about 2.6 g/L and about 3.0 g/L, between about 2.6 g/L and about 2.9 g/L, between about 2.6 g/L and about 2.8 g/L, between about 2.7 g/L and about 3.3 g/L, between about 2.7 g/L and about 3.2 g/L, between about 2.7 g/L and about 3.1 g/L, between about 2.7 g/L and about 3.0 g/L, between about 2.7 g/L and about 2.9 g/L, between about 2.8 g/L and about 3.3 g/L, between about 2.8 g/L and about 3.2 g/L, between about 2.8 g/L and about 3.1 g/L, between about 2.8 g/L and about 3.0 g/L, between about 2.9 g/L and about 3.3 g/L, between about 2.9 g/L and about 3.2 g/L, between about 2.9 g/L and about 3.1 g/L, between about 3.0 g/L and about 3.3 g/L, between about 3.0 g/L and about 3.2 g/L, or between about 3.1 g/L and about 3.3 g/L), when the pore type is a drilled pore and the pore size is between about 750 m and about 1.5 mm (e.g., between about 750 m to about 1.45 mm, between about 750 m and about 1.40 mm, between about 750 m and about 1.35 mm, between about 750 m and about 1.30 mm, between about 750 m and about 1.25 mm, between about 750 m and about 1.20 mm, between about 750 m and about 1.15 mm, between about 750 m and about 1.10 mm, between about 750 m and about 1.05 mm, between about 750 m and about 1.00 mm, between about 750 m and about 950 m, between about 750 m and about 900 m, between about 750 m and about 850 m, between about 750 m and about 800 m, between about 800 m and about 1.5 mm, between about 800 m and about 1.45 mm, between about 800 m and about 1.40 mm, between about 800 m and about 1.35 mm, between about 800 m and about 1.30 mm, between about 800 m and about 1.25 mm, between about 800 m and about 1.20 mm, between about 800 m and about 1.15 mm, between about 800 m and about 1.10 mm, between about 800 m and about 1.05 mm, between about 800 m and about 1.00 mm, between about 800 m and about 950 m, between about 800 m and about 900 m, between about 800 m and about 850 m, between about 850 m and about 1.5 mm, between about 850 m and about 1.45 mm, between about 850 m and about 1.40 mm, between about 850 m and about 1.35 mm, between about 850 m and about 1.30 mm, between about 850 m and about 1.25 mm, between about 850 m and about 1.20 mm, between about 850 m and about 1.15 mm, between about 850 m and about 1.10 mm, between about 850 m and about 1.05 mm, between about 850 m and about 1.00 mm, between about 850 m and about 950 m, between about 850 m and about 900 m, between about 900 m and about 1.5 mm, between about 900 m and about 1.45 mm, between about 900 m and about 1.40 mm, between about 900 m and about 1.35 mm, between about 900 m and about 1.30 mm, between about 900 m and about 1.25 mm, between about 900 m and about 1.20 mm, between about 900 m and about 1.15 mm, between about 900 m and about 1.10 mm, between about 900 m and about 1.05 mm, between about 900 m and about 1.00 mm, between about 900 m and about 950 m, between about 950 m and about 1.5 mm, between about 950 m and about 1.45 mm, between about 950 m and about 1.40 mm, between about 950 m and about 1.35 mm, between about 950 m and about 1.30 mm, between about 950 m and about 1.25 mm, between about 950 m and about 1.20 mm, between about 950 m and about 1.15 mm, between about 950 m and about 1.10 mm, between about 950 m and about 1.05 mm, between about 950 m and about 1.00 mm, between about 1.0 mm and about 1.5 mm, between about 1.0 mm and about 1.45 mm, between about 1.0 mm and about 1.40 mm, between about 1.0 mm and about 1.35 mm, between about 1.0 mm and about 1.30 mm, between about 1.0 mm and about 1.25 mm, between about 1.0 mm and about 1.20 mm, between about 1.0 mm and about 1.15 mm, between about 1.0 mm and about 1.10 mm, between about 1.0 mm and about 1.05 mm, between about 1.05 mm and about 1.5 mm, between about 1.05 mm and about 1.45 mm, between about 1.05 mm and about 1.40 mm, between about 1.05 mm and about 1.35 mm, between about 1.05 mm and about 1.30 mm, between about 1.05 mm and about 1.25 mm, between about 1.05 mm and about 1.20 mm, between about 1.05 mm and about 1.15 mm, between about 1.05 mm and about 1.10 mm, between about 1.10 mm and about 1.5 mm, between about 1.10 mm and about 1.45 mm, between about 1.10 mm and about 1.40 mm, between about 1.10 mm and about 1.35 mm, between about 1.10 mm and about 1.30 mm, between about 1.10 mm and about 1.25 mm, between about 1.10 mm and about 1.20 mm, between about 1.10 mm and about 1.15 mm, between about 1.15 mm and about 1.5 mm, between about 1.15 mm and about 1.45 mm, between about 1.15 mm and about 1.40 mm, between about 1.15 mm and about 1.35 mm, between about 1.15 mm and about 1.30 mm, between about 1.15 mm and about 1.25 mm, between about 1.15 mm and about 1.20 mm, between about 1.20 mm and about 1.5 mm, between about 1.20 mm and about 1.45 mm, between about 1.20 mm and about 1.40 mm, between about 1.20 mm and about 1.35 mm, between about 1.20 mm and about 1.30 mm, between about 1.20 mm and about 1.25 mm, between about 1.25 mm and about 1.5 mm, between about 1.25 mm and about 1.45 mm, between about 1.25 mm and about 1.40 mm, between about 1.25 mm and about 1.35 mm, between about 1.25 mm and about 1.30 mm, between about 1.30 mm and about 1.5 mm, between about 1.30 mm and about 1.45 mm, between about 1.30 mm and about 1.40 mm, between about 1.30 mm and about 1.35 mm, between about 1.35 mm and about 1.5 mm, between about 1.35 mm and about 1.45 mm, between about 1.35 mm and about 1.40 mm, between about 1.40 mm and about 1.5 mm, between about 1.40 mm and about 1.45 mm, or between about 1.45 mm and about 1.50).
(76) In some embodiments, the methods include selecting based on pore type and pore size a poloxamer-188 concentration of between about 3.3 g/L and about 4.3 g/L (e.g., between about 3.3 g/L and about 4.2 g/L, between about 3.3 g/L and about 4.1 g/L, between about 3.3 g/L and about 4.0 g/L, between about 3.3 g/L and about 3.9 g/L, between about 3.3 g/L and about 3.8 g/L, between about 3.3 g/L and about 3.7 g/L, between about 3.3 g/L and about 3.6 g/L, between about 3.3 g/L and about 3.5 g/L, between about 3.4 g/L and about 4.3 g/L, between about 3.4 g/L and about 4.2 g/L, between about 3.4 g/L and about 4.1 g/L, between about 3.4 g/L and about 4.0 g/L, between about 3.4 g/L and about 3.9 g/L, between about 3.4 g/L and about 3.8 g/L, between about 3.4 g/L and about 3.7 g/L, between about 3.4 g/L and about 3.6 g/L, between about 3.5 g/L and about 4.3 g/L, between about 3.5 g/L and about 4.2 g/L, between about 3.5 g/L and about 4.1 g/L, between about 3.5 g/L and about 4.0 g/L, between about 3.5 g/L and about 3.9 g/L, between about 3.5 g/L and about 3.8 g/L, between about 3.5 g/L and about 3.7 g/L, between about 3.6 g/L and about 4.3 g/L, between about 3.6 g/L and about 4.2 g/L, between about 3.6 g/L and about 4.1 g/L, between about 3.6 g/L and about 4.0 g/L, between about 3.6 g/L and about 3.9 g/L, between about 3.6 g/L and about 3.8 g/L, between about 3.7 g/L and about 4.3 g/L, between about 3.7 g/L and about 4.2 g/L, between about 3.7 g/L and about 4.1 g/L, between about 3.7 g/L and about 4.0 g/L, between about 3.7 g/L and about 3.9 g/L, between about 3.8 g/L and about 4.3 g/L, between about 3.8 g/L and about 4.2 g/L, between about 3.8 g/L and about 4.1 g/L, between about 3.8 g/L and about 4.0 g/L, between about 3.9 g/L and about 4.3 g/L, between about 3.9 g/L and about 4.2 g/L, between about 3.9 g/L and about 4.1 g/L, between about 4.0 g/L and about 4.3 g/L, between about 4.0 g/L and about 4.2 g/L, or between about 4.1 g/L and about 4.3 g/L), when the pore type is a drilled pore and the pore size is between about 250 m and about 750 m (e.g., between about 250 m and about 700 m, between about 250 m and about 650 m, between about 250 m and about 600 m, between about 250 m and about 550 m, between about 250 m and about 500 m, between about 250 m and about 450 m, between about 250 m and about 400 m, between about 250 m and about 350 m, between about 250 m and about 300 m, between about 300 m and about 750 m, between about 300 m and about 700 m, between about 300 m and about 650 m, between about 300 m and about 600 m, between about 300 m and about 550 m, between about 300 m and about 500 m, between about 300 m and about 450 m, between about 300 m and about 400 m, between about 300 m and about 350 m, between about 350 m and about 750 m, between about 350 m and about 700 m, between about 350 m and about 650 m, between about 350 m and about 600 m, between about 350 m and about 550 m, between about 350 m and about 500 m, between about 350 m and about 450 m, between about 350 m and about 400 m, between about 400 m and about 750 m, between about 400 m and about 700 m, between about 400 m and about 650 m, between about 400 m and about 600 m, between about 400 m and about 550 m, between about 400 m and about 500 m, between about 400 m and about 450 m, between about 450 m and about 750 m, between about 450 m and about 700 m, between about 450 m and about 650 m, between about 450 m and about 600 m, between about 450 m and about 550 m, between about 450 m and about 500 m, between about 500 m and about 750 m, between about 500 m and about 700 m, between about 500 m and about 650 m, between about 500 m and about 600 m, between about 500 m and about 550 m, between about 550 m and about 750 m, between about 550 m and about 700 m, between about 550 m and about 650 m, between about 550 m and about 600 m, between about 600 m and about 750 m, between about 600 m and about 700 m, between about 600 m and about 650 m, between about 650 m and about 750 m, between about 650 m and about 700 m, or between about 700 m and 750 m).
(77) The methods can include, e.g., selecting based on pore type and pore size poloxamer-188 at a greater concentration than about 4.3 g/L (e.g., a greater concentration than about 4.4 g/L, a greater concentration than about 4.5 g/L, a greater concentration than about 4.6 g/L, a greater concentration than about 4.7 g/L, a greater concentration than about 4.8 g/L, a greater concentration than about 4.9 g/L, a greater concentration than about 5.0 g/L, a greater concentration than about 5.1 g/L, a greater concentration than about 5.2 g/L, a greater concentration than about 5.3 g/L, a greater concentration than about 5.4 g/L, a greater concentration than about 5.5 g/L, a greater concentration than about 5.6 g/L, a greater concentration than about 5.7 g/L, a greater concentration than about 5.8 g/L, a greater concentration than about 5.9 g/L, a greater concentration than about 6.0 g/L, a greater concentration than about 6.1 g/L, a greater concentration than about 6.2 g/L, a greater concentration than about 6.3 g/L, a greater concentration than about 6.4 g/L, a greater concentration than about 6.5 g/L, a greater concentration than about 6.6 g/L, a greater concentration than about 6.7 g/L, a greater concentration than about 6.8 g/L, a greater concentration than about 6.9 g/L, a greater concentration than about 7.0 g/L, a greater concentration than about 7.1 g/L, a greater concentration than about 7.2 g/L, a greater concentration than about 7.3 g/L, a greater concentration than about 7.4 g/L, a greater concentration than about 7.5 g/L, a greater concentration than about 7.6 g/L, a greater concentration than about 7.7 g/L, a greater concentration than about 7.8 g/L, a greater concentration than about 7.9 g/L, a greater concentration than about 8.0 g/L, a greater concentration than about 8.1 g/L, a greater concentration than about 8.2 g/L, a greater concentration than about 8.2 g/L, a greater concentration than about 8.3 g/L, a greater concentration than about 8.4 g/L, a greater concentration than about 8.5 g/L, a greater concentration than about 8.6 g/L, a greater concentration than about 8.7 g/L, a greater concentration than about 8.8 g/L, a greater concentration than about 8.9 g/L, a greater concentration than about 9.0 g/L, a greater concentration than about 9.1 g/L, a greater concentration than about 9.2 g/L, a greater concentration than about 9.3 g/L, a greater concentration than about 9.4 g/L, a greater concentration than about 9.5 g/L, a greater concentration than about 9.6 g/L, a greater concentration than about 9.7 g/L, a greater concentration than about 9.8 g/L, a greater concentration than about 9.9 g/L, or a greater concentration than about 10.0 g/L), when the pore type is a drilled pore and the pore size is between about 1 nm and about 250 nm (e.g., between about 1 nm and about 200 nm, between about 1 nm and about 150 nm, between about 1 nm and about 100 nm, between about 1 nm and about 50 nm, between about 1 nm and about 25 nm, between about 10 nm and about 250 nm, between about 10 nm and about 200 m, between about 10 nm and about 150 nm, between about 10 nm and about 100 nm, between about 10 nm and about 50 nm, between about 10 nm and about 25 nm, between about 25 nm and about 250 nm, between about 25 nm and about 200 nm, between about 25 nm and about 150 nm, between about 25 nm and about 100 nm, between about 25 nm and about 50 nm, between about 50 nm and about 250 nm, between about 50 nm and about 200 nm, between about 50 nm and about 150 nm, between about 50 nm and about 100 nm, between about 100 nm and about 250 nm, between about 100 nm and about 200 m, between about 100 nm and about 150 nm, between about 150 nm and about 250 m, between about 150 nm and about 200 nm, or between about 200 nm and about 250 nm).
(78) In some examples, the methods include selecting based on pore type and pore size poloxamer-188 at a greater concentration than 4.3 g/L (e.g., a greater concentration than about 4.4 g/L, a greater concentration than about 4.5 g/L, a greater concentration than about 4.6 g/L, a greater concentration than about 4.7 g/L, a greater concentration than about 4.8 g/L, a greater concentration than about 4.9 g/L, a greater concentration than about 5.0 g/L, a greater concentration than about 5.1 g/L, a greater concentration than about 5.2 g/L, a greater concentration than about 5.3 g/L, a greater concentration than about 5.4 g/L, a greater concentration than about 5.5 g/L, a greater concentration than about 5.6 g/L, a greater concentration than about 5.7 g/L, a greater concentration than about 5.8 g/L, a greater concentration than about 5.9 g/L, a greater concentration than about 6.0 g/L, a greater concentration than about 6.1 g/L, a greater concentration than about 6.2 g/L, a greater concentration than about 6.3 g/L, a greater concentration than about 6.4 g/L, a greater concentration than about 6.5 g/L, a greater concentration than about 6.6 g/L, a greater concentration than about 6.7 g/L, a greater concentration than about 6.8 g/L, a greater concentration than about 6.9 g/L, a greater concentration than about 7.0 g/L, a greater concentration than about 7.1 g/L, a greater concentration than about 7.2 g/L, a greater concentration than about 7.3 g/L, a greater concentration than about 7.4 g/L, a greater concentration than about 7.5 g/L, a greater concentration than about 7.6 g/L, a greater concentration than about 7.7 g/L, a greater concentration than about 7.8 g/L, a greater concentration than about 7.9 g/L, a greater concentration than about 8.0 g/L, a greater concentration than about 8.1 g/L, a greater concentration than about 8.2 g/L, a greater concentration than about 8.2 g/L, a greater concentration than about 8.3 g/L, a greater concentration than about 8.4 g/L, a greater concentration than about 8.5 g/L, a greater concentration than about 8.6 g/L, a greater concentration than about 8.7 g/L, a greater concentration than about 8.8 g/L, a greater concentration than about 8.9 g/L, a greater concentration than about 9.0 g/L, a greater concentration than about 9.1 g/L, a greater concentration than about 9.2 g/L, a greater concentration than about 9.3 g/L, a greater concentration than about 9.4 g/L, a greater concentration than about 9.5 g/L, a greater concentration than about 9.6 g/L, a greater concentration than about 9.7 g/L, a greater concentration than about 9.8 g/L, a greater concentration than about 9.9 g/L, or a greater concentration than about 10.0 g/L), when the pore type is a drilled pore and the pore size is between about 160 m and about 190 m (e.g., between about 160 m and about 185 m, between about 160 m and about 180 m, between about 160 m and about 175 m, between about 160 m and about 170 m, between about 160 m and about 165 m, between about 165 m and about 190 m, between about 165 m and about 185 m, between about 165 m and about 180 m, between about 165 m and about 175 m, between about 165 m and about 170 m, between about 170 m and about 190 m, between about 170 m and about 185 m, between about 170 m and about 180 m, between about 170 m and about 175 m, between about 175 m and about 190 m, between about 175 m and about 185 m, between about 175 m and about 180 m, between about 180 m and about 190 m, between about 180 m and about 185 m, or between about 185 m and about 190 m).
(79) In some embodiments, the methods include selecting based on pore type and pore size poloxamer-188 at a concentration of between about 1.8 g/L and about 3.3 g/L (e.g., between about 1.8 g/L and about 3.2 g/L, between about 1.8 g/L and about 3.1 g/L, between about 1.8 g/L and about 3.0 g/L, between about 1.8 g/L and about 2.9 g/L, between about 1.8 g/L and about 2.8 g/L, between about 1.8 g/L and about 2.7 g/L, between about 1.8 g/L and about 2.6 g/L, between about 1.8 g/L and about 2.5 g/L, between about 1.8 g/L and about 2.4 g/L, between about 1.8 g/L and about 2.3 g/L, between about 1.8 g/L and about 2.2 g/L, between about 1.8 g/L and about 2.1 g/L, between about 1.8 g/L and about 2.0 g/L, between about 1.9 g/L and about 3.3 g/L, between about 1.9 g/L and about 3.2 g/L, between about 1.9 g/L and about 3.1 g/L, between about 1.9 g/L and about 3.0 g/L, between about 1.9 g/L and about 2.9 g/L, between about 1.9 g/L and about 2.8 g/L, between about 1.9 g/L and about 2.7 g/L, between about 1.9 g/L and about 2.6 g/L, between about 1.9 g/L and about 2.5 g/L, between about 1.9 g/L and about 2.4 g/L, between about 1.9 g/L and about 2.3 g/L, between about 1.9 g/L and about 2.2 g/L, between about 1.9 g/L and about 2.1 g/L, between about 2.0 g/L and about 3.3 g/L, between about 2.0 g/L and about 3.2 g/L, between about 2.0 g/L and about 3.1 g/L, between about 2.0 g/L and about 3.0 g/L, between about 2.0 g/L and about 2.9 g/L, between about 2.0 g/L and about 2.8 g/L, between about 2.0 g/L and about 2.7 g/L, between about 2.0 g/L and about 2.6 g/L, between about 2.0 g/L and about 2.5 g/L, between about 2.0 g/L and about 2.4 g/L, between about 2.0 g/L and about 2.3 g/L, between about 2.0 g/L and about 2.2 g/L, between about 2.1 g/L and about 3.3 g/L, between about 2.1 g/L and about 3.2 g/L, between about 2.1 g/L and about 3.1 g/L, between about 2.1 g/L and about 3.0 g/L, between about 2.1 g/L and about 2.9 g/L, between about 2.1 g/L and about 2.8 g/L, between about 2.1 g/L and about 2.7 g/L, between about 2.1 g/L and about 2.6 g/L, between about 2.1 g/L and about 2.5 g/L, between about 2.1 g/L and about 2.4 g/L, between about 2.1 g/L and about 2.3 g/L, between about 2.2 g/L and about 3.3 g/L, between about 2.2 g/L and about 3.2 g/L, between about 2.2 g/L and about 3.1 g/L, between about 2.2 g/L and about 3.0 g/L, between about 2.2 g/L and about 2.9 g/L, between about 2.2 g/L and about 2.8 g/L, between about 2.2 g/L and about 2.7 g/L, between about 2.2 g/L and about 2.6 g/L, between about 2.2 g/L and about 2.5 g/L, between about 2.2 g/L and about 2.4 g/L, between about 2.3 g/L and about 3.3 g/L, between about 2.3 g/L and about 3.2 g/L, between about 2.3 g/L and about 3.1 g/L, between about 2.3 g/L and about 3.0 g/L, between about 2.3 g/L and about 2.9 g/L, between about 2.3 g/L and about 2.8 g/L, between about 2.3 g/L and about 2.7 g/L, between about 2.3 g/L and about 2.6 g/L, between about 2.3 g/L and about 2.5 g/L, between about 2.4 g/L and about 3.3 g/L, between about 2.4 g/L and about 3.2 g/L, between about 2.4 g/L and about 3.1 g/L, between about 2.4 g/L and about 3.0 g/L, between about 2.4 g/L and about 2.9 g/L, between about 2.4 g/L and about 2.8 g/L, between about 2.4 g/L and about 2.7 g/L, between about 2.4 g/L and about 2.6 g/L, between about 2.5 g/L and about 3.3 g/L, between about 2.5 g/L and about 3.2 g/L, between about 2.5 g/L and about 3.1 g/L, between about 2.5 g/L and about 3.0 g/L, between about 2.5 g/L and about 2.9 g/L, between about 2.5 g/L and about 2.8 g/L, between about 2.5 g/L and about 2.7 g/L, between about 2.6 g/L and about 3.3 g/L, between about 2.6 g/L and about 3.2 g/L, between about 2.6 g/L and about 3.1 g/L, between about 2.6 g/L and about 3.0 g/L, between about 2.6 g/L and about 2.9 g/L, between about 2.6 g/L and about 2.8 g/L, between about 2.7 g/L and about 3.3 g/L, between about 2.7 g/L and about 3.2 g/L, between about 2.7 g/L and about 3.1 g/L, between about 2.7 g/L and about 3.0 g/L, between about 2.7 g/L and about 2.9 g/L, between about 2.8 g/L and about 3.3 g/L, between about 2.8 g/L and about 3.2 g/L, between about 2.8 g/L and about 3.1 g/L, between about 2.8 g/L and about 3.0 g/L, between about 2.9 g/L and about 3.3 g/L, between about 2.9 g/L and about 3.2 g/L, between about 2.9 g/L and about 3.1 g/L, between about 3.0 g/L and about 3.3 g/L, between about 3.0 g/L and about 3.2 g/L, or between about 3.1 g/L and about 3.3 g/L), when the pore type is a sintered pore and the pore size is greater than 150 m (e.g., greater than 155 m, greater than 160 m, greater than 165 m, greater than 170 m, greater than 175 m, greater than about 180 m, greater than 185 m, greater than about 190 m, greater than about 195 m, greater than about 200 m, greater than about 205 m, greater than about 210 m, greater than about 215 m, greater than about 220 m, greater than about 225 m, greater than about 230 m, greater than about 235 m, greater than about 240 m, greater than about 245 m, greater than about 250 m, greater than about 255 m, greater than about 260 m, greater than about 265 m, greater than about 270 m, greater than about 275 m, greater than about 280 m, greater than about 290 m, greater than about 300 m, greater than about 350 m, greater than about 400 m, greater than about 450 m, or greater than about 500 m).
(80) The methods can include, e.g., selecting based on pore type and pore size poloxamer-188 at a concentration of between about 3.3 g/L and about 4.3 g/L (e.g., between about 3.3 g/L and about 4.2 g/L, between about 3.3 g/L and about 4.1 g/L, between about 3.3 g/L and about 4.0 g/L, between about 3.3 g/L and about 3.9 g/L, between about 3.3 g/L and about 3.8 g/L, between about 3.3 g/L and about 3.7 g/L, between about 3.3 g/L and about 3.6 g/L, between about 3.3 g/L and about 3.5 g/L, between about 3.4 g/L and about 4.3 g/L, between about 3.4 g/L and about 4.2 g/L, between about 3.4 g/L and about 4.1 g/L, between about 3.4 g/L and about 4.0 g/L, between about 3.4 g/L and about 3.9 g/L, between about 3.4 g/L and about 3.8 g/L, between about 3.4 g/L and about 3.7 g/L, between about 3.4 g/L and about 3.6 g/L, between about 3.5 g/L and about 4.3 g/L, between about 3.5 g/L and about 4.2 g/L, between about 3.5 g/L and about 4.1 g/L, between about 3.5 g/L and about 4.0 g/L, between about 3.5 g/L and about 3.9 g/L, between about 3.5 g/L and about 3.8 g/L, between about 3.5 g/L and about 3.7 g/L, between about 3.6 g/L and about 4.3 g/L, between about 3.6 g/L and about 4.2 g/L, between about 3.6 g/L and about 4.1 g/L, between about 3.6 g/L and about 4.0 g/L, between about 3.6 g/L and about 3.9 g/L, between about 3.6 g/L and about 3.8 g/L, between about 3.7 g/L and about 4.3 g/L, between about 3.7 g/L and about 4.2 g/L, between about 3.7 g/L and about 4.1 g/L, between about 3.7 g/L and about 4.0 g/L, between about 3.7 g/L and about 3.9 g/L, between about 3.8 g/L and about 4.3 g/L, between about 3.8 g/L and about 4.2 g/L, between about 3.8 g/L and about 4.1 g/L, between about 3.8 g/L and about 4.0 g/L, between about 3.9 g/L and about 4.3 g/L, between about 3.9 g/L and about 4.2 g/L, between about 3.9 g/L and about 4.1 g/L, between about 4.0 g/L and about 4.3 g/L, between about 4.0 g/L and about 4.2 g/L, or between about 4.1 g/L and about 4.3 g/L), when the pore type is a sintered pore and the pore size is between about 80 m and about 150 m (e.g., between about 80 m and about 145 m, between about 80 m and about 140 m, between about 80 m and about 135 m, between about 80 m and about 130 m, between about 80 m and about 125 m, between about 80 m and about 120 m, between about 80 m and about 115 m, between about 80 m and about 110 m, between about 80 m and about 105 m, between about 80 m and about 100 m, between about 80 m and about 95 m, between about 80 m and about 90 m, between about 80 m and about 85 m, between about 85 m and about 150 m, between about 85 m and about 145 m, between about 85 m and about 140 m, between about 85 m and about 135 m, between about 85 m and about 130 m, between about 85 m and about 125 m, between about 85 m and about 120 m, between about 85 m and about 115 m, between about 85 m and about 110 m, between about 85 m and about 105 m, between about 85 m and about 100 m, between about 85 m and about 95 m, between about 85 m and about 90 m, between about 90 m and about 150 m, between about 90 m and about 145 m, between about 90 m and about 140 m, between about 90 m and about 135 m, between about 90 m and about 130 m, between about 90 m and about 125 m, between about 90 m and about 120 m, between about 90 m and about 115 m, between about 90 m and about 110 m, between about 90 m and about 105 m, between about 90 m and about 100 m, between about 90 m and about 95 m, between about 95 m and about 150 m, between about 95 m and about 145 m, between about 95 m and about 140 m, between about 95 m and about 135 m, between about 95 m and about 130 m, between about 95 m and about 125 m, between about 95 m and about 120 m, between about 95 m and about 115 m, between about 95 m and about 110 m, between about 95 m and about 105 m, between about 95 m and about 100 m, between about 100 m and about 150 m, between about 100 m and about 145 m, between about 100 m and about 140 m, between about 100 m and about 135 m, between about 100 m and about 130 m, between about 100 m and about 125 m, between about 100 m and about 120 m, between about 100 m and about 115 m, between about 100 m and about 110 m, between about 100 m and about 105 m, between about 105 m and about 150 m, between about 105 m and about 145 m, between about 105 m and about 140 m, between about 105 m and about 135 m, between about 105 m and about 130 m, between about 105 m and about 125 m, between about 105 m and about 120 m, between about 105 m and about 115 m, between about 105 m and about 110 m, between about 110 m and about 150 m, between about 110 m and about 145 m, between about 110 m and about 140 m, between about 110 m and about 135 m, between about 110 m and about 130 m, between about 110 m and about 125 m, between about 110 m and about 120 m, between about 110 m and about 115 m, between about 115 m and about 150 m, between about 115 m and about 145 m, between about 115 m and about 140 m, between about 115 m and about 135 m, between about 115 m and about 130 m, between about 115 nm and about 125 nm, between about 115 nm and about 120 nm, between about 120 nm and about 150 nm, between about 120 nm and about 145 nm, between about 120 nm and about 140 m, between about 120 nm and about 135 nm, between about 120 nm and about 130 m, between about 120 nm and about 125 nm, between about 125 nm and about 150 m, between about 125 nm and about 145 nm, between about 125 nm and about 140 nm, between about 125 nm and about 135 nm, between about 125 nm and about 130 m, between about 130 nm and about 150 nm, between about 130 nm and about 145 m, between about 130 nm and about 140 nm, between about 130 nm and about 135 m, between about 135 nm and about 150 nm, between about 135 nm and about 145 m, between about 135 nm and about 140 nm, between about 140 nm and about 150 m, between about 140 nm and about 145 nm, or between about 145 nm and about 150 nm).
(81) The methods can include, e.g., selecting based on pore type and pore size poloxamer-188 at a concentration of about 4.5 g/L or at a greater concentration than about 4.5 g/L (e.g., at a concentration of about 4.6 g/L or at a concentration of greater than 4.6 g/L, at a concentration of about 4.7 g/L or at a concentration of greater than 4.7 g/L, at a concentration of about 4.8 g/L or at a concentration of greater than 4.8 g/L, at a concentration of about 4.9 g/L or at a concentration of greater than 4.9 g/L, at a concentration of about 5.0 g/L or at a concentration of greater than 5.0 g/L, at a concentration of about 5.1 g/L or at a concentration of greater than 5.1 g/L, at a concentration of about 5.2 g/L or at a concentration of greater than 5.2 g/L, at a concentration of about 5.3 g/L or at a concentration of greater than 5.3 g/L, at a concentration of about 5.4 g/L or at a concentration of greater than 5.4 g/L, at a concentration of about 5.5 g/L or at a concentration of greater than 5.5 g/L, at a concentration of about 5.6 g/L or at a concentration of greater than 5.6 g/L, at a concentration of about 5.7 g/L or at a concentration of greater than 5.7 g/L, at a concentration of about 5.8 g/L or at a concentration of greater than 5.8 g/L, at a concentration of about 6.0 g/L or at a concentration of greater than 6.0 g/L, at a concentration of about 6.1 g/L or at a concentration of greater than 6.1 g/L, at a concentration of about 6.2 g/L or at a concentration of greater than 6.2 g/L, at a concentration of about 6.3 g/L or at a concentration of greater than 6.3 g/L, at a concentration of about 6.4 g/L or at a concentration of greater than 6.4 g/L, at a concentration of about 6.5 g/L or at a concentration of greater than 6.5 g/L, at a concentration of about 6.6 g/L or at a concentration of greater than 6.6 g/L, at a concentration of about 6.7 g/L or at a concentration of greater than 6.7 g/L, at a concentration of about 6.8 g/L or at a concentration of greater than 6.8 g/L, at a concentration of about 6.9 g/L or at a concentration of greater than 6.9 g/L, at a concentration of about 7.0 g/L or at a concentration of greater than 7.0 g/L, at a concentration of about 7.1 g/L or at a concentration of greater than 7.1 g/L, at a concentration of about 7.2 g/L or at a concentration of greater than 7.2 g/L, at a concentration of about 7.3 g/L or at a concentration of greater than 7.3 g/L, at a concentration of about 7.4 g/L or at a concentration of greater than 7.4 g/L, at a concentration of about 7.5 g/L or at a concentration of greater than 7.5 g/L, at a concentration of about 7.6 g/L or at a concentration of greater than 7.6 g/L, at a concentration of about 7.7 g/L or at a concentration of greater than 7.7 g/L, at a concentration of about 7.8 g/L or at a concentration of greater than 7.8 g/L, at a concentration of about 7.9 g/L or at a concentration of greater than 7.9 g/L, at a concentration of about 8.0 g/L or at a concentration of greater than 8.0 g/L, at a concentration of about 8.1 g/L or at a concentration of greater than 8.1 g/L, at a concentration of about 8.2 g/L or at a concentration of greater than 8.2 g/L, at a concentration of about 8.3 g/L or at a concentration of greater than 8.3 g/L, at a concentration of about 8.4 g/L or at a concentration of greater than 8.4 g/L, at a concentration of about 8.5 g/L or at a concentration of greater than 8.5 g/L, at a concentration of about 8.6 g/L or at a concentration of greater than 8.6 g/L, at a concentration of about 8.7 g/L or at a concentration of greater than 8.7 g/L, at a concentration of about 8.8 g/L or at a concentration of greater than 8.8 g/L, at a concentration of about 8.9 g/L or at a concentration of greater than 8.9 g/L, at a concentration of about 9.0 g/L or at a concentration of greater than 9.0 g/L, at a concentration of about 9.1 g/L or at a concentration of greater than 9.1 g/L, at a concentration of about 9.2 g/L or at a concentration of greater than 9.2 g/L, at a concentration of about 9.3 g/L or at a concentration of greater than 9.3 g/L, at a concentration of about 9.4 g/L or at a concentration of greater than 9.4 g/L, at a concentration of about 9.5 g/L or at a concentration of greater than 9.5 g/L, at a concentration of about 9.6 g/L or at a concentration of greater than 9.6 g/L, at a concentration of about 9.7 g/L or at a concentration of greater than 9.7 g/L, at a concentration of about 9.8 g/L or at a concentration of greater than 9.8 g/L, at a concentration of about 9.9 g/L or at a concentration of greater than 9.9 g/L, or at a concentration of about 10.0 g/L or at a concentration of greater than 10.0 g/L), when the pore type is a sintered pore and the pore size is between about 1 m and about 80 m (e.g., between about 1 m and about 50 m, between about 1 m and about 25 m, between about 1 m and about 10 m, between about 1 m and about 5 m, between about 5 m and about 80 m, between about 5 m and about 50 m, between about 5 m and about 25 m, between about 5 m and about 10 m, between about 10 m and about 80 m, between about 10 m and about 50 m, between about 10 m and about 25 m, between about 25 m and about 80 m, between about 25 m and about 50 m, or between about 50 m and about 80 m).
(82) When the culturing is perfusion culturing, the second liquid medium can include the selected poloxamer-188 concentration.
(83) Increasing Poloxamer-188 Concentration Based on Viable Cell Density
(84) Some embodiments of any of the methods described herein include increasing the poloxamer-188 concentration in the medium over time based on the viable cell density in the medium. For example, any of the methods described herein can include increasing the poloxamer-188 concentration in the medium to greater than 1.8 g/L (e.g., between about 1.8 g/L and about 3.0 g/L, between about 1.8 g/L and about 2.9 g/L, between about 1.8 g/L and about 2.8 g/L, between about 1.8 g/L and about 2.7 g/L, between about 1.8 g/L and about 2.5 g/L, between about 1.8 g/L and about 2.4 g/L, between about 1.8 g/L and about 2.3 g/L, between about 1.8 g/L and about 2.2 g/L, between about 1.8 g/L and about 2.1 g/L, between about 1.8 g/L and about 2.0 g/L, between about 1.9 g/L and about 3.0 g/L, between about 1.9 g/L and about 2.9 g/L, between about 1.9 g/L and about 2.8 g/L, between about 1.9 g/L and about 2.7 g/L, between about 1.9 g/L and about 2.6 g/L, between about 1.9 g/L and about 2.5 g/L, between about 1.9 g/L and about 2.4 g/L, between about 1.9 g/L and about 2.3 g/L, between about 1.9 g/L and about 2.2 g/L, between about 1.9 g/L and about 2.1 g/L, between about 2.0 g/L and about 3.0 g/L, between about 2.0 g/L and about 2.9 g/L, between about 2.0 g/L and about 2.8 g/L, between about 2.0 g/L and about 2.7 g/L, between about 2.0 g/L and about 2.6 g/L, between about 2.0 g/L and about 2.5 g/L, between about 2.0 g/L and about 2.4 g/L, between about 2.0 g/L and about 2.3 g/L, between about 2.0 g/L and about 2.2 g/L, between about 2.1 g/L and about 3.0 g/L, between about 2.1 g/L and about 2.9 g/L, between about 2.1 g/L and about 2.8 g/L, between about 2.1 g/L and about 2.7 g/L, between about 2.1 g/L and about 2.6 g/L, between about 2.1 g/L and about 2.5 g/L, between about 2.1 g/L and about 2.4 g/L, between about 2.1 g/L and about 2.3 g/L, between about 2.2 g/L and about 3.0 g/L, between about 2.2 g/L and about 2.9 g/L, between about 2.2 g/L and about 2.8 g/L, between about 2.2 g/L and about 2.7 g/L, between about 2.2 g/L and about 2.6 g/L, between about 2.2 g/L and about 2.5 g/L, between about 2.2 g/L and about 2.4 g/L, between about 2.3 g/L and about 3.0 g/L, between about 2.3 g/L and about 2.9 g/L, between about 2.3 g/L and about 2.8 g/L, between about 2.3 g/L and about 2.7 g/L, between about 2.3 g/L and about 2.6 g/L, between about 2.3 g/L and about 2.5 g/L, between about 2.4 g/L and about 3.0 g/L, between about 2.4 g/L and about 2.9 g/L, between about 2.4 g/L and about 2.8 g/L, between about 2.4 g/L and about 2.7 g/L, between about 2.4 g/L and about 2.6 g/L, between about 2.5 g/L and about 3.0 g/L, between about 2.5 g/L and about 2.9 g/L, between about 2.5 g/L and about 2.8 g/L, between about 2.5 g/L and about 2.7 g/L, between about 2.6 g/L and about 3.0 g/L, between about 2.6 g/L and about 2.9 g/L, between about 2.6 g/L and about 2.8 g/L, between about 2.7 g/L and about 3.0 g/L, between about 2.7 g/L and about 2.9 g/L, or between about 2.8 g/L and about 3.0 g/L) when the viable cell density in the medium is between about 2010.sup.6 cells/mL to about 6010.sup.6 cells/mL (e.g., between about 2010.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 2010.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 2010.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 2010.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 2010.sup.6 cells/mL and about 5010.sup.6 cells/mL, between about 2010.sup.6 cells/mL and about 4810.sup.6 cells/mL, between about 2010.sup.6 cells/mL and about 4610.sup.6 cells/mL, between about 2010.sup.6 cells/mL and about 4410.sup.6 cells/mL, between about 2010.sup.6 cells/mL and about 4210.sup.6 cells/mL, between about 2010.sup.6 cells/mL to about 4010.sup.6 cells/mL, between about 2010.sup.6 cells/mL and about 3810.sup.6 cells/mL, between about 2010.sup.6 cells/mL and about 3610.sup.6 cells/mL, between about 2010.sup.6 cells/mL and about 3410.sup.6 cells/mL, between about 2010.sup.6 cells/mL and about 3210.sup.6 cells/mL, between about 2010.sup.6 cells/mL and about 3010.sup.6 cells/mL, between about 2010.sup.6 cells/mL to about 2810.sup.6 cells/mL, between about 2010.sup.6 cells/mL and about 2610.sup.6 cells/mL, between about 2010.sup.6 cells/mL and about 2410.sup.6 cells/mL, between about 2010.sup.6 cells/mL and about 2210.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 2210.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 5010.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 4810.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 4610.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 4410.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 4210.sup.6 cells/mL, between about 2210.sup.6 cells/mL to about 4010.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 3810.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 3610.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 3410.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 3210.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 3010.sup.6 cells/mL, between about 2210.sup.6 cells/mL to about 2810.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 2610.sup.6 cells/mL, between about 2210.sup.6 cells/mL and about 2410.sup.6 cells/mL, between about 2410.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 2410.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 2410.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 2410.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 2410.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 2410.sup.6 cells/mL and about 5010.sup.6 cells/mL, between about 2410.sup.6 cells/mL and about 4810.sup.6 cells/mL, between about 2410.sup.6 cells/mL and about 4610.sup.6 cells/mL, between about 2410.sup.6 cells/mL and about 4410.sup.6 cells/mL, between about 2410.sup.6 cells/mL and about 4210.sup.6 cells/mL, between about 2410.sup.6 cells/mL to about 4010.sup.6 cells/mL, between about 2410.sup.6 cells/mL and about 3810.sup.6 cells/mL, between about 2410.sup.6 cells/mL and about 3610.sup.6 cells/mL, between about 2410.sup.6 cells/mL and about 3410.sup.6 cells/mL, between about 2410.sup.6 cells/mL and about 3210.sup.6 cells/mL, between about 2410.sup.6 cells/mL and about 3010.sup.6 cells/mL, between about 2410.sup.6 cells/mL to about 2810.sup.6 cells/mL, between about 2410.sup.6 cells/mL and about 2610.sup.6 cells/mL, between about 2610.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 2610.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 2610.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 2610.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 2610.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 2610.sup.6 cells/mL and about 5010.sup.6 cells/mL, between about 2610.sup.6 cells/mL and about 4810.sup.6 cells/mL, between about 2610.sup.6 cells/mL and about 4610.sup.6 cells/mL, between about 2610.sup.6 cells/mL and about 4410.sup.6 cells/mL, between about 2610.sup.6 cells/mL and about 4210.sup.6 cells/mL, between about 2610.sup.6 cells/mL to about 4010.sup.6 cells/mL, between about 2610.sup.6 cells/mL and about 3810.sup.6 cells/mL, between about 2610.sup.6 cells/mL and about 3610.sup.6 cells/mL, between about 2610.sup.6 cells/mL and about 3410.sup.6 cells/mL, between about 2610.sup.6 cells/mL and about 3210.sup.6 cells/mL, between about 2610.sup.6 cells/mL and about 3010.sup.6 cells/mL, between about 2610.sup.6 cells/mL to about 2810.sup.6 cells/mL, between about 2810.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 2810.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 2810.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 2810.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 2810.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 2810.sup.6 cells/mL and about 5010.sup.6 cells/mL, between about 2810.sup.6 cells/mL and about 4810.sup.6 cells/mL, between about 2810.sup.6 cells/mL and about 4610.sup.6 cells/mL, between about 2810.sup.6 cells/mL and about 4410.sup.6 cells/mL, between about 2810.sup.6 cells/mL and about 4210.sup.6 cells/mL, between about 2810.sup.6 cells/mL to about 4010.sup.6 cells/mL, between about 2810.sup.6 cells/mL and about 3810.sup.6 cells/mL, between about 2810.sup.6 cells/mL and about 3610.sup.6 cells/mL, between about 2810.sup.6 cells/mL and about 3410.sup.6 cells/mL, between about 2810.sup.6 cells/mL and about 3210.sup.6 cells/mL, between about 2810.sup.6 cells/mL and about 3010.sup.6 cells/mL, between about 3010.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 3010.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 3010.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 3010.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 3010.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 3010.sup.6 cells/mL and about 5010.sup.6 cells/mL, between about 3010.sup.6 cells/mL and about 4810.sup.6 cells/mL, between about 3010.sup.6 cells/mL and about 4610.sup.6 cells/mL, between about 3010.sup.6 cells/mL and about 4410.sup.6 cells/mL, between about 3010.sup.6 cells/mL and about 4210.sup.6 cells/mL, between about 3010.sup.6 cells/mL to about 4010.sup.6 cells/mL, between about 3010.sup.6 cells/mL and about 3810.sup.6 cells/mL, between about 3010.sup.6 cells/mL and about 3610.sup.6 cells/mL, between about 3010.sup.6 cells/mL and about 3410.sup.6 cells/mL, between about 3010.sup.6 cells/mL and about 3210.sup.6 cells/mL, between about 3210.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 3210.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 3210.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 3210.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 3210.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 3210.sup.6 cells/mL and about 5010.sup.6 cells/mL, between about 3210.sup.6 cells/mL and about 4810.sup.6 cells/mL, between about 3210.sup.6 cells/mL and about 4610.sup.6 cells/mL, between about 3210.sup.6 cells/mL and about 4410.sup.6 cells/mL, between about 3210.sup.6 cells/mL and about 4210.sup.6 cells/mL, between about 3210.sup.6 cells/mL to about 4010.sup.6 cells/mL, between about 3210.sup.6 cells/mL and about 3810.sup.6 cells/mL, between about 3210.sup.6 cells/mL and about 3610.sup.6 cells/mL, between about 3210.sup.6 cells/mL and about 3410.sup.6 cells/mL, between about 3410.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 3410.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 3410.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 3410.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 3410.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 3410.sup.6 cells/mL and about 5010.sup.6 cells/mL, between about 3410.sup.6 cells/mL and about 4810.sup.6 cells/mL, between about 3410.sup.6 cells/mL and about 4610.sup.6 cells/mL, between about 3410.sup.6 cells/mL and about 4410.sup.6 cells/mL, between about 3410.sup.6 cells/mL and about 4210.sup.6 cells/mL, between about 3410.sup.6 cells/mL to about 4010.sup.6 cells/mL, between about 3410.sup.6 cells/mL and about 3810.sup.6 cells/mL, between about 3410.sup.6 cells/mL and about 3610.sup.6 cells/mL, between about 3610.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 3610.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 3610.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 3610.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 3610.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 3610.sup.6 cells/mL and about 5010.sup.6 cells/mL, between about 3610.sup.6 cells/mL and about 4810.sup.6 cells/mL, between about 3610.sup.6 cells/mL and about 4610.sup.6 cells/mL, between about 3610.sup.6 cells/mL and about 4410.sup.6 cells/mL, between about 3610.sup.6 cells/mL and about 4210.sup.6 cells/mL, between about 3610.sup.6 cells/mL to about 4010.sup.6 cells/mL, between about 3610.sup.6 cells/mL and about 3810.sup.6 cells/mL, between about 3810.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 3810.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 3810.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 3810.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 3810.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 3810.sup.6 cells/mL and about 5010.sup.6 cells/mL, between about 3810.sup.6 cells/mL and about 4810.sup.6 cells/mL, between about 3810.sup.6 cells/mL and about 4610.sup.6 cells/mL, between about 3810.sup.6 cells/mL and about 4410.sup.6 cells/mL, between about 3810.sup.6 cells/mL and about 4210.sup.6 cells/mL, between about 3810.sup.6 cells/mL to about 4010.sup.6 cells/mL, between about 4010.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 4010.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 4010.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 4010.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 4010.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 4010.sup.6 cells/mL and about 5010.sup.6 cells/mL, between about 4010.sup.6 cells/mL and about 4810.sup.6 cells/mL, between about 4010.sup.6 cells/mL and about 4610.sup.6 cells/mL, between about 4010.sup.6 cells/mL and about 4410.sup.6 cells/mL, between about 4010.sup.6 cells/mL and about 4210.sup.6 cells/mL, between about 4210.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 4210.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 4210.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 4210.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 4210.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 4210.sup.6 cells/mL and about 5010.sup.6 cells/mL, between about 4210.sup.6 cells/mL and about 4810.sup.6 cells/mL, between about 4210.sup.6 cells/mL and about 4610.sup.6 cells/mL, between about 4210.sup.6 cells/mL and about 4410.sup.6 cells/mL, between about 4410.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 4410.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 4410.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 4410.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 4410.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 4410.sup.6 cells/mL and about 5010.sup.6 cells/mL, between about 4410.sup.6 cells/mL and about 4810.sup.6 cells/mL, between about 4410.sup.6 cells/mL and about 4610.sup.6 cells/mL, between about 4610.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 4610.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 4610.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 4610.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 4610.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 4610.sup.6 cells/mL and about 5010.sup.6 cells/mL, between about 4610.sup.6 cells/mL and about 4810.sup.6 cells/mL, between about 4810.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 4810.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 4810.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 4810.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 4810.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 4810.sup.6 cells/mL and about 5010.sup.6 cells/mL, between about 5010.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 5010.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 5010.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 5010.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 5010.sup.6 cells/mL and about 5210.sup.6 cells/mL, between about 5210.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 5210.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 5210.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 5210.sup.6 cells/mL and about 5410.sup.6 cells/mL, between about 5410.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 5410.sup.6 cells/mL and about 5810.sup.6 cells/mL, between about 5410.sup.6 cells/mL to about 5610.sup.6 cells/mL, between about 5610.sup.6 cells/mL and about 6010.sup.6 cells/mL, between about 5610.sup.6 cells/mL and about 5810.sup.6 cells/mL, or between about 5810.sup.6 cells/mL and about 6010.sup.6 cells/mL).
(85) Any of the methods described herein can include increasing the poloxamer-188 concentration in the medium to between about 3.0 g/L and about 6.0 g/L (e.g., between about 3.0 g/L and about 5.9 g/L, between about 3.0 g/L and about 5.8 g/L, between about 3.0 g/L and about 5.7 g/L, between about 3.0 g/L and about 5.6 g/L, between about 3.0 g/L and about 5.5 g/L, between about 3.0 g/L and about 5.4 g/L, between about 3.0 g/L and about 5.3 g/L, between about 3.0 g/L and about 5.2 g/L, between about 3.0 g/L and about 5.1 g/L, between about 3.0 g/L and about 5.0 g/L, between about 3.0 g/L and about 4.9 g/L, between about 3.0 g/L and about 4.8 g/L, between about 3.0 g/L and about 4.7 g/L, between about 3.0 g/L and about 4.6 g/L, between about 3.0 g/L and about 4.5 g/L, between about 3.0 g/L and about 4.4 g/L, between about 3.0 g/L and about 4.3 g/L, between about 3.0 g/L and about 4.2 g/L, between about 3.0 g/L and about 4.1 g/L, between about 3.0 g/L and about 4.0 g/L, between about 3.0 g/L and about 3.9 g/L, between about 3.0 g/L and about 3.8 g/L, between about 3.0 g/L and about 3.7 g/L, between about 3.0 g/L and about 3.6 g/L, between about 3.0 g/L and about 3.5 g/L, between about 3.0 g/L and about 3.4 g/L, between about 3.0 g/L and about 3.3 g/L, between about 3.0 g/L and about 3.2 g/L, between about 3.1 g/L and about 6.0 g/L, between about 3.1 g/L and about 5.9 g/L, between about 3.1 g/L and about 5.8 g/L, between about 3.1 g/L and about 5.7 g/L, between about 3.1 g/L and about 5.6 g/L, between about 3.1 g/L and about 5.5 g/L, between about 3.1 g/L and about 5.4 g/L, between about 3.1 g/L and about 5.3 g/L, between about 3.1 g/L and about 5.2 g/L, between about 3.1 g/L and about 5.1 g/L, between about 3.1 g/L and about 5.0 g/L, between about 3.1 g/L and about 4.9 g/L, between about 3.1 g/L and about 4.8 g/L, between about 3.1 g/L and about 4.7 g/L, between about 3.1 g/L and about 4.6 g/L, between about 3.1 g/L and about 4.5 g/L, between about 3.1 g/L and about 4.4 g/L, between about 3.1 g/L and about 4.3 g/L, between about 3.1 g/L and about 4.2 g/L, between about 3.1 g/L and about 4.1 g/L, between about 3.1 g/L and about 4.0 g/L, between about 3.1 g/L and about 3.9 g/L, between about 3.1 g/L and about 3.8 g/L, between about 3.1 g/L and about 3.7 g/L, between about 3.1 g/L and about 3.6 g/L, between about 3.1 g/L and about 3.5 g/L, between about 3.1 g/L and about 3.4 g/L, between about 3.1 g/L and about 3.3 g/L, between about 3.2 g/L and about 6.0 g/L, between about 3.2 g/L and about 5.9 g/L, between about 3.2 g/L and about 5.8 g/L, between about 3.2 g/L and about 5.7 g/L, between about 3.2 g/L and about 5.6 g/L, between about 3.2 g/L and about 5.5 g/L, between about 3.2 g/L and about 5.4 g/L, between about 3.2 g/L and about 5.3 g/L, between about 3.2 g/L and about 5.2 g/L, between about 3.2 g/L and about 5.1 g/L, between about 3.2 g/L and about 5.0 g/L, between about 3.2 g/L and about 4.9 g/L, between about 3.2 g/L and about 4.8 g/L, between about 3.2 g/L and about 4.7 g/L, between about 3.2 g/L and about 4.6 g/L, between about 3.2 g/L and about 4.5 g/L, between about 3.2 g/L and about 4.4 g/L, between about 3.2 g/L and about 4.3 g/L, between about 3.2 g/L and about 4.2 g/L, between about 3.2 g/L and about 4.1 g/L, between about 3.2 g/L and about 4.0 g/L, between about 3.2 g/L and about 3.9 g/L, between about 3.2 g/L and about 3.8 g/L, between about 3.2 g/L and about 3.7 g/L, between about 3.2 g/L and about 3.6 g/L, between about 3.2 g/L and about 3.5 g/L, between about 3.2 g/L and about 3.4 g/L, between about 3.3 g/L and about 6.0 g/L, between about 3.3 g/L and about 5.9 g/L, between about 3.3 g/L and about 5.8 g/L, between about 3.3 g/L and about 5.7 g/L, between about 3.3 g/L and about 5.6 g/L, between about 3.3 g/L and about 5.5 g/L, between about 3.3 g/L and about 5.4 g/L, between about 3.3 g/L and about 5.3 g/L, between about 3.3 g/L and about 5.2 g/L, between about 3.3 g/L and about 5.1 g/L, between about 3.3 g/L and about 5.0 g/L, between about 3.3 g/L and about 4.9 g/L, between about 3.3 g/L and about 4.8 g/L, between about 3.3 g/L and about 4.7 g/L, between about 3.3 g/L and about 4.6 g/L, between about 3.3 g/L and about 4.5 g/L, between about 3.3 g/L and about 4.4 g/L, between about 3.3 g/L and about 4.3 g/L, between about 3.3 g/L and about 4.2 g/L, between about 3.3 g/L and about 4.1 g/L, between about 3.3 g/L and about 4.0 g/L, between about 3.3 g/L and about 3.9 g/L, between about 3.3 g/L and about 3.8 g/L, between about 3.3 g/L and about 3.7 g/L, between about 3.3 g/L and about 3.6 g/L, between about 3.3 g/L and about 3.5 g/L, between about 3.4 g/L and about 6.0 g/L, between about 3.4 g/L and about 5.9 g/L, between about 3.4 g/L and about 5.8 g/L, between about 3.4 g/L and about 5.7 g/L, between about 3.4 g/L and about 5.6 g/L, between about 3.4 g/L and about 5.5 g/L, between about 3.4 g/L and about 5.4 g/L, between about 3.4 g/L and about 5.3 g/L, between about 3.4 g/L and about 5.2 g/L, between about 3.4 g/L and about 5.1 g/L, between about 3.4 g/L and about 5.0 g/L, between about 3.4 g/L and about 4.9 g/L, between about 3.4 g/L and about 4.8 g/L, between about 3.4 g/L and about 4.7 g/L, between about 3.4 g/L and about 4.6 g/L, between about 3.4 g/L and about 4.5 g/L, between about 3.4 g/L and about 4.4 g/L, between about 3.4 g/L and about 4.3 g/L, between about 3.4 g/L and about 4.2 g/L, between about 3.4 g/L and about 4.1 g/L, between about 3.4 g/L and about 4.0 g/L, between about 3.4 g/L and about 3.9 g/L, between about 3.4 g/L and about 3.8 g/L, between about 3.4 g/L and about 3.7 g/L, between about 3.4 g/L and about 3.6 g/L, between about 3.5 g/L and about 6.0 g/L, between about 3.5 g/L and about 5.9 g/L, between about 3.5 g/L and about 5.8 g/L, between about 3.5 g/L and about 5.7 g/L, between about 3.5 g/L and about 5.6 g/L, between about 3.5 g/L and about 5.5 g/L, between about 3.5 g/L and about 5.4 g/L, between about 3.5 g/L and about 5.3 g/L, between about 3.5 g/L and about 5.2 g/L, between about 3.5 g/L and about 5.1 g/L, between about 3.5 g/L and about 5.0 g/L, between about 3.5 g/L and about 4.9 g/L, between about 3.5 g/L and about 4.8 g/L, between about 3.5 g/L and about 4.7 g/L, between about 3.5 g/L and about 4.6 g/L, between about 3.5 g/L and about 4.5 g/L, between about 3.5 g/L and about 4.4 g/L, between about 3.5 g/L and about 4.3 g/L, between about 3.5 g/L and about 4.2 g/L, between about 3.5 g/L and about 4.1 g/L, between about 3.5 g/L and about 4.0 g/L, between about 3.5 g/L and about 3.9 g/L, between about 3.5 g/L and about 3.8 g/L, between about 3.5 g/L and about 3.7 g/L, between about 3.6 g/L and about 6.0 g/L, between about 3.6 g/L and about 5.9 g/L, between about 3.6 g/L and about 5.8 g/L, between about 3.6 g/L and about 5.7 g/L, between about 3.6 g/L and about 5.6 g/L, between about 3.6 g/L and about 5.5 g/L, between about 3.6 g/L and about 5.4 g/L, between about 3.6 g/L and about 5.3 g/L, between about 3.6 g/L and about 5.2 g/L, between about 3.6 g/L and about 5.1 g/L, between about 3.6 g/L and about 5.0 g/L, between about 3.6 g/L and about 4.9 g/L, between about 3.6 g/L and about 4.8 g/L, between about 3.6 g/L and about 4.7 g/L, between about 3.6 g/L and about 4.6 g/L, between about 3.6 g/L and about 4.5 g/L, between about 3.6 g/L and about 4.4 g/L, between about 3.6 g/L and about 4.3 g/L, between about 3.6 g/L and about 4.2 g/L, between about 3.6 g/L and about 4.1 g/L, between about 3.6 g/L and about 4.0 g/L, between about 3.6 g/L and about 3.9 g/L, between about 3.6 g/L and about 3.8 g/L, between about 3.7 g/L and about 6.0 g/L, between about 3.7 g/L and about 5.9 g/L, between about 3.7 g/L and about 5.8 g/L, between about 3.7 g/L and about 5.7 g/L, between about 3.7 g/L and about 5.6 g/L, between about 3.7 g/L and about 5.5 g/L, between about 3.7 g/L and about 5.4 g/L, between about 3.7 g/L and about 5.3 g/L, between about 3.7 g/L and about 5.2 g/L, between about 3.7 g/L and about 5.1 g/L, between about 3.7 g/L and about 5.0 g/L, between about 3.7 g/L and about 4.9 g/L, between about 3.7 g/L and about 4.8 g/L, between about 3.7 g/L and about 4.7 g/L, between about 3.7 g/L and about 4.6 g/L, between about 3.7 g/L and about 4.5 g/L, between about 3.7 g/L and about 4.4 g/L, between about 3.7 g/L and about 4.3 g/L, between about 3.7 g/L and about 4.2 g/L, between about 3.7 g/L and about 4.1 g/L, between about 3.7 g/L and about 4.0 g/L, between about 3.7 g/L and about 3.9 g/L, between about 3.8 g/L and about 6.0 g/L, between about 3.8 g/L and about 5.9 g/L, between about 3.8 g/L and about 5.8 g/L, between about 3.8 g/L and about 5.7 g/L, between about 3.8 g/L and about 5.6 g/L, between about 3.8 g/L and about 5.5 g/L, between about 3.8 g/L and about 5.4 g/L, between about 3.8 g/L and about 5.3 g/L, between about 3.8 g/L and about 5.2 g/L, between about 3.8 g/L and about 5.1 g/L, between about 3.8 g/L and about 5.0 g/L, between about 3.8 g/L and about 4.9 g/L, between about 3.8 g/L and about 4.8 g/L, between about 3.8 g/L and about 4.7 g/L, between about 3.8 g/L and about 4.6 g/L, between about 3.8 g/L and about 4.5 g/L, between about 3.8 g/L and about 4.4 g/L, between about 3.8 g/L and about 4.3 g/L, between about 3.8 g/L and about 4.2 g/L, between about 3.8 g/L and about 4.1 g/L, between about 3.8 g/L and about 4.0 g/L, between about 3.9 g/L and about 6.0 g/L, between about 3.9 g/L and about 5.9 g/L, between about 3.9 g/L and about 5.8 g/L, between about 3.9 g/L and about 5.7 g/L, between about 3.9 g/L and about 5.6 g/L, between about 3.9 g/L and about 5.5 g/L, between about 3.9 g/L and about 5.4 g/L, between about 3.9 g/L and about 5.3 g/L, between about 3.9 g/L and about 5.2 g/L, between about 3.9 g/L and about 5.1 g/L, between about 3.9 g/L and about 5.0 g/L, between about 3.9 g/L and about 4.9 g/L, between about 3.9 g/L and about 4.8 g/L, between about 3.9 g/L and about 4.7 g/L, between about 3.9 g/L and about 4.6 g/L, between about 3.9 g/L and about 4.5 g/L, between about 3.9 g/L and about 4.4 g/L, between about 3.9 g/L and about 4.3 g/L, between about 3.9 g/L and about 4.2 g/L, between about 3.9 g/L and about 4.1 g/L, between about 4.0 g/L and about 6.0 g/L, between about 4.0 g/L and about 5.9 g/L, between about 4.0 g/L and about 5.8 g/L, between about 4.0 g/L and about 5.7 g/L, between about 4.0 g/L and about 5.6 g/L, between about 4.0 g/L and about 5.5 g/L, between about 4.0 g/L and about 5.4 g/L, between about 4.0 g/L and about 5.3 g/L, between about 4.0 g/L and about 5.2 g/L, between about 4.0 g/L and about 5.1 g/L, between about 4.0 g/L and about 5.0 g/L, between about 4.0 g/L and about 4.9 g/L, between about 4.0 g/L and about 4.8 g/L, between about 4.0 g/L and about 4.7 g/L, between about 4.0 g/L and about 4.6 g/L, between about 4.0 g/L and about 4.5 g/L, between about 4.0 g/L and about 4.4 g/L, between about 4.0 g/L and about 4.3 g/L, between about 4.0 g/L and about 4.2 g/L, between about 4.1 g/L and about 6.0 g/L, between about 4.1 g/L and about 5.9 g/L, between about 4.1 g/L and about 5.8 g/L, between about 4.1 g/L and about 5.7 g/L, between about 4.1 g/L and about 5.6 g/L, between about 4.1 g/L and about 5.5 g/L, between about 4.1 g/L and about 5.4 g/L, between about 4.1 g/L and about 5.3 g/L, between about 4.1 g/L and about 5.2 g/L, between about 4.1 g/L and about 5.1 g/L, between about 4.1 g/L and about 5.0 g/L, between about 4.1 g/L and about 4.9 g/L, between about 4.1 g/L and about 4.8 g/L, between about 4.1 g/L and about 4.7 g/L, between about 4.1 g/L and about 4.6 g/L, between about 4.1 g/L and about 4.5 g/L, between about 4.1 g/L and about 4.4 g/L, between about 4.1 g/L and about 4.3 g/L, between about 4.2 g/L and about 6.0 g/L, between about 4.2 g/L and about 5.9 g/L, between about 4.2 g/L and about 5.8 g/L, between about 4.2 g/L and about 5.7 g/L, between about 4.2 g/L and about 5.6 g/L, between about 4.2 g/L and about 5.5 g/L, between about 4.2 g/L and about 5.4 g/L, between about 4.2 g/L and about 5.3 g/L, between about 4.2 g/L and about 5.2 g/L, between about 4.2 g/L and about 5.1 g/L, between about 4.2 g/L and about 5.0 g/L, between about 4.2 g/L and about 4.9 g/L, between about 4.2 g/L and about 4.8 g/L, between about 4.2 g/L and about 4.7 g/L, between about 4.2 g/L and about 4.6 g/L, between about 4.2 g/L and about 4.5 g/L, between about 4.2 g/L and about 4.4 g/L, between about 4.3 g/L and about 6.0 g/L, between about 4.3 g/L and about 5.9 g/L, between about 4.3 g/L and about 5.8 g/L, between about 4.3 g/L and about 5.7 g/L, between about 4.3 g/L and about 5.6 g/L, between about 4.3 g/L and about 5.5 g/L, between about 4.3 g/L and about 5.4 g/L, between about 4.3 g/L and about 5.3 g/L, between about 4.3 g/L and about 5.2 g/L, between about 4.3 g/L and about 5.1 g/L, between about 4.3 g/L and about 5.0 g/L, between about 4.3 g/L and about 4.9 g/L, between about 4.3 g/L and about 4.8 g/L, between about 4.3 g/L and about 4.7 g/L, between about 4.3 g/L and about 4.6 g/L, between about 4.3 g/L and about 4.5 g/L, between about 4.4 g/L and about 6.0 g/L, between about 4.4 g/L and about 5.9 g/L, between about 4.4 g/L and about 5.8 g/L, between about 4.4 g/L and about 5.7 g/L, between about 4.4 g/L and about 5.6 g/L, between about 4.4 g/L and about 5.5 g/L, between about 4.4 g/L and about 5.4 g/L, between about 4.4 g/L and about 5.3 g/L, between about 4.4 g/L and about 5.2 g/L, between about 4.4 g/L and about 5.1 g/L, between about 4.4 g/L and about 5.0 g/L, between about 4.4 g/L and about 4.9 g/L, between about 4.4 g/L and about 4.8 g/L, between about 4.4 g/L and about 4.7 g/L, between about 4.4 g/L and about 4.6 g/L, between about 4.5 g/L and about 6.0 g/L, between about 4.5 g/L and about 5.9 g/L, between about 4.5 g/L and about 5.8 g/L, between about 4.5 g/L and about 5.7 g/L, between about 4.5 g/L and about 5.6 g/L, between about 4.5 g/L and about 5.5 g/L, between about 4.5 g/L and about 5.4 g/L, between about 4.5 g/L and about 5.3 g/L, between about 4.5 g/L and about 5.2 g/L, between about 4.5 g/L and about 5.1 g/L, between about 4.5 g/L and about 5.0 g/L, between about 4.5 g/L and about 4.9 g/L, between about 4.5 g/L and about 4.8 g/L, between about 4.5 g/L and about 4.7 g/L, between about 4.6 g/L and about 6.0 g/L, between about 4.6 g/L and about 5.9 g/L, between about 4.6 g/L and about 5.8 g/L, between about 4.6 g/L and about 5.7 g/L, between about 4.6 g/L and about 5.6 g/L, between about 4.6 g/L and about 5.5 g/L, between about 4.6 g/L and about 5.4 g/L, between about 4.6 g/L and about 5.3 g/L, between about 4.6 g/L and about 5.2 g/L, between about 4.6 g/L and about 5.1 g/L, between about 4.6 g/L and about 5.0 g/L, between about 4.6 g/L and about 4.9 g/L, between about 4.6 g/L and about 4.8 g/L, between about 4.7 g/L and about 6.0 g/L, between about 4.7 g/L and about 5.9 g/L, between about 4.7 g/L and about 5.8 g/L, between about 4.7 g/L and about 5.7 g/L, between about 4.7 g/L and about 5.6 g/L, between about 4.7 g/L and about 5.5 g/L, between about 4.7 g/L and about 5.4 g/L, between about 4.7 g/L and about 5.3 g/L, between about 4.7 g/L and about 5.2 g/L, between about 4.7 g/L and about 5.1 g/L, between about 4.7 g/L and about 5.0 g/L, between about 4.7 g/L and about 4.9 g/L, between about 4.8 g/L and about 6.0 g/L, between about 4.8 g/L and about 5.9 g/L, between about 4.8 g/L and about 5.8 g/L, between about 4.8 g/L and about 5.7 g/L, between about 4.8 g/L and about 5.6 g/L, between about 4.8 g/L and about 5.5 g/L, between about 4.8 g/L and about 5.4 g/L, between about 4.8 g/L and about 5.3 g/L, between about 4.8 g/L and about 5.2 g/L, between about 4.8 g/L and about 5.1 g/L, between about 4.8 g/L and about 5.0 g/L, between about 4.9 g/L and about 6.0 g/L, between about 4.9 g/L and about 5.9 g/L, between about 4.9 g/L and about 5.8 g/L, between about 4.9 g/L and about 5.7 g/L, between about 4.9 g/L and about 5.6 g/L, between about 4.9 g/L and about 5.5 g/L, between about 4.9 g/L and about 5.4 g/L, between about 4.9 g/L and about 5.3 g/L, between about 4.9 g/L and about 5.2 g/L, between about 4.9 g/L and about 5.1 g/L, between about 5.0 g/L and about 6.0 g/L, between about 5.0 g/L and about 5.9 g/L, between about 5.0 g/L and about 5.8 g/L, between about 5.0 g/L and about 5.7 g/L, between about 5.0 g/L and about 5.6 g/L, between about 5.0 g/L and about 5.5 g/L, between about 5.0 g/L and about 5.4 g/L, between about 5.0 g/L and about 5.3 g/L, between about 5.0 g/L and about 5.2 g/L, between about 5.1 g/L and about 6.0 g/L, between about 5.1 g/L and about 5.9 g/L, between about 5.1 g/L and about 5.8 g/L, between about 5.1 g/L and about 5.7 g/L, between about 5.1 g/L and about 5.6 g/L, between about 5.1 g/L and about 5.5 g/L, between about 5.1 g/L and about 5.4 g/L, between about 5.1 g/L and about 5.3 g/L, between about 5.2 g/L and about 6.0 g/L, between about 5.2 g/L and about 5.9 g/L, between about 5.2 g/L and about 5.8 g/L, between about 5.2 g/L and about 5.7 g/L, between about 5.2 g/L and about 5.6 g/L, between about 5.2 g/L and about 5.5 g/L, between about 5.2 g/L and about 5.4 g/L, between about 5.3 g/L and about 6.0 g/L, between about 5.3 g/L and about 5.9 g/L, between about 5.3 g/L and about 5.8 g/L, between about 5.3 g/L and about 5.7 g/L, between about 5.3 g/L and about 5.6 g/L, between about 5.3 g/L and about 5.5 g/L, between about 5.4 g/L and about 6.0 g/L, between about 5.4 g/L and about 5.9 g/L, between about 5.4 g/L and about 5.8 g/L, between about 5.4 g/L and about 5.7 g/L, between about 5.4 g/L and about 5.6 g/L, between about 5.5 g/L and about 6.0 g/L, between about 5.5 g/L and about 5.9 g/L, between about 5.5 g/L and about 5.8 g/L, between about 5.5 g/L and about 5.7 g/L, between about 5.6 g/L and about 6.0 g/L, between about 5.6 g/L and about 5.9 g/L, between about 5.6 g/L and about 5.8 g/L, between about 5.7 g/L and about 6.0 g/L, between about 5.7 g/L and about 5.9 g/L, or between about 5.8 g/L and about 6.0 g/L) when the viable cell density in the medium is between about 6010.sup.6 cells/mL to about 9010.sup.6 cells/mL (e.g., between about 6010.sup.6 cells/mL and about 8810.sup.6 cells/mL, between about 6010.sup.6 cells/mL to about 8610.sup.6 cells/mL, between about 6010.sup.6 cells/mL and about 8410.sup.6 cells/mL, between about 6010.sup.6 cells/mL and about 8210.sup.6 cells/mL, between about 6010.sup.6 cells/mL and about 8010.sup.6 cells/mL, between about 6010.sup.6 cells/mL and about 7810.sup.6 cells/mL, between about 6010.sup.6 cells/mL and about 7610.sup.6 cells/mL, between about 6010.sup.6 cells/mL and about 7410.sup.6 cells/mL, between about 6010.sup.6 cells/mL and about 7210.sup.6 cells/mL, between about 6010.sup.6 cells/mL to about 7010.sup.6 cells/mL, between about 6010.sup.6 cells/mL and about 6810.sup.6 cells/mL, between about 6010.sup.6 cells/mL and about 6610.sup.6 cells/mL, between about 6010.sup.6 cells/mL and about 6410.sup.6 cells/mL, between about 6010.sup.6 cells/mL and about 6210.sup.6 cells/mL, between about 6210.sup.6 cells/mL and about 9010.sup.6 cells/mL, between about 6210.sup.6 cells/mL and about 8810.sup.6 cells/mL, between about 6210.sup.6 cells/mL to about 8610.sup.6 cells/mL, between about 6210.sup.6 cells/mL and about 8410.sup.6 cells/mL, between about 6210.sup.6 cells/mL and about 8210.sup.6 cells/mL, between about 6210.sup.6 cells/mL and about 8010.sup.6 cells/mL, between about 6210.sup.6 cells/mL and about 7810.sup.6 cells/mL, between about 6210.sup.6 cells/mL and about 7610.sup.6 cells/mL, between about 6210.sup.6 cells/mL and about 7410.sup.6 cells/mL, between about 6210.sup.6 cells/mL and about 7210.sup.6 cells/mL, between about 6210.sup.6 cells/mL to about 7010.sup.6 cells/mL, between about 6210.sup.6 cells/mL and about 6810.sup.6 cells/mL, between about 6210.sup.6 cells/mL and about 6610.sup.6 cells/mL, between about 6210.sup.6 cells/mL and about 6410.sup.6 cells/mL, between about 6410.sup.6 cells/mL and about 9010.sup.6 cells/mL, between about 6410.sup.6 cells/mL and about 8810.sup.6 cells/mL, between about 6410.sup.6 cells/mL to about 8610.sup.6 cells/mL, between about 6410.sup.6 cells/mL and about 8410.sup.6 cells/mL, between about 6410.sup.6 cells/mL and about 8210.sup.6 cells/mL, between about 6410.sup.6 cells/mL and about 8010.sup.6 cells/mL, between about 6410.sup.6 cells/mL and about 7810.sup.6 cells/mL, between about 6410.sup.6 cells/mL and about 7610.sup.6 cells/mL, between about 6410.sup.6 cells/mL and about 7410.sup.6 cells/mL, between about 6410.sup.6 cells/mL and about 7210.sup.6 cells/mL, between about 6410.sup.6 cells/mL to about 7010.sup.6 cells/mL, between about 6410.sup.6 cells/mL and about 6810.sup.6 cells/mL, between about 6410.sup.6 cells/mL and about 6610.sup.6 cells/mL, between about 6610.sup.6 cells/mL and about 9010.sup.6 cells/mL, between about 6610.sup.6 cells/mL and about 8810.sup.6 cells/mL, between about 6610.sup.6 cells/mL to about 8610.sup.6 cells/mL, between about 6610.sup.6 cells/mL and about 8410.sup.6 cells/mL, between about 6610.sup.6 cells/mL and about 8210.sup.6 cells/mL, between about 6610.sup.6 cells/mL and about 8010.sup.6 cells/mL, between about 6610.sup.6 cells/mL and about 7810.sup.6 cells/mL, between about 6610.sup.6 cells/mL and about 7610.sup.6 cells/mL, between about 6610.sup.6 cells/mL and about 7410.sup.6 cells/mL, between about 6610.sup.6 cells/mL and about 7210.sup.6 cells/mL, between about 6610.sup.6 cells/mL to about 7010.sup.6 cells/mL, between about 6610.sup.6 cells/mL and about 6810.sup.6 cells/mL, between about 6810.sup.6 cells/mL and about 9010.sup.6 cells/mL, between about 6810.sup.6 cells/mL and about 8810.sup.6 cells/mL, between about 6810.sup.6 cells/mL to about 8610.sup.6 cells/mL, between about 6810.sup.6 cells/mL and about 8410.sup.6 cells/mL, between about 6810.sup.6 cells/mL and about 8210.sup.6 cells/mL, between about 6810.sup.6 cells/mL and about 8010.sup.6 cells/mL, between about 6810.sup.6 cells/mL and about 7810.sup.6 cells/mL, between about 6810.sup.6 cells/mL and about 7610.sup.6 cells/mL, between about 6810.sup.6 cells/mL and about 7410.sup.6 cells/mL, between about 6810.sup.6 cells/mL and about 7210.sup.6 cells/mL, between about 6810.sup.6 cells/mL to about 7010.sup.6 cells/mL, between about 7010.sup.6 cells/mL and about 9010.sup.6 cells/mL, between about 7010.sup.6 cells/mL and about 8810.sup.6 cells/mL, between about 7010.sup.6 cells/mL to about 8610.sup.6 cells/mL, between about 7010.sup.6 cells/mL and about 8410.sup.6 cells/mL, between about 7010.sup.6 cells/mL and about 8210.sup.6 cells/mL, between about 7010.sup.6 cells/mL and about 8010.sup.6 cells/mL, between about 7010.sup.6 cells/mL and about 7810.sup.6 cells/mL, between about 7010.sup.6 cells/mL and about 7610.sup.6 cells/mL, between about 7010.sup.6 cells/mL and about 7410.sup.6 cells/mL, between about 7010.sup.6 cells/mL and about 7210.sup.6 cells/mL, between about 7210.sup.6 cells/mL and about 9010.sup.6 cells/mL, between about 7210.sup.6 cells/mL and about 8810.sup.6 cells/mL, between about 7210.sup.6 cells/mL to about 8610.sup.6 cells/mL, between about 7210.sup.6 cells/mL and about 8410.sup.6 cells/mL, between about 7210.sup.6 cells/mL and about 8210.sup.6 cells/mL, between about 7210.sup.6 cells/mL and about 8010.sup.6 cells/mL, between about 7210.sup.6 cells/mL and about 7810.sup.6 cells/mL, between about 7210.sup.6 cells/mL and about 7610.sup.6 cells/mL, between about 7210.sup.6 cells/mL and about 7410.sup.6 cells/mL, between about 7410.sup.6 cells/mL and about 9010.sup.6 cells/mL, between about 7410.sup.6 cells/mL and about 8810.sup.6 cells/mL, between about 7410.sup.6 cells/mL to about 8610.sup.6 cells/mL, between about 7410.sup.6 cells/mL and about 8410.sup.6 cells/mL, between about 7410.sup.6 cells/mL and about 8210.sup.6 cells/mL, between about 7410.sup.6 cells/mL and about 8010.sup.6 cells/mL, between about 7410.sup.6 cells/mL and about 7810.sup.6 cells/mL, between about 7410.sup.6 cells/mL and about 7610.sup.6 cells/mL, between about 7610.sup.6 cells/mL and about 9010.sup.6 cells/mL, between about 7610.sup.6 cells/mL and about 8810.sup.6 cells/mL, between about 7610.sup.6 cells/mL to about 8610.sup.6 cells/mL, between about 7610.sup.6 cells/mL and about 8410.sup.6 cells/mL, between about 7610.sup.6 cells/mL and about 8210.sup.6 cells/mL, between about 7610.sup.6 cells/mL and about 8010.sup.6 cells/mL, between about 7610.sup.6 cells/mL and about 7810.sup.6 cells/mL, between about 7810.sup.6 cells/mL and about 9010.sup.6 cells/mL, between about 7810.sup.6 cells/mL and about 8810.sup.6 cells/mL, between about 7810.sup.6 cells/mL to about 8610.sup.6 cells/mL, between about 7810.sup.6 cells/mL and about 8410.sup.6 cells/mL, between about 7810.sup.6 cells/mL and about 8210.sup.6 cells/mL, between about 7810.sup.6 cells/mL and about 8010.sup.6 cells/mL, between about 8010.sup.6 cells/mL and about 9010.sup.6 cells/mL, between about 8010.sup.6 cells/mL and about 8810.sup.6 cells/mL, between about 8010.sup.6 cells/mL to about 8610.sup.6 cells/mL, between about 8010.sup.6 cells/mL and about 8410.sup.6 cells/mL, between about 8010.sup.6 cells/mL and about 8210.sup.6 cells/mL, between about 8210.sup.6 cells/mL and about 9010.sup.6 cells/mL, between about 8210.sup.6 cells/mL and about 8810.sup.6 cells/mL, between about 8210.sup.6 cells/mL to about 8610.sup.6 cells/mL, between about 8210.sup.6 cells/mL and about 8410.sup.6 cells/mL, between about 8410.sup.6 cells/mL and about 9010.sup.6 cells/mL, between about 8410.sup.6 cells/mL and about 8810.sup.6 cells/mL, between about 8410.sup.6 cells/mL to about 8610.sup.6 cells/mL, between about 8610.sup.6 cells/mL and about 9010.sup.6 cells/mL, between about 8610.sup.6 cells/mL and about 8810.sup.6 cells/mL, or between about 8810.sup.6 cells/mL and about 9010.sup.6 cells/mL).
(86) Any of the methods described herein can include increasing the poloxamer-188 concentration in the medium to greater than 6.0 g/L (e.g., greater than 6.1 g/L, greater than 6.2 g/L, greater than 6.3 g/L, greater than 6.4 g/L, greater than 6.5 g/L, greater than 6.6 g/L, greater than 6.7 g/L, greater than 6.8 g/L, greater than 6.9 g/L, greater than 7.0 g/L, greater than 7.1 g/L, greater than 7.2 g/L, greater than 7.3 g/L, greater than 7.4 g/L, greater than 7.5 g/L, greater than 7.6 g/L, greater than 7.7 g/L, greater than 7.8 g/L, greater than 7.9 g/L, greater than 8.0 g/L, greater than 8.1 g/L, greater than 8.2 g/L, greater than 8.3 g/L, greater than 8.4 g/L, greater than 8.5 g/L, greater than 8.6 g/L, greater than 8.7 g/L, greater than 8.8 g/L, greater than 8.9 g/L, greater than 9.0 g/L, greater than 9.1 g/L, greater than 9.2 g/L, greater than 9.3 g/L, greater than 9.4 g/L, greater than 9.5 g/L, greater than 9.6 g/L, greater than 9.7 g/L, greater than 9.8 g/L, greater than 9.9 g/L, greater than 10.0 g/L, greater than about 10.1 g/L, greater than about 10.2 g/L, greater than about 10.3 g/L, greater than about 10.4 g/L, greater than about 10.5 g/L, greater than about 10.6 g/L, greater than about 10.7 g/L, greater than about 10.8 g/L, greater than about 10.9 g/L, or greater than about 11.0 g/L) poloxamer-188, when the viable cell density in the medium is greater than between about 9010.sup.6 cells/mL (e.g., greater than about 9110.sup.6 cells/mL, greater than about 9210.sup.6 cells/mL, greater than about 9310.sup.6 cells/mL, greater than about 9410.sup.6 cells/mL, greater than about 9510.sup.6 cells/mL, greater than about 9610.sup.6 cells/mL, greater than about 9710.sup.6 cells/mL, greater than about 9810.sup.6 cells/mL, greater than about 9910.sup.6 cells/mL, greater than about 10010.sup.6 cells/mL, greater than about 10110.sup.6 cells/mL, greater than about 10210.sup.6 cells/mL, greater than about 10310.sup.6 cells/mL, greater than about 10410.sup.6 cells/mL, greater than about 10510.sup.6 cells/mL, greater than about 10610.sup.6 cells/mL, greater than about 10710.sup.6 cells/mL, greater than about 10810.sup.6 cells/mL, greater than about 10910.sup.6 cells/mL, greater than about 11010.sup.6 cells/mL, greater than about 11110.sup.6 cells/mL, greater than about 11210.sup.6 cells/mL, greater than about 11310.sup.6 cells/mL, greater than about 11410.sup.6 cells/mL, greater than about 11510.sup.6 cells/mL, greater than about 11610.sup.6 cells/mL, greater than about 11710.sup.6 cells/mL, greater than about 11810.sup.6 cells/mL, greater than about 11910.sup.6 cells/mL, or greater than about 12010.sup.6 cells/mL).
(87) In some embodiments, the methods include: increasing the poloxamer-188 concentration in the medium to between about 1.8 g/L to about 3.0 g/L (e.g., any of the ranges therebetween described herein) when the viable cell density in the medium is between about 3510.sup.6 cells/mL to about 6010.sup.6 cells/mL (e.g., or any of the ranges therebetween described herein); increasing the poloxamer-188 concentration in the medium to between about 3.0 g/L to about 6.0 g/L (e.g., any of the ranges therebetween described herein) when the viable cell concentration density in the medium is between about 6010.sup.6 cells/mL to about 9010.sup.6 cells/mL (e.g., any of the ranges therebetween described herein); and increasing the poloxamer-188 concentration in the medium to greater than 6.0 g/L (e.g., any of the concentrations greater than 6.0 g/L described herein) when the viable cell density concentration in the medium is greater than 9010.sup.6 cells/mL (e.g., any of the viable cell densities greater than 9010.sup.6 cells/mL described herein). Some embodiments of any of the methods described herein further include determining the viable cell density in the medium at one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty) time points during the culturing.
(88) Poloxamer-188 to AntiFoam Ratio
(89) In any of the embodiments described herein, the liquid medium comprises antifoam (e.g., antifoam-c) and the ratio of antifoam (g/L) to poloxamer-188 (g/L) in the medium is between about 0.5% to about 6.0% (e.g., between about 0.5% and about 5.5%, between about 0.5% and about 5.0%, between about 0.5% and about 4.5%, between about 0.5% and about 4.0%, between about 0.5% and about 3.5%, between about 0.5% and about 3.0%, between about 0.5% and about 2.5%, between about 0.5% and about 2.0%, between about 0.5% and about 1.5%, between about 0.5% and about 1.0%, between about 1.0% and about 6.0%, between about 1.0% and about 5.5%, between about 1.0% and about 5.0%, between about 1.0% and about 4.5%, between about 1.0% and about 4.0%, between about 1.0% and about 3.5%, between about 1.0% and about 3.0%, between about 1.0% and about 2.5%, between about 1.0% and about 2.0%, between about 1.0% and about 1.5%, between about 2.0% and about 6.0%, between about 2.0% and about 5.5%, between about 2.0% and about 5.0%, between about 2.0% and about 4.5%, between about 2.0% and about 4.0%, between about 2.0% and about 3.5%, between about 2.0% and about 3.0%, between about 2.0% and about 2.5%, between about 2.5% and about 6.0%, between about 2.5% and about 5.5%, between about 2.5% and about 5.0%, between about 2.5% and about 4.5%, between about 2.5% and about 4.0%, between about 2.5% and about 3.5%, between about 2.5% and about 3.0%, between about 3.0% and about 6.0%, between about 3.0% and about 5.5%, between about 3.0% and about 5.0%, between about 3.0% and about 4.5%, between about 3.0% and about 4.0%, between about 3.0% and about 3.5%, between about 3.5% and about 6.0%, between about 3.5% and about 5.5%, between about 3.5% and about 5.0%, between about 3.5% and about 4.5%, between about 3.5% and about 4.0%, between about 4.0% and about 6.0%, between about 4.0% and about 5.5%, between about 4.0% and about 5.0%, between about 4.0% and about 4.5%, between about 4.5% and about 6.0%, between about 4.5% and about 5.5%, between about 4.5% and about 5.0%, between about 5.0% and about 6.0%, between about 5.0% and about 5.5%, or between about 5.5% and about 6.0%).
(90) Culture Media
(91) Liquid culture media are known in the art. The liquid culture media (e.g., a first and/or second liquid medium) can be supplemented with a mammalian serum (e.g., fetal calf serum and bovine serum), and/or a growth hormone or growth factor (e.g., insulin, transferrin, and epidermal growth factor). Alternatively or in addition, the liquid culture media (e.g., a first and/or second liquid medium) can be a chemically-defined liquid culture medium, an animal-derived component free liquid culture medium, a serum-free liquid culture medium, or a serum-containing liquid culture medium. Non-limiting examples of chemically-defined liquid culture media, animal-derived component free liquid culture media, serum-free liquid culture media, and serum-containing liquid culture media are commercially available.
(92) A liquid medium typically contains an energy source (e.g., a carbohydrate, such as glucose), essential amino acids (e.g., the basic set of twenty amino acids plus cysteine), vitamins and/or other organic compounds required at low concentrations, free fatty acids, and/or trace elements. The liquid culture media (e.g., a first and/or second liquid medium) can, if desired, be supplemented with, e.g., a mammalian hormone or growth factor (e.g., insulin, transferrin, or epidermal growth factor), salts and buffers (e.g., calcium, magnesium, and phosphate salts), nucleosides and bases (e.g., adenosine, thymidine, and hypoxanthine), protein and tissue hydrolysates, and/or any combination of these additives.
(93) A wide variety of different liquid culture media that can be used to culture cells (e.g., mammalian cells) in any of the methods described herein are known in the art. Medium components that also may be useful in the present processes include, but are not limited to, chemically-defined (CD) hydrolysates, e.g., CD peptone, CD polypeptides (two or more amino acids), and CD growth factors. Additional examples of liquid medium and medium components are known in the art.
(94) Skilled practitioners will appreciate that the first liquid medium and the second or feed liquid medium described herein can be the same type of media or different media.
(95) Liquid medium obtained from a cell culture can be filtered or clarified to obtain a liquid medium that is substantially free of cells and/or viruses. Methods for filtering or clarifying a liquid medium in order to remove cells are known in the art (e.g., 0.2-m filtration and filtration using an Alternating Tangential Flow (ATFTM) system). Mammalian cells can also be removed from liquid medium using centrifugation and removing the supernatant that is liquid medium that is substantially free of cells, or by allowing the cells to settle to the gravitational bottom of a vessel or bioreactor containing the liquid medium, and removing the liquid medium (the liquid medium that is substantially free of cells) that is distant from the settled recombinant cells.
(96) Temperature
(97) The step of culturing of mammalian cells can be performed at a temperature of about 31 C. to about 40 C. Skilled practitioners will appreciate that the temperature can be changed at specific time point(s) in during the culturing step, e.g., on an hourly or daily basis. For example, the temperature can be changed or shifted (e.g., increased or decreased) at about one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, fourteen days, fifteen days, sixteen days, seventeen days, eighteen days, nineteen days, or about twenty days or more after the initial seeding of the bioreactor with the cell (e.g., mammalian cell). For example, the temperature can be shifted upwards (e.g., a change of up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to or about 20 degrees C.). For example, the temperature can be shifted downwards (e.g., a change of up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to or about 20 C.).
(98) CO.sub.2
(99) The culturing step described herein can further include exposing the liquid medium in the bioreactor to an atmosphere containing at most or about 15% CO.sub.2 (e.g., at most or about 14% CO.sub.2, 12% CO.sub.2, 10% CO.sub.2, 8% CO.sub.2, 6% CO.sub.2, 5% CO.sub.2, 4% CO.sub.2, 3% CO.sub.2, 2% CO.sub.2, or at most or about 1% CO.sub.2).
(100) Recombinant Proteins
(101) Non-limiting examples of recombinant proteins that can be produced by the methods provided herein include immunoglobulins (including light and heavy chain immunoglobulins, antibodies, or antibody fragments (e.g., any of the antibody fragments described herein), enzymes (e.g., a galactosidase (e.g., an alpha-galactosidase), Myozyme, or Cerezyme), proteins (e.g., human erythropoietin, tumor necrosis factor (TNF), or an interferon alpha or beta), or immunogenic or antigenic proteins or protein fragments (e.g., proteins for use in a vaccine). The recombinant protein can be an engineered antigen-binding polypeptide that contains at least one multifunctional recombinant protein scaffold (see, e.g., the recombinant antigen-binding proteins described in Gebauer et al., Current Opin. Chem. Biol. 13:245-255, 2009; and U.S. Patent Application Publication No. 2012/0164066 (herein incorporated by reference in its entirety)). Non-limiting examples of recombinant proteins that are antibodies include: panitumumab, omalizumab, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, amatuximab, anatumomab, anrukinzumab, apolizumab, arcitumomab, atinumab, tocilizumab, basilizimab, bectumomab, belimumab, bevacizumab, besilesomab, bezlotoxumab, biciromab, canakinumab, certolizumab, cetuximab, cixutumumab, daclizumab, denosumab, densumab, eculizumab, edrecolomab, efalizumab, efungumab, epratuzumab, ertumaxomab, etaracizumab, figitumumab, golimumab, ibritumomab tiuxetan, igovomab, imgatuzumab, infliximab, inolimomab, inotuzumab, labetuzumab, lebrikizumab, moxetumomab, natalizumab, obinutuzumab, oregovomab, palivizumab, panitumumab, pertuzumab, ranibizumab, rituximab, tocilizumab, tositumomab, tralokinumab, tucotuzumab, trastuzumab, veltuzumab, zalutumumab, and zatuximab. Additional examples of recombinant antibodies that can be produced by the methods described herein are known in the art. Additional non-limiting examples of recombinant proteins that can be produced by the present methods include: alglucosidase alfa, laronidase, abatacept, galsulfase, lutropin alfa, antihemophilic factor, agalsidase beta, interferon beta-la, darbepoetin alfa, tenecteplase, etanercept, coagulation factor IX, follicle stimulating hormone, interferon beta-la, imiglucerase, dornase alfa, epoetin alfa, insulin or insulin analogs, mecasermin, factor VIII, factor VIIa, anti-thrombin III, protein C, human albumin, erythropoietin, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, interleukin-11, laronidase, idursuphase, galsulphase, -1-proteinase inhibitor, lactase, adenosine deaminase, tissue plasminogen activator, thyrotropin alpha (e.g., Thyrogen) and alteplase. Additional examples of recombinant proteins that can be produced by the present methods include acid a-glucosidase, alglucosidase alpha (e.g., Myozyme and Lumizyme), -L-iduronidase (e.g., Aldurazyme), iduronate sulfatase, heparan N-sulfatase, galactose-6-sulfatase, acid -galactosidase, -glucoronidase, N-acetylglucosamine-1-phosphotransferase, -N-acetylgalactosaminidase, acid lipase, lysosomal acid ceramidase, acid sphingomyelinase, -glucosidase (e.g., Cerezyme and Ceredase), galactosylceramidase, -galactosidase-A (e.g., Fabrazyme), acid -galactosidase, -galactosidase, neuraminidase, hexosaminidase A, and hexosaminidase B.
(102) A secreted, soluble recombinant protein can be recovered from the liquid medium (e.g., a first and/or second liquid medium) by removing or otherwise physically separating the liquid medium from the cells (e.g., mammalian cells). A variety of different methods for removing liquid medium from cells (e.g., mammalian cells) are known in the art, including, for example, centrifugation, filtration, pipetting, and/or aspiration. The secreted recombinant protein can then be recovered and further purified from the liquid medium using a variety of biochemical techniques including various types of chromatography (e.g., affinity chromatography, molecular sieve chromatography, cation exchange chromatography, or anion exchange chromatography) and/or filtration (e.g., molecular weight cut-off filtration).
(103) Collecting the Recombinant Protein
(104) Some embodiments further include collecting the recombinant protein (e.g., any of the recombinant proteins described herein). In some examples, the collecting includes lysing the mammalian cells. In other examples, collecting can include collecting the recombinant protein from the medium (e.g., one or both of the first and second liquid medium when perfusion culturing is performed, or one or both of the first liquid medium and the feed liquid medium when batch culturing is performed).
(105) Collecting can be performed after the culturing achieves a viable cell density of, e.g., greater than about 3010.sup.6 cells/mL, greater than about 3210.sup.6 cells/mL, greater than about 3410.sup.6 cells/mL, greater than about 3610.sup.6 cells/mL, greater than about 3810.sup.6 cells/mL, greater than about 4010.sup.6 cells/mL, greater than about 4210.sup.6 cells/mL, greater than about 4410.sup.6 cells/mL, greater than about 4610.sup.6 cells/mL, greater than about 4810.sup.6 cells/mL, greater than about 5010.sup.6 cells/mL, greater than about 5210.sup.6 cells/mL, greater than about 5410.sup.6 cells/mL, greater than about 5610.sup.6 cells/mL, greater than about 5810.sup.6 cells/mL, greater than about 6010.sup.6 cells/mL, greater than about 6210.sup.6 cells/mL, greater than about 6410.sup.6 cells/mL, greater than about 6610.sup.6 cells/mL, greater than about 6810.sup.6 cells/mL, greater than about 7010.sup.6 cells/mL, greater than about 7210.sup.6 cells/mL, greater than about 7410.sup.6 cells/mL, greater than about 7610.sup.6 cells/mL, greater than about 7810.sup.6 cells/mL, greater than about 8010.sup.6 cells/mL, greater than about 8210.sup.6 cells/mL, greater than about 8410.sup.6 cells/mL, greater than about 8610.sup.6 cells/mL, greater than about 8810.sup.6 cells/mL, greater than about 9010.sup.6 cells/mL, greater than about 9210.sup.6 cells/mL, greater than about 9410.sup.6 cells/mL, greater than about 9610.sup.6 cells/mL, greater than about 9810.sup.6 cells/mL, greater than about 10010.sup.6 cells/mL, greater than about 10210.sup.6 cells/mL, greater than about 10410.sup.6 cells/mL, greater than about 10610.sup.6 cells/mL, greater than about 10810.sup.6 cells/mL, greater than about 11010.sup.6 cells/mL, greater than about 11210.sup.6 cells/mL, greater than about 11410.sup.6 cells/mL, greater than about 11610.sup.6 cells/mL, greater than about 11810.sup.6 cells/mL, greater than about 12010.sup.6 cells/mL, greater than about 12210.sup.6 cells/mL, greater than about 12410.sup.6 cells/mL, greater than about 12610.sup.6 cells/mL, greater than about 12810.sup.6 cells/mL, greater than about 13010.sup.6 cells/mL, greater than about 13210.sup.6 cells/mL, greater than about 13410.sup.6 cells/mL, greater than about 13610.sup.6 cells/mL, greater than about 13810.sup.6 cells/mL, greater than about 14010.sup.6 cells/mL, greater than about 14210.sup.6 cells/mL, greater than about 14410.sup.6 cells/mL, greater than about 14610.sup.6 cells/mL, greater than about 14810.sup.6 cells/mL, greater than about 15010.sup.6 cells/mL, greater than about 15210.sup.6 cells/mL, greater than about 15410.sup.6 cells/mL, greater than about 15610.sup.6 cells/mL, greater than about 15810.sup.6 cells/mL, greater than about 16010.sup.6 cells/mL, greater than about 16210.sup.6 cells/mL, greater than about 16410.sup.6 cells/mL, greater than about 16610.sup.6 cells/mL, greater than about 16810.sup.6 cells/mL, greater than about 17010.sup.6 cells/mL, greater than about 17210.sup.6 cells/mL, greater than about 17410.sup.6 cells/mL, greater than about 17610.sup.6 cells/mL, greater than about 17810.sup.6 cells/mL, greater than about 18010.sup.6 cells/mL, greater than about 18210.sup.6 cells/mL, greater than about 18410.sup.6 cells/mL, greater than about 18610.sup.6 cells/mL, greater than about 18810.sup.6 cells/mL, greater than about 19010.sup.6 cells/mL, greater than about 19210.sup.6 cells/mL, greater than about 19410.sup.6 cells/mL, greater than about 19610.sup.6 cells/mL, greater than about 19810.sup.6 cells/mL, or greater than about 20010.sup.6 cells/mL).
(106) Formulating the Recombinant Protein
(107) Some embodiments of any of the methods described herein further include a step of formulating the recombinant protein (e.g., collected recombinant protein) into a pharmaceutical composition. For example, formulating can include adding a pharmaceutically acceptable excipient to the recombinant protein (e.g., collected recombinant protein). Formulating can include mixing a pharmaceutically acceptable excipient with the recombinant protein (e.g., collected recombinant protein). Examples of pharmaceutically acceptable excipients (e.g., non-naturally occurring pharmaceutically acceptable excipients) are well known in the art. In some embodiments, the recombinant protein (e.g., collected recombinant protein) is formulated for intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular administration.
EXAMPLES
Example 1. Effect of Increased Concentrations of Poloxamer-188 on a Perfusion Cell Culture Producing a Secreted Recombinant Antibody
(108) This set of experiments was performed to test the effect of increasing concentrations of poloxamer-188 on cell growth and recombinant antibody production in a perfusion cell culture including mammalian cells containing a nucleic acid encoding the recombinant antibody. In these experiments, a first perfusion cell culture run was performed which used CD-CHO media containing 1.8 g/L over the entire culture period and a second perfusion cell culture run was performed which used CD-CHO media containing increasing concentrations of poloxamer-188 over the culture period (increasing from 1.8 g/L to 6.8 g/L poloxamer-188 over the culture period). For each cell culture run, perfusion was performed at a rate of 4 reactor volumes (RVs)/day on day 12 through day 33 of the culture, switching to a rate of 6 RVs/day on day 33 through day 63 of the culture, and switching to a rate of 7 RVs/day on day 63 through day 72 of the culture. The second cell culture run was performed using CD-CHO medium containing 1.8 g/L poloxamer-188 on day 0 through day 14 of the culture, CD-CHO medium, containing 2.8 g/L poloxamer-188 on day 15 through day 34 of the culture, CD-CHO medium containing 3.8 g/L poloxamer-188 on day 35 through day 39 of the culture, CD-CHO medium containing 4.8 g/L poloxamer-188 on day 40 through day 56 of the culture, and CD-CHO medium containing 5.8 g/L poloxamer-188 on day 57 through day 72 of the culture.
(109) The viable cell density, the specific growth rate, the percentage of viable cells, the volumetric productivity rate, and the specific productivity rate were determined each day for both the first and second perfusion cultures using well-known methods.
(110) The data show that viable cell densities exceeding 10010.sup.6 cells/mL are achieved by establishing a poloxamer-188 concentration of greater than 1.8 g/L in the culture (
(111) The data in
Example 2. Effect of Increased Concentrations of Poloxamer-188 on Stability of a Recombinant Antibody in Liquid Medium
(112) A set of experiments was performed to determine whether increased concentrations of poloxamer-188 would inhibit degradation of a recombinant protein present in a clarified liquid medium after harvest from a cell culture. In these experiments, a perfusion cell culture run similar to the second perfusion cell culture run described in Example 1 was performed, except after the culture achieved a concentration of 6.8 g/L poloxamer-188, the poloxamer-188 concentration in the culture was reduced to 3.95 g/L poloxamer-188, and then reduced once again to 3.0 g/L poloxamer-188. In these experiments, two or three samples of the liquid medium were removed from the culture at the following time points: a first time point when the culture contained 6.8 g/L poloxamer-188, a second time point when the culture contained a concentration of 3.95 g/L poloxamer-188, and a third time point when the culture contained a concentration of 3.0 g/L poloxamer-188. One sample collected from each of the time points was stored at 4 C., one sample collected from each of the time points was incubated for 7 days at room temperature prior to storage at 4 C., and for each of the second and third time points, a sample was supplemented with poloxamer-188 to achieve a concentration of 6.8 g/L polaxmer-188 in each sample, and then each sample was incubated for 9 days at room temperature prior to storage at 4 C. The resulting untreated and treated samples were run on a 4-20% Bis-Tris Gel and stained with Coomassie Blue.
(113) The data in
Example 3. Effect of Sparger Pore Type and Size on Amount of Poloxamer-188 Necessary to Achieve Beneficial Effect on Cell Culture Growth and Productivity
(114) A set of experiments was performed to test the effect of sparger pore type and size on the amount of poloxamer-188 necessary to achieve beneficial growth and productivity properties in a perfusion cell culture. In these experiments, different perfusion cell culture runs were performed using different spargers: a sparger having a sintered pore having a size of 20 m (1 reactor); a sparger having a drilled pore having a size of 200 m (2 reactors); or a sparger having a drilled pore having a size of 500 m (1 reactor). Each perfusion culture initially used CD CHO medium containing 1.8 g/L poloxamer-188 and poloxamer-188 was added to the culture in increasing increments in order to maintain cell viability and minimize cell lysis as necessary. The viable cell density, the specific growth rate, the specific lactate dehydrogenase production rate, the percentage of viable cells, the volumetric productivity rate, and the aerobic glucose consumption for each cell culture was performed at different time points during the culture period using well-known methods.
(115) The data show that a concentration of 3.8 g/L poloxamer-188 in the culture was able to maintain the viable cell density in the perfusion cell culture run performed using a sparger having a drilled pore having a size of 500 m, a concentration of 4.8 g/L poloxamer-188 was able to maintain the viable cell density in the perfusion cell culture run performed using a sparger having a drilled pore having a size of 200 m, and a concentration of 5.8 g/L poloxamer-188 was able to maintain the viable cell density in the perfusion cell culture run performed using a sparger having a sintered pore having a size of 20 m (
(116) The data also show that similar specific growth rates (
(117) The data show that all the tested spargers can be used to achieve a viable cell density of 6010.sup.6 cells/mL, when poloxamer-188 is present in a concentration of greater than 1.8 g/L in the culture. For example, a viable cell density of greater than 6010.sup.6 cells/mL can be achieved in: a perfusion cell culture run using a sparger with a drilled pore having a size of 1 mm by using 2.8 g/L poloxamer-188 in the culture, a perfusion cell culture run using a sparger with a drilled pore having a size of 500 m by using 3.8 g/L poloxamer-188 in the culture, a perfusion cell culture run using a sparger with a drilled pore having a size of 200 m by using 4.8 g/L poloxamer-188 in the culture, a perfusion cell culture run using a sparger with a sintered pore having a size of 100 m by using 3.8 g/L poloxamer-188 in the culture, and a perfusion culture using a sparger with a sintered pore having a size of 20 m by using 5.8 g/L poloxamer-188 in the culture. The data indicate that the smaller the pore size, the more poloxamer-188 is required to maintain a healthy culture.
Example 4. Effect of Sparger Pore Type, Pore Size, and Antifoam to Poloxamer-188 Ratio on Perfusion Culture Cell Growth
(118) An additional set of experiments was performed to determine the steady-state cell growth in perfusion cultures using spargers with different pore types and sizes and different antifoam to poloxamer-188 ratios in the culture (shown in Table 1 below).
(119) The data in Table 1 show that a smaller pore size requires that more antifoam-c needs to be added to the culture to mitigate the generation of foam, that additional antifoam-c is needed as the sparge rate increases, and that the optimal ratio of antifoam-c to poloxamer-188 may be between 0 and <5% (e.g., between about 1% to about 3%).
(120) TABLE-US-00001 TABLE 1 Steady State Viable Cell Density Achieved for Tested Perfusion Cultures Antifoam Antifoam Steady-State Additional Antifoam addition Sparge Rate addition Viable Cell Pluronic (g/L) Addition to total (vol gas per to sparge Perfusion Sparger Density (CD-CHO = rate pluronic ratio min to volume rate ratio Rate Exp Type (10e.sup.6 cells/mL) 1.8 g/L) (ppm/day) (%/day) of reactor) (ppm) (RV/day) 2 20 m 60 4 43 0.7 0.03 1 4 drilled hole 1 100 m 110 4 160 2.8 0.43 0.26 6 sintered 2 200 m 60 3 77-116 1.6-2.4 0.16-0.19 0.33-0.42 4 drilled hole 2 500 m 60 2 69 1.8 0.15 0.32 4 drilled hole 1 1 mm 65 1 64 2.3 0.69 0.06 4 drilled hole
Example 5. Effect of Antifoam-C to Poloxamer-188 Ratio on Cell Growth in Batch Cell Culture Runs
(121) A set of experiments was performed to test the effect of antifoam-c to poloxamer-188 ratio on cell growth in a batch shake flask cell culture runs using CD-CHO medium containing 0% antifoam-c to poloxamer-188 ratio, a 5.6% antifoam-c to poloxamer-188 ratio, an 11.1% antifoam-c to poloxamer-188 ratio, a 27.8% antifoam-c to poloxamer-188 ratio, or a 55.6% antifoam-c to poloxamer-188 ratio. The shake flask cell cultures were baffled to passively generate bubbles to simulate a bioreactor. The viable cell density and specific lactate dehydrogenase production rate in each cell culture run were determined each day over the culture period using well-known methods.
(122) The data show that lower antifoam-c to poloxamer-188 ratios demonstrate improved viable cell densities (
Other Embodiments
(123) It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.