Method for improving cell viability in production bioreactor

Abstract

The invention relates to a method for increasing cell viability in a production bioreactor, methods of predicting the effect of sensitizer concentration on cell viability in a production bioreactor, methods of increasing cell viability in a production bioreactor, methods of predicting cell viability in a production bioreactor, and methods for culturing cells in a production bioreactor are provided.

Description

[0001] This invention application is a divisional application based on an invention patent application with an application date of March 15, 2019, application number 201980031298.3 (international application number PCT / US2019 / 022530), and titled “Method for Improving Cell Viability in a Production Bioreactor”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 644,339 filed on March 16, 2018 and U.S. Provisional Patent Application Serial No. 62 / 645,755 filed on March 20, 2018; the entire contents of each of these applications are incorporated herein by reference. Technical Field

[0004] The present disclosure relates to methods of increasing cell viability in production bioreactors and to the fields of biotechnology and cell biology. Background Art

[0005] Process intensification offers many advantages for the production of recombinant therapeutic proteins. One of the strategies for process intensification is to maintain high cell density to improve recombinant protein production, which also requires high oxygen demand. Oxygen sparging in production bioreactors leads to foaming in production bioreactors. Foaming often occurs in intensified fed-batch and perfusion processes. Therefore, antifoaming agents are often added in these processes. However, the addition and accumulation of antifoaming agents may make cells shear-sensitive, leading to increased cell death.

[0006] Therefore, there is a need to develop a model to characterize acceptable cell culture conditions between various cell culture parameters (e.g., shear, media components and additives, and antifoam concentration) in production bioreactors. Summary of the invention

[0007] The present invention is based on, for example, the discovery that the presently claimed methods utilizing data obtained in test culture vessels (e.g., baffled shake flasks) can be used to accurately predict the effect of sensitizer concentration on cell viability in a production bioreactor, accurately select conditions that are optimal for cell viability in a production bioreactor, predict cell viability in a production bioreactor, and accurately identify a set of conditions that can be used to culture cells in a production bioreactor.

[0008] In one aspect, the present disclosure relates to a method for predicting the effect of sensitizer concentration on cell viability in a production bioreactor. The method involves (a) determining cell viability under multiple test conditions in a test culture vessel, wherein the multiple test conditions are defined by a set of parameters; and (b) predicting the effect of sensitizer concentration on cell viability in a production bioreactor based on the cell viability in the test culture vessel under the multiple conditions.

[0009] In some embodiments, the set of parameters includes shear stress, concentration of a sensitizer, and concentration of a protectant.

[0010] In some embodiments, the plurality of test conditions are selected based on a full factorial screening and / or an endocentric composite design.

[0011] In some embodiments, the sensitizer is a defoaming agent.

[0012] In some embodiments, the antifoaming agent is a polydimethylsiloxane-based antifoaming agent or dimethicone.

[0013] In some embodiments, the test culture container has a volume of about 5 mL to about 5 L, or has a volume of about 10 mL to about 500 mL. In some embodiments, the test culture container is a baffled shake flask.

[0014] In some embodiments, the production bioreactor has a volume of about 5 L to about 20,000 L, or has a volume of about 50 L to about 10,000 L. In some embodiments, the production bioreactor is a perfusion bioreactor or a fed-batch bioreactor.

[0015] In some embodiments, the method further comprises culturing the cells in the production bioreactor using a concentration of a sensitizer predicted to have a specific cell mortality rate of less than 40% per day during culturing of the cells in the production bioreactor.

[0016] In some embodiments, the method further comprises culturing the cells in the production bioreactor using a concentration of a sensitizer predicted to have a specific cell mortality rate of less than 20% per day during culturing of the cells in the production bioreactor.

[0017] In some embodiments, the method further comprises culturing the cells in the production bioreactor using a concentration of a sensitizer predicted to result in a cell specific LDH production of less than 150 nU / cell / day during culturing of the cells in the production bioreactor.

[0018] In some embodiments, the method further comprises culturing the cells in the production bioreactor using a concentration of a sensitizer predicted to result in a cell-specific LDH production of less than 100 nU / cell / day during culturing of the cells in the production bioreactor.

[0019] In some embodiments, the method further comprises culturing the cells in the production bioreactor using a concentration of a sensitizer predicted to result in an apparent growth rate of greater than 1% per day during culturing of the cells in the production bioreactor.

[0020] In some embodiments, the method further comprises culturing the cells in the production bioreactor using a sensitizer at a concentration predicted to result in an apparent growth rate of greater than 5% per day during culturing of the cells in the production bioreactor.

[0021] In some embodiments, the cells contain a nucleic acid encoding a recombinant therapeutic protein.

[0022] In some embodiments, the method further comprises collecting the recombinant therapeutic protein. In some embodiments, the method further comprises purifying the recombinant therapeutic protein. In some embodiments, the method further comprises formulating the purified recombinant therapeutic protein.

[0023] In some embodiments, the cell is a bacterial, yeast, or mammalian cell.

[0024] In some embodiments, cell viability is determined by measuring the concentration of lactate dehydrogenase (LDH) in the test culture vessel.

[0025] In some embodiments, the test culture vessel is a baffled shake flask and the plurality of test conditions include one or more of the following:

[0026] (a) rotary agitation at about 125 RPM to about 400 RPM;

[0027] (b) a sensitizer concentration of about 0 ppm to about 5000 ppm; and

[0028] (c) a protectant concentration of about 1 g / L to about 10 g / L.

[0029] In some embodiments, the protective agent is a poloxamer, a poloxamine, or a pluronic polyol.

[0030] In some embodiments, the poloxamer is poloxamer-188, poloxamer-401, poloxamer-402, or poloxamer-407.

[0031] In some embodiments, the poloxamine is poloxamine-904 or poloxamine-908.

[0032] In another aspect, the present disclosure relates to a method for increasing cell viability in a production bioreactor. The method relates to

[0033] (a) determining cell viability under a plurality of test conditions in a test culture vessel, wherein the plurality of conditions are defined by a set of parameters;

[0034] (b) selecting conditions that are optimal for cell viability in a production bioreactor based on the cell viability in the test culture vessel under the plurality of conditions; and

[0035] (c) cultivating the cells in the production bioreactor under selected conditions.

[0036] In some embodiments, the set of parameters includes shear stress, concentration of a sensitizer, and concentration of a protectant.

[0037] In some embodiments, the plurality of test conditions are selected based on a full factorial screening and / or an endocentric composite design.

[0038] In some embodiments, the sensitizer is a defoamer. In some embodiments, the defoamer is a polydimethylsiloxane-based defoamer or dimethicone.

[0039] In some embodiments, the test culture vessel has a volume of about 5 mL to about 5L.

[0040] In some embodiments, the test culture vessel has a volume of about 10 mL to about 500 mL. In some embodiments, the test culture vessel is a baffled shake flask.

[0041] In some embodiments, the production bioreactor has a volume of about 5 L to about 20,000 L. In some embodiments, the production bioreactor has a volume of about 50 L to about 10,000 L. In some embodiments, the production bioreactor is a perfusion bioreactor or a fed-batch bioreactor.

[0042] In some embodiments, step (c) results in a specific cell mortality rate of less than 40% per day during the process of culturing cells in the production bioreactor. In some embodiments, step (c) results in a specific cell mortality rate of less than 20% per day during the process of culturing cells in the production bioreactor. In some embodiments, step (c) results in a specific LDH production of cells less than 150nU / cell / day during the process of culturing cells in the production bioreactor. In some embodiments, step (c) results in a specific LDH production of cells less than 100nU / cell / day during the process of culturing cells in the production bioreactor. In some embodiments, step (c) results in an apparent growth rate greater than 1% per day during the process of culturing cells in the production bioreactor. In some embodiments, step (c) results in an apparent growth rate greater than 5% per day during the process of culturing cells in the production bioreactor.

[0043] In some embodiments, the cells contain a nucleic acid encoding a recombinant therapeutic protein.

[0044] In some embodiments, the method further comprises collecting the recombinant therapeutic protein. In some embodiments, the method further comprises purifying the recombinant therapeutic protein. In some embodiments, the method further comprises formulating the purified recombinant therapeutic protein.

[0045] In some embodiments, the cell is a bacterial, yeast or mammalian cell.In some embodiments, cell viability is determined by measuring the concentration of lactate dehydrogenase (LDH) in the test culture vessel.

[0046] In some embodiments, the test culture vessel is a baffled shake flask and the plurality of test conditions include one or more of the following:

[0047] (a) rotary agitation at about 125 RPM to about 400 RPM;

[0048] (b) a sensitizer concentration of about 0 ppm to about 5000 ppm; and

[0049] (c) a protectant concentration of about 1 g / L to about 10 g / L.

[0050] In some embodiments, the protective agent is a poloxamer, a poloxamine, or a pluronic polyol. In some embodiments, the poloxamer is poloxamer-188, poloxamer-401, poloxamer-402, or poloxamer-407. In some embodiments, the poloxamine is poloxamer-904 or poloxamer-908.

[0051] In another aspect, the present disclosure relates to a method for predicting cell viability in a production bioreactor. The method relates to

[0052] (a) selecting a set of parameters comprising at least two parameters;

[0053] (b) determining cell viability under a plurality of test conditions as defined by said set of parameters in a test culture vessel; and

[0054] (c) predicting the cell viability in the production bioreactor based on the cell viability in the test culture vessel under the plurality of conditions.

[0055] In some embodiments, the set of parameters includes shear stress, concentration of a sensitizer, and concentration of a protectant.

[0056] In some embodiments, the plurality of test conditions are selected based on a full factorial screening and / or an endocentric composite design.

[0057] In some embodiments, the sensitizer is a defoaming agent.

[0058] In some embodiments, the defoaming agent is a polydimethylsiloxane-based defoaming agent. In some embodiments, the defoaming agent is dimethicone.

[0059] In some embodiments, the test culture vessel has a volume of about 5 mL to about 5L.

[0060] In some embodiments, the test culture vessel has a volume of about 10 mL to about 500 mL.

[0061] In some embodiments, the test culture vessel is a baffled shake flask.

[0062] In some embodiments, the production bioreactor has a volume of about 5L to about 20,000L, or has a volume of about 50L to about 10,000L.

[0063] In some embodiments, the production bioreactor is a perfusion bioreactor or a fed-batch bioreactor.

[0064] In some embodiments, the method further comprises culturing the cells in the production bioreactor using conditions predicted to result in a specific cell mortality rate of less than 40% per day during culturing of the cells in the production bioreactor.

[0065] In some embodiments, the method further comprises culturing the cells in the production bioreactor using conditions predicted to result in a specific cell mortality rate of less than 20% per day during culturing of the cells in the production bioreactor.

[0066] In some embodiments, the method further comprises culturing the cells in the production bioreactor using conditions predicted to result in a cell-specific LDH production of less than 150 nU / cell / day during culturing of the cells in the production bioreactor.

[0067] In some embodiments, the method further comprises culturing the cells in the production bioreactor using conditions predicted to result in a cell-specific LDH production of less than 100 nU / cell / day during culturing of the cells in the production bioreactor.

[0068] In some embodiments, the method further comprises culturing the cells in the production bioreactor using conditions predicted to result in an apparent growth rate of greater than 1% per day during culturing of the cells in the production bioreactor.

[0069] In some embodiments, the method further comprises culturing the cells in the production bioreactor using conditions predicted to result in an apparent growth rate of greater than 5% per day during culturing of the cells in the production bioreactor.

[0070] In some embodiments, the cells contain a nucleic acid encoding a recombinant therapeutic protein.

[0071] In some embodiments, the method further comprises collecting the recombinant therapeutic protein. In some embodiments, the method further comprises purifying the recombinant therapeutic protein. In some embodiments, the method further comprises formulating the purified recombinant therapeutic protein.

[0072] In some embodiments, the cell is a bacterial, yeast, or mammalian cell.

[0073] In some embodiments, cell viability is determined by measuring the concentration of lactate dehydrogenase (LDH) in the test culture vessel.

[0074] In some embodiments, the test culture vessel is a baffled shake flask and the plurality of test conditions include one or more of the following:

[0075] (a) rotary agitation at about 125 RPM to about 400 RPM;

[0076] (b) a sensitizer concentration of about 0 ppm to about 5000 ppm; and

[0077] (c) a protectant concentration of about 1 g / L to about 10 g / L.

[0078] In some embodiments, the protective agent is a poloxamer, a poloxamine, or a pluronic polyol. In some embodiments, the poloxamer is poloxamer-188, poloxamer-401, poloxamer-402, or poloxamer-407. In some embodiments, the poloxamine is poloxamer-904 or poloxamer-908.

[0079] In another aspect, the present disclosure also provides a method for culturing cells in a production bioreactor, the method comprising culturing the cells in a liquid culture medium under one or more of the following conditions:

[0080] a) P / V (W / m 3 ) is from about 1.0 to about 110.0 (e.g., from about 10.0 to about 100.0, from about 20.0 to about 80.0, from about 40.0 to about 60.0, or about 6.0);

[0081] b) The air volume per minute per liquid volume (VVM) of the production bioreactor (min -1 ) is from about 0.01 to about 0.10 (e.g., from about 0.02 to about 0.08, from about 0.04 to about 0.06, or about 0.06);

[0082] c) the culture medium comprises a sensitizer at a concentration of about 0 ppm to about 5000 ppm (e.g., about 50 ppm to about 4000 ppm, about 1000 ppm to about 2000 ppm, or about 100 ppm); and

[0083] d) the liquid culture medium comprises a protectant at a concentration of about 1 g / L to about 15 g / L (e.g., about 2 g / L to about 10 g / L, about 3 g / L to about 7 g / L, about 4 g / L to about 6 g / L, or about 5 g / L).

[0084] In some embodiments, the sensitizer is a defoaming agent.

[0085] In some embodiments, the defoaming agent is a polydimethylsiloxane-based defoaming agent. In some embodiments, the defoaming agent is dimethicone.

[0086] In some embodiments, the protective agent is a poloxamer, a poloxamine, or a pluronic polyol.

[0087] In some embodiments, the poloxamer is poloxamer-188, poloxamer-401, poloxamer-402, or poloxamer-407. In some embodiments, the poloxamine is poloxamine-904 or poloxamine-908.

[0088] In some embodiments, the production bioreactor has a volume of about 5 L to about 20,000 L. In some embodiments, the production bioreactor has a volume of about 50 L to about 10,000 L. In some embodiments, the production bioreactor is a perfusion bioreactor or a fed-batch bioreactor.

[0089] In some embodiments, the method results in a specific cell mortality rate of less than 40% per day during the process of culturing cells in the production bioreactor. In some embodiments, the method results in a specific cell mortality rate of less than 20% per day during the process of culturing cells in the production bioreactor. In some embodiments, the method results in a specific LDH production of cells less than 150nU / cell / day during the process of culturing cells in the production bioreactor. In some embodiments, the method results in a specific LDH production of cells less than 100nU / cell / day during the process of culturing cells in the production bioreactor. In some embodiments, the method results in an apparent growth rate of greater than 1% per day during the process of culturing cells in the production bioreactor. In some embodiments, the method results in an apparent growth rate of greater than 5% per day during the process of culturing cells in the production bioreactor.

[0090] In some embodiments, the cells contain a nucleic acid encoding a recombinant therapeutic protein.

[0091] In some embodiments, the method further comprises collecting the recombinant therapeutic protein. In some embodiments, the method further comprises purifying the recombinant therapeutic protein. In some embodiments, the method further comprises formulating the purified recombinant therapeutic protein.

[0092] In some embodiments, the cell is a bacterial, yeast or mammalian cell.In some embodiments, cell viability is determined by measuring the concentration of lactate dehydrogenase (LDH) in the test culture vessel.

[0093] As used herein, the word "a" or "an" before a noun means one or more of the specific noun. For example, the phrase "a cell" means "one or more cells".

[0094] The term "protective agent" means a reagent that reduces the sensitivity of cells to shear stress (e.g., the sensitivity of mammalian cells) during cultivation in a container (e.g., a test culture container or a production bioreactor). The non-limiting example of a protective agent includes, for example, poloxamers (e.g., poloxamer-188, poloxamer-401, poloxamer-402, and poloxamer-407), poloxamines (e.g., poloxamer-904 or poloxamer-908), and pluronic polyols. Other examples of protective agents are described herein and are known in the art.

[0095] The term "sensitizer" means an agent added to and / or present in a liquid culture medium of a cell (e.g., a mammalian cell culture) that increases the sensitivity of the cell to shear stress during culture in a container (e.g., a test culture container or a production bioreactor). Non-limiting examples of sensitizers include defoamers (e.g., polydimethylsiloxane-based defoamers or dimethicone). Additional examples of sensitizers are described herein and are known in the art.

[0096] The term "mammalian cell" means any cell from or derived from any mammal (e.g., human, hamster, mouse, green monkey, rat, pig, cow, or rabbit). For example, a mammalian cell can be an immortalized cell. In some embodiments, the mammalian cell is a differentiated cell. In some embodiments, the mammalian cell is an undifferentiated cell. Non-limiting examples of mammalian cells are described herein. Other examples of mammalian cells are known in the art.

[0097] The terms "culturing" or "cell culture" mean the maintenance or proliferation of mammalian cells under a controlled set of physical conditions.

[0098] The term "culture of cells" or "cell culture" means a liquid medium containing a plurality of cells that are maintained or proliferated under a controlled set of physical conditions.

[0099] The term "liquid culture medium" means a fluid containing enough nutrients to allow cells (e.g., mammalian cells) to grow or proliferate in vitro. For example, the liquid culture medium may contain one or more of the following: amino acids (e.g., 20 kinds of amino acids), purines (e.g., hypoxanthine), pyrimidines (e.g., thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, nicotinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate. In some embodiments, the liquid culture medium may contain serum from mammals. In some embodiments, the liquid culture medium does not contain serum or other extracts (liquid culture medium of determined components) from mammals. In some embodiments, the liquid culture medium may contain trace metals, mammalian growth hormones, and / or mammalian growth factors. Another example of a liquid culture medium is a minimal culture medium (e.g., a culture medium containing only inorganic salts, a carbon source, and water). Non-limiting examples of liquid culture medium are described herein. Other examples of liquid culture medium are known in the art and are commercially available. The liquid culture medium may contain mammalian cells of any density. For example, as used herein, a volume of liquid culture medium removed from a bioreactor can be substantially free of mammalian cells.

[0100] The term "animal-derived component-free liquid culture medium" means a liquid culture medium that does not contain any components (eg, proteins or serum) derived from mammals.

[0101] The term "serum-free liquid medium" means a liquid medium that does not contain mammalian serum.

[0102] The term "serum-containing liquid medium" means a liquid medium containing mammalian serum.

[0103] The term "chemically defined liquid medium" is a term of art and means a liquid medium in which all chemical components are known. For example, a chemically defined liquid medium does not contain fetal bovine serum, bovine serum albumin, or human serum albumin, as these preparations typically contain a complex mixture of albumin and lipids.

[0104] The term "protein-free liquid culture medium" means a liquid culture medium that does not contain any protein (eg, any detectable protein).

[0105] The term "perfusion bioreactor" means a bioreactor containing a plurality of cells (e.g., mammalian cells) in a first liquid culture medium, wherein the culture of the cells present in the bioreactor includes periodically or continuously removing the first liquid culture medium and simultaneously or shortly thereafter adding a second liquid culture medium of substantially the same volume to the bioreactor. In some examples, the second liquid culture medium may be the same as the first liquid culture medium. In some examples, there is an incremental change (e.g., increase or decrease) in the volume of the first liquid culture medium removed and added over an incremental time period (e.g., a time period of about 24 hours, a time period between about 1 minute and about 24 hours, or a time period greater than 24 hours) during the culture time period (e.g., a daily re-feed rate of the culture medium). The fraction of the culture medium removed and replaced each day can vary according to the specific cells, initial inoculation density, and cell density at a specific time of the culture. "RV" or "reactor volume" means the volume of the culture medium present at the beginning of the culture process (e.g., the total volume of the culture medium present after inoculation).

[0106] The term "fed-batch bioreactor" is a term of art and means a bioreactor containing a plurality of cells (e.g., mammalian cells) in a first liquid culture medium, wherein the cultivation of the cells present in the bioreactor includes the periodic or continuous addition of a second liquid culture medium to the first liquid culture medium without substantially or significantly removing the first liquid culture medium or the second liquid culture medium from the cell culture. The second liquid culture medium may be the same as the first liquid culture medium. In some examples of fed-batch culture, the second liquid culture medium is a concentrated form of the first liquid culture medium. In some examples of fed-batch culture, the second liquid culture medium is added as a dry powder. In some examples of fed-batch culture, the second liquid culture medium is a completely different formulation than the first liquid culture medium.

[0107] The term "concentrated fed-batch bioreactor" is a term of art and means a bioreactor containing a plurality of cells (e.g., mammalian cells) in a first liquid culture medium, wherein the culturing of the cells present in the bioreactor includes periodically or continuously adding a second liquid culture medium to the first liquid culture medium while substantially or significantly removing the first liquid culture medium or a second liquid culture medium from the cell culture without removing the product from the bioreactor. The second liquid culture medium may be the same as the first liquid culture medium. In some examples of concentrated fed-batch culturing, the second liquid culture medium is a concentrated form of the first liquid culture medium. In some examples of concentrated fed-batch culturing, the second liquid culture medium is added as a dry powder. In some examples of concentrated fed-batch culturing, the second liquid culture medium is a completely different formulation of the first liquid culture medium.

[0108] "Rotational agitation" is a term well known in the art and refers to the movement of a shake flask in a generally circular manner (e.g., clockwise or counterclockwise) so as to, for example, increase the concentration of dissolved O2 in a liquid medium contained therein. Agitation can be performed using any method known in the art (e.g., an instrument that moves the shake flask in a circular or elliptical motion, such as a rotary shaker). Exemplary devices that can be used to perform rotational agitation are described herein. Additional examples of such devices are also known in the art and are commercially available.

[0109] The term "shake flask" means a container (e.g., a sterile container) that can hold a certain volume of liquid culture medium, has at least one gas permeable surface (e.g., one end with a gas permeable element such as a membrane, which can also serve as a sterile barrier) and / or at least one vented cover, and at least a portion of its shape is approximately truncated conical. For example, the shake flask can be a baffled shake flask or an Erlenmeyer flask, or any art-recognized modified form thereof.

[0110] The term "production bioreactor" is a term of art and refers to a large-scale bioreactor (e.g., having an internal volume of more than 5 L, 1,000 L, 5,000 L, 10,000 L, 20,000 L, 50,000 L, or 100,000 L). For example, the production bioreactor can be a perfusion bioreactor.

[0111] The term "test culture vessel" is a term of art and means a small-scale bioreactor (e.g., relative to a production bioreactor) having a volume of about 2 mL to about 500 mL. In some examples, the aspect ratio of the test culture vessel corresponds approximately to the aspect ratio of the production bioreactor in the methods described herein. In some embodiments, the test culture vessel is a shake flask (e.g., a baffled shake flask). Additional examples of test culture vessels are described herein and are known in the art.

[0112] The term "batch culture" is a term of art and means a container (e.g., a bioreactor) containing a plurality of cells (e.g., mammalian cells) in a liquid culture medium, wherein the culturing of the cells present in the container (e.g., a bioreactor) does not include the addition of substantial or significant amounts of fresh liquid culture medium to the cell culture and does not include the removal of substantial or significant amounts of liquid culture medium from the cell culture during the course of the culturing.

[0113] The term "fed-batch culture" is a term of art, and means a container (e.g., a production bioreactor) comprising a plurality of cells (e.g., mammalian cells) in a liquid culture medium, wherein the culture of the cells present in the container (e.g., a production bioreactor) includes adding fresh liquid culture medium to the container periodically or continuously during the culture process without removing the liquid culture medium in large quantities or significantly from the container. The fresh liquid culture medium may be the same as the liquid culture medium present in the container at the beginning of the culture. In some examples of fed-batch culture, the fresh liquid culture medium is a concentrated form of liquid culture medium present in the container at the beginning of the culture. In some examples of fed-batch culture, the fresh culture medium is added as a dry powder. In some examples of fed-batch culture, the fresh culture medium is a completely different formulation than the liquid culture medium present in the container at the beginning of the culture.

[0114] The approach described herein offers several advantages. For example, it can match different scales, predict delta parameters, and optimize existing design spaces. It can also improve the predictability / matching of performance across scales, create more representative small-scale models, increase process robustness, improve shear tolerance for process intensification, and provide guidance on the addition of sensitizers and protectants across different shear profiles ( Fig.12 ).

[0115] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, data entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification, including definitions, will prevail.

[0116] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims.

[0117] Specifically, the present invention includes but is not limited to the following:

[0118] 1. A method for predicting the effect of sensitizer concentration on cell viability in a production bioreactor, the method comprising:

[0119] (a) determining cell viability under a plurality of test conditions in a test culture vessel, wherein the plurality of test conditions are defined by a set of parameters; and

[0120] (b) predicting the effect of sensitizer concentration on cell viability in a production bioreactor based on the cell viability in the test culture vessel under the plurality of conditions.

[0121] 2. A method according to item 1, wherein the parameter group includes shear stress, concentration of sensitizer and concentration of protective agent.

[0122] 3. The method according to item 1 or item 2, wherein the multiple test conditions are selected based on full factorial screening and / or an inscribed central composite design.

[0123] 4. The method according to any one of items 1 to 3, wherein the sensitizer is a defoaming agent.

[0124] 5. The method according to item 4, wherein the defoaming agent is a polydimethylsiloxane-based defoaming agent.

[0125] 6. The method of claim 4, wherein the defoaming agent is dimethicone.

[0126] 7. The method of any one of items 1-6, wherein the volume of the test culture container is about 5 mL to about 5 L.

[0127] 8. The method of claim 7, wherein the volume of the test culture container is from about 10 mL to about 500 mL.

[0128] 9. The method according to any one of items 1 to 8, wherein the test culture vessel is a baffled shake flask.

[0129] 10. The method of any one of items 1-9, wherein the production bioreactor has a volume of about 5 L to about 20,000 L.

[0130] 11. A method according to claim 10, wherein the volume of the production bioreactor is about 50L to about 10,000L.

[0131] 12. A method according to any one of items 1 to 11, wherein the production bioreactor is a perfusion bioreactor.

[0132] 13. A method according to any one of items 1-11, wherein the production bioreactor is a fed-batch bioreactor.

[0133] 14. A method according to any one of items 1-13, which also includes culturing cells in the production bioreactor using a certain concentration of a sensitizer, wherein the concentration of the sensitizer is predicted to have a specific cell mortality rate of less than 40% per day during the process of culturing the cells in the production bioreactor.

[0134] 15. A method according to any one of items 1-13, which also includes culturing cells in the production bioreactor using a certain concentration of a sensitizer, wherein the concentration of the sensitizer is predicted to have a specific cell mortality rate of less than 20% per day during the process of culturing the cells in the production bioreactor.

[0135] 16. A method according to any one of items 1-13, which also includes culturing cells in the production bioreactor using a certain concentration of a sensitizer, wherein the concentration of the sensitizer is predicted to result in a cell-specific LDH production of less than 150nU / cell / day during the process of culturing the cells in the production bioreactor.

[0136] 17. A method according to any one of items 1-13, which also includes culturing cells in the production bioreactor using a certain concentration of a sensitizer, wherein the concentration of the sensitizer is predicted to result in a cell-specific LDH production of less than 100 nU / cell / day during the process of culturing the cells in the production bioreactor.

[0137] 18. A method according to any one of items 1-13, which also includes culturing cells in the production bioreactor using a certain concentration of a sensitizer, wherein the concentration of the sensitizer is predicted to result in an apparent growth rate of greater than 1% per day during the process of culturing the cells in the production bioreactor.

[0138] 19. A method according to any one of items 1-13, which also includes culturing cells in the production bioreactor using a certain concentration of a sensitizer, wherein the concentration of the sensitizer is predicted to result in an apparent growth rate of greater than 5% per day during the process of culturing the cells in the production bioreactor.

[0139] 20. A method according to any one of items 14-19, wherein the cell contains a nucleic acid encoding a recombinant therapeutic protein.

[0140] 21. The method of claim 20, further comprising collecting the recombinant therapeutic protein.

[0141] 22. The method of claim 21, further comprising purifying the recombinant therapeutic protein.

[0142] 23. The method of claim 22, further comprising formulating the purified recombinant therapeutic protein.

[0143] 24. A method according to any one of items 1-23, wherein the cell is a bacterial, yeast or mammalian cell.

[0144] 25. The method according to any one of items 1 to 24, wherein cell viability is determined by measuring the concentration of lactate dehydrogenase (LDH) in the test culture vessel.

[0145] 26. The method according to any one of items 1-8 and 10-25, wherein the test culture vessel is a baffled shake flask and the plurality of test conditions comprises one or more of the following:

[0146] (a) rotary agitation at about 125 RPM to about 400 RPM;

[0147] (b) a sensitizer concentration of about 0 ppm to about 120 ppm; and

[0148] (c) a protectant concentration of about 1 g / L to about 10 g / L.

[0149] 27. The method of item 2 or 26, wherein the protective agent is a poloxamer, a poloxamine or a pluronic polyol.

[0150] 28. The method of claim 27, wherein the poloxamer is poloxamer-188, poloxamer-401, poloxamer-402 or poloxamer-407.

[0151] 29. The method of claim 27, wherein the poloxamine is poloxamine-904 or poloxamine-908.

[0152] 30. A method for increasing cell viability in a production bioreactor, the method comprising:

[0153] (a) determining cell viability under a plurality of test conditions in a test culture vessel, wherein the plurality of conditions are defined by a set of parameters;

[0154] (b) selecting conditions that are optimal for cell viability in a production bioreactor based on the cell viability in the test culture vessel under the plurality of conditions; and

[0155] (d) cultivating the cells in the production bioreactor under selected conditions.

[0156] 31. A method according to item 30, wherein the parameter group includes shear stress, concentration of sensitizer and concentration of protective agent.

[0157] 32. A method according to item 30 or item 31, wherein the multiple test conditions are selected based on full factorial screening and / or an inscribed central composite design.

[0158] 33. A method according to item 31 or 32, wherein the sensitizer is a defoaming agent.

[0159] 34. A method according to item 33, wherein the defoaming agent is a polydimethylsiloxane-based defoaming agent.

[0160] 35. A method according to claim 33, wherein the defoaming agent is dimethicone.

[0161] 36. A method according to any one of items 30-35, wherein the volume of the test culture container is about 5 mL to about 5 L.

[0162] 37. A method according to claim 36, wherein the volume of the test culture container is about 10 mL to about 500 mL.

[0163] 38. A method according to any one of items 30 to 37, wherein the test culture vessel is a baffled shake flask.

[0164] 39. A method according to any one of items 30-38, wherein the volume of the production bioreactor is about 5L to about 20,000L.

[0165] 40. A method according to claim 39, wherein the volume of the production bioreactor is about 50L to about 10,000L.

[0166] 41. A method according to any one of items 30-40, wherein the production bioreactor is a perfusion bioreactor.

[0167] 42. A method according to any one of items 30-40, wherein the production bioreactor is a fed-batch bioreactor.

[0168] 43. A method according to any one of items 30-42, wherein step (c) results in a specific cell mortality rate of less than 40% per day during cultivation of the cells in the production bioreactor.

[0169] 44. A method according to any one of items 30-42, wherein step (c) results in a specific cell mortality rate of less than 20% per day during cultivation of the cells in the production bioreactor.

[0170] 45. A method according to any one of items 30-42, wherein step (c) results in a cell-specific LDH production of less than 150 nU / cell / day during culturing of the cells in the production bioreactor.

[0171] 46. ​​A method according to any one of items 30-42, wherein step (c) results in a cell-specific LDH production of less than 100 nU / cell / day during cultivation of the cells in the production bioreactor.

[0172] 47. A method according to any one of items 30-42, wherein step (c) results in an apparent growth rate of greater than 1% per day during cultivation of the cells in the production bioreactor.

[0173] 48. A method according to any one of items 30-42, wherein step (c) results in an apparent growth rate of greater than 5% per day during cultivation of the cells in the production bioreactor.

[0174] 49. A method according to any one of items 30-48, wherein the cell contains a nucleic acid encoding a recombinant therapeutic protein.

[0175] 50. The method of claim 49, further comprising collecting the recombinant therapeutic protein.

[0176] 51. The method of claim 50, further comprising purifying the recombinant therapeutic protein.

[0177] 52. The method of claim 51, further comprising formulating the purified recombinant therapeutic protein.

[0178] 53. A method according to any one of items 30-52, wherein the cell is a bacterial, yeast or mammalian cell.

[0179] 54. A method according to any one of items 30-53, wherein cell viability is determined by measuring the concentration of lactate dehydrogenase (LDH) in the test culture vessel.

[0180] 55. The method according to any one of items 30-37 and 39-54, wherein the test culture vessel is a baffled shake flask and the plurality of test conditions comprises one or more of the following:

[0181] (a) rotary agitation at about 125 RPM to about 400 RPM;

[0182] (b) a sensitizer concentration of about 0 ppm to about 120 ppm; and

[0183] (c) a protectant concentration of about 1 g / L to about 10 g / L.

[0184] 56. A method according to item 31 or 55, wherein the protective agent is a poloxamer, a poloxamine or a pluronic polyol.

[0185] 57. A method according to claim 56, wherein the poloxamer is poloxamer-188, poloxamer-401, poloxamer-402 or poloxamer-407.

[0186] 58. The method of claim 56, wherein the poloxamine is poloxamine-904 or poloxamine-908.

[0187] 59. A method for predicting cell viability in a production bioreactor, the method comprising:

[0188] (a) selecting a set of parameters comprising at least two parameters;

[0189] (b) determining cell viability under a plurality of test conditions as defined by said set of parameters in a test culture vessel; and

[0190] (c) predicting the cell viability in the production bioreactor based on the cell viability in the test culture vessel under the plurality of conditions.

[0191] 60. A method according to claim 59, wherein the parameter group includes shear stress, concentration of sensitizer and concentration of protective agent.

[0192] 61. A method according to claim 59 or claim 60, wherein the multiple test conditions are selected based on full factorial screening and / or an inscribed central composite design.

[0193] 62. A method according to item 60 or 61, wherein the sensitizer is a defoaming agent.

[0194] 63. A method according to item 62, wherein the defoaming agent is a polydimethylsiloxane-based defoaming agent.

[0195] 64. A method according to claim 62, wherein the defoaming agent is dimethicone.

[0196] 65. A method according to any one of items 59-64, wherein the volume of the test culture container is about 5 mL to about 5 L.

[0197] 66. A method according to claim 65, wherein the volume of the test culture container is about 10 mL to about 500 mL.

[0198] 67. A method according to any one of items 59-66, wherein the test culture vessel is a baffled shake flask.

[0199] 68. A method according to any one of items 59-67, wherein the volume of the production bioreactor is about 5L to about 20,000L.

[0200] 69. A method according to claim 68, wherein the volume of the production bioreactor is about 50L to about 10,000L.

[0201] 70. A method according to any one of items 59-69, wherein the production bioreactor is a perfusion bioreactor.

[0202] 71. A method according to any one of items 59-69, wherein the production bioreactor is a fed-batch bioreactor.

[0203] 72. A method according to any one of items 59-71, further comprising culturing cells in the production bioreactor using certain conditions, wherein the conditions are predicted to result in a specific cell mortality rate of less than 40% per day during the process of culturing the cells in the production bioreactor.

[0204] 73. A method according to any one of items 59-71, further comprising culturing cells in the production bioreactor using certain conditions, wherein the conditions are predicted to result in a specific cell mortality rate of less than 20% per day during the process of culturing the cells in the production bioreactor.

[0205] 74. A method according to any one of items 59-71, further comprising culturing cells in the production bioreactor using certain conditions, wherein the conditions are predicted to result in a cell-specific LDH production of less than 150 nU / cell / day during the process of culturing the cells in the production bioreactor.

[0206] 75. A method according to any one of items 59-71, further comprising culturing cells in the production bioreactor using certain conditions, wherein the conditions are predicted to result in a cell-specific LDH production of less than 100 nU / cell / day during the process of culturing the cells in the production bioreactor.

[0207] 76. A method according to any one of items 59-71, further comprising culturing cells in the production bioreactor using certain conditions, wherein the conditions are predicted to result in an apparent growth rate greater than 1% per day during the process of culturing the cells in the production bioreactor.

[0208] 77. A method according to any one of items 59-71, further comprising culturing cells in the production bioreactor using certain conditions, wherein the conditions are predicted to result in an apparent growth rate greater than 5% per day during the process of culturing the cells in the production bioreactor.

[0209] 78. A method according to any one of items 72-77, wherein the cell contains a nucleic acid encoding a recombinant therapeutic protein.

[0210] 79. The method of claim 78, further comprising collecting the recombinant therapeutic protein.

[0211] 80. The method of claim 79, further comprising purifying the recombinant therapeutic protein.

[0212] 81. The method of claim 80, further comprising formulating the purified recombinant therapeutic protein.

[0213] 82. A method according to any one of items 59-81, wherein the cell is a bacterial, yeast or mammalian cell.

[0214] 83. A method according to any one of items 59-82, wherein cell viability is determined by measuring the concentration of lactate dehydrogenase (LDH) in the test culture vessel.

[0215] 84. The method of any one of items 59-66 and 68-83, wherein the test culture vessel is a baffled shake flask and the plurality of test conditions comprises one or more of the following:

[0216] (a) rotary agitation at about 125 RPM to about 400 RPM;

[0217] (b) a sensitizer concentration of about 0 ppm to about 120 ppm; and

[0218] (c) a protectant concentration of about 1 g / L to about 10 g / L.

[0219] 85. A method according to item 60 or 84, wherein the protective agent is a poloxamer, a poloxamine or a pluronic polyol.

[0220] 86. A method according to claim 85, wherein the poloxamer is poloxamer-188, poloxamer-401, poloxamer-402 or poloxamer-407.

[0221] 87. A method according to claim 85, wherein the poloxamine is poloxamine-904 or poloxamine-908.

[0222] 88. A method for culturing cells in a production bioreactor, the method comprising culturing the cells in a liquid culture medium under one or more of the following conditions:

[0223] e) P / V (kW / m 3 ) is from about 1.0 to about 110 (e.g., from about 3.0 to about 8.0, from about 3.0 to about 7.0, from about 4.0 to about 6.0, from about 5.0 to about 6.0, or about 6.0);

[0224] f) The air volume per minute per liquid volume (VVM) of the production bioreactor (min -1 ) is from about 0.01 to about 0.10 (e.g., from about 0.02 to about 0.08, from about 0.04 to about 0.06, or about 0.06);

[0225] g) the culture medium comprises a sensitizer at a concentration of about 0 ppm to about 5,000 ppm (e.g., about 0 ppm to about 120 ppm, about 50 ppm to about 110 ppm, about 80 ppm to about 100 ppm, or about 100 ppm); and

[0226] h) the liquid culture medium comprises a protectant at a concentration of about 1 g / L to about 15 g / L (e.g., about 2 g / L to about 10 g / L, about 3 g / L to about 7 g / L, about 4 g / L to about 6 g / L, or about 5 g / L).

[0227] 89. A method according to claim 88, wherein the sensitizer is a defoaming agent.

[0228] 90. A method according to claim 89, wherein the defoaming agent is a polydimethylsiloxane-based defoaming agent.

[0229] 91. A method according to claim 89, wherein the defoaming agent is dimethicone.

[0230] 92. A method according to any one of items 88-91, wherein the protective agent is a poloxamer, a poloxamine or a pluronic polyol.

[0231] 93. A method according to item 92, wherein the poloxamer is poloxamer-188, poloxamer-401, poloxamer-402 or poloxamer-407.

[0232] 94. A method according to claim 92, wherein the poloxamine is poloxamine-904 or poloxamine-908.

[0233] 95. A method according to any one of items 88-94, wherein the volume of the production bioreactor is about 5L to about 20,000L.

[0234] 96. A method according to claim 95, wherein the volume of the production bioreactor is about 50L to about 10,000L.

[0235] 97. A method according to any one of items 88-96, wherein the production bioreactor is a perfusion bioreactor.

[0236] 98. A method according to any one of items 88-96, wherein the production bioreactor is a fed-batch bioreactor.

[0237] 99. A method according to any one of items 88-98, wherein the method results in a specific cell mortality rate of less than 40% per day during cultivation of the cells in the production bioreactor.

[0238] 100. A method according to any one of items 88-98, wherein the method results in a specific cell mortality rate of less than 20% per day during cultivation of the cells in the production bioreactor.

[0239] 101. A method according to any one of items 88-98, wherein the method results in a cell-specific LDH production of less than 150 nU / cell / day during cultivation of the cells in the production bioreactor.

[0240] 102. A method according to any one of items 88-98, wherein the method results in a cell-specific LDH production of less than 100 nU / cell / day during cultivation of the cells in the production bioreactor.

[0241] 103. A method according to any one of items 88-98, wherein the method results in an apparent growth rate of greater than 1% per day during cultivation of the cells in the production bioreactor.

[0242] 104. A method according to any one of items 88-98, wherein the method results in an apparent growth rate of greater than 5% per day during cultivation of the cells in the production bioreactor.

[0243] 105. A method according to any one of items 88-98, wherein the cell contains a nucleic acid encoding a recombinant therapeutic protein.

[0244] 106. The method according to claim 105, further comprising collecting the recombinant therapeutic protein.

[0245] 107. The method according to claim 106, further comprising purifying the recombinant therapeutic protein.

[0246] 108. The method of claim 107, further comprising formulating the purified recombinant therapeutic protein.

[0247] 109. A method according to any one of items 88-108, wherein the cell is a bacterial, yeast or mammalian cell.

[0248] 110. A method according to any one of items 88-109, wherein cell viability is determined by measuring the concentration of lactate dehydrogenase (LDH) in the test culture vessel. BRIEF DESCRIPTION OF THE DRAWINGS

[0249] Figure 1 is a diagram showing how bubbles in cell culture medium can cause cell death.

[0250] Figure 2 is a graph illustrating that the addition of a defoamer (an exemplary sensitizer) can move operating conditions out of the acceptable operating space.

[0251] Figure 3 It is a graph showing the relationship between specific lactate dehydrogenase (LDH) production and cell death rate.

[0252] Figure 4 is a graph showing the relationship between a protectant, a sensitizer, and rotational agitation, and the effects of the protectant, the sensitizer, and agitation on specific LDH production in cell culture.

[0253] Figure 5 It is a graph showing that the specific LDH production is a result of the sensitizer concentration, the protective agent concentration and the rotary stirring (shear stress).

[0254] Figure 6 The figure above shows a full factorial design (2 3 ), where the response is specific LDH production (mortality), and the factors are shear, protectant concentration, and sensitizer concentration. The middle graph shows that the failure leads to an extreme value, there is a large range between the failure point and the rest of the data, the failure point is difficult to model, and the extreme value is excluded (inoperable area). The lower graph shows that the center point is poorly predicted (indicating curvature, the response may be nonlinear, and there is no linear growth with increasing RPM).

[0255] Figure 7 is a graph showing the full factorial design combined with the inscribed central composite design for selected sensitizer concentration, protectant concentration, and rotational agitation (shear stress) parameters.

[0256] Figure 8 is a plot showing the advantage of combining a full factorial design with an internal central composite design.

[0257] Fig. 9 is a graph illustrating that the method described herein can be used to identify areas of poor performance (light shading) and areas of operational failure (dark shading).

[0258] Fig.10 Graph illustrating a method of normalizing shear stress across scales via specific LDH production and a method of predicting the impact of parameter changes (delta parameter).

[0259] Fig.11 is a diagram illustrating how the model described herein can be used to optimize operating conditions of a production bioreactor. The data show that the operating conditions (rectangles with filled open squares) are currently effective but can be further optimized (light shaded rectangles to the right of the large arrow) (e.g., increasing the concentration range of sensitizer tolerance in the process and improving resilience to foaming out of the vessel).

[0260] Fig.12 The capabilities and utility of the models described herein are listed. The models are able to match different scales, predict parameter changes, and optimize existing design spaces. The models also provide an increase in the predictability / matching of performance across scales (e.g., creating more representative small-scale models based on traditional methods and enhancing the understanding of shear across scales). The models also provide increased process robustness (e.g., improved shear tolerance for process intensification and improved guidance on sensitizer concentrations and protective agent addition concentrations across different shear distributions).

[0261] Fig.13A is a graph comparing the observed specific LDH production values ​​in the tested benchtop vessels with the predicted specific LDH production values.

[0262] Fig. 13B Data from the experiments described in Example 2 are depicted.

[0263] Fig. 13C Observed and predicted specific LDH production values ​​are listed.

[0264] Fig.14 The observed and predicted effects of defoamers on LDH production were compared.

[0265] Fig.15A is a graph comparing the observed specific LDH production values ​​in the production bioreactors tested with the predicted values.

[0266] Fig. 15BThe specific LDH production values ​​observed in the production bioreactors tested are listed along with the predicted values. DETAILED DESCRIPTION

[0267] The present disclosure relates to methods for optimizing process intensification for producing proteins or polypeptides. In one aspect, the present disclosure relates to small-scale test culture vessel (e.g., baffled shake flask) models that can characterize the operating space defined by various operating parameters (e.g., protectant (e.g., poloxamer-188) concentration, sensitizer (e.g., defoamer), shear stress (e.g., stirring rate or oxygen sparge rate), culture medium components and additives, pH and time, etc.). In particular, the methods described herein can be used to predict the effects of these operating parameters on cell culture growth and viability in production bioreactors. Orthogonal methods have been used to correlate shear stress between small-scale and larger-scale models, and the predicted operating space has been verified by data at benchtop and production scales.

[0268] Therefore, provided herein are methods for predicting the effect of sensitizer concentration on cell viability in a production bioreactor, methods for improving cell viability in a production bioreactor, methods for predicting cell viability in a production bioreactor, and methods for culturing cells in a production bioreactor. Non-limiting aspects of these methods are described herein, and the methods may be combined without limitation.

[0269] Process Intensification

[0270] Process intensification refers to improving manufacturing and processing by optimizing existing operating protocols so that the manufacturing process can be more precise and efficient. These improvements can bring a variety of advantages, such as increasing biomass, improving volumetric productivity, optimizing facility capacity utilization, reducing commodity costs, maintaining or improving product quality consistency, reducing facility footprint and / or reducing unit operations. At the molecular level, process intensification can also involve enhancing mixing, improving reaction kinetics and / or increasing yield and specificity.

[0271] The present disclosure relates to process intensification for producing proteins or polypeptides (e.g., recombinant therapeutic proteins, recombinant proteins, or recombinant polypeptides). One strategy to increase the production of recombinant therapeutic proteins or polypeptides is to increase cell density. High cell density generally requires high oxygen consumption, and the required oxygen sparging often results in foaming problems (i.e., bubbles). Figure 1 As shown, bubbles can cause cell death, for example, through cell-bubble contact, cell-bubble attachment, or lethal forces from bubble collapse. Equation 1 shows the relationship between cell death rate, volumetric aeration, and bubble size.

[0272]

[0273] Therefore, defoamers are usually added to the cell culture process. However, the addition and / or accumulation of defoamers may lead to a negative impact on cell culture performance (e.g., mammalian cell culture performance). In some cases, the addition and accumulation of defoamers may lead to an increase in cell mortality, particularly at a production scale (production bioreactor scale). Therefore, it is important to evaluate the impact of operating parameters or changes in these operating parameters (e.g., defoamers or some other parameters) at a production scale (production bioreactor scale).

[0274] Sensitizers, stirring and protective agents

[0275] Because it is extremely expensive to test the effects of various operating parameters at production scale (production bioreactor scale), pilot experiments are usually first performed in test culture vessels (e.g., shake flasks, laboratory fermenters, or pilot fermenters). These test culture vessels can be operated to test the effects of various parameters (e.g., stirring, sensitizer concentration, protectant concentration, temperature, pH, nutrient composition, biomass load, and feeding strategy) at a small scale, and the observed effects are associated with the effects at production scale (production bioreactor scale). In many traditional methods, power / volume (P / V), volumetric mass transfer coefficient (kLa), tip speed, mixing time, computational fluid dynamics (CFD) modeling are typically used to associate the results in the test culture vessels with the effects at production scale (production bioreactor scale).

[0276] Among these various parameters, the sensitizer parameter (e.g., the concentration of the sensitizer) is particularly difficult to scale up. In some embodiments of any of the methods described herein, the sensitizer can be a defoaming agent (e.g., dimethicone, dimethicone emulsion, or polydimethylsiloxane (PDMS)-based oil). A description of defoaming agents can be found, for example, in et al., "Mechanism of antifoaming action of simethicone," J. Appl. Toxicol. 14(3):207-211, 1994; and Bergeron et al., "Polydimethylsiloxane (PDMS)-based antifoams," Colloids and Surfaces A: Physicochemical and Engineering Aspects 122(1-3):103-120, 1997; both of which are incorporated herein by reference in their entirety.

[0277] Other examples of sensitizers include, for example, organic oil-based sensitizers, silicone oil-based sensitizers, and mixed liquid-solid mixtures / emulsified sensitizers (e.g., fumed silica, oil combination defoamers). The sensitizer may be a silicone foam control agent, such as ACP-0100 defoamer compound, ACP-1500 defoamer compound, AFE-1510 defoamer emulsion, AFE-1520 defoamer emulsion, AFE-1530 defoamer emulsion, ACP-1400 defoamer compound, AFE-0700 defoamer emulsion, ACP-1920 powder defoamer and PMX-200 silicone fluid. Additional examples of sensitizers include Dow Defoamer C emulsion, Defoamer 204 (Sigma Aldrich), Defoamer A (Sigma Aldrich), Defoamer B emulsion (Sigma Aldrich), Defoamer C emulsion (Sigma Aldrich), Defoamer Y-30 emulsion (Sigma Aldrich), Defoamer O-30 (Sigma Aldrich) and Defoamer SE-15 (Sigma Aldrich). A non-limiting example of an organic, non-silicone polypropylene-based polyether sensitizer is Defoamer 204. A non-limiting example of a dimethicone emulsion is Defoamer EX-Cell. Non-limiting examples of mixed solid-liquid defoamers include silicone oil (PDMS), Compound A, modified CA (MCA), Emulsion A (EA) and EA analogs (AEA) described in Marinova et al., Langmuir 17: 2426-2436, 2001. Non-limiting examples of antifoaming agents based on polydimethylsiloxane (PMSA) are described in Bergeron et al., Colloids and Surfaces A: Physiochemical and Engineering Aspects 122: 103-120, 1997.

[0278] Other examples of sensitizers are known in the art. See, for example, Rashmika et al., Biotechnol. Prog. 34(1):262-270, 2018.

[0279] Several other parameters (e.g., baffles, flask size, stirring) also have an impact on the sensitivity of cells to shear. Due to the interaction with other parameters, these sensitizer parameters (e.g., antifoam concentration) often have nonlinear relationships during scale-up and are geometrically independent across different scales. Therefore, traditional models are often powerless to predict the impact of sensitizer parameters at production scale.

[0280] What makes predictions more challenging is that sensitizer parameters can change over the production time period. For example, during production (e.g., culturing cells in a production bioreactor), cell density increases and requires higher oxygen sparging. Therefore, more defoamer is required and added to the production bioreactor. The increased defoamer concentration makes the cells more sensitive to shear stress and quickly moves the operating conditions out of the acceptable operating space ( Figure 2 ).

[0281] In addition, protective agent can protect cells from increased shear stress, and is usually added to liquid culture medium. The increased protective agent concentration may be necessary to alleviate the negative effects of sensitizers, but the interaction of protective agent with other parameters makes it difficult to determine the exact amount of required protective agent. Some common protective agents include, for example, poloxamer, poloxamine and / or pluronic polyols. In some embodiments, the protective agent is poloxamer (for example, poloxamer-188, poloxamer-401, poloxamer-402 or poloxamer-407). In some other embodiments, the protective agent is poloxamine (for example, poloxamer-904 or poloxamer-908). In some additional embodiments, the protecting agent is a Pluronic polyol (e.g., L-61, L-121, P-103, P-123, P-84, P-104, P-65, P-105, L-35, F-127, F-38, F-68, or F-108) or a reverse Pluronic polyol (e.g., 25R-2, 25R-4, 25R-5, 25R-8, 10R-5, or 10R-8). Detailed descriptions of exemplary protective agents can be found, for example, in Moghimi et al., "Poloxamers and poloxamines in nanoparticle engineering and experimental medicine," Trends in Biotechnology 18(10):412-420, 2000; Murhammer et al., "Structural features of nonionic polyglycol polymer molecules responsible for the protective effect in sparged animal cell bioreactors," Biotechnology Progress 6(2):142-148, 1990; Murhammer, "Pluronic polyols, cell protection," Encyclopedia of Bioprocess Technology (1999); each of which is incorporated herein by reference in its entirety.

[0282] Modeling Based on Factorial and Central Composite Designs

[0283] The present disclosure provides a modeling approach that captures the effects of multiple operating parameters (e.g., protectants (media components / additives, such as Pluronic), sensitizers (e.g., antifoaming agents), and shear stress (agitation)) on, for example, cell viability, cell mortality, recombinant therapeutic protein production or yield, with the goal of optimizing reactors at any scale based on results from testing culture vessel models.

[0284] In one aspect, the methods described herein can be used to predict cell viability in a production bioreactor. The method involves selecting a set of parameters including at least two (e.g., three, four, five, or six) parameters; determining the cell viability under multiple test conditions defined by the parameter set in a test culture container (e.g., any test culture container described herein); and predicting the cell viability in the production bioreactor based on the cell viability in the test culture container under the multiple conditions. Based on the prediction, conditions with increased cell viability can be identified. Also provided herein is a method for predicting the effect of sensitizer concentration on cell viability in a production bioreactor. The method involves determining the cell viability under multiple test conditions in a test culture container, wherein the multiple test conditions are defined by a set of parameters; and predicting the effect of sensitizer concentration on cell viability in a production bioreactor based on the cell viability in the test culture container under the multiple conditions.

[0285] The test culture container can be a rotary tube (e.g., a baffled rotary tube), a flask (e.g., a shake flask with a volume of about 3 L) (e.g., a baffled shake flask), an automated laboratory fermenter (Ambr 15 and Amber 250 from Sartorius), a laboratory fermenter, or a pilot fermenter. In some embodiments, the volume of the test culture container can be less than 5 L, less than 3 L, less than 1 L, less than 500 ml, or less than 100 ml (e.g., less than 1 L or 50 ml to 500 ml). In some embodiments, the volume (internal volume) of the test culture container is about 5 mL to about 5 L (e.g., about 10 mL to about 500 mL or about 2.5 L to about 3.5 L). For example, the volume (internal volume) of the test culture container can be about 5 mL to about 5 L, about 5 mL to about 4.5 L, about 5 mL to about 4.0 L, about 5 mL to about 3.5 L, about 5 mL to about 3.0 L, about 5 mL to about 2.5 L, about 5 mL to about 2 L, about 5 mL to about 1.5 L, about 5 mL to about 1 L, about 5 mL to about 900 mL, about 5 mL to about 800 mL, about 5 mL to about 700 mL, about 5 mL to about 600 mL, about 5 mL to about 500 mL, about 5 mL to about 450 mL, about 5 mL to about 400 mL, about 5 mL to about 350 mL, about 5 mL to about 300 mL, about 5 mL to about 2 50mL, about 5mL to about 200mL, about 5mL to about 150mL, about 5mL to about 100mL, about 5mL to about 80mL, about 5mL to about 60mL, about 5mL to about 50mL, about 5mL to about 40mL, about 5mL to about 30mL, about 5mL to about 25mL, about 5mL to about 20mL, about 5mL to about 15mL, about 5mL to about 10mL, about 10mL to about 5L, about 10mL to about 4.5L, about 10mL to about 4.0L, about 10mL to about 3.5L, about 10mL to about 3.0L, about 10mL to about 2.5L, about 10mL to about 2L, about 10mL to about 1.5L, about 10mL to about 1L, about 10mL to about 900mL, about 10mL to about 800mL, about 10mL to about 700mL, about 10mL to about 600mL, about 10mL to about 500mL, about 10mL to about 450mL, about 10mL to about 400mL, about 10mL to about 350mL, about 10mL to about 300mL, about 10mL to about 250mL, about 10mL to about 200mL, about 10mL to about 150mL, about 10mL to about 100mL, about 10mL to about 80mL, about 10mL to about 60mL, about 10mL to about 50mL, about 10mL to about 40mL, about 10mL to about 30mL, about 10mL to about about 25mL, about 10mL to about 20mL, about 10mL to about 15mL, about 15mL to about 5L, about 15mL to about 4.5L, about 15mL to about 4.0L, about 15mL to about 3.5L, about 15mL to about 3.0L, about 15mL to about 2.5L, about 15mL to about 2L, about 15mL to about 1.5L, about 15mL to about 1L, about 15mL to about 900mL, about 15mL to about 800mL, about 15mL to about 700mL, about 15mL to about 600mL, about 15mL to about 500mL, about 15mL to about 450mL, about 15mL to about 400mL, about 15mL to about 350mL, about 15mL to about 300mL, about 15 mL to about 250 mL, about 15 mL to about 200 mL, about 15 mL to about 150 mL, about 15 mL to about 100 mL, about 15 mL to about 80 mL, about 15 mL to about 60 mL, about 15 mL to about 50 mL, about 15 mL to about 40 mL, about 15 mL to about 30 mL, about 15 mL to about 25 mL, about 15 mL to about 20 mL, about 20 mL to about 5 L, about 20 mL to about 4.5 L, about 20 mL to about 4.0 L, about 20 mL to about 3.5 L, about 20 mL to about 3.0 L, about 20 mL to about 2.5 L, about 20 mL to about 2 L, about 20 mL to about 1.5 L, about 20 mL to about 1 L, about 20 mL to about 900 mL L, about 20mL to about 800mL, about 20mL to about 700mL, about 20mL to about 600mL, about 20mL to about 500mL, about 20mL to about 450mL, about 20mL to about 400mL, about 20mL to about 350mL, about 20mL to about 300mL, about 20mL to about 250mL, about 20mL to about 200mL, about 20mL to about 150mL, about 20mL to about 100mL, about 20mL to about 80mL, about 20mL to about 60mL, about 20mL to about 50mL, about 20mL to about 40mL, about 20mL to about 30mL, about 20mL to about 25mL, about 25mL to about 5L, about 25mL to about 4.5L, about 25mL to about 4.0L, about 25mL to about 3.5L, about 25mL to about 3.0L, about 25mL to about 2.5L, about 25mL to about 2L, about 25mL to about 1.5L, about 25mL to about 1L, about 25mL to about 900mL, about 25mL to about 800mL, about 25mL to about 700mL, about 25mL to about 600mL, about 25mL to about 500mL, about 25mL to about 450mL, about 25mL to about 400mL, about 25mL to about 350mL, about 25mL to about 300mL, about 25mL to about 250mL, about 25mL to about 200mL, about 25mL to about 150mL, about 25mL to about 10 0mL, about 25mL to about 80mL, about 25mL to about 60mL, about 25mL to about 50mL, about 25mL to about 40mL, about 25mL to about 30mL, about 30mL to about 5L, about 30mL to about 4.5L, about 30mL to about 4.0L, about 30mL to about 3.5L, about 30mL to about 3.0L, about 30mL to about 2.5L, about 30mL to about 2L, about 30mL to about 1.5L, about 30mL to about 1L, about 30mL to about 900mL, about 30mL to about 800mL, about 30mL to about 700mL, about 30mL to about 600mL, about 30mL to about 500mL, about 30mL to about 450mL, about 30mL to about L to about 400mL, about 30mL to about 350mL, about 30mL to about 300mL, about 30mL to about 250mL, about 30mL to about 200mL, about 30mL to about 150mL, about 30mL to about 100mL, about 30mL to about 80mL, about 30mL to about 60mL, about 30mL to about 50mL, about 30mL to about 40mL, about 40mL to about 5L, about 40mL to about 4.5L, about 40mL to about 4.0L, about 40mL to about 3.5L, about 40mL to about 3.0L, about 40mL to about 2.5L, about 40mL to about 2L, about 40mL to about 1.5L, about 40mL to about 1L, about 40mL to about 900mL L, about 40mL to about 800mL, about 40mL to about 700mL, about 40mL to about 600mL, about 40mL to about 500mL, about 40mL to about 450mL, about 40mL to about 400mL, about 40mL to about 350mL, about 40mL to about 300mL, about 40mL to about 250mL, about 40mL to about 200mL, about 40mL to about 150mL, about 40mL to about 100mL, about 40mL to about 80mL, about 40mL to about 60mL, about 40mL to about 50mL, about 50mL to about 5L, about 50mL to about 4.5L, about 50mL to about 4.0L, about 50mL to about 3.5L, about 50mL to about 3.0L, about 50mL to about 2.5L, about 50mL to about 2L, about 50mL to about 1.5L, about 50mL to about 1L, about 50mL to about 900mL, about 50mL to about 800mL, about 50mL to about 700mL, about 50mL to about 600mL, about 50mL to about 500mL, about 50mL to about 450mL, about 50mL to about 400mL, about 50mL to about 350mL, about 50mL to about 300mL, about 50mL to about 250mL, about 50mL to about 200mL, about 50mL to about 150mL, about 50mL to about 100mL, about 50mL to about about 80mL, about 50mL to about 60mL, about 60mL to about 5L, about 60mL to about 4.5L, about 60mL to about 4.0L, about 60mL to about 3.5L, about 60mL to about 3.0L, about 60mL to about 2.5L, about 60mL to about 2L, about 60mL to about 1.5L, about 60mL to about 1L, about 60mL to about 900mL, about 60mL to about 800mL, about 60mL to about 700mL, about 60mL to about 600mL, about 60mL to about 500mL, about 60mL to about 450mL, about 60mL to about 400mL, about 60mL to about 350mL , about 60mL to about 300mL, about 60mL to about 250mL, about 60mL to about 200mL, about 60mL to about 150mL, about 60mL to about 100mL, about 60mL to about 80mL, about 80mL to about 5L, about 80mL to about 4.5L, about 80mL to about 4.0L, about 80mL to about 3.5L, about 80mL to about 3.0L, about 80mL to about 2.5L, about 80mL to about 2L, about 80mL to about 1.5L, about 80mL to about 1L, about 80mL to about 900mL, about 80mL to about 800mL, about 80mL to about 700mL, about 80mL to about L to about 600mL, about 80mL to about 500mL, about 80mL to about 450mL, about 80mL to about 400mL, about 80mL to about 350mL, about 80mL to about 300mL, about 80mL to about 250mL, about 80mL to about 200mL, about 80mL to about 150mL, about 80mL to about 100mL, about 100mL to about 5L, about 100mL to about 4.5L, about 100mL to about 4.0L, about 100mL to about 3.5L, about 100mL to about 3.0L, about 100mL to about 2.5L, about 100mL to about 2L, about 100mL to about 1.5L, about 100mL to about 1L, about 100mL to about 900mL, about 100mL to about 800mL, about 100mL to about 700mL, about 100mL to about 600mL, about 100mL to about 500mL, about 100mL to about 450mL, about 100mL to about 400mL, about 100mL to about 350mL, about 100mL to about 300mL, about 100mL to about 250mL, about 100mL to about 200mL, about 100mL to about 150mL, about 150mL to about 5L, about 150mL to about 4.5L, about 150mL to about 4.0L, about 150mL to about 3.5L, about 150mL to about 3.0L, about 15 0mL to about 2.5L, about 150mL to about 2L, about 150mL to about 1.5L, about 150mL to about 1L, about 150mL to about 900mL, about 150mL to about 800mL, about 150mL to about 700mL, about 150mL to about 600mL, about 150mL to about 500mL, about 150mL to about 450mL, about 150mL to about 400mL, about 150mL to about 350mL, about 150mL to about 300mL, about 150mL to about 250mL, about 150mL to about 200mL, about 200mL to about 5L, about 200mL to about 4.5L, about 200mL to about 4.0L, about 200mL to about 3.5L, From about 200 mL to about 3.0 L, from about 200 mL to about 2.5 L, from about 200 mL to about 2 L, from about 200 mL to about 1.5 L, from about 200 mL to about 1 L, from about 200 mL to about 900 mL, from about 200 mL to about 800 mL, from about 200 mL to about 700 mL, from about 200 mL to about 600 mL, from about 200 mL to about 500 mL, from about 200 mL to about 450 mL, from about 200 mL to about 400 mL, from about 200 mL to about 350 mL, from about 200 mL to about 300 mL, from about 200 mL to about 250 mL, from about 250 mL to about 5 L, from about 250 mL to about 4.5 L, from about 250 mL to about 4.0 L, from about 250 mL to about 3.5 L, about 250mL to about 3.0L, about 250mL to about 2.5L, about 250mL to about 2L, about 250mL to about 1.5L, about 250mL to about 1L, about 250mL to about 900mL, about 250mL to about 800mL, about 250mL to about 700mL, about 250mL to about 600mL, about 250mL to about 500mL, about 250mL to about 450mL, about 250mL to about 400mL, about 250mL to about 350mL, about 250mL to about 300mL, about 300mL to about 5L, about 300mL to about 4.5L, about 300mL to about 4.0L, about 300mL to about 3.5L, about 300mL to about 3.0L, about 300mL to about 2.5L, about 300mL to about 2L, about 300mL to about 1.5L, about 300mL to about 1L, about 300mL to about 900mL, about 300mL to about 800mL, about 300mL to about 700mL, about 300mL to about 600mL, about 300mL to about 500mL, about 300mL to about 450mL, about 300mL to about 400mL, about 300mL to about 350mL, about 350mL to about 5L, about 350mL to about 4.5L, about 350mL to about 4.0L, about 350mL to about 3.5L, about 350mL to about 3.0L, about 350mL to about 2.5L, about 350mL to about 2L, about 350mL to about 1.5L, about 350mL to about 1L, about 350mL to about 900mL, about 350mL to about 800mL, about 350mL to about 700mL, about 350mL to about 600mL, about 350mL to about 500mL, about 350mL to about 450mL, about 350mL to about 400mL, about 400mL to about 5L, about 400mL to about 4.5L, about 400mL to about 4.0L, about 400mL to about 3.5L, about 400mL to about 3.0L, about 400mL to about 2.5L, about 400mL to about 2L, about 400mL to about 1.5L, about 400mL to about 1L, about 400mL to about 900mL , about 450mL to about 1L, about 450mL to about 900mL, about 450mL to about 800mL, about 400mL to about 700mL, about 400mL to about 600mL, about 400mL to about 500mL, about 400mL to about 450mL, about 450mL to about 5L, about 450mL to about 4.5L, about 450mL to about 4.0L, about 450mL to about 3.5L, about 450mL to about 3.0L, about 450mL to about 2.5L, about 450mL to about 2L, about 450mL to about 1.5L, about 450mL to about 1L, about 450mL to about 900mL, about 450mL to about 800mL, about 450mL to about 700mL, about 450mL to about 600mL, about 450mL to about about 500mL, about 500mL to about 5L, about 500mL to about 4.5L, about 500mL to about 4.0L, about 500mL to about 3.5L, about 500mL to about 3.0L, about 500mL to about 2.5L, about 500mL to about 2L, about 500mL to about 1.5L, about 500mL to about 1L, about 500mL to about 900mL, about 500mL to about 800mL, about 500mL to about 700mL, about 500mL to about 600mL, about 600mL to about 5L, about 600mL to about 4.5L, about 600mL to about 4.0L, about 600mL to about 3.5L, about 600mL to about 3.0L, about 600mL to about 2.5L, about 600mL to about 2L, about 600mL to about 1.5L, about 600mL to about 1L, about 600mL to about 900mL, about 600mL to about 800mL, about 600mL to about 700mL, about 700mL to about 5L, about 700mL to about 4.5L, about 700mL to about 4.0L, about 700mL to about 3.5L, about 700mL to about 3.0L, about 700mL to about 2.5L, about 700mL to about 2L, about 700mL to about 1.5L, about 700mL to about 1L, about 700mL to about 900mL, about 700mL to about 800mL L to about 800mL, about 800mL to about 5L, about 800mL to about 4.5L, about 800mL to about 4.0L, about 800mL to about 3.5L, about 800mL to about 3.0L, about 800mL to about 2.5L, about 800mL to about 2L, about 800mL to about 1.5L, about 800mL to about 1L, about 800mL to about 900mL, about 900mL to about 5L, about 900mL to about 4.5L, about 900mL to about 4.0L, about 900mL to about 3.5L, about 900mL to about 3.0L, about 900mL to about 2.5L, From about 900 mL to about 2 L, from about 900 mL to about 1.5 L, from about 900 mL to about 1 L, from about 1 L to about 5 L, from about 1 L to about 4.5 L, from about 1 L to about 4.0 L, from about 1 L to about 3.5 L, from about 1 L to about 3.0 L, from about 1 L to about 2.5 L, from about 1 L to about 2 L, from about 1 L to about 1.5 L, from about 1.5 L to about 5 L, from about 1.5 L to about 4.5 L, from about 1.5 L to about 4.0 L, from about 1.5 L to about 3.5 L, from about 1.5 L to about 3.0 L, from about 1.5 L to about 2.5 L, from about 1.5 L to about 2 L, from about 2 L to about 5 L, from about 2 L to about 4.5 L, In some embodiments, the test culture vessel has a height to width ratio that is approximately the same as the height to width ratio of the production bioreactor. .

[0286] In some embodiments, the volume of the liquid culture medium placed in the test culture container (during the culture period) can be in the range of about 0.5 mL to about 1.5 L, about 0.5 mL to about 1.4 L, about 0.5 mL to about 1.3 L, about 0.5 mL to about 1.2 L, about 0.5 mL to about 1.1 L, about 0.5 mL to about 1.0 L, about 0.5 mL to about 900 mL, about 0.5 mL to about 800 mL, about 0.5 mL to about 700 mL, about 0.5 mL to about 600 mL, about 0.5 mL to about 500 mL, about 0.5 mL to about 400 mL. , about 0.5mL to about 300mL, about 0.5mL to about 250mL, about 0.5mL to about 200mL, about 0.5mL to about 150mL, about 0.5mL to about 100mL, about 0.5mL to about 80mL, about 0.5mL to about 60mL, about 0.5mL to about 50mL, about 0.5mL to about 40mL, about 0.5mL to about 30mL, about 0.5mL to about 25mL, about 0.5mL to about 20mL, about 0.5mL to about 10mL, about 0.5mL to about 5mL, about 0.5mL to about 2.5mL, about 2. 5mL to about 1.5L, about 2.5mL to about 1.4L, about 2.5mL to about 1.3L, about 0.5mL to about 1.2L, about 0.5mL to about 1.1L, about 0.5mL to about 1.0L, about 2.5mL to about 900mL, about 2.5mL to about 800mL, about 2.5mL to about 700mL, about 2.5mL to about 600mL, about 2.5mL to about 500mL, about 2.5mL to about 400mL, about 2.5mL to about 300mL, about 2.5mL to about 250mL, about 2.5mL to about 200mL, about 2.5mL to about 350mL, about 2.5mL to about 400mL. 5mL to about 150mL, about 2.5mL to about 100mL, about 2.5mL to about 80mL, about 2.5mL to about 60mL, about 2.5mL to about 50mL, about 2.5mL to about 40mL, about 2.5mL to about 30mL, about 2.5mL to about 25mL, about 2.5mL to about 20mL, about 2.5mL to about 10mL, about 2.5mL to about 5mL, about 5mL to about 1.5L, about 5mL to about 1.4L, about 5mL to about 1.3L, about 5mL to about 1.2L, about 5mL to about 1.1L, about 5mL to about 1.0L, about 5mL to about 900mL, about 5mL to about 800mL, about 5mL to about 700mL, about 5mL to about 600mL, about 5mL to about 500mL, about 5mL to about 400mL, about 5mL to about 300mL, about 5mL to about 250mL, about 5mL to about 200mL, about 5mL to about 150mL, about 5mL to about 100mL, about 5mL to about 80mL, about 5mL to about 60mL, about 5mL to about 50mL, about 5mL to about 40mL, about 5mL to about 30mL, about 5mL to about 25mL, about 5mL to about 20mL, about 5mL to about 150mL, 0mL, about 10mL to about 1.5L, about 10mL to about 1.4L, about 10mL to about 1.3L, about 10mL to about 1.2L, about 10mL to about 1.1L, about 10mL to about 1.0L, about 10mL to about 900mL, about 10mL to about 800mL, about 10mL to about 700mL, about 10mL to about 600mL, about 10mL to about 500mL, about 10mL to about 400mL, about 10mL to about 300mL, about 10mL to about 250mL, about 10mL to about 200mL, about 10mL to about 150mL, about 10mL to about 100mL, about 10mL to about 80mL, about 10mL to about 60mL, about 10mL to about 50mL, about 10mL to about 40mL, about 10mL to about 30mL, about 10mL to about 25mL, about 10mL to about 20mL, about 20mL to about 1.5L, about 20mL to about 1.4L, about 20mL to about 1.3L, about 20mL to about 1.2L, about 20mL to about 1.1L, about 20mL to about 1.0L, about 20mL to about 900mL, about 20mL to about 800mL, about 20mL to about 700mL, about 20mL to about 600mL, about 20mL to about 500mL , about 20mL to about 400mL, about 20mL to about 300mL, about 20mL to about 250mL, about 20mL to about 200mL, about 20mL to about 150mL, about 20mL to about 100mL, about 20mL to about 80mL, about 20mL to about 60mL, about 20mL to about 50mL, about 20mL to about 40mL, about 20mL to about 30mL, about 20mL to about 25mL, about 25mL to about 1.5L, about 25mL to about 1.4L, about 25mL to about 1.3L, about 25mL to about 1.2L, about 25mL to about 1.1L, about 25mL to about 1.0L, about 25mL to about 900mL, about 25mL to about 800mL, about 25mL to about 700mL, about 25mL to about 600mL, about 25mL to about 500mL, about 25mL to about 400mL, about 25mL to about 300mL, about 25mL to about 250mL, about 25mL to about 200mL, about 25mL to about 150mL, about 25mL to about 100mL, about 25mL to about 80mL, about 25mL to about 60mL, about 25mL to about 50mL, about 25mL to about 40mL, about 25mL to about 30mL, about 30mL to about 1.5L, about 30mL to about 1.4L, about 30mL to about 1.3L, about 30mL to about about 1.2L, about 30mL to about 1.1L, about 30mL to about 1.0L, about 30mL to about 900mL, about 30mL to about 800mL, about 30mL to about 700mL, about 30mL to about 600mL, about 30mL to about 500mL, about 30mL to about 400mL, about 30mL to about 300mL, about 30mL to about 250mL, about 30mL to about 200mL, about 30mL to about 150mL, about 30mL to about 100mL, about 30mL to about 80mL, about 30mL to about 60mL, about 30mL to about 50mL, about 30mL to about 40mL, about 40mL to about 1.5L, about 40mL to about 1.4L, about 40 40mL to about 1.3L, about 40mL to about 1.2L, about 40mL to about 1.1L, about 40mL to about 1.0L, about 40mL to about 900mL, about 40mL to about 800mL, about 40mL to about 700mL, about 40mL to about 600mL, about 40mL to about 500mL, about 40mL to about 400mL, about 40mL to about 300mL, about 40mL to about 250mL, about 40mL to about 200mL, about 40mL to about 150mL, about 40mL to about 100mL, about 40mL to about 80mL, about 40mL to about 60mL, about 40mL to about 50mL, about 50mL to about 1.5L, about 50mL to about 1.4L, From about 50 mL to about 1.3 L, from about 50 mL to about 1.2 L, from about 50 mL to about 1.1 L, from about 50 mL to about 1.0 L, from about 50 mL to about 900 mL, from about 50 mL to about 800 mL, from about 50 mL to about 700 mL, from about 50 mL to about 600 mL, from about 50 mL to about 500 mL, from about 50 mL to about 400 mL, from about 50 mL to about 300 mL, from about 50 mL to about 250 mL, from about 50 mL to about 200 mL, from about 50 mL to about 150 mL, from about 50 mL to about 100 mL, from about 50 mL to about 80 mL, from about 50 mL to about 60 mL, from about 60 mL to about 1.5 L, from about 60 mL to about 1.4 L, from about 60 mL to about 1.3L, about 60mL to about 1.2L, about 60mL to about 1.1L, about 60mL to about 1.0L, about 60mL to about 900mL, about 60mL to about 800mL, about 60mL to about 700mL, about 60mL to about 600mL, about 60mL to about 500mL, about 60mL to about 400mL, about 60mL to about 300mL, about 60mL to about 250mL, about 60mL to about 200mL, about 60mL to about 150mL, about 60mL to about 100mL, about 60mL to about 80mL, about 80mL to about 1.5L, about 80mL to about 1.4L, about 80mL to about 1 .3L, about 80mL to about 1.2L, about 80mL to about 1.1L, about 80mL to about 1.0L, about 80mL to about 900mL, about 80mL to about 800mL, about 80mL to about 700mL, about 80mL to about 600mL, about 80mL to about 500mL, about 80mL to about 400mL, about 80mL to about 300mL, about 80mL to about 250mL, about 80mL to about 200mL, about 80mL to about 150mL, about 80mL to about 100mL, about 100mL to about 1.5L, about 100mL to about 1.4L, about 100mL to about 1.3L, about 100mL to about L to about 1.2L, about 100mL to about 1.1L, about 100mL to about 1.0L, about 100mL to about 900mL, about 100mL to about 800mL, about 100mL to about 700mL, about 100mL to about 600mL, about 100mL to about 500mL, about 100mL to about 400mL, about 100mL to about 300mL, about 100mL to about 250mL, about 100mL to about 200mL, about 100mL to about 150mL, about 150mL to about 1.5L, about 150mL to about 1.4L, about 150mL to about 1.3L, about 150mL to about 1.2L, From about 150 mL to about 1.1 L, from about 150 mL to about 1.0 L, from about 150 mL to about 900 mL, from about 150 mL to about 800 mL, from about 150 mL to about 700 mL, from about 150 mL to about 600 mL, from about 150 mL to about 500 mL, from about 150 mL to about 400 mL, from about 150 mL to about 300 mL, from about 150 mL to about 250 mL, from about 150 mL to about 200 mL, from about 200 mL to about 1.5 L, from about 200 mL to about 1.4 L, from about 200 mL to about 1.3 L, from about 200 mL to about 1.2 L, from about 200 mL to about 1.1 L, from about 200 mL to about 1.0L, about 200mL to about 900mL, about 200mL to about 800mL, about 200mL to about 700mL, about 200mL to about 600mL, about 200mL to about 500mL, about 200mL to about 400mL, about 200mL to about 300mL, about 200mL to about 250mL, about 250mL to about 1.5L, about 250mL to about 1.4L, about 250mL to about 1.3L, about 250mL to about 1.2L, about 250mL to about 1.1L, about 250mL to about 1.0L, about 250mL to about 900mL, about 250mL to about 800mL, about 250mL to about 700mL, about 250mL to about 600mL, about 250mL to about 500mL, about 250mL to about 400mL, about 250mL to about 300mL, about 300mL to about 1.5L, about 300mL to about 1.4L, about 300mL to about 1.3L, about 300mL to about 1.2L, about 300mL to about 1.1L, about 300mL to about 1.0L, about 300mL to about 900mL, about 300mL to about 800mL, about 300mL to about 700mL, about 300mL to about 600mL, about 300mL to about 500mL, about 300mL to about 400mL, about 400mL to about 1.5L, about 400mL to about 1.4L, about 400mL to about 1.3L, about 400mL To about 1.2L, about 400mL to about 1.1L, about 400mL to about 1.0L, about 400mL to about 900mL, about 400mL to about 800mL, about 400mL to about 700mL, about 400mL to about 600mL, about 400mL to about 500mL, about 500mL to about 1.5L, about 500mL to about 1.4L, about 500mL to about 1.3L, about 500mL to about 1.2L, about 500mL to about 1.1L, about 500mL to about 1.0L, about 500mL to about 900mL, about 500mL to about 800mL, about 500mL to about 700mL, about 500mL to about 600mL, about 600mL to about 1.5L , about 600mL to about 1.4L, about 600mL to about 1.3L, about 600mL to about 1.2L, about 600mL to about 1.1L, about 600mL to about 1.0L, about 600mL to about 900mL, about 600mL to about 800mL, about 600mL to about 700mL, about 700mL to about 1.5L, about 700mL to about 1.4L, about 700mL to about 1.3L, about 700mL to about 1.2L, about 700mL to about 1.1L, about 700mL to about 1.0L, about 700mL to about 900mL, about 700mL to about 800mL, about 800mL to about 1.5L, about 800mL to about 1.4L, about 800mL to about 1.3L, about 700mL to about 1.2L, about 700mL to about 1.1L, about 700mL to about 1.0L, about 700mL to about 900mL, about 700mL to about 800mL, about 800mL to about 1.5L, about 800mL to about 1.4L, about 800mL to about3L, about 800mL to about 1.2L, about 800mL to about 1.1L, about 800mL to about 1.0L, about 800mL to about 900mL, about 900mL to about 1.5L, about 900mL to about 1.4L, about 900mL to about 1.3L, about 900mL to about 1.2L, about 900mL to about 1.1L, about 900mL to about 1.0L, about 1.0L to about 1.5L, about 1.0L to about 1.4L , about 1.0L to about 1.3L, about 1.0L to about 1.2L, about 1.0L to about 1.1L, about 1.1L to about 1.5L, about 1.1L to about 1.4L, about 1.1L to about 1.3L, about 1.1L to about 1.2L, about 1.2L to about 1.5L, about 1.2L to about 1.4L, about 1.2L to about 1.3L, about 1.3L to about 1.5L, about 1.3L to about 1.4L, or about 1.4L to about 1.5L. .

[0287] In some embodiments, the test culture container is a shake flask, such as a shake flask with baffles. The volume of the shake flask can be, for example, about 250 mL to about 300 mL, about 300 mL to about 400 mL, or about 400 mL to about 500 mL. The shake flask can include at least one breathable surface (e.g., at least one surface with a breathable membrane, which can also be used as a sterile barrier) and / or at least one vent cap. The shake flask can have a structure on its outer surface that allows the shake flask to be stably placed in a tissue culture box (e.g., a rotary incubator).

[0288] The inner surface of the shake flask can have at least one coating (e.g., at least one gelatin, collagen, poly-L-ornithine, polystyrene, and laminin coating). Exemplary shake flasks that can be used for any of the methods described herein are available from Corning Inc. (Tewksbury, Massachusetts), Presens (Brundby, Denmark), Nalge-Nunc International (Rochester, New York). Exemplary shake flasks include baffled shake flasks or conical flasks, or any art-recognized modifications thereof. Additional examples of shake flasks (e.g., different shapes and sizes of shake flasks) and inner surface coatings of shake flasks are known in the art and can be used in the methods of the present invention.

[0289] In some embodiments, the rotational agitation of the test culture container can be about 125RPM to about 400RPM (e.g., about 150RPM to about 350RPM or about 200RPM to about 300RPM). For example, the rotational agitation of the test culture container can be about 125RPM to about 400RPM, about 125RPM to about 380RPM, about 125RPM to about 360RPM, about 125RPM to about 340RPM, about 125RPM to about 320RPM, about 125RPM to about 300RPM, about 125RPM to about 280RPM, about 125RPM to about 260RPM, about 125RPM to about 240RPM, about 125RPM to about 220RPM, about 125RPM to about 200RPM, about 125RPM to about 180RPM, about 125RPM to about 160RPM, about The present invention relates to a method for preparing an air flowmeter having a plurality of moving parts and a plurality of moving parts, wherein the moving parts are selected from the group consisting of: 125RPM to about 140RPM, about 140RPM to about 400RPM, about 140RPM to about 380RPM, about 140RPM to about 360RPM, about 140RPM to about 340RPM, about 140RPM to about 320RPM, about 140RPM to about 300RPM, about 140RPM to about 280RPM, about 140RPM to about 260RPM, about 140RPM to about 240RPM, about 140RPM to about 220RPM, about 140RPM to about 200RPM, about 140RPM to about 180RPM, about 140RPM to about 160RPM, about 160RPM to about 180RPM. RPM is about 400RPM, about 160RPM to about 380RPM, about 160RPM to about 360RPM, about 160RPM to about 340RPM, about 160RPM to about 320RPM, about 160RPM to about 300RPM, about 160RPM to about 280RPM, about 160RPM to about 260RPM, about 160RPM to about 240RPM, about 160RPM to about 220RPM, about 160RPM to about 200RPM, about 160RPM to about 180RPM, about 180RPM to about 400RPM, about 180RPM to about 380RPM, about 180RPM to about 360RPM 0RPM, about 180RPM to about 340RPM, about 180RPM to about 320RPM, about 180RPM to about 300RPM, about 180RPM to about 280RPM, about 180RPM to about 260RPM, about 180RPM to about 240RPM, about 180RPM to about 220RPM, about 180RPM to about 200RPM, about 200RPM to about 400RPM, about 200RPM to about 380RPM, about 200RPM to about 360RPM, about 200RPM to about 340RPM, about 200RPM to about 320RPM, about 200RPM to about 300RPM,From about 200RPM to about 280RPM, from about 200RPM to about 260RPM, from about 200RPM to about 240RPM, from about 200RPM to about 220RPM, from about 220RPM to about 400RPM, from about 220RPM to about 380RPM, from about 220RPM to about 360RPM, from about 220RPM to about 340RPM, from about 220RPM to about 320RPM, from about 220RPM to about 300RPM, from about 220RPM to about 280RPM, from about 220RPM to about 26 ... RPM to about 240RPM, about 240RPM to about 400RPM, about 240RPM to about 380RPM, about 240RPM to about 360RPM, about 240RPM to about 340RPM, about 240RPM to about 320RPM, about 240RPM to about 300RPM, about 240RPM to about 280RPM, about 240RPM to about 260RPM, about 260RPM to about 400RPM, about 260RPM to about 380RPM, about 260RPM to about 360RPM, about 260RPM to about about 340RPM, about 260RPM to about 320RPM, about 260RPM to about 300RPM, about 260RPM to about 280RPM, about 280RPM to about 400RPM, about 280RPM to about 380RPM, about 280RPM to about 360RPM, about 280RPM to about 340RPM, about 280RPM to about 320RPM, about 280RPM to about 300RPM, about 300RPM to about 400RPM, about 300RPM to about 380RPM, about 280RPM to about 360RPM, about 280RPM to about 340RPM, about 280RPM to about 320RPM, about 280RPM to about 300RPM, about 300RPM to about 400RPM, about 300RPM to about 380RPM, about 300RPM to about 360 RPM, about 300RPM to about 340RPM, about 300RPM to about 320RPM, about 320RPM to about 400RPM, about 320RPM to about 380RPM, about 320RPM to about 360RPM, about 320RPM to about 340RPM, about 340RPM to about 400RPM, about 340RPM to about 380RPM, about 340RPM to about 360RPM, about 360RPM to about 400RPM, about 360RPM to about 380RPM, or about 380RPM to about 400RPM.

[0290] In some embodiments, the rotary stirring of the test culture container can be performed using a rotary incubator with a throw (orbit) diameter between about 25 mm and about 50 mm. In some embodiments, the sensitizer concentration of the test culture container can be about 0 ppm to about 5,000 ppm (e.g., about 0 ppm to about 120 ppm, about 30 ppm to about 120 ppm, or about 50 ppm to about 100 ppm). In some embodiments, the sensitizer (e.g., defoamer, such as any defoamer described herein) concentration of the test culture container can be 0 ppm to about 200 ppm, 0 ppm to about 190 ppm, 0 ppm to about 180 ppm, 0 ppm to about 170 ppm, 0 ppm to about 160 ppm, 0 ppm to about 150 ppm, 0 ppm to about 140 ppm, 0 ppm to about 130 ppm, 0 ppm to about 120 ppm, 0 ppm to about 110 ppm, 0 ppm to about 100 ppm, 0 ppm to about 90 ppm, 0 ppm to about 80 ppm, 0 ppm to about 70 ppm, 0 ppm to about 60 ppm, 0 ppm to about 50 ppm, 0 ppm to about 40 ppm, 0 ppm to about 30 ppm, 0 ppm to about 20 ppm, 0 ppm to about 10 ppm, about 10 ppm to about 200 ppm, about 10 ppm to about 190 ppm, about 10 ppm to about 180 ppm, about 10 ppm to about 170 ppm, about 10 ppm to about 160 ppm, about 10 ppm to about 150 ppm, about 10 ppm to about 140 ppm, about 10 ppm to about 130 ppm, about 10 ppm to about 1 20ppm, about 10ppm to about 110ppm, about 10ppm to about 100ppm, about 10ppm to about 90ppm, about 10ppm to about 80ppm, about 10ppm to about 70ppm, about 10ppm to about 60ppm, about 10ppm to about 50ppm, about 10ppm to about 40ppm, about 10ppm to about 30ppm, about 10ppm to about 20ppm, about 20ppm to about 200ppm, about 20ppm to about 190ppm, about 20ppm to about 180ppm, about 20ppm to about 170ppm, about 20ppm to about 180ppm m to about 160ppm, about 20ppm to about 150ppm, about 20ppm to about 140ppm, about 20ppm to about 130ppm, about 20ppm to about 120ppm, about 20ppm to about 110ppm, about 20ppm to about 100ppm, about 20ppm to about 90ppm, about 20ppm to about 80ppm, about 20ppm to about 70ppm, about 20ppm to about 60ppm, about 20ppm to about 50ppm, about 20ppm to about 40ppm, about 20ppm to about 30ppm, about 30ppm to about 200ppm,about 30 ppm to about 190 ppm, about 30 ppm to about 180 ppm, about 30 ppm to about 170 ppm, about 30 ppm to about 160 ppm, about 30 ppm to about 150 ppm, about 30 ppm to about 140 ppm, about 30 ppm to about 130 ppm, about 30 ppm to about 120 ppm, about 30 ppm to about 110 ppm, about 30 ppm to about 100 ppm, about 30 ppm to about 90 ppm, about 30 ppm to about 80 ppm, about 30 ppm to about 70 ppm, about 30 ppm to about 60 ppm, about 30 ppm to about 50 ppm, about 30 ppm to about 40 ppm, about 40 ppm to about 200 ppm ppm, about 40 ppm to about 190 ppm, about 40 ppm to about 180 ppm, about 40 ppm to about 170 ppm, about 40 ppm to about 160 ppm, about 40 ppm to about 150 ppm, about 40 ppm to about 140 ppm, about 40 ppm to about 130 ppm, about 40 ppm to about 120 ppm, about 40 ppm to about 110 ppm, about 40 ppm to about 100 ppm, about 40 ppm to about 90 ppm, about 40 ppm to about 80 ppm, about 40 ppm to about 70 ppm, about 40 ppm to about 60 ppm, about 40 ppm to about 50 ppm, about 50 ppm to about 200 ppm, about 50 ppm to about 1 90ppm, about 50ppm to about 180ppm, about 50ppm to about 170ppm, about 50ppm to about 160ppm, about 50ppm to about 150ppm, about 50ppm to about 140ppm, about 50ppm to about 130ppm, about 50ppm to about 120ppm, about 50ppm to about 110ppm, about 50ppm to about 100ppm, about 50ppm to about 90ppm, about 50ppm to about 80ppm, about 50ppm to about 70ppm, about 50ppm to about 60ppm, about 60ppm to about 200ppm, about 60ppm to about 190ppm, about 60ppm to about 180ppm, about 60ppm to about m to about 170ppm, about 60ppm to about 160ppm, about 60ppm to about 150ppm, about 60ppm to about 140ppm, about 60ppm to about 130ppm, about 60ppm to about 120ppm, about 60ppm to about 110ppm, about 60ppm to about 100ppm, about 60ppm to about 90ppm, about 60ppm to about 80ppm, about 60ppm to about 70ppm, about 70ppm to about 200ppm, about 70ppm to about 190ppm, about 70ppm to about 180ppm, about 70ppm to about 170ppm, about 70ppm to about 160ppm, about 70ppm to about 150ppm,from about 70 ppm to about 140 ppm, from about 70 ppm to about 130 ppm, from about 70 ppm to about 120 ppm, from about 70 ppm to about 110 ppm, from about 70 ppm to about 100 ppm, from about 70 ppm to about 90 ppm, from about 70 ppm to about 80 ppm, from about 80 ppm to about 200 ppm, from about 80 ppm to about 190 ppm, from about 80 ppm to about 180 ppm, from about 80 ppm to about 170 ppm, from about 80 ppm to about 160 ppm, from about 80 ppm to about 150 ppm, from about 80 ppm to about 140 ppm, from about 80 ppm to about 130 ppm, from about 80 ppm to about 120 ppm, from about 80 ppm to about 1 ppm, about 90 ppm to about 150 ppm, about 90 ppm to about 140 ppm, about 90 ppm to about 130 ppm, about 90 ppm to about 120 ppm, about 90 ppm to about 110 ppm, about 90 ppm to about 100 ppm, about 100 ppm to about 200 ppm, about 100 ppm to about 190 ppm, about 90 ppm to about 180 ppm, about 90 ppm to about 170 ppm, about 90 ppm to about 160 ppm, about 90 ppm to about 150 ppm, about 90 ppm to about 140 ppm, about 90 ppm to about 130 ppm, about 90 ppm to about 120 ppm, about 90 ppm to about 110 ppm, about 90 ppm to about 100 ppm, about 100 ppm to about 200 ppm, about 100 ppm to about 190 ppm, about 100 ppm to about 180 ppm, From about 100 ppm to about 170 ppm, from about 100 ppm to about 160 ppm, from about 100 ppm to about 150 ppm, from about 100 ppm to about 140 ppm, from about 100 ppm to about 130 ppm, from about 100 ppm to about 120 ppm, from about 100 ppm to about 110 ppm, from about 110 ppm to about 200 ppm, from about 110 ppm to about 190 ppm, from about 110 ppm to about 180 ppm, from about 110 ppm to about 170 ppm, from about 110 ppm to about 160 ppm, from about 110 ppm to about 150 ppm, from about 110 ppm to about 140 ppm, from about 110 ppm to about 130 ppm, from about 110 ppm to about 120 ppm about 120 ppm, about 120 ppm to about 200 ppm, about 120 ppm to about 190 ppm, about 120 ppm to about 180 ppm, about 120 ppm to about 170 ppm, about 120 ppm to about 160 ppm, about 120 ppm to about 150 ppm, about 120 ppm to about 140 ppm, about 120 ppm to about 130 ppm, about 130 ppm to about 200 ppm, about 130 ppm to about 190 ppm, about 130 ppm to about 180 ppm, about 130 ppm to about 170 ppm, about 130 ppm to about 160 ppm, about 130 ppm to about 150 ppm, about 130 ppm to about 140 ppm,about 140 ppm to about 200 ppm, about 140 ppm to about 190 ppm, about 140 ppm to about 180 ppm, about 140 ppm to about 170 ppm, about 140 ppm to about 160 ppm, about 140 ppm to about 150 ppm, about 150 ppm to about 200 ppm, about 150 ppm to about 190 ppm, about 150 ppm to about 180 ppm, about 150 ppm to about 170 ppm, about 150 ppm to about 160 ppm, about 160 ppm to about 200 ppm ppm, about 160 ppm to about 190 ppm, about 160 ppm to about 180 ppm, about 160 ppm to about 170 ppm, about 170 ppm to about 200 ppm, about 170 ppm to about 190 ppm, about 170 ppm to about 180 ppm, about 180 ppm to about 200 ppm, about 180 ppm to about 190 ppm, or about 190 ppm to about 200 ppm (wherein the sensitizer is present in a liquid culture medium placed in a test culture vessel, such as for a period of time during the culture).

[0291] In some embodiments, the concentration of the sensitizer (e.g., an antifoaming agent, such as any of the antifoaming agents described herein) in the test culture container can be 0 ppm to about 5,000 ppm, 0 ppm to about 4,500 ppm, 0 ppm to about 4,000 ppm, 0 ppm to about 3,500 ppm, 0 ppm to about 3,000 ppm, 0 ppm to about 2,500 ppm, 0 ppm to about 2,000 ppm, 0 ppm to about 1,500 ppm, 0 ppm to about 1,000 ppm, 0 ppm to about 800 ppm, 0 ppm to about 600 ppm, 0 ppm to about 500 ppm, 0 ppm to about 450 ppm, 0 ppm to about 400 ppm, 0 ppm to about 500 ppm, 0 ppm to about 600 ppm, 0 ppm to about 700 ppm, 0 ppm to about 8 ... ppm, about 10 ppm to about 5,000 ppm, about 10 ppm to about 4,500 ppm, about 10 ppm to about 5,000 ppm, about 10 ppm to about 3,000 ppm, about 10 ppm to about 5,000 ppm, about 10 ppm to about 4,500 ppm, about 10 ppm to about 5,000 ppm, about 10 ppm to about 3,500 ppm, about 10 ppm to about 5,000 ppm, about 10 ppm to about 4,000 ppm, about 10 ppm to about 3,500 ppm, about 10 ppm to about 5,000 ppm, about 10 ppm to about 3,500 ppm, about 10 ppm to about 5,000 ppm, about 10 ppm to about 4,000 ppm, about 10 ppm to about 3,500 ppm ppm, about 10 ppm to about 3,000 ppm, about 10 ppm to about 2,500 ppm, about 10 ppm to about 2,000 ppm, about 10 ppm to about 1,500 ppm, about 10 ppm to about 1,000 ppm, about 10 ppm to about 800 ppm, about 10 ppm to about 600 ppm, about 10 ppm to about 500 ppm, about 10 ppm to about 450 ppm, about 10 ppm to about 400 ppm, about 10 ppm to about 350 ppm, about 10 ppm to about 300 ppm, about 10 ppm to about 250 ppm, about 10 ppm to about 200 ppm, about 10 ppm to about 150 ppm, about 10 ppm to about 1 ppm, about 10 ppm to about 120 ppm, about 10 ppm to about 100 ppm, about 10 ppm to about 80 ppm, about 10 ppm to about 60 ppm, about 10 ppm to about 50 ppm, about 10 ppm to about 40 ppm, about 10 ppm to about 30 ppm, about 10 ppm to about 20 ppm, about 20 ppm to about 5,000 ppm, about 20 ppm to about 4,500 ppm, about 20 ppm to about 4,000 ppm, about 20 ppm to about 3,500 ppm, about 20 ppm to about 3,000 ppm, about 20 ppm to about 2,500 ppm, about 20 ppm to about 2,000 ppm, about 20 ppm to about 1,500ppm, about 20ppm to about 1,000ppm, about 20ppm to about 800ppm, about 20ppm to about 600ppm, about 20ppm to about 500ppm, about 20ppm to about 450ppm, about 20ppm to about 400ppm, about 20ppm to about 350ppm, about 20ppm to about 300ppm, about 20ppm to about 250ppm, about 20ppm to about 200ppm, about 20ppm to about 150ppm, about 20ppm to about 140ppm, about 20ppm to about 120ppm, about 20ppm to about 150ppm, about 20ppm to about 140ppm, about 20ppm to about 150ppm, about 20ppm to about 160ppm, about 20ppm to about 170ppm, about 20ppm to about 180ppm, about 20ppm to about 190ppm, about 20ppm to about 210ppm, about 20ppm to about 250ppm, about 20ppm to about 200ppm m to about 100 ppm, about 20 ppm to about 80 ppm, about 20 ppm to about 60 ppm, about 20 ppm to about 50 ppm, about 20 ppm to about 40 ppm, about 20 ppm to about 30 ppm, about 30 ppm to about 5,000 ppm, about 30 ppm to about 4,500 ppm, about 30 ppm to about 4,000 ppm, about 30 ppm to about 3,500 ppm, about 30 ppm to about 3,000 ppm, about 30 ppm to about 2,500 ppm, about 30 ppm to about 2,000 ppm, about 30 ppm to about 1,5 ppm, about 30 ppm to about 1,000 ppm, about 30 ppm to about 800 ppm, about 30 ppm to about 600 ppm, about 30 ppm to about 500 ppm, about 30 ppm to about 450 ppm, about 30 ppm to about 400 ppm, about 30 ppm to about 350 ppm, about 30 ppm to about 300 ppm, about 30 ppm to about 250 ppm, about 30 ppm to about 200 ppm, about 30 ppm to about 150 ppm, about 30 ppm to about 140 ppm, about 30 ppm to about 120 ppm, about 30 ppm to about about 100 ppm, about 30 ppm to about 80 ppm, about 30 ppm to about 60 ppm, about 30 ppm to about 50 ppm, about 30 ppm to about 40 ppm, about 40 ppm to about 5,000 ppm, about 40 ppm to about 4,500 ppm, about 40 ppm to about 4,000 ppm, about 40 ppm to about 3,500 ppm, about 40 ppm to about 3,000 ppm, about 40 ppm to about 2,500 ppm, about 40 ppm to about 2,000 ppm, about 40 ppm to about 1,500 ppm, about 40 ppm to about 1,ppm, about 40 ppm to about 800 ppm, about 40 ppm to about 600 ppm, about 40 ppm to about 500 ppm, about 40 ppm to about 450 ppm, about 40 ppm to about 400 ppm, about 40 ppm to about 350 ppm, about 40 ppm to about 300 ppm, about 40 ppm to about 250 ppm, about 40 ppm to about 200 ppm, about 40 ppm to about 150 ppm, about 40 ppm to about 140 ppm, about 40 ppm to about 120 ppm, about 40 ppm to about 100 ppm, about 40 ppm to about 80 ppm, about 40 ppm to about 60 ppm, about 40 ppm to about 50 ppm, about 50 ppm ppm to about 5,000 ppm, about 50 ppm to about 4,500 ppm, about 50 ppm to about 4,000 ppm, about 50 ppm to about 3,500 ppm, about 50 ppm to about 3,000 ppm, about 50 ppm to about 2,500 ppm, about 50 ppm to about 2,000 ppm, about 50 ppm to about 1,500 ppm, about 50 ppm to about 1,000 ppm, about 50 ppm to about 800 ppm, about 50 ppm to about 600 ppm, about 50 ppm to about 500 ppm, about 50 ppm to about 450 ppm, about 50 ppm to about 400 ppm, about 50 ppm to about 350 ppm, about 50 ppm to about 3 00ppm, about 50ppm to about 250ppm, about 50ppm to about 200ppm, about 50ppm to about 150ppm, about 50ppm to about 140ppm, about 50ppm to about 120ppm, about 50ppm to about 100ppm, about 50ppm to about 80ppm, about 50ppm to about 60ppm, about 60ppm to about 5,000ppm, about 60ppm to about 4,500ppm, about 60ppm to about 4,000ppm, about 60ppm to about 3,500ppm, about 60ppm to about 3,000ppm, about 60ppm to about 2,500ppm, about 60ppm to about 2,000ppm, about 60ppm to about ppm, about 60 ppm to about 1,500 ppm, about 60 ppm to about 1,000 ppm, about 60 ppm to about 800 ppm, about 60 ppm to about 600 ppm, about 60 ppm to about 500 ppm, about 60 ppm to about 450 ppm, about 60 ppm to about 400 ppm, about 60 ppm to about 350 ppm, about 60 ppm to about 300 ppm, about 60 ppm to about 250 ppm, about 60 ppm to about 200 ppm, about 60 ppm to about 150 ppm, about 60 ppm to about 140 ppm, about 60 ppm to about 120 ppm, about 60 ppm to about 100 ppm, about 60 ppm to about 80 ppm, about 80 ppm to about 5,ppm, about 80 ppm to about 4,000 ppm, about 80 ppm to about 3,500 ppm, about 80 ppm to about 3,000 ppm, about 80 ppm to about 2,500 ppm, about 80 ppm to about 2,000 ppm, about 80 ppm to about 1,500 ppm, about 80 ppm to about 1,000 ppm, about 80 ppm to about 800 ppm, about 80 ppm to about 600 ppm, about 80 ppm to about 500 ppm, about 80 ppm to about 450 ppm, about 80 ppm to about ppm, about 80 ppm to about 350 ppm, about 80 ppm to about 300 ppm, about 80 ppm to about 250 ppm, about 80 ppm to about 200 ppm, about 80 ppm to about 150 ppm, about 80 ppm to about 140 ppm, about 80 ppm to about 120 ppm, about 80 ppm to about 100 ppm, about 100 ppm to about 5,000 ppm, about 100 ppm to about 4,500 ppm, about 100 ppm to about 4,000 ppm, about 100 ppm to about 3,500 ppm, about 100 ppm to about 3,000 ppm 0ppm, about 100ppm to about 2,500ppm, about 100ppm to about 2,000ppm, about 100ppm to about 1,500ppm, about 100ppm to about 1,000ppm, about 100ppm to about 800ppm, about 100ppm to about 600ppm, about 100ppm to about 500ppm, about 100ppm to about 450ppm, about 100ppm to about 400ppm, about 100ppm to about 350ppm, about 100ppm to about 300ppm, about 100ppm to about 250ppm, about 100ppm to about ppm, about 120 ppm to about 3,000 ppm, about 120 ppm to about 2,500 ppm, about 120 ppm to about 2,000 ppm, about 120 ppm to about 1,500 ppm, about 120 ppm to about 1,500 ppm, about 120 ppm to about 2,000 ppm, about 120 ppm to about 1,500 ppm, about 120 ppm to about 1,ppm, about 120 ppm to about 800 ppm, about 120 ppm to about 600 ppm, about 120 ppm to about 500 ppm, about 120 ppm to about 450 ppm, about 120 ppm to about 400 ppm, about 120 ppm to about 350 ppm, about 120 ppm to about 300 ppm, about 120 ppm to about 250 ppm, about 120 ppm to about 200 ppm, about 120 ppm to about 150 ppm, about 120 ppm to about 140 ppm, about 140 ppm to about 5,000 ppm, about 140 ppm to about 4,500 ppm, about 140 ppm to about 4,000 ppm, about 140 ppm to about ppm to about 3,500 ppm, about 140 ppm to about 3,000 ppm, about 140 ppm to about 2,500 ppm, about 140 ppm to about 2,000 ppm, about 140 ppm to about 1,500 ppm, about 140 ppm to about 1,000 ppm, about 140 ppm to about 800 ppm, about 140 ppm to about 600 ppm, about 140 ppm to about 500 ppm, about 140 ppm to about 450 ppm, about 140 ppm to about 400 ppm, about 140 ppm to about 350 ppm, about 140 ppm to about 300 ppm, about 140 ppm to about 250 ppm, about 140 ppm to about 2 ppm, about 150 ppm to about 5,000 ppm, about 150 ppm to about 4,500 ppm, about 150 ppm to about 4,000 ppm, about 150 ppm to about 3,500 ppm, about 150 ppm to about 3,000 ppm, about 150 ppm to about 2,500 ppm, about 150 ppm to about 2,000 ppm, about 150 ppm to about 1,500 ppm, about 150 ppm to about 1,000 ppm, about 150 ppm to about 800 ppm, about 150 ppm to about 600 ppm, about 150 ppm to about 500 ppm, about 150 ppm to about ppm, about 150 ppm to about 400 ppm, about 150 ppm to about 350 ppm, about 150 ppm to about 300 ppm, about 150 ppm to about 250 ppm, about 150 ppm to about 200 ppm, about 200 ppm to about 5,000 ppm, about 200 ppm to about 4,500 ppm, about 200 ppm to about 4,000 ppm, about 200 ppm to about 3,500 ppm, about 200 ppm to about 3,000 ppm, about 200 ppm to about 2,500 ppm, about 200 ppm to about 2,000 ppm, about 200 ppm to about 1,500 ppm, about 200 ppm to about 1,ppm, about 200 ppm to about 800 ppm, about 200 ppm to about 600 ppm, about 200 ppm to about 500 ppm, about 200 ppm to about 450 ppm, about 200 ppm to about 400 ppm, about 200 ppm to about 350 ppm, about 200 ppm to about 300 ppm, about 200 ppm to about 250 ppm, about 250 ppm to about 5,000 ppm, about 250 ppm to about 4,500 ppm, about 250 ppm to about 4,000 ppm, about 250 ppm to about 3,500 ppm, about 250 ppm to about 3,000 ppm, about 250 ppm to about 2,500 ppm, about 250 ppm to about 3,000 ppm ppm to about 2,000 ppm, about 250 ppm to about 1,500 ppm, about 250 ppm to about 1,000 ppm, about 250 ppm to about 800 ppm, about 250 ppm to about 600 ppm, about 250 ppm to about 500 ppm, about 250 ppm to about 450 ppm, about 250 ppm to about 400 ppm, about 250 ppm to about 350 ppm, about 250 ppm to about 300 ppm, about 300 ppm to about 5,000 ppm, about 300 ppm to about 4,500 ppm, about 300 ppm to about 4,000 ppm, about 300 ppm to about 3,500 ppm, about 300 ppm to about 3,000 ppm ppm, about 300 ppm to about 2,500 ppm, about 300 ppm to about 2,000 ppm, about 300 ppm to about 1,500 ppm, about 300 ppm to about 1,000 ppm, about 300 ppm to about 800 ppm, about 300 ppm to about 600 ppm, about 300 ppm to about 500 ppm, about 300 ppm to about 450 ppm, about 300 ppm to about 400 ppm, about 300 ppm to about 350 ppm, about 350 ppm to about 5,000 ppm, about 350 ppm to about 4,500 ppm, about 350 ppm to about 4,000 ppm, about 350 ppm to about 3,500 ppm, about 350 ppm to about ppm to about 3,000 ppm, about 350 ppm to about 2,500 ppm, about 350 ppm to about 2,000 ppm, about 350 ppm to about 1,500 ppm, about 350 ppm to about 1,000 ppm, about 350 ppm to about 800 ppm, about 350 ppm to about 600 ppm, about 350 ppm to about 500 ppm, about 350 ppm to about 450 ppm, about 350 ppm to about 400 ppm, about 400 ppm to about 5,000 ppm, about 400 ppm to about 4,500 ppm, about 400 ppm to about 4,000 ppm, about 400 ppm to about 3,500 ppm, about 400 ppm to about 3,ppm, about 400 ppm to about 2,500 ppm, about 400 ppm to about 2,000 ppm, about 400 ppm to about 1,500 ppm, about 400 ppm to about 1,000 ppm, about 400 ppm to about 800 ppm, about 400 ppm to about 600 ppm, about 400 ppm to about 500 ppm, about 400 ppm to about 450 ppm, about 450 ppm to about 5,000 ppm, about 450 ppm to about 4,500 ppm, about 450 ppm to about 4,000 ppm, about 450 ppm to about 3,500 ppm, about 450 ppm to about 3,000 ppm, about 450 ppm to about 2,500 ppm ppm, about 450 ppm to about 2,000 ppm, about 450 ppm to about 1,500 ppm, about 450 ppm to about 1,000 ppm, about 450 ppm to about 800 ppm, about 450 ppm to about 600 ppm, about 450 ppm to about 500 ppm, about 500 ppm to about 5,000 ppm, about 500 ppm to about 4,500 ppm, about 500 ppm to about 4,000 ppm, about 500 ppm to about 3,500 ppm, about 500 ppm to about 3,000 ppm, about 500 ppm to about 2,500 ppm, about 500 ppm to about 2,000 ppm, about 500 ppm to about 1,500 ppm, about 50 0 ppm to about 1,000 ppm, about 500 ppm to about 800 ppm, about 500 ppm to about 600 ppm, about 600 ppm to about 5,000 ppm, about 600 ppm to about 4,500 ppm, about 600 ppm to about 4,000 ppm, about 600 ppm to about 3,500 ppm, about 600 ppm to about 3,000 ppm, about 600 ppm to about 2,500 ppm, about 600 ppm to about 2,000 ppm, about 600 ppm to about 1,500 ppm, about 600 ppm to about 1,000 ppm, about 600 ppm to about 800 ppm, about 800 ppm to about 5,000 ppm, about 800 ppm ppm to about 4,500 ppm, about 800 ppm to about 4,000 ppm, about 800 ppm to about 3,500 ppm, about 800 ppm to about 3,000 ppm, about 800 ppm to about 2,500 ppm, about 800 ppm to about 2,000 ppm, about 800 ppm to about 1,500 ppm, about 800 ppm to about 1,000 ppm, about 1,000 ppm to about 5,000 ppm, about 1,000 ppm to about 4,500 ppm, about 1,000 ppm to about 4,000 ppm, about 1,000 ppm to about 3,500 ppm, about 1,000 ppm to about 3,000 ppm, about 1,000 ppm to about 2,ppm, about 1,500 ppm to about 2,000 ppm, about 1,000 ppm to about 1,500 ppm, about 1,500 ppm to about 5,000 ppm, about 1,500 ppm to about 4,500 ppm, about 1,500 ppm to about 4,000 ppm, about 1,500 ppm to about 3,500 ppm, about 1,500 ppm to about 3,000 ppm, about 1,500 ppm to about 2,500 ppm, about 1,500 ppm to about 2,000 ppm, about 2,000 ppm to about 5,000 ppm, about 2,000 ppm to about 4,500 ppm, about 2,000 ppm to about 4,000 ppm, about 2,000 ppm to about 3,500 ppm, about 2,000 ppm to about 3,000 ppm, about 2,000 ppm to about 2,500 ppm, about 2,500 ppm to about 5,000 ppm ppm, about 2,500 ppm to about 4,500 ppm, about 2,500 ppm to about 4,000 ppm, about 2,500 ppm to about 3,500 ppm, about 2,500 ppm to about 3,000 ppm, about 3,000 ppm to about 5,000 ppm, about 3,000 ppm to about 4,500 ppm, about 3,000 ppm to about 4,000 ppm, about 3,000 ppm to about 3,500 ppm, about 3,500 ppm to about 5,000 ppm, about 3,500 ppm to about 4,500 ppm, about 3,500 ppm to about 5,000 ppm, about 3,500 ppm to about 4,500 ppm, about 3,500 ppm to about 4,000 ppm, about 4,000 ppm to about 5,000 ppm, about 4,000 ppm to about 4,500 ppm, or about 4,500 ppm to about 5,000 ppm (wherein the sensitizer is present in a liquid culture medium placed in a test culture vessel, such as for a period of time during the culture).

[0292] In some embodiments, the protectant concentration of the test culture container can be about 1 g / L to about 10 g / L (e.g., about 2 g / L to about 8 g / L, about 3 g / L to about 7 g / L, about 4 g / L to about 6 g / L, or about 5 g / L). In some embodiments, the protective agent (e.g., any protective agent described herein or known in the art) concentration of the test culture container can be about 1.0 g / L to about 10.0 g / L, about 1.0 g / L to about 9.5 g / L, about 1.0 g / L to about 9.0 g / L, about 1.0 g / L to about 8.5 g / L, about 1.0 g / L to about 8.0 g / L, about 1.0 g / L to about 7.5 g / L, about 1.0 g / L to about 7.0 g / L, about 1.0 g / L to about 6.5 g / L, about 1.0 g / L to about 6.0 g / L, about 1.0 g / L to about 5.5 g / L, about 1.0 g / L to about 5.0 g / L, about 1.0 g / L to about g / L, about 1.0 g / L to about 4.0 g / L, about 1.0 g / L to about 3.5 g / L, about 1.0 g / L to about 3.0 g / L, about 1.0 g / L to about 2.5 g / L, about 1.0 g / L to about 2.0 g / L, about 1.0 g / L to about 1.5 g / L, about 1.5 g / L to about 10.0 g / L, about 1.5 g / L to about 9.5 g / L, about 1.5 g / L to about 9.0 g / L, about 1.5 g / L to about 8.5 g / L, about 1.5 g / L to about 8.0 g / L, about 1.5 g / L to about 7.5 g / L, about 1.5 g / L to about 7.0 g / L, about 1.5 g / L to about 6. g / L, about 1.5 g / L to about 6.0 g / L, about 1.5 g / L to about 5.5 g / L, about 1.5 g / L to about 5.0 g / L, about 1.5 g / L to about 4.5 g / L, about 1.5 g / L to about 4.0 g / L, about 1.5 g / L to about 3.5 g / L, about 1.5 g / L to about 3.0 g / L, about 1.5 g / L to about 2.5 g / L, about 1.5 g / L to about 2.0 g / L, about 2.0 g / L to about 10.0 g / L, about 2.0 g / L to about 9.5 g / L, about 2.0 g / L to about 9.0 g / L, about 2.0 g / L to about 8.5 g / L, about 2.0 g / L to about 8.0 g / L , about 2.0 g / L to about 7.5 g / L, about 2.0 g / L to about 7.0 g / L, about 2.0 g / L to about 6.5 g / L, about 2.0 g / L to about 6.0 g / L, about 2.0 g / L to about 5.5 g / L, about 2.0 g / L to about 5.0 g / L, about 2.0 g / L to about 4.5 g / L, about 2.0 g / L to about 4.0 g / L, about 2.0 g / L to about 3.5 g / L, about 2.0 g / L to about 3.0 g / L, about 2.0 g / L to about 2.5 g / L, about 2.5 g / L to about 10.0 g / L, about 2.5 g / L to about 9.5 g / L, about 2.5 g / L to about 9.0 g / L, about 2.g / L, about 2.5 g / L to about 8.5 g / L, about 2.5 g / L to about 8.0 g / L, about 2.5 g / L to about 7.5 g / L, about 2.5 g / L to about 7.0 g / L, about 2.5 g / L to about 6.5 g / L, about 2.5 g / L to about 6.0 g / L, about 2.5 g / L to about 5.5 g / L, about 2.5 g / L to about 5.0 g / L, about 2.5 g / L to about 4.5 g / L, about 2.5 g / L to about 4.0 g / L, about 2.5 g / L to about 3.5 g / L, about 2.5 g / L to about 3.0 g / L, about 3.0 g / L to about 10.0 g / L, about 3.0 g / L to about 9.5 g / L, about 3.0 g / L to about 9.0 g / L, about 3.0 g / L to about 8. 5g / L, about 3.0g / L to about 8.0g / L, about 3.0g / L to about 7.5g / L, about 3.0g / L to about 7.0g / L, about 3.0g / L to about 6.5g / L, about 3.0g / L to about 6.0g / L, about 3.0g / L to about 5.5g / L, about 3.0g / L to about 5.0g / L, about 3.0g / L to about 4.5g / L, about 3.0g / L to about 4.0g / L, about 3.0g / L to about 3.5g / L, about 3.5g / L to about 10.0g / L, about 3.5g / L to about 9.5g / L, about 3.5g / L to about 9.0g / L, about 3.5g / L to about 8.5g / L, about 3.5g / L to about 8.0g / L, about 3. g / L, about 3.5 g / L to about 7.5 g / L, about 3.5 g / L to about 7.0 g / L, about 3.5 g / L to about 6.5 g / L, about 3.5 g / L to about 6.0 g / L, about 3.5 g / L to about 5.5 g / L, about 3.5 g / L to about 5.0 g / L, about 3.5 g / L to about 4.5 g / L, about 3.5 g / L to about 4.0 g / L, about 4.0 g / L to about 10.0 g / L, about 4.0 g / L to about 9.5 g / L, about 4.0 g / L to about 9.0 g / L, about 4.0 g / L to about 8.5 g / L, about 4.0 g / L to about 8.0 g / L, about 4.0 g / L to about 7.5 g / L, about 4.0 g / L to about 7.0 g / L, about 4.0 g / L to about 6. g / L, about 4.0 g / L to about 6.0 g / L, about 4.0 g / L to about 5.5 g / L, about 4.0 g / L to about 5.0 g / L, about 4.0 g / L to about 4.5 g / L, about 4.5 g / L to about 10.0 g / L, about 4.5 g / L to about 9.5 g / L, about 4.5 g / L to about 9.0 g / L, about 4.5 g / L to about 8.5 g / L, about 4.5 g / L to about 8.0 g / L, about 4.5 g / L to about 7.5 g / L, about 4.5 g / L to about 7.0 g / L, about 4.5 g / L to about 6.5 g / L, about 4.5 g / L to about 6.0 g / L, about 4.5 g / L to about 5.5 g / L, about 4.5 g / L to about 5.0 g / L, about 5.g / L, about 5.0 g / L to about 9.5 g / L, about 5.0 g / L to about 9.0 g / L, about 5.0 g / L to about 8.5 g / L, about 5.0 g / L to about 8.0 g / L, about 5.0 g / L to about 7.5 g / L, about 5.0 g / L to about 7.0 g / L, about 5.0 g / L to about 6.5 g / L, about 5.0 g / L to about 6.0 g / L, about 5.0 g / L to about 5.5 g / L, about 5.5 g / L to about 10.0 g / L, about 5.5 g / L to about 9.5 g / L, about 5.5 g / L to about 9.0 g / L, about 5.5 g / L to about 8.5 g / L, about 5.5 g / L to about about 8.0 g / L, about 5.5 g / L to about 7.5 g / L, about 5.5 g / L to about 7.0 g / L, about 5.5 g / L to about 6.5 g / L, about 5.5 g / L to about 6.0 g / L, about 6.0 g / L to about 10.0 g / L, about 6.0 g / L to about 9.5 g / L, about 6.0 g / L to about 9.0 g / L, about 6.0 g / L to about 8.5 g / L, about 6.0 g / L to about 8.0 g / L, about 6.0 g / L to about 7.5 g / L, about 6.0 g / L to about 7.0 g / L, about 6.0 g / L to about 6.5 g / L, about 6.5 g / L to about 10.0 g / L, about 6.5 g / L to about 9.5 g / L / L, about 6.5g / L to about 9.0g / L, about 6.5g / L to about 8.5g / L, about 6.5g / L to about 8.0g / L, about 6.5g / L to about 7.5g / L, about 6.5g / L to about 7.0g / L, about 7.0g / L to about 10.0g / L, about 7.0g / L to about 9.5g / L, about 7.0g / L to about 9.0g / L, about 7.0g / L to about 8.5g / L, about 7.0g / L to about 8.0g / L, about 7.0g / L to about 10.0g / L, about 7.5g / L to about 9.5g / L, about 7.0g / L to about 9.0g / L, about 7.5g / L to about 8.0g / L, .5 g / L to about 8.5 g / L, about 7.5 g / L to about 8.0 g / L, about 8.0 g / L to about 10.0 g / L, about 8.0 g / L to about 9.5 g / L, about 8.0 g / L to about 9.0 g / L, about 8.0 g / L to about 8.5 g / L, about 8.5 g / L to about 10.0 g / L, about 8.5 g / L to about 9.5 g / L, about 8.5 g / L to about 9.0 g / L, about 9.0 g / L to about 10.0 g / L, about 9.0 g / L to about 9.5 g / L, or about 9.5 g / L to about 10.0 g / L (where the protectant is present in the liquid culture medium placed in the test culture vessel, such as for a period of time during the culture).

[0293] The production bioreactor can be a perfusion bioreactor, a fed-batch bioreactor, or a concentrated fed-batch bioreactor. In some embodiments, the volume of the production bioreactor can be from about 5L to about 20,000L (e.g., from about 50L to about 10,000L). In some embodiments, the volume of the production bioreactor can be greater than 5L, greater than 10L, greater than 50L, greater than 100L, greater than 1000L, greater than 2000L, greater than 5000L, greater than 20000L, greater than 100000L, or greater than 150000L (e.g., greater than 1000L). In some embodiments, the volume of the production bioreactor can be about 5 L to about 20,000 L, about 5 L to about 18,000 L, about 5 L to about 16,000 L, about 5 L to about 14,000 L, about 5 L to about 12,000 L, about 5 L to about 10,000 L, about 5 L to about 9,500 L, about 5 L to about 9,000 L, about 5 L to about 8,500 L, about 5 L to about 8,000 L, about 5 L to about 7,500 L, about 5 L to about 7,000 L, about 5 L to about 6,500 L, about 5 L to about 6,000 L, or about 5 L to about 8,500 L. From about 5L to about 5,500L, from about 5L to about 5,000L, from about 5L to about 4,500L, from about 5L to about 4,000L, from about 5L to about 3,500L, from about 5L to about 3,000L, from about 5L to about 2,500L, from about 5L to about 2,000L, from about 5L to about 1,500L, from about 5L to about 1,000L, from about 5L to about 900L, from about 5L to about 800L, from about 5L to about 700L, from about 5L to about 600L, from about 5L to about 500L, from about 5L to about 400L, from about 5L to about 300L, from about 5L to about 2 50L, about 5L to about 200L, about 5L to about 150L, ​​about 5L to about 100L, about 5L to about 80L, about 5L to about 60L, about 5L to about 50L, about 5L to about 25L, about 5L to about 10L, about 10L to about 20,000L, about 10L to about 18,000L, about 10L to about 16,000L, about 10L to about 14,000L, about 10L to about 12,000L, about 10L to about 10,000L, about 10L to about 9,500L, about 10L to about 9,000L, about 10L to about 8 ,500L, about 10L to about 8,000L, about 10L to about 7,500L, about 10L to about 7,000L, about 10L to about 6,500L, about 10L to about 6,000L, about 10L to about 5,500L, about 10L to about 5,000L, about 10L to about 4,500L, about 10L to about 4,000L, about 10L to about 3,500L, about 10L to about 3,000L, about 10L to about 2,500L, about 10L to about 2,000L, about 10L to about 1,500L, about 10L to about 1,000L, about 10L to about 900L, about 10L to about 800L, about 10L to about 700L, about 10L to about 600L, about 10L to about 500L, about 10L to about 400L, about 10L to about 300L, about 10L to about 250L, about 10L to about 200L, about 10L to about 150L, ​​about 10L to about 100L, about 10L to about 80L, about 10L to about 60L, about 10L to about 50L, about 10L to about 25L, about 25L to about 20,000L, about 25L to about 18,000L, about 25L to about 16,000L, about 25L to about 14,000L, about 25L to about 12,000L, about 25L to about 10,000L, about 25L to about 9,500L, about 25L to about 9,000L, about 25L to about 8,500L, about 25L to about 8,000L, about 25L to about 7,500L, about 25L to about 7,000L, about 25L to about 6,500L, about 25L to about 6,000L, about 25L to about 5,500L, about 25L to about 5,000L, about 25L to about 4,500L, about 25L to about 4,000L, about 25L to about 3,500L, about 25L to about 3,000L, about 25L to about 2,500L, about 25L to about 2,000L, about 25L to about 1,500L, about 25 L to about 1,000L, about 25L to about 900L, about 25L to about 800L, about 25L to about 700L, about 25L to about 600L, about 25L to about 500L, about 25L to about 400L, about 25L to about 300L, about 25L to about 250L, about 25L to about 200L, about 25L to about 150L, ​​about 25L to about 100L, about 25L to about 80L, about 25L to about 60L, about 25L to about 50L, about 50L to about 20,000L, about 50L to about 18,000L, about 50L to about 16,000L, about 50L to about 14,000L, about 50L to about 12,000L, about 50L to about 10,000L, about 50L to about 9,500L, about 50L to about 9,000L, about 50L to about 8,500L, about 50L to about 8,000L, about 50L to about 7,500L, about 50L to about 7,000L, about 50L to about 6,500L, about 50L to about 6,000L, about 50L to about 5,500L, about 50L to about 5,000L, about 50L to about 4,500L, about 50L to about 4,000L, about 50L to about 3,500L, about 50L to about 3,000L, about 50L to about 2,500L, about 50L to about 2,000L, about 50L to about 1,500L, about 50L to about 1,000L, about 50L to about 900L, about 50L to about 800L, about 50L to about 700L, about 50L to about 600L, about 50L to about 500L, about 50L to about 400L, about 50L to about 300L, about 50L to about 250L, about 50L to about 200L, about 50L to about 150L, ​​about 50L to about 100L, about 50L to about 80L, about 50L to about 60L, about 60L to about 20,000L, about 60L to about 18,000L, about 60L to about 16,000L, about 60L to about 14,000L, about 60L to about 12,000L, about 60L to about 10,000L 0L, about 60L to about 9,500L, about 60L to about 9,000L, about 60L to about 8,500L, about 60L to about 8,000L, about 60L to about 7,500L, about 60L to about 7,000L, about 60L to about 6,500L, about 60L to about 6,000L, about 60L to about 5,500L, about 60L to about 5,000L, about 60L to about 4,500L, about 60L to about 4,000L, about 60L to about 3,500L, about 60L to about 3,000L, about 60L to about 2,500L, about 60L to about 2,000L, about 60L to about 1,500L, about 60 L to about 1,000L, about 60L to about 900L, about 60L to about 800L, about 60L to about 700L, about 60L to about 600L, about 60L to about 500L, about 60L to about 400L, about 60L to about 300L, about 60L to about 250L, about 60L to about 200L, about 60L to about 150L, ​​about 60L to about 100L, about 60L to about 80L, about 80L to about 20,000L, about 80L to about 18,000L, about 80L to about 16,000L, about 80L to about 14,000L, about 80L to about 12,000L, about 80L to about 10,000L, about 8 0L to about 9,500L, about 80L to about 9,000L, about 80L to about 8,500L, about 80L to about 8,000L, about 80L to about 7,500L, about 80L to about 7,000L, about 80L to about 6,500L, about 80L to about 6,000L, about 80L to about 5,500L, about 80L to about 5,000L, about 80L to about 4,500L, about 80L to about 4,000L, about 80L to about 3,500L, about 80L to about 3,000L, about 80L to about 2,500L, about 80L to about 2,000L, about 80L to about 1,500L, about 80L to about 1,000L, about 80L to about 900L, about 80L to about 800L, about 80L to about 700L, about 80L to about 600L, about 80L to about 500L, about 80L to about 400L, about 80L to about 300L, about 80L to about 250L, about 80L to about 200L, about 80L to about 150L, ​​about 80L to about 100L, about 100L to about 20,000L, about 100L to about 18,000L, about 100L to about 16,000L, about 100L to about 14,000L, about 100L to about 12,000L, about 100L to about 10,000L, about 100L to about 9,500L, about 100L to about 9,000L, about 100L to about 8,500L, about 100L to about 8,000L, about 100L to about 7,500L, about 100L to about 7,000L, about 100L to about 6,500L, about 100L to about 6,000L, about 100L to about 5,500L, about 100L to about 5,000L, about 100L to about 4,500L, about 100L to about 4,000L, about 100L to about 3,500L, about 100L to about 3,000L, about 100L to about 2,500L, about 100L to about 2,000L, about 100L to about 1,500L, about 100L to about 1 ,000L, about 100L to about 900L, about 100L to about 800L, about 100L to about 700L, about 100L to about 600L, about 100L to about 500L, about 100L to about 400L, about 100L to about 300L, about 100L to about 250L, about 100L to about 200L, about 100L to about 150L, ​​about 150L to about 20,000L, about 150L to about 18,000L, about 150L to about 16,000L, about 150L to about 14,000L, about 150L to about 12,000L, about 150L to about 10,000L, about 150L to about 9,500L, about 150L to about 9,000L, about 150L to about 8,500L, about 150L to about 8,000L, about 150L to about 7,500L, about 150L to about 7,000L, about 150L to about 6,500L, about 150L to about 6,000L, about 150L to about 5,500L, about 150L to about 5,000L, about 150L to about 4,500L, about 150L to about 4,000L, about 150L to about 3,500L, about 150L to about 3,000L, about 150L to about 2,500L, about 150L to about 2,000L, about 150L to about 1,500L, about 150L to about 1,000L, about 150L to about 900L, about 150L to about 800L, about 150L to about 700L, about 150L to about 600L, about 150L to about 500L, about 150L to about 400L, about 150L to about 300L, about 150L to about 250L, about 150L to about 200L, about 200L to about 20,000L, about 200L to about 18,000L, about 200L to about 16,000L, about 200L to about 14,000L, about 200L to about 12,000L, about 200L to about 10,000L, about 200L to about 9,500L, about 200L to about 9,000L, about 200L to about 8,500L, about 200L to about 8,000L, about 200L to about 7,500L, about 200L to about 7,000L, about 200L to about 6,500L, about 200L to about 6,000L, about 200L to about 5,500L, about 200L to about 5,000L, about 200L to about 4,500L, about 200L to about 4,000L, about 200L to about 3,500L, about 200L to about 3,000L, about 200L to about 2,500L, about 200L to about 2,000L, about 200L to about 1,500L, about 200L to about 1,000L, about 200L to about 900L, about 200L to about 800L, about 200L to about 700L, about 2 00L to about 600L, about 200L to about 500L, about 200L to about 400L, about 200L to about 300L, about 200L to about 250L, about 250L to about 20,000L, about 250L to about 18,000L, about 250L to about 16,000L, about 250L to about 14,000L, about 250L to about 12,000L, about 250L to about 10,000L, about 250L to about 9,500L, about 250L to about 9,000L, about 250L to about 8,500L, about 250L to about 8,000L, about 250L to about 7,500L, about 250L to about 7,000L, about 250L to about 6,500L, about From about 250L to about 6,000L, from about 250L to about 5,500L, from about 250L to about 5,000L, from about 250L to about 4,500L, from about 250L to about 4,000L, from about 250L to about 3,500L, from about 250L to about 3,000L, from about 250L to about 2,500L, from about 250L to about 2,000L, from about 250L to about 1,500L, from about 250L to about 1,000L, from about 250L to about 900L, from about 250L to about 800L, from about 250L to about 700L, from about 250L to about 600L, from about 250L to about 500L, from about 250L to about 400L, from about 250L to about 300L, from about 300L to about 20,000L, about 300L to about 18,000L, about 300L to about 16,000L, about 300L to about 14,000L, about 300L to about 12,000L, about 300L to about 10,000L, about 300L to about 9,500L, about 300L to about 9,000L, about 300L to about 8,500L, about 300L to about 8,000L, about 300L to about 7,500L, about 300L to about 7,000L, about 300L to about 6,500L, about 300L to about 6,000L, about 300L to about 5,500L, about 300L to about 5,000L, about 300L to about 4,500L, about 300L to about 4,000L , about 300L to about 3,500L, about 300L to about 3,000L, about 300L to about 2,500L, about 300L to about 2,000L, about 300L to about 1,500L, about 300L to about 1,000L, about 300L to about 900L, about 300L to about 800L, about 300L to about 700L, about 300L to about 600L, about 300L to about 500L, about 300L to about 400L, about 400L to about 20,000L, about 400L to about 18,000L, about 400L to about 16,000L, about 400L to about 14,000L, about 400L to about 12,000L, about 400L to about 10,000L, From about 400L to about 9,500L, from about 400L to about 9,000L, from about 400L to about 8,500L, from about 400L to about 8,000L, from about 400L to about 7,500L, from about 400L to about 7,000L, from about 400L to about 6,500L, from about 400L to about 6,000L, from about 400L to about 5,500L, from about 400L to about 5,000L, from about 400L to about 4,500L, from about 400L to about 4,000L, from about 400L to about 3,500L, from about 400L to about 3,000L, from about 400L to about 2,500L, from about 400L to about 2,000L, from about 400L to about 1,500L, from about 400L to about 1, 000L, about 400L to about 900L, about 400L to about 800L, about 400L to about 700L, about 400L to about 600L, about 400L to about 500L, about 500L to about 20,000L, about 500L to about 18,000L, about 500L to about 16,000L, about 500L to about 14,000L, about 500L to about 12,000L, about 500L to about 10,000L, about 500L to about 9,500L, about 500L to about 9,000L, about 500L to about 8,500L, about 500L to about 8,000L, about 500L to about 7,500L, about 500L to about 7,000L, about 500L to about 6,500L, about 500L to about 6,000L, about 500L to about 5,500L, about 500L to about 5,000L, about 500L to about 4,500L, about 500L to about 4,000L, about 500L to about 3,500L, about 500L to about 3,000L, about 500L to about 2,500L, about 500L to about 2,000L, about 500L to about 1,500L, about 500L to about 1,000L, about 500L to about 900L, about 500L to about 800L, about 500L to about 700L, about 500L to about 600L, about 600L to about 20,000L, about 600L to about 18,000L, about 600L to about 16, 000L, about 600L to about 14,000L, about 600L 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[0294] In some embodiments, the operating parameter can be any parameter related to cell viability or cell death (e.g., shear stress, sensitizer concentration, protectant concentration, temperature, pH, nutrient composition, biomass load, and feeding strategy). Cell viability or cell death rate can be measured by using cells that can express lactate dehydrogenase (LDH). When cells that can express LDH die, the LDH in the cells is released into the liquid culture medium. Therefore, cell viability or cell death rate can be measured orthogonally to LDH and is proportional to specific LDH production (sLDHp) ( Figure 3 ).

[0295] Figure 4 It shows how pluronic (eg, protectant), sensitizer, and shear (eg, stirring) can affect specific LDH production. Figure 4 As shown, protectants can reduce cell bubble attachment and increase cell durability, thereby maintaining high cell viability and protecting cells from death. In contrast, sensitizers can increase cell bubble attachment and reduce cell durability, thereby reducing cell viability. As these parameters are interrelated with each other, it is often difficult to predict the exact impact of sensitizers, agitation, and / or protectants through traditional models.

[0296] Figure 5 The specific LDH output in the small-scale model is shown to be a function of sensitizer concentration, protective agent concentration and stirring. Due to the interaction between these parameters, the specific LDH output does not have a linear relationship with any of these parameters usually. The impact of these parameters on the specific LDH output can be determined by factorial design (e.g., full factorial design or fractional factorial design).

[0297] Factorial design involves conducting an experiment under a set of conditions, and the conditions are defined by a set of parameters (e.g., operating parameters). In the set of conditions, each parameter can have two or more levels. The purpose of factorial design is to study the main effects and interaction effects between different parameters (e.g., sensitizer concentration, protectant concentration, and stirring). The term "full factorial design" refers to an experimental design that includes all possible combinations of different levels across all parameters. For example, for three parameters (i.e., factors) each with two levels, a full factorial design will have a total of 8 combinations, and is usually called a 2 3 Factorial Design ( Figure 6 ). In contrast, the term "fractional factorial design" refers to an experimental design that consists of carefully selected subsets (parts) of the experimental runs of the full factorial design. The subsets are chosen so as to exploit the sparsity-of-effect principle to reveal information about the most important features of the problem under investigation, while using a fraction of the work of the full factorial design in terms of experimental runs and resources. By performing a factorial design, it is possible to identify inoperable regions (e.g., combinations of parameter values ​​that make an operation fail). Factorial designs are known in the art and are described, for example, in Box, GE; Hunter, WG; Hunter, JS (2005). Statistics for Experimenters: Design, Innovation, and Discovery (2nd ed.). Wiley. ISBN 0-471-71813-0; Vicente, Gemma et al., "Application of the factorial design of experiments and response surface methodology to optimize biodiesel production." Industrial crops and products 8.1 (1998): 29-35; Vandervoort, Jo and Annick Ludwig. "Biocompatible stabilizers in the preparation of PLGA nanoparticles: a factorial design study." International journal of pharmaceuticals 238.1-2 (2002): 77-92; each of these references is incorporated herein by reference in its entirety.

[0298] In a typical factorial design, the central point (e.g., the target region) is often poorly predicted. Therefore, in some embodiments, the factorial design can be used in combination with a central composite design (e.g., an inner central composite design, an outer central composite design, or a face-centered composite design) ( Figure 7 ). A central composite design is an experimental design consisting of an embedded factor or fractional factorial design containing a central point, enhanced by a set of axial points, allowing for the estimation of curvature. Composite designs are known in the art, and a detailed description of central composite designs can be found in, for example, Box, GE; Hunter, WG; Hunter, JS (2005). Statistics for Experimenters: Design, Innovation, and Discovery (2nd Edition). Wiley. ISBN 0-471-71813-0; Ahmadi, M. et al., "Application of the central composite design and response surface methodology to the advanced treatment of olive oil processing wastewater using Fenton's peroxidation." Journal of Hazardous Materials 123.1-3 (2005): 187-195; each of these documents is incorporated herein by reference in its entirety.

[0299] In some embodiments, the test conditions are selected based on full factorial screening and central composite design (e.g., inscribed central composite design). By combining full factorial screening and central composite design, the selected test conditions can maximize the target area and minimize the potential failure area ( Figure 8 and Fig. 9 ).

[0300] The experimental results of these test conditions in the test culture vessel provide estimated contours that illustrate the relationship between the selected parameters. Once the contours of the small-scale model have been determined, the small-scale model can be used to predict the missing values. For example, based on the knowledge of defoamer concentration or shear stress, it can be predicted how much Pluronic is needed for operation. This relationship is determined by correlating cell growth rate and mortality across different scales (e.g., small scale, benchtop, production, etc.).

[0301] In some embodiments, the estimated contours of the small-scale model can be used to match data points collected at a different scale (e.g., production scale). Since the concentrations of the sensitizer and protectant are known, the shear stress in the small-scale model can be determined from the contours, which corresponds to the shear stress in the production bioreactor ( Fig.10 Based on the shear stress and the sensitizer concentration corresponding to the shear stress in the production bioreactor, the effect of a change in protectant concentration (Δ[protectant]) can be determined from the estimated contours ( Fig.11 ). For example, Fig.11 As shown, the leftmost point (circle), the left center point (circle), the right center point (circle), and the rightmost point (circle) all have the same shear stress and sensitizer concentration. The shaded area is the inoperable space. By increasing the protective agent concentration (from the right center point (circle) to the rightmost point (circle), an arrow is depicted between the two points), the acceptable sensitizer concentration range can be increased, resulting in better resilience to the sensitizer.

[0302] Thus, in some embodiments, a model as described herein can be used to determine the mutual influence between different parameters. Traditional models typically study the influence of each parameter individually. A model as described herein can study the influence of three parameters at once, rather than studying the influence of each parameter individually. Thus, such a model as described herein can predict the expected cell culture performance when any single parameter is changed.

[0303] Furthermore, those traditional scale-up parameters generally do not provide predictions of cell culture performance. The model described herein can provide better guidance for predicting the operating range, which leads to a lower probability of failure when moving to production scale and predicts cell culture performance once other traditional parameters (P / V, tip speed, mixing time, K) have been met. L a) Provides the ability to amplify.

[0304] In some other embodiments, the model as described herein can be used to establish the operational design space of the reactor at any scale given any single parameter or combination of parameters (e.g., Pluronic, defoaming and shearing). The model can be used to determine the amount of the maximum defoamer that a cell culture can tolerate before failure for a given shear condition.

[0305] In some embodiments, the model can be used to determine the maximum amount of antifoam agent that the pluronic concentration can maintain before it fails for given shear conditions.

[0306] In some embodiments, the model can be used to test different types of antifoams (Antifoam C, autoclaved antifoam, irradiated antifoam, etc.) against different Pluronics.

[0307] In some embodiments, the model can be cell density range independent and is not necessarily applicable only to intensified perfusion cultures because it uses LDH for normalization across scales.

[0308] In some embodiments, the model can also test antifoam concentrations using full addition (1 bolus at the beginning) or daily addition.

[0309] In some embodiments, the model can be used to test the kinetics behind the inactivation of antifoam agents and their impact on cell culture.

[0310] cell

[0311] The methods provided herein can be used to culture a variety of different cells (eg, mammalian cells, bacterial cells, and yeast cells).

[0312] Non-limiting examples of bacterial cells include E. coli, Clostridium difficile, Clostridium sporogenes, Clostridium beijerinkii, and Clostridium acetobutylicum. Exemplary commercially available bacterial cells that can be used in any of the methods described herein include BL21 (DE3) (Novagen), BL21 (DE3)-pLysS (Novagen), BL21 Star-pLysS (Invitrogen), BL21-SI (Invitrogen), BL21-AI (Invitrogen), Tuner (Novagen), TunerpLysS (Novagen), Origami (Novagen), Origami D (Novagen), Origami B pLysS (Novagen), Rosetta (Novagen), Rosetta pLysS(Novagen), Rosetta-gami-pLysS(Novagen), BL21CodonPlus(Stratagene), AD494(Novagen), BL21trxB(Novagen), HMS174(Novagen), NovaBlue(DE3)(Novagen), BLR(Novagen), C41(DE3)(Lucigen), C43(DE3)(Lucigen), Lemo21(DE3)(New England BioLabs), SHuffle T7 (New England BioLabs), ArticExpress (Agilent Technologies), and Artic Express (DE3) (Agilent Technologies).

[0313] Non-limiting examples of yeast cells include: Saccharomyces cerevisiae, S. exiguus, and Pichia pastoris. Additional examples of yeast cells that can be used for any of the methods described herein include those from Saccharomyces, Pichia, Kluyveromyces, Hansenula, and Yarrowia. Additional examples of yeast cells that can be used for any of the methods described herein are described in Nielsen, Methods in Enzymology 536: 133-147, 2014.

[0314] Non-limiting examples of mammalian cells that can be cultured using any of the methods described herein include: Chinese hamster ovary (CHO) cells (e.g., CHO DG44 cells, CHO-K1s cells, and CHO-DXB11 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 HEK293F), African green monkey kidney epithelial (Vero) cells, and Madin-Darby canine (Cocker Spaniel) kidney epithelial (MDCK) cells. Additional mammalian cells that can be cultured using the methods described herein are known in the art. In non-limiting examples of any of the methods described herein, the concentration of mammalian cells present in the test culture vessel and / or production bioreactor at the start of the culture is about 0.1 x 10 6 cells / ml to about 10x10 6 cells / mL (e.g., about 0.1 x 10 6 cells / ml to about 9x10 6 cells / ml, about 0.1x10 6 cells / ml is about 8x10 6 cells / ml, about 0.1x10 6 cells / ml is about 7x10 6 cells / ml, about 0.1x10 6 cells / ml to about 6x10 6 cells / ml, about 0.1x10 6 cells / ml to about 5x10 6 cells / ml, about 0.1x10 6 cells / ml is about 4x10 6 cells / ml, about 0.1x10 6 cells / ml to about 3x10 6cells / ml, about 0.1x10 6 cells / ml to about 2x10 6 cells / ml, about 0.1x10 6 cells / ml to about 1.5x10 6 cells / ml, about 0.1x10 6 cells / ml to about 1.0x10 6 cells / ml, about 0.1x10 6 cells / ml is about 0.8x10 6 cells / ml, about 0.1x10 6 cells / ml to about 0.5x10 6 cells / ml, about 0.1x10 6 cells / ml is about 0.3x10 6 cells / ml, about 0.2x10 6 cells / ml to about 10x10 6 cells / ml, about 0.2x10 6 cells / ml to about 9x10 6 cells / ml, about 0.2x10 6 cells / ml is about 8x10 6 cells / ml, about 0.2x10 6 cells / ml is about 7x10 6 cells / ml, about 0.2x10 6 cells / ml to about 6x10 6 cells / ml, about 0.2x10 6 cells / ml to about 5x10 6 cells / ml, about 0.2x10 6 cells / ml is about 4x10 6 cells / ml, about 0.2x10 6 cells / ml to about 3x10 6 cells / ml, about 0.2x10 6 cells / ml to about 2x10 6 cells / ml, about 0.2x10 6 cells / ml to about 1.5x10 6 cells / ml, about 0.2x10 6 cells / ml to about 1.0x10 6 cells / ml, about 0.2x10 6 cells / ml is about 0.8x10 6 cells / ml, about 0.2x10 6 cells / ml to about 0.5x106 cells / ml, about 0.3x10 6 cells / ml to about 10x10 6 cells / ml, about 0.3x10 6 cells / ml to about 9x10 6 cells / ml, about 0.3x10 6 cells / ml is about 8x10 6 cells / ml, about 0.3x10 6 cells / ml is about 7x10 6 cells / ml, about 0.3x10 6 cells / ml to about 6x10 6 cells / ml, about 0.3x10 6 cells / ml to about 5x10 6 cells / ml, about 0.3x10 6 cells / ml is about 4x10 6 cells / ml, about 0.3x10 6 cells / ml to about 3x10 6 cells / ml, about 0.3x10 6 cells / ml to about 2x10 6 cells / ml, about 0.3x10 6 cells / ml to about 1.5x10 6 cells / ml, about 0.3x10 6 cells / ml to about 1.0x10 6 cells / ml, about 0.3x10 6 cells / ml is about 0.8x10 6 cells / ml, about 0.3x10 6 cells / ml to about 0.5x10 6 cells / ml, about 0.5x10 6 cells / ml to about 10x10 6 cells / ml, about 0.5x10 6 cells / ml to about 9x10 6 cells / ml, about 0.5x10 6 cells / ml is about 8x10 6 cells / ml, about 0.5x10 6 cells / ml is about 7x10 6 cells / ml, about 0.5x10 6 cells / ml to about 6x10 6 cells / ml, about 0.5x10 6 cells / ml to about 5x106 cells / ml, about 0.5x10 6 cells / ml is about 4x10 6 cells / ml, about 0.5x10 6 cells / ml to about 3x10 6 cells / ml, about 0.5x10 6 cells / ml to about 2x10 6 cells / ml, about 0.5x10 6 cells / ml to about 1.5x10 6 cells / ml, about 0.5x10 6 cells / ml to about 1.0x10 6 cells / ml, about 0.5x10 6 cells / ml is about 0.8x10 6 cells / ml, about 0.8x10 6 cells / ml to about 10x10 6 cells / ml, about 0.8x10 6 cells / ml to about 9x10 6 cells / ml, about 0.8x10 6 cells / ml is about 8x10 6 cells / ml, about 0.8x10 6 cells / ml is about 7x10 6 cells / ml, about 0.8x10 6 cells / ml to about 6x10 6 cells / ml, about 0.8x10 6 cells / ml to about 5x10 6 cells / ml, about 0.8x10 6 cells / ml is about 4x10 6 cells / ml, about 0.8x10 6 cells / ml to about 3x10 6 cells / ml, about 0.8x10 6 cells / ml to about 2x10 6 cells / ml, about 0.8x10 6 cells / ml to about 1.5x10 6 cells / ml, about 0.8x10 6 cells / ml to about 1.0x10 6 cells / ml, about 1.0x10 6 cells / ml to about 10x10 6 cells / ml, about 1.0x10 6 cells / ml to about 9x106 cells / ml, about 1.0x10 6 cells / ml is about 8x10 6 cells / ml, about 1.0x10 6 cells / ml is about 7x10 6 cells / ml, about 1.0x10 6 cells / ml to about 6x10 6 cells / ml, about 1.0x10 6 cells / ml to about 5x10 6 cells / ml, about 1.0x10 6 cells / ml is about 4x10 6 cells / ml, about 1.0x10 6 cells / ml to about 3x10 6 cells / ml, about 1.0x10 6 cells / ml to about 2x10 6 cells / ml, about 1.0x10 6 cells / ml to about 1.5x10 6 cells / ml, about 1.5x10 6 cells / ml to about 10x10 6 cells / ml, about 1.5x10 6 cells / ml to about 9x10 6 cells / ml, about 1.5x10 6 cells / ml is about 8x10 6 cells / ml, about 1.5x10 6 cells / ml is about 7x10 6 cells / ml, about 1.5x10 6 cells / ml to about 6x10 6 cells / ml, about 1.5x10 6 cells / ml to about 5x10 6 cells / ml, about 1.5x10 6 cells / ml is about 4x10 6 cells / ml, about 1.5x10 6 cells / ml to about 3x10 6 cells / ml, about 1.5x10 6 cells / ml to about 2x10 6 cells / ml, about 2x10 6 cells / ml to about 10x10 6 cells / ml, about 2x10 6 cells / ml to about 9x10 6cells / ml, about 2x10 6 cells / ml is about 8x10 6 cells / ml, about 2x10 6 cells / ml is about 7x10 6 cells / ml, about 2x10 6 cells / ml to about 6x10 6 cells / ml, about 2x10 6 cells / ml to about 5x10 6 cells / ml, about 2x10 6 cells / ml is about 4x10 6 cells / ml, about 2x10 6 cells / ml to about 3x10 6 cells / ml, about 3x10 6 cells / ml to about 10x10 6 cells / ml, about 3x10 6 cells / ml to about 9x10 6 cells / ml, about 3x10 6 cells / ml is about 8x10 6 cells / ml, about 3x10 6 cells / ml is about 7x10 6 cells / ml, about 3x10 6 cells / ml to about 6x10 6 cells / ml, about 3x10 6 cells / ml to about 5x10 6 cells / ml, about 3x10 6 cells / ml is about 4x10 6 cells / ml, about 4x10 6 cells / ml to about 10x10 6 cells / ml, about 4x10 6 cells / ml to about 9x10 6 cells / ml, about 4x10 6 cells / ml is about 8x10 6 cells / ml, about 4x10 6 cells / ml is about 7x10 6 cells / ml, about 4x10 6 cells / ml to about 6x10 6 cells / ml, about 4x10 6 cells / ml to about 5x10 6 cells / ml, about 5x10 6 cells / ml to about 10x10 6cells / ml, about 5x10 6 cells / ml to about 9x10 6 cells / ml, about 5x10 6 cells / ml is about 8x10 6 cells / ml, about 5x10 6 cells / ml is about 7x10 6 cells / ml, about 5x10 6 cells / ml to about 6x10 6 cells / ml, about 6x10 6 cells / ml to about 10x10 6 cells / ml, about 6x10 6 cells / ml to about 9x10 6 cells / ml, about 6x10 6 cells / ml is about 8x10 6 cells / ml, about 6x10 6 cells / ml is about 7x10 6 cells / ml, about 7x10 6 cells / ml to about 10x10 6 cells / ml, about 7x10 6 cells / ml to about 9x10 6 cells / ml, about 7x10 6 cells / ml is about 8x10 6 cells / ml, about 8x10 6 cells / ml to about 10x10 6 cells / ml, about 8x10 6 cells / ml to about 9x10 6 cells / mL or approximately 9x10 6 cells / ml to about 10x10 6 cells / mL).

[0315] The cell (e.g., a mammalian cell) may contain a recombinant nucleic acid (e.g., a nucleic acid stably integrated into the genome of the cell) encoding a recombinant therapeutic protein (e.g., a recombinant therapeutic protein secreted by the cell). Non-limiting examples of recombinant therapeutic proteins are described herein and are known in the art.

[0316] Recombinant therapeutic proteins

[0317] Recombinant therapeutic proteins can be secretory proteins that are released by the cells (e.g., mammalian cells) into the liquid culture medium of the cell culture. For example, the nucleic acid sequence encoding the soluble recombinant protein can contain a sequence encoding a secretion signal peptide at the N or C terminus of the recombinant protein, which is cleaved by an enzyme present in the cells (e.g., mammalian cells) and subsequently released into the liquid culture medium of the cell culture. In other cases, the recombinant protein is a soluble protein that is not secreted, and the recombinant protein is recovered from the cells (e.g., mammalian cells).

[0318] Non-limiting examples of recombinant proteins include immunoglobulins (including light and heavy chain immunoglobulins, antibodies or antibody fragments (e.g., any antibody fragment described herein)), enzymes (e.g., β-galactosidase (e.g., α-galactosidase), Myozyme or Cerezyme), proteins (e.g., human erythropoietin, tumor necrosis factor (TNF) or interferon α or β), or immunogenic or antigenic proteins or protein fragments (e.g., proteins for vaccines). In some embodiments, the recombinant protein is an engineered antigen-binding polypeptide containing at least one multifunctional recombinant protein scaffold (see, e.g., Gebauer et al., Current Opin. Chem. Biol. 13: 245-255, 2009; and recombinant antigen-binding proteins described in U.S. Patent Application Publication No. 2012 / 0164066 (incorporated herein by reference in its entirety)). Non-limiting examples of recombinant proteins that are antibodies include panitumumab, omalizumab, abavoumab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab, alacizumab, alemtuzumab, alirocumab, atumomab, amatuximab, anatumomab, apolizumab, atinumumab, and dapoxetine. In some embodiments, the present invention relates to an antibody that is directed against a monoclonal antibody or a combination of the following: a monoclonal antibody, ... Additional non-limiting examples of recombinant proteins include: alglucosidase alfa, laronidase, abatacept, galactosidase, luteinizing hormone alfa, antihemophilic factor, agalsidase beta, interferon beta-1a, darbepoetin alfa, tenecteplase, etanercept, coagulation factor IX, follicle stimulating hormone, interferon beta-1a, imiglucerase, dornase alfa, epoetin alfa, and alteplase.

[0319] The secreted soluble recombinant protein can be recovered from the liquid culture medium. A variety of different methods for removing the liquid culture medium from cells (e.g., mammalian cells) are known in the art, including, for example, centrifugation, filtration, pipetting and / or suction. The secreted recombinant protein can then be recovered from the liquid culture medium and further purified using a variety of biochemical techniques, including various types of chromatography (e.g., affinity chromatography, molecular sieve chromatography, cation exchange chromatography or anion exchange chromatography) and / or filtration (e.g., molecular weight cut-off filtration).

[0320] In order to recycle the intracellular recombinant protein, the cell (for example, mammalian cell) can be cracked. The various methods for lysing cells (for example, mammalian cells) are known in the art, including for example ultrasonic treatment and / or detergent, enzymatic and / or chemical lysis. Various biochemical methods known in the art can be used to purify recombinant proteins from cell lysates (for example, mammalian cell lysates), usually starting from a centrifugal step to remove cell debris, and then carrying out one or more other steps (for example, one or more types of chromatography (for example, affinity chromatography, molecular sieve chromatography, cation exchange chromatography or anion exchange chromatography) and / or filtration (for example, molecular weight cut-off filtration)).

[0321] In some embodiments, the recovered recombinant protein is at least or about 50% pure by weight, e.g., at least or about 55% pure by weight, at least 60% pure by weight, at least 65% pure by weight, at least 70% pure by weight, at least 75% pure by weight, at least 80% pure by weight, at least 85% pure by weight, at least 90% pure by weight, at least 95% pure by weight, at least 96% pure by weight, at least 97% pure by weight, at least 98% pure by weight, or at least or about 99% pure by weight, or greater than 99% pure by weight.

[0322] In some embodiments, the recovered recombinant protein is a recombinant human therapeutic protein that has one or more different biophysical properties compared to the same native protein in the human body (e.g., differences in the type or amount of glycosylation, differences in phosphorylation, differences in acylation, differences in metalation or metal stoichiometry, and / or differences in cofactor binding).

[0323] Also provided herein are recombinant therapeutic proteins produced by any of the methods described herein.

[0324] Liquid culture medium

[0325] Liquid culture medium is known in the art. The liquid culture medium can be supplemented with mammalian serum (for example, fetal bovine serum and bovine serum) and / or growth hormone or growth factor (for example, insulin, transferrins and epidermal growth factor). Alternatively or additionally, liquid culture medium can be a liquid culture medium determined by chemical composition, a liquid culture medium without animal-derived components, a serum-free liquid culture medium or a liquid culture medium containing serum. The non-limiting examples of liquid culture medium determined by chemical composition, a liquid culture medium without animal-derived components, a serum-free liquid culture medium and a liquid culture medium containing serum are commercially available.

[0326] Liquid culture media typically contain an energy source (e.g., carbohydrates such as glucose), essential amino acids (e.g., a basic set of twenty amino acids plus cysteine), vitamins and / or other organic compounds, free fatty acids and / or trace elements as required at low concentrations. If desired, the liquid culture media may be supplemented with, for example, mammalian hormones or growth factors (e.g., insulin, transferrin or epidermal growth factor), salts and buffers (e.g., calcium, magnesium and phosphate), nucleosides and bases (e.g., adenosine, thymidine and hypoxanthine), proteins and tissue hydrolysates and / or any combination of these or other additives.

[0327] Non-limiting examples of liquid culture media that are particularly useful in the presently described methods include, for example, CD CHO, Opti CHO, and Forti CHO (all available from Life Technologies; Grand Island, New York), Hycell CHO medium (Thermo Fisher Scientific, Inc.; Waltham, Massachusetts), Ex-cell CD CHO Fusion medium (Sigma-Aldrich Co.; St. Louis, Missouri), and PowerCHO medium (Lonza Group, Ltd.; Basel, Switzerland). Media components that can also be used in the methods of the present invention include, but are not limited to, chemically defined (CD) hydrolysates, such as CD peptones, CD polypeptides (two or more amino acids), and CD growth factors. Other examples of liquid tissue culture media and media components are known in the art.

[0328] Perfusion culture

[0329] The cells can be cultured using perfusion culture. Cultivating cells (e.g., mammalian cells) in a container by perfusion includes removing a first volume of a first liquid culture medium (e.g., a first volume of a first liquid culture medium containing any concentration of mammalian cells, such as substantially no cells) from the container, and adding a second volume of a second liquid culture medium to the first liquid culture medium. The removal and addition can be performed simultaneously or sequentially, or a combination of the two. Furthermore, the removal and addition can be performed continuously (e.g., at a rate of removing and replacing between 0.1% and 800% (e.g., 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%, and between 4% and 30%) of the volume of the container or the volume of the first liquid culture medium over any given period of time (e.g., over a period of 24 hours, over an incremental period of time from about 1 hour to about 24 hours, or over an incremental period of time greater than 24 hours)) or periodically (e.g., once every three days, once every other day, once a day, twice a day, three times a day, four times a day, or five times a day), or any combination thereof. In the case of periodicity, the volume removed or replaced (e.g., within a period of about 24 hours, within an incremental period of about 1 hour to about 24 hours, or within an incremental period of greater than 24 hours) can be, for example, between 0.1% and 800% (e.g., between 1% and 700%, between 1% and 600%, between 1% and 500%, between 1% and 400%, between 1% and 300%, between 1% and 200%, between 1% and 100%, between 100% and 200%, between 5% and 150%, between 10% and 50%, between 15% and 40%, between 8% and 80%, and between 4% and 30%) of the volume of the container or the volume of the first liquid culture medium. In some cases, the first volume of the first liquid culture medium removed and the second volume of the second liquid culture medium added over a period of every 24 hours (or alternatively, an incremental period of about 1 hour to about 24 hours or an incremental period of greater than 24 hours) over the entire or partial culture period can remain approximately the same. As is known in the art, the rate (volume / time unit) at which the first volume of the first liquid culture medium is removed and the rate (volume / time unit) at which the second volume of the second liquid culture medium is added can vary. The rate (volume / time unit) at which the first volume of the first liquid culture medium is removed and the rate (volume / time unit) at which the second volume of the second liquid culture medium is added can be approximately the same or can be different.

[0330] Alternatively, the volume removed and added can be varied (e.g., increased stepwise) over each 24-hour period (or alternatively, an incremental period of between 1 hour and about 24 hours or an incremental period of greater than 24 hours) during the culture period. For example, the volume of the first liquid culture medium removed and the volume of the second liquid culture medium added can be increased (e.g., stepwise or by staggered increments) from a volume between 0.5% to about 20% of the volume of the container or the first liquid culture medium to about 25% to about 150% of the volume of the container or the first liquid culture medium over each 24-hour period (or alternatively, an incremental period of between about 1 hour and about 24 hours or an incremental period of greater than 24 hours) during the culture period.

[0331] Skilled practitioners will appreciate that the first liquid culture medium and the second liquid culture medium can be the same type of culture medium. In other cases, the first liquid culture medium and the second liquid culture medium can be different.

[0332] The first volume of the first liquid culture medium can be removed, for example, by a mechanical system that can remove the first volume of the first liquid culture medium (e.g., the first volume of the first liquid culture medium substantially free of cells from the bioreactor) from the container. Alternatively or additionally, the first volume of the first liquid culture medium can be removed by osmosis or gravity flow through a sterile membrane having a molecular weight cutoff that excludes cells (e.g., mammalian cells).

[0333] The second volume of the second liquid culture medium can be added to the first liquid culture medium in an automated manner (eg, by a perfusion pump).

[0334] In some cases, removing the first volume of the first liquid culture medium (e.g., the first volume of the first liquid culture medium that is substantially free of mammalian cells) and adding the second volume of the second liquid culture medium to the first liquid culture medium does not occur within at least 1 hour (e.g., within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 12 hours, within 14 hours, within 16 hours, within 18 hours, within 24 hours, within 36 hours, within 48 hours, within 72 hours, within 96 hours, or within 96 hours) of inoculating the container with the cells (e.g., mammalian cells.

[0335] Fed-batch culture

[0336] The culture of cells (e.g., mammalian cells) can be carried out using fed-batch culture. Fed-batch culture of cells (e.g., mammalian cells) includes adding (e.g., regularly or continuously adding) a second volume of a second liquid culture medium to a first liquid culture medium over most of the culture time period. The addition of the second liquid culture medium can be continuously (e.g., at a rate of adding a volume of 0.1% to 300% (e.g., 1% to 250%, 1% to 100%, 100% to 200%, 5% to 150%, 10% to 50%, 15% to 40%, 8% to 80%, and 4% to 30%) of the volume of the container or the first liquid culture medium over any given time period (e.g., 24 hours, about 1 hour to about 24 hours, or about 24 hours) or regularly (e.g., once every three days, every other day, once a day, twice a day, three times a day, four times a day, or five times a day), or any combination thereof. In the case of periodic addition, the volume added (e.g., within a period of about 24 hours, within an incremental period of about 1 hour to about 24 hours, or within an incremental period of greater than 24 hours) can be, for example, between 0.1% and 300% of the volume of the container or the volume of the first liquid culture medium (e.g., between 1% and 200%, between 1% and 100%, between 100% and 200%, between 5% and 150%, between 10% and 50%, between 15% and 40%, between 8% and 80%, and between 4% and 30%). In some cases, the second volume of the second liquid culture medium added over a period of every 24 hours (or alternatively, an incremental period of about 1 hour to about 24 hours or an incremental period of greater than 24 hours) over the entire or partial culture period can remain approximately the same. As known in the art, the rate (volume / time unit) at which the second volume of the second liquid culture medium is added can vary over the entire or partial culture period. For example, the volume of the second liquid culture medium added over each 24 hour period (or alternatively, an incremental period of between 1 hour and about 24 hours or an incremental period of greater than 24 hours) during the culture period can vary (e.g., increase stepwise). For example, the volume of the second liquid culture medium added over each 24 hour period (or alternatively, an incremental period of between about 1 hour and about 24 hours or an incremental period of greater than 24 hours) during the culture period can increase (e.g., stepwise or by staggered increments) from a volume between 0.5% to about 20% of the volume of the container or the volume of the first liquid culture medium to about 25% to about 150% of the volume of the container or the volume of the first liquid culture medium. The rate (volume / time unit) of adding the second volume of the second liquid culture medium can be approximately the same over the entire or a portion of the culture period.

[0337] Skilled practitioners will appreciate that the first liquid culture medium and the second liquid culture medium can be the same type of culture medium. In other cases, the first liquid culture medium and the second liquid culture medium can be different. The volume of the second liquid culture medium can be added to the first liquid culture medium in an automated manner (e.g., by a perfusion pump).

[0338] In some cases, adding the second volume of the second liquid culture medium to the first liquid culture medium does not occur within at least 1 hour (e.g., within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 12 hours, within 14 hours, within 16 hours, within 18 hours, within 24 hours, within 36 hours, within 48 hours, within 72 hours, within 96 hours, or within 96 hours) of inoculating the container with cells (e.g., mammalian cells. Cell culture medium in a fed-batch culture is typically harvested at the end of the culture period and used in any of the processes described herein, however, cell culture medium in a fed-batch culture can also be harvested at one or more time points during the culture period and used in any of the processes described herein.

[0339] A skilled practitioner will appreciate that various culture parameters (e.g., vessel, volume, rate or frequency of changing culture volume, frequency of agitation, temperature, medium, and CO) may affect the culture. 2 Any of the above (or any combination) can be used in performing these methods.

[0340] temperature

[0341] Cultivation can be carried out at a temperature of 32°C to about 39°C (e.g., about 32°C to about 37°C). Skilled practitioners will appreciate that the temperature can be changed at a specific one or more time points (e.g., within one or more time periods in the first time period, the second time period, and the third time period) in the culture method, such as by hourly or daily calculation. For example, the temperature can be changed or moved (e.g., increased or decreased) at about one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or about twenty days or more after the initial inoculation of the shake flask with mammalian cells or at any time point within the first, second, and / or third time period described herein. For example, the temperature can be shifted upward (e.g., by up to or about 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C, 1.0°C, 1.5°C, 2.0°C, 2.5°C, 3.0°C, 3.5°C, 4.0°C, 4.5°C, 5.0°C, 5.5°C, 6.0°C, 6.5°C, 7.0°C, 7.5°C, 8.0°C, 8.5°C, 9.0°C, 9.5°C, or 10.0°C). For example, the temperature can be shifted downward (e.g., by up to or about 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C, 1.0°C, 1.5°C, 2.0°C, 2.5°C, 3.0°C, 3.5°C, 4.0°C, 4.5°C, 5.0°C, 5.5°C, 6.0°C, 6.5°C, 7.0°C, 7.5°C, 8.0°C, 8.5°C, 9.0°C, 9.5°C, or 10°C).

[0342] CO 2

[0343] The culture may include an atmosphere containing up to or about 1% to 15% CO 2 (e.g., up to or about 14% CO 2 , 12% CO 2 , 10% CO 2 , 8% CO 2 , 6% CO 2 , 5% CO 2 , 4% CO 2 , 3% CO 2 , 2% CO 2 or at most or about 1% CO 2 ). Culturing can include incubating the cell culture in a humidified atmosphere (e.g., at least or about 20%, 30%, 40%, 50%, 60%, 70%, 85%, 80%, 85%, 90%, or at least or about 95% humidity or about 100% humidity).

[0344] Cell viability measurement

[0345] A variety of methods and assays that can be used to measure cell viability in cell cultures (e.g., mammalian cell cultures) are known in the art. For example, lactate dehydrogenase (LDH) can be detected in a liquid culture medium of a cell culture. Commercial kits for detecting LDH in a liquid culture medium of a cell culture are known in the art.

[0346] Cell viability is determined using a variety of different dyes that are excluded by viable cells but selectively taken up by non-viable cells. Non-limiting examples of these dyes include propidium iodide, trypan blue, and 7-aminoactinomycin D. Cell viability is determined by visualizing or otherwise detecting staining of the cells after incubation with the dye.

[0347] Additional assays for determining cell viability in cell culture include detecting mitochondrial activity or caspase activity in cells (eg, Resazurin and Formazan). Additional methods for determining cell viability in cell culture are known in the art.

[0348] Method for predicting the effect of sensitizer concentration on cell viability in a production bioreactor

[0349] Provided herein is a method for predicting the effect of a sensitizer (e.g., any sensitizer described herein or known in the art) concentration on cell viability in a production bioreactor, comprising: (a) determining cell viability under multiple test conditions in a test culture vessel (e.g., any test culture vessel described herein), wherein the multiple test conditions are defined by a set of parameters; and (b) predicting the effect of a sensitizer concentration on cell viability in a production bioreactor (e.g., any production bioreactor described herein) based on the cell viability in the test culture vessel under the multiple conditions.

[0350] In some embodiments, the parameter set includes, consists of, or consists essentially of shear stress, sensitizer concentration, and protectant (e.g., any protectant described herein) concentration. In some embodiments of these methods, the plurality of test conditions are selected based on full factorial screening and / or endocentric composite design. In some embodiments of any of these methods, the sensitizer is a defoamer (e.g., a polydimethylsiloxane-based defoamer, dimethicone, or any other defoamer described herein or known in the art).

[0351] In some embodiments, the test culture container (e.g., any exemplary test culture container described herein or known in the art) has a volume of about 5 mL to about 5 L (or any sub-range of this range described herein). In some embodiments, the test culture container is a shake flask, such as a baffled shake flask.

[0352] In some embodiments, the volume of the production bioreactor (e.g., any exemplary production bioreactor described herein or known in the art) is about 5 L to about 20,000 L (or any subrange of this range described herein). In some examples, the production bioreactor is a perfusion bioreactor. In other examples, the production bioreactor is a fed-batch bioreactor.

[0353] As used herein, the specific cell death rate is defined by the formula:

[0354] Specific cell death rate = (specific LDH production) / (LDH content per cell).

[0355] Some embodiments of these methods further comprise culturing cells (e.g., any of the cells described herein) in the production bioreactor using a concentration of a sensitizer predicted to have a specific cell death rate of less than 40% per day, less than 39% per day, less than 38% per day, less than 37% per day, less than 36% per day, less than 35% per day, less than 34% per day, less than 33% per day, less than 32% per day, less than 31% per day, less than 30% per day, less than 29% per day, less than 20% per day, less than 21% per day, less than 22% per day, less than 23% per day, less than 24% per day, less than 25% per day, less than 26% per day, less than 27% per day, less than 28% per day, less than 29% per day, less than 29% per day, less than 28 ... 8%, less than 27% per day, less than 26% per day, less than 25% per day, less than 24% per day, less than 23% per day, less than 22% per day, less than 21% per day, less than 20% per day, less than 19% per day, less than 18% per day, less than 17% per day, less than 16% per day, less than 15% per day, less than 14% per day, less than 13% per day, less than 12% per day, less than 11% per day, less than 10% per day, less than 9% per day, less than 8% per day, less than 7% per day, less than 6% per day, less than 5% per day, less than 4% per day, less than 3% per day, less than 2% per day or less than 1% per day. Some embodiments of any of the methods described herein further comprise culturing cells (e.g., any of the cells described herein) in the production bioreactor using a concentration of a sensitizer predicted to have a specific cell death rate during culturing of cells (e.g., any of the cells described herein) in the production bioreactor of about 0.1% to about 40% per day, about 0.1% to about 38% per day, about 0.1% to about 36% per day, about 0.1% to about 34% per day, about 0.1% to about 32% per day, about 0.1% to about 30% per day, about 0.1% to about 28% per day, about 0.1% to about 26% per day, about 0.1% to about 24% per day, about 0.1% to about 22% per day, about 0.1% to about 20% per day, about 0.1% to about 18% per day, about 0.1% to about 16% per day, about 0.1% to about 18% per day, about 0.1% to about 19% per day, about 0.1% to about 20% per day .1% to about 14% per day, about 0.1% to about 12% per day, about 0.1% to about 10% per day, about 0.1% to about 8% per day, about 0.1% to about 6% per day, about 0.1% to about 5% per day, about 0.1% to about 4.5% per day, about 0.1% to about 4.0% per day, about 0.1% to about 3.5% per day, about 0.1% to about 3.0% per day, about 0.1% to about 2.5% per day, about 0.1% to about 2.0%, about 0.1% to about 1.5% per day, about 0.1% to about 1.0% per day, about 0.1% to about 0.5% per day, about 0.5% to about 40% per day, about 0.5% to about 38% per day, about 0.5% to about 36% per day, about 0.5% to about 34% per day, about 0.5% to about 32% per day, about 0.5% to about 30% per day, about 0.5% to about 28% per day,From about 0.5% to about 26% per day, from about 0.5% to about 24% per day, from about 0.5% to about 22% per day, from about 0.5% to about 20% per day, from about 0.5% to about 18% per day, from about 0.5% to about 16% per day, from about 0.5% to about 14% per day, from about 0.5% to about 12% per day, from about 0.5% to about 10% per day, from about 0.5% to about 8% per day, from about 0.5% to about 6% per day, from about 0.5% to about 5% per day, from about 0.5% to about 4.5% per day, from about 0.5% to about 4.0% per day, from about 0.5% to about 3.5% per day, from about 0.5% to about 3.0% per day, from about 0.5% to about 2.5% per day, from about 0.5% to about 2.0% per day, from about 0.5% to about 1.5% per day , about 0.5% to about 1.0% per day, about 1.0% to about 40% per day, about 1.0% to about 38% per day, about 1.0% to about 36% per day, about 1.0% to about 34% per day, about 1.0% to about 32% per day, about 1.0% to about 30% per day, about 1.0% to about 28% per day, about 1.0% to about 26% per day, about 1.0% to about 24% per day, about 1.0% to about 22% per day, about 1.0% to about 20% per day, about 1.0% to about 18% per day, about 1.0% to about 16% per day, about 1.0% to about 14% per day, about 1.0% to about 12% per day, about 1.0% to about 10% per day, about 1.0% to about 8% per day, about 1.0% to about 6% per day, about 1.0% to about 5%, about 1.0% to about 4.5% per day, about 1.0% to about 4.0% per day, about 1.0% to about 3.5% per day, about 1.0% to about 3.0% per day, about 1.0% to about 2.5% per day, about 1.0% to about 2.0% per day, about 1.0% to about 1.5% per day, about 1.5% to about 40% per day, about 1.5% to about 38% per day, about 1.5% to about 36% per day, about 1.5% to about 34% per day, about 1.5% to about 32% per day, about 1.5% to about 30% per day, about 1.5% to about 28% per day, about 1.5% to about 26% per day, about 1.5% to about 24% per day, about 1.5% to about 22% per day, about 1.5% to about 20% per day, about 1.5% to about 18% per day, about 1.5% to about 16% per day, about 1.5% to about 14% per day, about 1.5% to about 12% per day, about 1.5% to about 10% per day, about 1.5% to about 8% per day, about 1.5% to about 6% per day, about 1.5% to about 5% per day, about 1.5% to about 4.5% per day, about 1.5% to about 4.0% per day, about 1.5% to about 3.5% per day, about 1.5% to about 3.0% per day, about 1.5% to about 2.5% per day, about 1.5% to about 2.0% per day, about 2.0% to about 40% per day, about 2.0% to about 38% per day, about 2.0% to about 36% per day, about 2.0% to about 34% per day, about 2.0% to about 32% per day,From about 2.0% to about 30% per day, from about 2.0% to about 28% per day, from about 2.0% to about 26% per day, from about 2.0% to about 24% per day, from about 2.0% to about 22% per day, from about 2.0% to about 20% per day, from about 2.0% to about 18% per day, from about 2.0% to about 16% per day, from about 2.0% to about 14% per day, from about 2.0% to about 12% per day, from about 2.0% to about 10% per day, from about 2.0% to about 8% per day, from about 2.0% to about 6% per day, from about 2.0% to about 5% per day, from about 2.0% to about 4.5% per day, from about 2.0% to about 4.0% per day, from about 2.0% to about 3.5% per day, from about 2.0% to about 3.0% per day, from about 2.0% to about 2.5% per day %, about 2.5% to about 40% per day, about 2.5% to about 38% per day, about 2.5% to about 36% per day, about 2.5% to about 34% per day, about 2.5% to about 32% per day, about 2.5% to about 30% per day, about 2.5% to about 28% per day, about 2.5% to about 26% per day, about 2.5% to about 24% per day, about 2.5% to about 22% per day, about 2.5% to about 20% per day, about 2.5% to about 18% per day, about 2.5% to about 16% per day, about 2.5% to about 14% per day, about 2.5% to about 12% per day, about 2.5% to about 10% per day, about 2.5% to about 8% per day, about 2.5% to about 6% per day, about 2.5% to about 5% per day, about 2.5% to about 10% per day From about 2.5% to about 4.5%, from about 2.5% to about 4.0% per day, from about 2.5% to about 3.5% per day, from about 2.5% to about 3.0% per day, from about 3.0% to about 40% per day, from about 3.0% to about 38% per day, from about 3.0% to about 36% per day, from about 3.0% to about 34% per day, from about 3.0% to about 32% per day, from about 3.0% to about 30% per day, from about 3.0% to about 28% per day, from about 3.0% to about 26% per day, from about 3.0% to about 24% per day, from about 3.0% to about 22% per day, from about 3.0% to about 20% per day, from about 3.0% to about 18% per day, from about 3.0% to about 16% per day, from about 3.0% to about 14% per day, from about 3.0% to about 12% per day , about 3.0% to about 10% per day, about 3.0% to about 8% per day, about 3.0% to about 6% per day, about 3.0% to about 5% per day, about 3.0% to about 4.5% per day, about 3.0% to about 4.0% per day, about 3.0% to about 3.5% per day, about 3.5% to about 40% per day, about 3.5% to about 38% per day, about 3.5% to about 36% per day, about 3.5% to about 34% per day, about 3.5% to about 32% per day, about 3.5% to about 30% per day, about 3.5% to about 28% per day, about 3.5% to about 26% per day, about 3.5% to about 24% per day, about 3.5% to about 22% per day, about 3.5% to about 20% per day, about 3.5% to about 18% per day,From about 3.5% to about 16% per day, from about 3.5% to about 14% per day, from about 3.5% to about 12% per day, from about 3.5% to about 10% per day, from about 3.5% to about 8% per day, from about 3.5% to about 6% per day, from about 3.5% to about 5% per day, from about 3.5% to about 4.5% per day, from about 3.5% to about 4.0% per day, from about 4.0% to about 40% per day, from about 4.0% to about 38% per day, from about 4.0% to about 36% per day, from about 4.0% to about 34% per day, from about 4.0% to about 32% per day, from about 4.0% to about 30% per day, from about 4.0% to about 28% per day, from about 4.0% to about 26% per day, from about 4.0% to about 24% per day, from about 4.0% to about 22% per day, and from about 4 .0% to about 20% per day, about 4.0% to about 18% per day, about 4.0% to about 16% per day, about 4.0% to about 14% per day, about 4.0% to about 12% per day, about 4.0% to about 10% per day, about 4.0% to about 8% per day, about 4.0% to about 6% per day, about 4.0% to about 5% per day, about 4.0% to about 4.5% per day, about 4.5% to about 40% per day, about 4.5% to about 38% per day, about 4.5% to about 36% per day, about 4.5% to about 34% per day, about 4.5% to about 32% per day, about 4.5% to about 30% per day, about 4.5% to about 28% per day, about 4.5% to about 26% per day, about 4.5% to about 24% per day, about 4.5% to about 22%, about 4.5% to about 20% per day, about 4.5% to about 18% per day, about 4.5% to about 16% per day, about 4.5% to about 14% per day, about 4.5% to about 12% per day, about 4.5% to about 10% per day, about 4.5% to about 8% per day, about 4.5% to about 6% per day, about 4.5% to about 5% per day, about 5% to about 40% per day, about 5% to about 38% per day, about 5% to about 36% per day, about 5% to about 34% per day, about 5% to about 32% per day, about 5% to about 30% per day, about 5% to about 28% per day, about 5% to about 26% per day, about 5% to about 24% per day, about 5% to about 22% per day, about 5% to about 20% per day, about 5% to about 18% per day, From about 5% to about 16% per day, from about 5% to about 14% per day, from about 5% to about 12% per day, from about 5% to about 10% per day, from about 5% to about 8% per day, from about 5% to about 6% per day, from about 6% to about 40% per day, from about 6% to about 38% per day, from about 6% to about 36% per day, from about 6% to about 34% per day, from about 6% to about 32% per day, from about 6% to about 30% per day, from about 6% to about 28% per day, from about 6% to about 26% per day, from about 6% to about 24% per day, from about 6% to about 22% per day, from about 6% to about 20% per day, from about 6% to about 18% per day, from about 6% to about 16% per day, from about 6% to about 14% per day, from about 6% to about 12% per day, from about 6% to about 10% per day, from about 6% to about 8% per day,From about 8% to about 40% per day, from about 8% to about 38% per day, from about 8% to about 36% per day, from about 8% to about 34% per day, from about 8% to about 32% per day, from about 8% to about 30% per day, from about 8% to about 28% per day, from about 8% to about 26% per day, from about 8% to about 24% per day, from about 8% to about 22% per day, from about 8% to about 20% per day, from about 8% to about 18% per day, from about 8% to about 16% per day, from about 8% to about 14% per day, from about 8% to about 12% per day, from about 8% to about 10% per day, from about 10% to about 40% per day, from about 10% to about 38% per day, from about 10% to about 36% per day, from about 10% to about 34% per day, from about 10% to about 32% per day, from about 10% to about 30% per day, From about 10% to about 28% per day, from about 10% to about 26% per day, from about 10% to about 24% per day, from about 10% to about 22% per day, from about 10% to about 20% per day, from about 10% to about 18% per day, from about 10% to about 16% per day, from about 10% to about 14% per day, from about 10% to about 12% per day, from about 12% to about 40% per day, from about 12% to about 38% per day, from about 12% to about 36% per day, from about 12% to about 34% per day, from about 12% to about 32% per day, from about 12% to about 30% per day, from about 12% to about 28% per day, from about 12% to about 26% per day, from about 12% to about 24% per day, from about 12% to about 22% per day, from about 12% to about 20% per day, from about 12% to about 18% per day, about 12% to about 16% per day, about 12% to about 14% per day, about 14% to about 40% per day, about 14% to about 38% per day, about 14% to about 36% per day, about 14% to about 34% per day, about 14% to about 32% per day, about 14% to about 30% per day, about 14% to about 28% per day, about 14% to about 26% per day, about 14% to about 24% per day, about 14% to about 22% per day, about 14% to about 20% per day, about 14% to about 18% per day, about 14% to about 16% per day, about 16% to about 40% per day, about 16% to about 38% per day, about 16% to about 36% per day, about 16% to about 34% per day, about 16% to about 32% per day, about 1 6% to about 30% per day, about 16% to about 28% per day, about 16% to about 26% per day, about 16% to about 24% per day, about 16% to about 22% per day, about 16% to about 20% per day, about 16% to about 18% per day, about 18% to about 40% per day, about 18% to about 38% per day, about 18% to about 36% per day, about 18% to about 34% per day, about 18% to about 32% per day, about 18% to about 30% per day, about 18% to about 28% per day, about 18% to about 26% per day, about 18% to about 24% per day, about 18% to about 22% per day, about 18% to about 20% per day, about 20% to about 40% per day, about 20% to about 38% per day, about 20% to about 36% per day,From about 20% to about 34% per day, from about 20% to about 32% per day, from about 20% to about 30% per day, from about 20% to about 28% per day, from about 20% to about 26% per day, from about 20% to about 24% per day, from about 20% to about 22% per day, from about 22% to about 40% per day, from about 22% to about 38% per day, from about 22% to about 36% per day, from about 22% to about 34% per day, from about 22% to about 32% per day, from about 22% to about 30% per day, From about 22% to about 28% per day, from about 22% to about 26% per day, from about 22% to about 24% per day, from about 24% to about 40% per day, from about 24% to about 38% per day, from about 24% to about 36% per day, from about 24% to about 34% per day, from about 24% to about 32% per day, from about 24% to about 30% per day, from about 24% to about 28% per day, from about 24% to about 26% per day, from about 26% to about 40% per day, from about 26% to about 38% per day, From about 26% to about 36% per day, from about 26% to about 34% per day, from about 26% to about 32% per day, from about 26% to about 30% per day, from about 26% to about 28% per day, from about 28% to about 40% per day, from about 28% to about 38% per day, from about 28% to about 36% per day, from about 28% to about 34% per day, from about 28% to about 32% per day, from about 28% to about 30% per day, from about 30% to about 40% per day, from about 30% to about 38% per day, From about 30% to about 36% per day, from about 30% to about 34% per day, from about 30% to about 32% per day, from about 32% to about 40% per day, from about 32% to about 38% per day, from about 32% to about 36% per day, from about 32% to about 34% per day, from about 34% to about 40% per day, from about 34% to about 38% per day, from about 34% to about 36% per day, from about 36% to about 40% per day, from about 36% to about 38% per day, or from about 38% to about 40% per day.

[0356] Some embodiments of these methods further comprise culturing cells (e.g., any of the cells described herein) in the production bioreactor (e.g., any of the production bioreactors described herein) using a concentration of a sensitizer predicted to produce a specific LDH production of less than 150 nU / cell / day, less than 145 nU / cell / day, less than 140 nU / cell / day, less than 135 nU / cell / day, less than 130 nU / cell / day, less than 125 nU / cell / day, less than 120 nU / cell / day, less than 115 nU / cell / day, less than 150 nU / cell / day, less than 110 nU / cell / day, less than 105 nU / cell / day, less than 110 nU / cell / day, less than 120 nU / cell / day, less than 135 nU / cell / day, less than 130 nU / cell / day, less than 125 nU / cell / day, less than 120 nU / cell / day, less than 115 nU / cell / day, less than 150 nU / cell / day, less than 110 nU / cell / day, less than 105 nU / cell / day, less than 130 nU / cell / day, less than 13 ...5 nU / cell / day, less than 100nU / cell / day, less than 95nU / cell / day, less than 90nU / cell / day, less than 85nU / cell / day, less than 80nU / cell / day, less than 75nU / cell / day, less than 70nU / cell / day, less than 65nU / cell / day, less than 60nU / cell / day, less than 55nU / cell / day, less than 50nU / cell / day, less than 45nU / cell / day, less than 45nU / cell / day, less than 40nU / cell / day, less than 35nU / cell / day, less than 30nU / cell / day, less than 25nU / cell / day, less than 20nU / cell / day, less than 15nU / cell / day, less than 10nU / cell / day, less than 5nU / cell / day or less than 1nU / cell / day. Some embodiments of these methods further comprise culturing cells (e.g., any of the cells described herein) in the production bioreactor (e.g., any of the production bioreactors described herein) using a concentration of a sensitizer predicted to produce a specific LDH production of about 0.1 nU / cell / day to about 150 nU / cell / day, about 0.1 nU / cell / day to about 140 nU / cell / day, about 0.1 nU / cell / day to about 130 nU / cell / day, about 0.1 nU / cell / day to about 120 nU / cell / day, about 0.1 nU / cell / day to about 110 nU / cell / day, about 0.1 nU / cell / day to about 100 nU / cell / day, about 0.1 nU / cell / day to about 95 nU / cell / day, about 0.1 nU / cell / day to about 90 nU / cell / day, about 0.1 nU / cell / day to about 85 nU / cell / day, about 0.1 nU / cell / day to about 80 nU / cell / day, about 0.1 nU / cell / day to about 75 nU / cell / day, about 0.1 nU / cell / day to about 70 nU / cell / day, about 0.1 nU / cell / day to about 65 nU / cell / day, about 0.1 nU / cell / day to about 60 nU / cell / day, about 0.1 nU / cell / day to about 55 nU / cell / day,about 0.1 nU / cell / day to about 50 nU / cell / day, about 0.1 nU / cell / day to about 45 nU / cell / day, about 0.1 nU / cell / day to about 40 nU / cell / day, about 0.1 nU / cell / day to about 35 nU / cell / day, about 0.1 nU / cell / day to about 30 nU / cell / day, about 0.1 nU / cell / day to about 25 nU / cell / day, about 0.1 nU / cell / day to about 20 nU / cell / day, about 0.1 nU / cell / day to about 15 nU / cell / day, about 0.1 nU / cell / day to about 10 nU / cell / day, about 0.1 nU / cell / day to about 5 nU / cell / day, about 0.1 nU / cell / day to about 2.5 nU / cell / day, about 0.1 nU / cell / day to about 1.0 nU / cell / day, about 1.0 nU / cell / day to about 150 nU / cell / day, about 1.0 nU / cell / day to about 140 nU / cell / day, about 1.0 nU / cell / day to about 130 nU / cell / day, about 1.0 nU / cell / day to about 120 nU / cell / day, about 1.0 nU / cell / day to about 110 nU / cell / day, about 1.0 nU / cell / day to about 100 nU / cell / day, about 1.0 nU / cell / day to about 95 nU / cell / day, about 1.0 nU / cell / day to about 90 nU / cell / day, about 1.0 nU / cell / day to about 85 nU / cell / day, about 1.0 nU / cell / day to about day to about 80 nU / cell / day, about 1.0 nU / cell / day to about 75 nU / cell / day, about 1.0 nU / cell / day to about 70 nU / cell / day, about 1.0 nU / cell / day to about 65 nU / cell / day, about 1.0 nU / cell / day to about 60 nU / cell / day, about 1.0 nU / cell / day to about 55 nU / cell / day, about 1.0 nU / cell / day to about 50 nU / cell / day, about 1.0 nU / cell / day to about 45 nU / cell / day, about 1.0 nU / cell / day to about 40 nU / cell / day, about 1.0 nU / cell / day to about 35 nU / cell / day, about 1.0 nU / cell / day to about 30 nU / cell / day, about 1.0 nU / cell / day to about From about 1 to about 25 nU / cell / day, from about 1.0 nU / cell / day to about 20 nU / cell / day, from about 1.0 nU / cell / day to about 15 nU / cell / day, from about 1.0 nU / cell / day to about 10 nU / cell / day, from about 1.0 nU / cell / day to about 5 nU / cell / day, from about 1.0 nU / cell / day to about 2.5 nU / cell / day, from about 2.5 nU / cell / day to about 150 nU / cell / day, from about 2.5 nU / cell / day to about 140 nU / cell / day, from about 2.5 nU / cell / day to about 130 nU / cell / day, from about 2.5 nU / cell / day to about 120 nU / cell / day, from about 2.5 nU / cell / day to about 110 nU / cell / day,about 2.5nU / cell / day to about 100nU / cell / day, about 2.5nU / cell / day to about 95nU / cell / day, about 2.5nU / cell / day to about 90nU / cell / day, about 2.5nU / cell / day to about 85nU / cell / day, about 2.5nU / cell / day to about 80nU / cell / day, about 2.5nU / cell / day to about 75nU / cell / day, about 2.5nU / cell / day to about 70nU / cell / day, about 2.5nU / cell / day to about 65nU / cell / day, about 2.5nU / cell / day to about 60nU / cell / day, about 2.5nU / cell / day to about 55nU / cell / day, about 2.5nU / cell / day to about 50nU / cell / day, about 2.5nU / cell / day to about 45nU / cell / day, about 2.5nU / cell / day to about 40nU / cell / day, about 2.5nU / cell / day to about 35nU / cell / day, about 2.5nU / cell / day to about 30nU / cell / day, about 2.5nU / cell / day to about 25nU / cell / day, about 2.5nU / cell / day to about 20nU / cell / day, about 2.5nU / cell / day to about 15nU / cell / day, about 2.5nU / cell / day to about 10nU / cell / day, about 2.5nU / cell / day to about 5nU / cell / day, about 5nU / cell / day to about 150nU / cell / day, about 5nU / cell / day to about 140 nU / cell / day, about 5nU / cell / day to about 130nU / cell / day, about 5nU / cell / day to about 120nU / cell / day, about 5nU / cell / day to about 110nU / cell / day, about 5nU / cell / day to about 100nU / cell / day, about 5nU / cell / day to about 95nU / cell / day, about 5nU / cell / day to about 90nU / cell / day, about 5nU / cell / day to about 85nU / cell / day, about 5nU / cell / day to about 80nU / cell / day, about 5nU / cell / day to about 75nU / cell / day, about 5nU / cell / day to about 70nU / cell / day, about 5nU / cell / day to about 65nU / cell / day, about 5nU / cell / day to about From about 5 to about 60 nU / cell / day, from about 5 to about 55 nU / cell / day, from about 5 to about 50 nU / cell / day, from about 5 to about 45 nU / cell / day, from about 5 to about 40 nU / cell / day, from about 5 to about 35 nU / cell / day, from about 5 to about 30 nU / cell / day, from about 5 to about 25 nU / cell / day, from about 5 to about 20 nU / cell / day, from about 5 to about 15 nU / cell / day, from about 5 to about 10 nU / cell / day, from about 10 to about 150 nU / cell / day,about 10 nU / cell / day to about 140 nU / cell / day, about 10 nU / cell / day to about 130 nU / cell / day, about 10 nU / cell / day to about 120 nU / cell / day, about 10 nU / cell / day to about 110 nU / cell / day, about 10 nU / cell / day to about 100 nU / cell / day, about 10 nU / cell / day to about 95 nU / cell / day, about 10 nU / cell / day to about 90 nU / cell / day, about 10 nU / cell / day to about 85 nU / cell / day, about 10 nU / cell / day to about 80 nU / cell / day, about 10 nU / cell / day to about 75 nU / cell / day, about 10 nU / cell / day to about 70 nU / cell / day, about 10 nU / cell / day to about 65 nU / cell / day, about 10 nU / cell / day to about 60 nU / cell / day, about 10 nU / cell / day to about 55 nU / cell / day, about 10 nU / cell / day to about 50 nU / cell / day, about 10 nU / cell / day to about 45 nU / cell / day, about 10 nU / cell / day to about 40 nU / cell / day, about 10 nU / cell / day to about 35 nU / cell / day, about 10 nU / cell / day to about 30 nU / cell / day, about 10 nU / cell / day to about 25 nU / cell / day, about 10 nU / cell / day to about 20 nU / cell / day, about 10 nU / cell / day to about 15 nU / cell / day, about 15nU / cell / day to about 150nU / cell / day, about 15nU / cell / day to about 140nU / cell / day, about 15nU / cell / day to about 130nU / cell / day, about 15nU / cell / day to about 120nU / cell / day, about 15nU / cell / day to about 110nU / cell / day, about 15nU / cell / day to about 100nU / cell / day, about 15nU / cell / day to about 95nU / cell / day, about 15nU / cell / day to about 90nU / cell / day, about 15nU / cell / day to about 85nU / cell / day, about 15nU / cell / day to about 80nU / cell / day, about 15nU / cell / day to about 75nU / cell / day, about 15nU / cell / day to about 70nU / cell / day, about 15nU / cell / day to about 65nU / cell / day, about 15nU / cell / day to about 60nU / cell / day, about 15nU / cell / day to about 55nU / cell / day, about 15nU / cell / day to about 50nU / cell / day, about 15nU / cell / day to about 45nU / cell / day, about 15nU / cell / day to about 40nU / cell / day, about 15nU / cell / day to about 35nU / cell / day, about 15nU / cell / day to about 30nU / cell / day, about 15nU / cell / day to about 25nU / cell / day, about 15nU / cell / day to about 20nU / cell / day,about 20nU / cell / day to about 150nU / cell / day, about 20nU / cell / day to about 140nU / cell / day, about 20nU / cell / day to about 130nU / cell / day, about 20nU / cell / day to about 120nU / cell / day, about 20nU / cell / day to about 110nU / cell / day, about 20nU / cell / day to about 100nU / cell / day, about 20nU / cell / day to about 95nU / cell / day, about 20nU / cell / day to about 90nU / cell / day, about 20nU / cell / day to about 85nU / cell / day, about 20nU / cell / day to about 80nU / cell / day, about 20nU / cell / day to about 75nU / cell / day, about 20nU / cell / day to about 70nU / cell / day, about 20nU / cell / day to about 65nU / cell / day, about 20nU / cell / day to about 60nU / cell / day, about 20nU / cell / day to about 55nU / cell / day, about 20nU / cell / day to about 50nU / cell / day, about 20nU / cell / day to about 45nU / cell / day, about 20nU / cell / day to about 40nU / cell / day, about 20nU / cell / day to about 35nU / cell / day, about 20nU / cell / day to about 30nU / cell / day, about 20nU / cell / day to about 25nU / cell / day, about 25nU / cell / day to about 150nU / cell / day, about 25nU / cell / day to about 140nU / cell / day, about 25nU / cell / day to about 130nU / cell / day, about 25nU / cell / day to about 120nU / cell / day, about 25nU / cell / day to about 110nU / cell / day, about 25nU / cell / day to about 100nU / cell / day, about 25nU / cell / day to about 95nU / cell / day, about 25nU / cell / day to about 90nU / cell / day, about 25nU / cell / day to about 85nU / cell / day, about 25nU / cell / day to about 80nU / cell / day, about 25nU / cell / day to about 75nU / cell / day, about 25nU / cell / day to about 70nU / cell / day , about 25 nU / cell / day to about 65 nU / cell / day, about 25 nU / cell / day to about 60 nU / cell / day, about 25 nU / cell / day to about 55 nU / cell / day, about 25 nU / cell / day to about 50 nU / cell / day, about 25 nU / cell / day to about 45 nU / cell / day, about 25 nU / cell / day to about 40 nU / cell / day, about 25 nU / cell / day to about 35 nU / cell / day, about 25 nU / cell / day to about 30 nU / cell / day, about 30 nU / cell / day to about 150 nU / cell / day, about 30 nU / cell / day to about 140 nU / cell / day, about 30 nU / cell / day to about 130 nU / cell / day,about 30nU / cell / day to about 120nU / cell / day, about 30nU / cell / day to about 110nU / cell / day, about 30nU / cell / day to about 100nU / cell / day, about 30nU / cell / day to about 95nU / cell / day, about 30nU / cell / day to about 90nU / cell / day, about 30nU / cell / day to about 85nU / cell / day, about 30nU / cell / day to about 80nU / cell / day, about 30nU / cell / day to about 75nU / cell / day, about 30nU / cell / day to about 70nU / cell / day, about 30nU / cell / day to about 65nU / cell / day, about 30nU / cell / day to about 60nU / cell / day, about 30nU / cell / day to about 55nU / cell / day, about 30nU / cell / day to about 50nU / cell / day, about 30nU / cell / day to about 45nU / cell / day, about 30nU / cell / day to about 40nU / cell / day, about 30nU / cell / day to about 35nU / cell / day, about 35nU / cell / day to about 150nU / cell / day, about 35nU / cell / day to about 140nU / cell / day, about 35nU / cell / day to about 130nU / cell / day, about 35nU / cell / day to about 120nU / cell / day, about 35nU / cell / day to about 110nU / cell / day, about 35nU / cell / day to about 100nU / cell / day day, about 35 nU / cell / day to about 95 nU / cell / day, about 35 nU / cell / day to about 90 nU / cell / day, about 35 nU / cell / day to about 85 nU / cell / day, about 35 nU / cell / day to about 80 nU / cell / day, about 35 nU / cell / day to about 75 nU / cell / day, about 35 nU / cell / day to about 70 nU / cell / day, about 35 nU / cell / day to about 65 nU / cell / day, about 35 nU / cell / day to about 60 nU / cell / day, about 35 nU / cell / day to about 55 nU / cell / day, about 35 nU / cell / day to about 50 nU / cell / day, about 35 nU / cell / day to about 45 nU / cell / day, about 3 From about 5 to about 40 nU / cell / day, from about 40 to about 150 nU / cell / day, from about 40 to about 140 nU / cell / day, from about 40 to about 130 nU / cell / day, from about 40 to about 120 nU / cell / day, from about 40 to about 110 nU / cell / day, from about 40 to about 100 nU / cell / day, from about 40 to about 95 nU / cell / day, from about 40 to about 90 nU / cell / day, from about 40 to about 85 nU / cell / day, from about 40 to about 80 nU / cell / day,about 40nU / cell / day to about 75nU / cell / day, about 40nU / cell / day to about 70nU / cell / day, about 40nU / cell / day to about 65nU / cell / day, about 40nU / cell / day to about 60nU / cell / day, about 40nU / cell / day to about 55nU / cell / day, about 40nU / cell / day to about 50nU / cell / day, about 40nU / cell / day to about 45nU / cell / day, about 45nU / cell / day to about 150nU / cell / day, about 45nU / cell / day to about 140nU / cell / day, about 45nU / cell / day to about 130nU / cell / day, about 45nU / cell / day to about 120nU / cell / day, about 45nU / cell / day to about 110nU / cell / day, about 45nU / cell / day to about 100nU / cell / day, about 45nU / cell / day to about 95nU / cell / day, about 45nU / cell / day to about 90nU / cell / day, about 45nU / cell / day to about 85nU / cell / day, about 45nU / cell / day to about 80nU / cell / day, about 45nU / cell / day to about 75nU / cell / day, about 45nU / cell / day to about 70nU / cell / day, about 45nU / cell / day to about 65nU / cell / day, about 45nU / cell / day to about 60nU / cell / day, about 45nU / cell / day to about 55nU / cell / day, about 45 nU / cell / day to about 50nU / cell / day, about 50nU / cell / day to about 150nU / cell / day, about 50nU / cell / day to about 140nU / cell / day, about 50nU / cell / day to about 130nU / cell / day, about 50nU / cell / day to about 120nU / cell / day, about 50nU / cell / day to about 110nU / cell / day, about 50nU / cell / day to about 100nU / cell / day, about 50nU / cell / day to about 95nU / cell / day, about 50nU / cell / day to about 90nU / cell / day, about 50nU / cell / day to about 85nU / cell / day, about 50nU / cell / day to about 80nU / cell / day, about 50nU / cell / day to about 75nU / cell / day, about 50nU / cell / day to about 70nU / cell / day, about 50nU / cell / day to about 65nU / cell / day, about 50nU / cell / day to about 60nU / cell / day, about 50nU / cell / day to about 55nU / cell / day, about 55nU / cell / day to about 150nU / cell / day, about 55nU / cell / day to about 140nU / cell / day, about 55nU / cell / day to about 130nU / cell / day, about 55nU / cell / day to about 120nU / cell / day, about 55nU / cell / day to about 110nU / cell / day, about 55nU / cell / day to about 100nU / cell / day,about 55nU / cell / day to about 95nU / cell / day, about 55nU / cell / day to about 90nU / cell / day, about 55nU / cell / day to about 85nU / cell / day, about 55nU / cell / day to about 80nU / cell / day, about 55nU / cell / day to about 75nU / cell / day, about 55nU / cell / day to about 70nU / cell / day, about 55nU / cell / day to about 65nU / cell / day, about 55nU / cell / day to about 60nU / cell / day, about 60nU / cell / day to about 150nU / cell / day, about 60nU / cell / day to about 140nU / cell / day, about 60nU / cell / day to about 130nU / cell / day, about The invention relates to a method for preparing an aqueous solution of at least one molecule of the present invention and to a method for preparing the aqueous solution of at least one molecule of the present invention. The method comprises: a) using a method for preparing the aqueous solution of at least one molecule of the present invention and to ... 5nU / cell / day to about 140nU / cell / day, about 65nU / cell / day to about 130nU / cell / day, about 65nU / cell / day to about 120nU / cell / day, about 65nU / cell / day to about 110nU / cell / day, about 65nU / cell / day to about 100nU / cell / day, about 65nU / cell / day to about 95nU / cell / day, about 65nU / cell / day to about 90nU / cell / day, about 65nU / cell / day to about 85nU / cell / day, about 65nU / cell / day to about 80nU / cell / day, about 65nU / cell / day to about 75nU / cell / day, about 65nU / cell / day to about 70nU / cell / day, about 70nU / cell / day to about 150nU / cell / day, about 70nU / cell / day to about 140nU / cell / day, about 70nU / cell / day to about 130nU / cell / day, about 70nU / cell / day to about 120nU / cell / day, about 70nU / cell / day to about 110nU / cell / day, about 70nU / cell / day to about 100nU / cell / day, about 70nU / cell / day to about 95nU / cell / day, about 70nU / cell / day to about 90nU / cell / day, about 70nU / cell / day to about 85nU / cell / day, about 70nU / cell / day to about 80nU / cell / day, about 70nU / cell / day to about 75nU / cell / day,about 75 nU / cell / day to about 150 nU / cell / day, about 75 nU / cell / day to about 140 nU / cell / day, about 75 nU / cell / day to about 130 nU / cell / day, about 75 nU / cell / day to about 120 nU / cell / day, about 75 nU / cell / day to about 110 nU / cell / day, about 75 nU / cell / day to about 100 nU / cell / day, about 75 nU / cell / day to about 95 nU / cell / day, about 75 nU / cell / day to about 90 nU / cell / day, about 75 nU / cell / day to about 85 nU / cell / day, about 75 nU / cell / day to about 80 nU / cell / day, about 80 nU / cell / day to about 150 nU / cell The invention relates to an amount of about 80 nU / cell / day to about 140 nU / cell / day, about 80 nU / cell / day to about 130 nU / cell / day, about 80 nU / cell / day to about 120 nU / cell / day, about 80 nU / cell / day to about 110 nU / cell / day, about 80 nU / cell / day to about 100 nU / cell / day, about 80 nU / cell / day to about 95 nU / cell / day, about 80 nU / cell / day to about 90 nU / cell / day, about 80 nU / cell / day to about 85 nU / cell / day, about 85 nU / cell / day to about 150 nU / cell / day, about 85 nU / cell / day to about 140 nU / cell / day, about 85 nU / cell / day to about 130 nU / cell / day, about 85nU / cell / day to about 120nU / cell / day, about 85nU / cell / day to about 110nU / cell / day, about 85nU / cell / day to about 100nU / cell / day, about 85nU / cell / day to about 95nU / cell / day, about 85nU / cell / day to about 90nU / cell / day, about 90nU / cell / day to about 150nU / cell / day, about 90nU / cell / day to about 140nU / cell / day, about 90nU / cell / day to about 130nU / cell / day, about 90nU / cell / day to about 120nU / cell / day, about 90nU / cell / day to about 110nU / cell / day, about 90nU / cell / day to about about 95 nU / cell / day to about 110 nU / cell / day, about 95 nU / cell / day to about 100 nU / cell / day, about 90 nU / cell / day to about 95 nU / cell / day, about 95 nU / cell / day to about 150 nU / cell / day, about 95 nU / cell / day to about 140 nU / cell / day, about 95 nU / cell / day to about 130 nU / cell / day, about 95 nU / cell / day to about 120 nU / cell / day, about 95 nU / cell / day to about 110 nU / cell / day, about 95 nU / cell / day to about 100 nU / cell / day, about 100 nU / cell / day to about 150 nU / cell / day, about 100 nU / cell / day to about 140 nU / cell / day, about 100 nU / cell / day to about 130 nU / cell / day,about 100 nU / cell / day to about 120 nU / cell / day, about 100 nU / cell / day to about 110 nU / cell / day, about 110 nU / cell / day to about 150 nU / cell / day, about 110 nU / cell / day to about 140 nU / cell / day, about 110 nU / cell / day to about 130 nU / cell / day, about 110 nU / cell / day to about 120 nU / cell / day, About 120nU / cell / day to about 150nU / cell / day, about 120nU / cell / day to about 140nU / cell / day, about 120nU / cell / day to about 130nU / cell / day, about 130nU / cell / day to about 150nU / cell / day, about 130nU / cell / day to about 140nU / cell / day, or about 140nU / cell / day to about 150nU / cell / day.

[0357] The growth rate is a compound growth calculation which can be determined using the equation below.

[0358] Final cell density = initial density x e kt ,

[0359] where k is the growth constant and t is the time in days.

[0360] The apparent growth rate is the sum of the calculated mortality rate plus the growth rate.

[0361] Some embodiments of any of the methods described herein further comprise culturing cells (e.g., any of the cells described herein) in the production bioreactor (e.g., any of the production bioreactors described herein) with a concentration of a sensitizer predicted to produce an apparent growth rate of greater than 1% per day, greater than 1.5% per day, greater than 2% per day, greater than 2.5% per day, greater than 3% per day, greater than 3.5% per day, greater than 4.0% per day, greater than 4.5% per day, greater than 5.0% per day, greater than 5.5% per day, greater than 6.0% per day, greater than 6.5% per day, greater than 7.0% per day, greater than 7.5% per day, greater than 8.0% per day, greater than 8.5% per day, greater than 9.0% per day, greater than 9.5% per day, greater than 10.0% per day, greater than 10.5% per day, greater than 11.0% per day, greater than 11.5% per day, greater than 12.0% per day, greater than 12.5% ​​per day, greater than 13.0% per day, greater than 13.5% per day, greater than 14.0% per day, greater than 14.5% per day, greater than 15.0% per day, greater than 15.5% per day, greater than 16.0% per day, greater than 16.5% per day, greater than 17.0% per day, greater than 17.5% per day, greater than 18.0% per day, greater than 18.5% per day, greater than 19.0% per day, greater than 19.5% per day, greater than 20.0% per day, greater than 21.0% per day, greater than 22.0% per day, greater than 23.5% per day, greater than 2 13.0% per day, greater than 13.5% per day, greater than 14.0% per day, greater than 14.5% per day, greater than 15.0% per day, greater than 16% per day, greater than 17% per day, greater than 18% per day, greater than 19% per day, greater than 20% per day, greater than 21% per day, greater than 22% per day, greater than 23% per day, greater than 24% per day, greater than 25% per day, greater than 30% per day, greater than 35% per day, greater than 40% per day, greater than 45% per day, greater than 50% per day, greater than 55% per day, greater than 60% per day, greater than 65% per day, greater than 70% per day, greater than 75% per day, greater than 80% per day, greater than 85% per day, greater than 90% per day, greater than 95% per day, greater than 100% per day, greater than 105% per day, greater than 110% per day, greater than 115% per day, greater than 120% per day, greater than 125% per day, greater than 130% per day, greater than 135% per day, greater than 140% per day, greater than 145% per day or greater than 150% per day. Some embodiments of any of the methods described herein further comprise culturing cells (e.g., any of the cells described herein) in the production bioreactor (e.g., any of the production bioreactors described herein) with a concentration of a sensitizer predicted to produce an apparent growth rate during culturing of the cells in the production bioreactor of about 0.1% to about 150% per day, about 0.1% to about 140% per day, about 0.1% to about 135% per day, about 0.1% to about 130% per day, about 0.1% to about 125% per day, about 0.1% to about 120% per day, about 0.1% to about 115% per day, about 0.1% to about 110% per day, about 0.1% to about 105% per day, about 0.1% to about 100% per day, about 0.1% to about 95% per day, about 0.1% to about 90% per day, about 0.1% to about 85% per day,From about 0.1% to about 80% per day, from about 0.1% to about 75% per day, from about 0.1% to about 70% per day, from about 0.1% to about 65% per day, from about 0.1% to about 60% per day, from about 0.1% to about 55% per day, from about 0.1% to about 50% per day, from about 0.1% to about 45% per day, from about 0.1% to about 40% per day, from about 0.1% to about 35% per day, from about 0.1% to about 30% per day, from about 0.1% to about 25% per day, from about 0.1% to about 20% per day, from about 0.1% to about 18% per day, from about 0.1% to about 16% per day, from about 0.1% to about 14% per day, from about 0.1% to about 16% per day about 12%, about 0.1% to about 10% per day, about 0.1% to about 9.0% per day, about 0.1% to about 8.0% per day, about 0.1% to about 7.0% per day, about 0.1% to about 6.0% per day, about 0.1% to about 5.0% per day, about 0.1% to about 4.5% per day, about 0.1% to about 4.0% per day, about 0.1% to about 3.5% per day, about 0.1% to about 3.0% per day, about 0.1% to about 2.5% per day, about 0.1% to about 2.0% per day, about 0.1% to about 1.5% per day, about 0.1% to about 1.0% per day, about 0.1% to about 0.5% per day, about 0.5% per day % to about 140% per day, about 0.5% per day to about 135% per day, about 0.5% per day to about 130% per day, about 0.5% per day to about 125% per day, about 0.5% per day to about 120% per day, about 0.5% per day to about 115% per day, about 0.5% per day to about 110% per day, about 0.5% per day to about 105% per day, about 0.5% per day to about 100% per day, about 0.5% per day to about 95% per day, about 0.5% per day to about 90% per day, about 0.5% per day to about 85% per day, about 0.5% per day to about 80% per day, about 0.5% per day to about 75% per day, about 0.5% per day to about 70% per day, about 0.5% per day to about 65% per day, about 0.5% per day to about 60% per day, about 0.5% per day to about 55% per day, about 0.5% per day to about 50% per day, about 0.5% per day to about 45% per day, about 0.5% per day to about 40% per day, about 0.5% per day to about 35% per day, about 0.5% per day to about 30% per day, about 0.5% per day to about 25% per day, about 0.5% per day to about 20% per day, about 0.5% per day to about 18% per day, about 0.5% per day to about 16% per day, about 0.5% per day to about 14% per day, about 0.5% per day to about 12% per day, about 0.5% per day to about 10% per day, about 0.5% per day to about 9.0% per day, about 0.5% per day to about 8.0% per day, about 0.5% per day to about 7.0% per day,About 0.5% to about 6.0% per day, about 0.5% to about 5.0% per day, about 0.5% to about 4.5% per day, about 0.5% to about 4.0% per day, about 0.5% to about 3.5% per day, about 0.5% to about 3.0% per day, about 0.5% to about 2.5% per day, about 0.5% to about 2.0% per day, about 0.5% to about 1.5% per day, about 0.5% to about 1.0% per day, about 1.0% to about 140% per day, about 1.0% to about 135% per day, about 1.0% to about 130% per day, about 1.0% to about 125% per day, about 1.0% to about 120% per day, about 1.0% to about 1 about 1.0% to about 115% per day, about 1.0% to about 110% per day, about 1.0% to about 105% per day, about 1.0% to about 100% per day, about 1.0% to about 95% per day, about 1.0% to about 90% per day, about 1.0% to about 85% per day, about 1.0% to about 80% per day, about 1.0% to about 75% per day, about 1.0% to about 70% per day, about 1.0% to about 65% per day, about 1.0% to about 60% per day, about 1.0% to about 55% per day, about 1.0% to about 50% per day, about 1.0% to about 45% per day, about 1.0% to about 40% per day, about 1.0% to about 35% per day, about 1.0% to about 45% per day From about 1.0% to about 30% per day, from about 1.0% to about 25% per day, from about 1.0% to about 20% per day, from about 1.0% to about 18% per day, from about 1.0% to about 16% per day, from about 1.0% to about 14% per day, from about 1.0% to about 12% per day, from about 1.0% to about 10% per day, from about 1.0% to about 9.0% per day, from about 1.0% to about 8.0% per day, from about 1.0% to about 7.0% per day, from about 1.0% to about 6.0% per day, from about 1.0% to about 5.0% per day, from about 1.0% to about 4.5% per day, from about 1.0% to about 4.0% per day, from about 1.0% to about 3.5% per day, from about 1.0% to about 3.0% per day, about 1.0% to about 2.5% per day, about 1.0% to about 2.0% per day, about 1.0% to about 1.5% per day, about 1.5% to about 140% per day, about 1.5% to about 135% per day, about 1.5% to about 130% per day, about 1.5% to about 125% per day, about 1.5% to about 120% per day, about 1.5% to about 115% per day, about 1.5% to about 110% per day, about 1.5% to about 105% per day, about 1.5% to about 100% per day, about 1.5% to about 95% per day, about 1.5% to about 90% per day, about 1.5% to about 85% per day,From about 1.5% to about 80% per day, from about 1.5% to about 75% per day, from about 1.5% to about 70% per day, from about 1.5% to about 65% per day, from about 1.5% to about 60% per day, from about 1.5% to about 55% per day, from about 1.5% to about 50% per day, from about 1.5% to about 45% per day, from about 1.5% to about 40% per day, from about 1.5% to about 35% per day, from about 1.5% to about 30% per day, from about 1.5% to about 25% per day, from about 1.5% to about 20% per day, from about 1.5% to about 18% per day, from about 1.5% to about 16% per day, from about 1.5% to about 14% per day, from about 1.5% to about 14% per day about 12%, about 1.5% to about 10% per day, about 1.5% to about 9.0% per day, about 1.5% to about 8.0% per day, about 1.5% to about 7.0% per day, about 1.5% to about 6.0% per day, about 1.5% to about 5.0% per day, about 1.5% to about 4.5% per day, about 1.5% to about 4.0% per day, about 1.5% to about 3.5% per day, about 1.5% to about 3.0% per day, about 1.5% to about 2.5% per day, about 1.5% to about 2.0% per day, about 2.0% to about 140% per day, about 2.0% to about 135% per day, about 2.0% to about 130% per day, about 2.0% per day % to about 125% per day, about 2.0% per day to about 120% per day, about 2.0% per day to about 115% per day, about 2.0% per day to about 110% per day, about 2.0% per day to about 105% per day, about 2.0% per day to about 100% per day, about 2.0% per day to about 95% per day, about 2.0% per day to about 90% per day, about 2.0% per day to about 85% per day, about 2.0% per day to about 80% per day, about 2.0% per day to about 75% per day, about 2.0% per day to about 70% per day, about 2.0% per day to about 65% per day, about 2.0% per day to about 60% per day, about 2.0% per day to about 55% per day, about 2.0% per day to about 50% per day, about 2.0% per day to about about 45%, about 2.0% to about 40% per day, about 2.0% to about 35% per day, about 2.0% to about 30% per day, about 2.0% to about 25% per day, about 2.0% to about 20% per day, about 2.0% to about 18% per day, about 2.0% to about 16% per day, about 2.0% to about 14% per day, about 2.0% to about 12% per day, about 2.0% to about 10% per day, about 2.0% to about 9.0% per day, about 2.0% to about 8.0% per day, about 2.0% to about 7.0% per day, about 2.0% to about 6.0% per day, about 2.0% to about 5.0% per day, about 2.0% to about 4.5% per day,About 2.0% to about 4.0% per day, about 2.0% to about 3.5% per day, about 2.0% to about 3.0% per day, about 2.0% to about 2.5% per day, about 2.5% to about 140% per day, about 2.5% to about 135% per day, about 2.5% to about 130% per day, about 2.5% to about 125% per day, about 2.5% to about 120% per day, about 2.5% to about 115% per day, about 2.5% to about 110% per day, about 2.5% to about 105% per day, about 2.5% to about 100% per day, about 2.5% to about 95% per day, about 2.5% to about 90% per day, about 2.5% to about 100% per day about 85% per day, about 2.5% to about 80% per day, about 2.5% to about 75% per day, about 2.5% to about 70% per day, about 2.5% to about 65% per day, about 2.5% to about 60% per day, about 2.5% to about 55% per day, about 2.5% to about 50% per day, about 2.5% to about 45% per day, about 2.5% to about 40% per day, about 2.5% to about 35% per day, about 2.5% to about 30% per day, about 2.5% to about 25% per day, about 2.5% to about 20% per day, about 2.5% to about 18% per day, about 2.5% to about 16% per day, about 2.5% to about 14% per day, about 2.5% to about 16% per day From about 2.5% to about 12% per day, from about 2.5% to about 10% per day, from about 2.5% to about 9.0% per day, from about 2.5% to about 8.0% per day, from about 2.5% to about 7.0% per day, from about 2.5% to about 6.0% per day, from about 2.5% to about 5.0% per day, from about 2.5% to about 4.5% per day, from about 2.5% to about 4.0% per day, from about 2.5% to about 3.5% per day, from about 2.5% to about 3.0% per day, from about 3.0% to about 140% per day, from about 3.0% to about 135% per day, from about 3.0% to about 130% per day, from about 3.0% to about 125% per day, from about 3.0% to about 1 20%, about 3.0% to about 115% per day, about 3.0% to about 110% per day, about 3.0% to about 105% per day, about 3.0% to about 100% per day, about 3.0% to about 95% per day, about 3.0% to about 90% per day, about 3.0% to about 85% per day, about 3.0% to about 80% per day, about 3.0% to about 75% per day, about 3.0% to about 70% per day, about 3.0% to about 65% per day, about 3.0% to about 60% per day, about 3.0% to about 55% per day, about 3.0% to about 50% per day, about 3.0% to about 45% per day, about 3.0% to about 40% per day,About 3.0% to about 35% per day, about 3.0% to about 30% per day, about 3.0% to about 25% per day, about 3.0% to about 20% per day, about 3.0% to about 18% per day, about 3.0% to about 16% per day, about 3.0% to about 14% per day, about 3.0% to about 12% per day, about 3.0% to about 10% per day, about 3.0% to about 9.0% per day, about 3.0% to about 8.0% per day, about 3.0% to about 7.0% per day, about 3.0% to about 6.0% per day, about 3.0% to about 5.0% per day, about 3.0% to about 4.5% per day, about 3.0% to about 4.0% per day, about 3.0% to about 5.0% per day about 3.0% to about 3.5% per day, about 3.5% to about 140% per day, about 3.5% to about 135% per day, about 3.5% to about 130% per day, about 3.5% to about 125% per day, about 3.5% to about 120% per day, about 3.5% to about 115% per day, about 3.5% to about 110% per day, about 3.5% to about 105% per day, about 3.5% to about 100% per day, about 3.5% to about 95% per day, about 3.5% to about 90% per day, about 3.5% to about 85% per day, about 3.5% to about 80% per day, about 3.5% to about 75% per day, about 3.5% to about 70% per day , about 3.5% to about 65% per day, about 3.5% to about 60% per day, about 3.5% to about 55% per day, about 3.5% to about 50% per day, about 3.5% to about 45% per day, about 3.5% to about 40% per day, about 3.5% to about 35% per day, about 3.5% to about 30% per day, about 3.5% to about 25% per day, about 3.5% to about 20% per day, about 3.5% to about 18% per day, about 3.5% to about 16% per day, about 3.5% to about 14% per day, about 3.5% to about 12% per day, about 3.5% to about 10% per day, about 3.5% to about 9.0% per day, about 3.5% per day % to about 8.0% per day, about 3.5% to about 7.0% per day, about 3.5% to about 6.0% per day, about 3.5% to about 5.0% per day, about 3.5% to about 4.5% per day, about 3.5% to about 4.0% per day, about 4.0% to about 140% per day, about 4.0% to about 135% per day, about 4.0% to about 130% per day, about 4.0% to about 125% per day, about 4.0% to about 120% per day, about 4.0% to about 115% per day, about 4.0% to about 110% per day, about 4.0% to about 105% per day, about 4.0% to about 100% per day, about 4.0% to about 95% per day,From about 4.0% to about 90% per day, from about 4.0% to about 85% per day, from about 4.0% to about 80% per day, from about 4.0% to about 75% per day, from about 4.0% to about 70% per day, from about 4.0% to about 65% per day, from about 4.0% to about 60% per day, from about 4.0% to about 55% per day, from about 4.0% to about 50% per day, from about 4.0% to about 45% per day, from about 4.0% to about 40% per day, from about 4.0% to about 35% per day, from about 4.0% to about 30% per day, from about 4.0% to about 25% per day, from about 4.0% to about 20% per day, from about 4.0% to about 18% per day, from about 4.0% to about 18% per day, about 16% per day, about 4.0% to about 14% per day, about 4.0% to about 12% per day, about 4.0% to about 10% per day, about 4.0% to about 9.0% per day, about 4.0% to about 8.0% per day, about 4.0% to about 7.0% per day, about 4.0% to about 6.0% per day, about 4.0% to about 5.0% per day, about 4.0% to about 4.5% per day, about 4.5% to about 140% per day, about 4.5% to about 135% per day, about 4.5% to about 130% per day, about 4.5% to about 125% per day, about 4.5% to about 120% per day, about 4.5% to about 115% per day, about 4.5% per day to about 110% per day, about 4.5% per day to about 105% per day, about 4.5% per day to about 100% per day, about 4.5% per day to about 95% per day, about 4.5% per day to about 90% per day, about 4.5% per day to about 85% per day, about 4.5% per day to about 80% per day, about 4.5% per day to about 75% per day, about 4.5% per day to about 70% per day, about 4.5% per day to about 65% per day, about 4.5% per day to about 60% per day, about 4.5% per day to about 55% per day, about 4.5% per day to about 50% per day, about 4.5% per day to about 45% per day, about 4.5% per day to about 40% per day, about 4.5% per day to about 35% per day, about 4.5% per day to about 30% per day , about 4.5% to about 25% per day, about 4.5% to about 20% per day, about 4.5% to about 18% per day, about 4.5% to about 16% per day, about 4.5% to about 14% per day, about 4.5% to about 12% per day, about 4.5% to about 10% per day, about 4.5% to about 9.0% per day, about 4.5% to about 8.0% per day, about 4.5% to about 7.0% per day, about 4.5% to about 6.0% per day, about 4.5% to about 5.0% per day, about 5.0% to about 140% per day, about 5.0% to about 135% per day, about 5.0% to about 130% per day, about 5.0% to about 125% per day,From about 5.0% to about 120% per day, from about 5.0% to about 115% per day, from about 5.0% to about 110% per day, from about 5.0% to about 105% per day, from about 5.0% to about 100% per day, from about 5.0% to about 95% per day, from about 5.0% to about 90% per day, from about 5.0% to about 85% per day, from about 5.0% to about 80% per day, from about 5.0% to about 75% per day, from about 5.0% to about 70% per day, from about 5.0% to about 65% per day, from about 5.0% to about 60% per day, from about 5.0% to about 55% per day, from about 50% per day, from about 5.0% to about 45% per day, from about 5.0% per day to about 50% per day .0% to about 40% per day, about 5.0% to about 35% per day, about 5.0% to about 30% per day, about 5.0% to about 25% per day, about 5.0% to about 20% per day, about 5.0% to about 18% per day, about 5.0% to about 16% per day, about 5.0% to about 14% per day, about 5.0% to about 12% per day, about 5.0% to about 10% per day, about 5.0% to about 9.0% per day, about 5.0% to about 8.0% per day, about 5.0% to about 7.0% per day, about 5.0% to about 6.0% per day, 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18% per day, about 6.0% to about 16% per day, about 6.0% to about 14% per day, about 6.0% to about 12% per day, about 6.0% to about 10% per day, about 6.0% to about 9.0% per day, about 6.0% to about 8.0% per day, about 6.0% to about 7.0% per day, about 7.0% to about 140% per day, about 7.0% to about 135% per day,From about 7.0% to about 130% per day, from about 7.0% to about 125% per day, from about 7.0% to about 120% per day, from about 7.0% to about 115% per day, from about 7.0% to about 110% per day, from about 7.0% to about 105% per day, from about 7.0% to about 100% per day, from about 7.0% to about 95% per day, from about 7.0% to about 90% per day, from about 7.0% to about 85% per day, from about 7.0% to about 80% per day, from about 7.0% to about 75% per day, from about 7.0% to about 70% per day, from about 7.0% to about 65% per day, from about 7.0% to about 60% per day, from about 7.0% to about 55% per day, from about 7.0% to about 50% per day, about 7.0% to about 45% per day, about 7.0% to about 40% per day, about 7.0% to about 35% per day, about 7.0% to about 30% per day, about 7.0% to about 25% per day, about 7.0% to about 20% per day, about 7.0% to about 18% per day, about 7.0% to about 16% per day, about 7.0% to about 14% per day, about 7.0% to about 12% per day, about 7.0% to about 10% per day, about 7.0% to about 9.0% per day, about 7.0% to about 8.0% per day, about 8.0% to about 140% per day, about 8.0% to about 135% per day, about 8.0% to about 140% per day About 130% per day, about 8.0% per day to about 125% per day, about 8.0% per day to about 120% per day, about 8.0% per day to about 115% per day, about 8.0% per day to about 110% per day, about 8.0% per day to about 105% per day, about 8.0% per day to about 100% per day, about 8.0% per day to about 95% per day, about 8.0% per day to about 90% per day, about 8.0% per day to about 85% per day, about 8.0% per day to about 80% per day, about 8.0% per day to about 75% per day, about 8.0% per day to about 70% per day, about 8.0% per day to about 65% per day, about 8.0% per day to about 60% per day, about 8.0% per day to about 55% per day, about 8.0% per day to about 5 0%, about 8.0% to about 45% per day, about 8.0% to about 40% per day, about 8.0% to about 35% per day, about 8.0% to about 30% per day, about 8.0% to about 25% per day, about 8.0% to about 20% per day, about 8.0% to about 18% per day, about 8.0% to about 16% per day, about 8.0% to about 14% per day, about 8.0% to about 12% per day, about 8.0% to about 10% per day, about 8.0% to about 9.0% per day, about 9.0% to about 140% per day, about 9.0% to about 135% per day, about 9.0% to about 130% per day, about 9.0% to about 125% per day,From about 9.0% to about 120% per day, from about 9.0% to about 115% per day, from about 9.0% to about 110% per day, from about 9.0% to about 105% per day, from about 9.0% to about 100% per day, from about 9.0% to about 95% per day, from about 9.0% to about 90% per day, from about 9.0% to about 85% per day, from about 9.0% to about 80% per day, from about 9.0% to about 75% per day, from about 9.0% to about 70% per day, from about 9.0% to about 65% per day, from about 9.0% to about 60% per day, from about 9.0% to about 55% per day, from about 9.0% to about 50% per day, from about 9.0% to about 45% per day, from about 9.0% to about 9.0% per day. 0% to about 40% per day, about 9.0% to about 35% per day, about 9.0% to about 30% per day, about 9.0% to about 25% per day, about 9.0% to about 20% per day, about 9.0% to about 18% per day, about 9.0% to about 16% per day, about 9.0% to about 14% per day, about 9.0% to about 12% per day, about 9.0% to about 10% per day, about 10% to about 140% per day, about 10% to about 135% per day, about 10% to about 130% per day, about 10% to about 125% per day, about 10% to about 120% per day, about 10% to about 115% per day, about 10% to about 110% per day, About 10% to about 105% per day, about 10% to about 100% per day, about 10% to about 95% per day, about 10% to about 90% per day, about 10% to about 85% per day, about 10% to about 80% per day, about 10% to about 75% per day, about 10% to about 70% per day, about 10% to about 65% per day, about 10% to about 60% per day, about 10% to about 55% per day, about 10% to about 50% per day, about 10% to about 45% per day, about 10% to about 40% per day, about 10% to about 35% per day, about 10% to about 30% per day, about 10% to about 25% per day, about 10% to about 20%, about 10% to about 18% per day, about 10% to about 16% per day, about 10% to about 14% per day, about 10% to about 12% per day, about 12% to about 140% per day, about 12% to about 135% per day, about 12% to about 130% per day, about 12% to about 125% per day, about 12% to about 120% per day, about 12% to about 115% per day, about 12% to about 110% per day, about 12% to about 105% per day, about 12% to about 100% per day, about 12% to about 95% per day, about 12% to about 90% per day, about 12% to about 85% per day, about 12% to about 80% per day,From about 12% to about 75% per day, from about 12% to about 70% per day, from about 12% to about 65% per day, from about 12% to about 60% per day, from about 12% to about 55% per day, from about 12% to about 50% per day, from about 12% to about 45% per day, from about 12% to about 40% per day, from about 12% to about 35% per day, from about 12% to about 30% per day, from about 12% to about 25% per day, from about 12% to about 20% per day, from about 12% to about 18% per day, from about 12% to about 16% per day, from about 12% to about 14% per day, from about 14% to about 140% per day, from about 14% to about 135% per day, from about 14% to about 1 130%, about 14% to about 125% per day, about 14% to about 120% per day, about 14% to about 115% per day, about 14% to about 110% per day, about 14% to about 105% per day, about 14% to about 100% per day, about 14% to about 95% per day, about 14% to about 90% per day, about 14% to about 85% per day, about 14% to about 80% per day, about 14% to about 75% per day, about 14% to about 70% per day, about 14% to about 65% per day, about 14% to about 60% per day, about 14% to about 55% per day, about 14% to about 50% per day, about 14% to about 4 ... 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40% per day, about 16% to about 35% per day, about 16% to about 30% per day, about 16% to about 25% per day, about 16% to about 20% per day, about 16% to about 18% per day, about 18% to about 140% per day, about 18% to about 135% per day,From about 18% to about 130% per day, from about 18% to about 125% per day, from about 18% to about 120% per day, from about 18% to about 115% per day, from about 18% to about 110% per day, from about 18% to about 105% per day, from about 18% to about 100% per day, from about 18% to about 95% per day, from about 18% to about 90% per day, from about 18% to about 85% per day, from about 18% to about 80% per day, from about 18% to about 75% per day, from about 18% to about 70% per day, from about 18% to about 65% per day, from about 18% to about 60% per day, from about 18% to about 55% per day, from about 18% to about 50% per day, from about 18% to about 50% per day, from about 18% to about 95% per day From about 18% to about 45% per day, from about 18% to about 40% per day, from about 18% to about 35% per day, from about 18% to about 30% per day, from about 18% to about 25% per day, from about 18% to about 20% per day, from about 20% to about 140% per day, from about 20% to about 135% per day, from about 20% to about 130% per day, from about 20% to about 125% per day, from about 20% to about 120% per day, from about 20% to about 115% per day, from about 20% to about 110% per day, from about 20% to about 105% per day, from about 20% to about 100% per day, from about 20% to about 95% per day, from about 20% to about 90% per day, from about 20% to about 85% per day, about 20% to about 80% per day, about 20% to about 75% per day, about 20% to about 70% per day, about 20% to about 65% per day, about 20% to about 60% per day, about 20% to about 55% per day, about 20% to about 50% per day, about 20% to about 45% per day, about 20% to about 40% per day, about 20% to about 35% per day, about 20% to about 30% per day, about 20% to about 25% per day, about 25% to about 140% per day, about 25% to about 135% per day, about 25% to about 130% per day, about 25% to about 125% per day, about 25% to about 140% per day about 120% per day, about 25% per day to about 115% per day, about 25% per day to about 110% per day, about 25% per day to about 105% per day, about 25% per day to about 100% per day, about 25% per day to about 95% per day, about 25% per day to about 90% per day, about 25% per day to about 85% per day, about 25% per day to about 80% per day, about 25% per day to about 75% per day, about 25% per day to about 70% per day, about 25% per day to about 65% per day, about 25% per day to about 60% per day, about 25% per day to about 55% per day, about 25% per day to about 50% per day, about 25% per day to about 45% per day, about 25% per day to about 40% per day, about 25% per day to about 35% per day,About 25% per day to about 30% per day, about 30% per day to about 140% per day, about 30% per day to about 135% per day, about 30% per day to about 130% per day, about 30% per day to about 125% per day, about 30% per day to about 120% per day, about 30% per day to about 115% per day, about 30% per day to about 110% per day, about 30% per day to about 105% per day, about 30% per day to about 100% per day, about 30% per day to about 95% per day, about 30% per day to about 90% per day, about 30% per day to about 85% per day, about 30% per day to about 80% per day, about 30% per day to about 75% per day, about 30% per day to about 70% per day, about 30% per day to about 65% per day, about 100% per day From about 30% to about 60% per day, from about 30% to about 55% per day, from about 30% to about 50% per day, from about 30% to about 45% per day, from about 30% to about 40% per day, from about 30% to about 35% per day, from about 35% to about 140% per day, from about 35% to about 135% per day, from about 35% to about 130% per day, from about 35% to about 125% per day, from about 35% to about 120% per day, from about 35% to about 115% per day, from about 35% to about 110% per day, from about 35% to about 105% per day, from about 35% to about 100% per day, from about 35% to about 95% per day, from about 35% to about 90% per day, from about 35% per day % to about 85% per day, about 35% per day to about 80% per day, about 35% per day to about 75% per day, about 35% per day to about 70% per day, about 35% per day to about 65% per day, about 35% per day to about 60% per day, about 35% per day to about 55% per day, about 35% per day to about 50% per day, about 35% per day to about 45% per day, about 35% per day to about 40% per day, about 40% per day to about 140% per day, about 40% per day to about 135% per day, about 40% per day to about 130% per day, about 40% per day to about 125% per day, about 40% per day to about 120% per day, about 40% per day to about 115% per day, about 40% per day to about 110% per day, about 40% per day to about about 105%, about 40% per day to about 100% per day, about 40% per day to about 95% per day, about 40% per day to about 90% per day, about 40% per day to about 85% per day, about 40% per day to about 80% per day, about 40% per day to about 75% per day, about 40% per day to about 70% per day, about 40% per day to about 65% per day, about 40% per day to about 60% per day, about 40% per day to about 55% per day, about 40% per day to about 50% per day, about 40% per day to about 45% per day, about 45% per day to about 140% per day, about 45% per day to about 135% per day, about 45% per day to about 130% per day, about 45% per day to about 125% per day, about 45% per day to about 120% per day,About 45% to about 115% per day, about 45% to about 110% per day, about 45% to about 105% per day, about 45% to about 100% per day, about 45% to about 95% per day, about 45% to about 90% per day, about 45% to about 85% per day, about 45% to about 80% per day, about 45% to about 75% per day, about 45% to about 70% per day, about 45% to about 65% per day, about 45% to about 60% per day, about 45% to about 55% per day, about 45% to about 50% per day, about 50% to about 140% per day, about 50% to about 135% per day, about 50% to about 130% per day, about From about 50% to about 125% per day, from about 50% to about 120% per day, from about 50% to about 115% per day, from about 50% to about 110% per day, from about 50% to about 105% per day, from about 50% to about 100% per day, from about 50% to about 95% per day, from about 50% to about 90% per day, from about 50% to about 85% per day, from about 50% to about 80% per day, from about 50% to about 75% per day, from about 50% to about 70% per day, from about 50% to about 65% per day, from about 50% to about 60% per day, from about 50% to about 55% per day, from about 55% to about 140% per day, from about 55% to about 135% per day, From about 55% to about 130% per day, from about 55% to about 125% per day, from about 55% to about 120% per day, from about 55% to about 115% per day, from about 55% to about 110% per day, from about 55% to about 105% per day, from about 55% to about 100% per day, from about 55% to about 95% per day, from about 55% to about 90% per day, from about 55% to about 85% per day, from about 55% to about 80% per day, from about 55% to about 75% per day, from about 55% to about 70% per day, from about 55% to about 65% per day, from about 55% to about 60% per day, from about 60% to about 140% per day, from about 60% to about 135% per day, from about 140% per day about 60% to about 130% per day, about 60% to about 125% per day, about 60% to about 120% per day, about 60% to about 115% per day, about 650% to about 110% per day, about 60% to about 105% per day, about 60% to about 100% per day, about 60% to about 95% per day, about 60% to about 90% per day, about 60% to about 85% per day, about 60% to about 80% per day, about 60% to about 75% per day, about 60% to about 70% per day, about 60% to about 65% per day, about 65% to about 140% per day, about 65% to about 135% per day, about 65% to about 130% per day,About 65% to about 125% per day, about 65% to about 120% per day, about 65% to about 115% per day, about 65% to about 110% per day, about 65% to about 105% per day, about 65% to about 100% per day, about 65% to about 95% per day, about 65% to about 90% per day, about 65% to about 85% per day, about 65% to about 80% per day, about 65% to about 75% per day, about 65% to about 70% per day, about 70% to about 140% per day, about 70% to about 135% per day, about 70% to about 130% per day, about 70% to about 125% per day, about 70% to about 120% per day, about About 70% to about 115% per day, about 70% to about 110% per day, about 70% to about 105% per day, about 70% to about 100% per day, about 70% to about 95% per day, about 70% to about 90% per day, about 70% to about 85% per day, about 70% to about 80% per day, about 70% to about 75% per day, about 75% to about 140% per day, about 75% to about 135% per day, about 75% to about 130% per day, about 75% to about 125% per day, about 75% to about 120% per day, about 75% to about 115% per day, about 75% to about 110% per day, about 75% to about 105% per day, about From about 75% to about 100% per day, from about 75% to about 95% per day, from about 75% to about 90% per day, from about 75% to about 85% per day, from about 75% to about 80% per day, from about 80% to about 140% per day, from about 80% to about 135% per day, from about 80% to about 130% per day, from about 80% to about 125% per day, from about 80% to about 120% per day, from about 80% to about 115% per day, from about 80% to about 110% per day, from about 80% to about 105% per day, from about 80% to about 100% per day, from about 80% to about 95% per day, from about 80% to about 90% per day, from about 80% to about 85% per day, from about 85% to about 140% per day, about 85% to about 135% per day, about 85% to about 130% per day, about 85% to about 125% per day, about 85% to about 120% per day, about 85% to about 115% per day, about 85% to about 110% per day, about 85% to about 105% per day, about 85% to about 100% per day, about 85% to about 95% per day, about 85% to about 90% per day, about 90% to about 140% per day, about 90% to about 135% per day, about 90% to about 130% per day, about 90% to about 125% per day, about 90% to about 120% per day, about 90% to about 115% per day,From about 90% to about 110% per day, from about 90% to about 105% per day, from about 90% to about 100% per day, from about 90% to about 95% per day, from about 95% to about 140% per day, from about 95% to about 135% per day, from about 95% to about 130% per day, from about 95% to about 125% per day, from about 95% to about 120% per day, from about 95% to about 115% per day, from about 95% to about 110% per day, from about 95% to about 105% per day, from about 95% to about 100% per day %, about 100% to about 140% per day, about 100% to about 135% per day, about 100% to about 130% per day, about 100% to about 125% per day, about 100% to about 120% per day, about 100% to about 115% per day, about 100% to about 110% per day, about 100% to about 105% per day, about 105% to about 140% per day, about 105% to about 135% per day, about 105% to about 130% per day, about 105% to about 125% per day , about 105% to about 120% per day, about 105% to about 115% per day, about 105% to about 110% per day, about 110% to about 140% per day, about 110% to about 135% per day, about 110% to about 130% per day, about 110% to about 125% per day, about 110% to about 120% per day, about 110% to about 115% per day, about 115% to about 140% per day, about 115% to about 135% per day, about 115% to about 130% per day , about 115% to about 125% per day, about 115% to about 120% per day, about 120% to about 140% per day, about 120% to about 135% per day, about 120% to about 130% per day, about 120% to about 125% per day, about 125% to about 140% per day, about 125% to about 135% per day, about 125% to about 130% per day, about 130% to about 140% per day, about 130% to about 135% per day, or about 135% to about 140% per day. ,

[0362] In some embodiments of these methods, the cell contains a nucleic acid encoding a recombinant therapeutic protein. Some embodiments of these methods further include collecting or recovering the recombinant therapeutic protein. Some embodiments of these methods further include purifying the recombinant therapeutic protein. Some embodiments of these methods further include formulating a purified recombinant therapeutic protein.

[0363] In some embodiments of these methods, the cell is a bacterium (e.g., any exemplary bacterium described herein or known in the art), a yeast (e.g., any exemplary yeast cell described herein or known in the art), or a mammalian cell (e.g., any exemplary mammalian cell described herein or known in the art).

[0364] In some embodiments of these methods, cell viability is determined by measuring the concentration of lactate dehydrogenase (LDH) in the test culture vessel.

[0365] In some embodiments, the test culture container is a baffled shake flask (e.g., any baffled shake flask described herein or known in the art), and the multiple test conditions include one or more of the following: (a) rotational agitation of about 125RPM to about 400RPM (or any sub-range of this range described herein); (b) a sensitizer (e.g., any sensitizer described herein or known in the art) concentration of about 0ppm to about 120ppm (or any sub-range of this range described herein); and (c) a protective agent (e.g., any protective agent described herein or known in the art) concentration of about 1g / L to about 10g / L (or any sub-range of this range described herein). In some embodiments, the protective agent is a poloxamer (e.g., poloxamer-188, poloxamer-401, poloxamer-402, or poloxamer-407), a poloxamine (e.g., poloxamer-904 or poloxamer-908), or a pluronic polyol.

[0366] Methods for improving cell viability in production bioreactors

[0367] Also provided herein is a method for increasing cell viability in a production bioreactor (e.g., any production bioreactor described herein), comprising: (a) determining cell viability under multiple test conditions in a test culture vessel, wherein the multiple conditions are defined by a set of parameters; (b) based on the cell viability in the test culture vessel under the multiple conditions, selecting conditions that are optimal for cell viability in a production bioreactor; and (c) culturing cells in the production bioreactor under the selected conditions.

[0368] In some embodiments of these methods, the parameter set includes, consists essentially of, or consists of shear stress, sensitizer (e.g., any sensitizer described herein or known in the art) concentration, and protectant (e.g., any protectant described herein or known in the art) concentration. In some embodiments of these methods, the plurality of test conditions are selected based on full factorial screening and / or endocentric composite design. In some embodiments of these methods, the sensitizer is a defoamer (e.g., a polydimethylsiloxane-based defoamer, dimethicone, or any other defoamer described herein or known in the art).

[0369] In some embodiments of these methods, the test culture container (e.g., any test culture container described herein or known in the art) has a volume of about 5 mL to about 5 L (or any subrange of this range described herein). In some embodiments of these methods, the test culture container is a shake flask, such as a baffled shake flask (e.g., any baffled shake flask described herein or known in the art).

[0370] In some embodiments of these methods, the volume of the production bioreactor (e.g., any production bioreactor described herein) is about 5 L to about 20,000 L (or any subrange of this range described herein). In some examples, the production bioreactor is a perfusion bioreactor. In other examples, the production bioreactor is a fed-batch bioreactor.

[0371] In some embodiments, step (c) results in a specific cell death rate of less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% per day during culturing of cells in the production bioreactor. In some embodiments, step (c) results in a specific cell death rate of about 0.1% to about 40% (or any subrange of this range described herein) per day during culturing of cells in the production bioreactor.

[0372] In some embodiments, step (c) results in a specific LDH production of less than 150 nU / cell / day, less than 145 nU / cell / day, less than 140 nU / cell / day, less than 135 nU / cell / day, less than 130 nU / cell / day, less than 125 nU / cell / day, less than 120 nU / cell / day, less than 115 nU / cell / day, less than 150 nU / cell / day, less than 110 nU / cell / day, less than 105 nU / cell / day, less than 100 nU / cell / day, less than 95 nU / cell / day, less than 9 ...125 nU / cell / day, less than 120 nU / cell / day, less than 125 nU / cell / day, less than 120 nU / cell / day, less than 125 nU / cell / day, less than 120 nU / cell / day, less than 125 nU / cell / day, less than 120 nU / cell / day, less than 12 Less than 85nU / cell / day, less than 80nU / cell / day, less than 75nU / cell / day, less than 70nU / cell / day, less than 65nU / cell / day, less than 60nU / cell / day, less than 55nU / cell / day, less than 50nU / cell / day, less than 45nU / cell / day, less than 45nU / cell / day, less than 40nU / cell / day, less than 35nU / cell / day, less than 30nU / cell / day, less than 25nU / cell / day, less than 20nU / cell / day, less than 15nU / cell / day, less than 10nU / cell / day, less than 5nU / cell / day or less than 1nU / cell / day. In some embodiments of these methods, step (c) results in a cell-specific LDH production of about 0.1nU / cell / day to about 150nU / cell / day (or any subrange of this range described herein) during culturing of the cells in the production bioreactor.

[0373] In some embodiments, step (c) results in an apparent growth rate of greater than 1% per day, greater than 1.5% per day, greater than 2% per day, greater than 2.5% per day, greater than 3% per day, greater than 3.5% per day, greater than 4.0% per day, greater than 4.5% per day, greater than 5.0% per day, greater than 5.5% per day, greater than 6.0% per day, greater than 6.5% per day, greater than 7.0% per day, greater than 7.5% per day, greater than 8.0% per day, greater than 8.5% per day, greater than 9.0% per day, greater than 9.5% per day, greater than 10.0% per day, greater than 10.5% per day, greater than 11.0% per day, greater than 11.5% per day, greater than 12.0% per day, greater than 12.5% ​​per day, greater than 13.0% per day, greater than 13.5% per day, greater than 14.0% per day, greater than 14.5% per day, greater than 15.0% per day, greater than 15.5% per day, greater than 16.0% per day, greater than 16.5% per day, greater than 17. greater than 15.0% per day, greater than 16% per day, greater than 17% per day, greater than 18% per day, greater than 19% per day, greater than 20% per day, greater than 21% per day, greater than 22% per day, greater than 23% per day, greater than 24% per day, greater than 25% per day, greater than 30% per day, greater than 35% per day, greater than 40% per day, greater than 45% per day, greater than 50% per day, greater than 55% per day, greater than 60% per day, greater than 65% per day, greater than 70% per day, greater than 75% per day, greater than 80% per day, greater than 85% per day, greater than 90% per day, greater than 95% per day, greater than 100% per day, greater than 105% per day, greater than 110% per day, greater than 115% per day, greater than 120% per day, greater than 125% per day, greater than 130% per day, greater than 135% per day, greater than 140% per day, greater than 145% per day or greater than 150% per day. In some embodiments of these methods, step (c) results in an apparent growth rate of about 0.1% per day to about 150% per day (or any subrange of this range described herein) during culturing of the cells in the production bioreactor.

[0374] In some embodiments of these methods, the cell (e.g., any cell described herein) contains a nucleic acid encoding a recombinant therapeutic protein (e.g., any recombinant therapeutic protein described herein). Some embodiments of these methods also include collecting or recovering the recombinant therapeutic protein. Some embodiments of these methods also include purifying the recombinant therapeutic protein. Some embodiments of these methods also include formulating a purified recombinant therapeutic protein. In some embodiments of these methods, the cell is a bacterium (e.g., any exemplary bacterium described herein or known in the art), a yeast cell (e.g., any exemplary yeast cell described herein or known in the art), or a mammalian cell (e.g., any exemplary mammalian cell described herein or known in the art).

[0375] In some embodiments of these methods, cell viability is determined by measuring the lactate dehydrogenase (LDH) concentration in the test culture container. In some embodiments of these methods, the test culture container is a baffled shake flask, and the multiple test conditions include (or consist of) one or more (e.g., two or three) of the following: (a) rotational agitation of about 125RPM to about 400RPM (or any sub-range of this range as described herein); (b) a sensitizer (e.g., any sensitizer described herein or known in the art) concentration of about 0ppm to about 120ppm (or any sub-range of this range as described herein); and (c) a protective agent (e.g., any protective agent described herein or known in the art) concentration of about 1g / L to about 10g / L (or any sub-range of this range as described herein). In some embodiments of these methods, the protective agent is a poloxamer (eg, poloxamer-188, poloxamer-401, poloxamer-402, or poloxamer-407), a poloxamine (eg, poloxamine-904 or poloxamine-908), or a pluronic polyol.

[0376] Method for predicting cell viability in production bioreactors

[0377] Also provided herein is a method for predicting cell viability in a production bioreactor (e.g., any production bioreactor described herein), comprising: (a) selecting a set of parameters comprising at least two parameters (e.g., 2, 3, 4, 5, or 6); (b) determining the cell viability under multiple test conditions as defined by the parameter set in a test culture vessel (e.g., any test culture vessel described herein); and (c) predicting the cell viability in the production bioreactor based on the cell viability in the test culture vessel under the multiple conditions.

[0378] In some embodiments of these methods, the parameter set includes, consists essentially of, or consists of shear stress, sensitizer (e.g., any exemplary sensitizer described herein or known in the art) concentration, and protectant (e.g., any protectant described herein or known in the art) concentration. In some embodiments of these methods, the plurality of test conditions are selected based on full factorial screening and / or endocentric composite design.

[0379] In some embodiments of these methods, the sensitizer is an antifoaming agent (eg, a polydimethylsiloxane-based antifoaming agent, dimethicone, or any other antifoaming agent described herein or known in the art).

[0380] In some embodiments of these methods, the test culture container (e.g., any test culture container described herein or known in the art) has a volume of about 5 mL to about 5 L (or any subrange of this range described herein). In some embodiments of these methods, the test culture container is a shake flask, such as a baffled shake flask (e.g., any baffled shake flask described herein or known in the art).

[0381] In some embodiments of these methods, the volume of the production bioreactor (e.g., any production bioreactor described herein) is about 5 L to about 20,000 L (or any subrange of this range described herein). In some examples, the production bioreactor is a perfusion bioreactor. In other examples, the production bioreactor is a fed-batch bioreactor.

[0382] Some embodiments of these methods also include culturing cells in the production bioreactor using conditions predicted to result in a specific cell mortality rate of less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% per day during culturing cells in the production bioreactor. Some embodiments of these methods also include culturing cells in the production bioreactor using conditions predicted to result in a specific cell mortality rate of about 0.1% to about 40% (or any subrange of this range described herein) per day during culturing cells in the production bioreactor.

[0383] Some embodiments of these methods further comprise culturing the cells in the production bioreactor using conditions predicted to result in the following specific LDH production by the cells during culturing of the cells in the production bioreactor: less than 150 nU / cell / day, less than 145 nU / cell / day, less than 140 nU / cell / day, less than 135 nU / cell / day, less than 130 nU / cell / day, less than 125 nU / cell / day, less than 120 nU / cell / day, less than 115 nU / cell / day, less than 150 nU / cell / day, less than 110 nU / cell / day, less than 105 nU / cell / day, less than 100 nU / cell / day, less than 95 nU / cell / day, less than 90nU / cell / day, less than 85nU / cell / day, less than 80nU / cell / day, less than 75nU / cell / day, less than 70nU / cell / day, less than 65nU / cell / day, less than 60nU / cell / day, less than 55nU / cell / day, less than 50nU / cell / day, less than 45nU / cell / day, less than 45nU / cell / day, less than 40nU / cell / day, less than 35nU / cell / day, less than 30nU / cell / day, less than 25nU / cell / day, less than 20nU / cell / day, less than 15nU / cell / day, less than 10nU / cell / day, less than 5nU / cell / day or less than 1nU / cell / day. Some embodiments of these methods further comprise culturing the cells in the production bioreactor using conditions predicted to result in a cell-specific LDH production of about 0.1 nU / cell / day to about 150 nU / cell / day (or any subrange of this range described herein) during culturing of the cells in the production bioreactor.

[0384] Some embodiments of these methods further comprise culturing the cells in the production bioreactor using conditions predicted to result in an apparent growth rate of greater than 1% per day, greater than 1.5% per day, greater than 2% per day, greater than 2.5% per day, greater than 3% per day, greater than 3.5% per day, greater than 4.0% per day, greater than 4.5% per day, greater than 5.0% per day, greater than 5.5% per day, greater than 6.0% per day, greater than 6.5% per day, greater than 7.0% per day, greater than 7.5% per day, greater than 8.0% per day, greater than 8.5% per day, greater than 9.0% per day, greater than 9.5% per day, greater than 10.0% per day, greater than 10.5% per day, greater than 11.0% per day, greater than 11.5% per day, greater than 12.0% per day, greater than 12.5% ​​per day, greater than 13.0% per day, greater than 13.5% per day, greater than 14.0% per day, greater than 14.5% per day, greater than 15.0% per day, greater than 15.5% per day, greater than 16.0% per day, greater than 16.5% per day, greater than 17.0% per day, greater than 17.5% per day, greater than 18.0% per day, greater than 18.5% per day, greater than 19.0% per day, greater than 19.5% per day, greater than 10.0% per day, greater than 10.5% per day, greater than 11.0% per day, greater than 11.5% per day, greater than 12.0% per day, greater than 12.5% ​​per day, greater than 13.0% per day, greater than 13.5% per day, .0%, greater than 14.5% per day, greater than 15.0% per day, greater than 16% per day, greater than 17% per day, greater than 18% per day, greater than 19% per day, greater than 20% per day, greater than 21% per day, greater than 22% per day, greater than 23% per day, greater than 24% per day, greater than 25% per day, greater than 30% per day, greater than 35% per day, greater than 40% per day, greater than 45% per day, greater than 50% per day, greater than 55% per day, greater than 60% per day, greater than 65% per day, greater than 70% per day, greater than 75% per day, greater than 80% per day, greater than 85% per day, greater than 90% per day, greater than 95% per day, greater than 100% per day, greater than 105% per day, greater than 110% per day, greater than 115% per day, greater than 120% per day, greater than 125% per day, greater than 130% per day, greater than 135% per day, greater than 140% per day, greater than 145% per day, or greater than 150% per day. Some embodiments of these methods further comprise culturing the cells in the production bioreactor using conditions predicted to result in an apparent growth rate of about 0.1% per day to about 150% per day (or any sub-range of this range described herein) during culturing of the cells in the production bioreactor.

[0385] In some embodiments of these methods, the cell (e.g., any cell described herein) contains a nucleic acid encoding a recombinant therapeutic protein (e.g., any recombinant therapeutic protein described herein). Some embodiments of these methods also include collecting or recovering the recombinant therapeutic protein. Some embodiments of these methods also include purifying the recombinant therapeutic protein. Some embodiments of these methods also include formulating a purified recombinant therapeutic protein. In some embodiments of these methods, the cell is a bacterium (e.g., any exemplary bacterium described herein or known in the art), a yeast cell (e.g., any exemplary yeast cell described herein or known in the art), or a mammalian cell (e.g., any exemplary mammalian cell described herein or known in the art).

[0386] In some embodiments of these methods, cell viability is determined by measuring the lactate dehydrogenase (LDH) concentration in the test culture container. In some embodiments of these methods, the test culture container is a baffled shake flask, and the multiple test conditions include (or consist of) one or more (e.g., two or three) of the following: (a) rotational agitation of about 125RPM to about 400RPM (or any sub-range of this range as described herein); (b) a sensitizer (e.g., any sensitizer described herein or known in the art) concentration of about 0ppm to about 120ppm (or any sub-range of this range as described herein); and (c) a protective agent (e.g., any protective agent described herein or known in the art) concentration of about 1g / L to about 10g / L (or any sub-range of this range as described herein). In some embodiments of these methods, the protective agent is a poloxamer (eg, poloxamer-188, poloxamer-401, poloxamer-402, or poloxamer-407), a poloxamine (eg, poloxamine-904 or poloxamine-908), or a pluronic polyol.

[0387] Method for culturing cells in a production bioreactor

[0388] Also provided herein are methods of culturing cells (e.g., any of the cells described herein) in a production bioreactor, comprising culturing the cells in a liquid culture medium under one or more (e.g., two, three, or four) of the following conditions:

[0389] (a) P / V (W / m 3) is from about 1.0 to about 110 (e.g., from about 3.0 to about 7.0, from about 4.0 to about 6.0, from about 5.0 to about 6.0, or about 6.0) (e.g., from about 1.0 to about 110, from about 1.0 to about 95, from about 1.0 to about 90, from about 1.0 to about 85, from about 1.0 to about 80, from about 1.0 to about 75, from about 1.0 to about 70, from about 1.0 to about 65, from about 1.0 to about 60, from about 1.0 to about 55, from about 1.0 to about 50, from about 1.0 to about 45, from about 1.0 to about 40, from about 1.0 to about 35, from about 1.0 to about 30, from about 1.0 to about 25, from about 1.0 to about 20, from about 1.0 to about 18, from about 1.0 to about 16, from about 1.0 to about 14, from about 1.0 to about 12 about 10, about 1.0 to about 9.0, about 1.0 to about 8.0, about 1.0 to about 7.0, about 1.0 to about 6.0, about 1.0 to about 5.0, about 1.0 to about 4.0, about 1.0 to about 3.0, about 1.0 to about 2.0, about 2.0 to about 110, about 2.0 to about 95, about 2.0 to about 90, about 2.0 to about 85, about 2.0 to about 80, about 2.0 to about 75, about 2.0 to about 70, about 2.0 to about 65, about 2.0 to about 60, about 2.0 to about 55, about 2.0 to about 50, about 2.0 to about 45, about 2.0 to about 40, about 2.0 to about 35, about 2.0 to about 30, about 2.0 to about 25, about 20, about 2.0 to about 18, about 2 .0 to about 16, about 2.0 to about 14, about 2.0 to about 12, about 2.0 to about 10, about 2.0 to about 9.0, about 2.0 to about 8.0, about 2.0 to about 7.0, about 2.0 to about 6.0, about 2.0 to about 5.0, about 2.0 to about 4.0, about 2.0 to about 3.0, about 3.0 to about 110, about 3.0 to about 95, about 3.0 to about 90, about 3.0 to about 85, about 3.0 to about 80, about 3.0 to about 75, about 3.0 to about 70, about 3.0 to about 65, about 3.0 to about 60, about 3.0 to about 55, about 3.0 to about 50, about 3.0 to about 45, about 3.0 to about 40, about 3.0 to about 35, about 30, about 3.0 to about 25, about 3.0 to about 20, about 3.0 to about 18, about 3.0 to about 16, about 3.0 to about 14, about 3.0 to about 12, about 3.0 to about 10, about 3.0 to about 9.0, about 3.0 to about 8.0, about 3.0 to about 7.0, about 3.0 to about 6.0, about 3.0 to about 5.0, about 3.0 to about 4.0, about 4.0 to about 110, about 4.0 to about 95, about 4.0 to about 90, about 4.0 to about 85, about 4.0 to about 80, about 4.0 to about 75, about 4.0 to about 70, about 4.0 to about 65, about 4.0 to about 60, about 4.0 to about 55, about 4.0 to about 50, about 4.0 to about 45, about 4.0 to about 40, about 4.0 to about 35, about 4.0 to about 30,about 4.0 to about 25, about 4.0 to about 20, about 4.0 to about 18, about 4.0 to about 16, about 4.0 to about 14, about 4.0 to about 12, about 4.0 to about 10, about 4.0 to about 9.0, about 4.0 to about 8.0, about 4.0 to about 7.0, about 4.0 to about 6.0, about 4.0 to about 5.0, about 5.0 to about 110, about 5.0 to about 95, about 5.0 to about 90, about 5.0 to about 85, about 5.0 to about 80, about 5.0 to about 75, about 5.0 to about 70, about 5.0 to about 65, about 5.0 to about 60, about 5.0 to about 55, about 5.0 to about 50, about 5.0 to about 45, about 5.0 to about 40, about 5.0 to about 35, about 5.0 to about 30, about 5.0 to about 25, about 5.0 to about 20, about 5.0 to about 18, about 5.0 to about 16, about 5.0 to about 14, about 5.0 to about 12, about 5.0 to about 10, about 5.0 to about 9.0, about 5.0 to about 8.0, about 5.0 to about 7.0, about 5.0 to about 6.0, about 6.0 to about 110, about 6.0 to about 95, about 6.0 to about 90, about 6.0 to about 85, about 6.0 to about 80, about 6.0 to about 75, about 6.0 to about 70, about 6.0 to about 65, about 6.0 to about 60, about 6.0 to about 55, about 6.0 to about 50, about 6.0 to about 45, about 6.0 to about 40, about 6.0 to about 35, about 6.0 to about 30, about 6.0 to about 25, about 6 .0 to about 20, about 6.0 to about 18, about 6.0 to about 16, about 6.0 to about 14, about 6.0 to about 12, about 6.0 to about 10, about 6.0 to about 9.0, about 6.0 to about 8.0, about 6.0 to about 7.0, about 7.0 to about 110, about 7.0 to about 95, about 7.0 to about 90, about 7.0 to about 85, about 7.0 to about 80, about 7.0 to about 75, about 70, about 70 to about 65, about 7.0 to about 60, about 7.0 to about 55, about 7.0 to about 50, about 7.0 to about 45, about 7.0 to about 40, about 7.0 to about 35, about 7.0 to about 30, about 7.0 to about 25, about 7.0 to about 20, about 7.0 to about 18, about 7.0 to about about 16, about 7.0 to about 14, about 7.0 to about 12, about 7.0 to about 10, about 7.0 to about 9.0, about 7.0 to about 8.0, about 8.0 to about 110, about 8.0 to about 95, about 8.0 to about 90, about 8.0 to about 85, about 80, about 8.0 to about 75, about 8.0 to about 70, about 8.0 to about 65, about 8.0 to about 60, about 8.0 to about 55, about 8.0 to about 50, about 8.0 to about 45, about 8.0 to about 40, about 8.0 to about 35, about 8.0 to about 30, about 8.0 to about 25, about 8.0 to about 20, about 8.0 to about 18, about 8.0 to about 16, about 8.0 to about 14, about 8.0 to about 12, about 8.0 to about 10,from about 8.0 to about 9.0, from about 9.0 to about 110, from about 9.0 to about 95, from about 9.0 to about 90, from about 9.0 to about 85, from about 9.0 to about 80, from about 9.0 to about 75, from about 9.0 to about 70, from about 9.0 to about 65, from about 9.0 to about 60, from about 9.0 to about 55, from about 9.0 to about 50, from about 9.0 to about 45, from about 9.0 to about 40, from about 9.0 to about 35, from about 9.0 to about 30, from about 9.0 to about 25, from about 9.0 to about 20, from about 9.0 to about 18, from about 9.0 to about 16, from about 9.0 to about 14, from about 9.0 to about 12, from about 9.0 to about 10, from about 10 to about 110, from about 10 to about 95, from about 10 to about 90, from about 10 to about 85, from about 10 to about 80 , about 10 to about 75, about 10 to about 70, about 10 to about 65, about 12 to about 60, about 10 to about 55, about 12 to about 50, about 10 to about 45, about 10 to about 40, about 10 to about 35, about 10 to about 30, about 10 to about 25, about 10 to about 20, about 10 to about 18, about 10 to about 16, about 10 to about 14, about 10 to about 12, about 12 to about 110, about 12 to about 95, about 12 to about 90, about 12 to about 85, about 12 to about 80, about 12 to about 75, about 12 to about 70, about 12 to about 65, about 12 to about 60, about 12 to about 55, about 12 to about 50, about 12 to about 45, about 12 to about 40, about 12 to about 35, about 12 to about 30, about 12 to about 25, about 12 to about 20, about 12 to about 18, about 12 to about 16, about 12 to about 14, about 14 to about 110, about 14 to about 95, about 14 to about 90, about 14 to about 85, about 14 to about 80, about 14 to about 75, about 14 to about 70, about 14 to about 65, about 14 to about 60, about 14 to about 55, about 14 to about 50, about 14 to about 45, about 14 to about 40, about 14 to about 35, about 14 to about 30, about 14 to about 25, about 14 to about 20, about 14 to about 18, about 14 to about 16, about 16 to about 110, about 16 to about 95, about 16 to about 90, about 16 to about 85, about 16 to about 80, about 16 to about 75, about 16 to about 70, about 16 to about 65, about 16 to about 60, about 16 to about 55, about 16 to about 50, about 16 to about 45, about 16 to about 40, about 16 to about 35, about 16 to about 30, about 16 to about 25, about 16 to about 20, about 16 to about 18, about 18 to about 110, about 18 to about 95, about 18 to about 90, about 18 to about 85, about 18 to about 80, about 18 to about 75, about 18 to about 70, about 18 to about 65, about 18 to about 60, about 18 to about 55, about 18 to about 50, about 18 to about 45, about 18 to about 40, about 18 to about 35, about 18 to about 30, about 18 to about 25, about 18 to about 20, about 20 to about 110, about 20 to about 95, about 20 to about 90,about 20 to about 85, about 20 to about 80, about 20 to about 75, about 20 to about 70, about 20 to about 65, about 20 to about 60, about 20 to about 55, about 20 to about 50, about 20 to about 45, about 20 to about 40, about 20 to about 35, about 20 to about 30, about 20 to about 25, about 25 to about 110, about 25 to about 95, about 25 to about 90, about 25 to about 85, about 25 to about 80, about 25 to about 75, about 25 to about 70, about 25 to about 65, about 25 to about 60, about 25 to about 55, about 25 to about 50, about 25 to about 45, about 25 to about 40, about 25 to about 35, about 25 to about 30, about 30 to about 110, about 30 to about 95, about 30 to about 95 about 35 to about 95, about 35 to about 90, about 35 to about 85, about 35 to about 80, about 35 to about 75, about 35 to about 70, about 35 to about 65, about 35 to about 60, about 35 to about 55, about 35 to about 50, about 35 to about 45, about 35 to about 40, about 30 to about 35, about 35 to about 110, about 35 to about 95, about 35 to about 90, about 35 to about 85, about 35 to about 80, about 35 to about 75, about 35 to about 70, about 35 to about 65, about 35 to about 60, about 35 to about 55, about 35 to about 50, about 35 to about 45, about 35 to about 40, about 40 to about 110, about 40 to about 95, about 40 to about 90, about 40 to about 85, about 40 to about 80, about 40 to about 75, about 40 to about about 45 to about 80, about 45 to about 75, about 45 to about 70, about 45 to about 65, about 45 to about 60, about 45 to about 55, about 45 to about 50, about 40 to about 45, about 45 to about 110, about 45 to about 95, about 45 to about 90, about 45 to about 85, about 45 to about 80, about 45 to about 75, about 45 to about 70, about 45 to about 65, about 45 to about 60, about 45 to about 55, about 45 to about 50, about 50 to about 110, about 50 to about 95, about 50 to about 90, about 50 to about 85, about 50 to about 80, about 50 to about 75, about 50 to about 70, about 50 to about 65, about 50 to about 60, about 50 to about 55, about 55 to about 110, about 55 to about 95, about 55 to about 90, about 55 to about 75, about 55 to about 70, about 55 to about 65, about 55 to about 60, about 60 to about 110, about 60 to about 95, about 60 to about 90, about 60 to about 85, about 60 to about 80, about 60 to about 75, about 60 to about 70, about 60 to about 65, about 65 to about 110, about 65 to about 95, about 65 to about 90, about 65 to about 85, about 65 to about 80, about 65 to about 75, about 65 to about 70, about 70 to about 110, about 70 to about 95, about 70 to about 90, about 70 to about 85, about 70 to about 80, about 70 to about 75, about 75 to about 110, about 75 to about 95, about 75 to about 90, about 75 to about 85, about 75 to about 80,from about 80 to about 110, from about 80 to about 95, from about 80 to about 90, from about 80 to about 85, from about 85 to about 110, from about 85 to about 95, from about 85 to about 90, from about 90 to about 110, from about 90 to about 95, from about 95 to about 110, from about 95 to about 105, from about 95 to about 100, from about 100 to about 110, from about 100 to about 105, or from about 105 to about 110);

[0390] (b) The air volume per minute per liquid volume (VVM) of the production bioreactor (min -1 ) is from about 0.01 to about 0.10 (e.g., from about 0.02 to about 0.08, from about 0.04 to about 0.06, or about 0.06) (e.g., from about 0.01 to about 0.10, from about 0.01 to about 0.09, from about 0.01 to about 0.08, from about 0.01 to about 0.07, from about 0.01 to about 0.06, from about 0.01 to about 0.05, from about 0.01 to about 0.04, from about 0.01 to about 0.03, from about 0.01 to about 0.02, about 0.02 to about 0.10, about 0.02 to about 0.09, about 0.02 to about 0.08, about 0.02 to about 0.07, about 0.02 to about 0.06, about 0.02 to about 0.05, about 0.02 to about 0.04, about 0.02 to about 0.03, about 0.03 to about 0.10, about 0.03 to about 0.09, about 0.03 to about 0.08, about 0.03 to about 0.07, about 0.03 to about 0.06, about 0.03 to about 0.05, about 0.03 to about 0.04, about 0.04 to about 0.10, about 0.04 to about 0.09, about 0.04 to about 0.08, about 0.04 to about 0.07, about 0.04 to about 0.06, about 0.04 to about 0.05, about 0.05 to about 0.10, about 0.05 to about 0.09, about 0.05 to about 0. 08, about 0.05 to about 0.07, about 0.05 to about 0.06, about 0.06 to about 0.10, about 0.06 to about 0.09, about 0.06 to about 0.08, about 0.06 to about 0.07, about 0.07 to about 0.10, about 0.07 to about 0.09, about 0.07 to about 0.08, about 0.08 to about 0.10, about 0.08 to about 0.09, or about 0.09 to about 0.10);

[0391] (c) the culture medium comprises a sensitizer (e.g., any sensitizer described herein or known in the art) at a concentration of about 0 ppm to about 5,000 ppm (or any subrange of this range described herein); and

[0392] (d) the liquid culture medium comprises a protectant (e.g., any protectant described herein or known in the art) at a concentration of about 1 g / L to about 15 g / L (e.g., any sub-range of this range described herein, such as about 1 g / L to about 15 g / L, about 1 g / L to about 14 g / L, about 1 g / L to about 13 g / L, about 1 g / L to about 12 g / L, about 1 g / L to about 11 g / L, about 2 g / L to about 15 g / L, about 2 g / L to about 14 g / L, about 2 g / L to about 13 g / L, about 2 g / L to about 12 g / L, about 2 g / L to about 11 g / L, about 3 g / L to about 15 g / L g / L, about 3g / L to about 14g / L, about 3g / L to about 13g / L, about 3g / L to about 12g / L, about 3g / L to about 11g / L, about 4g / L to about 15g / L, about 4g / L to about 14g / L, about 4g / L to about 13g / L, about 4g / L to about 12g / L, about 4g / L to about 11g / L, about 5g / L to about 15g / L, about 5g / L to about 14g / L, about 5g / L to about 13g / L, about 5g / L to about 12g / L, about 5g / L to about 11g / L, about 6g / L to about 15g / L, about 6g / L to about 14g / L, about 6g / L to about 13g / L, about 6g / L to about 12g / L, about 6g / L to about 11g / L, about 7g / L to about 15g / L, about 7g / L to about 14g / L, about 7g / L to about 13g / L, about 7g / L to about 12g / L, about 7g / L to about 11g / L, about 8g / L to about 15g / L, about 8g / L to about 14g / L, about 8g / L to about 13g / L, about 8g / L to about 12g / L, about 8g / L to about 11g / L, about 9g / L to about 15g / L, about 9g / L to about 14g / L, about 9g / L to about 13g / L, about 9g / L to about 12g / L, about 9g / L g / L, about 11 g / L to about 12 g / L, about 10 g / L to about 11 g / L, about 11 g / L to about 15 g / L, about 11 g / L to about 14 g / L, about 11 g / L to about 13 g / L, about 11 g / L to about 12 g / L, about 12 g / L to about 15 g / L, about 12 g / L to about 14 g / L, about 12 g / L to about 13 g / L, about 13 g / L to about 15 g / L, about 13 g / L to about 14 g / L, or about 14 g / L to about 15 g / L).

[0393] Under optimized conditions, the specific cell death rate can be less than or predicted to be less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% per day. Under optimized conditions, the specific cell death rate can be about 0.1% to about 40% (or any subrange of this range described herein).

[0394] In some embodiments, cell viability can be determined by measuring lactate dehydrogenase (LDH) concentration. Cell specific LDH output (nU / cell / day) can be lower than or predicted to be lower than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 (e.g., 150). In some embodiments, LDH production (nU / cell / day) can be from about 0.1 to about 200 (e.g., any subrange of this range described herein, such as from about 0.1 to about 200, from about 0.1 to about 190, from about 0.1 to about 180, from about 0.1 to about 170, from about 0.1 to about 160, from about 0.5 to about 200, from about 0.5 to about 190, from about 0.5 to about 180, from about 0.5 to about 170, from about 0.1 to about 160, 5 to about 160, about 1.0 to about 200, about 1.0 to about 190, about 1.0 to about 180, about 1.0 to about 170, about 1.0 to about 160, about 2.5 to about 200, about 2.5 to about 190, about 2.5 to about 180, about 2.5 to about 170, about 2.5 to about 160, about 5.0 to about 200, about 5.0 to about 190, about 5.0 to about 180, about 5.0 to about 170, about 5.0 to about 160, about 10 to about 200, about 10 to about 190, about 10 to about 180, about 10 to about 170, about 10 to about 160, about 20 to about 200, about 20 to about 190, about 20 to about 180, about 20 to about 170, about 20 to about 160, about 30 to about 200, about 30 to about 190, about 30 to about 180, about 30 to about 170, about 30 to about 160, about 40 to about 200, about 40 to about 190, about 40 to about 180, about 40 to about 170, about 40 to about 160, about 50 to about 200, about 50 to about 190 0, about 50 to about 180, about 50 to about 170, about 50 to about 160, about 60 to about 200, about 60 to about 190, about 60 to about 180, about 60 to about 1...

Claims

1. A method for predicting the effect of sensitizer concentration on cell viability in a production bioreactor, the method comprising: (a) determining cell viability under a plurality of test conditions in a test culture vessel, wherein the plurality of test conditions are defined by a set of parameters; as well as (b) predicting the effect of sensitizer concentration on cell viability in a production bioreactor based on the cell viability in the test culture vessel under the plurality of conditions.

2. The method of claim 1, wherein the set of parameters comprises shear stress, concentration of a sensitizer, and concentration of a protective agent.

3. The method according to claim 1 or claim 2, wherein the multiple test conditions are selected based on full factorial screening and / or an inscribed central composite design.

4. The method according to any one of claims 1 to 3, wherein the sensitizer is a defoaming agent.

5. The method of claim 4, wherein the defoaming agent is a polydimethylsiloxane-based defoaming agent.

6. The method of claim 4, wherein the defoaming agent is dimethicone.

7. The method of any one of claims 1-6, wherein the volume of the test culture container is about 5 mL to about 5 L.

8. The method of claim 7, wherein the volume of the test culture container is about 10 mL to about 500 mL.

9. The method according to any one of claims 1 to 8, wherein the test culture vessel is a baffled shake flask.

10. The method of any one of claims 1-9, wherein the production bioreactor has a volume of about 5 L to about 20,000 L.

Images