Reduced copper loss

Abstract

Provided herein is a method for preventing copper loss in a solution. The solution is a cell culture medium, cell culture supply, or cell culture additive containing copper(II) and cysteine. The method includes having substantially the same amount of soluble copper before and after filtering the solution. Also provided is a method for preparing a cell culture medium, cell culture supply, or cell culture additive containing copper(II), cysteine, and other components without loss of soluble copper. Also provided is a method for culturing cells or producing a biological product, comprising using a medium prepared by the method for preventing copper loss or the method for preparing the medium. Further provided are a medium prepared by the method and uses of such a medium.

Description

Technical Field

[0001] Cross - Reference to Related Applications

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 010,532, filed on April 15, 2020, the content of which is incorporated herein by reference in its entirety.

[0002]

[0002] This disclosure relates, in one aspect, to a method for preventing copper loss in solution. A method for preparing a cell culture medium, cell culture feed, or cell culture additive, including copper(II), cysteine, and other components, without loss of soluble copper is also provided. A cell culture medium, cell culture feed, or cell culture additive can be obtained or may be obtained by these methods. Cells may be cultured in a medium prepared by such methods, and biological products may be produced by said cells.

Background Art

[0003]

[0003] Cell culture may be used to create products such as recombinant proteins, and in fact, the cultured cells themselves may be the product. Cell culture may use either eukaryotic cells such as mammalian cells or prokaryotic cells such as bacterial cells.

[0004]

[0004] Cell culture relies on the use of culture media to support and grow cells under controlled conditions. Culture media and associated supplies, supplements, and additives require the presence of many different components for successful cell culture. The development of chemically defined culture media (CDMs) for animal cell culture that can support the commercial production of recombinant proteins has been a significant milestone for the biotechnology industry. CDM eliminates the variability inherent in culture media containing serum / lysates (Price PJ, Gregory EA. Relationship between in vitro growth promotion and biophysical and biochemical properties of the serum supplement, In Vitro Cellular & Developmental Biology-Plant, 1982;18(6). The entire text is incorporated herein by reference) and drastically reduces the risks associated with atypical drugs (van der Valk J, Mellor D, Brands R, et al., The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture, Toxicology in Vitro. 2004;18(1):1-12. The entire text is incorporated herein by reference). Prior to CDM, serum or lysates were relied upon to deliver various nutrients and components necessary for cell proliferation.

[0005]

[0005] CDMs offer an opportunity to study the interactions of culture medium components compared to undefined media (e.g., those containing serum or hydrolysates). However, one advantage of media containing serum / lysates is that the compounds are usually already in a bioavailable form. For example, iron is an essential cofactor for many cellular functions, but it can be toxic to cells. In undefined media, biological chelating agents such as transferrin are relied upon to deliver iron without toxicity (Bjare U. Serum-free cell culture. Pharmacology & Therapeutics. 1992;53(3):355-374, the whole is incorporated herein by reference). In CDMs, non-protein chelating agents such as citrates need to be studied and implemented to prevent iron toxicity. CDMs typically contain many components, and these components can interact in various ways that are not always readily understood. Successful implementation of CDMs requires understanding how the components chemically interact and how to maintain them in a bioavailable form. Therefore, understanding how components interact is also important in obtaining optimal media and supplements, including undefined media.

[0006]

[0006] Therefore, improved methods are needed for preparing cell culture media, feeds, and supplements in order to reduce potential problems caused by interactions between components. [Overview of the Initiative]

[0007]

[0007] We have confirmed that in cell culture media, feeds, or additives containing cysteine, oxidation of cysteine ​​to cystine readily occurs in the presence of trace metal catalysts, primarily copper and iron. When copper catalyzes the oxidation of cysteine, it is converted to an insoluble form of copper(I). Once there is no more cysteine ​​to be oxidized, the copper usually returns to its soluble form. However, if the solution is sterile filtered while the oxidation of cysteine ​​is occurring, at least some of the copper usually becomes insoluble and is removed from the medium, reducing the level of copper in the medium. This is undesirable because copper is a trace metal necessary for many cell cultures, and copper loss from the solution means that the resulting medium provides the cultured cells with less copper than intended. This usually results in reduced productivity. Therefore, the object of this disclosure is to minimize and / or avoid copper loss from a solution (cell culture medium, cell culture feed, or cell culture additive) during filtration.

[0008]

[0008] A first aspect of the present disclosure provides a method for preventing copper loss in a solution. The solution is a cell medium, cell culture feed, or cell culture additive containing copper(II) and cysteine. The method includes having substantially the same amount of soluble copper before and after filtration of the solution.

[0009]

[0009] In one embodiment, filtration is carried out using a solution containing at least a predetermined level of dissolved oxygen (dO2). Maintaining the oxygen level at at least a predetermined level allows for rapid conversion of copper(I) to soluble copper(II) and prevents the accumulation of large amounts of insoluble copper(I). The predetermined level may be at least about 6% dO2. The predetermined level may be at least about 8% dO2, for example, at least about 10% dO2, or at least about 12% dO2. Filtration may be carried out using a solution containing a dO2 level of at least about 6% dissolved oxygen (dO2). The method may further include maintaining the dO2 level of the solution at at least about 8% dO2 (for example, at least about 10% dO2, or at least about 12% dO2) for at least about 10 minutes before starting filtration. The method may further include maintaining the dO2 level of the solution at at least about 6% dO2 for at least about 10 minutes before starting the filtration. The method may further include maintaining the dO2 level of the solution at at least about 8% dO2 (e.g., a level of at least about 10% dO2, or at least about 12% dO2) for at least about 15 minutes before starting the filtration. The method may further include maintaining the dO2 level of the solution at at least about 6% dO2 for at least about 15 minutes before starting the filtration.

[0010]

[0010] The predetermined level may be a dO2 level of at least about 0.4 mg / L, for example, a dO2 level of at least about 0.6 mg / L, or a dO2 level of at least about 0.8 mg / L. Filtration may be carried out using a solution containing a dO2 level of at least about 0.3 mg / L of dissolved oxygen (dO2). The method may further include maintaining the dO2 level of the solution at at least about 0.4 mg / L dO2 (for example, a dO2 level of at least about 0.6 mg / L, or a dO2 level of at least about 0.8 mg / L) for at least about 10 minutes before starting filtration. The method may further include maintaining the dO2 level of the solution at at least about 0.3 mg / L dO2 for at least about 10 minutes before starting filtration. The method may further include maintaining the dO2 level of the solution at at least about 0.4 mg / L dO2 (e.g., a level of at least about 0.6 mg / L dO2, or at least about 0.8 mg / L dO2) for at least about 15 minutes before starting the filtration. The method may further include maintaining the dO2 level of the solution at at least about 0.3 mg / L dO2 for at least about 15 minutes before starting the filtration.

[0011]

[0011] In one embodiment, the dO2 level is maintained at least the required level by optimizing the oxygenation and / or filtration of the solution. The required level may be about 6% dO2, about 8% dO2, about 10% dO2, or about 12% dO2. The required level may be about 8% dO2. The required level may be about 0.3 mg / L dO2, about 0.4 mg / L dO2, about 0.6 mg / L dO2, or about 0.8 mg / L dO2. The required level may be about 0.4 mg / L dO2.

[0012]

[0012] Oxygenation may include sparging the solution with an oxygen-containing gas. Oxygenation may include stirring and / or mixing the solution to increase contact between the oxygen-containing gas and the solution. Oxygenation may include sparging the solution with an oxygen-containing gas and stirring or mixing the solution to increase contact between the oxygen-containing gas and the solution. The oxygen-containing gas may be air.

[0013]

[0013] Optimizing filtration may include increasing the filtration rate and / or filter size and / or filter volume so that the dO2 level does not fall below a predetermined level during filtration. The predetermined level may be about 6% dO2, about 8% dO2, about 10% dO2, or about 12% dO2. The predetermined level may be about 8% dO2. The predetermined level may be about 0.3 mg / L dO2, about 0.4 mg / L dO2, about 0.6 mg / L dO2, or about 0.8 mg / L dO2. The predetermined level may be about 0.4 mg / L dO2.

[0014]

[0014] Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 10%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 15%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 20%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 25%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 30%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 50%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 75%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 100%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 10%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 15%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 20%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 25%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 30%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 50%. Increasing this may involve increasing the filter size (and optionally the filter capacity) by at least approximately 75%.Increasing may include increasing the filter size (and optionally the filter capacity) by at least approximately 100%. Increasing may include increasing the filter capacity by at least approximately 10%. Increasing may include increasing the filter capacity by at least approximately 15%. Increasing may include increasing the filter capacity by at least approximately 20%. Increasing may include increasing the filter capacity by at least approximately 25%. Increasing may include increasing the filter capacity by at least approximately 30%. Increasing may include increasing the filter capacity by at least approximately 50%. Increasing may include increasing the filter capacity by at least approximately 75%. Increasing may include increasing the filter capacity by at least approximately 100%.

[0015]

[0015] Filtration can be completed within approximately 24 hours after adding copper(II) to the solution. Filtration can be completed within approximately 18 hours after adding copper(II) to the solution. Filtration can be completed within approximately 12 hours after adding copper(II) to the solution. Filtration can be completed within approximately 9 hours after adding copper(II) to the solution. Filtration can be completed within approximately 6 hours after adding copper(II) to the solution. Filtration can be completed within approximately 5 hours after adding copper(II) to the solution. Filtration can be completed within approximately 4 hours after adding copper(II) to the solution. Filtration can be completed within approximately 3 hours after adding copper(II) to the solution. Filtration can be completed within approximately 2 hours after adding copper(II) to the solution. Filtration can be completed within approximately 1 hour after adding copper(II) to the solution. Filtration can be completed within approximately 30 minutes after adding copper(II) to the solution. Filtration can be completed within approximately 20 minutes after adding copper(II) to the solution. Filtration can be completed within approximately 15 minutes after adding copper(II) to the solution. Filtration can also be completed within approximately 10 minutes after adding copper(II) to the solution.

[0016]

[0016] Filtration can be carried out in a volume of approximately 100,000 L or less. Filtration can be carried out in a volume of approximately 50,000 L or less. Filtration can be carried out in a volume of approximately 25,000 L or less. Filtration can be carried out in a volume of approximately 25,000 L or less. Filtration can be carried out in a volume of approximately 15,000 L or less. Filtration can be carried out in a volume of approximately 10,000 L or less. Filtration can be carried out in a volume of approximately 5,000 L or less. Filtration can be carried out in a volume of approximately 4,000 L or less. Filtration can be carried out in a volume of approximately 3,000 L or less. Filtration can be carried out in a volume of approximately 2,000 L or less. Filtration can be carried out in a volume of approximately 1,000 L or less. Filtration can be carried out in a volume of approximately 500 L or less. Filtration can be carried out in a volume of approximately 250 L or less. Filtration can be carried out in a volume of approximately 100 L or less.

[0017]

[0017] Filtration may be carried out in a volume of at least about 50 L. Filtration may be carried out in a volume of at least about 100 L. Filtration may be carried out in a volume of about 100 L to about 2,000 L, or filtration may be carried out in a volume of about 100 L to about 25,000 L. Filtration may be carried out in a volume of at least 1,000 L. For example, filtration may be carried out in a volume of about 1,000 L to about 2,000 L, or filtration may be carried out in a volume of about 1,000 L to about 25,000 L.

[0018]

[0018] Filtration can be completed within approximately 18 hours (for example, within approximately 12 hours, within approximately 9 hours, or within approximately 6 hours) after adding copper(II) to a solution containing a volume of approximately 25,000 L or less. Filtration can be completed within approximately 12 hours (for example, within approximately 9 hours, within approximately 6 hours, or within approximately 3 hours) after adding copper(II) to a solution containing a volume of approximately 15,000 L or less. Filtration can be completed within approximately 9 hours (for example, within approximately 6 hours, within approximately 3 hours, or within approximately 1 hour) after adding copper(II) to a solution containing a volume of approximately 10,000 L or less. Filtration can be completed within approximately 9 hours (for example, within approximately 6 hours, within approximately 3 hours, or within approximately 1 hour) after adding copper(II) to a solution containing a volume of approximately 5,000 L or less. Filtration can be completed within approximately one hour (for example, within approximately 30 minutes or within approximately 20 minutes) after adding copper(II) to a solution containing a volume of approximately 2,000 L or less. Filtration can be completed within approximately 30 minutes (for example, within approximately 20 minutes or within approximately 10 minutes) after adding copper(II) to a solution containing a volume of approximately 1,000 L or less.

[0019]

[0019] Filtration may be carried out at a rate of at least about 5 L / min, 10 L / min, about 20 L / min, about 30 L / min, about 40 L / min, about 50 L / min, about 60 L / min, about 70 L / min, or about 80 L / min. Filtration may be carried out at a rate of at least 10 L / min. Filtration may be carried out at a rate of at least 20 L / min. Filtration may be carried out at a rate of at least 30 L / min. Filtration may be carried out at a rate of at least 40 L / min. Filtration may be carried out at a rate of at least 50 L / min.

[0020]

[0020] Filtration can be carried out at a rate of approximately 170 L / min, approximately 150 L / min, approximately 130 L / min, approximately 110 L / min, approximately 100 L / min, approximately 90 L / min, or within approximately 80 L / min. Filtration can be carried out at a rate of approximately 170 L / min or less. Filtration can be carried out at a rate of approximately 150 L / min or less. Filtration can be carried out at a rate of approximately 130 L / min or less. Filtration can be carried out at a rate of approximately 110 L / min or less. Filtration can be carried out at a rate of approximately 100 L / min or less.

[0021]

[0021] Filtration can be carried out at a rate of approximately 5 L / min to approximately 170 L / min. Filtration can be carried out at a rate of approximately 10 L / min to approximately 150 L / min. Filtration can be carried out at a rate of approximately 20 L / min to approximately 150 L / min. Filtration can be carried out at a rate of approximately 30 L / min to approximately 150 L / min. Filtration can be carried out at a rate of approximately 40 L / min to approximately 150 L / min. Filtration can be carried out at a rate of approximately 50 L / min to approximately 150 L / min. Filtration can be carried out at a rate of approximately 10 L / min to approximately 130 L / min. Filtration can be carried out at a rate of approximately 20 L / min to approximately 130 L / min. Filtration can be carried out at a rate of approximately 30 L / min to approximately 130 L / min. Filtration can be carried out at a rate of approximately 40 L / min to approximately 130 L / min. Filtration can be carried out at a rate of approximately 50 L / min to approximately 130 L / min. Filtration can be carried out at a rate of approximately 10 L / min to approximately 100 L / min. Filtration can be carried out at a rate of approximately 20 L / min to approximately 100 L / min. Filtration can be carried out at a rate of approximately 30 L / min to approximately 100 L / min. Filtration can be carried out at a rate of approximately 40 L / min to approximately 100 L / min. Filtration can be carried out at a rate of approximately 50 L / min to approximately 100 L / min.

[0022]

[0022] Filtration may be carried out at a rate of at least 0.1% of the total volume per minute. Filtration may be carried out at a rate of at least 0.25% of the total volume per minute. Filtration may be carried out at a rate of at least 0.5% of the total volume per minute. Filtration may be carried out at a rate of at least 1% of the total volume per minute. Filtration may be carried out at a rate of at least 2.5% of the total volume per minute. Filtration may be carried out at a rate of at least 5% of the total volume per minute.

[0023]

[0023] The method may include monitoring the oxygen level before starting filtration, for example, monitoring the oxygen level for at least about 10 minutes (e.g., at least about 15 minutes) before starting filtration. The method may include monitoring the dO2 level during filtration. The method may include monitoring the oxygen level before starting filtration and monitoring the dO2 level during filtration. When the method includes monitoring the oxygen level, maintaining the oxygen level at least at a predetermined level may include adding oxygen to the solution in response to the monitored oxygen level approaching a predetermined level. When the method includes monitoring the oxygen level, maintaining the oxygen level at at least at a predetermined level may include increasing the oxygenation of the solution and / or optimizing the filtration in response to the monitored oxygen level approaching a predetermined level. The dO2 level may be monitored using any suitable technique. The dO2 level may be monitored using an electrochemical oxygen sensor or an optical oxygen sensor. The dO2 level may be monitored using an electrochemical oxygen sensor. The dO2 level may be monitored using an optical oxygen sensor.

[0024]

[0024] In embodiments, the method comprises providing a cysteine ​​solution and a copper(II) solution, wherein the filtration comprises filtering the cysteine ​​solution and the copper(II) solution separately, and then combining the filtered cysteine ​​solution and the filtered copper(II) solution to form a cell medium, cell culture feed, or cell culture additive. By keeping the copper(II) solution and the cysteine ​​solution separate until after filtration, the copper is retained as soluble copper(II) before and during filtration.

[0025]

[0025] The cysteine solution can contain at least a majority of the components of a cell culture medium, a cell culture supply, or a cell culture additive other than copper. The cysteine solution can contain substantially all of the components of a cell culture medium, a cell culture supply, or a cell culture additive other than copper and other trace elements such as iron. The cysteine solution can contain substantially all of the components of a cell culture medium, a cell culture supply, or a cell culture additive other than copper and iron. The cysteine solution can contain substantially all of the components of a cell culture medium, a cell culture supply, or a cell culture additive other than copper.

[0026]

[0026] The copper solution can contain at least a majority of the components of a cell culture medium, a cell culture supply, or a cell culture additive other than cysteine.

[0027]

[0027] The concentration of copper in the copper(II) solution can range from about 0.005 μM to about 1.5 M, such as from 0.005 μM to about 1 M. The upper limit of this range can be temperature-dependent. Thus, a solution intended for use at a temperature of at least about 30 °C can have a concentration of up to about 1.5 M, while a compound intended for use or storage at a temperature below room temperature (e.g., a temperature below about 20 °C, such as a temperature below about 10 °C) can have a concentration of up to about 1 M. When the final solution is a cell culture medium, the concentration of copper in the copper(II) solution can range from about 0.005 μM to about 5 μM. When the final solution is a cell culture supply, the concentration of copper in the copper(II) solution can range from about 0.1 mM to about 150 mM. When the final solution is a cell culture additive, the concentration of copper in the copper(II) solution can range from about 1 mM to about 1.5 M, such as from about 1 mM to about 1 M.

[0028]

[0028] In embodiments, filtration includes sterile filtration. Sterile filtration may include the use of a filter medium having a pore size of about 0.5 μm or less. Sterile filtration may include the use of a filter medium having a pore size of about 0.2 μm or less. Sterile filtration may include the use of a filter medium having a pore size of about 0.1 μm or less. Sterile filtration may include the use of a filter medium having a pore size of about 0.1 μm or less. In embodiments, the filter medium includes one or more membranes, hollow fibers, columns, helical membranes, tubes, capillaries, capsules, cassettes, disks, frits, or plugs.

[0029]

[0029] In the embodiments, cysteine ​​is substantially provided as a cysteine ​​derivative that does not contain sulfhydryl groups. For example, cysteine ​​may contain less than about 1 mM of free cysteine. For example, cysteine ​​may contain less than 0.5 mM of free cysteine ​​(or less than 0.1 mM of free cysteine). Cysteine ​​derivatives that do not contain sulfhydryl groups do not react with copper(II) to form insoluble copper(I).

[0030]

[0030] Cysteine ​​can be substantially converted to a cysteine ​​derivative that does not contain a sulfhydryl group before copper(II) is added to the solution. The cysteine ​​derivative may contain a disulfide and / or thiazolidinedione moiety.

[0031]

[0031] Cysteine ​​derivatives may include disulfides. For example, cysteine ​​derivatives may include cysteine, S-sulfocysteine, S-sulfocysteinylglycine, L-cysteine ​​mixed disulfides, and / or S-sulfoglutathione. Cysteine ​​derivatives may also include cystine.

[0032]

[0032] Cysteine ​​derivatives may include a thiazolidinedione moiety. For example, cysteine ​​derivatives may include 4-carboxy-2-methylthiazolidinedion-2-carboxylate, 2-methyl-1,3-thiazolidinedion-2,4-dicarboxylic acid, 2-(2-carboxyethyl)21,3-thiazolidinedion-2,4-dicarboxylic acid, L-2-oxothiazolidinedion-4-carboxylic acid, 2-alkyl-thiazolidinedion-4(R)-carboxylic acid, 2-aryl-thiazolidinedion-4(R)-carboxylic acid, and carbohydrate-based thiazolidinediones (e.g., D-ribose-L-cysteine). Cysteine ​​derivatives may include 4-carboxy-2-methylthiazolidinedion-2-carboxylate.

[0033]

[0033] Cysteine ​​derivatives include cystine, 4-carboxy-2-methylthiazolidined-2-carboxylate, S-sulfocysteine, S-sulfocysteinylglycine, and cysteine. It may contain at least one of the following: S-linked N-acetylglucosamine (GlcNAC-cys), homocystine, L-cysteine ​​mixed disulfide, L-cysteine ​​mixed peptide, S-alkylated cysteine, cysteine ​​having a thiol protecting group (e.g., FMOC-protected cysteine), 2-methyl-1,3-thiazolidine-2,4-dicarboxylic acid, 2-(2-carboxyethyl)21,3-thiazolidine-2,4-dicarboxylic acid, reduced and oxidized glutathione (GSH), S-sulfoglutathione, S-acyl-GSH, S-carboxy-L-cysteine, L-2-oxothiazolidine-4-carboxylic acid, 2-alkyl-thiazolidine-4(R)-carboxylic acid, 2-aryl-thiazolidine-4(R)-carboxylic acid, or carbohydrate-based thiazolidine (e.g., D-ribose-L-cysteine).

[0034]

[0034] In embodiments, copper may be in the form of a copper(II) chelating agent. The chelating agent may comprise at least one chelating agent selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), neocuprione, bathocuprione, D-penicillamine, triethylenetetramine (TETA), dimercaprol (BAL), 8-hydroxyquinoline, cryoquinol, and 5,7-dichloro-2-[(dimethylamino)methyl]-8-quinolinol (PBT2).

[0035]

[0035] In a second aspect, the present disclosure provides a method for preparing a cell medium, cell culture feed, or cell culture additive containing copper(II), cysteine, and other components without loss of soluble copper. The method comprises providing an aqueous solution containing cysteine ​​and other components; providing an aqueous solution containing copper(II); filtering the aqueous solution containing cysteine ​​and other components separately from the aqueous solution containing copper(II); and combining the filtered solutions to provide a cell medium, cell culture feed, or cell culture additive.

[0036]

[0036] Preparation of an aqueous solution containing cysteine ​​and other components may involve dissolving and / or solubilizing cysteine ​​and other components in an aqueous solvent system (e.g., water). Preparation of a solution containing cysteine ​​and other components may involve dissolving and / or solubilizing cysteine ​​and other components in a first solvent system containing an acid, a base, or a water-miscible organic solvent, and then diluting the first solvent system with water and optionally other components to form an aqueous solution.

[0037]

[0037] Preparation of an aqueous solution containing copper(II) may involve dissolving a copper(II) salt in an aqueous solvent system (e.g., water). Preparation of a copper(II) solution may involve dissolving and / or solubilizing a copper(II) salt in a first solvent system containing an acid, a base, or a water-miscible organic solvent, and then diluting the first solvent system with water and optionally other components to form an aqueous solution.

[0038]

[0038] Acids may be inorganic or organic acids. Exemplary inorganic acids include hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monocarbonate, phosphoric acid, monohydrogen phosphoric acid, dihydrogen phosphoric acid, sulfuric acid, monohydrogen sulfuric acid, hydroiodic acid, or phosphorous acid. Exemplary organic acids include relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, and methanesulfone. Exemplary bases include ammonia; hydroxides such as sodium, potassium, calcium, ammonium, organic amino acids, or magnesium; and carbonates or bicarbonates of sodium, potassium, or ammonium. Exemplary water-miscible organic solvents include ethanol, methanol, acetone, dimethyl sulfoxide (DMSO), and dimethylformadine.

[0039]

[0039] The concentration of copper in the copper(II) solution may range from about 0.005 μM to about 1 M. If the final solution is cell culture medium, the concentration of copper in the copper(II) solution may range from about 0.005 μM to about 5 μM. If the final solution is cell culture feed, the concentration of copper in the copper(II) solution may range from about 0.1 mM to about 150 mM. If the final solution is a cell culture additive, the concentration of copper in the copper(II) solution may range from about 1 mM to about 1 M.

[0040]

[0040] In one embodiment, filtration is carried out using a solution containing at least a predetermined level of dissolved oxygen (dO2). Maintaining the oxygen level at at least a predetermined level allows for rapid conversion of copper(I) to soluble copper(II) and prevents the accumulation of large amounts of insoluble copper(I). The predetermined level may be at least about 6% dO2. The predetermined level may be at least about 8% dO2, for example, at least about 10% dO2, or at least about 12% dO2. Filtration may be carried out using a solution containing a dO2 level of at least about 6% dissolved oxygen (dO2). The method may further include maintaining the dO2 level of the solution at at least about 8% dO2 (for example, at least about 10% dO2, or at least about 12% dO2) for at least about 10 minutes before starting filtration. The method may further include maintaining the dO2 level of the solution at at least about 6% dO2 for at least about 10 minutes before starting the filtration. The method may further include maintaining the dO2 level of the solution at at least about 8% dO2 (e.g., a level of at least about 10% dO2, or at least about 12% dO2) for at least about 15 minutes before starting the filtration. The method may further include maintaining the dO2 level of the solution at at least about 6% dO2 for at least about 15 minutes before starting the filtration.

[0041]

[0041] The predetermined level may be a dO2 level of at least about 0.4 mg / L, for example, a dO2 level of at least about 0.6 mg / L, or a dO2 level of at least about 0.8 mg / L. Filtration may be carried out using a solution containing a dO2 level of at least about 0.3 mg / L of dissolved oxygen (dO2). The method may further include maintaining the dO2 level of the solution at at least about 0.4 mg / L dO2 (for example, a dO2 level of at least about 0.6 mg / L, or a dO2 level of at least about 0.8 mg / L) for at least about 10 minutes before starting filtration. The method may further include maintaining the dO2 level of the solution at at least about 0.3 mg / L dO2 for at least about 10 minutes before starting filtration. The method may further include maintaining the dO2 level of the solution at at least about 0.4 mg / L dO2 (e.g., a level of at least about 0.6 mg / L dO2, or at least about 0.8 mg / L dO2) for at least about 15 minutes before starting the filtration. The method may further include maintaining the dO2 level of the solution at at least about 0.3 mg / L dO2 for at least about 15 minutes before starting the filtration.

[0042]

[0042] In one embodiment, the dO2 level is maintained at least a required level by optimizing the oxygenation and / or filtration of the solution. The required level may be about 6% dO2, about 8% dO2, about 10% dO2, or about 12% dO2. The required level may be about 8% dO2. The required level may be about 0.3 mg / L dO2, about 0.4 mg / L dO2, about 0.6 mg / L dO2, or about 0.8 mg / L dO2. The required level may be about 0.4 mg / L dO2.

[0043]

[0043] Oxygenation may include sparging the solution with an oxygen-containing gas. Oxygenation may include stirring and / or mixing the solution to increase contact between the oxygen-containing gas and the solution. Oxygenation may include sparging the solution with an oxygen-containing gas and stirring or mixing the solution to increase contact between the oxygen-containing gas and the solution. The oxygen-containing gas may be air.

[0044]

[0044] Optimizing filtration may include increasing the filtration rate and / or filter size and / or filter volume so that the dO2 level does not fall below a predetermined level during filtration. The predetermined level may be about 6% dO2, about 8% dO2, about 10% dO2, or about 12% dO2. The predetermined level may be about 8% dO2. The predetermined level may be about 0.3 mg / L dO2, about 0.4 mg / L dO2, about 0.6 mg / L dO2, or about 0.8 mg / L dO2. The predetermined level may be about 0.4 mg / L dO2.

[0045]

[0045] Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 10%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 15%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 20%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 25%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 30%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 50%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 75%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 100%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 10%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 15%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 20%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 25%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 30%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 50%. Increasing this may involve increasing the filter size (and optionally the filter capacity) by at least approximately 75%.Increasing may include increasing the filter size (and optionally the filter capacity) by at least approximately 100%. Increasing may include increasing the filter capacity by at least approximately 10%. Increasing may include increasing the filter capacity by at least approximately 15%. Increasing may include increasing the filter capacity by at least approximately 20%. Increasing may include increasing the filter capacity by at least approximately 25%. Increasing may include increasing the filter capacity by at least approximately 30%. Increasing may include increasing the filter capacity by at least approximately 50%. Increasing may include increasing the filter capacity by at least approximately 75%. Increasing may include increasing the filter capacity by at least approximately 100%.

[0046]

[0046] The method may include monitoring the oxygen level before starting filtration, for example, monitoring the oxygen level for at least about 10 minutes (e.g., at least about 15 minutes) before starting filtration. The method may include monitoring the dO2 level during filtration. The method may include monitoring the oxygen level before starting filtration and monitoring the dO2 level during filtration. When the method includes monitoring the oxygen level, maintaining the oxygen level at least at a predetermined level may include adding oxygen to the solution in response to the monitored oxygen level approaching a predetermined level. When the method includes monitoring the oxygen level, maintaining the oxygen level at at least at a predetermined level may include increasing the oxygenation of the solution and / or optimizing the filtration in response to the monitored oxygen level approaching a predetermined level. The dO2 level may be monitored using any suitable technique. The dO2 level may be monitored using an electrochemical oxygen sensor or an optical oxygen sensor. The dO2 level may be monitored using an electrochemical oxygen sensor. The dO2 level may be monitored using an optical oxygen sensor.

[0047]

[0047] In the embodiments, cysteine ​​is substantially provided as a cysteine ​​derivative that does not contain sulfhydryl groups. For example, cysteine ​​may contain less than about 1 mM of free cysteine. For example, cysteine ​​may contain less than 0.5 mM of free cysteine ​​(or less than 0.1 mM of free cysteine). Cysteine ​​derivatives that do not contain sulfhydryl groups do not react with copper(II) to form insoluble copper(I).

[0048]

[0048] Cysteine ​​can be substantially converted to a cysteine ​​derivative that does not contain a sulfhydryl group before copper(II) is added to the solution. The cysteine ​​derivative may contain a disulfide and / or thiazolidinedione moiety.

[0049]

[0049] Cysteine ​​derivatives may include disulfides. For example, cysteine ​​derivatives may include cystine, S-sulfocysteine, S-sulfocysteinylglycine, L-cysteine ​​mixed disulfides, and / or S-sulfoglutathione. Cysteine ​​derivatives may include cystine.

[0050]

[0050] Cysteine ​​derivatives may include a thiazolidinedione moiety. For example, cysteine ​​derivatives may include 4-carboxy-2-methylthiazolidinedion-2-carboxylate, 2-methyl-1,3-thiazolidinedion-2,4-dicarboxylic acid, 2-(2-carboxyethyl)21,3-thiazolidinedion-2,4-dicarboxylic acid, L-2-oxothiazolidinedion-4-carboxylic acid, 2-alkyl-thiazolidinedion-4(R)-carboxylic acid, 2-aryl-thiazolidinedion-4(R)-carboxylic acid, and carbohydrate-based thiazolidinediones (e.g., D-ribose-L-cysteine). Cysteine ​​derivatives may include 4-carboxy-2-methylthiazolidinedion-2-carboxylate.

[0051]

[0051] Cysteine ​​derivatives include cystine, 4-carboxy-2-methylthiazolidined-2-carboxylate, S-sulfocysteine, S-sulfocysteinylglycine, and cysteine. It may contain at least one of the following: S-linked N-acetylglucosamine (GlcNAC-cys), homocystine, L-cysteine ​​mixed disulfide, L-cysteine ​​mixed peptide, S-alkylated cysteine, cysteine ​​having a thiol protecting group (e.g., FMOC-protected cysteine), 2-methyl-1,3-thiazolidine-2,4-dicarboxylic acid, 2-(2-carboxyethyl)21,3-thiazolidine-2,4-dicarboxylic acid, reduced and oxidized glutathione (GSH), S-sulfoglutathione, S-acyl-GSH, S-carboxy-L-cysteine, L-2-oxothiazolidine-4-carboxylic acid, 2-alkyl-thiazolidine-4(R)-carboxylic acid, 2-aryl-thiazolidine-4(R)-carboxylic acid, or carbohydrate-based thiazolidine (e.g., D-ribose-L-cysteine).

[0052]

[0052] In embodiments, copper may be in the form of a copper(II) chelate. The chelate may comprise at least one chelating agent selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), neocuproine, vasocuproine, D-penicillamine, triethylenetetramine (TETA), dimercaprol (BAL), 8-hydroxyquinoline, cryoquinol, and 5,7-chloro-2-[(dimethylamino)methyl]-8-quinolinol (PBT2).

[0053]

[0053] In a third aspect, the present disclosure provides a method for preparing a cell medium, cell culture feed, or cell culture additive containing copper(II), cysteine, and other components without loss of soluble copper. The method comprises filtering a solution containing copper(II), cysteine, and other components to provide a cell medium, cell culture feed, or cell culture additive.

[0054]

[0054] Filtration can be completed within approximately 24 hours after adding copper(II) to the solution. Filtration can be completed within approximately 18 hours after adding copper(II) to the solution. Filtration can be completed within approximately 12 hours after adding copper(II) to the solution. Filtration can be completed within approximately 9 hours after adding copper(II) to the solution. Filtration can be completed within approximately 6 hours after adding copper(II) to the solution. Filtration can be completed within approximately 5 hours after adding copper(II) to the solution. Filtration can be completed within approximately 4 hours after adding copper(II) to the solution. Filtration can be completed within approximately 3 hours after adding copper(II) to the solution. Filtration can be completed within approximately 2 hours after adding copper(II) to the solution. Filtration can be completed within approximately 1 hour after adding copper(II) to the solution. Filtration can be completed within approximately 30 minutes after adding copper(II) to the solution. Filtration can be completed within approximately 20 minutes after adding copper(II) to the solution. Filtration can be completed within approximately 15 minutes after adding copper(II) to the solution. Filtration can also be completed within approximately 10 minutes after adding copper(II) to the solution.

[0055]

[0055] Filtration can be carried out in a volume of approximately 100,000 L or less. Filtration can be carried out in a volume of approximately 50,000 L or less. Filtration can be carried out in a volume of approximately 25,000 L or less. Filtration can be carried out in a volume of approximately 25,000 L or less. Filtration can be carried out in a volume of approximately 15,000 L or less. Filtration can be carried out in a volume of approximately 10,000 L or less. Filtration can be carried out in a volume of approximately 5,000 L or less. Filtration can be carried out in a volume of approximately 4,000 L or less. Filtration can be carried out in a volume of approximately 3,000 L or less. Filtration can be carried out in a volume of approximately 2,000 L or less. Filtration can be carried out in a volume of approximately 1,000 L or less. Filtration can be carried out in a volume of approximately 500 L or less. Filtration can be carried out in a volume of approximately 250 L or less. Filtration can be carried out in a volume of approximately 100 L or less.

[0056]

[0056] Filtration may be carried out in a volume of at least about 50 L. Filtration may be carried out in a volume of at least about 100 L. Filtration may be carried out in a volume of about 100 L to about 2,000 L, or filtration may be carried out in a volume of about 100 L to about 25,000 L. Filtration may be carried out in a volume of at least 1,000 L. For example, filtration may be carried out in a volume of about 1,000 L to about 2,000 L, or filtration may be carried out in a volume of about 1,000 L to about 25,000 L.

[0057]

[0057] Filtration can be completed within approximately 18 hours (for example, within approximately 12 hours, within approximately 9 hours, or within approximately 6 hours) after adding copper(II) to a solution containing a volume of approximately 25,000 L or less. Filtration can be completed within approximately 12 hours (for example, within approximately 9 hours, within approximately 6 hours, or within approximately 3 hours) after adding copper(II) to a solution containing a volume of approximately 15,000 L or less. Filtration can be completed within approximately 9 hours (for example, within approximately 6 hours, within approximately 3 hours, or within approximately 1 hour) after adding copper(II) to a solution containing a volume of approximately 10,000 L or less. Filtration can be completed within approximately 9 hours (for example, within approximately 6 hours, within approximately 3 hours, or within approximately 1 hour) after adding copper(II) to a solution containing a volume of approximately 5,000 L or less. Filtration can be completed within approximately one hour (for example, within approximately 30 minutes or within approximately 20 minutes) after adding copper(II) to a solution containing a volume of approximately 2,000 L or less. Filtration can be completed within approximately 30 minutes (for example, within approximately 20 minutes or within approximately 10 minutes) after adding copper(II) to a solution containing a volume of approximately 1,000 L or less.

[0058]

[0058] Filtration may be carried out at a rate of at least about 5 L / min, 10 L / min, about 20 L / min, about 30 L / min, about 40 L / min, about 50 L / min, about 60 L / min, about 70 L / min, or about 80 L / min. Filtration may be carried out at a rate of at least 10 L / min. Filtration may be carried out at a rate of at least 20 L / min. Filtration may be carried out at a rate of at least 30 L / min. Filtration may be carried out at a rate of at least 40 L / min. Filtration may be carried out at a rate of at least 50 L / min.

[0059]

[0059] Filtration can be carried out at a rate of approximately 170 L / min, approximately 150 L / min, approximately 130 L / min, approximately 110 L / min, approximately 100 L / min, approximately 90 L / min, or within approximately 80 L / min. Filtration can be carried out at a rate of approximately 170 L / min or less. Filtration can be carried out at a rate of approximately 150 L / min or less. Filtration can be carried out at a rate of approximately 130 L / min or less. Filtration can be carried out at a rate of approximately 110 L / min or less. Filtration can be carried out at a rate of approximately 100 L / min or less.

[0060]

[0060] Filtration can be carried out at a rate of approximately 5 L / min to approximately 170 L / min. Filtration can be carried out at a rate of approximately 10 L / min to approximately 150 L / min. Filtration can be carried out at a rate of approximately 20 L / min to approximately 150 L / min. Filtration can be carried out at a rate of approximately 30 L / min to approximately 150 L / min. Filtration can be carried out at a rate of approximately 40 L / min to approximately 150 L / min. Filtration can be carried out at a rate of approximately 50 L / min to approximately 150 L / min. Filtration can be carried out at a rate of approximately 10 L / min to approximately 130 L / min. Filtration can be carried out at a rate of approximately 20 L / min to approximately 130 L / min. Filtration can be carried out at a rate of approximately 30 L / min to approximately 130 L / min. Filtration can be carried out at a rate of approximately 40 L / min to approximately 130 L / min. Filtration can be carried out at a rate of approximately 50 L / min to approximately 130 L / min. Filtration can be carried out at a rate of approximately 10 L / min to approximately 100 L / min. Filtration can be carried out at a rate of approximately 20 L / min to approximately 100 L / min. Filtration can be carried out at a rate of approximately 30 L / min to approximately 100 L / min. Filtration can be carried out at a rate of approximately 40 L / min to approximately 100 L / min. Filtration can be carried out at a rate of approximately 50 L / min to approximately 100 L / min.

[0061]

[0061] Filtration may be carried out at a rate of at least 0.1% of the total volume per minute. Filtration may be carried out at a rate of at least 0.25% of the total volume per minute. Filtration may be carried out at a rate of at least 0.5% of the total volume per minute. Filtration may be carried out at a rate of at least 1% of the total volume per minute. Filtration may be carried out at a rate of at least 2.5% of the total volume per minute. Filtration may be carried out at a rate of at least 5% of the total volume per minute.

[0062]

[0062] In the embodiments, filtration includes sterile filtration. Sterile filtration may include the use of a filter medium having a pore size of about 0.5 μm or less. Sterile filtration may include the use of a filter medium having a pore size of about 0.2 μm or less. Sterile filtration may include the use of a filter medium having a pore size of about 0.1 μm or less. Sterile filtration may include the use of a filter medium having a pore size of about 0.1 μm or less.

[0063]

[0063] In one embodiment, filtration is carried out using a solution containing at least a predetermined level of dissolved oxygen (dO2). Maintaining the oxygen level at at least a predetermined level allows for rapid conversion of copper(I) to soluble copper(II) and prevents the accumulation of large amounts of insoluble copper(I). The predetermined level may be at least about 6% dO2. The predetermined level may be at least about 8% dO2, for example, at least about 10% dO2, or at least about 12% dO2. Filtration may be carried out using a solution containing a dO2 level of at least about 6% dissolved oxygen (dO2). The method may further include maintaining the dO2 level of the solution at at least about 8% dO2 (for example, at least about 10% dO2, or at least about 12% dO2) for at least about 10 minutes before starting filtration. The method may further include maintaining the dO2 level of the solution at at least about 6% dO2 for at least about 10 minutes before starting the filtration. The method may further include maintaining the dO2 level of the solution at at least about 8% dO2 (e.g., a level of at least about 10% dO2, or at least about 12% dO2) for at least about 15 minutes before starting the filtration. The method may further include maintaining the dO2 level of the solution at at least about 6% dO2 for at least about 15 minutes before starting the filtration.

[0064]

[0064] The predetermined level may be a dO2 level of at least about 0.4 mg / L, for example, a dO2 level of at least about 0.6 mg / L, or a dO2 level of at least about 0.8 mg / L. Filtration may be carried out using a solution containing a dO2 level of at least about 0.3 mg / L of dissolved oxygen (dO2). The method may further include maintaining the dO2 level of the solution at at least about 0.4 mg / L dO2 (for example, a dO2 level of at least about 0.6 mg / L, or a dO2 level of at least about 0.8 mg / L) for at least about 10 minutes before starting filtration. The method may further include maintaining the dO2 level of the solution at at least about 0.3 mg / L dO2 for at least about 10 minutes before starting filtration. The method may further include maintaining the dO2 level of the solution at at least about 0.4 mg / L dO2 (e.g., a level of at least about 0.6 mg / L dO2, or at least about 0.8 mg / L dO2) for at least about 15 minutes before starting the filtration. The method may further include maintaining the dO2 level of the solution at at least about 0.3 mg / L dO2 for at least about 15 minutes before starting the filtration.

[0065]

[0065] In one embodiment, the dO2 level is maintained at least a required level by optimizing the oxygenation and / or filtration of the solution. The required level may be about 6% dO2, about 8% dO2, about 10% dO2, or about 12% dO2. The required level may be about 8% dO2. The required level may be about 0.3 mg / L dO2, about 0.4 mg / L dO2, about 0.6 mg / L dO2, or about 0.8 mg / L dO2. The required level may be about 0.4 mg / L dO2.

[0066]

[0066] Oxygenation may include sparging the solution with an oxygen-containing gas. Oxygenation may include stirring and / or mixing the solution to increase contact between the oxygen-containing gas and the solution. Oxygenation may include sparging the solution with an oxygen-containing gas and stirring or mixing the solution to increase contact between the oxygen-containing gas and the solution. The oxygen-containing gas may be air.

[0067]

[0067] Optimizing filtration may include increasing the filtration rate and / or filter size and / or filter volume so that the dO2 level does not fall below a predetermined level during filtration. The predetermined level may be about 6% dO2, about 8% dO2, about 10% dO2, or about 12% dO2. The predetermined level may be about 8% dO2. The predetermined level may be about 0.3 mg / L dO2, about 0.4 mg / L dO2, about 0.6 mg / L dO2, or about 0.8 mg / L dO2. The predetermined level may be about 0.4 mg / L dO2.

[0068]

[0068] Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 10%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 15%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 20%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 25%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 30%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 50%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 75%. Increasing may include increasing the filtration rate (and optionally, the filter size, and / or optionally, the filter capacity) by at least about 100%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 10%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 15%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 20%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 25%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 30%. Increasing may include increasing the filter size (and optionally, the filter capacity) by at least about 50%. Increasing this may involve increasing the filter size (and optionally the filter capacity) by at least approximately 75%.Increasing may include increasing the filter size (and optionally the filter capacity) by at least approximately 100%. Increasing may include increasing the filter capacity by at least approximately 10%. Increasing may include increasing the filter capacity by at least approximately 15%. Increasing may include increasing the filter capacity by at least approximately 20%. Increasing may include increasing the filter capacity by at least approximately 25%. Increasing may include increasing the filter capacity by at least approximately 30%. Increasing may include increasing the filter capacity by at least approximately 50%. Increasing may include increasing the filter capacity by at least approximately 75%. Increasing may include increasing the filter capacity by at least approximately 100%.

[0069]

[0069] The method may include monitoring the oxygen level before starting filtration, for example, monitoring the oxygen level for at least about 10 minutes (e.g., at least about 15 minutes) before starting filtration. The method may include monitoring the dO2 level during filtration. The method may include monitoring the oxygen level before starting filtration and monitoring the dO2 level during filtration. When the method includes monitoring the oxygen level, maintaining the oxygen level at least at a predetermined level may include adding oxygen to the solution in response to the monitored oxygen level approaching a predetermined level. When the method includes monitoring the oxygen level, maintaining the oxygen level at at least at a predetermined level may include increasing the oxygenation of the solution and / or optimizing the filtration in response to the monitored oxygen level approaching a predetermined level. The dO2 level may be monitored using any suitable technique. The dO2 level may be monitored using an electrochemical oxygen sensor or an optical oxygen sensor. The dO2 level may be monitored using an electrochemical oxygen sensor. The dO2 level may be monitored using an optical oxygen sensor.

[0070]

[0070] In the embodiments, cysteine ​​is substantially provided as a cysteine ​​derivative that does not contain sulfhydryl groups. For example, cysteine ​​may contain less than about 1 mM of free cysteine. For example, cysteine ​​may contain less than 0.5 mM of free cysteine ​​(or less than 0.1 mM of free cysteine). Sulfhydryl group-free cysteine ​​derivatives do not react with copper(II) to form insoluble copper(I).

[0071]

[0071] Cysteine ​​can be substantially converted to a cysteine ​​derivative that does not contain a sulfhydryl group before copper(II) is added to the solution. The cysteine ​​derivative may contain a disulfide and / or thiazolidined moiety.

[0072]

[0072] Cysteine ​​derivatives may include disulfides. For example, cysteine ​​derivatives may include cystine, S-sulfocysteine, S-sulfocysteinylglycine, L-cysteine ​​mixed disulfides, and / or S-sulfoglutathione. Cysteine ​​derivatives may include cystine.

[0073]

[0073] Cysteine ​​derivatives may include a thiazolidinedione moiety. For example, cysteine ​​derivatives may include 4-carboxy-2-methylthiazolidinedione-2-carboxylate, 2-methyl-1,3-thiazolidinedione-2,4-dicarboxylic acid, 2-(2-carboxyethyl)21,3-thiazolidinedione-2,4-dicarboxylic acid, L-2-oxothiazolidinedione-4-carboxylic acid, 2-alkyl-thiazolidinedione-4(R)-carboxylic acid, 2-aryl-thiazolidinedione-4(R)-carboxylic acid, and carbohydrate-based thiazolidinediones (e.g., D-ribose-L-cysteine). Cysteine ​​derivatives may include 4-carboxy-2-methylthiazolidinedione-2-carboxylate.

[0074]

[0074] Cysteine ​​derivatives include cystine, 4-carboxy-2-methylthiazolidined-2-carboxylate, S-sulfocysteine, S-sulfocysteinylglycine, and cysteine. It may contain at least one of the following: S-linked N-acetylglucosamine (GlcNAC-cys), homocystine, L-cysteine ​​mixed disulfide, L-cysteine ​​mixed peptide, S-alkylated cysteine, cysteine ​​having a thiol protecting group (e.g., FMOC-protected cysteine), 2-methyl-1,3-thiazolidine-2,4-dicarboxylic acid, 2-(2-carboxyethyl)21,3-thiazolidine-2,4-dicarboxylic acid, reduced and oxidized glutathione (GSH), S-sulfoglutathione, S-acyl-GSH, S-carboxy-L-cysteine, L-2-oxothiazolidine-4-carboxylic acid, 2-alkyl-thiazolidine-4(R)-carboxylic acid, 2-aryl-thiazolidine-4(R)-carboxylic acid, or carbohydrate-based thiazolidine (e.g., D-ribose-L-cysteine).

[0075]

[0075] In embodiments, copper may be in the form of a copper(II) chelate. The chelate may comprise at least one chelating agent selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), neocuproine, vasocuproine, D-penicillamine, triethylenetetramine (TETA), dimercaprol (BAL), 8-hydroxyquinoline, cryoquinol, and 5,7-chloro-2-[(dimethylamino)methyl]-8-quinolinol (PBT2).

[0076]

[0076] In any embodiment of the first, second, or third aspect, the other component comprises at least one carbon source, and / or at least one nitrogen source, and / or at least one vitamin, and / or at least one buffer, and / or at least one inorganic salt, and / or at least one trace metal other than copper, and / or at least one polyamine, and / or at least one (essential) fatty acid, and / or at least one antioxidant, and / or at least one corticosteroid, and / or at least one chelating agent, and / or at least one lipase inhibitor. The other component may comprise at least one carbon source, and / or at least one nitrogen source, and / or at least one vitamin, and / or at least one buffer, and / or at least one inorganic salt, and / or at least one trace metal other than copper, and / or at least one polyamine, and / or at least one (essential) fatty acid. The other component may comprise at least one carbon source. The other component may comprise at least one nitrogen source. Other components may include at least one vitamin. Other components may include at least one buffer. Other components may include at least one inorganic salt. Other components may include at least one trace metal other than copper. Other components may include at least one polyamine. Other components may include at least one (essential) fatty acid. Other components may include at least one carbon source, at least one nitrogen source, at least one vitamin, at least one buffer, at least one inorganic salt, at least one trace metal other than copper, at least one polyamine, and at least one (essential) fatty acid.

[0077]

[0077] At least one carbon source may include sugars, optionally monosaccharides or disaccharides. The sugars may include at least one (e.g., two or more) glucose, galactose, maltose, fructose, ribose, and deoxyribose. The sugars may include glucose and / or deoxyribose.

[0078]

[0078] At least one nitrogen source (for example, two or more nitrogen sources) may contain at least one amino acid and / or ammonia / ammonium. At least one nitrogen source may contain at least one amino acid. The at least one amino acid may be selected from L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

[0079]

[0079] At least one vitamin (for example, two or more vitamins) may be selected from ascorbic acid (e.g., magnesium ascorbate), biotin, choline (e.g., choline chloride), D-Ca2+-pantothenate, folic acid, inositol, menadione, niacinamide, nicotinic acid, para-aminobenzoic acid (PABA), pyridoxal, pyridoxine, riboflavin, thiamine, vitamin A acetate, vitamin B12, vitamin D2, and substances having vitamin-like activity (e.g., alpha-lipoic acid or dihydrolipoic acid (DHLA)).

[0080]

[0080] At least one buffer is Good's buffer (e.g., as disclosed in NE Good, et al., “Hydrogen Ion Buffers for Biological Research”, Biochemistry (1966), 5(2), 467-477; NE Good and S. Izawa, “Hydrogen Ion Buffers, Methods in Enzymology, (1972), Vol. 24, 53-68; and WJ Ferguson, et al., “Hydrogen Ion Buffers for Biological Research”, Analytical Biochemistry, (1980), 104(2)). The present invention may include at least one buffer (e.g., two or more buffers) capable of buffering at a pH in the range of about 6 to about 8, such as those disclosed in 300-310. The at least one buffer may include carbonates, bicarbonates, phosphates, hydrogen phosphates, dihydrogen phosphates, lactates, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), N,N-bis(2-hydroxyethyl)-2-aminesulfonic acid. It may contain at least one buffer (e.g., two or more buffers) selected from at least one buffer containing noethanesulfonic acid (BES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino]ethanesulfonic acid (TES), 2-(N-morpholino)ethanesulfonic acid (MES), and 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (bis-Tris).

[0081]

[0081] At least one inorganic salt (for example, two or more inorganic salts) may include at least one soluble salt of calcium, potassium, magnesium, sodium, or iron. The soluble salt may be a salt having a solubility of at least 1 g / L in water at 25°C. At least one inorganic salt (for example, two or more inorganic salts) may include at least one of CaCl2, KCl, MgCl2, MgSO4, NaCl, NaHCO3, Na2HPO4, NaH2PO4, and iron citrate chelate or iron sulfate chelate.

[0082]

[0082] The at least one trace metal other than copper (for example, two or more trace metals) may include at least one ion from among barium, bromine, cobalt, iodine, manganese, chromium, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, and aluminum.

[0083]

[0083] At least one polyamine (for example, two or more polyamines) may include at least one of putrescine, cadaverine, spermine, and spermidine.

[0084]

[0084] At least one (essential) fatty acid (e.g., at least two fatty acids) may be selected from n-3, n-6, n-9 fatty acids and cholesterol. At least one (essential) fatty acid (e.g., at least two fatty acids) may be selected from linoleic acid (ALA), linolenic acid (LA), docosahexenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (AA). For example, at least one essential fatty acid (e.g., at least two fatty acids) may be selected from ALA and linolenic acid LA.

[0085]

[0085] At least one antioxidant (for example, two or more antioxidants) may include at least one of ascorbic acid, taurine, hypotaurine, tocopherol, and tocotrienol.

[0086]

[0086] At least one corticosteroid (e.g., two or more corticosteroids) may include at least one glucocorticoid (e.g., hydrocortisone) or mineralocorticoid (e.g., aldosterone).

[0087]

[0087] At least one metal chelating agent (for example, two or more chelating agents) may include at least one of the following: ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), neocuproine, vasocuproine, D-penicillamine, triethylenetetramine (TETA), dimercaprol (BAL), 8-hydroxyquinoline, cryoquinol, and 5,7-dichloro-2-[(dimethylamino)methyl]-8-quinolinol (PBT2), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), and deferoxamine mesylate.

[0088]

[0088] In a fourth aspect, the present disclosure provides a method for culturing cells in a culture medium. The method includes preventing copper loss in the culture medium by the method of the first aspect, or preparing the culture medium by the method of the second or third aspect; bringing cells into contact with the culture medium; and culturing the cells in the culture medium.

[0089]

[0089] The cells may be eukaryotic cells, prokaryotic cells, or archaeal cells. Eukaryotic cells may be mammalian cells. The cells may be selected from hybridoma cells, CHO cells, COS cells, VERO cells, HeLa cells, HEK 293 cells, PER-C6 cells, K562 cells, MOLT-4 cells, Ml cells, NS-1 cells, COS-7 cells, MDBK cells, MDCK cells, MRC-5 cells, WI-38 cells, WEHI cells, SP2 / 0 cells, BHK cells (including BHK-21 cells), and their derivatives.

[0090]

[0090] Culturing cells in a culture medium may include growing the cells. The method may further include isolating and / or purifying the grown cells.

[0091]

[0091] In a fifth aspect, the present disclosure provides a method for producing a biological product. The method includes preventing copper loss in the culture medium according to the first aspect or preparing the culture medium according to the second or third aspect; contacting cells configured to express the biological product with the culture medium; and culturing the cells in the culture medium under conditions such that the cells express the biological product.

[0092]

[0092] The biological product may be a polypeptide, protein, peptide, hormone, virus or virus-like particle, nucleic acid or fragment thereof; optionally, an antibody or a biologically functional fragment of an antibody.

[0093]

[0093] The cells may be eukaryotic cells, prokaryotic cells, or archaeal cells. Eukaryotic cells may be mammalian cells. The cells may be selected from hybridoma cells, CHO cells, COS cells, VERO cells, HeLa cells, HEK 293 cells, PER-C6 cells, K562 cells, MOLT-4 cells, Ml cells, NS-1 cells, COS-7 cells, MDBK cells, MDCK cells, MRC-5 cells, WI-38 cells, WEHI cells, SP2 / 0 cells, BHK cells (including BHK-21 cells), and their derivatives.

[0094]

[0094] The method may further include isolating and / or purifying the product.

[0095]

[0095] In the sixth aspect, the method provides a cell culture medium, cell culture feed, or cell culture additive that can be obtained or are obtained by the method of the second or third aspect.

[0096]

[0096] In a seventh aspect, the present disclosure provides a sixth aspect of the use of a cell medium, cell culture feed, or cell culture additive for culturing cells. Culturing may include producing a biological product from the cells. Culturing may include growing the cells. Culturing may include growing the cells and producing a biological product from the cells. The use may further include isolating the biological product.

[0097]

[0097] Embodiments of the present invention will be further described below with reference to the attached drawings. [Brief explanation of the drawing]

[0098] [Figure 1] The time course of copper removal by filtration in Genentech Essential Medium is illustrated. Aliquots (n=3) were filtered at each time point, and the copper content was compared to that of the unfiltered medium. [Figure 2] This shows how several parameters changed over time in an exemplary culture medium after the addition of trace metals. a) provides the cysteine ​​monomer concentration profile, b) provides the dissolved oxygen (dO2) concentration profile compared to the saturation percentage, c) provides the redox potential, and d) superimposes the results for copper removal, cysteine ​​monomer, and dissolved oxygen, allowing for a comparison of the relative levels of each of these parameters. [Figure 3] a) Increase in starting cysteine ​​concentration in the non-mixed system, b) Increase in starting cysteine ​​concentration in the mixed system, c) Nitrogen sparging after cysteine ​​depletion, and d) Expansion of copper removal by nitrogen sparging before cysteine ​​depletion. [Figure 4] This shows the formation of 2,4-thiazolidine through the condensation reaction of cysteine ​​and pirubate. [Figure 5]This report provides cysteine ​​monomer profiles measured with Elman's reagent in a simple culture medium. Four conditions were compared: a simple culture medium without metal ions or pirubate, a simple culture medium containing pirubate, a simple culture medium containing metal ions, and a simple culture medium containing both pirubate and metal ions. [Figure 6] This provides replicated data demonstrating the consistency of copper removal, cysteine ​​monomers, dissolved oxygen, and redox profiles in cysteine-containing cell media (comparison with Figures 1 and 2). [Modes for carrying out the invention]

[0099]

[0098] Throughout this specification and the claims, the words “comprise” and “contain” and their variations mean “comprise but not limited to,” and they are not intended (and do not exclude) other parts, additives, ingredients, integers or processes. Throughout this specification and the claims, unless otherwise required by context, the singular form includes the plural form. In particular, where the indefinite article is used, this specification shall be understood to intend the plural form as well as the singular form unless otherwise required by context.

[0100]

[0099] Features, integers, characteristics, compounds, chemical parts, or groups described in relation to a particular aspect, embodiment, or example of this disclosure should be understood to be applicable to any other aspect, embodiment, or example described herein, unless they are incompatible. All features disclosed herein (including any appended claims, abstract, and drawings) and / or all steps of any method or process so so disclosed may be combined in any combination, except for combinations in which at least some of such features and / or steps are mutually exclusive. This disclosure is not limited to the details of any of the embodiments described above. This disclosure extends to any novel features or any novel combination of features disclosed herein (including any appended claims, abstract, and drawings), or any novel steps or any novel combination of steps of any method or process so so disclosed.

[0101]

[0100] The reader's attention is directed to all papers and documents filed concurrently with or prior to this application in connection with this specification and published publicly with this specification, the contents of all such papers and documents are incorporated herein by reference.

[0102]

[0101] All publications, patent applications, patents, and other references referred to herein are incorporated in their entirety by reference. In case of any conflict, this specification, including definitions, shall prevail.

[0103] definition

[0102] The following explanations of terms and methods are provided to better describe this disclosure and to guide those skilled in the art in implementing this disclosure.

[0104]

[0103] As used herein, the term “soluble copper” includes references to solvated copper ions, i.e., copper ions in a solution that are surrounded or complexed by solvent molecules. The solution may optionally be an aqueous solution containing a polar organic solvent (such as ethanol). Soluble copper may include or consist of copper(II) ions, for example, copper(II) salts having solubility in water greater than 1 g per 100 mL at 20°C.

[0105]

[0104] As used herein, the term “insoluble” includes references to solids having a solubility in water of less than 0.05 g per 100 mL at 20°C (e.g., species containing copper(I)).

[0106]

[0105] As used herein, the term “solution” includes references to a liquid phase containing multiple substances unless otherwise required by context. When the solution is a cell medium, cell culture feed, or cell culture additive, the solvent is typically water, and the other substances in the solution are typically solutes. Solutes may be lost from the solution by precipitation.

[0107]

[0106] As used herein, the term “filtration” includes reference to the process by which a solution passes through a porous material (filter). Solid particles present in the solution are retained on the filter, especially when the particles are larger than the pores of the filter. Filtration of cell culture media, cell culture feed, or cell culture additives may be carried out using filters with pore sizes up to approximately 0.5 μm, such as up to approximately 0.2 μm (e.g., up to approximately 0.1 μm). As used herein, the term “sterile filtration” refers to filtration carried out using filters with pore sizes small enough to remove microorganisms from a solution, such as viruses, bacteria, fungi, mycoplasmas, and pathogens of transmissible spongiform encephalopathy. Sterile filtration may use nanofilters with pore sizes up to approximately 0.1 μm, such as up to 0.05 μm. Sterile filtration may provide a logarithmic reduction value (LRV) of related microorganisms greater than approximately 4 LRV.

[0108]

[0107] The term "filterable" with respect to solution components such as copper means that the component is completely solvated or has a particle size small enough for the particles to quantitatively pass through the pores of the filter medium during filtration. Filterable components may include particles with a maximum dimension of up to 90% (e.g., up to 70%) of the filter's pore size. Filterable components may consist of or include completely solvated species and optionally particles with a maximum dimension of up to 90% (e.g., up to 70%) of the filter's pore size.

[0109]

[0108] As used herein, the term “dissolved oxygen” (dO2) includes a reference to the level of oxygen dissolved in a solution. dO2 may also be provided in relative units of “% dO2,” which represents the saturation percentage of O2 in water based on an O2 partial pressure of 212.2 mbar at the temperature of the solution (e.g., temperatures ranging from about 8°C to about 40°C). For example, dO2 may be at least about 8% dO2. dO2 may also be provided in absolute units of “mg / L.” For example, dO2 may be at least about 1 mg / L at a temperature of 8°C, and / or dO2 may be at least about 0.3 mg / L at a temperature of 40°C.

[0110]

[0109] As used herein, the term “atmosphere” includes references to air having a composition similar to that of the Earth’s atmosphere at sea level, including nitrogen, oxygen, water vapor, argon, carbon dioxide, and trace gases. The oxygen level in dry air is typically about 20 to about 21 volume percent.

[0111]

[0110] As used herein, the term “chelate” refers to a complex comprising a metal ion and at least one polydentate ligand (e.g., bidentate, tridentate, quadentate, quinate, hexate, etc.). Exemplary metal ions in copper(II). Exemplary polydentate ligands include ethylenediaminetetraacetate (EDTA), diethylenetriaminepentaacetate (DTPA), neocuproine, vasocuproine, D-penicillamine, triethylenetetramine (TETA), dimercaprol (BAL), 8-hydroxyquinoline, cryoquinol, and 5,7-dichloro-2-[(dimethylamino)methyl]-8-quinolinol (PBT2). While the exemplary polydentate ligands are shown in the form of specific acids or bases, it will be understood that ligands may be provided in the form of any suitable conjugate acid or conjugate base.

[0112]

[0111] As used herein, the term “cysteine” includes references to the amino acid L-cysteine, its tautomers, geometric isomers, salts, or solvates. As used herein, the term “cysteine ​​derivative” includes references to derivatives of cysteine ​​in which a hydrogen atom of a disulfide is replaced by a bond to another moiety, and therefore cysteine ​​derivatives do not contain a sulfhydryl group. For example, cysteine ​​derivatives may contain a disulfide or thiazolidined moiety.

[0113]

[0112] As used herein, the term “cell medium” refers to a nutrient solution for culturing cells. “Cell culture feed” and “cell culture additive” refer to nutrient supplements that may be added to a cell medium to improve its performance. For example, cell culture feed and / or cell culture additives may be added to a cell medium during batch culture of cells. A cell medium may contain chemically defined or undefined components.

[0114]

[0113] As used herein, the term “contact” includes placing cells to be cultured in vitro into a culture vessel together with the medium in which the cells are cultured. The term “contact” includes mixing the cells with the medium, pipetting the medium onto the cells in the culture vessel, and immersing the cells in the medium.

[0115]

[0114] As used herein, the term “combining” includes references to mixing or blending raw materials in a cell culture medium preparation.

[0116]

[0115] As used herein, a "chemically defined" medium is a medium in which all components are known. Chemically defined media are distinguished from serum, embryo extracts, and hydrolysates that contain unknown components. The cell media of this disclosure may be chemically defined media. The cell culture feeds of this disclosure may be chemically defined. The cell culture additives of this disclosure may be chemically defined.

[0117]

[0116] As used herein, “undefined medium” or “medium containing an undefined component” includes reference to a medium containing one or more unknown raw materials. Undefined components may be provided, for example, by serum, peptone, hydrolysates (such as yeast, plant, or serum hydrolysates), and embryo extracts.

[0118]

[0117] As used herein, the term “raw material” means any compound, whether of chemical or biological origin, that can be used in cell culture media, feeds, or additives to maintain or promote cell growth or proliferation, or the expression of products by cells. The terms “component,” “nutrient,” and “raw material” may be used interchangeably and all mean such compounds. Typical raw materials used in cell culture media include carbon sources (such as monosaccharides), nitrogen sources (such as amino acids), vitamins, buffers, inorganic salts, trace metals, polyamines, lipids, and fatty acids. Exemplary raw materials include amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, etc. Other raw materials that promote or maintain the ex vivo culture of cells may be selected by those skilled in the art according to specific needs.

[0119]

[0118] Cell culture media are composed of many raw materials, and these raw materials vary depending on the medium. As used herein, “1× preparation” means any aqueous solution containing some or all of the raw materials found in cell culture media at working concentrations. “1× preparation” can refer, for example, to a cell culture medium or any subgroup of the materials of that medium. The concentrations of the raw materials in a 1× solution are approximately the same as the concentrations of the raw materials found in cell culture preparations used to maintain or culture cells in vitro. A cell culture medium used for in vitro culture of cells is a 1× preparation by definition. When many raw materials are present, each raw material in a 1× preparation has a concentration approximately equal to the concentration of those raw materials in the cell culture medium. Thus, when referring to a “1× preparation,” it is intended that each raw material in the solution has the same or approximately the same concentration as found in the cell culture medium being described. The concentrations of the raw materials in 1× preparations of cell culture media are well known to those skilled in the art, and the relevant components and their concentrations depend on the cell type or application. See, for example, Methods For Preparation of Media, Supplements and Substrate For Serum-Free Animal Cell Culture, New York: Allen R. Liss (1984); HJ Morton, "A survey of commercially available tissue culture media", In Vitro (1970), 6(2), 89-108; and J. Van der Valk, et al., (2010), "Optimization of chemically defined cell culture media-replacing fetal bovine serum in mammalian in vitro methods", Toxicology in vitro, 24(4), 1053-1063; all of these references are incorporated herein by reference in their entirety. However, 1× formulations may have different osmotic pressures and / or pH compared to culture media, especially when they contain fewer components.

[0120]

[0119] As used herein, “nn × formulation” (where “nn” is an integer greater than 1) refers to a solution in which each ingredient in the solution is provided at a concentration approximately nn times higher than the corresponding ingredient in a 1 × formulation. Cell culture feeds and cell culture additives are 1 × 1.5 × 10 9 × formulations may be supplied, for example, as 1× to 10,000× formulations. For example, cell culture feeds may be supplied as 1× to 1,000× formulations. As those skilled in the art will understand, the upper limit of "nn×" for a given formulation depends on the solubility of each component in the formulation and the required concentration of each component in the corresponding 1× formulation. Therefore, feeds and additives containing only components with relatively high solubility that are required even at relatively low concentrations in the corresponding 1× formulation can usually achieve higher "nn".

[0121]

[0120] As used herein, the term “cell” includes references to eukaryotic cells, prokaryotic cells, and archaeal cells. Unless otherwise required by context, references to cells may include references to plural(cells). Eukaryotic cells may be mammalian cells, e.g., hybridoma, CHO cells, COS cells, VERO cells, HeLa cells, HEK 293 cells, PER-C6 cells, K562 cells, MOLT-4 cells, Ml cells, NS-1 cells, COS-7 cells, MDBK cells, MDCK cells, MRC-5 cells, WI-38 cells, WEHI cells, SP2 / 0 cells, BHK cells (including BHK-21 cells), and their derivatives. Prokaryotic cells may be Escherichia coli, Pseudomonas species, Bacillus species, Streptomyces species, Lactobacillus species, Lactococcus species, and their derivatives. Archaeal cells may be Haloarchaea and their derivatives.

[0122] cell culture medium

[0121] Cell culture media contain many components. Cell culture media provide the nutrients necessary to maintain and grow cells in a controlled, artificial, in vitro environment. The properties and composition of cell culture media vary depending on the specific cell requirements. Important parameters include osmotic pressure, pH, and nutrient formulations.

[0123]

[0122] The culture medium contains a mixture of amino acids, glucose, salts, vitamins, and other nutrients and is available from suppliers in either powder or liquid form. The requirements for these components vary depending on the cell line. pH adjustment is important for optimal culture conditions and is generally achieved using a suitable buffer system. CDM is preferred for therapeutic and related applications because it provides a reproducible contamination-free medium when prepared and used under sterile conditions, although some cell types may require the use of a medium containing serum, proteins, or other biological extracts (such as yeast extracts or enzymatic digests of plant or animal material).

[0124]

[0123] Cell culture has also been performed using extremely simple definition media consisting essentially of vitamins, amino acids, organic salts, inorganic salts, and buffers. However, such media (often called "basal media") are usually severely deficient in the nutrients required by most animal cells. Therefore, these media often need to be supplemented with feed or other additives, for example, to form a complete medium. Furthermore, in batch culture systems, the medium is often periodically supplemented with concentrated feed or additives to maintain the viability of cultured cells and / or the production of biological products such as polypeptides (e.g., antibodies or biologically functional fragments of antibodies), proteins, peptides, hormones, viruses or virus-like particles, nucleic acids, or fragments thereof.

[0125]

[0124] The raw materials that may be present in the basal medium include amino acids (nitrogen source), vitamins, inorganic salts, sugars (carbon source), buffer salts, and lipids. A basal medium for use with a certain mammalian cell culture system may contain ethanolamine, D-glucose, N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] (HEPES), linoleic acid, lipoic acid, phenol red, PLURON1C F68, putrescine, and sodium pyruvate.

[0126]

[0125] The amino acid raw materials that may be included in the culture medium include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and their derivatives. These amino acids can be commercially available, for example, from Sigma (Saint Louis, Missouri).

[0127]

[0126] Vitamin raw materials that may be included in the culture medium include biotin, choline chloride, and D-Ca 2+ - It contains pantothenate, folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine, and vitamin B12. These vitamins can be commercially available, for example, from Sigma (Saint Louis, Missouri).

[0128]

[0127] Inorganic salt raw materials that can be used in culture media include one or more calcium salts (e.g., CaCl2), Fe(NO3)3, KCl, one or more magnesium salts (e.g., MgCl2 and / or MgSO4), one or more manganese salts (e.g., MnCl2), NaCl, NaHCO3, N2HPO4, and ions of trace elements selenium, vanadium, zinc, and copper. These trace elements can be provided in various forms, preferably in the form of salts such as Na2SeO3, NH4VO3, ZnSO4, and CuSO4. These inorganic salts can be commercially available, for example, from Sigma (Saint Louis, Missouri).

[0129]

[0128] Examples of culture media useful for mammalian culture include commercially available media such as Ham F10 (Sigma), Minimum Essential Medium ((MEM)Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma), which are suitable for culturing host cells. Furthermore, any culture medium described in Ham and Wallace (1979), Meth. in Enz. 58:44; Barnes and Sato (1980), Anal. Biochem. 102:255; U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, or 4,560,655; International Publication Nos. 90 / 03430, 87 / 00195; U.S. Reissue Patent No. 30,985; or U.S. Patent No. 5,122,469 (all of these disclosures are incorporated herein by reference) may be used as a culture medium for host cells. One of these culture media may contain, as needed, hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphates), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), and antibiotics (such as gentamycin). TMDrugs, trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or equivalent energy sources may be supplemented. Any other necessary supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature and pH, are those already used in host cells selected for expression and will be apparent to those skilled in the art. Exemplary culture conditions include M. Takagi and K. Ueda, "Comparison of the optimal culture conditions for cell growth and tissue plasminogen activator production by human embryo lung cells on microcarriers", Biotechnology, (1994), 41, 565-570; HJ Morton, "A survey of commercially available tissue culture media", In Vitro (1970), 6(2), 89-108; J. Van der Valk, et al., (2010), "Optimization of chemically defined cell culture media-replacing fetal bovine serum in mammalian in vitro methods", Toxicology in vitro, 24(4), 1053-1063; RJ Graham et al., "Consequences of trace metal variability and supplementation on Chinese hamster ovary (CHO) cell culture performance: A review of key mechanisms and considerations", BIOtechnol. Bioeng. (2019), 116(12), 3446-3456; S. Janoschek et al.These are provided in "A protocol to transfer a fed-batch platform process into semi-perfusion mode: The benefit of automated small-scale bioreactors compared to shake flasks as scale-down model," Biotechnol. Prog., (2019), 35(2), e2757; and M. Kuiper et al., "Repurposing fed-batch media and feeds for highly productive CHO perfusion processes," Biotechnology Progress, 15 April 2019, https: / / doi.org / 10.1002 / btpr.2821, and all references to these are incorporated herein by reference.

[0130]

[0129] The cell culture media, feeds, or additives of this disclosure and the present invention contain copper(II) and cysteine. Table 1 shows the amounts of copper(II) and cysteine ​​(including cysteine ​​derivatives) present in exemplary culture media, feeds, and additives. JPEG2023522037000003.jpg44170 Cysteine ​​may be provided as L-cysteine ​​and in the form of several hydrates and / or salts. These cysteine-containing forms may be solubilized in water, organic solvents, basic solvents, or acidic solvents to increase solubility, provided that the solvent at the process-implementation concentration that comes into contact with the culture and / or product does not affect cell culture performance or product quality. For certain cell types, at least some of the cysteine ​​(e.g., substantially all of the cysteine) may be provided as cysteine ​​derivatives. For example, as disclosed herein, substantially all of the cysteine ​​may be provided as cysteine ​​derivatives that do not contain sulfhydryl groups. Copper(II) may be provided in the form of several hydrates and / or salts (typically sulfates containing some water). These copper-containing forms may be solubilized in water, organic solvents, basic solvents, or acidic solvents to increase solubility, provided that the solvent at the process-implementation concentration that comes into contact with the culture and / or product does not affect cell culture performance or product quality.

[0131]

[0130] Other components of the cell culture medium, feed, and additives include at least one carbon source and / or at least one nitrogen source and / or at least one vitamin and / or at least one buffer and / or at least one inorganic salt and / or at least one trace metal other than copper and / or at least one polyamine and / or at least one (essential) fatty acid. Other components may include at least one carbon source. Other components may include at least one nitrogen source. Other components may include at least one vitamin. Other components may include at least one buffer. Other components may include at least one inorganic salt. Other components may include at least one trace metal other than copper. Other components may include at least one polyamine. Other components may include at least one (essential) fatty acid. Other components may include at least one carbon source, at least one nitrogen source, at least one vitamin, at least one buffer, at least one inorganic salt, at least one non-copper trace metal, at least one polyamine, and at least one (essential) fatty acid.

[0132]

[0131] At least one carbon source may include sugars, optionally monosaccharides or disaccharides. The sugars may include at least one (e.g., two or more) glucose, galactose, maltose, fructose, ribose, and deoxyribose. The sugars may include glucose and / or deoxyribose.

[0133]

[0132] At least one nitrogen source (e.g., two or more nitrogen sources) may contain at least one amino acid and / or ammonia / ammonium. At least one nitrogen source may contain at least one amino acid. The at least one amino acid may be selected from L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, or derivatives thereof. In embodiments, at least one amino acid includes, but is not limited to, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, or derivatives thereof.

[0134]

[0133] At least one vitamin (for example, two or more vitamins) may be selected from ascorbic acid (e.g., magnesium ascorbate), biotin, choline (e.g., choline chloride), D-Ca2+-pantothenate, folic acid, inositol, menadione, niacinamide, nicotinic acid, para-aminobenzoic acid (PABA), pyridoxal, pyridoxine, riboflavin, thiamine, vitamin A acetate, vitamin B12, and vitamin D2.

[0135]

[0134] At least one buffering agent is Good's buffering agent (e.g., as disclosed in NE Good, et al., “Hydrogen Ion Buffers for Biological Research”, Biochemistry (1966), 5(2), 467-477; NE Good and S. Izawa, “Hydrogen Ion Buffers, Methods in Enzymology, (1972), Vol. 24, 53-68; and WJ Ferguson, et al., “Hydrogen Ion Buffers for Biological Research”, Analytical Biochemistry, (1980), 104(2)). The invention may include at least one buffer (e.g., two or more buffers) capable of buffering at a pH in the range of about 6 to about 8, such as those disclosed in 300-310. The at least one buffer may include carbonates, bicarbonates, phosphates, hydrogen phosphates, dihydrogen phosphates, lactates, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), N,N-bis(2-hydroxyethyl)-2-A It may contain at least one buffer (e.g., two or more buffers) selected from at least one buffer containing minoethanesulfonic acid (BES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino]ethanesulfonic acid (TES), 2-(N-morpholino)ethanesulfonic acid (MES), and 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (bis-Tris).

[0136]

[0135] At least one inorganic salt (e.g., two or more inorganic salts) may include at least one soluble salt of calcium, potassium, magnesium, sodium, or iron. The soluble salt may be a salt having a solubility of at least 1 g / L in water at 25°C. At least one inorganic salt (e.g., two or more inorganic salts) may include at least one of CaCl2, KCl, MgCl2, MgSO4, NaCl, NaHCO3, Na2HPO4, NaH2PO4, and iron citrate chelate or iron sulfate chelate. At least one trace metal other than copper (e.g., two or more trace metals) may include at least one ion of barium, bromine, cobalt, iodine, manganese, chromium, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, and aluminum.

[0137]

[0136] At least one polyamine (for example, two or more polyamines) may include at least one of putrescine, cadaverine, spermine, and spermidine.

[0138]

[0137] At least one (essential) fatty acid (e.g., at least two fatty acids) may include, but are not limited to, n-3, n-6, n-9 fatty acids and cholesterol. At least one (essential) fatty acid (e.g., at least two fatty acids) may include, but are not limited to, linoleic acid (ALA), linolenic acid (LA), docosahexenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (AA), etc. At least one essential fatty acid (e.g., at least two fatty acids) may be selected from linoleic acid and linolenic acid.

[0139]

[0138] One embodiment provides a cell culture medium, cell culture feed, or cell culture additive comprising copper(II) and cysteine, which can be obtained or obtained by the method of the present invention.

[0140] Method for preventing copper loss and / or preparing a culture medium.

[0139] Cell culture media, feeds, or additives may lose copper during preparation. In particular, when cell culture media, feeds, or additives are filtered (e.g., sterile filtration), soluble copper species (e.g., copper(I) species) may be retained on the filter. This is problematic because filtration, especially sterile filtration, is a widely used method for providing sterile culture media, feeds, and additives, and these compositions typically contain components that are degraded by terminal sterilization.

[0141]

[0140] We found that cysteine, which is often present in cell culture media, plays a significant role in causing copper filtration removal. For example, when we tested modified media without cysteine, no copper loss was observed. Even when using media with cystine (in its oxidized dimer form) instead of cysteine, no copper removal during filtration was observed. We also observed that the transient change in copper loss over time was influenced by iron and pirubate. Copper loss was pH-dependent, occurring most severely around neutral pH, but was not limited to any particular buffer type. It should be noted that because cysteine ​​is typically present in significantly excess amounts compared to copper, relatively modest cysteine ​​loss from media / supplements / additives typically has little effect on the overall level of cysteine ​​in solution, while copper loss typically has a much greater effect.

[0142]

[0141] Cysteine ​​is oxidized to cystine, a dimer, in the presence of a metal ion catalyst and oxygen (Cavallini D, et al., The copper catalyzed oxidation of cysteine ​​to cystine. Archives of biochemistry and biophysics. 1969;130(1):354-361, the entire article is incorporated herein by reference). The oxidation of cysteine ​​occurs as follows: JPEG2023522037000004.jpg20170 However, the actual response: In JPEG2023522037000005.jpg10170, "RSH" is cysteine, and "RSSR" is cystine.

[0143]

[0142] In the first reaction, cysteine ​​reacts with oxygen to produce cystine and hydrogen peroxide, and in the second reaction, hydrogen peroxide reacts with a second equivalent of cysteine ​​to produce an additional equivalent of cystine and two equivalents of water. Both copper and iron catalyze the oxidation of cysteine, but copper has been reported to be a more active catalyst (Munday R, et al., Inhibition of copper-catalyzed cysteine ​​oxidation by nanomolar concentrations of iron salts. Free Radical Biology and Medicine. 2004;36(6):757-764; Ehrenberg L, et al., Kinetics of the copper-and iron-catalyzed oxidation of cysteine ​​by dioxygen. Acta Chemica Scandinavica. 1989;43, these are incorporated herein by reference in their entirety). The first step of the catalytic reaction is thought to be the reductive chelation of the metal ion by cysteine, yielding a cysteine-copper(I) or cysteine-iron(II) complex (Pecci L, et al., Novel findings on the copper catalysed oxidation of cysteine. Amino Acids. 1997;13(3-4):355-367, which is incorporated herein by reference in its entirety). Therefore, in the presence of a cysteine ​​monomer to be oxidized, the metal ion is reduced to the copper(I) state in the case of copper.

[0144]

[0143] Thus, oxidation of cysteine ​​leads to the formation of soluble copper(I) species. This occurrence in cell culture was confirmed by copper(I) assays. Furthermore, we found that in the presence of sufficient amounts of dO2, relatively insoluble copper(I) is readily oxidized back to copper(II), which represents soluble copper.

[0145]

[0144] Known chemistry is shown by the following reactions: JPEG2023522037000006.jpg21170 provides a net response: JPEG2023522037000007.jpg10170

[0146]

[0145] Without being bound by theory, it is thought that the same reaction as with free Cu(I) occurs with the Cu(I)-cysteine ​​complex, and the catalyst decomposes to release Cu(II). This mechanism can explain why dO2 needs to be low for copper removal to occur during filtration, and why oxygen is needed for the copper to become filterable again.

[0147]

[0146] Therefore, one way to avoid insoluble copper(I) is to ensure that there is sufficient dO2 in the solution to prevent the precipitation of copper(I) species. This can be achieved in many ways. For example, before and / or during filtration, the oxygen level may be kept at a predetermined level sufficiently high to ensure the quantitative oxidation of copper(I) to copper(II). The predetermined level may be at least about 6% dO2, at least about 8% dO2, at least about 10% dO2, or at least about 12% dO2. The predetermined level may be about 0.3 mg / L dO2, about 0.4 mg / L dO2, about 0.6 mg / L dO2, or about 0.8 mg / L dO2. Furthermore (or), filtration may be optimized so that filtration is completed while sufficient dO2 remains in the solution to prevent precipitation of copper(I) species. For example, filtration may be optimized by increasing the filtration rate and / or filter size and / or filter volume so that the dO2 level does not fall below a predetermined level during filtration.

[0148]

[0147] Filtration can be optimized by manipulating the operating variables of the filtration unit to satisfy the need for a constant filtration time for a given volume to be processed (e.g., manipulation of the filter area, flow rate, etc., based on the filtration method (e.g., dead-end membrane)), and by characterizing the system empirically or by an empirical and theoretical approach using techniques known to those skilled in the art. See, for example, Ozturk S and Hu W, Cell Culture Technology for Pharmaceutical and Cell-based Therapies, 2005; Rajniak P et al., Sterilizing filtration - Principles and practice for successful scale-up to manufacturing, 2008. These disclosures as a whole are incorporated herein by reference.

[0149]

[0148] For example, the optimal filter size can be determined using empirical filter sizing studies, which can be carried out based on various endpoints (Pmax (pressure) or Tmax (turbidity) for a constant flow rate, Vmax (volume) for a constant pressure) as a function of the relevant large-scale filtration unit operation. This makes it possible to define the filter area required to maintain the flux to process a batch amount within a given time. Of course, there are realistic limitations in production sites / lines based on space, cost, etc. Typical standardized (unitless) flow rates used for scaling up / scale down used in scaling on the industry scale range from 10 to 8000, and can cover processing volumes from 50 L to 25000 L. Also, these methods may be extrapolated to cover larger or smaller volumes as needed.

[0150] Filtration may be carried out using filters with pore sizes up to approximately 0.5 μm, such as up to approximately 0.2 μm (e.g., up to approximately 0.1 μm). Filtration may include sterile filtration, which may be carried out using nanofilters with pore sizes up to approximately 0.1 μm, such as up to 0.05 μm. Sterile filtration can provide logarithmic reduction values ​​(LRV) of related microorganisms (e.g., viruses) greater than approximately 4 LRV. Nanofiltration of cell culture media is an important approach to reduce microbial contamination in upstream processes such as the preparation of cell media, feeds, and additives. For example, see Shirota, M. and Kiss, R., "Risk Mitigation in Preventing Adventitious Agent Contamination of Mammalian Cell Cultures," Adv. Biochem. Eng. Biotechnol., 2017, https: / / doi.org / 10.1007 / 10_2017_38; and Miesegaes, G., et al., "Virus retentive filters." In: Flickinger M (ed) Encyclopedia of industrial biotechnology: bioprocess, bioseparation, and cell technology, Wiley, 2010, pp 1–11. These are incorporated herein by reference in their entirety. Filters include, but are not limited to, membrane filters (see, for example, https: / / www.mrwa.com / WaterWorksMnl / Chapter%2019%20Membrane%20Filtration.pdf, which is incorporated herein by reference in its entirety), hollow fiber filters, column filters, helical membrane filters, tube filters, capillary filters, capsule filters, cassette filters, disk filters, frit filters, and plug filters.

[0151]

[0150] Any method to ensure that there is sufficient dO2 in the solution to prevent copper(I) precipitation may include monitoring the oxygen level before the start of filtration, for example, monitoring the oxygen level for at least about 10 minutes (e.g., at least about 15 minutes) before the start of filtration. The method may include monitoring the level of dO2 during filtration. The method may include monitoring the oxygen level before the start of filtration and monitoring the level of dO2 during filtration. When the method includes monitoring the oxygen level, maintaining the oxygen level at least at a predetermined level may include adding oxygen to the solution in response to the monitored oxygen level approaching a predetermined level. When the method includes monitoring the oxygen level, maintaining the oxygen level at at least at a predetermined level may include increasing the oxygenation of the solution and / or optimizing the filtration in response to the monitored oxygen level approaching a predetermined level. The level of dO2 may be monitored using any suitable technique. The level of dO2 may be monitored using an electrochemical oxygen sensor or an optical oxygen sensor. The level of dO2 may be monitored using an electrochemical oxygen sensor. The level of dO2 can be monitored using an optical oxygen sensor. Suitable oxygen sensors and methods for detecting dissolved oxygen are known to those skilled in the art. See, for example, A Guide to Oxygen Measurement: Theory and Practice of Oxygen Applications, Mettler-Toledo GmbH, 2016; Wei, Y., et al., "Review of dissolved oxygen detection technology: From laboratory analysis to online intelligent detection", Sensors, 2019, 19, 3995. These are incorporated herein by reference in their entirety.

[0152]

[0151] In addition to the oxidation of cysteine ​​to cystine, cysteine ​​in the culture medium is also consumed by the condensation of cysteine ​​and pirubate, and can produce 2,4-thiazolidinedione via a Schiff base intermediate (Sullivan MX and Hess WC, The Effect of Pyruvic Acid on the Estimation of Cystine and Cysteine, The Journal of Biological Chemistry, 1937, 122; Nishiuch Y, et al., Cytotoxicity of cysteine ​​in culture media, In Vitro, 1976, 12(9); Rohac R, et al., Carbon-sulfur bond-forming reaction catalysed by the radical SAM enzyme HydE, Nature Chemistry, 2016, 8(5), 491-500; these are incorporated herein by reference in their entirety). This reaction has been reported to occur in cell culture media containing cysteine ​​and pirubate, with the 2,4-thiazolidine product confirmed by mass spectrometry (Kuschelewski J, et al., Antioxidant effect of thiazolidine molecules in cell culture media improves stability and performance, Biotechnology progress, 2017; this is incorporated herein by reference in its entirety). This provides another approach that can be used to prevent copper loss. If cysteine ​​reacts in a competitive reaction, for example by condensation with pirubate, cysteine ​​is not available to reduce copper(II) to the insoluble form of copper(I). However, if cysteine ​​is present in the presence of both copper(II) and pirubate, both reactions may occur, and therefore some of the soluble copper(II) may still be lost from the solution.

[0153]

[0152] When a cell medium, feed, or additive is intended for use with cells that can efficiently metabolize cysteine ​​derivatives, it may be possible to avoid the loss of soluble copper by substantially providing cysteine ​​as a sulfhydryl group-free cysteine ​​derivative before adding copper(II) to the solution. For example, cysteine ​​may contain less than about 1 mM of free cysteine. For example, cysteine ​​may contain less than 0.5 mM of free cysteine ​​(or less than 0.1 mM of free cysteine). Sulfhydryl group-free cysteine ​​derivatives do not react with copper(II) to form insoluble copper(I). However, this approach is not better than other methods disclosed herein for preventing the loss of soluble copper when the medium, feed, or additive is intended for use with cells that do not efficiently metabolize such cysteine ​​derivatives.

[0154]

[0153] Cysteine ​​derivatives may contain a disulfide, a thiazolidinedione moiety, a protective moiety, or a protective sulfur group. Cysteine ​​derivatives may contain a disulfide and / or a thiazolidinedione moiety. Cysteine ​​derivatives may contain a disulfide. For example, cysteine ​​derivatives may contain cysteine, S-sulfocysteine, S-sulfocysteinylglycine, L-cysteine ​​mixed disulfide, and / or S-sulfoglutathione. Cysteine ​​derivatives may contain cystine. Cysteine ​​derivatives may contain a thiazolidinedione moiety. For example, cysteine ​​derivatives may include 4-carboxy-2-methylthiazolidinedion-2-carboxylate, 2-methyl-1,3-thiazolidinedion-2,4-dicarboxylic acid, 2-(2-carboxyethyl)21,3-thiazolidinedion-2,4-dicarboxylic acid, L-2-oxothiazolidinedion-4-carboxylic acid, 2-alkyl-thiazolidinedion-4(R)-carboxylic acid, 2-aryl-thiazolidinedion-4(R)-carboxylic acid, and carbohydrate-based thiazolidinediones (e.g., D-ribose-L-cysteine). Cysteine ​​derivatives may include 4-carboxy-2-methylthiazolidinedion-2-carboxylate. Cysteine ​​derivatives include cystine, 4-carboxy-2-methylthiazolidinedion-2-carboxylate, S-sulfocysteine, S-sulfocysteinylglycine, and cysteine. It may contain at least one of the following: S-linked N-acetylglucosamine (GlcNAC-cys), homocystine, L-cysteine ​​mixed disulfide, L-cysteine ​​mixed peptide, S-alkylated cysteine, cysteine ​​having a thiol protecting group (e.g., FMOC-protected cysteine), 2-methyl-1,3-thiazolidine-2,4-dicarboxylic acid, 2-(2-carboxyethyl)21,3-thiazolidine-2,4-dicarboxylic acid, reduced and oxidized glutathione (GSH), S-sulfoglutathione, S-acyl-GSH, S-carboxy-L-cysteine, L-2-oxothiazolidine-4-carboxylic acid, 2-alkyl-thiazolidine-4(R)-carboxylic acid, 2-aryl-thiazolidine-4(R)-carboxylic acid, or carbohydrate-based thiazolidine (e.g., D-ribose-L-cysteine).

[0155]

[0154] Copper may be provided in the form of a copper(II) chelate. The chelate may contain at least one chelating agent selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), neocuproine, vasocuproine, D-penicillamine, triethylenetetramine (TETA), dimercaprol (BAL), 8-hydroxyquinoline, cryoquinol, and 5,7-dichloro-2-[(dimethylamino)methyl]-8-quinolinol (PBT2). Without wishing to be bound by any theory, it is thought that providing copper in the form of a copper(II) chelate prevents significant reduction from copper to copper(I), thereby avoiding the loss of soluble copper from the solution.

[0156]

[0155] A further approach to prevent the loss of copper in cell culture media, feed, or additives is to filter the copper(II) solution and the cysteine ​​solution separately and then combine the two filtered solutions to form a final solution. This can be done by providing the reagent and the majority of the volume to the cysteine ​​solution, filtering that solution, and then adding the filtered copper solution as a bolus. This may be advantageous because copper is typically a trace element provided at relatively low concentrations in the final solution. Another approach would be to provide the copper and the majority of the components to a single filtered solution and then add the filtered cysteine ​​solution to form a final solution. As those skilled in the art will understand, provided that the copper(II) solution and the cysteine ​​solution are filtered separately, various sequences and combinations of filtering separately and then combining the filtered solutions with the finally filtered culture media, feed, or additives can be used in such methodologies. Furthermore, if a sterile solution is ultimately desired, it is desirable that the filtered solutions be handled aseptically.

[0157]

[0156] This prevents the reaction between copper(II) and cysteine ​​before or during filtration, which would form an insoluble copper(I) species. Although copper(I) species may form after filtration, this will remain in the culture medium, feed, or additive. Furthermore, in cell culture, oxygen is added to the solution during culture, so the insoluble copper(I) will be oxidized to soluble copper(II) during culture. Therefore, all copper in the culture medium, feed, or additive becomes available to the cultured cells.

[0158]

[0157] An aspect of the present disclosure provides a method for preventing copper loss in a solution. The solution is a cell medium, cell culture feed, or cell culture additive containing copper(II) and cysteine. The method includes having substantially the same amount of soluble copper before and after filtration of the solution.

[0159]

[0158] Another aspect of the present disclosure provides a method for preparing a cell medium, cell culture feed, or cell culture additive containing copper(II), cysteine, and other components without loss of soluble copper. The method comprises providing an aqueous solution containing cysteine ​​and other components; providing an aqueous solution containing copper(II); filtering the aqueous solution containing cysteine ​​and other components separately from the aqueous solution containing copper(II); and combining the filtered solutions to provide a cell medium, cell culture feed, or cell culture additive.

[0160]

[0159] Further aspects of the present disclosure provide a method for preparing a cell medium, cell culture feed, or cell culture additive containing copper(II), cysteine, and other components without loss of soluble copper. The method comprises filtering a solution containing copper(II), cysteine, and other components to provide a cell medium, cell culture feed, or cell culture additive.

[0161] Methods for culturing and / or producing biological products

[0160] Methods for culturing cells and methods for producing biological products from cultured cells are also provided.

[0162]

[0161] Protocols for the generation, recovery, and purification of recombinant antibodies in mammalian cells such as CHO may include the following steps: Cells may be cultured in a stirred-tank bioreactor system, and a procedure called fed-batch culture is used. In a preferred fed-batch culture, mammalian host cells and culture medium are first supplied to the culture vessel, and additional culture nutrients are supplied to the culture continuously or individually during the culture, and before the end of the culture, cells and / or products are periodically recovered or not recovered. Fed-batch culture may include, for example, semi-continuous fed-batch culture in which the entire culture (including cells and culture medium) is periodically removed and replaced with fresh medium. Fed-batch culture is distinguished from simple batch culture in which all components for cell culture (including cells and all culture nutrients) are supplied to the culture vessel at the start of the culture process. Fed-batch culture can be further distinguished from perfusion culture insofar as the supernatant is not removed from the culture vessel during the process (in perfusion culture, cells are retained in the culture by means of filtration, mounting, anchoring to a microcarrier, etc., and the culture medium is introduced continuously or intermittently and removed from the culture vessel).

[0163]

[0162] Furthermore, the cultured cells may be grown according to any scheme or routine that is suitable for a particular host cell and a particular development plan to be intended. Thus, one-step or multi-step culture procedures may be used. In single-step culture, the host cells are seeded in a culture environment, and the process of this disclosure is used during a single development stage of cell culture. Alternatively, multi-step culture may be used. In multi-step culture, cells may be cultured in a number of steps or stages. For example, cells may be grown in a culture in the first step or growth phase. At this stage, cells, which may have been removed from storage, are seeded in a medium suitable for promoting growth and high viability. By adding fresh medium to the host cell culture, the cells can be maintained in the growth phase for a suitable period.

[0164]

[0163] During the proliferation phase of cell culture, fed-batch or continuous cell culture conditions may be devised to promote the growth of mammalian cells. During the proliferation phase, cells grow under conditions and for a period of time that are maximized for proliferation. Culture conditions, such as temperature, pH, and dissolved oxygen (dO2), are used for specific hosts and will be obvious to those skilled in the art. Generally, pH is adjusted to a level between about 6.5 and 7.5 using either an acid (e.g., CO2) or a base (e.g., Na2CO3 or NaOH). A suitable temperature range for culturing mammalian cells such as CHO cells is about 30-38°C, and a suitable dO2 is between 5% and 90% of the air saturation.

[0165] At certain stages, cells may be used to inoculate the generation stage or process of cell culture. Alternatively, as described above, the generation stage or process may be continuous with the seeding or growth stage or process.

[0166]

[0165] The cell culture environment in the production stage of cell culture is usually controlled. Therefore, when glycoproteins are produced, factors affecting the cell-specific productivity of mammalian host cells can be manipulated so that a desired sialic acid content is achieved in the resulting glycoprotein. In a preferred embodiment, the production stage of the cell culture process is preceded by a transition stage of cell culture in which parameters for the production stage of cell culture are engaged. Further details of this process are described in U.S. Patent No. 5,721,121 and Chaderjian et al., Biotechnol. Prog. 21(2):550-3 (2005), the entirety of which these disclosures are expressly incorporated herein by reference.

[0167]

[0166] After culturing for a sufficient period, the expressed biological products, such as proteins, can be purified. The procedure for purifying proteins or other products derived from cell debris initially depends on the site of protein expression. Some proteins or other products can be secreted directly from cells into the surrounding growth medium, while others are produced intracellularly. If produced intracellularly, the first step of the purification method involves cell lysis, which can be carried out by various methods including mechanical shearing, osmotic shock, or enzymatic treatment. Such disruption releases all of the cell's contents into the homogenate, as well as intracellular fragments that are difficult to remove due to their small size. These are generally removed by fractional centrifugation or filtration. Directly secreted proteins also face the same problems, albeit small, during the process of producing biological products (e.g., proteins), due to natural cell death and the release of intracellular host cell proteins and components.

[0168]

[0167] After a clarified solution containing the target biological product (e.g., protein) is obtained, it is usually attempted to separate it from other components produced by the cells using a combination of different chromatographic techniques. These techniques separate the mixture of products based on the charge, degree of hydrophobicity, or size of the products. Multiple different chromatographic resins are available for each of these techniques, allowing for precise adjustment of the purification scheme for the specific products involved. The basis of each of these separation methods is that proteins or similar species can be moved along a long column at different rates, so that they either physically separate as they move along the column or selectively adhere to the separation medium and are then eluted separately by different solvents. In some cases, the desired protein is separated from impurities when impurities adhere specifically to the column and the target protein does not, i.e., when the target protein is in a "flow-through" state. Thus, the purification of recombinant proteins from host cell cultures may involve one or more affinity (e.g., protein A) and / or ion-exchange chromatography steps.

[0169]

[0168] In addition to mammalian host cells, other eukaryotes can be used as host cells for the expression of biological products such as recombinant proteins. For expression in common baker's yeast or yeast host cells such as Saccharomyces cerevisiae, suitable vectors include 2-micron plasmid-based episomal replication vectors, accumulation vectors, and yeast artificial chromosome (YAC) vectors. Other yeasts suitable for the recombinant production of heterologous proteins include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 (1981); EP 139,383).Published May 2, 1985); Kluiveromyces host (US Patent No. 4,943,529; Fleer et al., Bio / Technology, 2: 968 975 (1991)), e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 737 (1983)), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al., Bio / Technology, 8: 135 (1990), K. thermotolerance, and K. marxianus; Yarowia (EP 402, 226); Pichia pastris (EP 183, 070; Sreekrishna et al., J. Basic Microbiol., 28: 265 278 (1988)); Candida; Trichoderma leucoma (EP 244, 234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76: 5259 5263 (1979)); Schwanniomyces, e.g., Schwanniomyces occidentalis (EP 394, 538 published 31 Oct.) 1990); and filamentous fungi, such as Neurospora, Penicillium, and Tripocladium (International Publication No. 91 / 00357).This includes Aspergillus hosts such as A. needlance (Ballance et al., Biochem. Biophys. Res. Commun., 112: 284 289 (1983); Tilburn et al., Gene, 26: 205 221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470 1474 (1984)), and A. niger (Kelly and Hynes, EMBO J., 4: 475 479 (1985)). These publications are incorporated herein by reference in their entirety. Methylotrophic yeasts are appropriate here and include, but are not limited to, yeasts that can grow on methanol, selected from genera consisting of Hansenula, Candida, Chloechera, Pichia, Saccharomyces, Torlopsis, and Rhodotorula. A list of specific species of exemplary yeasts of this class is given in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982), which is incorporated herein by reference in its entirety. The expression systems of the listed and other yeasts are well known in the art and commercially available.

[0170]

[0169] For expression in insect host cells, such as Sf9 cells, suitable vectors include baculovirus vectors. For expression in plant host cells, particularly in dicotyledonous plant hosts such as tobacco, suitable expression vectors include vectors derived from the Agrobacterium tumefaciens Ti plasmid.

[0171]

[0170] Appropriate methods also extend to the culture of prokaryotic or archaeal host cells. Prokaryotic and archaeal host cells suitable for expressing antibodies and other proteins to be protected by the means of the present disclosure include archaea and bacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia coli (e.g., E. coli), Bacillus (e.g., Bacillus subtilis), Enterobacter, Pseudomonas species (e.g., Pseudomonas aeruginosa), Salmonella typhimurium, Serratia marcescens, Klebsiella, Proteus, Sigella, Rhizobia, Vitreosilla, or Paracoccus. In one embodiment, Gram-negative cells are used. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and its derivatives (including strain 33D3 having genotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41 kanR (U.S. Patent No. 5,639,635)). Other strains and their derivatives include, for example, E. coli 294 (ATCC 31,446), E. coli B, E. coli λ1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative and not limiting. Methods for constructing derivatives of any of the above bacteria having defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). This document is incorporated herein by reference in its entirety. In general, appropriate bacterial selection is necessary, taking into account the replication potential of the replicon in bacterial cells. For example, when providing a replicon using known plasmids such as pBR322, pBR325, pACYC177, or pKN410, Escherichia coli, Serratia, or Salmonella species can be appropriately used as hosts.Typically, the host cells need to secrete minimal amounts of proteolytic enzymes, and it may be desirable to incorporate additional protease inhibitors into the cell culture.

[0172]

[0171] Methods for generating, recovering, and producing biological products such as recombinant proteins from non-mammalian host cell cultures are also well known in the art. When polypeptides are produced in non-mammalian cells, such as microorganisms like fungi or Escherichia coli, the polypeptides are recovered intracellularly or in the pericellular lumen (Kipriyanov and Little, Molecular Biotechnology, 12: 173 201 (1999); Skerra and Pluckthun, Science, 240: 1038 1040 (1988); these are incorporated herein by reference in their entirety). Therefore, it is necessary to release the proteins from the cells into the extracellular medium by extraction such as cell lysis. Such disruption releases all of the cell's contents into the homogenate, and furthermore, intracellular fragments that are difficult to remove due to their small size are generated. These are generally removed by fractionation centrifugation or filtration.

[0173]

[0172] Cell lysis is typically achieved using mechanical disruption techniques such as homogenization or head milling. While the target protein is generally released effectively, such techniques have several drawbacks (Engler, Protein Purification Process Engineering, Harrison eds., 37 55 (1994), the entirety of which is incorporated herein by reference). Temperature increases often occur during processing, which can lead to protein inactivation. Furthermore, the resulting suspension contains a wide range of contaminating proteins, nucleic acids, and polysaccharides. Nucleic acids and polysaccharides increase the viscosity of the solution, which can complicate subsequent processing by centrifugation, cross-flow filtration, or chromatography. Complex associations of these contaminants with the target protein can complicate the purification process and result in unacceptably low yields. Improved methods for purifying heterologous polypeptides from microbial fermentation broth or homogenates are described, for example, in U.S. Patent No. 7,169,908, the entire disclosure of which is expressly incorporated herein by reference.

[0174]

[0173] In one embodiment, the present disclosure provides a method for culturing cells in a culture medium. The method comprises: preventing copper loss in the culture medium by the method of the present invention, or preparing the culture medium by the method of the present invention; bringing cells into contact with the culture medium; and culturing the cells in the culture medium.

[0175]

[0174] In another aspect, the present disclosure provides a method for producing a biological product. The method comprises: preventing copper loss in a culture medium by the method of the present invention or preparing a culture medium by the method of the present invention; contacting cells configured to express the biological product with the culture medium; and culturing the cells in the culture medium under conditions such that the cells express the biological product.

[0176]

[0175] In a further embodiment, the use of the cell culture medium, cell culture feed, or cell culture additive of the present invention for culturing cells is provided.

[0177]

[0176] It is emphasized that the methods for culturing, fermenting, preparing, recovering, and purifying biological products described herein are for illustrative purposes only. The disclosed methods can be combined with any manufacturing processes developed for the production, recovery, and purification of recombinant proteins or other biological products.

[0178] Assay

[0177] Assay for the Quantification of Cysteine ​​(Monomer)

[0178] The concentration of cysteine ​​can be determined using the Elmann reagent assay for free thiols, a method commonly used to measure cysteine ​​in the context of cysteine ​​oxidation (Smith RC, et al., Oxidation Of Thiols By Copper(II). Phosphorus, Sulfur, and Silicon and the Related Elements. 1994;90(1-4):147-154; and Munday R, et al., Inhibition of copper-catalyzed cysteine ​​oxidation by nanomolar concentrations of iron salts. Free Radical Biology and Medicine. 2004;36(6):757-764. These are incorporated herein by reference in their entirety). This assay was performed by adding Elmann reagent (DTNB, Sigma Aldrich) to the culture medium and measuring the absorbance at 412 nm. Beer's law and 13,600 M -1 cm -1Using the extinction coefficient (according to Ellman GL, Tissue sulfhydryl groups, Archives of Biochemistry and Biophysics, 1959;82(1):70-77, the entire text is incorporated herein by reference), the readings were converted to the concentration of free thiols. If the medium does not contain other free thiols at significant concentrations, this provides the concentration of cysteine ​​monomer. If other free thiols are present, the obtained value needs to be corrected from non-cysteine ​​monomer thiols to provide the concentration of cysteine ​​monomer.

[0179]

[0179] Copper(I) basocupproine assay

[0180] This assay can be used to confirm the presence of copper(I). We used bathophenanthroline disulfonic acid, a water-soluble version of the chelating agent described in Blair D and Diehl H, Bathophenanthroline disulfonic acid and bathocuproine disulfonic acid, water-soluble reagents for iron and copper, Talanta. 1961;7(3-4) (Sigma Aldrich). Bathocuproine is a chelating agent that selectively binds to Cu(I) (see Smith G and Wilkins DH, New Colorimetric Reagent Specific for Copper. Analytical Chemistry, 1953;25(3)), preventing the oxidation of Cu(I) to Cu(II) by oxygen. The bathocuproine-Cu(I) complex has an extinction coefficient of 13,300 M. -1 cm -1 It can be quantified by its absorbance at 483 nm, thereby determining the level of Cu(I).

[0180]

[0181] Determination of pirate, acetate, and trace metals

[0182] Pirvate can be measured using a pirubate assay kit, such as the pirubate assay kit available for the Cedex Bio HT instrument. This assay is enzymatic, using lactate dehydrogenase and measuring the absorbance of NADH at 340 nm.

[0181]

[0183] Acetates can be measured using ion exchange chromatography. For example, acetates can be measured using a Dionex 3000 series HPLC with an RFIC IonPac AS11-HC column (Dionex) and conductivity detection. Acetates are eluted with a KOH gradient, and the sample results can be compared to a standard curve.

[0182]

[0184] Trace metals can be quantified by those skilled in the art using conventional methods of atomic detection, such as inductively coupled plasma-atomography (ICP-OES) or inductively coupled plasma-mass spectrometry (ICP-MS). In an exemplary ICP-OES method, the sample can be diluted with nitric acid and then drawn into an argon plasma where trace metals are ionized. Trace metals in the sample can be determined by spectrometer readings at specific wavelengths, and intensity readings were compared to a standard curve for quantification.

[0183]

[0185] Assay for copper removal by filtration

[0186] Filtration of solutions such as cell cultures, cell media, cell culture feeds, or cell culture additives can lead to the loss of insoluble copper on the filter. The amount of removed copper can be quantified after recovering the copper from the filter, for example, using the "trace metal determination" assay described above. Copper can be recovered from the filter by any suitable method.

[0184]

[0187] If the filter is made from a material that readily dissolves in a particular solvent system, the filter and the copper present on the filter can be dissolved in the appropriate solvent. For example, a polyethersulfone (PES) steriflip filter can be completely dissolved in DMSO. The DMSO solution was then diluted with 5% nitric acid. The copper content of the filtered medium, the unfiltered medium, and the dissolved filter solution was quantified by ICP-OES, thereby determining the amount of copper removed from the solution by filtration.

[0185]

[0188] Another approach to recovering the copper removed from the solution is to reverse the flow and flush the removed copper out of the filter. For example, if the filter area is 3.9 cm². 2 With the Millipore Millex PES 0.22 μm filter, 1 L of culture medium can be filtered, and then the filter can be flushed with a 50 mL backflow of 5% nitric acid. The copper content of the filtered medium, unfiltered medium, and 5% nitric acid washing solution was quantified by ICP-OES. This method can be readily applied to any filter that is compatible with backflow.

[0186]

[0189] An example of a specific assay that may be used is as follows: 1.5 L of medium was prepared in a 2 L glass beaker and covered with Parafilm to minimize oxygen transfer from the environment. The beaker was placed on a Corning PC-620D stirring plate with a calibrated RPM setting. The mixing was set to 60 RPM to maintain a homogeneous solution without generating vortices that increase gas transfer. A Mettler Toledo InPro 6860i optical dissolved oxygen probe and a Mettler Toledo InPro 3253i pH / ORP (oxidation-reduction potential) sensor were incorporated. The oxygen environment was manipulated by spurging either oxygen or nitrogen through a small diameter dip tube. Copper removal was measured by pipetting 40 ml of medium into a 50 ml conical tube, filtering half the volume through a steriflip, and quantifying copper removal by ICP-OES. [Examples]

[0187] Materials and methods

[0190] A dedicated CHO cell medium was used as the chemically defined medium 1 (CDM1). The medium is chemically defined, protein-free, and contains various amino acids, vitamins, glucose, bicarbonate buffer, salts, and trace elements. Two trace elements in the medium that are important for this study are iron and copper. Both are present at low μM concentrations, with iron being far more abundant than copper. Two components relevant to this study, cysteine ​​and pirubate, are in the low millimolar range (1–10 mM), with pirubate consistently exceeding cysteine. The medium was titrated to pH 7, and the osmotic pressure was approximately 300 mOsm.

[0188]

[0191] A fully chemically defined culture medium (CDM) recipe contains many components. Therefore, a simplified culture medium recipe was developed for studying the chemistry of cysteine ​​and copper. This medium maintains pH, buffering components, ionic strength, osmotic pressure, trace metal ions of copper and iron, and components known to interact with cysteine ​​and cysteine ​​(such as pirubate).

[0189]

[0192] The simplified medium used herein was a minimal viable product version of CDM1, possessing all the essential component chemistry, pH buffering, and redox active components present in CDM1. By performing experiments with both CDM1 and the simplified medium, it was possible to determine the effect with or without the full complexity of the CDM medium components.

[0190]

[0193] A high-throughput system was developed to test numerous conditions and time points. This method used 50 ml conical tubes and a Millipore (Waltham, MA) 0.22 μM PES Steriflip filter unit (unless otherwise specified). Culture media (CDM1 and simplified medium) were prepared, and 40 ml was divided equally into conical tubes. These were then stored under ambient conditions without agitation. At various time points after preparation, half the volume of one aliquot was filtered using Steriflip, leaving 20 ml unfiltered. The copper content of both filtered and unfiltered media was quantified by ICP-OES assay to determine the copper removed during filtration.

[0191] Example 1: Changes over time in copper removal by filtration

[0194] The removal of copper species by filtration after preparation of CDM1 medium is transient. The concentrations of cystine and copper in CDM1 were within the range of basal media defined in Table 1 of this specification. To characterize the time dependence, the medium was prepared and a series of samples were filtered every 45 minutes until no further copper removal was observed. The time-course profile of copper removal is shown in Figure 1. Initially, copper is completely dissolved and not removed by filtration. However, after approximately 1 hour, copper begins to be removed by filtration. After 12 hours, copper becomes completely filterable again. Between 1 hour and 12 hours, copper removal shows a U-shaped curve, with a maximum loss of approximately 80% occurring between 4 hours and 6 hours. This is a phenomenon specific to copper. No loss of other measurable trace metals in the medium was observed.

[0192]

[0195] To confirm that the unfilterable copper was in the form of Cu(I), the "basocupproine analysis of copper(I)" described above was used. Cysteine-containing medium 4 hours after copper addition was tested positive for Cu(I), but the control case of medium prepared with cystine was negative (no copper loss was observed). Stock solution of Cu(II)SO4 was also negative for Cu(I).

[0193] Example 2: Culture medium component knockout study

[0196] We conducted knockout studies of media components. These confirmed that cysteine ​​is the major component causing copper removal by filtration. When the modified medium did not contain cysteine, no copper loss was observed. No copper removal during filtration was observed in media using cystine (in its oxidized dimer form) instead of cysteine. We also observed that the transient change in copper loss over time was influenced by iron and pirubate. Copper loss was pH-dependent, occurring most severely around neutral pH, but was not limited to any particular buffer type.

[0194] Example 3: Inspection of the cysteine ​​oxidation process

[0197] The oxidation process of cysteine ​​was monitored by measuring the cysteine ​​monomer concentration, dO2, and redox potential over time after the addition of trace metals. Figure 2 shows the time course of cysteine ​​monomer, dO2, and redox potential, with an overlay of cysteine ​​monomer, dO2, and copper loss profiles. Based on these profiles, copper loss appears to occur only while cysteine ​​is being oxidized. These profiles are consistent across experiments.

[0195]

[0198] The addition of metal ions to the culture medium triggers a reaction that oxidizes cysteine ​​and consumes oxygen, causing an immediate decrease in dO2 and cysteine ​​monomers. When dO2 is limited, copper can no longer be filtered, and the rate of decrease in cysteine ​​monomers slows considerably. Until cysteine ​​monomers are limited, dO2 remains low and copper cannot be filtered. Once cysteine ​​monomers are depleted, dO2 begins to rise (as a result of ambient diffusion), and copper returns to a filterable form.

[0196]

[0199] The reaction of cysteine ​​monomers can also be tracked by measuring the redox potential. Cysteine ​​is a relatively potent reducing agent that produces a negative redox potential (Jocelyn PC, The Standard Redox Potential of Cysteine-Cystine from the Thiol-Disulphide Exchange Reaction with Glutathione and Lipoic Acid, European Journal of Biochemistry, 1967;2(3). The entire work is incorporated herein by reference). Figure 2c shows the redox potential profile. As oxygen is consumed first, the redox potential decreases, reaching a minimum when DO becomes zero. Subsequently, as cysteine ​​monomers are consumed, the redox potential decreases less. As the concentration of cysteine ​​monomers approaches zero, the redox potential stabilizes slightly above zero, indicating a mild oxidizing environment. This is thought to be a result of the presence of cystine in solution.

[0197]

[0200] The oxidation reaction of cysteine ​​to cystine has been reported to have a potential of -220mV at pH 7 (Jocelyn PC, ibid), which correlates well with the minimum observed potential of -200mV. While cell culture media typically contain other redox-active components (e.g., tyrosine), cysteine ​​appears to be the most influential component during the measurement period.

[0198]

[0201] In the example formulation, copper is added to the culture medium as Cu(II)SO4, the highly soluble form of copper(II). When catalyzing cysteine ​​oxidation, copper is converted to and retained as Cu(I), a significantly less soluble form. Without wishing to be bound by any theory, it is hypothesized that the copper(I) intermediate of cysteine ​​oxidation, which is likely a complex form with cysteine / cystine, has low solubility and forms a precipitate that is removed by filtration. This is consistent with reports that many copper thiolates have very low solubility and often form precipitates.

[0199]

[0202] The Pourbet (redox potential / pH) diagram describing the aqueous systems of copper, iron, and sulfur in Garrels RM, Christ CL, Eh-pH Diagrams. Harper and Row, 1965, at page 232 (the entire diagram is incorporated herein by reference) shows that at least six different forms exist for these three molecules under conditions observed in exemplary culture media (pH 7 and redox potentials ranging from 0 to -200 mV). While it cannot be simply assumed that replacing cysteine ​​with sulfur will produce the same species, the diagram illustrates the complexity of the interactions and the species transitions associated with changes in redox potential. From this, it is reasonable to assume that a similar number of copper-iron-thiol species can be produced during cysteine ​​oxidation. Without wishing to be bound by any theory, it is suggested that one or more of these forms are insoluble and cause copper-containing precipitates that can be removed by filtration. While no measurable loss of cysteine ​​or iron is observed, copper is typically present in cell media and associated compositions at significantly lower concentrations than cysteine ​​and iron. If either cysteine ​​or iron were removed at the same molar level as copper, the loss would usually not be significant enough to substantially affect the overall levels of cysteine ​​and iron in the culture medium, feed, or additive composition.

[0200] Example 4: Effect of increased cysteine ​​concentration

[0203] We investigated the time course of copper removal in media with two different initial cysteine ​​concentrations, 3 mM and 6 mM. Figure 3a shows the copper removal profiles at these different cysteine ​​concentrations using a high-throughput system (unmixed), and Figure 3b shows the profiles in a small-scale system (mixed). As the initial cysteine ​​monomer increased, the time it took for copper to be removed by filtration was extended.

[0201]

[0204] The time to low dO2 conditions and cysteine ​​monomer depletion was prolonged with copper removal. This supports the idea that the initial increase in cysteine ​​prolonged the copper removal period because cysteine ​​oxidation occurred for a longer time.

[0202]

[0205] The ability to control the timeframe of insoluble copper species by adjusting the concentration of cysteine ​​monomers further supports cysteine ​​oxidation as the cause of copper removal.

[0203] Example 5: Effects of low dO2

[0206] Examination of Figure 2d shows that oxidation of cysteine ​​begins immediately upon addition of metal ions to the culture medium, but copper becomes unfilterable until dO2 reaches a limit. Once the cysteine ​​monomer reaches its limit, dO2 begins to rise, and copper becomes filterable again. It appears that low dO2 is required for copper removal to occur, and oxygen is required for copper to return to a filterable form. This was supported by two experiments in which dO2 was kept at zero by sparging nitrogen into the system.

[0204]

[0207] In the first experiment, nitrogen sparging was initiated approximately 4 hours after cysteine ​​monomer depletion, and oxygen was slowly reintroduced to the system after 8 hours. Figure 3c shows that copper removal was extended while a zero dO2 environment was maintained by nitrogen sparging. As dO2 levels began to rise, copper was oxidized to the more soluble copper(II) oxidation state and became filterable again.

[0205]

[0208] In the second experiment, sparging was started shortly before the cysteine ​​monomer was depleted (depletion was later confirmed) and maintained for 11 hours. At this point, nitrogen sparging was stopped, and vigorous stirring was performed to rapidly increase dO2. Figure 3d shows that in this experiment, after sparging was started, the amount of copper removed increased to almost 100% and remained at that level until oxygen was reintroduced. The rapid increase in dO2 also rapidly restored the filterability of copper.

[0206]

[0209] These two experiments demonstrate that copper removal can be extended indefinitely by maintaining zero dO2, and that oxygen is necessary for copper to return to a filterable form. While this investigation cannot distinguish small changes at near-zero dO2, the second experiment also shows that copper can be quantitatively removed during filtration when a near-zero oxygen environment is reached using nitrogen sparging. This suggests that the reason only partial copper removal is seen in non-nitrogen sparging experiments is that cysteine ​​oxidation alone is not sufficient to reach zero dO2. Trace levels of oxygen may remain and convert some of the insoluble copper(I) into a filterable form of copper(II).

[0207] Example 6: Reaction of cysteine ​​and pirubate

[0210] In typical cell media, free cysteine ​​can be involved in two main reaction pathways. The first is oxidation to cystine. The second is the condensation of cysteine ​​and pirubate to 2,4-thiazolidine, as shown in Figure 4. These two reactions compete for cysteine ​​monomer, but a simplified medium formulation was used to control which reaction was occurring by removing metal ions to prevent the oxidation reaction, removing pirubate to prevent the condensation reaction, or removing both metal ions and pirubate to prevent both reactions. Next, the decrease in cysteine ​​monomer concentration was observed to understand the relative reaction rates. Figure 5 shows the cysteine ​​monomer profiles for the simplified medium with + / - metal ions and + / - pirubate.

[0208]

[0211] As shown in Figure 4, the condensation reaction of cysteine ​​and pirubate produces water molecules and hydrogen ions along with thiazolidinedione. When bicarbonate is used as a buffer species, it is difficult to measure the stoichiometric change in pH due to CO2 gas release. However, when bicarbonate was replaced with MOPS buffer in a simplified medium, a nearly stoichiometric decrease in pH due to the amount of cysteine ​​converted to thiazolidinedione could be measured. This indicates that thiazolidinedione formation is responsible for the pH decrease proportional to the cysteine ​​concentration, which is observed during medium retention of CDM containing cysteine ​​and pirubate.

[0209]

[0212] Furthermore, the pirubate concentration was measured before the addition of cysteine ​​and after the depletion of cysteine ​​monomers. In the absence of metal ions, the pirubate concentration decreased by 3.0 mM, which stoichiometrically correlated with a 3.0 mM decrease in cysteine, suggesting complete conversion to thiazolidinedione. In addition, in the absence of metal ions, dO2 remained unchanged, providing further evidence that cysteine ​​was not oxidized.

[0210]

[0213] When metal ions were present, the data for Pirvate supported competition for cysteine ​​monomers between the two reactions. The decrease in Pirvate concentration was small (1.7 mM), suggesting partial conversion to thiazolidinedione, and it is calculated that the remaining cysteine ​​(1.3 mM) was oxidized to cystine (0.65 mM). Furthermore, a decrease in dO2 characteristic of cysteine ​​oxidation was observed.

[0211]

[0214] Although pirubate is not directly involved in the copper mechanism that forms insoluble species, the presence of pirubate in the cell medium significantly influences the time course of copper removal. Insoluble copper species are maintained until all available cysteine ​​monomers for oxidation are depleted, and after oxygen begins to limit the cysteine ​​oxidation reaction, condensation with pirubate causes the consumption of much of the remaining monomers. In the absence of pirubate, oxidation becomes the sole pathway to cysteine ​​consumption. Because it is limited by oxygen availability, the oxidation reaction takes much longer to consume all the cysteine, resulting in a longer copper removal process.

[0212]

[0215] We found that when pirubate and metal ions are present in a simplified culture medium, a small amount of acetate (0.12 mM in the simplified medium) is produced. Acetate was not detected unless both pirubate ions and metal ions were present. As shown in Equation 1, the first step in the oxidation of cysteine ​​produces hydrogen peroxide, which can then oxidize additional cysteine ​​as shown in Equation 2. However, hydrogen peroxide is highly reactive and can interact with various culture medium components. One example is the oxidation from pirubate to acetate that we observed. Equation 7 shows how this reaction is typically represented (Nath KA, et al., alpha-Ketoacids scavenge H2O2 in vitro and in vivo and reduce menadione-induced DNA injury and cytotoxicity, American Journal of Physiology-Cell Physiology, 1995, 268(1); Giandomenico AR, et al., The importance of sodium pyruvate in assessing damage produced by hydrogen peroxide, Free Radical Biology and Medicine, 1997, 23(3), 426-434. These are incorporated herein by reference). JPEG2023522037000008.jpg9170

[0213]

[0216] The stoichiometry of cysteine ​​oxidation described in Equations 1-3 is 4:1 moles of oxidized cysteine ​​relative to the moles of oxygen consumed. This assumes a complete reaction between hydrogen peroxide and cysteine. In culture media, the stoichiometric ratio is less than 4:1 as a result of the tendency of hydrogen peroxide to react with other culture medium components such as pirubate.

[0214] Example 7: Effect of copper filter material and pore size

[0217] Examples 1-6, which used filters, utilized polyethersulfone (PES) membrane filters with a pore size of 0.22 μm. Polyvinylidene difluoride or polyvinylidene difluoride (PVDF) and nitrocellulose membranes with pore sizes ranging from 0.22 μm to 8 μm were also tested. It was found that copper removal during filtration was independent of the membrane material and pore size.

[0215]

[0218] Furthermore, it was demonstrated that the copper removed during filtration could be recovered from the filter material using the copper removal assay by filtration described herein. By dissolving the filter, 99% of the copper removed from the culture medium during filtration could be recovered, and by simply backflushing the filter, 89% of the removed copper could be recovered.

Claims

1. 1. A method for preventing copper loss in a solution, comprising: the solution is a cell culture medium, cell culture supply, or cell culture additive comprising copper(II) and cysteine; having substantially the same amount of soluble copper before and after filtering the solution; Filtration is performed to remove at least 6% dO ２ of dissolved oxygen (dO ２ ) level, method.

2. dO of the solution ２ 10. The method of claim 1, further comprising maintaining the level at at least 6% dO2 for at least 10 minutes prior to the start of filtration.

3. dO ２ The level is, a. oxygenating the solution; and / or b. optimizing filtration, wherein said optimizing filtration comprises optimizing dO during filtration. ２ optimizing filtration, including increasing filtration rate, filter size, and / or filter capacity so that levels do not fall below 6%; 3. The method of claim 1, wherein the amount of oxidized or oxidized cellulose is maintained at a required level by

4. Oxygenation is sparging the solution with an oxygen-containing gas; and / or agitating or mixing the solution to increase contact of the oxygen-containing gas with the solution; The method of claim 3, comprising:

5. Filtration is completed within 24 hours of adding copper(II) to the solution.

5. The method according to any one of claims 1 to 4.

6. providing a cysteine ​​solution and a copper(II) solution; and the filtering comprises separately filtering the cysteine ​​solution and the copper(II) solution, and then combining the filtered cysteine ​​solution and the filtered copper(II) solution to form the cell culture medium, cell culture supply, or cell culture additive.

6. The method according to any one of claims 1 to 5.

7. the cysteine ​​solution comprises at least a majority of the components of the cell culture medium, cell culture supplies, or cell culture additives other than copper; and / or the cysteine ​​solution comprises substantially all of the components of the cell culture medium, cell culture supplies, or cell culture additives, except for copper; and / or The concentration of copper in the copper(II) solution ranges from 0.005 μM to 1 M; The method of claim 6.

8. the filtration comprising sterile filtration; 8. The method according to any one of claims 1 to 7.

9. The cysteine ​​is substantially provided as a sulfhydryl-free cysteine ​​derivative; and / or Cysteine ​​is substantially converted to a cysteine ​​derivative that does not contain a sulfhydryl group before copper(II) is added to the solution; 9. The method according to any one of claims 1 to 8.

10. the cysteine ​​derivative is selected from the group consisting of cystine, 4-carboxy-2-methylthiazolidine-2-carboxylate, S-sulfocysteine, S-sulfocysteinylglycine, cysteine ​​S-linked N-acetylglucosamine (GlcNAC-cys), homocystine, L-cysteine ​​mixed disulfides, L-cysteine ​​mixed peptides, S-alkylated cysteine, cysteine ​​with a thiol-protecting group, 2-methyl-1,3-thiazolidine-2,4-dicarboxylic acid, 2-(2-carboxyethyl)21,3-thiazolidine-2,4-dicarboxylic acid, reduced and oxidized glutathione (GSH), S-sulfoglutathione, S-acyl-GSH, S-carboxy-L-cysteine, L-2-oxothiazolidine-4-carboxylic acid, 2-alkyl-thiazolidine-4(R)-carboxylic acid, 2-aryl-thiazolidine-4(R)-carboxylic acid, and carbohydrate-based thiazolidins; 10. The method of claim 9.

11. the copper is in the form of a copper(II) chelate; 11. The method according to any one of claims 1 to 10.

12. 1. A method of culturing cells in a medium, comprising: Preventing copper loss in the culture medium by the method according to any one of claims 1 to 11; contacting the cells with a medium; and Culturing the cells in a medium; A method comprising:

13. the cell is a eukaryotic cell, a prokaryotic cell, or an archaeal cell; The method of claim 12.

14. 1. A method of making a biological product, comprising: Preventing copper loss in cell culture media by the method of any one of claims 1 to 11, contacting a cell configured to express the biological product with a medium; and culturing the cells in a medium under conditions such that the cells express the biological product; A method comprising:

15. the biological product is a polypeptide, protein, peptide, hormone, virus or virus-like particle, nucleic acid, or fragment thereof; and / or the method further comprising isolating and / or purifying the biological product; 15. The method of claim 14.

16. The method of claim 13, wherein the cells are selected from hybridoma cells, CHO cells, COS cells, VERO cells, HeLa cells, HEK293 cells, PER-C6 cells, K562 cells, MOLT-4 cells, M1 cells, NS-1 cells, COS-7 cells, MDBK cells, MDCK cells, MRC-5 cells, WI-38 cells, WEHI cells, SP2 / 0 cells, BHK cells (including BHK-21 cells), and derivatives thereof.

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