Cell culture compositions and methods for polypeptide production

Abstract

The present invention provides cell culture media, such as chemically defined cell culture media, as well as methods of growing cells (i.e., culturing cells) and producing polypeptides (e.g., antibodies) using the media. The invention also provides compositions containing the polypeptides produced by these methods.

Description

[0001] This application is a divisional application of application number 202010250367.4, filed on April 24, 2013, with the invention name “Cell culture compositions and methods for polypeptide production”.

[0002] Cross-references to related patent applications

[0003] This application claims priority to U.S. Provisional Application Serial No. 61 / 637,778 filed on April 24, 2012, U.S. Provisional Application Serial No. 61 / 637,780 filed on April 24, 2012, and U.S. Provisional Application Serial No. 13 / 841,864 filed on March 15, 2013, the contents of each of which are hereby incorporated by reference in their entirety. Background Art

[0004] Methods for producing proteins in vitro using recombinant cell cultures are well known and are used for industrial-scale production of protein-based pharmaceutical products. However, significant challenges remain for the efficient production of proteins from recombinant cell cultures. For example, protein-based pharmaceutical products have certain quality attributes, such as size distribution, sequence integrity, and product color, that may be affected by the protein production method.

[0005] The color of protein drug products is a quality attribute of particular concern. In addition, regulatory requirements for acceptable color levels for protein-based drug products must also be met. Therefore, producing protein products with acceptable color (e.g., to meet regulatory requirements for product marketing) is an important aspect of drug manufacturing. Establishing product quality comparability with earlier clinical materials may also be critical.

[0006] The recent trend in subcutaneous delivery of monoclonal antibodies is accompanied by an increase in the concentration of the drug substance in the formulation (e.g., to ≥150 mg / mL). At these concentrations, the color of the drug product may be stronger, making it more difficult to produce a protein-based drug product with an acceptable color. Cell culture conditions may affect the quality attributes of protein-based drug products. The culture medium used to culture cells may have a particularly significant impact on protein production.

[0007] Recombinant DNA technology for in vitro production of proteins has historically used cell lines cultured in culture media, wherein the culture media are supplemented with varying, chemically undefined culture media components such as animal serum and peptone. Chemically undefined nutrients may lead to batch-to-batch variation, and products of animal origin may cause contamination of the culture media with undesirable components. Chemically defined cell culture media (CDM), whose composition is known and consistent between batches, have been developed to address these problems. Due to the various advantages associated with the use of CDM to produce proteins, the industry-wide trend is to convert from serum-containing and peptone-containing methods to methods utilizing CDM. Various CDMs have been described in the patent literature, such as U.S. Patent Nos. 4,767,704; 5,691,202; 6,048,728; 6,900,056; and 7,601,535, and US Patent Application Publication Nos. 20030087372 and 20110039330. However, chemically undefined culture media continue to be used in protein production.

[0008] There is a continuing need to provide improved, cost-effective in vitro production methods for proteins (e.g., antibodies) wherein the proteins have acceptable product quality attributes. Cell culture media that modulate one or more product quality attributes are desirable. Cell culture media having the following components, whether chemically undefined or chemically defined, would be useful in developing protein products, such as antibodies (e.g., for subcutaneous injection), wherein the components can consistently produce a protein product of lower color intensity while maintaining a desired protein concentration (e.g., ≥150 mg / mL). SUMMARY OF THE INVENTION

[0010] The present invention describes a cell culture medium that can provide a drug product with an acceptable color and a method for using the culture medium for cell growth (i.e., cell culture) and / or protein production. The present invention also provides such a culture medium that can provide a protein-based drug product with an acceptable color while maintaining a desired protein-based drug product concentration (e.g., ≥100 mg / mL or ≥150 mg / mL), which can be used in a protein production method, such as for producing antibodies for subcutaneous injection. The cell culture medium described herein can be chemically undefined or CDM. The present invention also contemplates a composition containing a culture medium described herein and a polypeptide (e.g., a polypeptide secreted into the culture medium by a host cell) and / or a cell containing an isolated nucleic acid encoding a polypeptide. The present invention provides a polypeptide prepared by the method described herein and a preparation containing the polypeptide and a carrier (e.g., a pharmaceutically acceptable carrier). The polypeptide preparation has an acceptable color on the one hand while maintaining a protein-based drug product concentration of at least 100 mg / mL or 150 mg / mL.

[0011] The cell culture media described herein generally contain one or more of the following components in amounts that achieve protein product quality attributes such as color: (a) cystine or cysteine; (b) vitamin B2, (c) vitamin B6 (pyridoxine and / or pyridoxal, which can be provided in the form of HCl salts), (d) vitamin B9, (e) vitamin B12, (f) an iron source such as ferric nitrate, ferric citrate or ferrous sulfate, and (g) hydrocortisone. In one variant, the cell culture medium contains 2 or 3 or 4 or 5 or 6 or each of the components (a), (b), (c), (d), (e), (f) and (g). It is understood that the cell culture media provided herein can contain any combination of the components (a), (b), (c), (d), (e), (f) and (g), just as if each combination was specifically and individually listed. In one aspect, the cell culture medium is CDM. In another aspect, the cell culture medium is chemically undefined. In one specific variation, the cell culture medium described herein comprises: (a) about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; (b) about 0.05 mg / L to about 1.0 mg / L vitamin B2; (c) about 0.05 mg / L to about 10.0 mg / L vitamin B6; (d) about 0.05 mg / L to about 12.0 mg / L vitamin B9; and (e) about 0.05 mg / L to about 2.5 mg / L vitamin B12, wherein the cell culture medium (a) can be CDM in one variation and / or (b) can further comprise one or more of the following components: (1) an iron source, such as ferric citrate or ferrous sulfate (which in one aspect is present at a concentration of about 2 μM to about 80 μM) and (2) hydrocortisone (which in one aspect is present at a concentration of about 0.05 μM to about 0.25 μM). In another variation, the cell culture medium described herein comprises: (a) about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; (b) about 2 μM to about 80 μM ferric citrate; and (c) about 0.05 μM to about 0.5 μM hydrocortisone, wherein the cell culture medium (1) can be CDM in one variation and / or (2) can further comprise one or more of the following components: (A) vitamin B2 (which is present in a concentration of about 0.05 mg / L to about 1.0 mg / L in one aspect); (B) vitamin B6 (which is present in a concentration of about 0.05 mg / L to about 10.0 mg / L in one aspect); (C) vitamin B9 (which is present in a concentration of about 0.05 mg / L to about 12.0 mg / L in one aspect); and (D) vitamin B12 (which is present in a concentration of about 0.05 mg / L to about 2.5 mg / L in one aspect).For any culture medium provided herein, in a variant, when the culture medium is used in a method for producing a polypeptide, the culture medium provided herein reduces the presence of charge variants (on the one hand, acidic charge variants) compared to the charge variants (on the one hand, acidic charge variants) obtained when producing polypeptides in different cell culture media (e.g., culture media containing different culture media components or containing the same culture media components in different amounts). In a variant of the compositions and methods provided herein, charge variants (on the one hand, acidic charge variants) constitute less than 25% or 20% or 18% or 15% or 10% of the polypeptide product. In another variant of the compositions and methods provided herein, at least 75% or 80% or 85% or 90% or 95% or more of the polypeptide product is the main type of protein. In some variants, the main type of protein is a protein that is dominant in quantity, as identified by the amino acid sequence, secondary structure and / or tertiary structure of the protein. In some variants, the main type of protein is a protein that is dominant in quantity, which can be identified by one or more post-translational modifications. In some variants, the post-translational modification is glycosylation. It will be appreciated that the percentages of a polypeptide product described herein can be determined before purification of the polypeptide product, after purification of the polypeptide product, or at any step during the polypeptide purification process.

[0012] Acidic variants can be evaluated by various methods, but preferably such methods include one, two, three, four or five of the following: ion exchange chromatography (IEC), wherein the composition is treated with a sialidase before, after and / or during IEC (e.g., to evaluate sialylated variants); reducing CE-SDS (e.g., to evaluate disulfide bridge reduced variants); non-reducing CE-SDS (e.g., to evaluate non-reduced variants); boric acid chromatography (e.g., to evaluate glycosylated variants); and peptide mapping (e.g., to evaluate deamidated variants). In one variation, the acidic variants are evaluated by ion exchange chromatography, e.g., using a weak cation exchanger and / or a cation exchanger having a carboxylic acid functional group (e.g., using a DIONEX PROPAC TM WCX-10 column) to evaluate overall acidic variants.

[0013] In another aspect of the invention, the cell culture medium described herein generally comprises one or more of the following components in an amount that achieves a quality attribute of a protein product, such as color: (a) cystine; (b) vitamin B1; (c) vitamin B2; (d) vitamin B3; (e) vitamin B5; (f) vitamin B6 (pyridoxine and / or pyridoxal, which can be provided in the form of an HCl salt); (g) vitamin B7; (h) vitamin B9; (i) vitamin B12; and (j) an iron source such as ferric nitrate, ferric citrate, or ferrous sulfate. In one variant, the cell culture medium contains 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or each of components (a), (b), (c), (d), (e), (f), (g), (h), (i), and (j). It is understood that the cell culture medium provided herein can contain any combination of components (a), (b), (c), (d), (e), (f), (g), (h), (i) and (j), just as if each combination was specifically and individually listed. In one aspect, the cell culture medium is CDM. In another aspect, the cell culture medium is chemically undefined. In a specific variation, the cell culture medium described herein comprises: (a) about 0.8 mM (in some embodiments, 0.7 mM) to about 2.5 mM cystine; (b) about 0.11 μM to about 0.72 μM vitamin B2; (c) about 4.5 μM to about 30.0 μM vitamin B6; (c) about 3.4 μM to about 22.0 μM vitamin B9; and (d) about 0.2 μM to about 1.5 μM vitamin B12, wherein the cell culture medium (a) can be CDM in one variation and / or (b) can further comprise one or more The following ingredients: (1) an iron source, such as ferric citrate or ferrous sulfate (which is present in a concentration of about 11.0 μM to about 36.0 μM in one aspect), (2) vitamin B1 (which is present in a concentration of about 2.0 μM to about 14.0 μM in one aspect), (3) vitamin B3 (which is present in a concentration of about 11.0 μM to about 72.0 μM in one aspect), (4) vitamin B5 (which is present in a concentration of about 6.8 μM to about 44.0 μM in one aspect) and (5) vitamin B7 (which is present in a concentration of about 0.02 μM to about 0.24 μM in one aspect).

[0014] Compared to a protein product produced in a cell culture medium having a different composition (e.g., a culture medium that does not contain the culture medium components and / or component amounts described herein), the use of the cell culture medium described herein can increase the stability (e.g., physical stability and / or chemical stability) of the protein product, for example, by reducing oxidation of the major species of protein. Compared to a polypeptide product produced in a culture medium having a different composition (e.g., a culture medium that does not contain the culture medium components and / or component amounts described herein), the use of the cell culture medium described herein can also reduce colored forms of the polypeptide product. Compared to a polypeptide product produced in a culture medium having a different composition (e.g., a culture medium that does not contain the culture medium components and / or component amounts described herein), the use of the cell culture medium described herein can also reduce the binding of the polypeptide product to other substances (e.g., adducts) in the cell culture vessel. The present invention contemplates methods of increasing the stability of polypeptide compositions, and also contemplates methods of reducing the presence and / or amount of colored forms of the polypeptide product and reducing the binding of the polypeptide product to other substances (e.g., adducts) in the cell culture vessel.

[0015] The invention also provides methods for preparing a formulation comprising a polypeptide (eg, an antibody), comprising the steps of producing the polypeptide by any of the methods described herein, and combining the polypeptide with one or more formulation ingredients, such as a pharmaceutically acceptable carrier or excipient.

[0016] The present invention also provides preparations comprising polypeptides (e.g., antibodies) produced by any method described herein. Preparations can include polypeptides and pharmaceutically acceptable carriers or excipients. The polypeptide preparations that can be suitable for administration to individuals can include separated and / or purified antibodies and have one or more desired product quality attributes, such as acceptable colors. In one aspect, preparations containing polypeptide products obtained by any method provided herein include polypeptides at a concentration of at least 100 mg / ml or at least 150 mg / ml. Preparations containing at least 100 mg / ml or at least 150 mg / ml polypeptide products (e.g., antibodies, such as IgG1 antibodies) can also have acceptable colors. The preparations can be suitable for injection, such as subcutaneous injection into individuals, which are people on the one hand. On the one hand, polypeptide drug products suitable for injection have a concentration greater than at least 100 mg / ml, at least 125 mg / ml, or at least 150 mg / ml, and according to the measurement of the COC test, have a color intensity greater than B3, B4, B5, B6, B7, B8, or B9. It is understood that the color intensity value determined by the COC test can be, but is not limited to, any of brown (B), brownish yellow (BY), yellow (Y), greenish yellow (GY), or red (R), wherein a higher value indicates a lighter color intensity. In some aspects, a polypeptide drug product suitable for injection has a concentration greater than at least 100 mg / ml, at least 125 mg / ml, or at least 150 mg / ml, and has a color intensity value less than the color intensity value of a reference solution as measured by a color test (e.g., a total color test or a NIFTY test). The formulations provided herein can include a polypeptide product, wherein no more than 25% or 20% or 18% or 15% or 10% of the polypeptide product is a polypeptide charge variant (which is an acidic charge variant on the one hand). The formulations described herein can also include a polypeptide product, wherein at least 75% or 80% or 85% or 90% or 95% or more of the polypeptide product is a major species protein. In one particular variation, a formulation comprising a polypeptide (e.g., an antibody) product is provided, wherein the formulation comprises the polypeptide product at a concentration greater than 100 mg / ml or greater than 125 mg / ml or greater than 150 mg / ml, and wherein the formulation has an acceptable color, wherein no more than 25% or 20% or 18% or 15% or 10% of the polypeptide product is a polypeptide charge variant (which in one aspect is an acidic charge variant).

[0017] The present invention also provides a composition comprising a cell culture medium and one or more other ingredients, such as cells and / or desired polypeptides (e.g., antibodies). The composition encompasses any and all stages of cell culture, such as inoculation, cell growth, and cell production / maintenance. In a variant, a composition is provided, comprising (a) cells containing isolated nucleic acids encoding polypeptides, and (b) cell culture medium provided herein. In a variant, a composition is provided, comprising: (a) polypeptides; and (b) cell culture medium provided herein, wherein on the one hand the polypeptide is secreted into the culture medium by cells comprising isolated nucleic acids encoding polypeptides, or released into the culture medium by lysing cells comprising isolated nucleic acids encoding polypeptides. The cell of the composition can be any cell described herein (e.g., CHO cells), and the cell culture medium of the composition can be any culture medium described herein, as if each and all combinations of cells and culture medium are specifically and individually listed. Similarly, the polypeptide of the composition can be any polypeptide described herein, and the culture medium of the composition can be any culture medium described herein, as if each and all combinations of polypeptides and culture medium are specifically and individually listed.

[0018] The present invention provides methods of growing cells (ie, culturing cells) by contacting the cells with a culture medium as described herein. In a specific variation of the method of growing cells (ie, culturing cells), the cell culture medium is CDM. In one variation, a method of growing cells (i.e., culturing cells) comprises the steps of contacting the cells with a cell culture medium comprising: (a) about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; (b) about 0.05 mg / L to about 1.0 mg / L vitamin B2; (c) about 0.05 mg / L to about 10.0 mg / L vitamin B6; (d) about 0.05 mg / L to about 12.0 mg / L vitamin B9; and (e) about 0.05 mg / L to about 2.5 mg / L vitamin B12, wherein the cell culture medium may further comprise one or more of the following components: (1) an iron source, such as ferric citrate or ferrous sulfate (which in one aspect is present at a concentration of about 2 μM to about 80 μM) and (2) hydrocortisone (which in one aspect is present at a concentration of about 0.05 μM to about 0.25 μM). In another variation, a method of growing cells (i.e., culturing cells) is provided, the method comprising the steps of contacting the cells with a cell culture medium comprising: (a) about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; (b) about 2 μM to about 80 μM ferric citrate; and (c) about 0.05 μM to about 0.5 μM hydrocortisone, wherein the cell culture medium may further comprise one or more of the following components: (1) vitamin B2 (which is present in one aspect at a concentration of about 0.05 mg / L to about 1.0 mg / L); (2) vitamin B6 (which is present in one aspect at a concentration of about 0.05 mg / L to about 10.0 mg / L); (3) vitamin B9 (which is present in one aspect at a concentration of about 0.05 mg / L to about 12.0 mg / L); and (4) vitamin B12 (which is present in one aspect at a concentration of about 0.05 mg / L to about 2.5 mg / L). In any of the methods of growing cells (i.e., culturing cells) provided herein, the cells can be contacted with a cell culture medium during a cell growth phase and / or a production phase. The present invention contemplates contacting cells with a culture medium described herein at any cell culture phase, such as during cell growth, production, and maintenance. It will be appreciated by those skilled in the art that cells are contacted with a culture medium described herein under conditions (e.g., temperature, pH, osmotic pressure, etc.) that promote cell maintenance and / or growth (including polypeptide production).

[0019] In another variation of the invention, a method of growing cells (i.e., culturing cells) comprises the steps of contacting the cells with a cell culture medium comprising: (a) about 0.8 mM (in some embodiments, 0.7 mM) to about 2.5 mM cystine; (b) about 0.11 μM to about 0.72 μM vitamin B2; (c) about 4.5 μM to about 30.0 μM vitamin B6; (d) about 3.4 μM to about 22.0 μM vitamin B9; and (d) about 0.2 μM to about 1.5 μM vitamin B12, and wherein the cell culture medium may further comprise a One or more of the following ingredients: (1) an iron source, such as ferric citrate or ferrous sulfate (which in one aspect is present at a concentration of about 11.0 μM to about 36.0 μM), (2) vitamin B1 (which in one aspect is present at a concentration of about 2.0 μM to about 14.0 μM), (3) vitamin B3 (which in one aspect is present at a concentration of about 11.0 μM to about 72.0 μM), (4) vitamin B5 (which in one aspect is present at a concentration of about 6.8 μM to about 44.0 μM) and (5) vitamin B7 (which in one aspect is present at a concentration of about 0.02 μM to about 0.24 μM).

[0020] In yet another variation of the invention, a method of growing cells (i.e., culturing cells) comprises the steps of contacting the cells with a cell culture medium comprising: about 0.7 mM to about 2.5 mM cystine; about 2 μM to about 80 μM ferric citrate; about 0.05 μM to about 0.5 μM hydrocortisone; about 0.11 μM to about 0.72 μM vitamin B2; about 4.5 μM to about 30.0 μM vitamin B6; about 3.4 μM to about 22.0 μM vitamin B9; and about 0.2 μM to about 1.5 μM pyridoxine. M vitamin B12, and wherein the cell culture medium may further comprise one or more of the following components: (1) vitamin B1 (which in one aspect is present at a concentration of about 2.0 μM to about 14.0 μM), (2) vitamin B3 (which in one aspect is present at a concentration of about 11.0 μM to about 72.0 μM), (3) vitamin B5 (which in one aspect is present at a concentration of about 6.8 μM to about 44.0 μM), and (4) vitamin B7 (which in one aspect is present at a concentration of about 0.02 μM to about 0.24 μM).

[0021] The present invention also provides a method for producing a polypeptide by growing cells in a cell culture medium (i.e., culturing the cells in a cell culture medium), the cells comprising an isolated nucleic acid encoding the polypeptide, wherein: (a) the cells express the polypeptide, (b) the cell culture medium comprises: (1) about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; (2) about 0.05 mg / L to about 1.0 mg / L vitamin B2; (3) about 0.05 mg / L to about 10.0 mg / L vitamin B6; (4) about 0.05 mg / L to about 12.0 mg / L vitamin B9; and (5) about 0.05 mg / L to about 2.5 mg / L vitamin B12, and wherein the cell culture medium may further comprise one or more of the following components: (A) an iron source, such as ferric citrate or ferrous sulfate (which in one aspect is present at a concentration of about 2 μM to about 80 μM) and (B) hydrocortisone (which in one aspect is present at a concentration of about 0.05 μM to about 0.25 μM). In another variation, a method is provided for producing a polypeptide by growing cells in a cell culture medium (i.e., culturing the cells in a cell culture medium), the cells comprising an isolated nucleic acid encoding the polypeptide, wherein: (a) the cells express the polypeptide, and (b) the cell culture medium comprises: (1) about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; (2) about 2 μM to about 80 μM ferric citrate; and (3) about 0.05 μM to about 0.5 μM hydrocortisone, wherein the cell culture medium It may further include one or more of the following ingredients: (A) vitamin B2 (which in one aspect is present at a concentration of about 0.05 mg / L to about 1.0 mg / L); (B) vitamin B6 (which in one aspect is present at a concentration of about 0.05 mg / L to about 10.0 mg / L); (C) vitamin B9 (which in one aspect is present at a concentration of about 0.05 mg / L to about 12.0 mg / L); and (D) vitamin B12 (which in one aspect is present at a concentration of about 0.05 mg / L to about 2.5 mg / L).

[0022] In another variation of the invention, the invention also provides a method for producing a polypeptide by growing cells in a cell culture medium (i.e., culturing cells in a cell culture medium), wherein the cells contain an isolated nucleic acid encoding the polypeptide, wherein: (a) the cells express the polypeptide, (b) the cell culture medium contains: (a) about 0.8 mM (in some embodiments, 0.7 mM) to about 2.5 mM cystine; (b) about 0.11 μM to about 0.72 μM vitamin B2; (c) about 4.5 μM to about 30.0 μM vitamin B6; (c) about 3.4 μM to about 22.0 μM vitamin B9; and (d) about 0.2 μM to about 1.5 μM vitamin B12. B12, and wherein the cell culture medium may further comprise one or more of the following components: (1) an iron source, such as ferric citrate or ferrous sulfate (which in one aspect is present at a concentration of about 11.0 μM to about 36.0 μM), (2) vitamin B1 (which in one aspect is present at a concentration of about 2.0 μM to about 14.0 μM), (3) vitamin B3 (which in one aspect is present at a concentration of about 11.0 μM to about 72.0 μM), (4) vitamin B5 (which in one aspect is present at a concentration of about 6.8 μM to about 44.0 μM) and (5) vitamin B7 (which in one aspect is present at a concentration of about 0.02 μM to about 0.24 μM).

[0023] In yet another variant of the invention, the invention also provides a method for producing a polypeptide by growing cells in a cell culture medium (i.e., culturing cells in a cell culture medium), wherein the cells contain an isolated nucleic acid encoding the polypeptide, wherein: (a) the cells express the polypeptide, (b) the cell culture medium contains: about 0.7 mM to about 2.5 mM cystine; about 2 μM to about 80 μM ferric citrate; about 0.05 μM to about 0.5 μM hydrocortisone; about 0.11 μM to about 0.72 μM vitamin B2; about 4.5 μM to about 30.0 μM vitamin B6; about 3.4 μM to about 22.0 μM vitamin B9; and about 0.2 μM to about 1.5 μM vitamin B12, and wherein the cell culture medium may further comprise one or more of the following components: (1) vitamin B1 (which in one aspect is present at a concentration of about 2.0 μM to about 14.0 μM), (2) vitamin B3 (which in one aspect is present at a concentration of about 11.0 μM to about 72.0 μM), (3) vitamin B5 (which in one aspect is present at a concentration of about 6.8 μM to about 44.0 μM) and (4) vitamin B7 (which in one aspect is present at a concentration of about 0.02 μM to about 0.24 μM).

[0024] The polypeptide produced by the methods described herein or present in the compositions described herein can be an antibody, such as an IgG1 antibody, in a variant. In one aspect, the polypeptide produced by the methods described herein or present in the compositions described herein is an anti-VEGF antibody, an anti-mesothelin antibody, an anti-PCSK9 antibody, or an anti-β7 antibody. The polypeptide produced according to the methods provided herein or present in the compositions described herein can be separated from the cell culture medium and can be further purified. The polypeptide (e.g., antibody) can also be concentrated to reach a desired concentration. Methods for concentrating polypeptides are well known in the art, for example, the concentration of the polypeptide or protein can be increased by applying ultrafiltration. In a specific variant, the polypeptide is separated at a concentration of at least 100 mg / ml or 150 mg / ml, and / or is presented as a colorless or slightly colored liquid. In a specific variant, the composition comprises an isolated polypeptide at a concentration of at least 100 mg / ml, and / or is presented as a colorless or slightly colored liquid. In one variant, the composition comprises an isolated polypeptide of at least 1mg / ml or 10mg / ml or 50mg / ml or 75mg / ml of concentration, and / or is presented as a colorless or slightly colored liquid. In another variant, the composition comprises an isolated polypeptide of at least about 1mg / ml or 10mg / ml or 50mg / ml or 75mg / ml of any concentration, and / or is presented as a colorless or slightly colored liquid. In another variant, the composition comprises an isolated polypeptide of at least about 1mg / ml or 10mg / ml or 50mg / ml or 75mg / ml to about 125mg / ml or to about 150mg / ml of any concentration, and / or is presented as a colorless or slightly colored liquid. The present invention also describes a composition comprising a polypeptide obtained by the method described herein and a pharmaceutically acceptable carrier.

[0025] The present invention also describes a kit for supplementing a cell culture medium with a chemically defined component, the kit comprising: (a) cystine in an amount that can provide about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine in the cell culture medium; (b) vitamin B2 in an amount that can provide about 0.05 mg / L to about 1.0 mg / L vitamin B2 in the cell culture medium; (c) vitamin B6 in an amount that can provide about 0.05 mg / L to about 10.0 mg / L vitamin B6 in the cell culture medium; (d) vitamin B12 in an amount that can provide about 0.05 mg / L to about 10.0 mg / L vitamin B12 in the cell culture medium; and (e) vitamin B13 in an amount that can provide about 0.05 mg / L to about 10.0 mg / L vitamin B13 in the cell culture medium. (e) vitamin B9 in an amount that can provide about 0.05 mg / L to about 12.0 mg / L vitamin B9 in the cell culture medium; and (e) vitamin B12 in an amount that can provide about 0.05 mg / L to about 2.5 mg / L vitamin B12 in the cell culture medium, wherein the kit may also include one or more of the following components: (1) an iron source, such as ferric citrate or ferrous sulfate (in one aspect, in an amount that can provide an iron source with a concentration of about 2 μM to about 80 μM) and (2) hydrocortisone (in one aspect, in an amount that can provide a concentration of about 0.05 μM to about 0.25 μM hydrocortisone). In another variation, the invention describes a kit for supplementing a cell culture medium with a chemically defined component, the kit comprising: (a) cystine in an amount that can provide about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine in the cell culture medium; (b) ferric citrate in an amount that can provide about 2 μM to about 80 μM ferric citrate in the cell culture medium; and (c) hydrocortisone in an amount that can provide about 0.05 μM to about 0.5 μM hydrocortisone in a cell growth (i.e., cell culture) medium, wherein the kit may also comprise one or more A plurality of the following ingredients: (1) vitamin B2 (in one aspect, an amount can provide about 0.05 mg / L to about 1.0 mg / L vitamin B2 in the cell culture medium); (2) vitamin B6 (in one aspect, an amount can provide about 0.05 mg / L to about 10.0 mg / L vitamin B6 in the cell culture medium); (3) vitamin B9 (in one aspect, an amount can provide about 0.05 mg / L to about 12.0 mg / L vitamin B9 in the cell culture medium); and (4) vitamin B12 (in one aspect, an amount can provide about 0.05 mg / L to about 2.5 mg / L vitamin B12 in the cell culture medium).Also provided herein are kits for supplementing a cell culture medium with chemically defined components, wherein the kits comprise: (a) cystine in an amount to provide about 0.8 mM (in some embodiments, 0.7 mM) to about 2.5 mM cystine in the cell culture medium; (b) vitamin B2 in an amount to provide about 0.11 μM to about 0.72 μM vitamin B2 in the cell culture medium; (c) vitamin B6 in an amount to provide about 4.5 μM to about 30.0 μM vitamin B6 in the cell culture medium; (d) vitamin B9 in an amount to provide about 3.4 μM to about 22.0 μM vitamin B9 in the cell culture medium; (e) vitamin B12 in an amount to provide about 0.2 μM to about 1.5 μM vitamin B12 in the cell culture medium; M vitamin B12, and wherein the kit may further include one or more of the following components: (1) an iron source, such as ferric citrate or ferrous sulfate (in one aspect, its amount can provide about 11.0 μM to about 36.0 μM in the cell culture medium), (2) vitamin B1 (in one aspect, its amount can provide about 2.0 μM to about 14.0 μM in the cell culture medium), (3) vitamin B3 (in one aspect, its amount can provide about 11.0 μM to about 72.0 μM in the cell culture medium), (4) vitamin B5 (in one aspect, its amount can provide about 6.8 μM to about 44.0 μM in the cell culture medium) and (5) vitamin B7 (in one aspect, its amount can provide about 0.02 μM to about 0.24 μM in the cell culture medium). The kit may also include instructions for use, such as instructions for preparing a culture medium (e.g., CDM) and / or producing polypeptides (including antibodies) from a cell culture system. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The COC test of antibody samples isolated from cell lines cultured under different cell culture conditions is described. From left to right, the vials contain preparations of antibodies isolated from cells, and the cells are cultured under the following conditions: I) chemically undefined medium; II) basal medium 1 and feed medium 2; III) basal medium 1 and feed medium 2; IV) basal medium 5 and feed medium 4; V) basal medium 5 and feed medium 2; VI) modified basal medium 3 and feed medium 4 containing cysteine ​​instead of cystine; VII) basal medium 3 and feed medium 4 and (VIII) modified basal medium 3 and feed medium 4 containing cysteine ​​instead of cystine. The COC value is described above the bottle. All vials contain about 150g / L protein. By NIFTY test determination, preparations III, IV and VI have color intensity values ​​of 1.59, 1.47 and 0.71 respectively. By total color test determination, preparations III, IV and VI have color intensity values ​​of 2.62, 2.04 and 1.00 respectively.

[0028] Figure 2 A series of graphs showing a slight decrease in cell number and antibody production in cell cultures incubated with basal medium 3 and feed medium 4 compared to basal medium 1 and feed medium 2. A and C) Cell number in culture during the duration of incubation as measured by collected cell volume (PCV), expressed as a percentage of the total culture volume. B and D) Antibody production in culture during the duration of incubation as measured by high performance liquid chromatography, expressed as antibody titer.

[0029] Figure 3 A series of graphs illustrate the effects of cell cultures grown in chemically defined medium (CDM) containing different levels of vitamin B2, vitamin B6 and vitamin B9 and vitamin B12 on productive cell biomass and antibody production. A) Productive biomass in culture measured by collected cell volume (PCV), expressed as a percentage of total culture volume. B) Antibody production of cells measured by high performance liquid chromatography, expressed as antibody titer. For vitamin B2, -1 indicates 0.25 mg / L base and 0 mg / L feed, 1 indicates 1.41 mg / L base and 10 mg / L feed; for vitamin B6, -1 indicates 5.35 mg / L base (pyridoxine) and 0 mg / L feed, 1 indicates 15.42 mg / L base and 7 mg / L feed of pyridoxine combined with 0 mg / L base and 60 mg / L feed of pyridoxal; for vitamin B9, -1 indicates 8.61 mg / L base and 0 mg / L feed, 1 indicates 9.93 mg / L base and 197 mg / L feed; for vitamin B12, -1 indicates 1.76 mg / L base and 0 mg / L feed, 1 indicates 3.05 mg / L base and 48 mg / L feed. The middle line indicates the predicted value calculated from the linear model derived from the data. The upper and lower lines indicate the 95% confidence interval of the prediction.

[0030] Figure 4A series of graphs showing the color intensity of antibodies isolated from cell cultures grown in CDM containing different levels of vitamin B2, vitamin B6, and vitamin B9 and vitamin B 12. Color intensity was determined using a color test, where higher values ​​indicate higher color intensity and lower values ​​indicate lower color intensity. For vitamin B2, -1 indicates 0.25 mg / L base and 0 mg / L feed, 1 indicates 1.41 mg / L base and 10 mg / L feed; for vitamin B6, -1 indicates 5.35 mg / L base (pyridoxine) and 0 mg / L feed, 1 indicates 15.42 mg / L base and 7 mg / L feed of pyridoxine combined with 0 mg / L base and 60 mg / L feed of pyridoxal; for vitamin B9, -1 indicates 8.61 mg / L base and 0 mg / L feed, 1 indicates 9.93 mg / L base and 197 mg / L feed; for vitamin B12, -1 indicates 1.76 mg / L base and 0 mg / L feed, 1 indicates 3.05 mg / L base and 48 mg / L feed.

[0031] Figure 5 A) A series of graphs showing the color intensity of the productive cell biomass, antibody production, and isolated antibodies from cell cultures grown in CDM containing increasing concentrations of ferrous sulfate. A) Over time, the productive biomass in the culture measured by collecting cell volume (IVPCV). B) Antibody production of cells measured by high performance liquid chromatography. C) Color intensity of antibodies isolated from cells measured by color test, wherein higher values ​​indicate higher color intensity and lower values ​​indicate lower color intensity. The upper and lower limits and mean values ​​of the bar indication data. D) Graph showing the color intensity of antibodies incubated in culture medium containing increasing concentrations of ferrous sulfate in an in vitro experiment.

[0032] Figure 6A series of graphs show the color intensity of the productive cell biomass, antibody production and separated antibodies from cell cultures, wherein the cell cultures are grown in CDM containing increasing concentrations of iron and different iron sources. A) Over time, the productive biomass (IVPCV) in the culture measured by collecting cell volume. B) Antibody production of cells measured by high performance liquid chromatography. C) Color intensity of antibodies separated from cells measured by color test, wherein higher numerical values ​​indicate higher color intensity, and lower numerical values ​​indicate lower color intensity. The upper and lower limits and mean values ​​of the bar indication data. D) Display the color intensity of antibody production and separated antibodies from cell cultures, wherein cell cultures are grown in CDM containing different iron sources of different concentrations and reduced levels of vitamin B. The plus sign represents low vitamin conditions, and the open circles represent high vitamin conditions. D) Antibody production of cells measured by high performance liquid chromatography. E) Color intensity of antibodies separated from cells measured by NIFTY test, wherein higher numerical values ​​represent higher color intensity, and lower numerical values ​​represent lower color intensity.

[0033] Figure 7 A series of graphs showing the color intensity of antibodies incubated in CDM containing different concentrations of ferrous sulfate and vitamin B2 in the absence or presence of catalase in an in vitro experiment. A) Color intensity of antibodies in the absence of catalase. B) Color intensity of antibodies in the presence of catalase. Color intensity is measured by a color test, where higher values ​​indicate higher color intensity and lower values ​​indicate lower color intensity. For ferrous sulfate, -1 indicates 18μM basal and 0μM feed, 1 indicates 75μM basal and 0μM feed; for vitamin B2, -1 indicates 0.25mg / L basal and 0mg / L feed, 1 indicates 1.41mg / L and 10mg / L feed. The center line indicates the predicted value calculated from the linear model derived from the data. The upper and lower lines indicate the 95% confidence interval of the prediction.

[0034] Figure 8A) The graph shows the correlation between reduced color intensity and reduced presence of acidic charge variants in antibody solutions obtained from cell cultures grown in modified basal medium 3 and feed medium 4 (plus signs) compared to modified basal medium 1 and feed medium 2 (open circles). Modified medium 1 contained 10 μM, 18 μM or 75 μM ferrous sulfate. Modified medium 3 contained 10 μM or 18 μM ferric citrate. B) The graph shows the correlation between reduced color intensity and reduced presence of acidic charge variants in antibody solutions obtained from cell cultures grown in medium containing 18 μM ferrous sulfate (open circles) relative to 75 μM ferrous sulfate (plus signs). C) The graph shows that there is no correlation between reduced color intensity and reduced presence of acidic charge variants in antibody solutions obtained from cell cultures grown in medium containing different levels of vitamins B2, B6, B9 and B12. Vitamin B levels were varied simultaneously to low concentration (open circles), medium concentration (plus sign), or high concentration (open diamonds). Color intensity was measured by a color test, where higher values ​​indicate higher color intensity and lower values ​​indicate lower color intensity. The percentage of acidic charge variants in the antibody solution was determined by ion exchange chromatography.

[0035] Fig. 9 A series of graphs are shown in the correlation between the color intensity reduced in the antibody solution obtained from the following cell culture and the presence of the reduced acidic charge variant, the cell culture is grown in a medium containing reduced concentrations of vitamin B2 and vitamin B6. A) The color intensity of the antibody separated from the cell measured by the color test, wherein a higher value indicates a higher color intensity and a lower value indicates a lower color intensity. B) The percentage of acidic charge variants in the antibody solution measured by ion exchange chromatography. For vitamin B2, -1 indicates 0.25mg / L basis and 0mg / L feed, 1 indicates 1.41mg / L basis and 10mg / L feed; for vitamin B6, -1 indicates 5.35mg / L basis (pyridoxine) and 0mg / L feed, 1 indicates 15.42mg / L basis and 7mg / L feed of pyridoxine combined with 0mg / L basis and 60mg / L feed of pyridoxal.

[0036] Fig.10A series of figures are shown in the presence of reduced color intensity and reduced acidic charge variants in antibody solutions obtained from cell cultures grown in basal medium containing ferric citrate (compared with containing ferrous sulfate) and with different concentrations of pyridoxal. A) Color intensity of antibodies separated from cells measured by color test, wherein higher numerical values ​​indicate higher color intensity and lower numerical values ​​indicate lower color intensity. B) Percentage of acidic charge variants in antibody solutions measured by ion exchange chromatography. For pyridoxal, -1 indicates 0mg / L basis and 0mg / L feed supplement, 1 indicates 0mg / L basis and 60mg / L feed supplement. Ferric citrate or ferrous sulfate are present in basal medium with 18 μM.

[0037] Fig.11 A series of figures show the color intensity and the level of acidic charge variants reduced in the antibody solution obtained from the following cell culture, wherein the cell culture is grown in a basal medium containing ferric citrate (compared with containing ferrous sulfate) and there are different concentrations of vitamin B2, B6, B9 and B12 in the feed medium. A) The color intensity of the antibody separated from the cells measured by the color test, wherein the higher numerical value indicates a higher color intensity and the lower numerical value indicates a lower color intensity. B) The percentage of acidic charge variants in the antibody solution measured by ion exchange chromatography. Level 1 indicates a feed medium without vitamin B2, B6, B9 and B12. Level 2 indicates a feed medium containing 10mg / L vitamin B2, 7mg / L pyridoxine, 60mg / L pyridoxal, 197mg / L vitamin B9 and 48mg / L vitamin B12. Level 3 indicates a feed medium containing 5 mg / L vitamin B2, 3.5 mg / L pyridoxine, 30 mg / L pyridoxal, 98.5 mg / L vitamin B9 and 24 mg / L vitamin B 12. Ferric citrate or ferrous sulfate is present in the basal medium at 18 μM.

[0038] Fig.12 A series of graphs showing reduced color intensity and reduced levels of acidic charge variants in antibody solutions obtained from cell cultures grown in a basal medium containing reduced concentrations of iron, wherein ferric citrate is preferably used instead of ferrous sulfate as the iron source. A) Color intensity of antibodies isolated from cells measured by color assay, wherein higher values ​​indicate higher color intensity and lower values ​​indicate lower color intensity. B) Percentage of acidic charge variants in antibody solutions measured by ion exchange chromatography.

[0039] Fig.13A series of graphs showing reduced color intensity and reduced levels of acidic charge variants in antibody solutions obtained from cell cultures grown in basal medium containing decreasing concentrations of ferric citrate. A) Color intensity of antibodies isolated from cells as determined by color assay, where higher values ​​indicate higher color intensity and lower values ​​indicate lower color intensity. B) Percentage of acidic charge variants in antibody solutions measured by ion exchange chromatography. * indicates basal medium 1 modified to contain ferric citrate at the indicated concentrations. Indicates modified basal medium 3 containing ferric citrate at the indicated concentration. ○ indicates an antibody solution isolated from cells cultured at 33°C. + indicates an antibody solution isolated from cells cultured at 37°C.

[0040] Fig.14 A series of graphs showing reduced color intensity and reduced levels of acidic charge variants in antibody solutions obtained from cell cultures grown in basal medium containing reduced concentrations of vitamins B2, B6, B9, and B12, with cystine added instead of cysteine, and in the presence of hydrocortisone. A) Color intensity of antibodies isolated from cells measured by color assay, where larger values ​​indicate higher color intensity and smaller values ​​indicate lower color intensity. B) Percentage of acidic charge variants in antibody solutions measured by ion exchange chromatography. * indicates basal medium containing 1.41 mg / L vitamin B2, 15.42 mg / L pyridoxine, 0 mg / L pyridoxal, 9.93 mg / L vitamin B9, and 3.05 mg / L vitamin B12. = represents a basal medium containing 0.7 mg / L vitamin B2, 7.7 mg / L pyridoxine, 0 mg / L pyridoxal, 4.9 mg / L vitamin B9 and 1.5 mg / L vitamin B12. ○ represents 480 mg / L cystine. Δ represents 525 mg / L cysteine. Hydrocortisone is present at 150 nM in the solution shown.

[0041] Fig.15A series of graphs showing the correlation between the total color test and NIFTY test measurements compared to the COC test measurements for color intensity. A) Total color and NIFTY values ​​are plotted, where the symbol represents the COC value. B) Color measurements obtained by the NIFTY test in the Protein A pool and in the corresponding drug substance formulation are plotted, where the symbol represents the COC value. C) Color measurements obtained by the total color test in the Protein A pool and in the corresponding drug substance formulation are plotted, where the symbol represents the COC value. Open circles represent COC values ​​≤ B3; open diamonds represent COC values ​​≤ B4 or BY4; plus signs represent COC values ​​≤ B5 or BY5; DS NIFTY and DS Total Color represent the corresponding test measurements in the final drug substance formulation; ProA NIFTY and ProA Total Color represent the corresponding test measurements in the Protein A pool. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention describes cell culture medium and methods for using the culture medium for cell growth (i.e., cell culture) and polypeptide production. The present invention also provides polypeptides produced by the described methods, including antibodies. The present invention also provides kits for preparing culture mediums and compositions containing culture mediums, and compositions containing cells and / or polypeptides produced by the described methods. The present invention describes pharmaceutical compositions comprising a concentration greater than at least 100 mg / mL, at least 125 mg / mL, or at least 150 mg / mL of polypeptides, and having a color intensity value greater than B3, B4, B5, B6, B7, B8 or B9 measured by color, opalescence and staining (COC) tests. The present invention also describes pharmaceutical compositions containing a concentration greater than at least 1 mg / ml, at least 10 mg / ml, at least 25 mg / ml, at least 50 mg / ml, or at least 75 mg / ml of polypeptides, and having a color intensity value greater than B3, B4, B5, B6, B7, B8 or B9 measured by the COC test. It is understood that the reference standard of the COC test can be, but is not limited to, any one of B, BY, Y, GY or R, wherein the larger value indicates a lighter color intensity. The present invention also provides a pharmaceutical composition comprising a polypeptide greater than at least 100 mg / ml, at least 125 mg / ml, or at least 150 mg / ml concentration, and having a color intensity value measured by a color test (e.g., total color test or NIFTY test) that is less than the color intensity value of a reference solution. The present invention also provides a pharmaceutical composition comprising a polypeptide greater than at least 1 mg / ml, at least 10 mg / ml, at least 25 mg / ml, at least 50 mg / ml, or at least 75 mg / ml concentration, and having a color intensity value measured by a color test (e.g., total color test or NIFTY test) that is less than the color intensity value of a reference solution. The present invention also provides a method for evaluating color in a solution containing a polypeptide (e.g., a solution containing an antibody). In a specific variant, the culture medium, method, kit, and composition comprise CDM.

[0044] Certain cell culture media (e.g., CDM) for polypeptide production have been found to provide polypeptide drug products with acceptable quality attributes. For example, certain CDMs have been found to regulate the color intensity of polypeptide drug products, wherein the polypeptides produced in the CDM provide acceptable colors (e.g., for use as injectable drug products) while maintaining the benefits associated with using the CDM. These CDMs, when used in polypeptide production methods, have been found to reduce the color intensity of polypeptide drug products compared to polypeptides produced in different culture media (e.g., culture media without culture media components and / or component amounts described herein). The cell culture media can be chemically defined or chemically undefined.

[0045] Without wishing to be bound by theory, it is believed that using specific culture medium components (such as cystine and / or cysteine, some vitamin B, hydrocortisone and / or iron source) at a certain concentration can produce polypeptide drug products with acceptable quality attributes, especially acceptable color. Although it is believed that the quality attributes of polypeptide drug products are particularly affected by using these culture medium components in the basal medium for cell growth, the culture medium provided herein can be considered to be used for cell growth, maintenance and any stage of polypeptide production process, including in basal medium and feed medium. The culture medium provided herein can be used in the method for growing cells (that is, culturing cells) and in the method for producing polypeptides, and can be used for the growth, maintenance and / or production stage of cells containing the isolated nucleic acid encoding the desired polypeptide. The culture medium described herein can be used to realize the improvement of one or more properties (such as, color, composition, purity, etc.) of polypeptide drug products or one or more aspects (such as, repeatability between batches, feasibility of production, production cost, etc.) of polypeptide production methods. The polypeptide (such as antibody) produced by the cell culture method using the cell base provided herein is described herein. In one aspect, the polypeptide has a concentration greater than at least 100 mg / ml, at least 125 mg / ml, or at least 150 mg / ml, with a color intensity value greater than B3, B4, B5, B6, B7, B8 or B9 as determined by the COC test. On the other hand, the polypeptide has a concentration greater than at least 1 mg / ml, at least 10 mg / ml, at least 25 mg / ml, at least 50 mg / ml or at least 75 mg / ml, and has a color intensity value greater than B3, B4, B5, B6, B7, B8 or B9 as determined by the COC test. In some aspects, the color intensity value determined by the COC test can be, but is not limited to, any one of B, BY, Y, GY or R, wherein the larger value indicates a lighter color intensity. The present invention also describes methods of applying the polypeptide products described herein, and articles comprising the polypeptide products produced herein.

[0046] definition

[0047] As used herein, unless expressly stated otherwise, the terms "a," "an," etc. are used to mean one or more.

[0048] Reference herein to "about" a value or parameter includes (and describes) embodiments involving that value or parameter itself. For example, reference to "about X" includes reference to "X." Numerical ranges encompass the numbers defining the range.

[0049] A "charge variant" is a variant of a main species of protein (eg, an antibody) that has a different charge than the main species of the protein.

[0050] "Acidic charge variants" are variants of a main species protein (e.g., an antibody) that are more acidic than the main species protein (e.g., an antibody). Acidic variants have gained a negative charge or lost a positive charge relative to the main species protein (e.g., an antibody). Acidic charge variants can be resolved using separation methods that separate proteins based on charge, such as ion exchange chromatography.

[0051] The term "major species protein" herein refers to the amino acid sequence structure of a protein (eg, antibody) in a composition, which is the protein (eg, antibody) molecule that predominates in quantity in the composition.

[0052] "Culturing" cells means contacting the cells with a cell culture medium under conditions suitable for the survival and / or growth of the cells.

[0053] "Batch culture" refers to a culture in which all components for cell culture (including cells and all culture nutrients) are added to the culture vessel at the beginning of the culture process.

[0054] As used herein, the phrase "fed-batch cell culture" refers to a batch culture in which cells and culture medium are initially provided to a culture vessel, other culture nutrients are fed to the culture continuously or in discontinuous additions during the culture, with or without periodic cell and / or product harvesting before termination of the culture.

[0055] "Perfusion culture" is a culture in which cells are confined in culture medium by means of, for example, filtration, encapsulation, anchoring on microcarriers, etc., and the culture medium is continuously or intermittently introduced into and removed from the culture vessel.

[0056] "Culture vessel" refers to a vessel for culturing cells. The culture vessel may be of any size as long as it can be used for culturing cells.

[0057] "Titer": The term "titer" is used herein to refer to the total amount of recombinantly expressed polypeptide produced by a cell culture divided by a given amount of culture medium volume. Titer is typically expressed in units of mg polypeptide / ml culture medium.

[0058] The terms "culture medium" and "cell culture medium" refer to a nutrient source used to grow or maintain cells. Those skilled in the art will appreciate that the nutrient source may contain components necessary for cell growth and / or survival, or may contain components that aid cell growth and / or survival. Vitamins, essential or non-essential amino acids, and trace elements are examples of culture medium components.

[0059] "Chemically defined cell culture medium" or "CDM" is a culture medium of defined composition that is free of products of animal origin, such as animal serum and peptone. The term also encompasses a culture medium of defined composition that is free of undefined or partially defined components (e.g., components such as animal serum, animal peptone, and plant peptone). It will be appreciated by those skilled in the art that a CDM can be used in a polypeptide production process, wherein cells are contacted with the CDM and the polypeptide is secreted into the CDM. Thus, it will be appreciated that a composition can contain a CDM and a polypeptide product, and the presence of the polypeptide product does not render the CDM chemically undefined.

[0060] "Chemically undefined cell culture medium" refers to a culture medium whose chemical composition cannot be clearly specified, which may contain one or more products of animal origin, such as animal serum and peptone. As will be appreciated by those skilled in the art, a chemically undefined cell culture medium may contain products of animal origin as a nutrient source. The term may also encompass cell culture media containing undefined or partially undefined components, such as, for example, components such as animal serum, animal peptone, and plant peptone.

[0061] The terms "polypeptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified by natural or human intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. Examples of polypeptides encompassed by this definition include: mammalian proteins, such as renin; growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1 antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors, such as factor VIIIC, factor IX, tissue factor, and von Willebrand factor; anticoagulant factors, such as protein C; atrial natriuretic peptide; pulmonary surfactant; plasminogen activators, such as Such as ureakinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factor; tumor necrosis factor α and β; enkephalinase; RANTES (regulator of activation expressed and secreted by normal T cells); human macrophage inflammatory protein (MIP-1α); serum albumin, such as human serum albumin; Mullerian inhibitory substance; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; microbial proteins, such as β-lactamase; DNA enzyme; IgE; cytotoxic T lymphocyte-associated antigen ( CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); hormone receptors or growth factor receptors; protein A or D; rheumatoid factor; neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophins 3, 4, 5, or 6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factors such as NGF-b; platelet-derived growth factor (PDGF); fibroblast growth factors such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF), For example, TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins (IGFBPs); CD proteins such as CD3, CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); interferons such as interferon α, β and γ;Colony stimulating factors (CSF), such as M-CSF, GM-CSF and G-CSF; interleukins (ILs), such as IL-1 to IL-10; superoxide dismutase; T cell receptors; surface membrane proteins; decay accelerating factors; viral antigens such as parts of the AIDS envelope; transport proteins; homing proteins; addressins; regulatory proteins; integrins, such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; tumor-associated antigens, such as CA125 (ovarian cancer antigen) or HER2, HER3 or HER4 receptors; immunoadhesins; and fragments and / or variants of any of the above proteins, and antibodies that bind to proteins (including, for example, any of the above proteins), including antibody fragments. ;

[0062] An "isolated polypeptide" is one that has been recovered from the cell or cell culture in which it was expressed.

[0063] "Nucleic acid", used interchangeably herein, refers to nucleotide polymers of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by a DNA or RNA polymer or by a synthetic reaction. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. Modifications to the nucleotide structure, if any, can occur before or after polymer assembly.

[0064] "Isolated nucleic acid" refers to and encompasses a non-naturally occurring, recombinant or naturally occurring sequence that is not in its normal environment or is separated from its normal environment.

[0065] A "purified" polypeptide is one that has been increased in purity so that it is present in a purer form than it is in its natural environment and / or when it was initially produced and / or synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily refer to absolute purity.

[0066] The term "antibody" is used in the broadest sense and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments.

[0067] "Antibody fragments" include portions of full-length antibodies, generally the antigen-binding or variable regions thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single-chain antibody molecules; diabodies; linear antibodies; and multispecific antibodies formed from antibody fragments.

[0068] The term "monoclonal antibody" is used herein to refer to antibodies obtained from a substantially homogeneous antibody population, that is, each individual antibody contained in the population is identical except for possible natural mutations (which may exist in a small number). Monoclonal antibodies are highly specific and are directed to a single antigenic site. In addition, unlike conventional (polyclonal) antibody preparations that typically include different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. The modifier "monoclonal" refers to the characteristic that an antibody is obtained from a substantially homogeneous antibody population, and should not be understood as requiring that the antibody be prepared in any particular manner. For example, the monoclonal antibodies used in accordance with the present disclosure may be prepared by the hybridoma method (first described by Kohler et al, Nature 256: 495 (1975)); or may be prepared by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described, for example, by Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0069] "Humanized" antibodies are chimeric antibody forms of non-human (e.g., rodent) antibodies that contain minimal sequences derived from the non-human antibodies. For the most part, humanized antibodies are human immunoglobulins (receptor antibodies), wherein residues from the hypervariable regions of the receptor are replaced with residues from the hypervariable regions of non-human species (donor antibodies) such as mice, rats, rabbits, or non-human primates, wherein the non-human species have desired antibody specificity, affinity, and ability. In some cases, the framework region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues. In addition, humanized antibodies may be included in residues that are not present in the receptor antibody or the donor antibody. These modifications may be performed to further improve antibody performance. Generally, humanized antibodies include at least one, typically two, substantially all of the variable regions, wherein all or substantially all of the hypervariable loops correspond to non-human immunoglobulins, and all or substantially all of the FRs are FRs of human immunoglobulin sequences. Humanized antibodies may optionally also include at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0070] A "species-dependent antibody" is an antibody that has a stronger binding affinity for an antigen from a first mammalian species than for a homolog of that antigen from a second mammalian species. Typically, a species-dependent antibody "binds specifically" (i.e., has no more than about 1x10 -7 M, not more than about 1x10 -8 M, or not more than approximately 1x10 -9 M), but the antibody has a binding affinity for a homolog of the antigen from a second non-human mammalian species that is at least about 50 times, or at least about 500 times, or at least about 1000 times weaker than its binding affinity for the human antigen. The species-dependent antibody may be any type of antibody as defined above, but is preferably a humanized or human antibody.

[0071] "Impurities" refer to substances that are different from the desired polypeptide product. Impurities include, but are not limited to: host cell materials, such as CHOP; leached protein A; nucleic acids; variants, fragments, aggregates or derivatives of the desired polypeptide; other polypeptides; endotoxins; viral contaminants; cell culture medium components, etc.

[0072] The term "pharmaceutical preparation" refers to a preparation which is in a form which permits the biological activity of the active ingredient to be effective and which contains no other ingredients which are unacceptably toxic to a subject to which the preparation is administered. Such preparations are sterile.

[0073] A "sterile" preparation is sterile, or free or substantially free of all viable microorganisms and their spores.

[0074] A "colorless or slightly colored" liquid refers to a liquid composition comprising a polypeptide that is measured by qualitative and / or quantitative analysis. Qualitative analysis includes visual inspection, such as comparing the composition comprising a polypeptide to a reference standard.

[0075] It is to be understood that whenever the expression "comprising" is used herein to describe an embodiment, similar embodiments described with the terms "consisting of" and / or "consisting essentially of are provided.

[0076] When aspects of the embodiments of the present invention are described in terms of Markush groups or other optional element groups, the present invention not only covers the entire group listed as a whole, as well as each member of the group individually and all possible subgroups in the main group, but also covers the main group with one or more of the components missing. The present invention also contemplates any one or more of the group members being explicitly excluded in the claimed invention.

[0077] Cell culture medium

[0078] The cell culture medium provided herein can be used in methods described herein (e.g., methods for growing cells (i.e., culturing cells) and producing polypeptides) and compositions. The culture medium components have been determined to provide polypeptide drug products with acceptable quality attributes, such as acceptable colors (e.g., for use as injectable drug products). Compared to polypeptides produced in different culture media, certain culture medium components reduce the color intensity of the polypeptide drug product, which may be particularly significant for polypeptide products prepared at a concentration greater than 100 mg / ml, 125 mg / ml, or 150 mg / ml. In some aspects, the polypeptide drug product has a concentration greater than at least 100 mg / ml, at least 125 mg / ml, or at least 150 mg / ml, and has a color intensity value greater than B3, B4, B5, B6, B7, B8, or B9 measured by the COC test. In some aspects, the polypeptide drug product has a concentration greater than at least 1 mg / ml, at least 10 mg / ml, at least 25 mg / ml, at least 50 mg / ml, or at least 75 mg / ml, and has a color intensity value greater than B3, B4, B5, B6, B7, B8, or B9 as measured by the COC test. In some aspects, the color intensity value determined by the COC test can be, but is not limited to, any of B, BY, Y, GY, or R, wherein a higher value indicates a lighter color intensity. In some aspects, the polypeptide drug product has a concentration greater than at least 100 mg / mL, at least 125 mg / mL, or at least 150 mg / mL, and has a color intensity value less than the color intensity value of a reference solution as measured by a color test (e.g., a total color test or a NIFTY test). In some respects, the polypeptide drug product has a concentration greater than at least 1 mg / ml, at least 10 mg / ml, at least 25 mg / ml, at least 50 mg / ml or at least 75 mg / ml, and has a color intensity value measured by a color test (e.g., total color test or NIFTY test) that is less than the color intensity value of a reference solution. Suitable cell culture media are described in detail throughout the specification, including the Summary of the Invention section and other places. Any culture medium described herein can be used for cell growth, maintenance and any stage of polypeptide production, and can be used in basal medium and / or feed medium. Culture medium described herein causes acceptable cell viability and antibody titer levels in a variant, and causes the polypeptide (e.g., antibody) separated from the cell culture grown in the culture medium to have acceptable color intensity.

[0079] The present invention provides a cell culture medium containing one or more of the following ingredients: (a) cystine and / or cysteine; (b) vitamin B2, (c) vitamin B6 (pyridoxine and / or pyridoxal), (d) vitamin B9, (e) vitamin B12, (f) an iron source such as ferric citrate, and (g) hydrocortisone. In a variant, the cell culture medium comprises 2 or 3 or 4 or 5 or 6 or each of the components (a), (b), (c), (d), (e), (f) and (g). It is understood that the cell culture medium provided herein may contain any combination of components (a), (b), (c), (d), (e), (f) and (g), just as if each and all combinations were specifically listed separately. For example, it is understood that a cell culture medium comprising 4 of the components (a), (b), (c), (d), (e), (f) and (g) may contain any combination of these components, as long as at least 4 of these components are present. In one aspect, the cell culture medium is CDM. On the other hand, cell culture medium is a chemically undefined cell culture medium.

[0080] The culture medium components can be added to the composition in a form known in the art. For example, vitamin B2 can be provided in the form of riboflavin powder, vitamin B6 can be provided in the form of pyridoxine HCl or in the form of pyridoxal HCl, vitamin B9 can be provided in the form of folic acid powder, vitamin B12 can be provided in the form of cyanocobalamin powder, cysteine ​​can be provided in the form of L-cysteine ​​monohydrochloride monohydrate powder, and cystine can be provided in the form of disodium salt monohydrate powder. In some embodiments, vitamin B6 is not provided in the form of pyridoxal HCl. In other non-limiting examples, vitamin B1 can be provided in the form of thiamine monohydrochloride, vitamin B3 can be provided in the form of nicotinamide, vitamin B5 can be provided in the form of D-calcium pantothenate, and vitamin B7 can be provided in the form of biotin. As another non-limiting example, iron can be added in different iron forms or iron sources. In some embodiments, the iron source is ferric citrate or ferrous sulfate. The culture medium components described herein can be provided in the form of salts, hydrates, salt hydrates, or in the form of solutions, extracts, or in solid forms.

[0081] In one variant, the culture medium comprises cystine and each of vitamin B2, B6, B9 and B12. In one variant, the culture medium comprises cystine, vitamin B2, B6, B9, B12 and an iron source such as ferric citrate. In another variant, cystine, vitamin B2, B6, B9, B12, an iron source such as ferric citrate and hydrocortisone are all included in the culture medium. In yet another variant, the culture medium comprises cystine, hydrocortisone and an iron source such as ferric citrate. In yet another variant, the culture medium comprises cystine, hydrocortisone, an iron source such as ferric citrate and vitamin B2, B6, B9 and B12 at least one. In yet another variant, the culture medium comprises cystine, hydrocortisone, an iron source such as ferric citrate and vitamin B2, B6, B9 and B12 at least 2 kinds. In yet another variant, the culture medium comprises cystine, hydrocortisone, an iron source such as ferric citrate and at least 3 of vitamins B2, B6, B9 and B12. In any culture medium described herein, on the one hand, the culture medium is CDM. On the one hand, the cell culture medium comprises cystine. In another variant, the cell culture medium comprises cystine but does not contain cysteine. In another variant, the cell culture medium comprises both cystine and cysteine.

[0082] In one variation, the culture medium is a cell culture medium comprising about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine, about 0.05 mg / L to about 1.0 mg / L vitamin B2, about 0.05 mg / L to about 10.0 mg / L vitamin B6, about 0.05 mg / L to about 12.0 mg / L vitamin B9, and about 0.05 mg / L to about 2.5 mg / L vitamin B12. In one variation, the concentration of vitamin B2 is about 0.05 mg / L to about 0.50 mg / L. In another variation, the concentration of vitamin B2 is about 0.05 mg / L to about 0.40 mg / L. In another variation, the concentration of vitamin B2 is about 0.05 mg / L to about 0.30 mg / L. In one variation, the concentration of vitamin B6 is about 0.05 mg / L to about 8.0 mg / L. In another variant, the concentration of vitamin B6 is about 0.05 mg / L to about 7.0 mg / L. In another variant, the concentration of vitamin B6 is about 0.05 mg / L to about 6.0 mg / L. In a variant, the cell culture medium comprises an iron source. In a variant, the iron source is ferric citrate or ferrous sulfate. In a variant, the cell culture medium comprises ferric citrate at a concentration of about 2 μM to about 80 μM. In any variant herein, the cell culture medium may also include hydrocortisone. In a variant, the concentration of hydrocortisone is about 0.05 μM to about 0.25 μM.

[0083] In another variation, the cell culture medium comprises one or more of: (a) about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine and / or cysteine ​​(which in one aspect is cystine); (b) about 0.05 mg / L to about 1.0 mg / L vitamin B2; (c) about 0.05 mg / L to about 10.0 mg / L vitamin B6 (which in one aspect is pyridoxine); (d) about 0.05 mg / L to about 12.0 mg / L vitamin B9; (e) about 0.05 mg / L to about 2.5 mg / L vitamin B12; (f) about 2 μM to about 80 μM iron source, such as ferric nitrate, ferric citrate, or ferric sulfate (which in one aspect is ferric citrate and / or ferrous sulfate); and (g) about 0.05 μM to about 0.25 μM hydrocortisone. In another variation, the cell culture medium comprises one or more of: (a) about 300 mg / L (in some embodiments, 200 mg / L) to about 600 mg / L cystine and / or cysteine ​​(which in one aspect is cystine); (b) about 0.05 mg / L to about 0.5 mg / L vitamin B2; (c) about 2.0 mg / L to about 8.0 mg / L vitamin B6 (which in one aspect is pyridoxine); (d) about 4.0 mg / L to about 12.0 mg / L vitamin B9; (e) about 1.0 to about 2.0 mg / L vitamin B12; (f) about 5 μM to about 25 μM iron source, such as ferric nitrate, ferric citrate or ferric sulfate (which in one aspect is ferric citrate and / or ferric sulfate); and (g) about 0.1 μM to about 0.2 μM hydrocortisone. In yet another variation, the cell culture medium comprises one or more of: (a) about 400 mg / L to about 500 mg / L cystine and / or cysteine ​​(which in one aspect is cystine); (b) about 0.1 mg / L to about 0.3 mg / L vitamin B2; (c) about 4.0 mg / L to about 6.0 mg / L vitamin B6 (which in one aspect is pyridoxine); (d) about 7.0 mg / L to about 10.0 mg / L vitamin B9; (e) about 1.5 to about 2.0 mg / L vitamin B12; (f) about 12 μM to about 20 μM iron source, such as ferric nitrate, ferric citrate or ferric sulfate (which in one aspect is ferric citrate and / or ferric sulfate); and (g) about 0.125 μM to about 0.175 μM hydrocortisone. In one variation, the cell culture medium comprises 2 or 3 or 4 or 5 or 6 or each of the components (a), (b), (c), (d), (e), (f) and (g) at the concentrations recited above. It will be appreciated that the cell culture medium can contain any combination of the components (a), (b), (c), (d), (e), (f) and (g) within the concentration ranges provided herein, as if each and every combination were specifically and individually listed.For example, it is understood that the culture medium may include components (a), (b), (c), (d) and (e) in one variant, and may optionally include components (f) and / or (g). It is also understood that in another variant, the culture medium may include components (a), (f) and (g), and may optionally include any one or more of components (b), (c), (d) and (e). In any variant in which the culture medium includes cystine or cysteine, on the one hand, the culture medium may include cystine (without cysteine ​​in a further variant). In any variant in which the culture medium includes vitamin B6, on the one hand, the culture medium may include pyridoxane. In any variant in which the culture medium includes an iron source, on the one hand, the iron source may be ferric citrate. Therefore, it is understood that in some variants, the culture medium may include cystine, pyridoxine and ferric citrate. On the one hand, the cell culture medium is CDM.

[0084] In one variation, the culture medium comprises (a) about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; (b) about 0.05 mg / L to about 1.0 mg / L vitamin B2; (c) about 0.05 mg / L to about 10.0 mg / L vitamin B6; (d) about 0.05 mg / L to about 12.0 mg / L vitamin B9; and (e) about 0.05 mg / L to about 2.5 mg / L vitamin B12. In one variation, the culture medium comprises (a) about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; (b) about 0.05 mg / L to about 1.0 mg / L vitamin B2; (c) about 0.05 mg / L to about 10.0 mg / L vitamin B6; (d) about 0.05 mg / L to about 12.0 mg / L vitamin B9; (e) about 0.05 mg / L to about 2.5 mg / L vitamin B12; and (f) about 2 μM to about 80 μM iron source, such as ferric citrate or ferrous sulfate. In another variation, the culture medium comprises each of the following components: (a) about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; (b) about 0.05 mg / L to about 1.0 mg / L vitamin B2; (c) about 0.05 mg / L to about 10.0 mg / L vitamin B6; (d) about 0.05 mg / L to about 12.0 mg / L vitamin B9; (e) about 0.05 mg / L to about 2.5 mg / L vitamin B12; (f) about 2 μM to about 80 μM iron source, such as ferric citrate or ferrous sulfate; and (g) about 0.05 μM to about 0.25 μM hydrocortisone. In yet another embodiment, the culture medium comprises: about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; about 2 μM to about 80 μM iron source, such as ferric citrate or ferrous sulfate; and about 0.05 μM to about 0.25 μM hydrocortisone. In yet another variant, the culture medium comprises: about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; about 2 μM to about 80 μM iron source, such as ferric citrate or ferrous sulfate; about 0.05 μM to about 0.25 μM hydrocortisone; and at least one or two or three of the following ingredients: about 0.05 mg / L to about 1.0 mg / L vitamin B2; about 0.05 mg / L to about 10.0 mg / L vitamin B6; about 0.05 mg / L to about 12.0 mg / L vitamin B9; and about 0.05 mg / L to about 2.5 mg / L vitamin B12. In any culture medium described herein, on the one hand, the culture medium is CDM. In any culture medium described herein, on the one hand, the culture medium is a chemically undefined cell culture medium.

[0085] In some other variations, the cell culture medium further comprises cysteine ​​in an amount as described in Table 1. For example, it is understood that a cell culture medium comprising (a) about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; (b) about 0.05 mg / L to about 1.0 mg / L vitamin B2; (c) about 0.05 mg / L to about 10.0 mg / L vitamin B6; (d) about 0.05 mg / L to about 12.0 mg / L vitamin B9; and (e) about 0.05 mg / L to about 2.5 mg / L vitamin B12 may further comprise about 80 mg / L to about 1500 mg / L cysteine. In one variation, a cell culture medium comprising (a) about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; (b) about 0.05 mg / L to about 1.0 mg / L vitamin B2; (c) about 0.05 mg / L to about 10.0 mg / L vitamin B6; (d) about 0.05 mg / L to about 12.0 mg / L vitamin B9; (e) about 0.05 mg / L to about 2.5 mg / L vitamin B12; and (f) about 2 μM to about 80 μM iron source, such as ferric citrate or ferrous sulfate, may further comprise about 80 mg / L to about 1500 mg / L cysteine. In some variations, a cell culture medium comprising (a) about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; (b) about 0.05 mg / L to about 1.0 mg / L vitamin B2; (c) about 0.05 mg / L to about 10.0 mg / L vitamin B6; (d) about 0.05 mg / L to about 12.0 mg / L vitamin B9; (e) about 0.05 mg / L to about 2.5 mg / L vitamin B12; (f) about 2 μM to about 80 μM iron source, such as ferric citrate or ferrous sulfate; and (g) about 0.05 μM to about 0.25 μM hydrocortisone may further comprise about 80 mg / L to about 1500 mg / L cysteine. In yet another variation, a cell culture medium comprising about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; about 2 μM to about 80 μM iron source, such as ferric citrate or ferrous sulfate; about 0.05 μM to about 0.25 μM hydrocortisone; and at least one or two or three of the following ingredients: about 0.05 mg / L to about 1.0 mg / L vitamin B2; about 0.05 mg / L to about 10.0 mg / L vitamin B6; about 0.05 mg / L to about 12.0 mg / L vitamin B9; and about 0.05 mg / L to about 2.5 mg / L vitamin B12, may further comprise about 80 mg / L to about 1500 mg / L cysteine.

[0086] Each culture medium component can be present in an amount that results in one or more beneficial properties (e.g., one or more acceptable product quality attributes). In one variant, the cell culture medium provided herein comprises the culture medium components of the amounts described in Table 1. It is understood that the culture medium can comprise any one or more culture medium components in Table 1 (e.g., any one or more of components (a)-(g), such as a culture medium comprising components (a), (b), (c), (d) and (e) or a culture medium comprising components (a), (f) and (g) or a culture medium comprising each of components (a)-(g)) in any amount listed in Table 1, as if each and all combinations of components and amounts were specifically listed separately. In a specific variant, the culture medium is CDM. In another specific variant, the culture medium is a chemically undefined cell culture medium. The culture medium (e.g., CDM) provided herein comprises pyridoxine in a variant and does not contain pyridoxal. A culture medium without pyridoxal can be used in a basal medium and / or a feed medium. In a variant, the basal medium does not contain pyridoxal and contains pyridoxine.

[0087] Table 1. Exemplary amounts of media components

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094] In some aspects, the present invention provides a cell culture medium comprising one or more of the following ingredients selected from the group consisting of: (a) vitamin B1; (b) vitamin B2; (c) vitamin B3; (d) vitamin B5; (e) vitamin B6; (f) vitamin B7; (g) vitamin B9; (h) vitamin B12; (i) an iron source such as ferric citrate; and (j) cystine. In some embodiments, the cell culture medium comprises 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or each of the ingredients (a), (b), (c), (d), (e), (f), (g), (h), (i) and (j). It is understood that the cell culture medium provided herein may contain any combination of the ingredients (a), (b), (c), (d), (e), (f), (g), (h), (i) and (j), just as if each and all combinations were specifically listed individually. For example, it is understood that a cell culture medium comprising eight of components (a), (b), (c), (d), (e), (f), (g), and (h) may comprise any combination of these components, as long as at least eight of these components are present. In some embodiments, the cell culture medium provided herein comprises components (b), (e), (g), (h), and (j). In some embodiments herein, the cell culture medium provided herein comprising components (b), (e), (g), (h), and (j) further comprises (a), (c), (d), and (f). In some embodiments herein, the cell culture medium provided herein comprising components (a), (b), (c), (d), (e), (f), (g), (h), and (j) further comprises (i).

[0095] In some aspects, the cell culture medium provided herein contains one or more culture medium components selected from the group consisting of: (a) vitamin B1; (b) vitamin B2; (c) vitamin B3; (d) vitamin B5; (e) vitamin B6; (f) vitamin B7; (g) vitamin B9; (h) vitamin B12; (i) an iron source such as ferric citrate; and (j) cystine in amounts as described in Table 1A. It is understood that the culture medium can contain any one or more of the culture medium components of Table 1A (e.g., any one or more of components (a)-(j), such as a culture medium comprising components (a), (b), (c), (d), (e), (f), (g), (h) and (i), or a culture medium comprising components (b), (e), (g), (h) and (j), or a culture medium comprising only one of components (a)-(j)) in any amount listed in Table 1A, as if each and all combinations of components and amounts were specifically listed individually. In some aspects, the cell culture medium comprises components (a), (b), (c), (d), (e), (f), (g), (h), and (i), wherein (a) is about 2 μM to about 14 μM vitamin B1, (b) is about 0.11 μM to about 0.72 μM vitamin B2, (c) is about 11 μM to about 72 μM vitamin B3, (d) is about 6.8 μM to about 44 μM vitamin B5, (e) is about 4.5 μM to about 30 μM vitamin B6, (f) is about 0.02 μM to about 0.14 μM vitamin B7, (g) is about 3.4 μM to about 22 μM vitamin B9, (h) is about 0.2 μM to about 1.5 μM vitamin B12, (i) is about 11 μM to about 36 μM ferric citrate, and (j) is about 0.9 mM to about 1.5 mM cystine.

[0096] In some other aspects, the cell culture medium further comprises cysteine ​​in an amount as described in Table 1A. For example, it is understood that a cell culture medium comprising (a) from about 2 μM to about 14 μM vitamin B1, (b) from about 0.11 μM to about 0.72 μM vitamin B2, (c) from about 11 μM to about 72 μM vitamin B3, (d) from about 6.8 μM to about 44 μM vitamin B5, (e) from about 4.5 μM to about 30 μM vitamin B6, (f) from about 0.02 μM to about 0.14 μM vitamin B7, (g) from about 3.4 μM to about 22 μM vitamin B9, (h) from about 0.2 μM to about 1.5 μM vitamin B12, (i) from about 11 μM to about 36 μM ferric citrate, and (j) from about 0.7 mM to about 2.0 mM cystine, may also comprise (k) from about 0.5 mM to about 2.0 mM cysteine.

[0097] Table 1A. Exemplary amounts of media components

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] The culture medium provided herein (e.g., CDM or chemically undefined culture medium) comprises cystine and does not contain cysteine ​​in a variant. The culture medium without cysteine ​​can be used in a basal medium or a feed medium. In a variant, the basal medium does not contain cysteine ​​but contains cystine. In a variant, a basal medium containing from about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine; from about 0.05 mg / L to about 1.0 mg / L vitamin B2; from about 0.05 mg / L to about 10.0 mg / L vitamin B6 (which is pyridoxine on the one hand); from about 0.05 mg / L to about 12.0 mg / L vitamin B9; and from about 0.05 mg / L to about 2.5 mg / L vitamin B12, which can be used in a basal medium. The basal medium further comprises any one or more of: (1) vitamin B1 (which is present in a concentration of from about 2.0 μM to about 14.0 μM in one aspect), (2) vitamin B3 (which is present in a concentration of from about 11.0 μM to about 72.0 μM in one aspect), (3) vitamin B5 (which is present in a concentration of from about 6.8 μM to about 44.0 μM in one aspect), and (4) vitamin B7 (which is present in a concentration of from about 0.02 μM to about 0.24 μM in one aspect), and the basal medium does not contain cysteine. In another variation, a basal medium comprising from about 0.8 mM (in some embodiments, 0.7 mM) to about 2.5 mM cystine; from about 0.11 μM to about 0.72 μM vitamin B2; from about 4.5 μM to about 30 μM vitamin B6 (which in one aspect is pyridoxine); from about 3.4 μM to about 22 μM vitamin B9; and from about 0.2 μM to about 1.5 μM vitamin B12, may further comprise any one or more of the following in one aspect: : (1) vitamin B1 (which is present in a concentration from about 2.0 μM to about 14.0 μM on the one hand), (2) vitamin B3 (which is present in a concentration from about 11.0 μM to about 72.0 μM on the one hand), (3) vitamin B5 (which is present in a concentration from about 6.8 μM to about 44.0 μM on the one hand), and (4) vitamin B7 (which is present in a concentration from about 0.02 μM to about 0.24 μM on the one hand), the basal medium does not contain cysteine. In any variant scheme herein, the basal medium may further include an iron source, such as ferric citrate or ferrous sulfate (which is present in a concentration from about 11.0 μM to about 36.0 μM on the one hand). In any variant scheme herein, the basal medium may also include hydrocortisone (which is present in a concentration from about 0.05 μM to about 0.5 μM on the one hand).

[0105] Substratum provided herein (e.g., CDM or chemically undefined substratum) comprises cysteine ​​in a variant scheme, does not contain cystine. Substratum without cystine can be used in basal medium or feed medium. In a variant scheme, feed medium does not contain cystine and comprises cysteine. In a variant scheme, feed medium comprises from about 80mg / L to about 1500mg / L cysteine. In another variant scheme, feed medium comprises from about 0.5mM to about 2.0mM cysteine. For example, a basal medium containing from about 300 mg / L (in some aspects, 200 mg / L) to about 1200 mg / L cystine; from about 0.05 mg / L to about 1.0 mg / L vitamin B2; from about 0.05 mg / L to about 10.0 mg / L vitamin B6 (which in one aspect is pyridoxine); from about 0.05 mg / L to about 12.0 mg / L vitamin B9; and from about 0.05 mg / L to about 2.5 mg / L vitamin B12, in one aspect, may further comprise any one or more of the following: (1) vitamin B1 (which in one aspect is from about 2. In one aspect, the basal medium may be supplemented with a feed medium comprising from about 80 mg / L to about 1500 mg / L cysteine ​​(in some aspects, 0.5 mM to about 2.0 mM cysteine). In another variation, the basal medium comprising from about 0.8 mM (in some aspects, 0.7 mM) to about 2.5 mM cystine; from about 0.11 μM to about 0.72 μM vitamin B2; from about 4.5 μM to about 30 μM vitamin B6 (which in one aspect is pyridoxine); from about 3.4 μM to about 22 μM vitamin B9; and from about 0.2 μM to about 1.5 μM vitamin B12, may further comprise any one or more of the following in one aspect: (1) vitamin B1 (which in one aspect is from about 2.0 μM to about 14.0 μM In one aspect, the basal medium may be supplemented with a feed medium comprising from about 80 mg / L to about 1500 mg / L cysteine ​​(in some aspects, 0.5 mM to about 2.0 mM cysteine).In any variation herein, the basal medium may also include a source of iron, such as ferric citrate or ferrous sulfate (which are present in a concentration of from about 11.0 μM to about 36.0 μM on the one hand). In any variation herein, the basal medium may also include hydrocortisone (which is present in a concentration of from about 0.05 μM to about 0.5 μM on the one hand).

[0106] Substratum provided herein (e.g., CDM or chemically undefined substratum) comprises ferric citrate in a variant scheme and does not contain ferrous sulfate. Substratum not containing ferrous sulfate can be used for basal medium or feed medium. In a variant scheme, basal medium does not contain ferrous sulfate and contains ferric citrate.

[0107] In a specific variation, the culture medium provided herein does not contain cysteine ​​and ferrous sulfate. In such a variation, the culture medium does not contain cysteine ​​and ferrous sulfate, but contains cystine and / or ferric citrate.

[0108] Substratum provided herein does not contain hydrocortisone in a variant. In another variant, substratum comprises hydrocortisone. On the one hand, substratum comprises hydrocortisone, does not contain cysteine ​​and / or ferrous sulfate. On the other hand, substratum comprises hydrocortisone and cystine and ferric citrate. In a specific variant, the substratum comprising hydrocortisone is a basal medium. In one variation, the basal medium comprises from about 0.8 mM (in some aspects, 0.7 mM) to about 2.5 mM cystine; from about 2 μM to about 80 μM ferric citrate; and from about 0.05 μM to about 0.5 μM hydrocortisone, wherein the basal medium may further comprise one or more of the following components: (1) vitamin B2 (which in one aspect is present at a concentration of from about 0.05 mg / L to about 1.0 mg / L); (2) vitamin B6 (which in one aspect is present at a concentration of from about 0.05 mg / L to about 10.0 mg / L); (3) vitamin B9 (which in one aspect is present at a concentration of from about 0.05 mg / L to about 12.0 mg / L); and (4) vitamin B12 (which in one aspect is present at a concentration of from about 0.05 mg / L to about 2.5 mg / L). In another variation, the basal medium comprises from about 300 mg / L (in some aspects, 200 mg / L) to about 1200 mg / L cystine; from about 2 μM to about 80 μM ferric citrate; and from about 0.05 μM to about 0.5 μM hydrocortisone, and wherein the basal medium may further comprise one or more of the following components: (1) vitamin B2 (which in one aspect is present at a concentration of from about 0.05 mg / L to about 1.0 mg / L); (2) vitamin B6 (which in one aspect is present at a concentration of from about 0.05 mg / L to about 10.0 mg / L); (3) vitamin B9 (which in one aspect is present at a concentration of from about 0.05 mg / L to about 12.0 mg / L); and (4) vitamin B12 (which in one aspect is present at a concentration of from about 0.05 mg / L to about 2.5 mg / L). In any of the variations herein, the basal medium may further comprise any one or more of: (1) vitamin B1 (which in one aspect is present at a concentration of from about 2.0 μM to about 14.0 μM), (2) vitamin B3 (which in one aspect is present at a concentration of from about 11.0 μM to about 72.0 μM), (3) vitamin B5 (which in one aspect is present at a concentration of from about 6.8 μM to about 44.0 μM), and (4) vitamin B7 (which in one aspect is present at a concentration of from about 0.02 μM to about 0.24 μM).

[0109] The present invention also provides a method for preparing a cell culture medium for culturing cells, wherein the method comprises: mixing any one or more culture medium components selected from the following group: (a) cystine and / or cysteine; (b) vitamin B2, (c) vitamin B6 (pyridoxine and / or pyridoxal), (d) vitamin B9, (e) vitamin B12, (f) an iron source such as ferric citrate and (g) hydrocortisone, wherein (a)-(g) are all provided in the amounts described in Table 1. The present invention also provides a method for preparing a cell culture medium for culturing cells, wherein the method comprises combining any one or more culture medium components selected from the following group in the amounts described in Table 1A: (a) vitamin B1; (b) vitamin B2; (c) vitamin B3; (d) vitamin B5; (e) vitamin B6; (f) vitamin B7; (g) vitamin B9; (h) vitamin B12; (i) an iron source such as ferric citrate; (j) cystine; and (k) cysteine. In one variation, the method includes adding any one or more culture medium components (e.g., Table 1 or Table 1A) described herein to a composition suitable for cell culture, wherein the one or more culture medium components can be added to the composition sequentially or simultaneously. In yet another variation, the method includes combining any one or more culture medium components (e.g., Table 1 or Table 1A) described herein in a composition suitable for cell culture in a first time period, wherein the method also includes adding a certain amount of one or more culture medium components in a second time period, such as at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, etc. in a cell culture cycle. In some embodiments, the cell culture cycle is at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or any number of days, wherein the cells can be maintained in cell culture and still remain viable. In one variation of the method of preparing a cell culture medium for culturing cells, cystine is added in an amount to provide about 300 mg / L (in some aspects, 200 mg / L) to about 1200 mg / L cystine in the cell culture medium, vitamin B2 is added in an amount to provide about 0.05 mg / L to about 1.0 mg / L vitamin B2 in the cell culture medium, vitamin B6 is added in an amount to provide about 0.05 mg / L to about 10.0 mg / L vitamin B6 in the cell culture medium, vitamin B9 is added in an amount to provide about 0.05 mg / L to about 12.0 mg / L vitamin B9 in the cell culture medium, and vitamin B12 is added in an amount to provide about 0.05 mg / L to about 2.5 mg / L vitamin B12 in the cell culture medium.In another variation of the method of preparing a cell culture medium for culturing cells, cystine is added in an amount to provide about 0.8 mM (in some methods, 0.7 mM) to about 2.5 mM cystine in the cell culture medium, vitamin B2 is added in an amount to provide about 0.11 μM to about 0.72 μM vitamin B2 in the cell culture medium, vitamin B6 is added in an amount to provide about 4.5 μM to about 30.0 μM vitamin B6 in the cell culture medium, vitamin B9 is added in an amount to provide about 3.4 μM to about 22.0 μM vitamin B9 in the cell culture medium, and vitamin B12 is added in an amount to provide about 0.2 μM to about 1.5 μM vitamin B12 in the cell culture medium.

[0110] In some variants herein, the cell culture medium is a basal cell culture medium. In other variants herein, the cell culture medium is a feed cell culture medium. In some variants herein, the cell culture medium is a basal cell culture medium, which comprises any one or more culture medium components selected from the group consisting of (a) cystine, (b) vitamin B2, (c) vitamin B6 (pyridoxine and / or pyridoxal), (d) vitamin B9, (e) vitamin B12, (f) iron source such as ferric citrate, and (g) hydrocortisone, wherein the basal culture medium supplements (e.g., in the time period after the start of the cell culture cycle, such as at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times in the cell culture cycle, etc.) a feed cell culture medium, wherein the feed cell culture medium comprises (a) cysteine ​​in the amount described in Table 1. In some variations herein, the cell culture medium is a basal cell culture medium comprising any one or more culture medium components selected from the group consisting of: (a) vitamin B1; (b) vitamin B2; (c) vitamin B3; (d) vitamin B5; (e) vitamin B6; (f) vitamin B7; (g) vitamin B9; (h) vitamin B12; (i) an iron source such as ferric citrate; and (j) cystine in the amounts described in Table 1A, wherein the basal cell culture medium is supplemented (e.g., within a period of time after the start of a cell culture cycle, such as any of at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, etc. during the cell culture cycle) with a feed cell culture medium comprising (k) cysteine ​​in the amounts described in Table 1A.

[0111] Those skilled in the art will appreciate that the cell culture medium described herein can (e.g., in addition to cystine, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, and vitamin B12, iron, and optionally one or more of hydrocortisone) contain other ingredients that can be used for cell culture. For example, it is understood that the cell culture medium can contain other ingredients, such as amino acids (e.g., glutamine, arginine, or asparagine), vitamins (including but not limited to ascorbic acid), trace elements, transition metals (including but not limited to nickel, copper or zinc), and other culture medium ingredients, such as, but not limited to, hydrolyzates derived from animals and / or plants. Any culture medium provided herein can also be supplemented with hormones and / or other growth factors (e.g., insulin, transferrins, or epidermal growth factor), ions (e.g., sodium, chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), and glucose or equivalent energy sources. Other cell culture medium components, such as those listed herein, can be included in the cell culture medium at various times during the cell culture cycle at appropriate concentrations, as will be apparent to those skilled in the art.

[0112] Compared with the quality attributes of polypeptides produced in different culture media, the culture media provided herein, when used in the polypeptide production method, causes one or more favorable product quality attributes on the one hand. The protein product (e.g., antibody product) produced with the charge variant distribution of change can affect the quality attributes of the protein product, such as the color of the protein product. In addition, the reactive oxygen (ROS) formed when using certain culture media components can oxidize specific amino acids to produce oxidized products. The presence of these product variants may also change the quality attributes of the protein product, such as color. The color of the composition (including the composition containing at least 100mg / ml, or 125mg / ml, or 150mg / ml polypeptides such as antibodies) comprising the polypeptide produced using the culture media described herein, on the one hand, has the color reference standard value described in Table 2. In certain variations, the color of a composition comprising a polypeptide produced using a culture medium as described herein (including a composition comprising at least 100 mg / ml, or 125 mg / ml, or 150 mg / ml polypeptide, such as an antibody) has, on the one hand, a color reference standard value selected from the group consisting of B3, B4, B5, B6, B7, B8, B9, BY3, BY4, BY5, BY6, BY7, Y3, Y4, Y5, Y6, Y7, GY3, GY4, GY5, GY6, GY7, R3, R4, R5, R6, and R7. On the other hand, the color of a composition comprising a polypeptide produced using a culture medium as described herein (including a composition comprising at least 1 mg / ml, or 25 mg / ml, or 50 mg / ml, or 75 mg / ml polypeptide, such as an antibody) has, on the one hand, a color reference standard value as described in Table 2. In some variations, the color of a composition comprising a polypeptide produced using the culture medium described herein (including compositions comprising at least 1 mg / ml, or 25 mg / ml, or 50 mg / ml or 75 mg / ml of a polypeptide, such as an antibody) has, on the one hand, a color reference standard value selected from the group consisting of: B3, B4, B5, B6, B7, B8, B9, BY3, BY4, BY5, BY6, BY7, Y3, Y4, Y5, Y6, Y7, GY3, GY4, GY5, GY6, GY7, R3, R4, R5, R6 and R7. For description of the color reference values ​​brown (B), brownish yellow (BY), yellow (Y), greenish yellow (GY) or red (R), see USP-24 Monograph 631 Color and Achromaticity. United States Pharmacopoeia Inc., 2000, p. 1926-1927, and Council of Europe. European Pharmacopoeia, 2008, 7th Ed. P. 22.In one variation, the culture medium provided herein reduces the presence of charge variants (e.g., acidic charge variants) when used in a polypeptide production method, compared to charge variants (e.g., acidic charge variants) obtained when producing polypeptides in different culture media. In another variation, the culture medium of the present invention reduces the presence of reactive oxygen species when used in a polypeptide production method, compared to reactive oxygen species obtained when producing polypeptides in different culture media. In another variation, the culture medium of the present invention reduces the presence of impurities when used in a polypeptide production method, compared to impurities obtained when producing polypeptides in different culture media.

[0113] As described herein, the invention provides various methods (eg, methods of culturing cells and methods of producing polypeptides) that use the cell culture media described in this section and elsewhere.

[0114] method

[0115] The cell culture media described herein (including any CDM or chemically undefined media described herein) can be used to culture cells for methods of producing polypeptides (including specific antibodies). The culture media can be used in methods of culturing (whether by batch culture, fed-batch culture or perfusion culture) cells, and can be used in methods of producing antibodies (including any aspects or variants or embodiments of the antibodies described herein).

[0116] The present invention provides a method of growing cells (i.e., culturing cells) by contacting the cells with a cell culture medium described herein. In one variant, the method comprises contacting the cells with a cell culture medium comprising one or more culture medium components described in Table 1 (e.g., the culture medium comprises components (a), (b), (c), (d) and (e) in any amount listed in Table 1, or the culture medium comprises components (a), (f) and (g), or the culture medium comprises each of components (a)-(g)). In one variant, the method comprises contacting the cells with a cell culture medium comprising one or more culture medium components described in Table 1A (e.g., the culture medium comprises components (a)-(j) in any amount listed in Table 1A, or the culture medium comprises components (a), (b), (c), (d), (e), (f), (g), (h) and (i), or the culture medium comprises components (b), (e), (g), (h) and (j), or the culture medium comprises only one of components (a)-(j)). In a specific variant of the method of growing cells (i.e., culturing cells), the cell culture medium is CDM. In one aspect of the method, cells are grown (i.e., cultured) in CDM basal medium. In another specific variant of the method for growing cells (i.e., culturing cells), the cell culture medium is a chemically undefined cell culture medium. In one aspect of the method, cells are grown (i.e., cultured) in a chemically undefined basal cell culture medium. In some aspects, cells are contacted with the cell culture medium in the cell growth phase. In some aspects, cells are contacted with the cell culture medium in the cell production phase. In some aspects, the method further comprises the step of adding cysteine ​​to the cell culture medium. In yet another aspect, cysteine ​​is added in an amount providing about 80 mg / L to about 1500 mg / L cysteine ​​in the cell culture medium. In another aspect, cysteine ​​is added in an amount providing about 1500 mg / L cysteine ​​in the cell culture medium. In yet another aspect, cysteine ​​is added in an amount providing about 140 mg / L cysteine ​​in the cell culture medium. In another aspect, cysteine ​​is added in an amount providing about 0.5 mM to about 2.0 mM cysteine ​​in the cell culture medium. In still other aspects, cysteine ​​is added in an amount to provide about 0.8 mM cysteine ​​in the cell culture medium.

[0117] The invention also provides methods for producing a polypeptide by growing (i.e., culturing in a cell culture medium) cells in a cell culture medium, wherein the cells contain an isolated nucleic acid encoding the polypeptide, wherein: (a) the cells express the polypeptide, and (b) the chemically defined cell culture medium contains one or more of the culture medium components described in Table 1 (e.g., the culture medium contains components (a), (b), (c), (d) and (e), or the culture medium contains components (a), (f) and (g), or the culture medium contains each of components (a)-(g) in any amount listed in Table 1). In another variation, a method for producing a polypeptide by growing (i.e., culturing) cells in a cell culture medium is provided, wherein the cells contain an isolated nucleic acid encoding the polypeptide, wherein: (a) the cells express the polypeptide, and (b) the cell culture medium contains one or more of the culture medium components described in Table 1A (e.g., the culture medium contains components (a)-(j), or the culture medium contains components (a), (b), (c), (d), (e), (f), (g), (h) and (i), or the culture medium contains components (b), (e), (g), (h) and (j), or the culture medium contains only one of components (a)-(j) in any amount listed in Table 1A). In a specific variation of the method for producing a polypeptide by growing (i.e., culturing in a cell culture medium) cells containing an isolated nucleic acid encoding the polypeptide in a cell culture medium, the cell culture medium is CDM. In one aspect of the method, the cells are grown (i.e., cultured) in a CDM basal medium. In another specific variant of the method for producing a polypeptide by growing (i.e., culturing in a cell culture medium) a cell containing an isolated nucleic acid encoding a polypeptide in a cell culture medium, the cell culture medium is a chemically undefined cell culture medium. In one aspect of the method, the cell is grown (i.e., cultured) in a chemically undefined basal cell culture medium. In some aspects, the culture is carried out in a cell growth phase. In some aspects, the culture is carried out in a cell production phase. In some variants, the method further comprises the step of adding cysteine ​​to the cell culture medium. In a further variant, cysteine ​​is added in an amount providing about 80 mg / L to about 1500 mg / L cysteine ​​in the cell culture medium. In another further variant, cysteine ​​is added in an amount providing about 1500 mg / L cysteine ​​in the cell culture medium. In yet another further variant, cysteine ​​is added in an amount providing about 140 mg / L cysteine ​​in the cell culture medium. In another further variant, cysteine ​​is added in an amount providing about 0.5 mM to about 2.0 mM cysteine ​​in the cell culture medium. In yet a further variation, cysteine ​​is added in an amount to provide about 0.8 mM cysteine ​​in the cell culture medium.

[0118] The present invention also provides a method for applying polypeptides described herein. For example, a method for applying a preparation comprising a polypeptide to an individual is provided, wherein the preparation has a concentration greater than at least 100 mg / mL, at least 125 mg / mL or at least 150 mg / mL of polypeptide, and has a color intensity value greater than B3, B4, B5, B6, B7, B8 or B9 measured by the COC test. In some aspects, the color intensity value measured by the COC test can be, but not limited to, any one of B, BY, Y, GY or R, wherein the higher value indicates a lighter color intensity. In another example, a method for applying a preparation comprising a polypeptide to an individual is provided, wherein the preparation has a concentration greater than at least 100 mg / mL, at least 125 mg / mL or at least 150 mg / mL of polypeptide, and has a color intensity value less than the color intensity value of a reference solution measured by a color test (e.g., total color test or NIFTY test). The polypeptide preparation can be applied in any suitable manner, including parenteral, intrapulmonary and intranasal, and if desired topical treatment, applied within the injury. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration.Administration can be carried out by any suitable approach, for example, by injection, for example intravenous or subcutaneous injection, and this part depends on whether administration is short-lived or long-term.Therefore, provided herein is polypeptide preparation containing and can be suitable for injection, for example, subcutaneous injection to individual (for example, subcutaneous injection to human body).In some respects, polypeptide preparation containing suitable for injection (for example, suitable for subcutaneous injection) has the concentration greater than at least 100mg / mL, at least 125mg / mL or at least 150mg / mL, and has the color intensity value greater than B3, B4, B5, B6, B7, B8 or B9 by COC test determination.In some respects, the color intensity value by COC test determination can be, but not limited to, any one of B, BY, Y, GY or R, wherein higher value indicates lighter color intensity. In some respects, the polypeptide preparation suitable for injection (e.g., suitable for subcutaneous injection) has a concentration greater than at least 100 mg / mL, at least 125 mg / mL, or at least 150 mg / mL, and has a color intensity value less than the color intensity value of a reference solution measured by a color test (e.g., total color test or NIFTY test). The present invention contemplates various dosing regimens, including, but not limited to, single administration or multiple administrations at different time points, bolus administration, and pulse infusion.

[0119] Other methods are provided throughout this specification, for example, in the Summary of the Invention section and elsewhere.

[0120] cell

[0121] The methods and compositions provided herein can use any cell suitable for growing and / or producing polypeptides (e.g., antibodies) in culture medium described herein, including animals, yeast, or insect cells. On the one hand, the cells of the methods and compositions are any mammalian cells or cell types suitable for cell culture and expression of polypeptides. The methods (e.g., methods for growing cells (i.e., culturing cells) and / or producing polypeptides) and compositions provided herein can therefore use any suitable cell type, including animal cells. On the one hand, the methods and compositions use mammalian cells. The methods and compositions can also use hybridoma cells. In a variant, the mammalian cell is a non-hybridoma mammalian cell that has been transformed with an external isolated nucleic acid encoding a desired polypeptide, such as an antibody, an antibody fragment (including a ligand-binding fragment), and a chimeric antibody. In one variation, the methods and compositions utilize mammalian cells selected from the group consisting of: human retinoblasts (PER.C6 (CruCell, Leiden, The Netherlands)); SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse supporting cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical cancer cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2). In a specific variant, the methods and compositions use CHO cells. In a specific variant, the CHO cell line is cultured and the polypeptide (e.g., antibody) is expressed from the CHO cell line.The polypeptide (eg, antibody) can be secreted into the culture medium (eg, CDM) from which it can be isolated and / or purified, or it can be released into the culture medium by lysing cells containing an isolated nucleic acid encoding the polypeptide.

[0122] Suitable methods, vectors and host cells adapted for synthesizing polypeptides of interest in recombinant vertebrate cell culture are known in the art and described, for example, in Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); Levinson et al.; EP 117,060; and EP 117,058. A plasmid particularly useful for expressing polypeptides in mammalian culture is pRK5 (European Patent Publication No. 307,247) or pSVI6B (PCT Publication No. WO 91 / 08291, published on June 13, 1991).

[0123] Host cells are transformed with expression or cloning vectors and cultured in a nutrient medium modified to induce promoters, select transformants, or amplify genes encoding the desired sequence. For mammalian cells, the calcium phosphate precipitation method of Graham and van der Erb, Virology, 52:456-457 (1978), or the lipofectamine method of Hawley-Nelson, Focus 15:73 (193) can be used. .TM. (Gibco BRL) method is preferred. General aspects of mammalian cell host system transformation are known in the art and have been described, for example, in U.S. Patent No. 4,399,216 to Axel (granted on August 16, 1983). For various techniques for transforming mammalian cells, see, for example, Keown et al., Methods in Enzymology (1989), Keown et al., Methods in Enzymology, 185:527-537 (1990), and Mansour et al., Nature, 336:348-352 (1988).

[0124] Methods and compositions also encompass the use of hybridomas that secrete monoclonal antibodies into cell culture. Monoclonal antibodies can be prepared by recovering immune cells (typically spleen cells or lymphocytes from lymph node tissue) from immunized animals, immortalizing immune cells in a conventional manner, such as by fusing with myeloma cells or by Epstein-Barr virus transformation, and screening clones expressing the desired antibody. Hybridoma technology was originally described by Kohler and Milstein, Eur. J. Immunol., 6: 511 (1976), and is also described in Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, NY, pp. 563-681 (1981), which has been widely used to produce hybrid cell lines that can secrete high levels of monoclonal antibodies against many specific antigens.

[0125] Peptides

[0126] The polypeptide produced by the compositions (cells) and methods described herein and present in the compositions provided herein can be homologous to the host cell, or preferably can be exogenous, i.e., for the host cell used, it is heterologous (i.e., foreign), such as human proteins produced by Chinese hamster ovary cells or yeast polypeptides produced by mammalian cells. In one variant, the polypeptide is a mammalian polypeptide (e.g., antibody) secreted directly into the culture medium by the host cell. In another variant, the polypeptide is released into the culture medium by cracking cells containing isolated nucleic acids encoding the polypeptide.

[0127] In one variation, the polypeptide is an amino acid sequence of sufficient length to produce higher order tertiary and / or quaternary structure. In one aspect, the polypeptide has a molecular weight of at least about 5-20 kD, or at least about 15-20 kD, preferably at least about 20 kD.

[0128] Any polypeptide that can be expressed in a host cell can be produced according to the disclosure, and can be present in the compositions provided herein. The polypeptide can be expressed from a gene that is endogenous to the host cell, or can be expressed from a gene that is introduced into the host cell by genetic engineering. The polypeptide can be a naturally occurring polypeptide, or can alternatively have a sequence that is engineered or artificially selected. The engineered polypeptide can be equipped from other naturally occurring polypeptide fragments of each other, or can include one or more non-naturally occurring fragments.

[0129] The polypeptide that may be desired to be expressed according to the present invention can often be selected based on the biological or chemical activity of interest. For example, the present invention can be used to express any pharmaceutically or commercially relevant enzyme, receptor, antibody, hormone, regulatory factor, antigen, binding agent, etc.

[0130] A variety of polypeptides can be produced according to the methods provided herein and are present in the compositions provided herein. Examples of bacterial polypeptides include, for example, alkaline phosphatase and beta-lactamase. Examples of mammalian polypeptides include molecules such as renin; growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1 antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor, and von Willebrand factor; anticoagulant factors such as protein C; atrial natriuretic peptide; pulmonary surfactant; plasminogen activators such as ureakinase or human urine or tissue plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor alpha and beta; enkephalinase; RANTES (regulator of activation expressed and secreted by normal T cells); human macrophage inflammatory protein (MIP-1α); serum albumin, such as human serum albumin; Mullerian inhibitory substance; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; microbial proteins, such as beta-lactamase; DNA enzymes; inhibins; activins; vascular endothelial growth factor (VEGF); hormone receptors or growth factor receptors; integrins; protein A or D; rheumatoid factor; neurotrophic factors, such as bone-derived neurotrophic factor (BDNF), neurotrophic factor (BNF), neurotrophic factor (BNF), and neurotrophic factor (BNF). trophin 3, 4, 5, or 6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factor such as NGF-b; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF), such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), Insulin-like growth factor binding protein; CD proteins such as CD3, CD4, CD8, and CD19; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); interferons such as interferon α, β and γ; colony stimulating factors (CSFs), such as M-CSF, GM-CSF and G-CSF; interleukins (ILs), such as IL-1 to IL-10; superoxide dismutase; T cell receptors; surface membrane proteins; decay accelerating factors; viral antigens such as parts of the AIDS envelope; transport proteins; homing proteins; addressins; regulatory proteins; antibodies; and fragments of any of the above polypeptides.

[0131] Antibodies are examples of mammalian polypeptides that can be present in the compositions provided herein and that are produced according to the methods provided herein. Antibodies are a class of preferred polypeptides that exhibit binding specificity to a specific antigen. Natural antibodies are typically heterotetrameric glycoproteins, approximately 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, while different numbers of disulfide bonds exist between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (V) at one end. H ), followed by several constant domains. Each light chain has a variable domain (V L ), with a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form the interface between the light chain and heavy chain variable domains.

[0132] Antibodies can be natural immunoglobulin molecules with different structures that are all based on the immunoglobulin fold. For example, an IgG antibody has two "heavy" chains and two "light" chains that are bound by disulfide bonds to form a functional antibody. Each heavy and light chain itself contains a "constant" (C) and a "variable" (V) region. The V region determines the antigen binding specificity of the antibody, while the C region provides structural support and has the function of non-antigen-specific interactions with immune effectors. The antigen binding specificity of an antibody or an antigen-binding fragment of an antibody is the ability of an antibody to specifically bind to a specific antigen.

[0133] The antigen binding specificity of an antibody is determined by the structural features of the V region. This variability is not evenly distributed over the 110 amino acid spans of the variable domain. On the contrary, the V region is composed of relatively constant segments of 15-30 amino acids called building blocks (FRs), which are separated by extremely variable shorter regions called "hypervariable regions" of 9-12 amino acids each. The variable regions of natural heavy and light chains each contain 4 FRs, which mainly adopt a β-folded configuration, connected by three hypervariable regions, wherein the hypervariable regions form loops connecting β-folded structures, and form a part of β-folded structures in some cases. In each chain, the hypervariable regions are close together through FRs, and together with the hypervariable regions from another chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant region is not directly involved in the binding of an antibody to an antigen, but exhibits various effector functions, such as participation in antibody-dependent cellular cytotoxicity (ADCC) of the antibody.

[0134] Each V region typically contains three complementarity determining regions ("CDRs", each containing a "hypervariable loop"), and four framework regions. The binding site of an antibody, the minimum structural unit required to bind to a specific desired antigen with substantial affinity, therefore typically includes three CDRs, and at least three, preferably four framework regions, wherein the framework regions are dispersed between the CDRs so that the CDRs maintain and present a suitable conformation. Classical four-chain antibodies have an antigen binding site defined by the cooperation of the VH and VL domains. Certain antibodies, such as camel and shark antibodies, lack light chains and rely solely on the binding site formed by the heavy chain. Single-domain engineered immunoglobulins can be prepared in which the binding site is formed by the heavy chain or the light chain alone, without the cooperation between VH and VL.

[0135] The term "variable" refers to that some parts of the variable domain have significantly different sequences between antibodies, and these parts are used for the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed over the entire variable domain of the antibody. It is concentrated in three segments called hypervariable regions in both the light chain and heavy chain variable domains. The more highly conserved regions in the variable domain are called framework regions (FRs). The variable domains of natural heavy and light chains each contain 4 FRs, which mainly adopt a β-folded configuration and are connected by three hypervariable regions, wherein the hypervariable regions form loops connecting the β-folded structure and form a part of the β-folded structure in some cases. The hypervariable regions in each chain are close together through FRs, and together with the hypervariable regions from another chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as participation in antibody-dependent cellular cytotoxicity (ADCC) of the antibody.

[0136] The term "hypervariable region" as used herein refers to the amino acid residues in an antibody that are responsible for antigen binding. The hypervariable region may include amino acid residues from a "complementarity determining region" or "CDR" (e.g., around residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in VL, around residues 31-35B (H1), 50-65 (H2), and 95-102 (H3) in VH (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or from a "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in VL, 26-32 (H1), 52A-55 (H2), and 96-101 (H3) in VH (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0137] "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0138] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments (each with one antigen-binding site), and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0139] "Fv" is the smallest antibody fragment containing complete antigen recognition and antigen binding sites. This region is composed of a dimer of a heavy chain and a light chain variable domain that are tightly non-covalently bound. The configuration it adopts allows the three hypervariable regions of each variable domain to interact with each other to define an antigen binding site on the surface of the VH-VL dimer. The six hypervariable regions collectively confer antibody antigen binding specificity. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) is also able to recognize and bind antigens, but it has a lower affinity than a complete binding site.

[0140] Fab fragments also contain the constant region of the light chain and the first constant region (CH1) of the heavy chain. The difference between Fab' fragments and Fab fragments is that they contain a few residues at the carboxyl end of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is used herein to refer to such Fab', the cysteine ​​residues in its constant domains carry at least one free sulfhydryl group. F(ab')2 antibody fragments were originally produced as Fab' fragment pairs, wherein there is a hinge cysteine ​​between the pair of Fab' fragments. Other chemical couplings of antibody fragments are also known.

[0141] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct classes, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0142] Based on the amino acid sequence of the constant domain of the heavy chain of the antibody, antibodies can be divided into different classes. There are 5 major classes of complete antibodies: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0143] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form a desired structure for antigen binding. For a review of scFv, see Plückthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0144] The term "diabody" refers to a small antibody fragment with two antigen binding sites, which fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains can be forced to pair with complementary domains on another chain, thereby generating two antigen binding sites. A more complete description of diabodies can be found in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0145] For purposes herein, a "complete antibody" is an antibody comprising heavy and light chain variable domains and an Fc region. These constant domains can be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the complete antibody has one or more effector functions.

[0146] "Native antibodies" are usually heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by one covalent disulfide bond, while the heavy chains of different immunoglobulin isotypes have different numbers of disulfide bonds. Each heavy chain and each light chain also have regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form the interface between the light chain and heavy chain variable domains.

[0147] A "naked antibody" is an antibody (as defined herein) that is not conjugated to a heterologous molecule, such as a cytotoxic moiety or radiolabel.

[0148] The antibody is directed to an antigen of interest. Preferably, the antigen is a biologically important polypeptide, and administration of the antibody to an individual suffering from a disease or condition can result in a therapeutic benefit in the mammal. However, antibodies directed to non-polypeptide antigens (e.g., tumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178) can also be used.

[0149] When the antigen is a polypeptide, it can be a transmembrane molecule (e.g., a receptor) or a ligand such as a growth factor. Exemplary antigens include molecules such as renin; growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyrotropin; lipoproteins; alpha-1 antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor, and von Willebrand factor; anticoagulant factors such as protein C; atrial natriuretic peptide; pulmonary surfactant; plasminogen activators such as ureakinase or human urine or tissue plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factor; tumor necrosis factor alpha and beta; enkephalinase; RANTES (activation regulatory factor expressed and secreted by normal T cells); human macrophage inflammatory protein (MIP-1α); serum albumin, such as human serum albumin; Mullerian inhibitory substance; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; microbial proteins, such as beta-lactamase; DNA enzymes; IgE; cytotoxic T lymphocyte-associated antigen (CTLA), such as CTLA-4; inhibins; activins; vascular endothelial growth factor (VEGF); hormone receptors or growth factor receptors; protein A or D; rheumatoid factor; neurotrophic factors, such as bone-derived neurotrophic factor (BDNF), neurotrophins 3, 4, 5, or 6 (NT-3, NT-4, NT-5, or NT-6). -6), or nerve growth factors such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factors such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factors (TGF), such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein; CD proteins such as CD3, CD4, CD8, CD18, CD19, CD20 and CD40; erythropoiesis factors; osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); interferons such as interferon α, β and γ; colony stimulating factors (CSFs), such as M-CSF, GM-CSF and G-CSF; interleukins (ILs), such as IL-1 to IL-10; superoxide dismutase; T cell receptors; surface membrane proteins; decay accelerating factors; viral antigens such as parts of the AIDS envelope; transporters; homing receptors; addressins; regulatory proteins; integrins, such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; tumor associated antigens, such as HER2, HER3 or HER4 receptors; and fragments of any of the above polypeptides.

[0150] Preferred molecular targets of the antibodies described herein include CD proteins, such as CD3, CD4, CD8, CD18, CD19, CD20, CD34 and CD40; members of the ErbB receptor family, such as EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules, such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, α4 / β7 integrin and αv / β3 integrin, including their α or β subunits (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors such as VEGF; tissue factor (TF); α interferon (α-IFN); interleukins such as IL-8; IgE; blood group antigens; flk2 / flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, etc.

[0151] Antibodies (including fragments thereof, including antigen-binding fragments) that can be produced by the methods described herein include, but are not limited to, anti-HER2, antibody 2C4, anti-VEGF, antibody C2B8, anti-CD11a, anti-tissue factor, IgG4b, anti-CD40, anti-CD20, anti-IgE, E25, E26, anti-PCSK9 and anti-β7.

[0152] Cell growth and peptide production

[0153] Generally, cells are combined (contacted) with any cell culture medium described herein under one or more conditions that promote cell growth, maintenance and / or polypeptide production. Methods for growing cells (i.e., culturing cells) and growing polypeptides use culture vessels (bioreactors) to accommodate cells and cell culture medium. The culture vessel can be composed of any material suitable for culturing cells, including glass, plastic or metal. Typically, the culture vessel is at least 1 liter and can be 10, 100, 250, 500, 1000, 2500, 5000, 8000, 10,000 liters or more. The culture conditions that can be adjusted during the culture process include, but are not limited to, pH and temperature.

[0154] Cell cultures are generally maintained during the initial growth phase under conditions conducive to the survival, growth and viability (maintenance) of the cell culture. The exact conditions vary with the cell type, the organism from which the cells are derived, and the nature and characteristics of the polypeptide being expressed.

[0155] The temperature of the cell culture in the initial growth phase is selected primarily based on the temperature range that can keep the cell culture alive. For example, CHO cells grow well at 37°C during the initial growth phase. In general, most mammalian cells grow well in the range of about 25°C to 42°C. Preferably, mammalian cells grow well in the range of about 35°C to 40°C. Depending on the needs of the cells and production requirements, one skilled in the art will be able to select one or more suitable temperatures for growing cells.

[0156] In one embodiment of the invention, the temperature of the initial growth stage is maintained at a constant temperature. In another embodiment, the temperature of the initial growth stage is maintained within a temperature range. For example, the temperature can be stably increased or decreased in the initial growth stage. Alternatively, the temperature can be increased or decreased by discrete amounts at different times in the initial growth stage. One of ordinary skill in the art can determine whether a single or multiple temperatures should be used and whether the temperature should be stably or adjusted by discrete amounts.

[0157] Cells can be grown for more or less time in the initial growth phase. In one variant, cells are grown for a length of time sufficient to reach a viable cell density that is a given percentage of a maximum viable cell density, wherein the maximum viable cell density is the density that the cells will eventually reach under conditions that allow the cells to grow undisturbed. For example, cells can be grown for a length of time sufficient to reach a desired viable cell density that is 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of the maximum viable cell density.

[0158] In another embodiment, the cells are allowed to grow for a specified period of time. For example, depending on the starting concentration of the cell culture, the temperature at which the cells are grown, and the intrinsic growth rate of the cells, the cells can be grown for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days. In some cases, the cells can be allowed to grow for a month or longer.

[0159] The cell culture may be stirred or shaken during the initial culture phase to increase oxygenation and diffusion of nutrients to the cells. According to the present invention, it will be apparent to those of ordinary skill in the art that it may be advantageous to control or adjust certain bioreactor internal conditions during the initial growth phase, including but not limited to pH, temperature, oxygenation, etc. For example, pH may be controlled by providing an appropriate amount of acid or base, and oxygenation may be controlled by a sparging device well known in the art.

[0160] The initial culture step is a growth phase, in which batch cell culture conditions can be modified to enhance the growth of recombinant cells, creating a seed train. The growth phase generally refers to the exponential growth phase, during which cells typically divide rapidly, e.g., grow. During this phase, cells are cultured under conditions optimal for cell growth for a period of time, typically 1-4 days, e.g., 1, 2, 3, or 4 days. The growth cycle of a host cell can be determined for a particular host cell by methods known to those skilled in the art.

[0161] In the growth phase, the basal cell culture medium and cells can be supplied to the culture vessel in batch mode. On the one hand, the culture medium contains less than about 5% or less than 1% or less than 0.1% serum and other animal-derived proteins. However, if desired, serum and animal-derived proteins can be used. In a specific variant, the basal culture medium is CDM. One or two times the amino acids, vitamins, trace elements and other culture medium components described in European Patent EP 307,247 or U.S. Patent No. 6,180,401 can be used, and these documents are hereby fully incorporated by reference.

[0162] Alternatively, commercially available culture media such as Ham's F10 (Sigma), Minimum Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) are suitable for culturing animal cells and can be supplemented with the chemically defined medium components described herein (e.g., by using the kits provided herein). In addition, any medium described in Ham and Wallace, Meth. Enz., 58: 44 (1979), Barnes and Sato, Anal. Biochem., 102: 255 (1980), US Pat. Nos. 4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90 / 03430; WO 87 / 00195; US Pat. No. Re. 30,985; or US Pat. No. 5,122,469 (the disclosures of all of which are hereby incorporated by reference in their entirety) can be used as culture medium for host cells, each of which can be supplemented with chemically defined medium components described herein (e.g., by using a kit provided herein). In one aspect, if the culture medium contains an animal-based product, the culture medium can be supplemented with CDM.

[0163] Any culture medium provided herein may also be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), ions (e.g., sodium, chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations, as will be known to those skilled in the art.

[0164] At a specific point in the growth of the cells, the cells can form an inoculum to inoculate the culture medium at the beginning of the production phase. Alternatively, the production phase can be continuous with the growth phase. The polypeptide production phase is generally followed by the cell growth phase.

[0165] In the polypeptide production stage, the cell culture can be maintained under a second set of culture conditions (relative to the growth stage) that are beneficial to cell culture survival and viability and suitable for expressing the desired polypeptide. For example, in the subsequent production stage, CHO cells express recombinant polypeptides and proteins well in the range of 25°C to 35°C. It is possible to migrate to increase cell density or viability or increase the expression of recombinant polypeptides or proteins by multiple discrete temperatures. On the one hand, compared to the impurities obtained when producing polypeptides in different culture media, the culture medium provided herein can reduce the presence of impurities when used in the method for increasing polypeptide production. In a variant, the impurity is a charge variant or reactive oxygen. On the one hand, compared to obtaining color intensity when producing polypeptides in different culture media, the culture medium provided herein reduces the color intensity of the polypeptide product when used in the method for increasing polypeptide production. In a variant, the method for increasing polypeptide production includes a temperature migration step in the polypeptide production stage. In further variations, the temperature shift step comprises a temperature shift from 31°C to 37°C, from 32°C to 37°C, from 33°C to 37°C, from 34°C to 37°C, from 35°C to 37°C, from 36°C to 37°C, from 31°C to 32°C, from 31°C to 33°C, from 31°C to 34°C, from 31°C to 35°C or from 31°C to 36°C.

[0166] Cells can be maintained in subsequent production stages until a desired cell density or output titer is reached. In one embodiment, cells are maintained in subsequent production stages until the titer of the recombinant polypeptide reaches a maximum. In other embodiments, the culture can be harvested before this point. For example, cells can be maintained long enough to allow the viable cell density to reach 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of the maximum viable cell density. In some cases, it may be desirable to allow the viable cell density to reach a maximum and then allow the viable cell density to drop to a certain level before harvesting the culture.

[0167] In some cases, it may be advantageous or necessary to supplement the cell culture with nutrients or other culture medium components that have been depleted or metabolized by the cells in the subsequent production phase. For example, it may be advantageous to supplement the cell culture with nutrients or other culture medium components that have been observed to have been depleted during monitoring of the cell culture. Alternatively or additionally, it may be advantageous or necessary to supplement the cell culture before the subsequent production phase. As a non-limiting example, it may be advantageous or necessary to supplement the cell culture with hormones and / or other growth factors, specific ions (e.g., sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds that are usually present at very low final concentrations), amino acids, lipids, or glucose or other energy sources.

[0168] Peptide purification

[0169] The polypeptide of interest is preferably recovered from the culture medium as a secreted polypeptide, but may also be recovered from host cell lysates when the polypeptide is directly expressed without a secretion signal. In one aspect, the polypeptide produced is an antibody, such as a monoclonal antibody.

[0170] The culture medium or lysate can be centrifuged to remove particulate cell debris. The polypeptide can then be purified from impurity soluble proteins and polypeptides, and the following schemes are examples of suitable purification schemes: fractionation on an immunoaffinity column or ion exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica gel or on a cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; and protein A Sepharose columns to remove impurities such as IgG. Protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) can be used to inhibit protein hydrolysis degradation during purification. It is clear to those skilled in the art that purification methods suitable for the polypeptide of interest may need to be modified to accommodate changes in the properties of the polypeptide when expressed in recombinant cell culture. Antibodies can generally be purified using chromatographic techniques (e.g., protein A, affinity chromatography plus a low pH elution step and ion exchange chromatography to remove process impurities). Purified protein can be concentrated to provide a concentrated protein drug product, such as a product having a protein concentration of at least 100 mg / mL or 125 mg / mL or 150 mg / mL, or a concentration of about 100 mg / mL or 125 mg / mL or 150 mg / mL. Purified protein can also be concentrated to provide a concentrated protein drug product, such as a product having a protein concentration of at least 1 mg / mL or 10 mg / mL or 25 mg / mL or 50 mg / mL or 75 mg / mL, or a product having a concentration of at least about 1 mg / mL or 10 mg / mL or 50 mg / mL or 75 mg / mL to about 125 mg / mL or to about 150 mg / mL. It is understood that the concentrated polypeptide product can be concentrated to a level allowed under the concentration conditions, such as a concentration at which the polypeptide is no longer soluble in solution. For example, a polypeptide purification method may include the steps of harvesting cell culture fluid from polypeptide producing cells, purifying the polypeptide by protein A affinity chromatography, further purifying by anion and cation exchange chromatography, filtering to remove viruses, and finally ultrafiltration and diafiltration steps for final formulation and concentration of the polypeptide. Non-limiting examples of methods for producing and purifying polypeptides for pharmaceutical formulation are described in Kelley, B. MAbs., 2009, 1(5): 443-452, which is hereby incorporated by reference in its entirety.

[0171] Peptide color evaluation

[0172] The polypeptide produced by the methods described herein and present in the compositions provided herein can be evaluated for color at any step of the protein purification method. The method for evaluating color can involve: harvesting cell culture fluid from cells grown in the culture medium described herein, purifying the polypeptide from the cell culture fluid to obtain a composition (e.g., solution) comprising the polypeptide, and evaluating the color of the solution comprising the polypeptide. In one variant, after purification using protein A affinity chromatography, the color of the composition comprising the polypeptide is evaluated. In a further variant, after purification by ion exchange chromatography, the color of the composition comprising the polypeptide is evaluated. In another variant, after purification by high performance liquid chromatography, the color of the composition comprising the polypeptide is evaluated. In yet another variant, after purification by hydrophobic interaction chromatography, the color of the composition comprising the polypeptide is evaluated. In yet another variant, after purification by size exclusion chromatography, the color of the composition comprising the polypeptide is evaluated. In one variant, after purification by filtration (including microfiltration or ultrafiltration), the color of the composition comprising the polypeptide is evaluated. In one variant, before evaluating the color, the composition comprising the polypeptide is concentrated (e.g., the composition may comprise at least 100 mg / mL, 125 mg / mL or 150 mg / mL polypeptide, such as an antibody). In some variants, before evaluating the color, the concentrated composition comprises at least 1 mg / mL, 25 mg / mL, 50 mg / mL or 75 mg / mL polypeptide (e.g., antibody). The composition comprising the polypeptide may be concentrated by centrifugation, a filter, a semipermeable membrane, dialysis, precipitation, ion exchange chromatography, affinity chromatography, high performance liquid chromatography, or hydrophobic interaction chromatography. In one variant, before evaluating the color, the polypeptide may be concentrated by freeze drying and resuspension. The composition comprising the polypeptide may be evaluated for color after being purified using one or more techniques described herein. It is contemplated herein that after the composition comprising the polypeptide undergoes one or more freeze-thaw cycles, the color evaluation of the composition is performed. It is also contemplated herein that a method for evaluating the color of a cell culture fluid comprising the polypeptide before purifying or concentrating the polypeptide.

[0173] The polypeptide produced by the methods described herein (or present in the compositions provided herein) using the culture medium described herein can be evaluated for color using one or more visual color standards. The method for color evaluation of the composition comprising the polypeptide includes the use of international or domestic color standards, such as, but not limited to, the United States Pharmacopoeia color standard and the European Pharmacopoeia color standard. See USP-24 monograph 631 Color and Achromaticity. United States Pharmacopoeia Inc., 2000, p. 1926-1927 and Council of Europe. European Pharmacopoeia, 2008, 7th edition, p. 22, which are hereby incorporated by reference in their entirety. For example, the color of the solution comprising the polypeptide can be evaluated using color, opalescence and staining (COC) tests. In a variant, a colorless, transparent, neutral glass tube with an identical 12 mm outer diameter is used to compare 2.0 mL of the composition containing the polypeptide and 2.0 mL of water or solvent or a reference solution specified in the monograph. Compare colors in diffuse daylight and observe horizontally relative to a white background to determine, measure or evaluate color. In another variant, the same flat bottom colorless transparent neutral glass tube with an inner diameter of 15 mm to 25 mm is used to compare the polypeptide-containing composition with water or a solvent or a reference solution specified in the monograph, wherein the layer depth is 40 mm. The colors are compared in diffuse daylight and observed vertically relative to a white background for color determination, measurement or evaluation. In one variant, the color determination, measurement or evaluation can be performed by human visual observation. In another variant, an automated method can be used for color determination, measurement or evaluation. For example, the tube can be loaded into a machine, and the machine images the tube so that the image is processed using an algorithm to determine, measure or evaluate the color. It is understood that the reference standard for the COC test can be, but is not limited to, any one of brown (B), brownish yellow (BY), yellow (Y), greenish yellow (GY) or red (R). The polypeptide-containing composition compared with the brown reference standard can be given a brown reference standard value of B1 (darkest), B2, B3, B4, B5, B6, B7, B8 or B9 (lightest). A polypeptide-containing composition compared to a tan reference standard can be assigned a tan reference standard value of BY1 (darkest), BY2, BY3, BY4, BY5, BY6 or BY7 (lightest). A polypeptide-containing composition compared to a yellow reference standard can be assigned a yellow reference standard value of Y1 (darkest), Y2, Y3, Y4, Y5, Y6 or Y7 (lightest). A polypeptide-containing composition compared to a greenish-yellow reference standard can be assigned a greenish-yellow reference standard value of GY1 (darkest), GY2, GY3, GY4, GY5, GY6 or GY7 (lightest).A composition containing a polypeptide compared to a red reference standard can be given a red reference standard value of R1 (darkest), R2, R3, R4, R5, R6 or R7 (lightest). In one aspect, an acceptable color can be any color other than the darkest color measured on the scale given herein (e.g., for a red reference standard value, except R1). In one variant, the color of a composition containing a polypeptide produced by cells cultured in a culture medium described herein has a reference standard value as described in Table 2. As described herein, it will be appreciated that, in one aspect, the culture medium that can be used in the methods and compositions herein results in a polypeptide composition (which in one variation is a composition comprising at least 100 mg / mL, or 125 mg / mL, or 150 mg / ml polypeptide) having a reference standard color value selected from the group consisting of: B3, B4, B5, B6, B7, B8, B9, BY3, BY4, BY5, BY6, BY7, Y3, Y4, Y5, Y6, Y7, GY3, GY4, GY5, GY6, GY7, R3, R4, R5, R6, and R7. In one aspect, the culture medium that can be used in the methods and compositions herein results in a polypeptide composition (which in one variation is a composition comprising at least 100 mg / mL or 125 mg / mL or 150 mg / ml polypeptide) having a reference standard color value greater than any one of B4, B5, B6, B7, B8, BY4, BY5, BY6, Y4, Y5, Y6, GY4, GY5, GY6, GY7, R3, R4, R5, and R6. It will be appreciated by those skilled in the art that the description of the reference standard color value is applicable to any culture medium, method, or composition described herein, and that the description of any culture medium, method, or composition described herein can be further modified.

[0174] Table 2. Exemplary reference standard values

[0175]

[0176]

[0177]

[0178] In another example, a polypeptide produced by the method described herein (or present in the composition provided herein) using the culture medium described herein can be evaluated for color using a quantitative test. In a variant, an automated method can be used for quantitative testing. In a variant, the quantitative test is a normalized fluorescence intensity (normalized fluorescene intensity, NIFTY) test or a total color test (Total Color assy) as described herein. For example, a solution containing a polypeptide produced by any method described herein can be evaluated for color intensity by a NIFTY test, wherein the following steps are implemented for a solution containing a polypeptide: 1) a size exclusion chromatography (SEC) is performed on a peptide test sample, wherein the mobile phase of the SEC comprises a buffer of a specific pH, and the column is maintained at a specific temperature; 2) the UV absorption of the SEC eluate at a specific wavelength (e.g., 280 nm) and the fluorescence at a specific excitation wavelength (e.g., 350 nm) and an emission wavelength (e.g., 425 nm) are monitored; 3) software known in the art (e.g., Agilent Chemstation software), integrating the polypeptide peak of SEC on the UV absorption and fluorescence emission chromatogram, and normalizing the fluorescence by dividing the fluorescence peak area of ​​the main peak by the UV absorption peak area of ​​the main peak; and 4) calculating the ratio of the normalized fluorescence of the polypeptide test sample to the normalized fluorescence of the polypeptide reference sample, wherein the polypeptide reference sample comprises a known COC value based on any of the reference standards disclosed herein (e.g., brown (B), brownish yellow (BY), yellow (Y), greenish yellow (GY), or red (R)), thereby obtaining a numerical value, wherein a higher numerical value (e.g., a higher NIFTY value) indicates a higher color intensity, and a lower numerical value (e.g., a lower NIFTY value) indicates a lower color intensity. In another example, a solution comprising a polypeptide produced by any method described herein is evaluated for color intensity by a total color test described herein.For example, a solution containing a polypeptide produced by any of the methods described herein can be evaluated for color intensity by a total color test, wherein the solution containing the polypeptide is subjected to the following steps: 1) using a spectrophotometer to measure the sample in the visible region (380-780 nm) to obtain an absorbance spectrum of the polypeptide test sample; 2) converting the absorbance spectrum to the CIE L*a*b* color scale as described in Standard Practice for Calculation of Color Tolerances and Color Differences from Instrumentally Measured Color Coordinates, Annual Book of ASTM Standards, Vol. 06.01, (2011); 3) obtaining a total color measurement, wherein the measurement represents ΔE, which corresponds to the total color intensity of the polypeptide in the three-dimensional CIE The Euclidean distance between the polypeptide test sample and water in L*a*b* color space; and 4) determining a color intensity value by calculating the ratio of the "total color" measurement of the polypeptide test sample to the "total color" measurement of a polypeptide reference sample, wherein the polypeptide reference sample contains a known COC value based on any of the reference standards disclosed herein (e.g., brown (B), brownish yellow (BY), yellow (Y), greenish yellow (GY), or red (R)), wherein a higher total color value indicates a higher color intensity and a lower total color value indicates a lower color intensity.

[0179] The color assays described herein can be used to evaluate the color of any solution (e.g., a solution containing a polypeptide), including, but not limited to, the color of the polypeptide compositions provided herein.

[0180] Evaluation of Peptide Charge Variants

[0181] The present invention provides methods for reducing the presence of charge variants (e.g., acidic charge variants), wherein the presence of charge variants (e.g., acidic charge variants) is reduced when the medium containing the components at the concentrations described herein is used in the polypeptide production method compared to the charge variants (e.g., acidic charge variants) obtained when the polypeptide is produced in a different culture medium (e.g., a culture medium containing components and / or component concentrations different from those described herein). It is understood by those skilled in the art that the description of the reference charge variant is applicable to any culture medium, method, or composition described herein, and can be further modified to the description of any culture medium, method, or composition described herein. It can also be understood that any variant scheme or embodiment of the culture medium provided in this section is also applicable to the description of the culture medium throughout the text. Here, the term "reducing the presence of charge variants" can refer to reducing the amount or presence of any type of charge variant (e.g., acidic charge variants, basic charge variants, and neutral charge variants), or reducing the amount or presence of specific charge variants such as acidic charge variants.

[0182] The present invention provides methods for reducing the presence of charge variants (e.g., acidic charge variants), and also provides compositions comprising reduced levels of charge variants (e.g., acidic charge variants), wherein the culture medium described herein reduces the presence of charge variants when used in a method for producing a polypeptide compared to the charge variants obtained when producing the polypeptide in a different culture medium. In one variant, the culture medium is a chemically undefined cell culture medium comprising from about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine, from about 0.05 mg / L to about 1.0 mg / L vitamin B2, from about 0.05 mg / L to about 10.0 mg / L vitamin B6, from about 0.05 mg / L to about 12.0 mg / L vitamin B9 and from about 0.05 mg / L to about 2.5 mg / L vitamin B12. In one variant, the concentration of vitamin B2 is from about 0.05 mg / L to about 0.50 mg / L. In another variation, the concentration of vitamin B2 is from about 0.05 mg / L to about 0.40 mg / L. In another variation, the concentration of vitamin B2 is from about 0.05 mg / L to about 0.30 mg / L. In one variation, the concentration of vitamin B6 is from about 0.05 mg / L to about 8.0 mg / L. In another variation, the concentration of vitamin B6 is from about 0.05 mg / L to about 7.0 mg / L. In another variation, the concentration of vitamin B6 is from about 0.05 mg / L to about 6.0 mg / L.

[0183] In a variant, the chemically undefined cell culture medium further comprises an iron source. In a variant, the iron source is ferric citrate or ferrous sulfate. In a variant, the chemically undefined cell culture medium comprises ferric citrate at a concentration from about 2 μM to about 80 μM. In any variant herein, the chemically undefined cell culture medium may also comprise hydrocortisone. In a variant, the concentration of hydrocortisone is from about 0.05 μM to about 0.25 μM.

[0184] The present invention provides methods for reducing the presence of charge variants (e.g., acidic charge variants), and the present invention also provides compositions comprising charge variants (e.g., acidic charge variants) of reduced levels, wherein the culture medium described herein reduces the presence of charge variants when used in a polypeptide production method compared to the charge variants obtained when producing polypeptides in different culture media. In one variant, the culture medium is a chemically defined cell culture medium, comprising from about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine, from about 2 μM to about 80 μM ferric citrate and from about 0.05 μM to about 0.5 μM hydrocortisone. In one variant, the chemically defined culture medium also comprises vitamin B2, vitamin B6, vitamin B9, and vitamin B12. In one variant, the concentration of vitamin B2 is from about 0.05 mg / L to about 1.0 mg / L. In another variant, the concentration of vitamin B2 is from about 0.05 mg / L to about 0.50 mg / L. In another variation, the concentration of vitamin B2 is from about 0.05 mg / L to about 0.40 mg / L. In another variation, the concentration of vitamin B2 is from about 0.05 mg / L to about 0.30 mg / L. In yet another variation, the concentration of vitamin B6 is from about 0.05 mg / L to about 10.0 mg / L. In yet another variation, the concentration of vitamin B6 is from about 0.05 mg / L to about 8.0 mg / L. In yet another variation, the concentration of vitamin B6 is from about 0.05 mg / L to about 7.0 mg / L. In yet another variation, the concentration of vitamin B6 is from about 0.05 mg / L to about 6.0 mg / L. In any variation described herein, the concentration of vitamin B6 is from about 0.05 mg / L to about 10.0 mg / L, from about 0.05 mg / L to about 8.0 mg / L, from about 0.05 mg / L to about 7.0 mg / L or from about 0.05 mg / L to about 6.0 mg / L. In yet another variation, the concentration of vitamin B9 is from about 0.05 mg / L to about 12.0 mg / L. In any variation described herein, the concentration of vitamin B9 is from about 0.05 mg / L to about 12.0 mg / L. In yet another variation, the concentration of vitamin B12 is from about 0.05 mg / L to about 2.5 mg / L. In any variation described herein, the concentration of vitamin B12 is from about 0.05 mg / L to about 2.5 mg / L.

[0185] The present invention provides methods for reducing the presence of charge variants (e.g., acidic charge variants), and the present invention also provides compositions comprising reduced levels of charge variants (e.g., acidic charge variants), wherein the culture medium described herein reduces the presence of charge variants when used in a polypeptide production method compared to the charge variants obtained when producing polypeptides in different culture media. In one variant, the culture medium is a chemically undefined cell culture medium, comprising from about 300 mg / L (in some embodiments, 200 mg / L) to about 1200 mg / L cystine, from about 2 μM to about 80 μM ferric citrate and from about 0.05 μM to about 0.5 μM hydrocortisone. In one variant, the chemically undefined culture medium also comprises vitamin B2, vitamin B6, vitamin B9, and vitamin B12. In one variant, the concentration of vitamin B2 is from about 0.05 mg / L to about 1.0 mg / L. In another variant, the concentration of vitamin B2 is from about 0.05 mg / L to about 0.50 mg / L. In another variation, the concentration of vitamin B2 is from about 0.05 mg / L to about 0.40 mg / L. In another variation, the concentration of vitamin B2 is from about 0.05 mg / L to about 0.30 mg / L. In yet another variation, the concentration of vitamin B6 is from about 0.05 mg / L to about 10.0 mg / L. In yet another variation, the concentration of vitamin B6 is from about 0.05 mg / L to about 8.0 mg / L. In yet another variation, the concentration of vitamin B6 is from about 0.05 mg / L to about 7.0 mg / L. In yet another variation, the concentration of vitamin B6 is from about 0.05 mg / L to about 6.0 mg / L. In any of the variations described herein, the concentration of vitamin B6 is from about 0.05 mg / L to about 10.0 mg / L, from about 0.05 mg / L to about 8.0 mg / L, from about 0.05 mg / L to about 7.0 mg / L, or from about 0.05 mg / L to about 6.0 mg / L. In any of the variations described herein, the concentration of vitamin B6 is from about 0.05 mg / L to about 10.0 mg / L. In yet another variation, the concentration of vitamin B9 is from about 0.05 mg / L to about 12.0 mg / L. In yet another variation, the concentration of vitamin B12 is from about 0.05 mg / L to about 2.5 mg / L. In any of the variations described herein, the concentration of vitamin B12 is from about 0.05 mg / L to about 2.5 mg / L.

[0186] The polypeptides (including compositions comprising the polypeptides) produced by the methods described herein can be evaluated for the presence of charge variants at any step of the protein purification process. The method for evaluating the presence of charge variants can involve: harvesting cell culture fluid from cells grown in a culture medium described herein; purifying the polypeptide from the cell culture fluid to obtain a composition comprising the polypeptide; evaluating the composition comprising the polypeptide, whether it has a reduced presence of charge variants compared to the presence of charge variants in a composition comprising the polypeptide produced in a different culture medium. In one variant, the composition comprising the polypeptide can contain an acidic charge variant or a basic charge variant. In another variant, the composition comprising the polypeptide contains an acidic charge variant. The formation of the charge variant may be caused by, but is not limited to, deamidation, sialylation, C-terminal lysine cleavage, saccharification, C-terminal lysine amidation, C-terminal glycine amidation, succinamide formation, amino acid oxidation, removal of sialic acid, or a combination thereof. The present invention also contemplates a method for evaluating the presence of charge variants in a cell culture fluid containing the polypeptide before purification or concentration of the polypeptide. Methods for detecting charge variants in a solution containing the polypeptide include: using a chromatographic technique, such as, but not limited to, ion exchange chromatography. See Khawli, LA, mAbs., 2010, 2(6):613-624, which is hereby incorporated by reference in its entirety.

[0187] In one variation of the compositions provided herein, charge variants (which are acidic charge variants in one aspect) account for less than 25% or 20% or 18% or 15% or 10% of the polypeptide product. In another variation of the compositions and methods provided herein, at least 75% or 80% or 85% or 90% or 95% or more of the polypeptide product is the main species protein. In this article, the term "main species protein" can further include proteins that are identified by the amino acid sequence, secondary structure, and / or tertiary structure of the protein, and any post-translational modifications such as glycosylation, and that are predominant in number.

[0188] Reagent test kit

[0189] The present invention describes a kit for supplementing a cell culture medium with chemically defined components. The kit may contain dry components to be reconstituted and may also contain instructions for use (e.g., for supplementing the kit components to the culture medium). The kit may contain the culture medium components provided herein in an amount suitable for supplementing the cell culture medium. In one variant, the kit comprises the culture medium components of Table 1. In another variant, the kit comprises the culture medium components of Table 1A. Various exemplary kit embodiments are provided in the "Exemplary Embodiments" section. In addition, the present invention also includes such variants, wherein the kit for supplementing the cell culture medium comprises an amount of any one or more culture medium components of Table 1 or Table 1A, wherein the amount can provide the one or more culture medium components at a concentration shown in Table 1 or Table 1A in the cell culture medium. In one variation, a kit for supplementing a cell culture medium comprises: cystine in an amount that can provide about 0.8 mM (in one aspect, 0.7 mM) to about 2.5 mM cystine in a cell culture medium; vitamin B2 in an amount that can provide from about 0.11 μM to about 0.72 μM vitamin B2 in a cell culture medium; vitamin B6 in an amount that can provide from about 4.5 μM to about 30.0 μM vitamin B6 in a cell culture medium; vitamin B9 in an amount that can provide from about 3.4 μM to about 22.0 μM vitamin B9 in a cell culture medium; and vitamin B12 in an amount that can provide from about 0.2 μM to about 1.5 μM vitamin B12 in a cell culture medium. In any variation herein, the kit may further comprise an iron source, such as ferric citrate or ferrous sulfate, in an amount that can provide from about 2 μM to about 80 μM (in some aspects, 11.0 μM to about 36.0 μM) of an iron source in a cell culture medium.

[0190] In another variation, a kit for supplementing a cell culture medium comprises: cystine in an amount that can provide from about 0.8 mM (in some aspects, 0.7 mM) to about 2.5 mM cystine in the cell culture medium; ferric citrate in an amount that can provide from about 2 μM to about 80 μM ferric citrate in the cell culture medium; hydrocortisone in an amount that can provide from about 0.05 μM to about 0.5 μM hydrocortisone in the cell culture medium; vitamin B2 in an amount that can provide from about 0.11 μM to about 0.72 μM vitamin B2 in the cell culture medium; vitamin B6 in an amount that can provide from about 4.5 μM to about 30.0 μM vitamin B6 in the cell culture medium; vitamin B9 in an amount that can provide from about 3.4 μM to about 22.0 μM vitamin B9 in the cell culture medium; and vitamin B12 in an amount that can provide from about 0.2 μM to about 1.5 μM vitamin B12 in the cell culture medium.

[0191] In any of the variations herein, the kit may further comprise any one or more of: vitamin B1 in an amount that can provide from about 2.0 μM to about 14.0 μM vitamin B1 in the cell culture medium; vitamin B3 in an amount that can provide from about 11.0 μM to about 72.0 μM vitamin B3 in the cell culture medium; vitamin B5 in an amount that can provide from about 6.8 μM to about 44.0 μM vitamin B5 in the cell culture medium; and vitamin B7 in an amount that can provide from about 0.02 μM to about 0.24 μM vitamin B7 in the cell culture medium.

[0192] In any of the variations herein, the kit can further comprise cysteine ​​in an amount that can provide about 80 mg / L to about 1500 mg / L cysteine ​​in the cell culture medium (in some aspects, 0.5 mM to about 2.0 mM).

[0193] In any of the variations herein, the cell culture medium can be CDM or a chemically defined cell culture medium.

[0194] Composition

[0195] The present invention also provides compositions, comprising cell culture medium and one or more other components, such as cells or desired polypeptides (such as antibodies). In one variant, a composition is provided, comprising: (a) cells comprising isolated nucleic acids encoding polypeptides; and (b) cell culture medium provided herein. In another variant, a composition is provided, comprising: (a) polypeptides; and (b) cell culture medium provided herein, wherein on the one hand, the polypeptide is secreted into the culture medium by cells comprising isolated nucleic acids encoding polypeptides. In another variant, a composition is provided, comprising: (a) polypeptides; and (b) cell culture medium provided herein, wherein the polypeptide is released into the culture medium by lysing cells comprising isolated nucleic acids encoding polypeptides. The cell of the composition can be any cell described herein (such as CHO cells), and the culture medium of the composition can be any culture medium described herein, such as a culture medium comprising culture medium components described in Table 1 or Table 1A (which can be CDM). Similarly, the polypeptide of the composition can be any polypeptide described herein, such as an antibody.

[0196] In one variant, the composition can have color. In one variant, the color is determined, measured or evaluated by using one or more visual color standards. The visual color standard can be an international or domestic color standard, such as, but not limited to, the United States Pharmacopoeia color standard and the European Pharmacopoeia color standard. See USP-24 monograph 631 Color and Achromaticity. United States Pharmacopoeia Inc., 2000, p. 1926-1927 and Council of Europe. European Pharmacopoeia, 2008, 7th edition, p. 22, which are hereby fully incorporated herein by reference. For example, the color, opalescence and coloration (COC) test can be used to determine, measure or evaluate the color of the composition. It is understood that the reference standard of the COC test can be, but not limited to, any one of brown (B), brownish yellow (BY), yellow (Y), greenish yellow (GY) or red (R). A polypeptide-containing composition compared to a brown reference standard may be assigned a brown reference standard value of B1 (darkest), B2, B3, B4, B5, B6, B7, B8 or B9 (lightest). A polypeptide-containing composition compared to a tan reference standard may be assigned a tan reference standard value of BY1 (darkest), BY2, BY3, BY4, BY5, BY6 or BY7 (lightest). A polypeptide-containing composition compared to a yellow reference standard may be assigned a yellow reference standard value of Y1 (darkest), Y2, Y3, Y4, Y5, Y6 or Y7 (lightest). A polypeptide-containing composition compared to a greenish-yellow reference standard may be assigned a greenish-yellow reference standard value of GY1 (darkest), GY2, GY3, GY4, GY5, GY6 or GY7 (lightest). The composition containing polypeptides compared to the red reference standard can be given a red reference standard value of R1 (darkest), R2, R3, R4, R5, R6 or R7 (lightest). The compositions described herein can have reference standard values ​​as described in Table 2 or as described in the entire text. In a specific variation, the compositions provided herein include polypeptides at a concentration of at least 100 mg / mL or 125 mg / mL or 150 mg / mL, or about 100 mg / mL or 125 mg / mL or 150 mg / mL or 175 mg / mL or 200 mg / mL. In another variation, the compositions provided herein include polypeptides at a concentration of at least 1 mg / mL or 10 mg / mL or 25 mg / mL or 50 mg / mL or 75 mg / mL, or about 1 mg / mL or 10 mg / mL or 25 mg / mL or 50 mg / mL or 75 mg / mL.In another variant, the compositions provided herein include at least about 1 mg / mL or 10 mg / mL or 50 mg / mL or 75 mg / mL to about 125 mg / mL or to about 150 mg / mL concentration of polypeptide. Any compositions provided herein can be up to the concentration of the solubility limit of the polypeptide, or with a concentration that can provide a therapeutically effective amount of polypeptide when administered to an individual, including the polypeptide. Herein, a "therapeutically effective amount" of a polypeptide (e.g., an antibody) refers to an amount effective for preventing or treating a disorder that a polypeptide can effectively treat. In yet another variant, the compositions provided herein have a color reference standard value selected from any of B4-B9, BY4-BY7, Y4-Y7, GY4-GY7, and R4-R7. The present invention also provides a composition comprising a polypeptide at a concentration of at least 100 mg / mL or 125 mg / mL or 150 mg / mL, or about 100 mg / mL or 125 mg / mL or 150 mg / mL or 175 mg / mL or 200 mg / mL, wherein the composition has a color reference standard value selected from any one of B4-B9, BY4-BY7, Y4-Y7, GY4-GY7 and R4-R7. Also provided herein are compositions comprising a polypeptide at a concentration of at least 1 mg / mL, or 10 mg / mL, or 25 mg / mL, or 50 mg / mL, or 75 mg / mL, or about 1 mg / mL, or 10 mg / mL, or 25 mg / mL, or 50 mg / mL, or 75 mg / mL, or at least about 1 mg / mL, or 10 mg / mL, or 50 mg / mL, or 75 mg / mL, to about 125 mg / mL, or to about 150 mg / mL, wherein the composition has a color reference standard value selected from any one of B4-B9, BY4-BY7, Y4-Y7, GY4-GY7, and R4-R7.

[0197] In another example, the color of the composition can be determined, measured or evaluated by using a quantitative test such as the NIFTY test or the total color test. In one variation, a higher value obtained by the quantitative test indicates a higher color intensity and a lower value indicates a lower color intensity.

[0198] Compositions (e.g., pharmaceutical preparations) of polypeptides (e.g., antibodies) produced by any of the methods described herein can be prepared by mixing the polypeptide having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of lyophilized preparations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations used, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 1 0 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGP and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more other glycosaminoglycanases such as chondroitinase. Exemplary lyophilized polypeptide formulations are described in U.S. Patent No. 6,267,958. Aqueous polypeptide formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including histidine-acetate buffer. Preparations for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtering through a sterile filtration membrane. In a specific variation, the pharmaceutical preparations provided herein include at least 100 mg / mL or 125 mg / mL or 150 mg / mL concentrations, or about 100 mg / mL or 125 mg / mL or 150 mg / mL or 175 mg / mL or 200 mg / mL concentrations of polypeptides. In another variation, the pharmaceutical preparations provided herein include at least 1 mg / mL or 10 mg / mL or 25 mg / mL or 50 mg / mL or 75 mg / mL concentrations, or about 1 mg / mL or 10 mg / mL or 25 mg / mL or 50 mg / mL or 75 mg / mL concentrations of polypeptides. In another variation, the pharmaceutical preparations provided herein include at least about 1 mg / mL or 10 mg / mL or 50 mg / mL or 75 mg / mL in any one to about 125 mg / mL or to about 150 mg / mL concentrations of polypeptides. In a variant embodiment, the pharmaceutical preparation comprising the polypeptide produced by the methods described herein uses a color test, such as, but not limited to, the COC test, the total color test or the NIFTY test, to evaluate the color intensity. In some aspects, the pharmaceutical preparation provided herein comprises a concentration greater than at least 100 mg / mL, at least 125 mg / mL or at least 150 mg / mL of polypeptide, and has a color intensity value greater than B3, B4, B5, B6, B7, B8 or B9 measured by the COC test. In some aspects, the pharmaceutical preparation provided herein comprises a concentration greater than at least 1 mg / mL, at least 10 mg / mL, at least 25 mg / mL, at least 50 mg / mL or at least 75 mg / mL of polypeptide, and has a color intensity value greater than B3, B4, B5, B6, B7, B8 or B9 measured by the COC test. In some aspects, the color intensity value measured by the COC test can be, but not limited to, any of B, BY, Y, GY or R, wherein a higher value indicates a lighter color intensity.In some aspects, the pharmaceutical preparations provided herein include a polypeptide having a concentration greater than at least 100 mg / mL, at least 125 mg / mL, or at least 150 mg / mL, and having a color intensity value less than the color intensity value of a reference solution as measured by a color test (e.g., a total test test or a NIFTY test). In some aspects, the pharmaceutical preparations provided herein include a polypeptide having a concentration greater than at least 1 mg / mL, at least 10 mg / mL, at least 25 mg / mL, at least 50 mg / mL, or at least 75 mg / mL, and having a color intensity value less than the color intensity value of a reference solution as measured by a color test (e.g., a total test test or a NIFTY test).

[0199] All references disclosed herein are incorporated by reference in their entirety.

[0200] Exemplary embodiments

[0201] 1. A method for culturing cells, comprising the steps of: contacting the cells with a cell culture medium, wherein the cell culture medium comprises:

[0202] From about 200 mg / L to about 1200 mg / L cystine;

[0203] From about 0.05 mg / L to about 1.0 mg / L vitamin B2;

[0204] From about 0.05 mg / L to about 10.0 mg / L vitamin B6;

[0205] From about 0.05 mg / L to about 12.0 mg / L vitamin B9; and

[0206] From about 0.05 mg / L to about 2.5 mg / L vitamin B12.

[0207] 2. The method of embodiment 1, comprising the step of contacting the cells with a cell culture medium comprising:

[0208] from about 0.7 mM to about 2.5 mM cystine;

[0209] From about 0.11 μM to about 0.72 μM vitamin B2;

[0210] From about 4.5 μM to about 30.0 μM vitamin B6;

[0211] From about 3.4 μM to about 22.0 μM vitamin B9; and

[0212] From about 0.2 μM to about 1.5 μM vitamin B12.

[0213] 3. The method according to embodiment 1 or 2, wherein the cell culture medium further comprises any one or more of vitamin B1, vitamin B3, vitamin B5 and vitamin B7.

[0214] 4. The method of embodiment 3, wherein the cell culture medium further comprises any one or more of the following:

[0215] From about 2.0 μM to about 14.0 μM vitamin B1;

[0216] From about 11.0 μM to about 72.0 μM vitamin B3;

[0217] From about 6.8 μM to about 44.0 μM vitamin B5; and

[0218] From about 0.02 μM to about 0.14 μM vitamin B7.

[0219] 5. The method of any one of embodiments 1-4, wherein the cell culture medium further comprises an iron source.

[0220] 6. The method of embodiment 5, wherein the iron source is ferric citrate or ferrous sulfate.

[0221] 7. The method of any one of embodiments 1-6, wherein the cell culture medium comprises ferric citrate at a concentration of from about 2 μM to about 80 μM.

[0222] 8. The method of any one of embodiments 1-7, wherein the cell culture medium comprises ferric citrate at a concentration of from about 11.0 μM to about 36.0 μM.

[0223] 9. The method of any one of embodiments 1-8, wherein the cell culture medium further comprises hydrocortisone.

[0224] 10. The method of embodiment 9, wherein the concentration of hydrocortisone in the cell culture medium is from about 0.05 μM to about 0.25 μM.

[0225] 11. The method of any one of embodiments 1-10, wherein the cell culture medium is a chemically defined cell culture medium.

[0226] 12. The method of any one of embodiments 1-10, wherein the cell culture medium is a chemically defined cell culture medium.

[0227] 13. The method of any one of embodiments 1-12, wherein the cells are contacted with the cell culture medium during a cell growth phase.

[0228] 14. The method of any one of embodiments 1-13, wherein the cells are contacted with the cell culture medium during a cell production phase.

[0229] 15. The method of embodiment 14, wherein the method further comprises the step of adding cysteine ​​to the cell culture medium.

[0230] 16. The method of embodiment 15, wherein cysteine ​​is added in an amount to provide from about 80 mg / L to about 1500 mg / L cysteine ​​in the cell culture medium.

[0231] 17. The method of embodiment 15, wherein cysteine ​​is added in an amount to provide about 1500 mg / L cysteine ​​in the cell culture medium.

[0232] 18. The method of embodiment 15, wherein cysteine ​​is added in an amount to provide about 140 mg / L cysteine ​​in the cell culture medium.

[0233] 19. A method for producing a polypeptide, comprising the steps of: culturing cells containing an isolated nucleic acid encoding the polypeptide in a cell culture medium, wherein:

[0234] (a) A cell culture medium comprising:

[0235] From about 200 mg / L to about 1200 mg / L cystine;

[0236] From about 0.05 mg / L to about 1.0 mg / L vitamin B2;

[0237] From about 0.05 mg / L to about 10.0 mg / L vitamin B6;

[0238] From about 0.05 mg / L to about 12.0 mg / L vitamin B9;

[0239] From about 0.05 mg / L to about 2.5 mg / L vitamin B12; and

[0240] (b) Cells express the polypeptide.

[0241] 20. The method of embodiment 19, wherein the cell culture medium comprises:

[0242] from about 0.7 mM to about 2.5 mM cystine;

[0243] From about 0.11 μM to about 0.72 μM vitamin B2;

[0244] From about 4.5 μM to about 30.0 μM vitamin B6;

[0245] From about 3.4 μM to about 22.0 μM vitamin B9; and

[0246] From about 0.2 μM to about 1.5 μM vitamin B12.

[0247] 21. The method of embodiment 19 or 20, wherein the cell culture medium further comprises any one or more of vitamin B1, vitamin B3, vitamin B5 and vitamin B7.

[0248] 22. The method of embodiment 21, wherein the cell culture medium further comprises any one or more of the following:

[0249] From about 2.0 μM to about 14.0 μM vitamin B1;

[0250] From about 11.0 μM to about 72.0 μM vitamin B3;

[0251] From about 6.8 μM to about 44.0 μM vitamin B5; and

[0252] From about 0.02 μM to about 0.14 μM vitamin B7.

[0253] 23. The method of any one of embodiments 19-22, wherein the cell culture medium further comprises an iron source.

[0254] 24. The method of embodiment 23, wherein the iron source is ferric citrate or ferrous sulfate.

[0255] 25. The method of any one of embodiments 19-24, wherein the cell culture medium comprises ferric citrate at a concentration of from about 2 μM to about 80 μM.

[0256] 26. The method of any one of embodiments 19-25, wherein the cell culture medium comprises ferric citrate at a concentration of from about 11.0 μM to about 36.0 μM.

[0257] 27. The method of any one of embodiments 19-26, wherein the cell culture medium further comprises hydrocortisone.

[0258] 28. The method of embodiment 27, wherein the concentration of hydrocortisone in the cell culture medium is from about 0.05 μM to about 0.25 μM.

[0259] 29. The method of any one of embodiments 19-28, wherein the cell culture medium is a chemically defined cell culture medium.

[0260] 30. The method of any one of embodiments 19-28, wherein the cell culture medium is a chemically defined cell culture medium.

[0261] 31. The method according to any one of embodiments 19-30, wherein the culturing is performed during a cell growth phase.

[0262] 32. The method according to any one of embodiments 19-31, wherein the culturing is performed in the cell production phase.

[0263] 33. The method of embodiment 32, wherein the method further comprises the step of adding cysteine ​​to the cell culture medium.

[0264] 34. The method of embodiment 33, wherein cysteine ​​is added in an amount to provide from about 80 mg / L to about 1500 mg / L cysteine ​​in the cell culture medium.

[0265] 35. The method of embodiment 33, wherein cysteine ​​is added in an amount to provide about 1500 mg / L cysteine ​​in the cell culture medium.

[0266] 36. The method of embodiment 33, wherein cysteine ​​is added in an amount to provide about 140 mg / L cysteine ​​in the cell culture medium.

[0267] 37. The method of any one of embodiments 19-36, wherein the polypeptide is an antibody.

[0268] 38. The method of embodiment 37, wherein the antibody is an IgG1 antibody.

[0269] 39. The method of embodiment 37 or 38, wherein the antibody is an anti-VEGF, anti-mesothelin, anti-PCSK9 or anti-β7 antibody.

[0270] 40. The method of any one of embodiments 19-39 further comprises the step of isolating the polypeptide from the cell culture medium.

[0271] 41. The method of embodiment 40, wherein the composition comprising the isolated polypeptide appears as a colorless or slightly colored liquid.

[0272] 42. The method of embodiment 41, wherein the composition comprises the isolated polypeptide at a concentration of at least 100 mg / mL.

[0273] 43. A polypeptide produced by the method of any one of embodiments 19-42.

[0274] 44. A pharmaceutical composition comprising the polypeptide of embodiment 43 and a pharmaceutically acceptable carrier.

[0275] 45. A kit for supplementing a cell culture medium with chemically defined components, the kit comprising:

[0276] Cystine in an amount to provide from about 200 mg / L to about 1200 mg / L cystine in the cell culture medium;

[0277] Vitamin B2 in an amount to provide from about 0.05 mg / L to about 1.0 mg / L vitamin B2 in the cell culture medium;

[0278] Vitamin B6 in an amount to provide from about 0.05 mg / L to about 10.0 mg / L vitamin B6 in the cell culture medium;

[0279] Vitamin B9 in an amount to provide from about 0.05 mg / L to about 12.0 mg / L vitamin B9 in the cell culture medium; and

[0280] Vitamin B12 in an amount to provide from about 0.05 mg / L to about 2.5 mg / L vitamin B12 in the cell culture medium.

[0281] 46. ​​The kit according to embodiment 45, comprising:

[0282] cystine in an amount to provide from about 0.7 mM to about 2.5 mM cystine in the cell culture medium;

[0283] Vitamin B2, in an amount to provide from about 0.11 μM to about 0.72 μM vitamin B2 in the cell culture medium;

[0284] Vitamin B6 in an amount to provide from about 4.5 μM to about 30.0 μM vitamin B6 in the cell culture medium;

[0285] Vitamin B9 in an amount to provide from about 3.4 μM to about 22.0 μM vitamin B9 in the cell culture medium; and

[0286] Vitamin B12 in an amount to provide from about 0.2 μM to about 1.5 μM vitamin B12 in the cell culture medium.

[0287] 47. The kit according to embodiment 45 or 46, wherein the kit further comprises any one or more of vitamin B1, vitamin B3, vitamin B5 and vitamin B7.

[0288] 48. The kit according to embodiment 47, wherein the kit further comprises any one or more of the following:

[0289] Vitamin B1, in an amount to provide from about 2.0 μM to about 14.0 μM vitamin B1 in the cell culture medium;

[0290] Vitamin B3 in an amount to provide from about 11.0 μM to about 72.0 μM vitamin B3 in the cell culture medium;

[0291] Vitamin B5 in an amount to provide from about 6.8 μM to about 44.0 μM vitamin B5 in the cell culture medium; and

[0292] Vitamin B7 in an amount to provide from about 0.02 μM to about 0.14 μM vitamin B7 in the cell culture medium.

[0293] 49. The kit of any one of embodiments 45-48, wherein the kit further comprises an iron source.

[0294] 50. The kit of embodiment 49, wherein the iron source is ferric citrate or ferrous sulfate.

[0295] 51. The kit of any one of embodiments 45-50, wherein the kit further comprises hydrocortisone.

[0296] 52. A cell culture medium comprising:

[0297] From about 200 mg / L to about 1200 mg / L cystine;

[0298] From about 0.05 mg / L to about 1.0 mg / L vitamin B2;

[0299] From about 0.05 mg / L to about 10.0 mg / L vitamin B6;

[0300] From about 0.05 mg / L to about 12.0 mg / L vitamin B9; and

[0301] From about 0.05 mg / L to about 2.5 mg / L vitamin B12.

[0302] 53. The culture medium of embodiment 52, comprising:

[0303] from about 0.7 mM to about 2.5 mM cystine;

[0304] From about 0.11 μM to about 0.72 μM vitamin B2;

[0305] From about 4.5 μM to about 30.0 μM vitamin B6;

[0306] From about 3.4 μM to about 22.0 μM vitamin B9; and

[0307] From about 0.2 μM to about 1.5 μM vitamin B12.

[0308] 54. The culture medium of embodiment 52 or 53 further comprises any one or more of vitamin B1, vitamin B3, vitamin B5 and vitamin B7.

[0309] 55. The culture medium of embodiment 54 further comprises any one or more of the following:

[0310] From about 2.0 μM to about 14.0 μM vitamin B1;

[0311] From about 11.0 μM to about 72.0 μM vitamin B3;

[0312] From about 6.8 μM to about 44.0 μM vitamin B5; and

[0313] From about 0.02 μM to about 0.14 μM vitamin B7.

[0314] 56. The culture medium of any one of embodiments 52-55, wherein the cell culture medium further comprises an iron source.

[0315] 57. The culture medium of embodiment 56, wherein the iron source is ferric citrate or ferrous sulfate.

[0316] 58. The culture medium of embodiment 57, wherein the cell culture medium comprises ferric citrate at a concentration of from about 2 μM to about 80 μM.

[0317] 59. The culture medium of embodiment 58, wherein the cell culture medium comprises ferric citrate at a concentration of from about 11.0 μM to about 36.0 μM.

[0318] 60. The culture medium of any one of embodiments 52-59, wherein the cell culture medium further comprises hydrocortisone.

[0319] 61. The culture medium of embodiment 60, wherein the cell culture medium comprises hydrocortisone at a concentration of from about 0.05 μM to about 0.25 μM.

[0320] 62. A method of culturing cells, comprising the steps of: contacting the cells with a cell culture medium, the cell culture medium comprising:

[0321] From about 200 mg / L to about 1200 mg / L cystine;

[0322] From about 2 μM to about 80 μM ferric citrate; and

[0323] From about 0.05 μM to about 0.5 μM hydrocortisone.

[0324] 63. The method of embodiment 62, wherein the cell culture medium further comprises vitamin B2, vitamin B6, vitamin B9 and / or vitamin B12.

[0325] 64. The method of embodiment 62 or 63, wherein the cell culture medium comprises from about 0.05 mg / L to about 1.0 mg / L vitamin B2.

[0326] 65. The method of any one of embodiments 62-64, wherein the cell culture medium comprises from about 0.05 mg / L to about 10.0 mg / L vitamin B6.

[0327] 66. The method of any one of embodiments 62-65, wherein the cell culture medium comprises from about 0.05 mg / L to about 12.0 mg / L vitamin B9.

[0328] 67. The method of any one of embodiments 62-66, wherein the cell culture medium comprises from about 0.05 mg / L to about 2.5 mg / L vitamin B12.

[0329] 68. The method of any one of embodiments 62-67, wherein the cell culture medium is a chemically defined cell culture medium.

[0330] 69. The method of any one of embodiments 62-67, wherein the cell culture medium is a chemically defined cell culture medium.

[0331] 70. The method of any one of embodiments 62-69, wherein the cells are contacted with the cell culture medium during a cell growth phase.

[0332] 71. The method of any one of embodiments 62-70, wherein the cells are contacted with the cell culture medium during a cell production phase.

[0333] 72. The method of embodiment 71, wherein the method further comprises the step of adding cysteine ​​to the cell culture medium.

[0334] 73. The method of embodiment 72, wherein cysteine ​​is added in an amount to provide from about 80 mg / L to about 1500 mg / L cysteine ​​in the cell culture medium.

[0335] 74. The method of embodiment 72, wherein cysteine ​​is added in an amount to provide about 1500 mg / L cysteine ​​in the cell culture medium.

[0336] 75. The method of embodiment 72, wherein cysteine ​​is added in an amount to provide about 140 mg / L cysteine ​​in the cell culture medium.

[0337] 76. A method for producing a polypeptide, comprising the steps of: culturing cells containing an isolated nucleic acid encoding the polypeptide in a cell culture medium, wherein:

[0338] (a) A cell culture medium comprising:

[0339] From about 200 mg / L to about 1200 mg / L cystine;

[0340] From about 2 μM to about 80 μM ferric citrate; and

[0341] From about 0.05 μM to about 0.5 μM hydrocortisone; and

[0342] (b) Cells express the polypeptide.

[0343] 77. The method of embodiment 76, wherein the cell culture medium further comprises vitamin B2, vitamin B6, vitamin B9 and / or vitamin B12.

[0344] 78. The method of embodiment 76 or 77, wherein the cell culture medium comprises from about 0.05 mg / L to about 1.0 mg / L vitamin B2.

[0345] 79. The method of any one of embodiments 76-78, wherein the cell culture medium comprises from about 0.05 mg / L to about 10.0 mg / L vitamin B6.

[0346] 80. The method of any one of embodiments 76-79, wherein the cell culture medium comprises from about 0.05 mg / L to about 12.0 mg / L vitamin B9.

[0347] 81. The method of any one of embodiments 76-80, wherein the cell culture medium comprises from about 0.05 mg / L to about 2.5 mg / L vitamin B12.

[0348] 82. The method of any one of embodiments 76-81, wherein the cell culture medium is a chemically defined cell culture medium.

[0349] 83. The method of any one of embodiments 76-81, wherein the cell culture medium is a chemically defined cell culture medium.

[0350] 84. The method of any one of embodiments 76-83, wherein the culturing is performed during a cell growth phase.

[0351] 85. The method of any one of embodiments 76-84, wherein the culturing is performed during the cell production phase.

[0352] 86. The method of embodiment 85, wherein the method further comprises the step of adding cysteine ​​to the cell culture medium.

[0353] 87. The method of embodiment 86, wherein cysteine ​​is added in an amount to provide from about 80 mg / L to about 1500 mg / L cysteine ​​in the cell culture medium.

[0354] 88. The method of embodiment 86, wherein cysteine ​​is added in an amount to provide about 1500 mg / L cysteine ​​in the cell culture medium.

[0355] 89. The method of embodiment 86, wherein cysteine ​​is added in an amount to provide about 140 mg / L cysteine ​​in the cell culture medium.

[0356] 90. The method of any one of embodiments 76-89, wherein the polypeptide is an antibody.

[0357] 91. The method of embodiment 90, wherein the antibody is an IgG1 antibody.

[0358] 92. The method of embodiment 90 or 91, wherein the antibody is an anti-VEGF, anti-mesothelin, anti-PCSK9 or anti-beta7 antibody.

[0359] 93. The method of any one of embodiments 76-92 further comprises the step of isolating the polypeptide from the cell culture medium.

[0360] 94. The method of embodiment 93, wherein the composition comprising the isolated polypeptide appears as a colorless or slightly colored liquid.

[0361] 95. The method of embodiment 94, wherein the composition comprises the isolated polypeptide at a concentration of at least 100 mg / mL.

[0362] 96. A polypeptide produced by the method of any one of embodiments 76-95.

[0363] 97. A pharmaceutical composition comprising the polypeptide of embodiment 96 and a pharmaceutically acceptable carrier.

[0364] 98. A kit for supplementing a cell culture medium with chemically defined components, the kit comprising:

[0365] Cystine in an amount to provide from about 200 mg / L to about 1200 mg / L cystine in the cell culture medium;

[0366] ferric citrate in an amount to provide a concentration of ferric citrate from about 2 μM to about 80 μM in the cell culture medium; and

[0367] Cortisol in an amount to provide a concentration of from about 0.05 μM to about 0.5 μM cortisol in the cell culture medium.

[0368] 99. The kit according to embodiment 98, wherein the kit further comprises vitamin B2, vitamin B6, vitamin B9 and / or vitamin B12.

[0369] 100. The kit of embodiment 98 or 99, wherein the kit comprises vitamin B2 in an amount to provide from about 0.05 mg / L to about 1.0 mg / L vitamin B2 in the cell culture medium.

[0370] 101. The kit of any one of embodiments 98-100, wherein the kit comprises vitamin B6 in an amount to provide from about 0.05 mg / L to about 10.0 mg / L vitamin B6 in the cell culture medium.

[0371] 102. The kit of any one of embodiments 98-101, wherein the kit comprises vitamin B9 in an amount to provide from about 0.05 mg / L to about 12.0 mg / L vitamin B9 in the cell culture medium.

[0372] 103. The kit of any one of embodiments 98-102, wherein the kit comprises vitamin B12 in an amount to provide from about 0.05 mg / L to about 2.5 mg / L vitamin B12 in the cell culture medium.

[0373] 104. A cell culture medium comprising:

[0374] From about 200 mg / L to about 1200 mg / L cystine;

[0375] From about 2 μM to about 80 μM ferric citrate; and

[0376] From about 0.05 μM to about 0.5 μM hydrocortisone.

[0377] 105. The culture medium of embodiment 104, wherein the culture medium further comprises vitamin B2, vitamin B6, vitamin B9 and / or vitamin B12.

[0378] 106. The culture medium of embodiment 104 or 105, wherein the culture medium comprises from about 0.05 mg / L to about 1.0 mg / L vitamin B2.

[0379] 107. The culture medium of any one of embodiments 104-106, wherein the culture medium comprises from about 0.05 mg / L to about 10.0 mg / L vitamin B6.

[0380] 108. The culture medium of any one of embodiments 104-107, wherein the culture medium comprises from about 0.05 mg / L to about 12.0 mg / L vitamin B9.

[0381] 109. The culture medium of any one of embodiments 104-108, wherein the culture medium comprises from about 0.05 mg / L to about 2.5 mg / L vitamin B12.

[0382] 110. A composition comprising (a) cells containing an isolated nucleic acid encoding a polypeptide; and (b) a culture medium according to any one of embodiments 52-61 and 104-109.

[0383] 111. A composition comprising: (a) a polypeptide; and (b) a culture medium according to any one of embodiments 52-61 and 104-109.

[0384] The following examples are provided to illustrate but not to limit the invention. Example

[0385] Substrates that can produce protein drug products with acceptable quality attributes such as color have been identified, especially substrata that produce protein products with acceptable quality attributes when they exist as concentrated solutions (e.g., concentrations reaching at least 100 mg / ml). In some aspects, the protein product exists in the form of a composition comprising at least 1 mg / ml. This article describes methods for culturing cells in a substratum provided herein and methods for producing polypeptides using the substratum. Substratum can comprise cystine and / or cysteine ​​and vitamin B2, B6, B9 and B12 on the one hand, wherein an iron source and hydrocortisone are optionally added. Substratum can comprise cystine and / or cysteine, hydrocortisone and an iron source on the other hand, wherein vitamin B2, B6, B9 and B12 are optionally added. Compositions containing cystine are particularly contemplated. Each substratum component can exist at any value provided herein in its entirety. Substratum can be chemically determined or chemically indeterminate. Compared with polypeptides produced in different substrata, substratum of the present invention can reduce the presence of polypeptide charge variants and / or reduce the presence of reactive oxygen when used in a polypeptide production method. The culture medium of the present invention can be used in any stage of cell culture and polypeptide production, and can be used in basal medium and / or feed medium. The present invention provides polypeptides produced by any method of the present invention and pharmaceutical compositions comprising the polypeptide products described in detail herein. On the one hand, the pharmaceutical composition comprises the polypeptide at a concentration of at least or about 100 mg / mL, 125 mg / mL and 150 mg / mL. On the other hand, the pharmaceutical composition comprises the polypeptide at a concentration of at least or about 1 mg / mL, 10 mg / mL, 25 mg / mL, 50 mg / mL and 75 mg / mL. Especially contemplated are methods for producing antibodies and compositions comprising antibodies. The present invention also describes a kit for supplementing chemically determined components to a cell culture medium.

[0386] In the whole process of process development of drug substances, it is important that product quality meets certain industry standards for clinical use. The recent trend of subcutaneous delivery of monoclonal antibodies requires that the concentration of drug substances in the formulation is increased to ≥100 mg / mL. However, at these high concentrations, the color of the drug substance is darker, which makes it more difficult to meet the quality expectations of the relevant product color established. As described herein, several modifications to culture media (whether chemically determined or chemically uncertain) have been identified that can reduce the color intensity of the drug substance to meet product quality standards.

[0387] The CHO cell line described in the examples was genetically engineered to secrete recombinant humanized antibodies (referred to herein as IgG1 monoclonal antibodies) using a dihydrofolate reductase (dhfr) / methotrexate selection method similar to that previously described by Kaufman et al., Mol. Cell. Biol., 2(11):1304-1319 (1982). The initial transfection used a GS selection system with methionine sulfoximine as the selection agent and a medium without glutamine. Subsequent supertransfections used a dhfr selection system and methotrexate as the selection agent. Before initiating the growth phase in a 2-liter stirred suspension bioreactor, cryogenically frozen ampoules of the CHO cell line were thawed and cultured in shake flasks in a chemically defined medium at 37°C in a humidified incubator with 5% CO2 for at least two weeks to obtain a cell culture suspension with good growth and viability properties.

[0388] Example 1: Color intensity displayed in a preparation containing antibodies isolated from antibody-producing cells

[0389] CHO cell lines capable of producing IgG1 monoclonal antibodies (anti-β7) were cultured in a chemically undefined medium containing peptone. The separated antibodies were purified and the color was evaluated using the standard "Clarity, Opalescence and Coloration" (COC) test (Council of Europe. European Pharmacopoeia., 2008, 7th edition, p. 22). In brief, the COC test was performed using the same flat-bottomed colorless, transparent neutral glass tube with an inner diameter of 15 mm to 25 mm. 150 g / L protein solution was added to the tube to a depth of 40 mm, and the protein solution was prepared from a purified concentrated cell culture fluid containing secreted IgG1 monoclonal antibodies. The tube containing the antibody solution was compared with 9 reference tubes by observing vertically against a white background in diffuse daylight, each of which was equipped with a reference solution from B1 (deepest) to B9 (shallowest). The IgG1 monoclonal antibody solution was determined to have a COC value of ≤B5 ( Figure 1 : Preparation I).

[0390] A CHO cell line producing an IgG1 monoclonal antibody (anti-β7) was cultured in CDM. For the preparation of the cell culture media, basal CDM (medium 1) and feed medium CDM (medium 2) solutions were prepared by combining the ingredients into a custom blended powder, dissolving the blended powder in water, and adjusting to a final pH and osmotic pressure that ensured optimal cell growth. Basal medium 1 and feed medium 2 each had over 20 ingredients, of which the ingredients of interest are listed in Table A. Some media ingredients, such as glucose, were not combined in the blended powder but were added separately during the medium preparation process. To initiate the growth phase of the cell culture, approximately 1.0 x 10 6 CHO cells were inoculated with 100 mL of feed medium 2 per liter of cell culture fluid at 3, 6, and 9 days. Cell culture was carried out in a batch feed mode, where 100 mL of feed medium 2 was added per liter of cell culture fluid for the initial production phase on days 3, 6, and 9. Glucose concentrations were analyzed daily and supplemented from 500 g / L glucose stock solution to prevent glucose depletion if the glucose concentration dropped below 3 g / L. The reactor was equipped with calibrated dissolved oxygen, pH, and temperature probes. Dissolved oxygen was controlled online by sparging air and / or oxygen. pH was controlled by adding CO2 or Na2CO3, and defoamers were added to the culture as needed. Cell culture was maintained at pH 7.0 and maintained at 37°C from day 0 to day 3, and then maintained at 33°C after day 3. Cell culture was stirred at 275 rpm, and dissolved oxygen levels were at 30% air saturation. Osmotic pressure was detected using an osmotic pressure agent from Advanced Instruments (Norwood, MA). In addition, offline pH and metabolite concentrations were determined daily using a Nova Bioprofile 400 (Nova Biomedical, Waltham, MA). Viable cell density (VCC) and cell viability were measured daily using Automatic cell counter (Beckman Coulter, Fullerton, CA) was used for measurement. The collected cell volume (PCV) was measured after centrifuging the cell suspension at 700 x g or 836 x g for 10 minutes using a graduated centrifuge tube (Kimble Science Products, Fullerton, CA). PCV was expressed as a percentage of total cell volume. When the protein content in the culture was about 2-10 g / L, at the end of the 14th day cell culture period, the cell culture fluid was harvested by centrifugation. The monoclonal antibodies in the harvested cell culture fluid were purified using protein A affinity chromatography. After purification, the concentration of protein in the eluted protein A pool was about 5-10 g / L. The protein A pool was further concentrated to 150 g / L using an Amicon Centricon centrifugal filter device (Millipore Corporation, Billerica, MA). Color was measured in the concentrated protein A pool using a standard COC test. Alternatively, the harvested cell culture fluid is purified using a standard antibody purification method, wherein the purification method includes affinity purification by protein A affinity chromatography, further purification by anion and cation exchange chromatography, filtration to remove viruses, and final ultrafiltration and diafiltration steps to finally prepare and concentrate the antibody, followed by color measurement using a standard COC test. See Kelley, B.mAbs., 2009, 1 (5): 443-452. Even though slightly higher, the color measured in the concentrated protein A pool generally indicates the expected color in the final antibody preparation produced by the standard antibody purification method. Before using high performance liquid chromatography purification, the cell culture fluid is collected daily by centrifugation of 1 mL of cell culture fluid for determining antibody titer. The COC test is performed using the same flat-bottomed, colorless, transparent, neutral glass tube with an inner diameter of 15 mm to 25 mm. Two tubes are each loaded with 150 g / L protein solution to a depth of 40 mm, and the protein solution is prepared from a purified concentrated cell culture fluid containing secreted IgG1 monoclonal antibodies. Each tube containing antibody solution was compared to nine reference tubes containing reference solutions from B1 (darkest) to B9 (lightest) by viewing vertically against a white background in diffuse daylight. Color analysis of the antibody-containing solution showed a COC value of ≤ B4 or ≤ B3 ( Figure 1 ; Formulations II and III, respectively).

[0391] Table A. Components of Interest

[0392]

[0393] Example 2: Reducing the color intensity of antibodies isolated from antibody-producing cell lines by changing specific components in the cell culture medium

[0394] Basal medium 1 and feed medium 2 were reformulated to contain reduced concentrations of several nutrients and used in cell culture experiments to determine whether the reformulated medium could reduce the color intensity of isolated monoclonal IgG1 antibodies (anti-β7) produced from CHO cell lines. Briefly, basal CDM (medium 3) and feed CDM (medium 4) solutions were prepared by combining the ingredients into a customized mixed powder, dissolving the powder in water, and adjusting to the final pH and osmotic pressure to ensure optimal cell growth. Basal medium 3 and feed medium 4 each have more than 20 ingredients, of which the ingredients of interest are listed in Table B. Some culture medium ingredients, such as glucose, are not combined in the mixed powder, but are added separately during the culture medium preparation process. Similarly, the culture medium ingredients that were changed for this study were not included in the mixed powder, but were added separately at appropriate levels during the culture medium preparation process (Table B). Ferric citrate was added from a 5 g / L ferric citrate stock solution, vitamin B2 was provided as riboflavin powder, vitamin B6 was provided as pyridoxine HCl or as pyridoxal HCl, vitamin B9 was provided as folic acid powder, vitamin B12 was provided as cyanocobalamin powder, cysteine ​​was provided as L-cysteine ​​monohydrochloride monohydrate powder, cystine was provided as disodium salt monohydrate powder, and hydrocortisone was added from a 150 μM stock solution.

[0395] Table B. Components of Interest

[0396]

[0397] a The iron source is ferrous sulfate

[0398] b The iron source is ferric citrate

[0399] To initiate the growth phase of cell culture, approximately 1.0 x 10 6Cells were inoculated with 100 mL of feed medium 4 per liter of cell culture fluid at 3, 6, and 9 days. The cells were cultured in a fed-batch mode, with 100 mL of feed medium 4 added per liter of cell culture fluid on days 3, 6, and 9 for the initial growth phase. Glucose concentration was analyzed daily and supplemented from a 500 g / L glucose stock solution to prevent glucose depletion if the glucose concentration dropped below 3 g / L. The reactor was equipped with calibrated dissolved oxygen, pH, and temperature probes. Dissolved oxygen was controlled online by sparging air and / or oxygen. pH was controlled by adding CO2 or Na2CO3, and defoamers were added to the culture as needed. The cell culture was maintained at pH 7.0 and maintained at 37°C from day 0 to day 3, and then maintained at 35°C after day 3. The cell culture was stirred at 275 rpm, and the dissolved oxygen level was 30% air saturation. Osmotic pressure was monitored using an osmometer from Advanced Instruments (Norwood, MA). In addition, offline pH and metabolite concentrations were measured daily using a Nova Bioprofile 400 (Nova Biomedical, Waltham, MA). Automatic cell counter (Beckman Coulter, Fullerton, CA) was used to measure the viable cell density (VCC) and cell viability daily. Using graduated centrifuge tubes (KimbleScience Products, Fullerton, CA), the collected cell volume (PCV) was measured after 10 minutes of centrifugal cell suspension at 700x g or 836x g. PCV was expressed as a percentage of total culture volume. When the protein amount in the culture was about 2-10g / L, at the end of the 14th day cell culture period, the cell culture fluid was harvested by centrifugation. Using protein A affinity chromatography, the monoclonal antibodies in the cell culture fluid harvested were purified. After purification, the protein concentration in the eluted protein A pool was about 5-10g / L. Using Amicon Centricon centrifugal filter device (Millipore Corporation, Billerica, MA), the protein A pool was concentrated to 150g / L. Using standard COC test, color was measured in concentrated protein A pools. The harvested cell culture fluid is also purified in parallel using a standard antibody purification method, which includes affinity purification by protein A affinity chromatography, further purification by anion and cation exchange chromatography, filtration to remove viruses, and final ultrafiltration and diafiltration steps to finally prepare and concentrate the antibody, followed by color measurement using a standard COC test. See Kelley, B.mAbs., 2009, 1 (5): 443-452. Even if slightly higher, the color measured in the concentrated protein A pool generally indicates the expected color in the final antibody preparation produced by the standard antibody purification method. Cell culture fluids are collected daily by centrifugation of 1 ml of cell culture fluid to determine antibody titers using high performance liquid chromatography. The COC test is performed using the same flat-bottomed, colorless, transparent, neutral glass tube with an inner diameter of 15 mm to 25 mm. A 150 g / L protein solution prepared from a purified, concentrated cell culture fluid containing secreted IgG1 monoclonal antibodies is loaded into the tube to a depth of 40 mm. The tube containing the antibody solution was compared to seven reference vials, each containing a reference solution from BY1 (darkest) to BY7 (lightest), by viewing vertically against a white background in diffuse daylight. The IgG1 monoclonal antibody solution was measured to have a COC value of ≤ BY5 ( Figure 1 ; Formulation VII). Cell cultures grown in basal medium 3 and feed medium 4 had slightly reduced harvested cell volumes (PCV) compared to cell cultures grown in basal medium 1 and feed medium 2 ( Figure 2 A). This decrease was associated with slightly reduced antibody production ( Figure 2B). Additional experiments to evaluate the effect of the media on PCV and antibody titers demonstrated that when cells were cultured using Basal Medium 3 and Feed Medium 4, PCV was reduced compared to cells cultured using Basal Medium 1 and Feed Medium 2 ( Figure 2 C). As before, a decrease in PCV was associated with decreased antibody production ( Figure 2 D).

[0400] These results indicate that the color intensity of the monoclonal IgG1 antibody produced by culturing CHO cells using basal medium 3 and feed medium 4 was reduced compared to the color intensity of the monoclonal IgG1 antibody produced by culturing cells in basal medium 1 and feed medium 2.

[0401] Example 3: Reduction of color intensity of antibodies isolated from antibody producing cell lines by changing vitamin B levels in cell culture medium

[0402] In order to determine the effect of the components changed in basal medium 3 and feed medium 4 on the color intensity of the isolated antibody, the levels of vitamins B2, B6, B9 and B12 were changed while the levels of other medium components were kept unchanged. The medium was prepared as described in Example 2. The medium components changed for this study were not included in the mixed powder, but were added separately at appropriate levels during the medium preparation process. Basal medium 5 was prepared to have a reduced vitamin level similar to basal medium 3 and all other components similar to basal medium 1 (Table C). In order to produce monoclonal IgG1 antibodies (anti-β7) from CHO cells, cell cultures were fed basal medium 5 for the initial growth phase and cultured with feed medium 2 or feed medium 4 on the 3rd, 6th and 9th days. As shown in Example 2, cell viability measurements and monoclonal IgG1 antibody separation were performed. After antibody purification, color intensity and antibody titer were also determined as described in Example 2. The IgG1 monoclonal antibody solution obtained from cells cultured using basal medium 5 and feed medium 2 was measured as a COC value of ≤B4 ( Figure 1 ; Formulation V). The IgG1 monoclonal antibody solution obtained from cells cultured with basal medium 5 and feed medium 4 was measured to have a COC value of ≤ B4 ( Figure 1 ; Formulation IV).

[0403] Table C. Culture media with reduced levels of vitamins B2, B6, B9 and B12

[0404]

[0405] Carry out full factorial study, to evaluate the contribution of each vitamin B component (vitamin B2, B6, B9 and B12) to the color intensity of separation antibody.When vitamin B2 level is as the factor treatment of separation, the level of pyridoxine and pyridoxal is combined into a single factor as vitamin B6, thus changing the concentration of these two nutrients simultaneously.Similarly, the level of vitamin B9 and B12 changes together as a single factor, thus changing the concentration of these two nutrients simultaneously.Several culture medium preparations have been prepared, including the culture medium listed in Table D, E and F.In order to produce monoclonal IgG1 antibody (anti-β7) from CHO cells, with fed batch mode, basal medium 5 and feed medium 4, basal medium 6 and feed medium 7, basal medium 8 and feed culture solution 9 or basal medium 10 and feed medium 11 are fed to cell culture. Other cell cultures were fed with a basal medium containing 1.41 mg / L vitamin B2 and / or 15.42 mg / L vitamin B6 (provided in the form of pyridoxine) and a feed medium containing 10 mg / L vitamin B2 and / or 76 mg / L vitamin B6 (7 mg / L pyridoxine and 60 mg / L pyridoxal). Cell viability measurements and monoclonal IgG1 antibody separation were performed as described in Example 2. After antibody purification, antibody titer was measured as described in Example 2. Color intensity was measured using a color test, where a higher numerical value indicated a higher color intensity, and a lower numerical value indicated a lighter color intensity. For this color test (referred to herein as a standardized fluorescence intensity (NIFTY) test), approximately 50 to 125 μg monoclonal antibody samples were analyzed by size exclusion chromatography (SEC) using a G3000SWXL post (TOSOH), wherein an isocratic flow rate of 0.5 ml / min was used. The mobile phase of SEC was 0.2 M potassium phosphate, 0.25 M potassium chloride, pH 6.2. The column temperature was controlled at 15 ° C. The UV absorption of the SEC eluate at 280nm, as well as the fluorescence at 350nm excitation wavelength and 425nm emission wavelength were monitored. The SEC peaks of the monoclonal antibody molecules were integrated on the UV absorption and fluorescence emission chromatograms using Agilent Chemstation software. For each monoclonal antibody sample, the fluorescence of the standardization was determined by dividing the fluorescence peak area of ​​the main peak by the UV absorption peak area of ​​the main peak (this corrected the fluorescence reaction by the antibody mass contribution). Subsequently, the color intensity value was determined by calculating the ratio of the standardized fluorescence of the monoclonal antibody sample tested to the standardized fluorescence of the reference monoclonal antibody sample containing a COC reading of ≤B5. The collected cell volume (IVPCV) and antibody titer levels that varied over time were analyzed using JMP 8.0.2 statistical software, which showed that although cell viability and antibody titer levels were only slightly affected by changing vitamin B2 and vitamin B6 levels in the basal and feed media ( Figure 3A and B; middle trend line), but increasing the levels of these two vitamins in the cell culture medium significantly increased the color intensity of the isolated antibodies ( Figure 4 ; middle trend line). In contrast, increasing levels of vitamin B9 and vitamin B12 had no significant effect on cell viability, antibody titer levels, and color intensity ( Figure 3 and 4 ; middle trend line). In addition, low levels of color intensity were observed for antibodies isolated from cell lines cultured using basal medium containing 0.25 mg / L vitamin B2 and 5.35 mg / L vitamin B6 (supplied as pyridoxine) and feed medium without vitamin B2 and vitamin B6.

[0406] The effects of vitamins B1, B3, B5, and B7 were also investigated; however, these nutrients had no significant effect on the color intensity of the antibody-containing solutions.

[0407] Table D. Culture media with changes in vitamin B2

[0408]

[0409]

[0410] Table E. Culture media with changes in vitamin B6

[0411]

[0412] Table F. Culture media with changes in vitamin B9 and B12

[0413]

[0414]

[0415] Example 4: Reduction of color intensity of antibodies isolated from antibody producing cell lines by changing iron sources and levels in cell culture medium

[0416] In order to evaluate the contribution of iron level to the color intensity of separated antibodies, the level of iron was changed while keeping other culture medium components levels unchanged. Culture medium was prepared as described in Example 2. The culture medium components changed for this study were not included in the mixed powder, and were added separately at a suitable level during the culture medium preparation process. Reproduce basal medium 1 with ferrous sulfate having a reduced level, to produce basal medium 12 containing 18 μM ferrous sulfate and basal medium 13 containing 10 μM ferrous sulfate. In order to produce monoclonal IgG1 antibody (anti-β7) from CHO cells, basal medium 1 and feed medium 2, basal medium 12 and feed medium 2, or basal medium 13 and feed medium 2 were fed to cell culture in fed batch mode. As described in Example 2, cell viability was measured and monoclonal IgG1 antibody was separated. After antibody purification, as described in Example 2, antibody titer was determined. Color intensity was determined with a color test, wherein a higher numerical value indicated a higher color intensity, and a lower numerical value indicated a lower color intensity. This color test (also referred to as the NIFTY test) was implemented as described in Example 3. The harvested cell volume (IVPCV) and antibody titer levels over time were analyzed using JMP 8.0.2 statistical software, which showed that although the use of lower iron concentrations relative to 75 μM iron concentrations reduced cell viability and antibody titer levels ( Figure 5 A and B), but lower iron concentrations in the cell culture medium significantly reduced the color intensity in the isolated antibody ( Figure 5 C).

[0417] In order to investigate whether the observed effects of cell culture medium conditions on color were due to intracellular or extracellular phenomena, a series of in vitro incubation experiments were performed. The monoclonal antibody was incorporated into freshly prepared cell culture medium in an amount of 1 g / L and incubated at 33°C for up to 6 days. The cell culture medium used for in vitro incubation had ferrous sulfate concentrations of 10, 18, and 75 μM. The vitamin B concentration remained unchanged, with reduced levels of 0.25 mg / L vitamin B2, 5.35 mg / L vitamin B6 (5.35 mg / L pyridoxine + 0 mg / L pyridoxal), 8.61 mg / L vitamin B9, and 1.76 mg / L vitamin B12. Samples were taken from the incubated mixture on days 0, 3, and 6, and the antibodies were purified by protein A chromatography. The color intensity of the solution containing the separated antibody was measured using the NIFTY test. In antibody-containing solutions isolated from cells grown in medium containing 75, 18, or 10 μM ferrous sulfate, respectively, an increase in color intensity from 1.0 unit on day 0 to 1.8, 1.44, or 1.27 units on day 6 was observed ( Figure 5 D) The increased color formation at higher iron concentrations mirrors the results obtained in cell culture experiments, suggesting that extracellular mechanisms play a role in antibody coloration.

[0418] In order to further evaluate the contribution of iron to the color intensity of the isolated antibodies, the source and level of iron were changed, while the levels of other culture medium components were kept unchanged. Culture medium was prepared as described in Example 2. The culture medium components changed for this study were not included in the mixed powder, but were added separately at appropriate levels during the culture medium preparation process. Basal medium 1 was re-formulated to have a reduced level of ferrous sulfate to produce a basic medium 12 containing 18 μM ferrous sulfate and a basic medium 13 containing 10 μM ferrous sulfate. In addition, basic medium 1 was re-formulated to have a reduced level of iron and different iron sources to produce a basic medium 14 containing 18 μM ferric nitrate, a basic medium 15 containing 18 μM ferric citrate, and a basic medium 16 containing 10 μM ferric citrate. To produce monoclonal IgG1 antibodies (anti-β7) from CHO cells, basal medium 1 and feed medium 2, basal medium 12 and feed medium 2, basal medium 13 and feed medium 2, basal medium 14 and feed medium 2, basal medium 15 and feed medium 2, or basal medium 16 and feed medium 2 were fed to the cell culture in a fed-batch mode. Cell viability measurements and monoclonal IgG1 antibody separations were performed as described in Example 2. After antibody purification, antibody titers were also measured as described in Example 2. Color intensity was determined using a color test, where higher values ​​indicate higher color intensity and lower values ​​indicate lower color intensity. The color test (also known as the NIFTY test) was performed as described in Example 3. The collected cell volume (IVPCV) and antibody titer levels that varied over time were analyzed using JMP 8.0.2 statistical software, which showed that the use of lower ferrous sulfate concentration levels reduced cell viability and antibody titer ( ) compared to 75 μM concentration of ferrous sulfate. Figure 6 A and B). Cell viability and antibody titers obtained from cell cultures using medium containing 18 μM ferric citrate were comparable to those obtained using medium containing 75 μM ferrous sulfate ( Figure 6 A and B). However, the use of 18 μM ferric citrate in the cell culture medium significantly reduced the color intensity of the isolated antibody compared to all tested concentrations of ferrous sulfate ( Figure 6 C). Cell viability, antibody titers, and color of isolated antibodies obtained from cell cultures grown in medium containing 18 μM ferric nitrate were comparable to those observed when using medium containing either 10 μM or 18 μM ferrous sulfate ( Figure 6 AC).

[0419] Reduced vitamin B levels and reduced iron concentrations were combined to examine whether the color beneficial effects due to vitamins and iron were additive. Basal medium 13, medium 14, medium 15, and medium 16 were re-formulated to have reduced levels of vitamin B levels, thereby producing basal medium 21, medium 22, medium 23, and culture fluid 25, respectively. In addition, basal medium 1 was re-formulated to have reduced vitamin B levels and 10 μM ferric nitrate as an iron source, thereby producing basal medium 24. The reduced vitamin levels in mediums 21 to 25 were as follows: 0.25 mg / L vitamin B2, 5.35 mg / L vitamin B6 (5.35 mg / L pyridoxine + 0 mg / L pyridoxal), 8.61 mg / L vitamin B9, and 1.76 mg / L vitamin B12. For the production of monoclonal IgG1 antibodies from CHO cells, basal medium 21, 22, 23, 24, or 25 and feed medium 2 were fed to the cell culture in fed-batch mode. Monoclonal IgG1 antibody isolation and antibody titer measurement were performed as described in Example 2. Color intensity was measured using the NIFTY assay. Analysis of antibody titer levels and color intensity showed that when the ferrous sulfate concentration was reduced from 75 μM to 18 μM and combined with lower vitamin concentrations, the resulting color and titer were lower ( 0.05 % ) than when either of these factors was reduced alone. Figure 6 D and E, plus signs). However, for either ferric nitrate or ferrous sulfate, reducing the iron concentration from 18 μM to 10 μM did not appear to provide any further benefit ( Figure 6 D and E, plus sign).

[0420] In order to study the contribution of reactive oxygen (ROS) to antibody color intensity, an in vitro experiment was performed in which 2g / L monoclonal IgG1 antibody samples were incorporated into a vial containing a cell culture medium supplemented with 75μM or 18μM ferrous sulfate. Other in vitro experiments were also performed in which 2g / L monoclonal IgG1 antibody (anti-β7) samples were incorporated into a vial containing a cell culture medium supplemented with 1.41mg / L vitamin B2 or 0.25mg / L vitamin B2. In the absence of any cells, with or without 203U / ml catalase, the samples were incubated at 37°C for 5 days. Color intensity was measured using a color test, in which a higher numerical value indicated a higher color intensity, and a lower numerical value indicated a lower color intensity. The color test, also referred to as the NIFTY test, is implemented as described in Example 3. The color level was analyzed with JMP 8.0.2 statistical software, and the analysis showed that although the increased iron level resulted in an antibody solution with increased color intensity ( Figure 7 A), but this color increase was reduced by the addition of catalase ( Figure 7 B; middle trend line). In contrast, although increasing levels of vitamin B2 resulted in antibody solutions with increased color intensity, this color intensity was not reduced by the addition of catalase ( Figure 7 A and B; middle trend line).

[0421] Example 5: Reducing the formation of acidic charge variants of antibodies isolated from antibody producing cell lines by changing specific components in the cell culture medium

[0422] In order to produce monoclonal IgG1 antibody (anti-β7) from CHO cells, cells are cultured in one of three reformulated basal medium 1 solutions, each of which contains 10 μM, 18 μM or 75 μM ferrous sulfate. Other cell cultures are fed (i.e., cultured) with one of two reformulated basal medium 3 solutions, wherein each of the two culture medium solutions contains 10 μM or 18 μM ferric citrate. All cell cultures are fed with iron-free feed medium in fed batch mode. As described in Example 2, monoclonal IgG1 antibodies are separated and antibody titers are determined. Color intensity is measured using a color test, wherein a higher value indicates a higher color intensity, and a lower value indicates a lower color intensity. This color test, also referred to as the NIFTY test, is implemented as described in Example 3. In order to check the acidic charge variants of antibodies in purified solutions, the charge heterogeneity of monoclonal antibodies is analyzed by ion exchange chromatography (IEC) using a Dionex ProPac WCX-10 (4x250 mm) column. The analysis was performed on an Agilent 1100HPLC system, with effluent monitored at 280nm. Mobile phase A was 25mM sodium phosphate (pH 6.6), and mobile phase B was 150mM sodium sulfate in mobile phase A. A linear gradient from 0 to 37% B was used in 45 minutes, with a flow rate of 0.5mL / min. The column temperature was controlled at 40°C. Before IEC analysis, the C-terminal lysine residue on the monoclonal antibody heavy chain was removed by carboxypeptidase B to reduce the complexity of charge heterogeneity in the alkaline region. The relationship between color intensity and the presence of acidic charge variants was analyzed using JMP 8.0.2 statistical software, which illustrates the strong correlation between high color intensity and increased levels of acidic charge variants in the antibody solution ( Figure 8 A). Furthermore, antibodies isolated from cell lines cultured using modified Medium 3 and Medium 4 had significantly reduced presence of acidic charge variants compared to cell lines cultured using Modified Medium 1 and Medium 2, with the greatest reduction observed at the lowest concentration of iron ( Figure 8 A).

[0423] The correlation between color intensity and acidic charge variant formation was examined in antibody-containing solutions obtained from cell lines cultured with medium containing different levels of iron or vitamin B. Monoclonal IgG1 antibodies were produced from CHO cells cultured in basal medium containing 18 μM or 75 μM ferrous sulfate, or in basal medium containing low, medium or high levels of vitamin B while the iron concentration was maintained at 18 μM. Low vitamin levels were: 0.25 mg / L vitamin B2, 5.35 mg / L vitamin B6 (5.35 mg / L pyridoxine + 0 mg / L pyridoxal), 8.61 mg / L vitamin B9 and 1.76 mg / L vitamin B12. Medium vitamin levels were: 0.70 mg / L vitamin B2, 7.7 mg / L vitamin B6 (7.7 mg / L pyridoxine + 0 mg / L pyridoxal), 4.9 mg / L vitamin B9 and 1.5 mg / L vitamin B12. The high vitamin levels were: 1.41 mg / L vitamin B2, 15.42 mg / L vitamin B6 (15.42 mg / L pyridoxine + 0 mg / L pyridoxal), 9.93 mg / L vitamin B9 and 3.05 mg / L vitamin B12. Monoclonal IgG1 antibodies were isolated and antibody titers were determined as described in Example 2. Color was determined using a total color test. For the total color test, a quantitative value for the relative color of the sample was obtained using the CIE color measurement system as described in Berns et al., Billmeyer and Saltzman's Principles of Color Technology, 3rd Edition, New York, NY, John Wiley & Sons, Inc., (2000). Briefly, the absorbance spectra of the neat test samples were measured in the visible region (380-780 nm) using a HP8453A spectrophotometer (1 cm path cuvette) after blanking with water. Then, according to Standard Practice for Calculation of Color Tolerances and Color Differences from Instrumentally Measured Color Coordinates, Annual Book of ASTM Standards, Vol. 06.01, (2011) previously described, the absorption spectrum is converted to the CIE L*a*b* color scale. For these calculations, an artificial flat spectrum in the visible region is used as the light source. "Total color" represents ΔE, which corresponds to the Euclidean distance between the test sample and water in the three-dimensional CIE L*a*b* color space. In addition, "total color" represents the overall color of the test monoclonal antibody sample, without distinguishing different hues.The color intensity value was then determined by calculating the ratio of the "total color" measurement of the test monoclonal antibody sample to the "total color" measurement of a reference monoclonal antibody sample, wherein the reference sample contained a COC reading of ≤ B5. A positive correlation was observed between increasing iron concentration and increasing color intensity (. Figure 8 B). In addition, increased iron concentrations resulted in increased levels of acidic charge variants. In contrast, simultaneously varying the levels of vitamins B2, B6, B9, and B12 in a medium containing a constant iron concentration showed no correlation between acidic charge variants and color intensity ( Figure 8 C).

[0424] Antibody separation from the cell line cultured from the cell culture medium containing the B2 and B6 of varying levels, the correlation of color intensity and the acidic charge variant formed is checked in the separated antibody. In order to produce monoclonal IgG1 antibody (anti-β7) from Chinese hamster ovary celI, with the fed-batch mode, the basis and feed medium containing the vitamin B2 and B6 of varying levels are fed to the cell culture. Subsequently, antibody separation and the existence of acidic charge variants are measured using the NIFTY test as described in Example 3. The relationship between color intensity and the presence of acidic charge variants is analyzed using JMP 8.0.2 statistical software, and analysis shows that in the antibody separated from the cell line cultured from the basal medium containing 1.41mg / L vitamin B2 and / or 15.42mg / L vitamin B6 (supply with pyridoxine) and the feed medium containing 10mg / L vitamin B2 and / or 76mg / L vitamin B6 (7mg / L pyridoxine and 60mg / L pyridoxal), there is a strong correlation ( Fig. 9 A and B). The lowest levels of color intensity and acidic charge variants were observed for antibodies isolated from cell lines cultured in basal medium containing 0.25 mg / L vitamin B2 and 5.35 mg / L vitamin B6 (supplied as pyridoxine) and in feed medium without vitamin B2 and vitamin B6 ( Fig. 9 A and B).

[0425] Carry out full factorial research, wherein in the presence of different iron sources, use the cell culture medium culture cell line containing the pyridoxal of variable level, separate antibody from cell line, determine the correlation between color intensity and acidic charge variant formation in antibody.In order to produce monoclonal IgG1 antibody (anti-β7) from Chinese hamster ovary syndrome (CHO) cells, use the basis and feed medium culture cell containing the pyridoxal of variable level.Ferric citrate or ferrous sulfate are provided in basal medium, and cell culture process is implemented in fed batch mode.Subsequently, separate antibody and use total color test to measure color intensity and measure the existence of acidic charge variant. The relationship between color intensity and the presence of acidic charge variants was analyzed using JMP 8.0.2 statistical software and showed a strong correlation between higher color intensity and increased levels of acidic charge variants for antibodies isolated from cell lines cultured in basal medium containing 0 mg / L pyridoxal and 18 μM ferrous sulfate and feed medium containing 0 mg / L or 60 mg / L pyridoxal, compared to antibodies isolated from cell lines cultured in basal medium containing 0 mg / L pyridoxal and 18 μM ferrous citrate and feed medium containing 0 mg / L or 60 mg / L pyridoxal ( Fig.10 A and B).

[0426] In the presence of different iron sources, the cell culture medium containing the vitamin B2, B6, B9 and B12 of varying levels is used to culture the cell line, and the correlation of color intensity and acidic charge variant formation is checked in the antibody separated from the cell line. In order to produce monoclonal IgG1 antibody (anti-β7) from Chinese hamster ovary celI, a basal medium containing 1.41mg / L vitamin B2, 15.42mg / L pyridoxine, 0mg / L pyridoxal, 9.93mg / L vitamin B9 and 3.05mg / L vitamin B12 and one of three different feed mediums are used to culture cells, wherein the three feed mediums contain vitamin B2, B6, B9 and B12 of varying levels respectively. Ferric citrate or ferrous sulfate is provided in the basal medium, and the cell culture process is implemented in fed batch mode. Antibody is subsequently separated, and the presence of color intensity and acidic charge variant is measured using the total color test. The relationship between color intensity and the presence of acidic charge variants was analyzed using JMP 8.0.2 statistical software and the analysis showed that culturing the cell lines in the presence of 18 μM ferrous sulfate or 18 μM ferric citrate using a feed medium containing 5 mg / L vitamin B2, 3.5 mg / L pyridoxine, 30 mg / L pyridoxal, 98.5 mg / L vitamin B9 and 24 mg / L vitamin B12 resulted in isolated antibodies with reduced color intensity compared to a feed medium containing 10 mg / L vitamin B2, 7 mg / L pyridoxine, 60 mg / L pyridoxal, 197 mg / L vitamin B9 and 48 mg / L vitamin B12 ( Fig.11A; vitamin level 2 and vitamin level 3, respectively). The greatest decrease in color intensity was observed in antibodies isolated from cells cultured in feed medium lacking vitamins B2, B6, B9, and B12 ( Fig.11 A; vitamin level 1). Although there was a significant decrease in antibody color intensity with decreasing concentrations of vitamins B2, B6, B9, and B12, preliminary results indicated no significant changes in the presence of acidic charge variants ( Fig.11 B).

[0427] Cell culture medium containing increasing concentrations of iron from different sources was used to culture cell lines to examine the correlation of color intensity and acidic charge variant formation in antibodies isolated from cell lines. In order to produce monoclonal IgG1 antibodies (anti-β7) from CHO cells, cells were cultured in fed-batch mode using basal medium 1 containing 75 μM ferrous sulfate, basal medium 12 containing 18 μM ferrous sulfate, or basal medium 13 containing 10 μM ferrous sulfate. Other cell cultures were fed with basal medium 15 containing 18 μM ferric citrate or basal medium 16 containing 10 μM ferric citrate. All cell cultures were fed with iron-free feed medium in fed-batch mode. As described in Example 3, color intensity was measured using the NIFTY test. In antibodies isolated from cells cultured with basal medium containing reduced levels of iron, the maximum reduction in color intensity ( Fig.12 A). Reduced color intensity correlates with reduced presence of acidic charge variants in separated antibody solutions ( Fig.12 B).

[0428] Cell lines were cultured with cell culture media containing increasing concentrations of ferric citrate, and the correlation between color intensity and acidic charge variant formation was examined in antibodies isolated from the cell lines. To produce monoclonal IgG1 antibodies (anti-β7) from CHO cells, cells were cultured in modified basal medium 1 or modified basal medium 3 containing 18 μM ferric citrate or 36 μM ferric citrate. All cell cultures were fed with iron-free feed medium in fed-batch mode. Monoclonal IgG1 antibodies were isolated from cell cultures incubated at 33°C or 37°C. Color intensity was measured using a color test, particularly a total color test, in which higher values ​​indicate higher color intensity and lower values ​​indicate lower color intensity. The greatest reduction in color intensity ( Fig.13 A). In the isolated antibody solution, the reduced color intensity correlates with the presence of reduced acidic charge variants. Antibodies isolated from cells cultured at 33°C showed the greatest reduction in acidic charge variants ( Fig.13B) Increasing the temperature from 33°C to 37°C resulted in a significant increase in antibody production from approximately 2500 mg / L to 4250 mg / L for antibody producing cells cultured in Modified Basal Medium 1 or Modified Basal Medium 3.

[0429] In order to determine the contribution of cysteine ​​to the color intensity of the isolated monoclonal IgG1 antibody (anti-β7) produced from the CHO cell line, the basal medium 3 was reformulated to replace 480 mg / L cystine and contain 525 mg / L cysteine ​​for cell culture experiments. In order to produce monoclonal IgG1 antibodies from CHO cells, cells were cultured in modified basal medium 3 and feed medium 4 containing 525 mg / L cysteine ​​in a fed-batch mode. The antibodies were separated and the color was measured using a standard COC test. The COC test was performed using a flat-bottomed, colorless, transparent, neutral glass tube with an identical inner diameter of 15 mm to 25 mm. Two tubes were each filled with a 150 g / L protein solution at a depth of 40 mm, which was prepared from a purified, concentrated cell culture fluid containing secreted IgG1 monoclonal antibodies. Each tube containing the antibody solution was compared to seven reference vials containing reference solutions from BY1 (darkest) to BY7 (lightest), or to nine reference vials containing reference solutions from B1 (darkest) to B9 (lightest), by vertical viewing against a white background in diffuse daylight. Color analysis of the antibody-containing solution showed a COC value of ≤ B5 or ≤ BY5 ( Figure 1 ; respectively preparations VI and VIII). Antibody preparation VI was further analyzed for color intensity using the above-mentioned total color test and the NIFTY test described in Example 3. When measured with the NIFTY test and the total color test, the color analysis of the antibody preparation showed color intensity values ​​of 0.71 and 1.00, respectively. The antibody preparation, compared with the color intensity of antibody preparation III (described in Example 1) and antibody preparation IV (described in Example 3), has a lighter color. When measured with the NIFTY test and the total color test, antibody preparation III showed color intensity values ​​of 1.59 and 2.61, respectively. When measured with the NIFTY test and the total color test, antibody preparation IV showed color intensity values ​​of 1.47 and 2.04, respectively.

[0430] A multivariate study was performed in which cell lines were cultured with cell culture media containing varying concentrations of vitamins B2, B6, B9, and B12 in the presence or absence of cystine or cysteine ​​and in the presence or absence of hydrocortisone to determine the correlation between color intensity and acidic charge variant formation in antibodies isolated from the cell lines. For the production of monoclonal IgG1 antibodies (anti-β7) from CHO cells, cells were cultured in a basal medium containing 1.41 mg / L vitamin B2, 15.42 mg / L pyridoxine, 0 mg / mL pyridoxal, 9.93 mg / L vitamin B9, and 3.05 mg / L vitamin B12, or a basal medium containing 0.7 mg / L vitamin B2, 7.7 mg / L pyridoxine, 0 mg / L pyridoxal, 4.9 mg / L vitamin B9, and 1.5 mg / L vitamin B12. In addition, the basal medium also contained 525 mg / L cysteine ​​or 480 mg / L cystine (Table G). Other basal media were prepared as shown in Table G and supplemented with 150 nM hydrocortisone. All cell cultures were fed with feed medium without iron in fed-batch mode.

[0431] Table G. Multivariate component experiments

[0432]

[0433]

[0434] Monoclonal IgG1 antibodies were isolated from cell cultures and the color intensity and presence of acidic charge variants were measured. Color intensity was determined using the total color test described above. The relationship between color intensity and the presence of acidic charge variants was analyzed using JMP 8.0.2 statistical software, which showed that there was a correlation between reduced color intensity and reduced levels of acidic charge variants in antibodies isolated from cell lines cultured in basal medium containing cystine compared to cysteine ​​( Fig.14 A and B). In addition, reduced vitamin B levels also resulted in reduced color intensity and acidic charge variant formation, and the addition of hydrocortisone enhanced this reduction ( Fig.14 A and B).

[0435] Example 6: Reduction of the color intensity of monoclonal IgG1 antibodies isolated from cell lines by changing specific components in the cell culture medium

[0436] Using a CHO cell line that produces different monoclonal IgG1 antibodies (mAb10), the effect of cystine on reducing color intensity when used in a basic cell culture medium was further studied. The cell line was cultured in a chemically uncertain basic medium, and the basic medium had a cysteine ​​amino acid (cysteine) in the form of a monomer in a concentration of 2.6mM, or a cysteine ​​amino acid (cysteine) in the form of a dimer in a concentration of 1.3mM. By inoculating cells in this chemically uncertain basic medium, the production of mAb10 was initiated in the cell culture, and batch feed medium was added to the bioreactor on the 3rd day in a 14-day cell culture cycle. The cells were cultured at 37°C on the 1st day, and the initial temperature migration occurred during the cell culture cycle. The culture medium was harvested, mAb10 was recovered in the form of a composition, and the color intensity was evaluated using the COC test afterwards. The composition containing mAb10 recovered from the cells cultured in the basic medium containing cysteine ​​was presented as a colorless or slightly colored liquid with a B7 color intensity value determined by the COC test. When cysteine ​​was replaced with cystine, the composition comprising mAb10 recovered from cells cultured in basal medium appeared as a colorless or slightly colored liquid with an improved color intensity COC value of B8.

[0437] In a multivariate study, four different schemes for producing mAb10 from CHO cells were used to evaluate the effects of certain culture medium components on the color intensity of antibody-containing compositions recovered from cell culture (Table H). The iron level and iron source in the chemically uncertain basal medium were different between the schemes. There were also different vitamin B levels and amino acid cysteine ​​in the chemically uncertain basal medium between the schemes-monomer form cysteine ​​amino acid (cysteine) or dimer form cysteine ​​amino acid (cystine). mAb10 was produced in cell culture by inoculating cells in a chemically uncertain basal medium, and batch feed medium was added to the bioreactor on the 3rd day in a 14-day cell culture cycle. The culture medium was harvested, mAb10 was harvested in the form of a composition, and the color intensity was evaluated using the COC test afterwards. The color intensity of the composition containing the recovered mAb was analyzed, and the analysis showed that compared with the color intensity of the antibody composition obtained using scheme 4 (COC value of B6), schemes 1, 2 and 3 resulted in an antibody composition with reduced color intensity (COC value of B7). These results show that the use of cystine instead of cysteine ​​in the basal medium, the reduction of vitamin B levels, and / or the reduction of iron and the change of the iron source lead to a reduction in color intensity in the mAb10 composition (Table H). For example, compared to Scheme 4, in Scheme 3, reducing the vitamin B level and using ferric citrate instead of ferrous sulfate at a lower concentration reduced the color intensity of the antibody composition without changing the use of cysteine ​​to cystine. Relative to Scheme 4, in Scheme 2, when ferric citrate replaced ferrous sulfate at a lower concentration and cystine was used instead of cysteine ​​without changing the vitamin B level, the same color intensity reduction effect was observed. Compared to the mAb10 composition obtained from Scheme 4, as seen in Scheme 1, the reduction of vitamin B levels, the reduction of iron levels, and the change of the iron source, as well as the use of cystine instead of cysteine, also led to a reduction in the color intensity of the mAb10 composition (Table H).

[0438] Table H. Summary of basal medium components

[0439]

[0440]

[0441] a Indicates ferric citrate as the iron source; b Indicates ferrous sulfate as the iron source

[0442] Example 7: NIFTY and Total Color Tests Compare to Standard COC Tests for Measurement of Color Intensity in Antibody-Containing Solutions

[0443] The recent emphasis on high concentration formulations has generally increased the color intensity of monoclonal antibody liquid formulations. Color is regarded as a quality attribute of the product, therefore, it is important to closely monitor the consistency of the color of the drug substance when developing and implementing the embodiment. One of the challenges faced when measuring color is to use a suitable test. Although the COC test is an industrial standard, the COC test is not completely quantitative and is susceptible to the influence of the individual's competent judgment of the test. In order to overcome this problem, two different color measurement methods have been developed, the total color test and the NIFTY test, and are used to measure color intensity in the examples.

[0444] The correlation between the three color measurement tests was determined by plotting total color values ​​on the abscissa and NIFTY values ​​on the ordinate, with data points based on actual COC measurements performed on antibody-containing solutions measured by the total color and NIFTY tests ( Fig.15 A). There was a good correlation between the quantitative measurements of the total color test and the NIFTY test (R 2 =0.75). Furthermore, it can be seen that these tests have a fairly good correlation with the actual COC measurements, indicating that the results obtained from the total color test and the NIFTY test can be used to predict the color intensity of the samples.

[0445] By two kinds of different methods from cell culture results and purification of antibodies, color intensity is measured afterwards.In one method, the monoclonal antibody in the cell culture of protein A affinity chromatography purification results is used.After purification, the concentration of protein is about 5-10g / L in the protein A pool of wash-out.Further use Amicon Centricon centrifugal filter device, protein A pool is concentrated to 150g / L.In concentrated protein A pool, use standard COC test, total color test or NIFTY test to measure color.In another method, use standard antibody purification method, the cell culture fluid of purification results, described standard method comprises: carry out affinity purification by protein A affinity chromatography, further purify by anion and cation exchange chromatography, filter to remove virus and final ultrafiltration and diafiltration step for final preparation and concentration of antibody, use standard COC test, total color test or NIFTY test to measure color afterwards.

[0446] Due to practical considerations, it was decided to use the color of the Protein A pool as a surrogate for the color of the final drug substance in several experiments described in the Examples. The predictive value of the color intensity measured from the antibody preparation prepared from the Protein A pool was evaluated against the color intensity obtained from the final fully purified antibody preparation. Fig.15 B) or total color test ( Fig.15C) The color intensity of the antibody-containing solution obtained from the Protein A pool was measured and compared with the color intensity measured in the final fully purified antibody preparation, which confirmed that there was a fairly good correlation between the total color measurement (R2=0.73) and the NIFTY measurement (R2=0.98). The total color comes from the absorption spectrum of the pool, so it can be expected that there will be higher color intensity in the earlier pools that are still in process due to the presence of non-antibody impurities or due to increased light scattering. In contrast, the NIFTY value is measured from the main peak of the size exclusion chromatography, so it can be expected that if the colored or non-colored protein molecules are not preferentially purified, the NIFTY value will remain constant during the purification process. Overall, a reasonable correlation of color measurements was observed between the Protein A pool and the formulated drug substance.

Claims

1. A method for culturing cells, comprising the steps of: contacting the cells with a cell culture medium, wherein the cell culture medium comprises: From about 200 mg / L to about 1200 mg / L cystine; From about 0.05 mg / L to about 1.0 mg / L vitamin B2; From about 0.05 mg / L to about 10.0 mg / L vitamin B6; From about 0.05 mg / L to about 12.0 mg / L vitamin B9; and From about 0.05 mg / L to about 2.5 mg / L vitamin B12.

2. A method for producing a polypeptide, comprising the steps of: culturing cells containing an isolated nucleic acid encoding the polypeptide in a cell culture medium, wherein: (a) A cell culture medium comprising: From about 200 mg / L to about 1200 mg / L cystine; From about 0.05 mg / L to about 1.0 mg / L vitamin B2; From about 0.05 mg / L to about 10.0 mg / L vitamin B6; From about 0.05 mg / L to about 12.0 mg / L vitamin B9; From about 0.05 mg / L to about 2.5 mg / L vitamin B12; and (b) Cells express the polypeptide.

3. A polypeptide produced by the method of claim 2.

4. A pharmaceutical composition comprising the polypeptide of claim 3 and a pharmaceutically acceptable carrier.

5. A kit for supplementing a cell culture medium with chemically defined components, the kit comprising: Cystine in an amount to provide from about 200 mg / L to about 1200 mg / L cystine in the cell culture medium; Vitamin B2 in an amount to provide from about 0.05 mg / L to about 1.0 mg / L vitamin B2 in the cell culture medium; Vitamin B6 in an amount to provide from about 0.05 mg / L to about 10.0 mg / L vitamin B6 in the cell culture medium; Vitamin B9 in an amount to provide from about 0.05 mg / L to about 12.0 mg / L vitamin B9 in the cell culture medium; and Vitamin B12 in an amount to provide from about 0.05 mg / L to about 2.5 mg / L vitamin B12 in the cell culture medium.

6. Cell culture medium, comprising: From about 200 mg / L to about 1200 mg / L cystine; From about 0.05 mg / L to about 1.0 mg / L vitamin B2; From about 0.05 mg / L to about 10.0 mg / L vitamin B6; From about 0.05 mg / L to about 12.0 mg / L vitamin B9; and From about 0.05 mg / L to about 2.5 mg / L vitamin B12.

7. A method for culturing cells, comprising the steps of: contacting the cells with a cell culture medium, wherein the cell culture medium comprises: From about 200 mg / L to about 1200 mg / L cystine; From about 2 μM to about 80 μM ferric citrate; and From about 0.05 μM to about 0.5 μM hydrocortisone.

8. A method for producing a polypeptide, comprising the steps of: culturing cells containing an isolated nucleic acid encoding the polypeptide in a cell culture medium, wherein: (a) A cell culture medium comprising: From about 200 mg / L to about 1200 mg / L cystine; From about 2 μM to about 80 μM ferric citrate; and From about 0.05 μM to about 0.5 μM hydrocortisone; and (b) Cells express the polypeptide.

9. A polypeptide produced by the method of claim 8.

10. A pharmaceutical composition comprising the polypeptide of claim 9 and a pharmaceutically acceptable carrier.

11. A kit for supplementing a cell culture medium with chemically defined components, the kit comprising: Cystine in an amount to provide from about 200 mg / L to about 1200 mg / L cystine in the cell culture medium; ferric citrate in an amount to provide a concentration of ferric citrate from about 2 μM to about 80 μM in the cell culture medium; and Cortisol in an amount to provide a concentration of from about 0.05 μM to about 0.5 μM cortisol in the cell culture medium.

12. Cell culture medium, comprising: From about 200 mg / L to about 1200 mg / L cystine; From about 2 μM to about 80 μM ferric citrate; and From about 0.05 μM to about 0.5 μM hydrocortisone.

13. A composition comprising (a) cells containing an isolated nucleic acid encoding a polypeptide; and (b) a culture medium according to any one of claims 6 and 12.

14. A composition comprising: (a) a polypeptide; and (b) a culture medium according to any one of claims 6 and 12.

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