HK1218141B - Cell culture compositions with antioxidants and methods for polypeptide production - Google Patents

Description

Cell culture composition with antioxidant and polypeptide production method

Cross-references to Related Patent Applications

This application claims priority to U.S. Provisional Application Serial No. 61/799,602, filed March 15, 2013, the contents of which are hereby incorporated by reference herein in their entirety.

Field of the Invention

The present invention relates to cell culture media comprising antioxidants, methods for cell culture and polypeptide production using the media, and compositions and kits comprising polypeptides produced by the methods provided herein.

Background Art

Cell culture production technology is widely used to produce protein-based products, such as pharmaceutical preparations of therapeutic proteins. The commercial production of protein-based products, such as antibody products, requires optimization of cell culture parameters so that cells produce enough protein products to meet production requirements. However, when optimizing cell culture parameters to improve the productivity of protein products, it is also necessary to maintain the desired quality attributes of the product, such as glycosylation profile, aggregate level, charge heterogeneity and amino acid sequence integrity (Li et al., mAbs, 2010, 2 (5): 466-477). Another quality attribute worthy of attention is the color of the protein product. For liquid formulations of therapeutic products for human use, regulatory requirements for acceptable color levels must be met (United States Pharmacopoeia Inc., 2000, p. 1926-1927 and Council of Europe. European Pharmacopoeia, 2008, ^{7 th} Ed. p. 22). Therefore, producing protein products with acceptable color is an important aspect of therapeutic protein production.

The recent trend toward subcutaneous delivery of therapeutic proteins, such as monoclonal antibodies, has been accompanied by increasing concentrations of formulated protein materials, for example, at concentrations of about 100 mg/mL or more (Daugherty et al., Adv Drug Deliver Rev, 2006, 58(5-6):686-706). A correlation has been observed between increasing amounts of therapeutic protein in a composition and increasing color intensity of the composition, which may be due to low levels of protein product variants that were previously unobservable by standard methods of monitoring color intensity of the formulated product.

Oxidation is a major chemical degradation pathway for protein pharmaceuticals. For example, methionine, cysteine, histidine, tryptophan, and tyrosine are amino acid residues that are susceptible to oxidation due to their reactivity with reactive oxygen species (ROS), and this oxidation is often observed during storage of pharmaceutical protein formulations. While it is known that cell culture conditions can affect the quality attributes of protein products (such as production of sufficient quantities for large-scale manufacturing), the impact of these conditions on the color intensity of the final protein product is unclear.

There is a continuing need to provide improved and cost-effective methods for producing proteins (e.g., antibodies) with acceptable product quality attributes (e.g., color intensity). Cell culture media, whether chemically undefined or chemically defined, having components that consistently deliver protein products at lower color intensity while maintaining a desired protein concentration (e.g., ≥100 mg/mL) can be used in the development of protein products, such as antibodies.

SUMMARY OF THE INVENTION

In some aspects, the present invention provides a method of culturing cells comprising a nucleic acid encoding a polypeptide, wherein the method comprises contacting the cells with a cell culture medium comprising hypotaurine or an analog thereof or a precursor thereof, wherein the cell culture medium comprising hypotaurine or an analog thereof or a precursor thereof reduces the color intensity of a composition comprising the polypeptide produced by the cells compared to a composition comprising the polypeptide produced by cells cultured in a cell culture medium without hypotaurine or an analog thereof or a precursor thereof. In some embodiments, the cell culture medium comprising hypotaurine or an analog thereof or a precursor thereof reduces the color intensity of the composition comprising the polypeptide produced by the cells by at least about 0.1% compared to a composition comprising the polypeptide produced by cells cultured in a cell culture medium without hypotaurine or an analog thereof or a precursor thereof. In some embodiments, the cell culture medium comprising hypotaurine or an analog thereof or a precursor thereof reduces the color intensity of the composition comprising the polypeptide produced by the cells by about 5% to about 50% compared to a composition comprising the polypeptide produced by cells cultured in a cell culture medium without hypotaurine or an analog thereof or a precursor thereof. In some embodiments herein, the cell culture medium comprises hypotaurine or an analog thereof or a precursor thereof at a concentration of at least about 0.0001 mM. In some embodiments herein, the cell culture medium comprises hypotaurine, or an analog or precursor thereof, at a concentration of about 0.0001 mM to about 500.0 mM. In some embodiments herein, the cell culture medium comprises hypotaurine, or an analog or precursor thereof, at a concentration of about 1.0 mM to about 40.0 mM. In some embodiments herein, the cell culture medium comprises hypotaurine, or an analog or precursor thereof, at a concentration of about 1.0 mM to about 10.0 mM. In some embodiments herein, the hypotaurine, or an analog or precursor thereof, is selected from hypotaurine, S-carboxymethylcysteine, cysteamine, cysteinesulfinic acid, and taurine. In some embodiments herein, the cell culture medium comprising hypotaurine, or an analog or precursor thereof, is a chemically defined cell culture medium. In some embodiments herein, the cell culture medium comprising hypotaurine, or an analog or precursor thereof, is a chemically undefined cell culture medium. In some embodiments herein, the cell culture medium comprising hypotaurine, or an analog or precursor thereof, is a basal cell culture medium. In some embodiments herein, the cell culture medium comprising hypotaurine, or an analog or precursor thereof, is a feed cell culture medium. In some embodiments herein, the cells are contacted with a cell culture medium comprising hypotaurine or its analog or precursor during the cell growth phase. In some embodiments herein, the cells are contacted with a cell culture medium comprising the hypotaurine or its analog or precursor during the cell production phase. In some embodiments herein, hypotaurine or its analog or precursor is added to the cell culture medium on at least one day of the cell culture cycle. In some embodiments herein, hypotaurine or its analog or precursor is added to the cell culture medium on day 0 of a 14-day cell culture cycle. In any embodiments herein, hypotaurine or its analog or precursor can be added to the cell culture medium on any day of the cell culture cycle. In some embodiments herein, the cells are mammalian cells. In some embodiments herein, the mammalian cells are Chinese hamster ovary (CHO) cells. In some embodiments herein, the polypeptide is an antibody or a fragment thereof.

In other aspects, the present invention provides a method of culturing a cell comprising a nucleic acid encoding a polypeptide, wherein the method comprises contacting the cell with a cell culture medium, wherein the cell culture medium comprises one or more of components (a)-(h): (a) hypotaurine; (b) s-carboxymethylcysteine; (c) carnosine; (d) anserine; (e) butylated hydroxyanisole; (f) lipoic acid; (g) quercetin hydrate; and (h) aminoguanidine; and wherein the cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of a composition comprising the polypeptide produced by the cell, compared to a composition comprising the polypeptide produced by cells cultured in a cell culture medium without the one or more of components (a)-(h). In some embodiments, the cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of the composition comprising the polypeptide produced by the cell by 0.1%, compared to a composition comprising the polypeptide produced by cells cultured in a cell culture medium without the one or more of components (a)-(h). In some embodiments, a cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of a composition comprising a polypeptide produced by a cell by about 5% to about 75%, compared to a composition comprising a polypeptide produced by cells cultured in a cell culture medium without one or more of components (a)-(h). In some embodiments, a cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of a composition comprising a polypeptide produced by a cell by about 5% to about 50%, compared to a composition comprising a polypeptide produced by cells cultured in a cell culture medium without one or more of components (a)-(h). In some embodiments herein, the cell culture medium comprising one or more of components (a)-(h) comprises one or more of the components (a)-(h) in an amount selected from the group consisting of: (a) hypotaurine at a concentration of at least about 0.0001 mM; (b) s-carboxymethylcysteine at a concentration of at least about 0.0001 mM; (c) carnosine at a concentration of at least about 0.0001 mM; (d) anserine at a concentration of at least about 0.0001 mM; (e) butylated hydroxyanisole at a concentration of at least about 0.0001 mM; (f) lipoic acid at a concentration of at least about 0.0001 mM; (g) quercetin hydrate at a concentration of at least about 0.0001 mM; and (h) aminoguanidine at a concentration of at least about 0.0003 mM. In further embodiments, the cell culture medium comprises hypotaurine at a concentration of about 2.0 mM to about 50.0 mM. In some embodiments herein, the cell culture medium comprises s-carboxymethylcysteine at a concentration of about 8.0 mM to about 12.0 mM. In some embodiments herein, the cell culture medium comprises carnosine at a concentration of about 8.0 mM to about 12.0 mM. In some embodiments herein, the cell culture medium comprises anserine at a concentration of about 3.0 mM to about 5.0 mM. In some embodiments herein, the cell culture medium comprises butylated hydroxyanisole at a concentration of about 0.025 mM to about 0.040 mM. In some embodiments herein, the cell culture medium comprises lipoic acid at a concentration of about 0.040 mM to about 0.060 mM. In some embodiments herein, the cell culture medium comprises quercetin hydrate at a concentration of about 0.010 mM to about 0.020 mM. In some embodiments, the cell culture medium comprises aminoguanidine at a concentration of about 0.0003 mM to about 245 mM. In some embodiments, the cell culture medium comprises aminoguanidine at a concentration of about 0.0003 mM to about 10 mM. In some embodiments herein, the cell culture medium is a chemically defined cell culture medium. In some embodiments herein, the cell culture medium is a chemically undefined cell culture medium. In some embodiments herein, the cell culture medium is a basal cell culture medium. In some embodiments herein, the cell culture medium is a feed cell culture medium. In some embodiments herein, the cells are contacted with the cell culture medium during the cell growth phase. In some embodiments herein, the cells are contacted with the cell culture medium during the cell production phase. In some embodiments herein, one or more of the components (a)-(h) are added to the cell culture medium on at least one day of the cell culture cycle. In some embodiments herein, one or more of the components (a)-(h) are added to the cell culture medium on day 0 of a 14-day cell culture cycle. In any embodiments herein, one or more of the components (a)-(h) can be added to the cell culture medium on any day of the cell culture cycle. In some embodiments herein, the cells are mammalian cells. In some embodiments herein, the mammalian cells are Chinese hamster ovary (CHO) cells. In some embodiments herein, the polypeptide is an antibody or a fragment thereof.

In some aspects, the present invention also provides a method for producing a polypeptide, comprising culturing cells comprising a nucleic acid encoding the polypeptide in a cell culture medium comprising hypotaurine or an analog or precursor thereof, and wherein the cell culture medium comprising hypotaurine or an analog or precursor thereof reduces the color intensity of the composition comprising the polypeptide produced by the cells compared to the color intensity of the composition comprising the polypeptide produced by cells cultured in a cell culture medium without hypotaurine or an analog or precursor thereof. In some embodiments, the cell culture medium comprising hypotaurine or an analog or precursor thereof reduces the color intensity of the composition comprising the polypeptide produced by the cells by at least about 0.1% compared to the composition comprising the polypeptide produced by cells cultured in a cell culture medium without hypotaurine or an analog or precursor thereof. In some embodiments, the cell culture medium comprising hypotaurine or an analog or precursor thereof reduces the color intensity of the composition comprising the polypeptide produced by the cells by about 5% to about 50% compared to the composition comprising the polypeptide produced by cells cultured in a cell culture medium without hypotaurine or an analog or precursor thereof. In some embodiments herein, the cell culture medium comprises hypotaurine or an analog or precursor thereof at a concentration of at least about 0.0001 mM. In some embodiments herein, the cell culture medium comprises hypotaurine, or an analog or precursor thereof, at a concentration of about 0.0001 mM to about 500.0 mM. In some embodiments herein, the cell culture medium comprises hypotaurine, or an analog or precursor thereof, at a concentration of about 1.0 mM to about 40.0 mM. In some embodiments herein, the cell culture medium comprises hypotaurine, or an analog or precursor thereof, at a concentration of about 1.0 mM to about 10.0 mM. In some embodiments herein, the hypotaurine, or an analog or precursor thereof, is selected from hypotaurine, S-carboxymethylcysteine, cysteamine, cysteinesulfinic acid, and taurine. In some embodiments herein, the cell culture medium is a chemically defined cell culture medium. In some embodiments herein, the cell culture medium is a chemically undefined cell culture medium. In some embodiments herein, the cell culture medium is a basal cell culture medium. In some embodiments herein, the cell culture medium is a fed cell culture medium. In some embodiments herein, the hypotaurine, or an analog or precursor thereof, is added to the cell culture medium on at least one day during the cell culture cycle. In some embodiments herein, hypotaurine or its analog or its precursor is added to the cell culture medium on day 0 of a 14-day cell culture cycle. In any embodiment herein, hypotaurine or its analog or its precursor can be added to the cell culture medium on any day of the cell culture cycle. In some embodiments herein, the cell is a mammalian cell. In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. In some embodiments herein, the polypeptide is an antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is secreted into the cell culture medium comprising hypotaurine or its analog or its precursor. In some embodiments, the method further comprises the step of recovering the polypeptide from the cell culture medium containing hypotaurine or its analog or its precursor. In some embodiments, the composition containing the recovered polypeptide is a liquid composition or a non-liquid composition. In some embodiments, the composition containing the recovered polypeptide appears as a colorless or slightly colored liquid.

In some aspects, the present invention provides a method of producing a polypeptide, comprising the step of culturing cells comprising a nucleic acid encoding the polypeptide in a cell culture medium, wherein the cell culture medium comprises one or more of components (a)-(h): (a) hypotaurine; (b) s-carboxymethylcysteine; (c) carnosine; (d) anserine; (e) butylated hydroxyanisole; (f) lipoic acid; (g) quercetin hydrate; and (h) aminoguanidine; and wherein the cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of a composition comprising the polypeptide produced by the cells compared to a composition comprising the polypeptide produced by cells cultured in a cell culture medium without the one or more of components (a)-(h). In some embodiments, the cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of the composition comprising the polypeptide produced by the cells by at least about 0.1% compared to a composition comprising the polypeptide produced by cells cultured in a cell culture medium without the one or more of components (a)-(h). In some embodiments, a cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of a composition comprising a polypeptide produced by a cell by about 5% to about 50%, compared to a composition comprising a polypeptide produced by cells cultured in a cell culture medium without one or more of components (a)-(h). In some embodiments, a cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of a composition comprising a polypeptide produced by a cell by about 5% to about 75%, compared to a composition comprising a polypeptide produced by cells cultured in a cell culture medium without one or more of components (a)-(h). In some embodiments herein, the cell culture medium comprises one or more of the components (a)-(h) in an amount selected from the group consisting of: (a) hypotaurine at a concentration of at least about 0.0001 mM; (b) s-carboxymethylcysteine at a concentration of at least about 0.0001 mM; (c) carnosine at a concentration of at least about 0.0001 mM; (d) anserine at a concentration of at least about 0.0001 mM; (e) butylated hydroxyanisole at a concentration of at least about 0.0001 mM; (f) lipoic acid at a concentration of at least about 0.0001 mM; (g) quercetin hydrate at a concentration of at least about 0.0001 mM; and (h) aminoguanidine at a concentration of at least about 0.0003 mM. In some embodiments herein, the cell culture medium comprises hypotaurine at a concentration of about 2.0 mM to about 50.0 mM. In some embodiments herein, the cell culture medium comprises s-carboxymethylcysteine at a concentration of about 8.0 mM to about 12.0 mM. In some embodiments herein, the cell culture medium comprises carnosine at a concentration of about 8.0 mM to about 12.0 mM. In some embodiments herein, the cell culture medium comprises anserine at a concentration of about 3.0 mM to about 5.0 mM. In some embodiments herein, the cell culture medium comprises butylated hydroxyanisole at a concentration of about 0.025 mM to about 0.040 mM. In some embodiments herein, the cell culture medium comprises lipoic acid at a concentration of about 0.040 mM to about 0.060 mM. In some embodiments herein, the cell culture medium comprises quercetin hydrate at a concentration of about 0.010 mM to about 0.020 mM. In some embodiments herein, the cell culture medium comprises aminoguanidine at a concentration of about 0.0003 mM to about 245 mM. In some embodiments herein, the cell culture medium comprises aminoguanidine at a concentration of about 0.0003 mM to about 10 mM. In some embodiments herein, the cell culture medium is a chemically defined cell culture medium. In some embodiments herein, the cell culture medium is a chemically undefined cell culture medium. In some embodiments herein, the cell culture medium is a basal cell culture medium. In some embodiments herein, the cell culture medium is a feed cell culture medium. In some embodiments herein, the cells are contacted with the cell culture medium during the cell growth phase. In some embodiments herein, the cells are contacted with the cell culture medium during the cell production phase. In some embodiments herein, one or more of the components (a)-(h) are added to the cell culture medium on at least one day of the cell culture cycle. In some embodiments herein, one or more of the components (a)-(h) are added to the cell culture medium on day 0 of a 14-day cell culture cycle. In any of the embodiments herein, one or more of the components (a)-(h) can be added to the cell culture medium on any day of the cell culture cycle. In some embodiments herein, the cells are mammalian cells. In some embodiments, the mammalian cells are Chinese hamster ovary (CHO) cells. In some embodiments herein, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is secreted into the cell culture medium. In some embodiments herein, the method further comprises the step of recovering the polypeptide from the cell culture medium comprising one or more of the components (a)-(h). In some embodiments, the composition comprising the recovered polypeptide is a liquid composition or a non-liquid composition. In some embodiments, the composition comprising the recovered polypeptide appears as a colorless or slightly colored liquid.In some embodiments herein, the polypeptide can be produced by any of the methods described herein.

In some aspects, the invention provides pharmaceutical compositions comprising a polypeptide produced by any of the methods described herein and a pharmaceutically acceptable carrier.

In some aspects, the present invention provides a kit for supplementing a cell culture medium with a chemically defined component, the kit comprising hypotaurine or an analog or precursor thereof at a concentration of at least about 0.0001 mM, and wherein the hypotaurine or an analog or precursor thereof is selected from the group consisting of hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulfinic acid, and taurine.

In other aspects, the present invention also provides a kit for supplementing a cell culture medium with a chemically defined component, the kit comprising one or more of the following: (a) hypotaurine in an amount to provide at least about 0.0001 mM hypotaurine in the cell culture medium; (b) s-carboxymethylcysteine in an amount to provide at least about 0.0001 mM s-carboxymethylcysteine in the cell culture medium; (c) carnosine in an amount to provide at least about 0.0001 mM carnosine in the cell culture medium; (d) anserine in an amount to provide at least about 0.0001 mM carnosine in the cell culture medium; (e) butylated hydroxyanisole in an amount to provide at least about 0.0001 mM of anserine in the cell culture medium; (f) lipoic acid in an amount to provide at least about 0.0001 mM of lipoic acid in the cell culture medium; (g) quercetin hydrate in an amount to provide at least about 0.0001 mM of quercetin hydrate in the cell culture medium; and (h) aminoguanidine in an amount to provide at least about 0.0003 mM of aminoguanidine in the cell culture medium.

In some aspects, the present invention provides a cell culture medium comprising at least about 0.0001 mM hypotaurine or an analog or precursor thereof selected from the group consisting of hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulfinic acid, and taurine.

In other aspects of the invention, a cell culture medium is provided comprising one or more of components (a)-(h): (a) at least about 0.0001 mM hypotaurine; (b) at least about 0.0001 mM s-carboxymethylcysteine; (c) at least about 0.0001 mM carnosine; (d) at least about 0.0001 mM anserine; (e) at least about 0.0001 mM butylated hydroxyanisole; (f) at least about 0.0001 mM lipoic acid; (g) at least about 0.0001 mM quercetin hydrate; and (h) at least about 0.0003 mM aminoguanidine.

In some aspects, the invention provides a composition comprising: (a) a cell comprising a nucleic acid encoding a polypeptide; and (b) any of the cell culture media described herein.

In some aspects of the present invention, a composition is provided comprising: (a) a polypeptide; and (b) any cell culture medium described herein. In some embodiments, the polypeptide is secreted into the cell culture medium by a cell comprising a nucleic acid encoding the polypeptide.

This description is considered sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art based on the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows representative compounds screened based on their effect on color intensity in representative cell culture media containing the antibody. Numerical results are normalized to a positive control, which was set to a 0% change in color intensity. Values above 0% indicate increased color intensity. Values below 0% indicate decreased color intensity.

Figure 2 is a subgraph of Figure 1 showing compounds that reduce color intensity in a representative cell culture medium containing an antibody. Numerical results are normalized to a positive control, where the value of the positive control is set to 0% color intensity change. Values below 0% indicate reduced color intensity.

Fig. 3 is a series of curve graphs showing that the shake flask cell culture model is comparable to the corresponding larger 2L cell culture model. A) Cell growth in the culture during the incubation period measured by viable cell density (VCC) is expressed as the number of cells per cell culture volume. B) Cell viability in the cell culture during the incubation period measured by viable cell number is expressed as the percentage of total cells. C) Antibody production in the cell culture during the incubation period measured by HPLC is expressed as antibody titer. SF represents the shake flask cell culture model. 2L represents a larger scale cell culture model.

FIG4 is a series of graphs showing that the addition of hypotaurine to cell culture media does not impair cell growth, cell viability, or antibody production. A) Cell growth in culture during the incubation period as measured by VCC and expressed as the number of cells per cell culture volume. B) Cell viability in cell culture during the incubation period as measured by viable cell count as a percentage of the total number of cells. C) Antibody production in cell culture during the incubation period as measured by HPLC as expressed as antibody titer.

Figure 5 shows the color intensity of antibody compositions isolated from cell cultures grown in medium supplemented with hypotaurine. 100%, 50%, or 25% represent Basal Medium 1 supplemented with 9.16 mM, 4.58 mM, or 2.29 mM hypotaurine, respectively. Filled circles represent color intensity values from cell culture experiments. Open circles represent color intensity values from incubation screening experiments. Numerical results are normalized to a positive control, where the value of the positive control was set to 0% color intensity change. Values below 0% indicate decreased color intensity.

Figure 6 shows the color intensity of antibody compositions isolated from cell cultures grown in medium supplemented with hypotaurine. 3X, 2X, or 1X represent Basal Medium 3 supplemented with 38.85 mM, 25.9 mM, or 12.95 mM hypotaurine, respectively. Solid circles represent color intensity values for cell culture experiments. Numerical results are normalized to a positive control, where the value of the positive control was set to 0% color intensity change. Values below 0% indicate decreased color intensity.

FIG7 contains graphs showing that the addition of hypotaurine or carboxymethylcysteine to the culture medium does not impair cell growth or cell viability. A) Cell growth in culture during the duration of incubation as measured by VCC and expressed as the number of cells per cell culture volume. B) Cell viability in culture during the duration of incubation as a percentage of the total culture volume.

Figure 8 shows that addition of hypotaurine or carboxymethylcysteine to the culture medium did not significantly reduce antibody production.Antibody production in the cell cultures was measured by high performance liquid chromatography over the duration of the incubation period and is expressed as antibody titer.

FIG9 shows the color intensity of antibody compositions isolated from cell cultures grown in medium supplemented with hypotaurine or carboxymethylcysteine. A and B) represent two different colorimetric assays used to measure color intensity. Numerical results are normalized to a positive control, where the value of the positive control is set to a 0% change in color intensity. Values below 0% indicate decreased color intensity.

Figure 10 shows the relative color intensity of antibody compositions isolated from cell cultures in medium supplemented with taurine, carnosine, aminoguanidine, a negative control, or a positive control.

Detailed Description of the Invention

I. Definition

The terms "culture medium" and "cell culture medium" refer to a nutrient source used to grow or maintain cells. As will be appreciated by those skilled in the art, the nutrient source can contain components necessary for cell growth and/or survival, or can contain components that aid in cell growth and/or survival. Vitamins, essential or nonessential amino acids, and trace elements are examples of culture medium components.

A "chemically defined cell culture medium" or "CDM" is a culture medium with a well-defined composition that is free of products of animal or plant origin, such as animal serum and plant peptones. Those skilled in the art will appreciate that a CDM can be used in a polypeptide production process, wherein cells are contacted with the CDM and secrete the polypeptide into the CDM. Thus, it will be understood 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.

A "chemically undefined cell culture medium" refers to a culture medium whose chemical composition cannot be determined, which may contain one or more products of animal or plant origin, such as animal serum and plant peptone. It will be appreciated by those skilled in the art that a chemically undefined cell culture medium may contain products of animal or plant origin as nutrient sources.

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

"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.

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

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

The "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.

As used herein, "hypotaurine analogues" refer to chemical compounds that are structurally similar to hypotaurine, but whose chemical composition differs from that of hypotaurine (e.g., the number, position, or chemical nature of the functional groups or substituents on the hypotaurine core differs). Hypotaurine analogues may or may not have different chemical or physical properties than hypotaurine, and may or may not have increased activity in cell culture medium compared to hypotaurine, for example, further reducing the color intensity of a polypeptide (e.g., an antibody) produced in cell culture medium compared to hypotaurine. For example, a hypotaurine analogue may be more hydrophilic or may have altered reactivity compared to hypotaurine. A hypotaurine analogue may mimic the chemical and/or biological activity of hypotaurine (i.e., it may have similar or identical activity), or, in some cases, may have increased or decreased activity compared to hypotaurine.

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.

"Nucleic acid," as used interchangeably herein, refers to polymers of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymers or by synthetic reactions. Polynucleotides 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.

"Isolated nucleic acid" refers to and encompasses a non-naturally occurring, recombinant, or naturally occurring sequence that is not in its usual environment or is separated from its usual environment. The isolated nucleic acid molecule exists in a form or environment that is different from its form or environment in nature. Thus, an isolated nucleic acid molecule can be distinguished from the nucleic acid molecule when present in a natural cell. However, an isolated nucleic acid molecule can include a nucleic acid molecule contained in a cell that normally expresses the protein, wherein, for example, the nucleic acid molecule is located in a chromosomal position that is different from the position in the natural cell.

An "isolated" protein (e.g., an isolated antibody) is a protein that has been identified and separated and/or recovered from a component of its natural environment. Impurity components in its natural environment are substances that would interfere with the research, diagnosis, or therapeutic use of the protein and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. An isolated protein includes an in situ protein in a recombinant cell because at least one component of the protein's natural environment is absent. However, typically, an isolated protein is prepared by at least one purification step.

A "purified" polypeptide is one that has been increased in purity so that it exists in a purer form than it is in its natural environment and/or as compared to 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.

"Impurities" refer to substances other than the desired polypeptide product. Impurities include, but are not limited to: host cell material, 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.

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 naturally or by 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 ureakinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor alpha and beta; enkephalinase; RANTES (normal activating regulatory factor (ACTI) expressed and secreted by T cells); human macrophage inflammatory protein (MIP-1α); serum albumin, such as human serum albumin; Müllerian 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), or nerve growth factor, such as TNF-α. 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), 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 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 (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 receptors; 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, including antibody fragments, that bind to proteins (including, for example, any of the above proteins).

The term "antibody" is used herein in the broadest sense and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. The antibody can be human, humanized, and/or affinity matured.

The term "monoclonal antibody" is used herein to refer to an antibody obtained from a substantially homogeneous antibody population, i.e., each individual antibody contained in the population is identical except for possible natural mutations (which may exist in small quantities). Monoclonal antibodies are highly specific, being directed to a single antigenic site. Furthermore, unlike polyclonal antibody preparations comprising different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to specificity, monoclonal antibodies are advantageous in that they can be synthesized without being contaminated by antibodies. The modifier "monoclonal" should not be construed as requiring that the antibody be prepared by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be prepared by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al.: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)); recombinant DNA methods in bacteria, eukaryotic or plant cells (see, e.g., U.S. Patent No. 4,816,567); phage display technology (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al.: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)); al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004); and techniques for producing human or human-like antibodies in animals having part or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc.Natl.Acad.Sci.USA 90:2551(1993); Jakobovits et al., Nature 362:255-258(1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al. al.,Nature and Lonberg and Lonberg. Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

The term "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective and that does not contain other ingredients that are unacceptably toxic to a subject to which the formulation is administered. Such formulations are sterile.

A "pharmaceutically acceptable" carrier, excipient, or stabilizer is one that is nontoxic to the cells or mammals to which it is exposed at the dosages and concentrations employed (Remington's Pharmaceutical Sciences ( ^20th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA). Typically, a physiologically acceptable carrier is a pH-buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween ^™ , polyethylene glycol (PEG), and Pluronics ^™ .

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

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 the polypeptide to a reference standard.

As used in this specification and the appended claims, the terms "a", "an", "the", etc. include plural references unless expressly stated otherwise. Thus, for example, reference to "a compound" optionally includes a combination of two or more such compounds, and so forth.

It is understood that aspects and embodiments of the present invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

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.

When embodiments or aspects of the invention are described in terms of Markush groups or other groups of alternative elements, the invention encompasses not only the entire group listed as a whole, and each member of the group individually and all possible subgroups within the main group, but also encompasses the main group with one or more of the components missing. The invention also contemplates any one or more of the group members being specifically excluded from the claimed invention.

II. Cell Culture Medium

The cell culture medium provided herein can be used in the methods described herein (e.g., methods for culturing cells and producing polypeptides) and compositions (e.g., pharmaceutical preparations). Culture medium components capable of providing polypeptide products (e.g., therapeutic proteins) with acceptable quality attributes, such as acceptable color intensity, have been identified. One or more of these identified culture medium components can be used to provide polypeptide products with acceptable color intensity. As used herein, the "acceptable color intensity" of a polypeptide product (e.g., a composition comprising a polypeptide) can refer to the color intensity required for obtaining regulatory approval, or the color intensity desired for use in evaluating the consistency of batches of polypeptide products. In some embodiments, the one or more culture medium components are antioxidants. In some embodiments, the one or more culture medium components are selected from hypotaurine, s-carboxymethylcysteine, anserine, butylated hydroxyanisole, carnosine, lipoic acid, quercetin hydrate, and aminoguanidine. In some embodiments, the one or more culture medium components are hypotaurine or its analogs or precursors. In some embodiments, the hypotaurine or its analog or precursor is selected from the group consisting of hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulfinic acid, and taurine. In some embodiments, the one or more culture medium components are taurine, reduced lipoic acid, or carvedilol.

Culture medium components can be added to the cell culture medium in a manner known in the art. For example, hypotaurine can be provided as a compound identified by CAS No. 300-84-5, s-carboxymethylcysteine can be provided as a compound identified by CAS No. 638-23-3, anserine can be provided as a compound identified by CAS No. 10030-52-1, butylated hydroxyanisole can be provided as a compound identified by CAS No. 25013-16-5, carnosine can be provided as a compound identified by CAS No. 305-84-0, lipoic acid can be provided as a compound identified by CAS No. 1200-22-2, and quercetin hydrate can be provided as a compound identified by CAS No. 522-12-3. As another example, an analog or precursor of hypotaurine can be provided, such as s-carboxymethylcysteine, cysteamine, cysteinesulfinic acid, and/or taurine. In some embodiments, s-carboxymethylcysteine is provided as a compound identified as CAS No. 638-23-3, cysteamine is provided as a compound identified as CAS No. 60-23-1, cysteinesulfinic acid is provided as a compound identified as CAS No. 1115-65-7, and taurine is provided as a compound identified as CAS No. 107-35-7. In some embodiments, the compounds listed in Table 4 are provided, such as reduced lipoic acid identified as CAS No. 462-20-4 or carvedilol identified as CAS No. 72956-09-3. In some embodiments, aminoguanidine is provided as aminoguanidine hydrochloride identified as CAS No. 1937-19-5. The culture medium components provided herein can be provided to the cell culture medium as salts, hydrates, or salt hydrates, or in any other form known to those skilled in the art. The culture medium components can also be provided to the cell culture medium as solutions, extracts, or in solid form. In some embodiments, the cell culture medium is a chemically defined medium. In other embodiments, the cell culture medium is a chemically undefined medium.

In some aspects, the present invention provides a cell culture medium comprising one or more of the following components: (a) hypotaurine; (b) s-carboxymethylcysteine; (c) carnosine; (d) anserine; (e) butylated hydroxyanisole; (f) lipoic acid; (g) quercetin hydrate; and (h) aminoguanidine. In some embodiments, the cell culture medium comprises 2 or 3 or 4 or 5 or 6 of components (a), (b), (c), (d), (e), (f), (g), and (h), or each of them. It is understood that the cell culture medium provided herein may contain any combination of components (a), (b), (c), (d), (e), (f), (g), and (h), just as if each combination were specifically and individually listed. For example, it will be understood that a cell culture medium comprising four of components (a), (b), (c), (d), (e), (f), (g) and (h) may include any combination of these components, as long as at least four of these components are present in the combination.

In some aspects, the cell culture medium provided herein comprises one or more culture medium components selected from the group consisting of (a) hypotaurine; (b) s-carboxymethylcysteine; (c) carnosine; (d) anserine; (e) butylated hydroxyanisole; (f) lipoic acid; (g) quercetin hydrate; and (h) aminoguanidine in the amounts described in Table 1. It should be understood that the culture medium can comprise any one or more of the culture medium components of Table 1 (e.g., any one or more of components (a)-(h), such as a culture medium comprising components (a), (b), (c), (d), and (e), or a culture medium comprising components (a), (b), and (g), or a culture medium comprising only one of components (a)-(h)) in any amount listed in Table 1, as if each and any combination of components and amounts were specifically and individually listed. In one aspect, the cell culture medium is a chemically defined culture medium. In another aspect, the cell culture medium is a chemically undefined culture medium. In some embodiments, the cell culture medium comprises one or more of components (a)-(h), wherein (a) is at least about 0.0001 mM hypotaurine, (b) is at least about 0.0001 mM s-carboxymethylcysteine, (c) is at least about 0.0001 mM carnosine, (d) is at least about 0.0001 mM anserine, (e) is at least about 0.0001 mM butylated hydroxyanisole, (f) is at least about 0.0001 mM lipoic acid, (g) is at least about 0.0001 mM quercetin hydrate, and (h) is at least about 0.0003 mM aminoguanidine. In some embodiments, the cell culture medium comprises one or more of components (a)-(h), wherein (a) is about 2.0 mM to about 50.0 mM hypotaurine, (b) is about 8.0 mM to about 12.0 mM s-carboxymethylcysteine, (c) is about 8.0 mM to about 12.0 mM carnosine, (d) is about 3.0 mM to about 5.0 mM anserine, (e) is about 0.025 mM to about 0.040 mM butylated hydroxyanisole, (f) is about 0.040 mM to about 0.060 mM lipoic acid, (g) is about 0.010 mM to about 0.020 mM quercetin hydrate, and (h) is about 0.0003 mM to about 10 mM aminoguanidine.

Table 1. Exemplary amounts of media components

In some aspects, the present invention provides a cell culture medium comprising a hypotaurine or an analog thereof or a precursor thereof selected from the group consisting of hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulfinic acid, and taurine. In some aspects, the cell culture medium comprises one or more of the following components: (a) hypotaurine; (b) s-carboxymethylcysteine; (c) cysteamine; (d) cysteinesulfinic acid; and (e) taurine. In some embodiments, the cell culture medium comprises two, three, or four, or each of (a), (b), (c), (d), and (e). It is understood that the cell culture medium provided herein may contain any combination of components (a), (b), (c), (d), and (e), as if each combination were specifically and individually listed. For example, it is understood that a cell culture medium comprising three of the components (a), (b), (c), (d), and (e) may contain any combination of these components, as long as at least three of these components are present in the combination. Taurine analogs include, for example, s-carboxymethylcysteine, cysteamine, cysteinesulfinic acid, and taurine. Examples of hypotaurine precursors are well known to those skilled in the art, and in some aspects, the hypotaurine precursor can be a hypotaurine analog.

In some aspects, the cell culture media provided herein comprise hypotaurine or an analog thereof or a precursor thereof in the amounts described in Table 2. It should be understood that the culture medium can comprise any one or more of the culture medium components of Table 2 (e.g., any one or more of components (a)-(e), e.g., a culture medium comprising components (a), (b), (c), and (d), or a culture medium comprising components (a), (b), and (c), or a culture medium comprising only one of components (a)-(e)) in any amount listed in Table 2, just as if each and any combination of components and amounts were specifically and individually listed. In some embodiments, the cell culture medium comprises hypotaurine or an analog thereof or a precursor thereof, e.g., hypotaurine, S-carboxymethylcysteine, cysteamine, cysteinesulfinic acid, and/or taurine, at a concentration of at least about 0.0001 mM. In some embodiments, the cell culture medium comprises hypotaurine or an analog or precursor thereof, such as hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulfinic acid, and/or taurine, at a concentration of about 0.5 mM to about 500.0 mM.

Table 2. Exemplary amounts of media components

In some aspects, the cell culture medium provided herein comprises reduced lipoic acid at a concentration of about 0.0001 mM to about 0.5 mM. In some aspects, the cell culture medium provided herein comprises carvedilol at a concentration of about 0.0001 mM to about 1.5 mM.

Each culture medium component provided herein can be present in an amount that results in one or more properties that are advantageous for culturing cells and/or producing polypeptides from the cell culture. Advantageous properties include, but are not limited to, reduced polypeptide oxidation in the cell culture and/or reduced color intensity of compositions comprising polypeptides produced by cells cultured in the cell culture medium of the present invention. Advantageous properties of the cell culture medium provided herein can also include reduced color intensity of compositions comprising polypeptides produced by cells cultured in the cell culture medium without affecting one or more product attributes, such as the amount of polypeptides produced by the cells (e.g., antibody titer), glycosylation (e.g., N-glycosylation) profile of the polypeptides, polypeptide charge heterogeneity in the compositions, or amino acid sequence integrity of the polypeptides. In some embodiments, one or more advantageous properties for culturing cells in the cell culture medium provided herein are reduced color intensity of compositions comprising polypeptides produced by the cells without affecting cell viability, the amount of polypeptides produced by the cells, glycosylation (e.g., N-glycosylation) profile of the polypeptides, polypeptide charge heterogeneity in the compositions, and/or amino acid sequence integrity of the polypeptides. In some embodiments, one or more advantageous properties for culturing cells in the cell culture media provided herein are: reduced color intensity of a composition comprising a polypeptide produced by the cells, and reduced oxidation of the polypeptide in cell culture. These advantageous properties also apply to methods for culturing cells comprising a nucleic acid encoding a polypeptide of interest and methods for producing a polypeptide of interest in cell culture, as described herein.

In some aspects, one or more culture medium components selected from the group consisting of hypotaurine, s-carboxymethylcysteine, anserine, butylated hydroxyanisole, carnosine, lipoic acid, quercetin hydrate, and aminoguanidine are provided herein in an amount that results in one or more properties that are advantageous for culturing cells and/or producing polypeptides from cell culture. In some embodiments, the amount of hypotaurine in the cell culture medium that can result in one or more advantageous properties is from about 0.5 mM to about 100 mM, about 1.6 mM to about 90 mM, about 1.7 mM to about 80 mM, about 1.8 mM to about 70 mM, about 1.9 mM to about 60 mM, about 2.0 mM to about 50 mM, or about 1.75 mM to about 50 mM. In some embodiments, the amount of s-carboxymethylcysteine in the cell culture medium that can result in one or more favorable properties is from about 0.5 mM to about 120 mM, from about 5.0 mM to about 15 mM, from about 6.0 mM to about 14 mM, from about 7.0 mM to about 13 mM, or from 8.0 mM to about 12 mM. In some embodiments, the amount of anserine in the cell culture medium that can result in one or more favorable properties is from about 0.5 mM to about 20 mM, from about 2.0 mM to about 10 mM, or from about 3.0 mM to about 5.0 mM. In some embodiments, the amount of butylated hydroxyanisole in the cell culture medium that can result in one or more favorable properties is from about 0.005 mM to about 0.2 mM, from about 0.02 mM to about 0.05 mM, or from about 0.025 mM to about 0.04 mM. In some embodiments, the amount of carnosine in the cell culture medium that can result in one or more favorable properties is from about 0.5 mM to about 20 mM, from about 6.0 mM to about 14 mM, or from about 8.0 mM to about 12 mM. In some embodiments, the amount of lipoic acid in the cell culture medium that can result in one or more favorable properties is from about 0.01 mM to about 1.5 mM, from about 0.036 mM to about 0.08 mM, from about 0.038 mM to about 0.07 mM, or from about 0.04 mM to about 0.06 mM. In some embodiments, the amount of quercetin hydrate in the cell culture medium that can result in one or more favorable properties is from about 0.005 mM to about 0.04 mM, from about 0.01 mM to about 0.03 mM, from about 0.015 mM to about 0.025 mM, or from about 0.01 mM to about 0.02 mM. In some embodiments, the amount of aminoguanidine in the cell culture medium that can result in one or more advantageous properties is from about 0.0003 mM to about 245 mM, about 0.003 mM to about 150 mM, about 0.03 mM to about 100 mM, about 0.03 mM to about 50 mM, about 0.03 mM to about 25 mM, or about 0.03 to about 10 mM. In some embodiments, the amount of one or more culture medium components selected from hypotaurine, s-carboxymethylcysteine, anserine, butylated hydroxyanisole, carnosine, lipoic acid, quercetin hydrate, and aminoguanidine that can result in one or more advantageous properties in the cell culture medium is provided in Table 1.

In some aspects, one or more culture medium components selected from the group consisting of hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulfinic acid, and taurine are provided herein in an amount that results in one or more properties that are advantageous for culturing cells and/or producing polypeptides from the cell culture. In some embodiments, the amount of hypotaurine in the cell culture medium that can result in one or more advantageous properties is from about 0.5 mM to about 100 mM, about 1.6 mM to about 90 mM, about 1.7 mM to about 80 mM, about 1.8 mM to about 70 mM, about 1.9 mM to about 60 mM, about 2.0 mM to about 50 mM, or about 1.75 mM to about 50 mM. In some embodiments, the amount of s-carboxymethylcysteine in the cell culture medium that can result in one or more advantageous properties is from about 0.5 mM to about 120 mM, about 5.0 mM to about 15 mM, about 6.0 mM to about 14 mM, about 7.0 mM to about 13 mM, or about 8.0 mM to about 12 mM. In some embodiments, the amount of cysteamine in the cell culture medium that can result in one or more advantageous properties is from about 0.01 mM to about 300 mM, about 0.02 mM to about 1 mM, about 0.04 mM to about 0.8 mM, about 0.06 mM to about 0.6 mM, about 0.08 mM to about 0.4 mM, or about 0.1 mM to about 0.2 mM. In some embodiments, the amount of cysteine sulfenic acid in the cell culture medium that can result in one or more favorable properties is from about 0.1 mM to 100 mM, from about 0.2 mM to about 10 mM, from about 0.3 mM to about 1 mM, from about 0.1 mM to about 1 mM, from about 0.2 mM to about 0.8 mM, or from about 0.3 mM to about 0.6 mM. In some embodiments, the amount of taurine in the cell culture medium that can result in one or more favorable properties is from about 0.5 mM to 500 mM, from about 4.0 mM to about 100 mM, or from about 1.0 mM to about 10 mM. In some embodiments, for one or more culture medium components selected from hypotaurine, s-carboxymethylcysteine, cysteamine, cysteine sulfenic acid, and taurine, the amounts in the cell culture medium that can result in one or more favorable properties are provided in Table 2.

In one aspect, compared to the quality attributes of the polypeptide produced in different culture media, the cell culture medium provided herein, when used in the method for producing polypeptide in cell culture, causes one or more favorable product quality attributes or favorable properties. The reactive oxygen species (ROS) formed by using certain culture medium components can oxidize the specific amino acids on the polypeptide and produce oxidized polypeptide products. The presence of the protein material of this oxidation can also change the product quality attributes of protein products, such as color intensity, which may be especially significant for a polypeptide product prepared at any concentration (such as, but not limited to, greater than about 1mg/mL, about 10mg/mL, about 25mg/mL, about 50mg/mL, or about 75mg/mL to any concentration of up to 100mg/mL). In some embodiments, the presence of the protein material of this oxidation can change the product quality attributes of protein products, such as color intensity, which may be especially significant for a polypeptide product prepared at any concentration of greater than about 100mg/mL, about 125mg/mL, about 150mg/mL, about 175mg/mL, about 200mg/mL or about 250mg/mL. The color intensity of compositions comprising polypeptides produced by the culture media detailed herein (including compositions comprising at least about 1 mg/mL, about 10 mg/mL, about 50 mg/mL, about 100 mg/mL, about 150 mg/mL, 200 mg/mL, or about 250 mg/mL of polypeptides (e.g., antibodies)) can be assessed using a colorimetric assay, such as the assays described herein, or assays described in, for example, but not limited to, the U.S. Pharmacopoeia color standards and the European Pharmacopoeia color standards. 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, which are incorporated herein by reference in their entireties. In any of the embodiments herein, the cell culture media provided herein can be used to prepare a composition comprising a polypeptide having a reduced color intensity as measured by a colorimetric assay compared to a reference solution. For example, the color intensity of a composition (e.g., a pharmaceutical formulation) comprising a polypeptide (e.g., a therapeutic polypeptide) produced using a cell culture medium provided herein can be reduced by any amount, including, but not limited to, at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5% or more, compared to a composition comprising the polypeptide produced using a cell culture medium that does not contain one or more of the components in Table 1 or Table 2.

Commercially available media suitable for culturing cells, such as, but not limited to, Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium ([DMEM], Sigma), Luria broth (LB) and Terrific broth (TB), can be supplemented with any of the media components detailed herein (e.g., by using the provided kits). In addition, reference may be made to Ham and Wallace, Meth. Enz., 58:44 (1979), Barnes and Sato, Anal. Biochem., 102:255 (1980), Vijayasankaran et al., Biomacromolecules., 6:605:611 (2005), Patkar et al., J Biotechnology, 93:217-229 (2002), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195; U.S. Pat. No. Re. 30,985; or U.S. Pat. No. 5,122,469, the disclosures of all of which are incorporated herein by reference in their entirety, supplemented with any of the medium components detailed herein (e.g., by using the provided kits).

In some embodiments, the cell culture medium provided herein comprises cystine and does not contain cysteine. In some embodiments, the cell culture medium provided herein comprises ferric citrate and does not contain ferrous sulfate. In some embodiments, the cell culture medium provided herein does not contain cysteine and ferrous sulfate. In some embodiments, the culture medium does not contain cysteine and ferrous sulfate and includes cystine and/or ferric citrate. In any embodiment herein, the cell culture medium can be a basal medium or a feed medium. Amino acids, vitamins, trace elements and other culture medium components can be used in one or two times the ranges described in European Patent EP 307,247 or U.S. Patent 6,180,401, which are incorporated herein by reference in their entirety.

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 typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. In some aspects, the cell culture medium provided herein comprises plant- or animal-derived proteins. In some embodiments, the cell culture medium provided herein does not contain plant- or animal-derived proteins. Any other necessary supplements may also be included at appropriate concentrations well known to those skilled in the art.

III. Methods and Applications of the Invention

The present invention provides methods for culturing cells in the cell culture medium provided herein for producing a polypeptide of interest. In some aspects, methods are provided for culturing cells comprising a nucleic acid encoding a polypeptide of interest, wherein the method comprises contacting the cells with a cell culture medium, wherein the cell culture medium comprises one or more components selected from the group consisting of hypotaurine, S-carboxymethylcysteine, carnosine, anserine, butylated hydroxyanisole, lipoic acid, and quercetin hydrate. In some embodiments, a method for culturing a cell comprising a nucleic acid encoding a polypeptide of interest is provided, wherein the method comprises contacting the cell with a cell culture medium, wherein the cell culture medium comprises one or more components selected from the group consisting of (a) hypotaurine, (b) s-carboxymethylcysteine, (c) carnosine, (d) anserine, (e) butylated hydroxyanisole, (f) lipoic acid; (g) quercetin hydrate; and (h) aminoguanidine, wherein the cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of a composition comprising the polypeptide produced by the cell compared to a composition comprising the polypeptide produced by cells cultured in a cell culture medium that does not contain one or more of the components (a)-(h). In some embodiments, the color intensity of the composition comprising the polypeptide is reduced by at least about 0.1%. In some embodiments, the color intensity of the composition comprising the polypeptide is reduced by at least about 5%. In some embodiments, the color intensity of the composition comprising the polypeptide is reduced by about 10% to about 30%. In some embodiments, the color intensity of the composition comprising the polypeptide is reduced by about 5% to about 75%. In some embodiments herein, the cell culture medium comprises one or more components in an amount selected from the group consisting of: (a) hypotaurine at a concentration of about 2.0 mM to about 50.0 mM, (b) s-carboxymethylcysteine at a concentration of about 8.0 mM to about 12.0 mM, (c) carnosine at a concentration of about 8.0 mM to about 12.0 mM, (d) anserine at a concentration of about 3.0 mM to about 5.0 mM, (e) butylated hydroxyanisole at a concentration of about 0.025 mM to about 0.040 mM, (f) lipoic acid at a concentration of about 0.040 mM to about 0.060 mM, (g) quercetin hydrate at a concentration of about 0.010 mM to about 0.020 mM, and (h) aminoguanidine at a concentration of about 0.0003 mM to about 20 mM. In some embodiments herein, the one or more components selected from (a) hypotaurine, (b) s-carboxymethylcysteine, (c) carnosine, (d) anserine, (e) butylated hydroxyanisole, (f) lipoic acid; (g) quercetin hydrate; and (h) aminoguanidine are added to the cell culture medium on day 0 of a 14-day cell culture cycle.

In some other aspects, a method for culturing a cell comprising a nucleic acid encoding a polypeptide of interest is provided, wherein the method includes contacting the cell with a cell culture medium comprising hypotaurine or its analog or precursor. In some embodiments, a method for culturing a cell comprising a nucleic acid encoding a polypeptide of interest is provided, wherein the method includes contacting the cell with a cell culture medium comprising hypotaurine or its analog or precursor, and wherein the cell culture medium comprising hypotaurine or its analog or precursor reduces the color intensity of the composition comprising the polypeptide produced by the cell compared to the color intensity of the composition comprising the polypeptide produced by cells cultured in a cell culture medium without hypotaurine or its analog or precursor. In some embodiments, the color intensity of the composition comprising the polypeptide is reduced by at least about 0.1%. In some embodiments, the color intensity of the composition comprising the polypeptide is reduced by at least about 5%. In some embodiments, the color intensity of the composition comprising the polypeptide is reduced by about 10% to about 30%. In some embodiments, the cell culture medium comprises hypotaurine or its analog or precursor at a concentration of at least about 0.0001 mM. In some embodiments, the cell culture medium comprises hypotaurine or its analog or precursor at a concentration of from about 0.5 mM to about 500 mM. In some embodiments, the cell culture medium comprises hypotaurine or its analog or precursor at a concentration of from about 1.0 mM to about 40 mM. In some embodiments, hypotaurine or its analog or precursor is selected from hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulfinic acid, and taurine. In some embodiments herein, hypotaurine or its analog or precursor is added to the cell culture medium on day 0 of a 14-day cell culture cycle. In some embodiments, hypotaurine or its analog or precursor is not added to the cell culture medium in an incremental manner during the cell culture cycle.

Also provided herein is a method for producing a polypeptide of interest, comprising the step of culturing cells comprising a nucleic acid encoding the polypeptide in a cell culture medium, wherein the cell culture medium comprises one or more components selected from the group consisting of (a) hypotaurine, (b) s-carboxymethylcysteine, (c) carnosine, (d) anserine, (e) butylated hydroxyanisole, (f) lipoic acid, (g) quercetin hydrate, and (h) aminoguanidine. In some embodiments, provided herein is a method for producing a polypeptide of interest, comprising culturing cells comprising a nucleic acid encoding the polypeptide in a cell culture medium, wherein the cell culture medium comprises one or more components selected from the group consisting of (a) hypotaurine, (b) s-carboxymethylcysteine, (c) carnosine, (d) anserine, (e) butylated hydroxyanisole, (f) lipoic acid, (g) quercetin hydrate, and (h) aminoguanidine, and wherein the cell culture medium comprising one or more of components (a)-(h) reduces the color intensity of a composition comprising the polypeptide produced by the cells compared to a composition comprising the polypeptide produced by cells cultured in a cell culture medium that does not contain one or more of the components (a)-(h). In some embodiments, the color intensity of the composition comprising the polypeptide is reduced by at least about 0.1%. In some embodiments, the color intensity of the composition comprising the polypeptide is reduced by at least about 5%. In some embodiments, the color intensity of the composition comprising the polypeptide is reduced by about 10% to about 30%. In some embodiments, the color intensity of the composition comprising the polypeptide is reduced by about 5% to about 75%. In some embodiments herein, the cell culture medium comprises one or more components in an amount selected from the group consisting of: (a) hypotaurine at a concentration of about 2.0 mM to about 50.0 mM, (b) s-carboxymethylcysteine at a concentration of about 8.0 mM to about 12.0 mM, (c) carnosine at a concentration of about 8.0 mM to about 12.0 mM, (d) anserine at a concentration of about 3.0 mM to about 5.0 mM, (e) butylated hydroxyanisole at a concentration of about 0.025 mM to about 0.040 mM, (f) lipoic acid at a concentration of about 0.040 mM to about 0.060 mM, (g) quercetin hydrate at a concentration of about 0.010 mM to about 0.020 mM, and (h) aminoguanidine at a concentration of about 0.0003 mM to about 20 mM. In some embodiments herein, the one or more components selected from (a) hypotaurine, (b) s-carboxymethylcysteine, (c) carnosine, (d) anserine, (e) butylated hydroxyanisole, (f) lipoic acid; (g) quercetin hydrate; and (h) aminoguanidine are added to the cell culture medium on day 0 of a 14-day cell culture cycle.

In another aspect, provided herein is a method for producing a polypeptide of interest, comprising culturing cells comprising a nucleic acid encoding the polypeptide in a cell culture medium. In some embodiments, provided herein is a method for producing a polypeptide of interest, comprising culturing cells comprising a nucleic acid encoding the polypeptide in a cell culture medium, wherein the cell culture medium comprises hypotaurine or an analog or precursor thereof, wherein the cell culture medium comprising hypotaurine or an analog or precursor thereof reduces the color intensity of the composition comprising the polypeptide produced by the cells compared to the color intensity of the composition comprising the polypeptide produced by cells cultured in a cell culture medium that does not contain hypotaurine or an analog or precursor thereof. In some embodiments, the color intensity of the composition comprising the polypeptide is reduced by at least about 0.1%. In some embodiments, the color intensity of the composition comprising the polypeptide is reduced by at least about 5%. In some embodiments, the color intensity of the composition comprising the polypeptide is reduced by about 10% to about 30%. In some embodiments, the cell culture medium comprises hypotaurine or an analog or precursor thereof at a concentration of at least about 0.0001 mM. In some embodiments, the cell culture medium comprises hypotaurine or an analog or precursor thereof at a concentration of about 0.5 mM to about 500 mM. In some embodiments, the cell culture medium comprises hypotaurine, or an analog or precursor thereof, at a concentration of about 1.0 mM to about 40 mM. In some embodiments, the hypotaurine, or an analog or precursor thereof, is selected from the group consisting of hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulfinic acid, and taurine. In some embodiments herein, the hypotaurine, or an analog or precursor thereof, is added to the cell culture medium on day 0 of a 14-day cell culture cycle. In some embodiments, the hypotaurine, or an analog or precursor thereof, is not added to the cell culture medium in an incremental manner during the cell culture cycle.

In any embodiment of this, the cell culture medium used in the methods described herein can be a chemically determined cell culture medium or a chemically uncertain cell culture medium. The cell culture medium provided herein can be used as a basal cell culture medium or a feed cell culture medium. In some embodiments, the cell culture medium provided herein is used in the method for culturing cells during the cell growth phase. In some embodiments, the cell culture medium provided herein is used in the method for culturing cells during the cell production phase. In any of these methods, the cell can be a mammalian cell, such as a Chinese hamster ovary celI. In some embodiments, the target polypeptide is an antibody or a fragment thereof.

In further embodiments herein, reclaim the polypeptide of interest. For compositions containing the recovered polypeptide, at least one purification step can be carried out before using quantitative or qualitative color determination as described herein to assess color intensity. In some embodiments, the compositions containing the recovered polypeptide are liquid compositions or non-liquid compositions. In some embodiments, the liquid compositions or non-liquid compositions containing the recovered polypeptide can be assessed by color determination as described herein or as known in the art. For example, the non-liquid compositions containing the recovered polypeptide can be lyophilized compositions, which can be reconstructed subsequently before measuring color intensity. In some embodiments herein, the color intensity of the compositions of the polypeptide produced by cells cultivated in a cell culture medium provided herein is reduced by at least about 0.1%, compared to the color intensity of the compositions of the polypeptide produced by cells cultivated in a cell culture medium that does not contain culture components as described herein (for example, hypotaurine or its analogs or precursors). In some embodiments, the color intensity is reduced by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.8%, or at least about 0.9% to about 1.0%. In some embodiments, the color intensity is reduced by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% to about 100%. In some embodiments, the color intensity is reduced by about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9% to about 1.0%. In some embodiments, the color intensity is reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90% to about 100%. In some embodiments, color intensity is reduced by about 1% to about 10%, about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 30%, about 5% to about 35%, about 5% to about 40%, about 5% to about 45%, about 5% to about 50%, about 10% to about 20%, or about 15% to about 25%. In some embodiments, the composition comprising the recovered polypeptide is displayed as a colorless or slightly colored liquid or composition. Liquid or composition can be determined as colorless or slightly colored using a color determination method as described herein or a color determination method known to those skilled in the art. In a further embodiment, the composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier as described herein.

The present invention also provides the method for using polypeptide as described herein.For example, provide for the method for using the preparation comprising polypeptide to individual, wherein the preparation has a concentration greater than at least about 100mg/mL, at least about 125mg/mL or at least about 150mg/mL polypeptide, and has the color intensity value greater than B3, B4, B5, B6, B7, B8 or B9 measured by COC assay.In some respects, the color intensity value measured by COC assay can be, but is not limited to, any one of B, BY, Y, GY or R, wherein the higher value indicates the lighter color intensity.The preparation comprising the polypeptide of interest can be suitable for injection, such as subcutaneous injection to individual (for example, subcutaneous injection to people).In some respects, the preparation comprising the polypeptide of interest suitable for injection (for example, suitable for subcutaneous injection) has a 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 measured by COC assay. In some aspects, the color intensity value determined by the COC assay can be, but is not limited to, any of B, BY, Y, GY, or R, where higher values indicate lighter color intensity.

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

Peptide production

The cell culture medium that this paper describes in detail can use in the method for culturing cells to produce polypeptide (comprising specific antibodies).This culture medium can use in the method for culturing cells by batch culture, fed batch culture or perfusion culture, and can use (comprising any aspect or embodiment of polypeptide as described herein) in the method for producing any polypeptide.For example, by the compositions that this paper describes in detail (, the cell cultivated in the cell culture medium that this paper provides) and method production and the polypeptide that is present in the compositions that this paper provides (for example, the cell culture medium that comprises the polypeptide produced) can be homologous with host cell, or preferably can be external source, this means that they are allos (i.e. external) with respect to used host cell, for example, the people's protein that produces by Chinese hamster ovary cell, or the yeast polypeptide that is produced by mammalian cell.In a variant form, polypeptide is directly secreted into the mammalian polypeptide (such as antibody) in the culture medium by host cell.In another variant form, by cracking the cell that comprises the nucleic acid of encoding this polypeptide, release polypeptide in culture medium.

Any polypeptide that is expressible in a host cell may be produced according to the present disclosure and may be present in the compositions of the present invention. The polypeptide may be expressed by a gene endogenous to the host cell, or by a gene introduced into the host cell through genetic engineering. The polypeptide may be naturally occurring, or alternatively may have an engineered or artificially selected sequence. Engineered polypeptides may be assembled from other naturally occurring polypeptide fragments, or may include one or more non-naturally occurring fragments.

About the polypeptide that may be expected to express according to the present invention, can often be selected based on the biological or chemical activity of purpose.For example, can adopt the present invention to express any pharmaceutical or commercial relevant enzyme, receptor, antibody, hormone, regulatory factor, antigen, binding agent etc.

Methods for producing polypeptides (e.g., antibodies) in cell culture are well known in the art. Non-limiting exemplary methods for producing antibodies (e.g., full-length antibodies, antibody fragments, and multispecific antibodies) in cell culture are provided herein. The methods herein can be adapted by those skilled in the art for the production of other proteins, such as protein-based inhibitors. For generally known and routinely employed techniques and procedures for producing proteins (e.g., therapeutic proteins), see Molecular Cloning: A Laboratory Manual (Sambrook et al., 4 ^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (FM Ausubel, et al. eds., 2003); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 2002); Current Protocols in Protein Science, (Horswill et al., 2006); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (RI Freshney, 6 ^th ed., J. Wiley and Sons, 2010), all of which are hereby incorporated by reference in their entirety.

(A) Antibody Preparation

Antibodies to an antigen of interest can be produced in cell culture using the cell culture media provided herein. Preferably, the antigen is a biologically important polypeptide and administration of a composition comprising the antibody to a mammal suffering from a disorder can produce a therapeutic benefit in the mammal.

(i) Antigen preparation

Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens to generate antibodies. For transmembrane molecules, such as receptors, fragments thereof (e.g., extracellular domains of receptors) can be used as immunogens. Alternatively, cells expressing transmembrane molecules can be used as immunogens. Such cells can be derived from natural sources (e.g., cancer cell lines), or can be cells transformed by recombinant technology to express transmembrane molecules. Other antigens and forms thereof that can be used to prepare antibodies will be apparent to those skilled in the art.

(ii) Certain Antibody-Based Methods

The monoclonal antibodies of interest can be prepared using the hybridoma method, which was first described in Kohler et al., Nature, 256:495 (1975) and further described in, for example, Hongo et al., Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al.: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) on human-human hybridomas. Other methods include those described in, for example, U.S. Pat. No. 7,189,826, which involves the production of monoclonal human natural IgM antibodies from hybridoma cell lines. Human hybridoma technology (Trioma technology) is described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

For various other hybridoma technologies, see, for example, US 2006/258841; US 2006/183887 (full-length human antibodies), US 2006/059575; US 2005/287149; US 2005/100546; US 2005/026229; and U.S. Patent Nos. 7,078,492 and 7,153,507. An exemplary protocol for producing monoclonal antibodies using the hybridoma method is described below. In one embodiment, mice or other suitable host animals, such as hamsters, are immunized to elicit lymphocytes that generate or are capable of generating antibodies that will specifically bind to the protein being immunized. Antibodies are elicited in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the polypeptide of interest or its fragment, and an adjuvant such as monophosphoryl lipid A (MPL)/trehalose dicyanomycolic acid (TDM) (Ribi Immunochem. Research, Inc., Hamilton, Mont.). Anti-antigen antibodies are measured in serum from the immunized animals, and booster immunizations are optionally administered. Lymphocytes are isolated from the animals that produce anti-antigen antibodies. Alternatively, lymphocytes can be immunized in vitro.

Lymphocytes can then be fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells. See, for example, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986). Myeloma cells that can effectively fuse, support stable and high-level production of antibodies by the selected antibody-producing cells, and are sensitive to culture medium (e.g., HAT culture medium) can be used. Exemplary myeloma cells include, but are not limited to, mouse myeloma lines, such as mouse myeloma lines derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human hybridoma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

The hybridoma cells thus prepared are inoculated into a suitable culture medium, for example, one that contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells, and grown. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium used for the hybridoma typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells. Preferably, a serum-free hybridoma cell culture method is used to reduce the use of animal-derived serum, such as fetal bovine serum, see, for example, Even et al., Trends in Biotechnology, 24(3), 105-108 (2006).

Oligopeptides are described by Franek in Trends in Monoclonal Antibody Research, 111-122 (2005) as tools for improving the productivity of hybridoma cell cultures. Specifically, certain amino acids (alanine, serine, asparagine, proline) or protein hydrolysate fractions can be added to standard culture media, and apoptosis can be significantly inhibited by synthetic oligopeptides consisting of three to six amino acid residues. These peptides can be present in millimolar or higher concentrations.

Monoclonal antibody production can be measured in culture medium growing hybridoma cells. The binding specificity of the monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, by Scatchard analysis. See, for example, Munson et al., Anal. Biochem., 107: 220 (1980).

After identifying hybridoma cells that produce antibodies with desired specificity, affinity and/or activity, the clones can be subcloned by limiting dilution procedures and grown by standard methods. See, for example, Goding, supra. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 culture media. In some embodiments, hybridoma cells are cultured in a cell culture medium provided herein. In some embodiments, hybridoma cells are cultured in a cell culture medium comprising one or more culture components selected from hypotaurine, s-carboxymethylcysteine, anserine, butylated hydroxyanisole, carnosine, lipoic acid, and quercetin hydrate. In some embodiments, the one or more culture components are hypotaurine or its analogs or precursors. In some embodiments, hypotaurine or its analogs or precursors are selected from hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulfinic acid, and taurine.

Recombinant methods can be used to produce antibodies. For the recombinant production of anti-antigen antibodies, the nucleic acid encoding the antibody is separated and inserted into a reproducible vector for further cloning (DNA amplification) or expression. Conventional methods can be used to easily separate and sequence the DNA encoding the antibody (for example, by using oligonucleotide probes that can specifically bind to the gene encoding the heavy chain and light chain of the antibody). Many vectors are available. Vector components generally include but are not limited to one or more of the following: signal sequence, origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

(iii) Certain library screening methods

Antibodies can be prepared by screening for antibodies with the desired activity (one or more) using combinatorial libraries. For example, various methods are known in the art for producing phage display libraries and screening such libraries for antibodies with the desired binding characteristics. These methods are generally described in Hoogenboom et al. Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001). For example, one method for producing an antibody of interest is by using a phage antibody library as described in Lee et al., J. Mol. Biol. (2004), 340 (5): 1073-93.

In principle, synthetic antibody clones are selected by screening a phage library containing phages that display various fragments of antibody variable regions (Fv) fused to phage coat proteins. By affinity chromatography against the desired antigen, the phage library is panned. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed onto the antigen, thereby separating them from unbound clones in the library. The bound clones are then eluted from the antigen and can be further enriched by additional antigen adsorption/elution cycles. Target phage clones can be selected by designing a suitable antigen screening program, followed by constructing full-length antibodies using the Fv sequence and suitable constant region (Fc) sequence from the target phage clones to obtain any target antibody, see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.

In certain embodiments, the antigen-binding domain of an antibody is composed of two variable (V) regions of approximately 110 amino acids, one from a light chain (VL) and one from a heavy chain (VH), each exhibiting three hypervariable loops (HVRs) or complementarity determining regions (CDRs). The variable domains can be functionally displayed on phage as single-chain Fv (scFv) fragments, wherein the VH and VL are covalently linked by a short flexible peptide, or as Fab fragments, wherein each can be fused to a constant domain and interact non-covalently, see Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, phage clones encoding scFv and phage clones encoding Fab are collectively referred to as "Fv phage clones" or "Fv clones".

Repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, after which clones that bind to the antigen can be retrieved from the library, see Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive libraries can be cloned without any immunization to provide a single source of human antibodies against a wide range of non-self and self antigens, see Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be prepared synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and performing in vitro rearrangement, see Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

In certain embodiments, filamentous phage is used to display antibody fragments fused to the minor coat protein pill. Antibody fragments can be displayed as single-chain Fv fragments, in which the VH and VL domains are linked to the same polypeptide chain by a flexible polypeptide spacer, see, e.g., Marks et al., J. Mol. Biol., 222:581-597 (1991); or as Fab fragments, in which one chain is fused to pill and the other chain is secreted into the bacterial host cell periplasm, where a Fab-coat protein structure is assembled and displayed on the phage surface by replacing some wild-type coat proteins, see, e.g., Hoogenboom et al., Nucl. Acids Res., 19:4133-4137 (1991).

In general, the nucleic acid encoding antibody gene fragment can be obtained from the immunocyte of human or animal harvest.If what is desired is to be biased towards the library of anti-antigen clone, then can use antigen immune subject to produce antibody response, and reclaim splenocyte and/or circulating B cell, other peripheral blood lymphocyte (PBL) for library construction.In one embodiment, by producing anti-antigen antibody response in the transgenic mouse carrying functional human immunoglobulin gene array (and lacking functional endogenous antibody production system), make antigen immunity cause the B cell of producing people's antibody for antigen, thus obtain the people's antibody gene fragment library that is biased towards anti-antigen clone.The generation of the transgenic mouse producing people's antibody is as described below.

Additional enrichment of anti-antigen reactive cell populations can be obtained by isolating B cells expressing antigen-specific membrane-bound antibodies using appropriate screening procedures, such as by using antigen affinity chromatography, or by adsorbing cells to fluorescent dye-labeled antigens followed by flow activated cell sorting (FACS) to separate the cells.

Alternatively, the use of splenocytes and/or B cells or other PBLs from unimmunized donors can better represent the potential antibody repertoire and also allow the use of any animal (human or non-human) species in which the antigen is not antigenic to construct the antibody library. For libraries incorporating in vitro antibody gene constructs, stem cells are harvested from the subject to provide nucleic acids encoding unrearranged antibody gene segments. Target immune cells can be obtained from various animal species, such as humans, mice, rats, rabbits, wolves, dogs, cats, pigs, cattle, horses, and avian species.

Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered from target cells and amplified. In the case of rearranged VH and VL gene libraries, genomic DNA or mRNA can be isolated from lymphocytes, followed by polymerase chain reaction (PCR) with primers matching the 5' and 3' ends of the rearranged VH and VL genes to obtain the desired DNA (see Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86: 3833-3837 (1989)), thereby preparing a diverse V gene library for expression. The V gene can be amplified from cDNA and genomic DNA using a reverse primer at the 5' end of the exon encoding the mature V-domain and a forward primer based on the J segment, see Orlandi et al. (1989) and Ward et al., Nature, 341: 544-546 (1989). However, for amplification from cDNA, the reverse primer can also be based on the leading exon, see Jones et al., Biotechnol., 9:88-89 (1991); the forward primer can be within the constant region, see Sastry et al., Proc. Natl. Acad. Sci. (USA), 86:5728-5732 (1989). To maximize complementarity, degeneracy can be incorporated into the primers, see Orlandi et al. (1989) or Sastry et al. (1989). In certain embodiments, library diversity is maximized by using PCR primers targeting each V gene family to amplify all available VH and VL rearrangements present in a sample of immune cell nucleic acid, e.g., as described in the method of Marks et al., J. Mol. Biol., 222:581-597 (1991) or as described in the method of Orum et al., Nucleic Acids Res., 21:4491-4498 (1993). To clone the amplified DNA into an expression vector, rare restriction sites can be introduced into the PCR primers as tags at one end (see Orlandi et al. (1989)), or further PCR amplification can be performed using tagged primers (see Clackson et al., Nature, 352:624-628 (1991)).

Repertoires of synthetic rearranged V genes can be derived in vitro from V gene segments. Most human VH gene segments have been cloned and sequenced (reported in Tom Linson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet., 3:88-94 (1993); diverse VH gene repertoires can be generated from these cloned segments (including all major conformations of the H1 and H2 loops) using PCR primers encoding H3 loops of diverse sequences and lengths, see Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be prepared in which all sequence diversity is concentrated in a single long H3 loop, see Barbas et al., Proc. Natl. Acad. Sci. USA, 89:4457-4461 (1992). Human Vκ and Vλ segments have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23: 1456-1461 (1993) and can be used to prepare synthetic light chain libraries. Based on a range of VH and VL folds and L3 and H3 lengths, synthetic V gene libraries will encode antibodies with considerable structural diversity. After amplifying the DNA encoding the V genes, the germline V gene segments can be rearranged in vitro according to the method of Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

Antibody fragment libraries can be constructed by combining VH and VL gene libraries in a variety of ways. Each library can be constructed in a different vector, and the vectors can be recombined in vitro (see, for example, Hogrefe et al., Gene, 128: 119-126 (1993)), or recombined in vivo by combinatorial infection, for example, the loxP system described by Waterhouse et al., Nucl. Acids Res., 21: 2265-2266 (1993). In vivo recombination methods use the double-stranded nature of Fab fragments to overcome the limitations imposed on library size by E. coli transformation efficiency. Naive VH and VL libraries are cloned separately, one clone into a phagemid and the other into a phage vector. The two libraries are then combined by phage infection of bacteria containing the phagemid, whereby each cell contains a different combination and the size of the library is limited only by the number of cells present (approximately 10 ¹² clones). Both vectors contain in vivo recombination signals that allow the VH and VL genes to be recombined on a single replicon and co-packaged into phage virions. These large libraries provide a large number of diverse antibodies with good affinity ( _Kd ^-1 is about ^10-8 M).

Alternatively, these libraries can be cloned sequentially into the same vector (see, e.g., Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991)), or assembled together by PCR and then cloned (see, e.g., Clackson et al., Nature, 352:624-628 (1991)). PCR assembly can also be used to link VH and VL DNAs together with DNA encoding a flexible peptide spacer to form a single-chain Fv (scFv) library. In yet another technique, "in-cell PCR assembly" is used to combine VH and VL genes by PCR within lymphocytes, followed by cloning of the linked gene library, see Embleton et al., Nucl. Acids Res., 20:3831-3837 (1992).

Antibodies generated from naive libraries (natural or synthetic) can have intermediate affinities ( _Kd ^-1 of approximately ¹⁰⁶ to ¹⁰⁷ M ^-1 ), but affinity maturation can also be simulated in vitro by constructing and reselecting from secondary libraries, see Winter et al. (1994) supra. For example, mutations can be introduced randomly in vitro using an error-prone polymerase (reported in Leung et al., Technique 1: 11-15 (1989)) as described in Hawkins et al., J. Mol. Biol., 226: 889-896 (1992) or in Gram et al., Proc. Natl. Acad. Sci USA, 89: 3576-3580 (1992). Alternatively, affinity maturation can be performed by randomly mutating one or more CDRs in each selected Fv clone (e.g., using primers carrying random sequences across the CDR of interest for PCR) and screening for clones with higher affinity. WO 9607754 (published on March 14, 1996) describes a method for inducing mutagenesis in the complementarity determining regions of immunoglobulin light chains to create a light chain gene library. Another effective method is to recombine the VH or VL domains selected by phage display with a library of natural V domain variants obtained from unimmunized donors and screen for higher affinity in several rounds of chain shuffling, see Marks et al., Biotechnol., 10: 779-783 (1992). This technology allows the production of antibodies and antibody fragments with affinities of about ^10-9 M or less.

Screening of the library can be accomplished by various techniques known in the art. For example, the antigen can be used to coat the wells of an adsorption plate, expressed on host cells attached to an adsorption plate, or used for cell sorting, or conjugated to biotin for capture by streptavidin-coated beads, or any other method for panning phage display libraries.

The phage library sample is contacted with the immobilized antigen under conditions suitable for binding of at least a portion of the phage particles to the adsorbent. Normally, conditions including pH, ionic strength, and temperature are selected to mimic physiological conditions. Phage bound to the solid phase are washed and then eluted with acid, as described, for example, in Barbas et al., Proc. Natl. Acad. Sci USA, 88:7978-7982 (1991), or with base elution, as described, for example, in Marks et al., J. Mol. Biol., 222:581-597 (1991), or by antigen competition elution, for example, using a procedure similar to the antigen competition method of Clackson et al., Nature, 352:624-628 (1991). Phage can be enriched 20-1,000-fold in a single round of selection. Alternatively, the enriched phage can be grown in bacterial culture and subjected to multiple rounds of selection.

The efficiency of selection depends on many factors, including the dissociation kinetics during washing, and whether multiple antibody fragments on a single phage can bind to antigen simultaneously. Antibodies with rapid dissociation kinetics (and weak binding affinity) can be retained by using a short time cleaning, polyvalent phage display and a high coating density of antigen in a solid phase. High density not only stabilizes phage by multivalent interactions, but is also conducive to rebinding the dissociated phage. The selection of antibodies with slow dissociation kinetics (and good binding affinity) can be promoted by using long time cleaning and monovalent phage display (see Bass et al., Proteins, 8: 309-314 (1990) and WO 92/09690) and a low coating density of antigen (with reference to Marks et al., Biotechnol., 10: 779-783 (1992)).

It is possible to select between phage antibodies with different affinities for the antigen (even with slightly different affinities). However, random mutation of a selected antibody (as performed in some affinity maturation techniques) is likely to produce many mutants, most of which bind to the antigen, while a few have higher affinities. Using a limited amount of antigen, rare high-affinity phage can outcompete. In order to retain all higher-affinity mutants, phage can be incubated with an excess of biotinylated antigen, but wherein the biotinylated antigen is used at a molar concentration lower than the target molar affinity constant of the antigen. High-affinity-bound phage can then be captured using streptavidin-coated paramagnetic beads. Such "equilibrium capture" allows antibodies to be selected based on their binding affinity, and its sensitivity allows mutant clones with only twice as high affinities to be isolated from a large excess of phage with lower affinities. The conditions for washing phage bound to the solid phase can also be manipulated to differentiate based on dissociation kinetics.

Anti-antigen clones can be selected based on activity. In certain embodiments, the present invention provides anti-antigen antibodies that can bind to living cells that naturally express the antigen, or to free-floating antigens, or to antigens attached to other cellular structures. Fv clones corresponding to such anti-antigen antibodies can be selected by: (1) isolating anti-antigen clones from a phage library as described above, and optionally expanding the population by growing the isolated phage clones in an appropriate bacterial host; (2) selecting an antigen and a second protein for which blocking and non-blocking activity, respectively, is desired; (3) adsorbing the anti-antigen phage clones to the immobilized antigen; (4) using an excess of the second protein to elute any unwanted clones that recognize antigen binding determinants that overlap or are in common with the binding determinants of the second protein; and (5) eluting clones that remain adsorbed after step (4). Optionally, clones with the desired blocking/non-blocking properties can be further enriched by repeating the screening procedure described herein one or more times.

DNA encoding the desired hybridoma-derived monoclonal antibody or phage-displayed Fv clone can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide primers designed to specifically amplify the desired heavy and light chain coding regions from the hybridoma or phage DNA template). Once isolated, the DNA can be placed in an expression vector and then transfected into a host cell, such as an E. coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not otherwise produce immunoglobulins, to obtain synthesis of the desired monoclonal antibody in the recombinant host cell. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs, 130: 151 (1992).

The DNA encoding the Fv clone can be combined with a known DNA sequence encoding the constant region of the heavy and/or light chain (e.g., appropriate DNA sequences can be obtained from Kabat et al., supra) to form a clone encoding a full-length or partial-length heavy and/or light chain. It should be understood that any isotype of constant region can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species. The definitions of "chimeric" and "hybrid" antibodies used herein include: an Fv clone derived from the variable domain DNA of one animal species (e.g., human) that is then fused to the constant region DNA of another animal species to form a coding sequence encoding a "hybrid" full-length heavy and/or light chain. In certain embodiments, an Fv clone from human variable DNA is fused to human constant region DNA to form a coding sequence encoding a full-length or partial-length human heavy and/or light chain.

The DNA encoding the anti-antigen antibody derived from the hybridoma can also be modified, for example, by substituting the coding sequences for the human heavy and light chain constant domains in place of the homologous murine sequences derived from the hybridoma clone (see, e.g., the method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The DNA encoding the antibody or fragment derived from the hybridoma or Fv clone can be further modified by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this manner, a "chimeric" or "hybrid" antibody is prepared that has the binding specificity of the antibody of interest derived from the Fv clone or hybridoma clone.

(iv) Humanized antibodies and human antibodies

Various methods for humanizing non-human antibodies are known in the art. For example, one or more amino acid residues in a humanized antibody are imported into the antibody from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from the "import" variable domain. Humanization can essentially follow the method of Winter and colleagues (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), wherein rodent CDRs or CDR sequences are substituted for the corresponding sequences of human antibodies. Therefore, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which substantially less than a complete human variable domain has been substituted with the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

In order to reduce antigenicity, the selection of the human variable domains (including light chain and heavy chain variable domains) used in the preparation of humanized antibodies is very important. According to the so-called "optimal" method, the variable domain sequences of rodent antibodies are screened for the entire library of known human variable domain sequences. The human sequence closest to rodents is then accepted as the human framework (FR) for humanized antibodies (Sims et al., J. Immunol., 151: 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)). Another method uses a specific framework derived from the consensus sequence of all human antibodies with a light chain or heavy chain of a specific subgroup. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that humanized antibodies maintain high affinity for antigens and other favorable biological properties. To achieve this goal, according to one embodiment of the method, humanized antibodies are prepared by analyzing the method for parental sequences and various conceptual humanized products using a three-dimensional model of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. A computer program illustrating and displaying the possible three-dimensional conformational structure of a selected candidate immunoglobulin sequence is available. The structure displayed is inspected to allow analysis of the possible effects of residues in the function of the candidate immunoglobulin sequence, that is, analysis of the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from receptor and input sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen. Generally speaking, hypervariable region residues directly and most substantially participate in influencing antigen binding.

The Fv clone variable domain sequence selected from the human phage display library can be combined with the known human constant domain sequence as described above to construct the target human antibody. Alternatively, the target human monoclonal antibody can be prepared by the hybridoma method. Human myeloma and mouse-human hybrid myeloma cell lines for producing human monoclonal antibodies are described in, for example, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).

Transgenic animals (e.g., mice) can be produced that are capable of producing a complete human antibody repertoire upon immunization and that do not have endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain joining region ( _JH ) gene in chimeric and germline mutant mice will result in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7: 33 (1993); and Duchosal et al. Nature 355: 258 (1992).

Gene shuffling can also be used to obtain human antibodies from non-human (e.g., rodent) antibodies, wherein the human antibodies have similar affinity and specificity to the initial non-human antibodies. According to this method (also referred to as "epitope imprinting"), the heavy chain or light chain variable region of the non-human antibody fragment obtained by phage display technology as described herein is replaced by a human V domain gene library to create a non-human chain/human chain scFv or Fab chimera population. Selection is carried out with antigen, resulting in the separation of non-human chain/human chain chimeric scFv or Fab, wherein the human chain recovers the antigen binding site that was destroyed when the corresponding non-human chain in the original phage display clone was removed, i.e., the selection of epitope control (imprinting) human chain partners. When the process is repeated to replace the remaining non-human chain, human antibodies are obtained (see PCT WO 93/06213, published on April 1, 1993). Unlike traditional non-human antibody humanization achieved by CDR transplantation, this technology provides fully human antibodies, without FR or CDR residues from non-human sources.

(v) Antibody fragments

Antibody fragments can be produced by traditional means such as enzymatic digestion, or by recombinant techniques. In some cases, there are advantages to using antibody fragments rather than intact antibodies. The smaller size of the fragments allows for rapid clearance and can result in improved access to solid tumors. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9: 129-134.

Various techniques have been developed for producing antibody fragments. Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed and secreted by E. coli, thus allowing these fragments to be easily produced in large quantities. Antibody fragments can be isolated from the antibody phage library discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab') ₂ fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another method, F(ab') ₂ fragments can be directly isolated from recombinant host cell cultures. Fab and F(ab') ₂ fragments containing salvage receptor binding epitope residues with increased in vivo half-life are described in U.S. Patent No. 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single-chain Fv fragment (scFv). See WO 93/16185; U.S. Patent Nos. 5,571,894 and 5,587,458. Fv and scFv are the only types that have complete binding sites but lack constant regions; therefore, they are suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins can be constructed to produce fusions of effector proteins fused to the amino or carboxyl termini of the scFv. See Antibody Engineering, ed. Borrebaeck, loc. cit. Antibody fragments can also be "linear antibodies," for example, as described in U.S. Patent No. 5,641,870. Such linear antibodies can be monospecific or bispecific.

(vi) Multispecific antibodies

Multispecific antibodies have binding specificity for at least two different epitopes, where the epitopes are typically from different antigens. Although such molecules typically only bind to two different epitopes (i.e., bispecific antibodies, BsAbs), when used herein, this term also encompasses antibodies with additional specificities, such as trispecific antibodies. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab') ₂ bispecific antibodies).

Methods for preparing bispecific antibodies are well known in the art. The production of traditional full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually carried out by affinity chromatography steps, which is quite cumbersome and the product yield is low. Similar methods are disclosed in WO 93/08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

According to a different approach, an antibody variable domain with desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant domain sequence. The fusion is preferably performed with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2, and CH3 regions. Typically, the first heavy chain constant region (CH1) comprising the light chain binding site is present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where the unequal ratios of the three polypeptide chains used to construct the optimal yield are provided, this provides great flexibility for adjusting the mutual ratios of the three polypeptide fragments. However, when expressing at least two polypeptide chains in equal proportions, resulting in high yields or when the ratio has no particular significance, the coding sequences of two or all three polypeptide chains can be inserted into one expression vector.

In one embodiment of the method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. It has been found that this asymmetric structure is convenient for separating the desired bispecific compound from the unwanted immunoglobulin chain combination, because the immunoglobulin light chain is only present in half of the bispecific molecule, which provides a convenient way for separation. This method is disclosed in WO 94/04690. For further details of producing bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

According to the another method described in WO96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. An interface comprises at least a portion of the _CH 3 domain of an antibody constant domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). On the interface of the second antibody molecule, a complementary " cavity " of the same or similar size as the large side chain is produced in the following manner: on the interface of the second antibody molecule, large amino acid side chains are replaced with smaller amino acid side chains (e.g., alanine or threonine). This provides a mechanism that improves the productive rate of heterodimers compared to other undesirable end products (e.g., homodimers).

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, in a heteroconjugate, one antibody can be coupled to avidin and the other to biotin. Such antibodies have been proposed, for example, for targeting immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treating HIV infection (WO 91/00360, WO 92/200373 and EP03089). Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980, along with many cross-linking techniques.

Techniques for preparing bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229:81 (1985) described a method in which intact antibodies are proteolytically cleaved to produce F(ab') ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize the dithiols in the adjacent position and prevent the formation of intermolecular disulfide bonds. The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then converted back into a Fab'-thiol by reduction with mercaptoethylamine and then mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibody can be used as a reagent for selectively immobilizing enzymes.

Recent advances have facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) described the production of fully humanized bispecific antibody F(ab') ₂ molecules. Each Fab' fragment was individually secreted from E. coli and subjected to directed chemical coupling in vitro to form a bispecific antibody.

Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been generated using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers are reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "dimer" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) provides another mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable region ( _VH ) connected to a light chain variable region ( _VL ) by a linker, wherein the linker is too short to allow pairing between the two functional domains on the same chain. Therefore, the _VH and _VL domains of one fragment are forced to pair with the complementary _VL and _VH domains of the other fragment, thereby forming two antigen-binding sites. Another strategy for preparing bispecific antibody fragments using single-chain Fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol, 152: 5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147:60 (1991).

(vii) Single domain antibodies

In some embodiments, the antibody of interest is a single domain antibody. A single domain antibody is a single polypeptide chain comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Patent No. 6,248,516 B1). In one embodiment, a single domain antibody consists of all or part of the heavy chain variable domain of an antibody.

(viii) Antibody variants

In some embodiments, it is contemplated that the amino acid sequence of an antibody described herein may be modified. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. An amino acid sequence variant of the antibody may be prepared by introducing a suitable variation into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, residue deletions and/or insertions and/or replacements in the amino acid sequence of the antibody. Any combination of deletions, insertions, and replacements may be performed to obtain the final construct, provided that the final construct has the desired characteristics. Amino acid changes may be introduced into the subject antibody amino acid sequence when preparing the sequence.

(B) Vectors, host cells, and recombination methods

The antibody produced by the cells cultivated in the cell culture medium provided by this paper can also be produced using recombinant methods. For the recombinant production of anti-antigen antibodies, the nucleic acid encoding the antibody is separated and inserted into a reproducible vector for further cloning (DNA amplification) or expression. Conventional methods can be used to easily separate and sequence the DNA encoding the antibody (for example, by using oligonucleotide probes that can specifically bind the heavy chain and light chain of the encoding antibody). Many vectors can be obtained. Vector components typically include but are not limited to one or more of the following: signal sequence, origin of replication, one or more marker genes, enhancer elements, promoters and transcription termination sequences.

(i) Signal sequence component

Antibodies can be produced recombinantly, either directly or as fusion polypeptides with heterologous polypeptides, preferably signal sequences or other polypeptides having specific cleavage sites at the N-terminus of mature proteins or polypeptides. The selected heterologous signal sequence is preferably a sequence that is recognized and processed (e.g., by a signal peptidase cleavage) by the host cell. For prokaryotic host cells that do not recognize and process native antibody signal sequences, the signal sequence can be replaced by a prokaryotic signal sequence, such as one selected from alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leader sequences. For yeast secretion, the native signal sequence can be replaced by, for example, a yeast invertase leader sequence, a factor leader sequence (including yeast and Kluyveromyces alpha factor leaders), an acid phosphatase leader sequence, a Candida albicans glucoamylase leader sequence, or a signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences and viral secretion leaders, such as the herpes simplex virus gD signal, are available.

(ii) Origin of replication

Both expression vectors and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Generally speaking, in a cloning vector, this sequence is a sequence that enables the vector to replicate independently of the host chromosomal DNA, including an origin of replication or an autonomously replicating sequence. This sequence is known for many bacteria, yeast, and viruses. The origin of replication of plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma virus, adenovirus, VSV, or BPV) are suitable for cloning vectors in mammalian cells. Typically, mammalian expression vectors do not require an origin of replication component (generally only the SV40 origin is used because it contains an early promoter).

(iii) Selecting gene modules

Expression and cloning vectors may contain a selection gene, also known as a selectable marker. Typical selection genes encode: (a) proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) proteins that compensate for auxotrophic deficiencies; or (c) proteins that provide key nutrients not found in complex culture media, such as the gene encoding D-alanine racemase for Bacillus.

An example of a selective method is the use of a drug that stops host cell growth. Cells that have been successfully transformed with the foreign gene produce a protein that confers drug resistance and can therefore survive the selection regimen. Examples of dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

Another example of suitable selectable markers for mammalian cells are those markers that allow identification of cells competent to take up the antibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein I and II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like.

For example, the cell that has transformed the DHFR gene can be identified by cultivating transformants in a culture medium that comprises methotrexate (Mtx), wherein methotrexate is a competitive antagonist of DHFR. Under these conditions, the DHFR gene is amplified together with any other co-converted nucleic acid. The Chinese hamster ovary (CHO) cell line (for example, ATCC CRL-9096) lacking endogenous DHFR activity can also be used.

Alternatively, cells transformed with the GS gene can be identified by culturing the transformants in a medium containing L-methionine sulfoximine (MSX), a GS inhibitor. Under these conditions, the GS gene is amplified along with any other co-transformed nucleic acids. The GS selection/amplification system can be used in combination with the DHFR selection/amplification system described above.

Alternatively, host cells (particularly wild-type hosts containing endogenous DHFR) transformed or co-transformed with a DNA sequence encoding the antibody of interest, a wild-type DHFR gene, and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH), can be selected by culturing in a medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic such as kanamycin, neomycin, or G418. See, U.S. Patent No. 4,965,199.

A suitable selection gene for yeast is the trpl gene present on the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). The trpl gene can provide a selection marker for yeast mutants that cannot grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977). Therefore, the presence of a trpl lesion within the yeast host cell genome provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) can be complemented by known plasmids carrying the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 can be used to transform Kluyveromyces. Alternatively, an expression system for large-scale production of recombinant bovine chymosin in Kluyveromyces lactis has been reported. Van den Berg, Bio/Technology, 8:135 (1990). Stable multicopy expression vectors for industrial strains of Kluyveromyces to secrete mature recombinant human serum albumin have also been disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991).

(iv) Promoter Component

Expression and cloning vectors generally contain a promoter that is recognized by the host organism and operably linked to the nucleic acid encoding the antibody. Promoters suitable for prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoters, tryptophan (trp) promoter systems, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are also suitable. Promoters suitable for bacterial systems may also contain an SD sequence (Shine-Dalgarno sequence) operably linked to the DNA encoding the antibody.

Promoter sequences for eukaryotic organisms are known. Almost all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site. Another sequence present 70 to 80 bases upstream of the transcription start site of many genes is the CNCAAT region, where N can be any nucleotide. The 3' end of most eukaryotic genes is the AATAAA sequence, which may be a signal that adds a polyadenylic acid tail to the 3' end of the coding sequence. All of these sequences are suitable for insertion into eukaryotic expression vectors.

Examples of suitable promoter sequences for yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters include the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothioneins, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. These promoters are inducible promoters with the added advantage that transcription can be controlled by culture conditions. Vectors and promoters suitable for yeast expression are further described in EP 73,657. Yeast promoters are preferably used in conjunction with yeast enhancers.

In mammalian host cells, antibody transcription from the vector can be controlled, for example, by promoters compatible with the host cell systems, derived from viral genomes (e.g., polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40)) as well as heterologous mammalian promoters (e.g., actin promoter or immunoglobulin promoter, heat shock protein promoter).

The early and late promoters of the SV40 virus are readily available as SV40 restriction fragments, which also contain the SV40 origin of replication. The immediate early promoter of the human cytomegalovirus is readily available as a Hind III E restriction fragment. U.S. Patent No. 4,419,446 discloses a system for expressing DNA in mammalian hosts using bovine papilloma virus as a vector. U.S. Patent No. 4,601,978 describes a modification of this system. See also Reyes et al., Nature 297:598-601 (1982), which describes the expression of human beta interferon cDNA in mouse cells under the control of the herpes simplex virus thymidine kinase promoter. Additionally, the Rous sarcoma virus long terminal repeat sequence can also be used as a promoter.

(v) Enhancer element components

Often, the transcription of the DNA encoding the antibody of the present invention in higher eukaryotic organisms can be enhanced by inserting an enhancer sequence into the vector. Many enhancer sequences from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin) are now known. However, enhancers from eukaryotic cell viruses are generally used. Examples include the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and the adenovirus enhancer located at the late side of the replication origin (100-270bp). See also the enhancer element for activating eukaryotic promoters described in Yaniv, Nature 297:17-18 (1982). The enhancer can be spliced into the 3' or 5' position of the antibody coding sequence in the vector, but is preferably located at the 5' position of the promoter.

(vi) Transcription termination component

Expression vectors used in eukaryotic host cells (nucleated cells of yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) also contain sequences necessary for terminating transcription and stabilizing the mRNA. Such sequences can typically be obtained from the 5' and, occasionally, 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments within the untranslated portion of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vectors disclosed therein.

(vii) Selection and transformation of host cells

Suitable host cells for cloning or expressing the DNA in the vectors herein include the prokaryotes, yeast, or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, and Bacilli, e.g., B. subtilis and B. licheniformis (e.g., DD 12, published April 12, 1989). 266,710), Pseudomonas such as Pseudomonas aeruginosa and Streptomyces. A preferred E. coli cloning host is E. coli 294 (ATCC 31,446), but other strains such as E. coli B, E. coli X1776 (ATCC 31,537) and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative and not limiting.

Full-length antibodies, antibody fusion proteins, and antibody fragments can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required, such as when therapeutic antibodies are conjugated to cytotoxic agents (e.g., toxins) that themselves show tumor cell destruction efficacy. Full-length antibodies have a greater circulation half-life. Production in E. coli is faster and more cost-effective. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent No. 5,648,237 (Carter et al.), U.S. Patent No. 5,789,199 (Joly et al.), U.S. Patent No. 5,840,523 (Simmons et al.), which describe translation initiation regions (TIRs) and signal sequences for optimizing expression and secretion. See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, which describe expression of antibody fragments in E. coli. After expression, antibodies can be isolated from the E. coli cell paste as a soluble fraction and, depending on the isotype, purified by, for example, a protein A or G column. Final purification can be performed by a similar process to that used to purify antibodies expressed, for example, in CHO cells.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or common baker's yeast is the most commonly used lower eukaryotic host microorganism. However, many other genera, species, and strains are also commonly available and can be used in the present invention, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium; and Aspergillus hosts such as A. nidulans and A. niger. For a review discussing the use of yeast and filamentous fungi for the production of therapeutic proteins, see, for example, Gerngross, Nat. Biotech. 22: 1409-1414 (2004).

Certain fungi and yeast strains whose glycosylation pathways have been "humanized" can be selected, resulting in the production of antibodies with partially or fully human glycosylation patterns. See, for example, Li et al., Nat. Biotech. 24:210-215 (2006) (describing humanization of the glycosylation pathway in Pichia pastoris); and Gerngross et al., supra.

Host cells suitable for expressing glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plants and insect cells. Many baculovirus strains and variants thereof and corresponding permissive insect host cells derived from hosts such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori have been identified. Many viral strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, which can be used, in particular, for transfection of Spodoptera frugiperda cells according to the present invention.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES ^™ technology for producing antibodies in transgenic plants).

Vertebrate cells can be used as hosts, and propagation of vertebrate cells in culture (tissue culture) has become routine. Examples of mammalian cells that can be used include SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or subcloned 293 cells for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (IM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor cell line (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and human hepatoma cell line (HepG2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NS0 and SP2/0. For review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268.

Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in a cell culture medium provided herein, which has been modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences.

Cell growth and peptide production

Generally, cells are combined (contacted) with any cell culture medium as described herein under one or more conditions that promote cell growth, maintenance and/or polypeptide production. The method for culturing cells and producing polypeptides uses a culture vessel (bioreactor) to accommodate cells and cell culture medium. The culture vessel can be made 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 larger. Culture conditions, such as temperature, pH, etc., can be the culture conditions previously used to select the host cell for expression, and will be apparent to those skilled in the art. The culture conditions that can be adjusted during the culture process include, but are not limited to, pH and temperature.

Cell cultures are generally maintained during the initial growth phase under conditions that are conducive to the viability, growth, and survival (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.

The temperature of the cell culture during the initial growth phase is selected primarily based on the temperature range that can keep the cell culture viable. For example, during the initial growth phase, CHO cells grow well at 37°C. Generally, most mammalian cells grow well within a range of approximately 25°C to 42°C. Preferably, mammalian cells grow well within a range of approximately 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 the appropriate temperature(s) for growing the cells.

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

Cells can be cultured for a longer or shorter time in the initial growth phase. In one variant, the cells are cultured for a length of time sufficient to achieve a viable cell density that is a given percentage of a maximum viable cell density, where the maximum viable cell density is the density that the cells will ultimately reach if the cells are allowed to grow undisturbed. For example, the cells can be cultured for a length of time sufficient to achieve 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.

In another embodiment, the cells are allowed to grow for a defined period of time. For example, depending on the starting concentration of the cell culture, the temperature of the cell culture, and the intrinsic growth rate of the cells, the cells can be cultured 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.

The cell culture can be stirred or shaken during the initial culture phase to increase oxygenation and diffusion of nutrients to the cells. In light of the present disclosure, it will be apparent to those skilled in the art that it may be advantageous to control or adjust certain internal bioreactor conditions during the initial growth phase, including but not limited to pH, temperature, oxygenation, and the like. For example, pH can be controlled by providing an appropriate amount of acid or base, and oxygenation can be controlled using a sparging device, as is well known in the art.

The initial culture step is the growth phase, during which batch cell culture conditions can be modified to enhance the growth of the recombinant cells and to produce 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, but not limited thereto. The growth cycle of host cells can be determined for a particular host cell by methods known to those skilled in the art.

During the growth phase, the basal cell culture medium and cells described herein can be supplied to the culture vessel in a batch mode. In one aspect, the culture medium contains less than about 5%, less than 1%, or less than 0.1% serum and other animal-derived proteins. However, serum and animal-derived proteins can be used if desired. At a specific point in the cell growth process, the cells can be used to form an inoculum to inoculate the culture medium at the start of the production phase. Alternatively, the production phase can be continuous with the growth phase. The polypeptide production phase generally follows the cell growth phase.

In the polypeptide production stage, 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 are suitable for expressing the desired polypeptide. For example, in the subsequent production stage, Chinese hamster ovary celI expresses recombinant polypeptides and proteins well in the range of 25 ℃ to 38 ℃. 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, provided herein is a culture medium that can reduce the presence of metabolic byproducts when used in the method for increasing polypeptide output. In one variation, the impurity is reactive oxygen. On the one hand, compared to the color intensity obtained when producing polypeptides in different culture media, provided herein is a culture medium that reduces the color intensity of polypeptide products when used in the method for increasing polypeptide output. In one variation, the method for increasing polypeptide output is included in a temperature migration step in the polypeptide production stage. In further variations, the temperature migration step includes a temperature migration from 31°C to 38°C, from 32°C to 38°C, from 33°C to 38°C, from 34°C to 38°C, from 35°C to 38°C, from 36°C to 38°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.

In some embodiments, the cell culture medium is grown in the presence of a plurality of cells of the present invention. The cell culture medium is grown in the presence of a plurality of cells of the present invention. The cell culture medium is grown in the presence of a plurality of cells of the present invention. The cell culture medium is grown in the presence of a plurality of cells of the present invention. The cell culture medium is grown in the presence of a plurality of cells of the present invention. The cell culture medium is grown in the presence of a plurality of cells of the present invention. The cell culture medium is grown in the presence of a plurality of cells of the present invention. The cell culture medium is grown in the presence of a plurality of cells of the present invention. The cell culture medium is grown in the presence of a plurality of cells of the present invention. The cell culture medium is grown in the presence of a plurality of cells of the present invention. The cell culture medium is grown in the presence of a plurality of cells of the present invention.

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 during the subsequent production phase. For example, it may be advantageous to supplement the cell culture with nutrients or other culture medium components that are 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 prior to the subsequent production phase. As non-limiting examples, 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 typically present at very low final concentrations), amino acids, lipids, or glucose or other energy sources.

Components provided herein (e.g., hypotaurine or its analogs or precursors) can be added to the cell culture medium at any time during the cell culture cycle. For example, hypotaurine can be added in any amount at any one or more days (e.g., any one or more days, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days) during a 14-day cell culture cycle, thereby providing a cell culture medium comprising hypotaurine at a concentration (e.g., at least 0.0001 mM) provided herein. Therefore, it is understood that for a 14-day cell culture cycle, hypotaurine can be added in any amount at any one or more days (e.g., any one or more days, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days) during a 0-14-day cell culture cycle. "Day 0" as used herein can refer to a cell culture medium supplemented with a component (e.g., hypotaurine) provided herein before cell culture medium is applied. It will be appreciated that the cell culture cycle can be any number of days, as long as the cells remain alive and/or produce sufficient levels of polypeptide (which can be determined by those skilled in the art). For example, the cell culture cycle can continue for at least 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 or 20 days. In some embodiments, the component provided herein (for example, hypotaurine or its analog or precursor) is added to the cell culture medium at least one day of the cell culture cycle.

Peptide purification

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.

The culture medium or lysate can be centrifuged to remove particulate cell debris. The polypeptide can then be purified from contaminating soluble proteins and polypeptides. The following are examples of suitable purification protocols: fractionation on an immunoaffinity column or ion exchange column; ethanol precipitation; reversed-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 proteolytic degradation during purification. Those skilled in the art will appreciate 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. Polypeptides can generally be purified using chromatographic techniques (e.g., Protein A, affinity chromatography with a low pH elution step and ion exchange chromatography to remove process impurities). For antibodies, the suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains ((Lindmark et al., J. Immunol. Meth. 62:1-13 (1983))). Protein G is recommended for all mouse isotypes and human γ3 ((Guss et al., EMBO J. 5:1567-1575 (1986))). The purified protein can be concentrated to provide a concentrated protein drug product, as described herein, for example, a product having a protein concentration of at least 1 mg/mL or 10 mg/mL or 50 mg/mL or 75 mg/mL or 100 mg/mL or 125 mg/mL or 150 mg/mL, or a concentration of about 1 mg/mL or 10 mg/mL or 50 mg/mL or 75 mg/mL or 100 mg/mL or 125 mg/mL or 150 mg/mL. It will be appreciated that the concentrated polypeptide product can be concentrated to provide a concentrated protein drug product. To a level permitted under the concentration conditions, for example, to a concentration at which the polypeptide is no longer soluble in solution. For example, a method for purifying a polypeptide may include the steps of harvesting cell culture fluid from polypeptide-producing cells, purifying the polypeptide by protein A affinity chromatography, further purification by anion and cation exchange chromatography, filtration to remove viruses, and a final ultrafiltration and diafiltration step 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.

Peptide color evaluation

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 cultivated in culture medium described herein, purifying 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 variation, after purification using protein A affinity chromatography, the color of the composition comprising the polypeptide is evaluated. In a further variation, after purification by ion exchange chromatography, the color of the composition comprising the polypeptide is evaluated. In another variation, after purification by high performance liquid chromatography, the color of the composition comprising the polypeptide is evaluated. In yet another variation, after purification by hydrophobic interaction chromatography, the color of the composition comprising the polypeptide is evaluated. In yet another variation, after purification by size exclusion chromatography, the color of the composition comprising the polypeptide is evaluated. In one variation, after purification by filtration (including microfiltration or ultrafiltration), the color of the composition comprising the polypeptide is evaluated. In one variation, before evaluating color, the composition comprising the polypeptide is concentrated (for example, the composition can comprise at least 1 mg/mL, 10 mg/mL, 50 mg/mL, 75 mg/mL, 100 mg/mL, 125 mg/mL or 150 mg/mL polypeptide, such as an antibody). The composition comprising the polypeptide can be concentrated by centrifugation, filtering device, semipermeable membrane, dialysis, precipitation, ion exchange chromatography, affinity chromatography, high performance liquid chromatography or hydrophobic interaction chromatography. In one variation, before evaluating color, the polypeptide can be concentrated by freeze-drying and resuspending. After purification using one or more techniques described herein, the composition comprising the polypeptide can be evaluated for color. It is contemplated herein that after the composition comprising the polypeptide has undergone one or more freeze-thaw cycles, the color evaluation of the composition can be performed. It is also contemplated herein that the cell culture fluid comprising the polypeptide can be evaluated for color before purification or concentration of the polypeptide.

The polypeptide produced by the methods described herein (or present in the compositions provided herein) using 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. Referring to 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, these documents are hereby incorporated herein 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 one variation, 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 the reference solution specified in the monograph. The colors are compared in diffuse daylight and observed horizontally relative to a white background to determine, measure or evaluate the color. In another variation, a polypeptide-containing composition is compared to water or a solvent or a monograph-specified reference solution using the same flat-bottomed, colorless, transparent, neutral glass tube with an inner diameter of 15 mm to 25 mm, with a depth of 40 mm. The color is compared in diffuse daylight, vertically observed against a white background for color determination, measurement, or evaluation. In one variation, color determination, measurement, or evaluation can be performed by human visual observation. In another variation, color determination, measurement, or evaluation can be performed using automated methods. For example, the tube can be loaded into a machine that images the tube so that the image is processed using an algorithm to determine, measure, or evaluate the color. It will be understood that the reference standard for the COC test can be, but is not limited to, any of brown (B), tan (BY), yellow (Y), green (GY), or red (R). The polypeptide-containing composition compared to the brown reference standard can 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 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 polypeptide-containing composition compared to a red reference standard can be assigned 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., except R1 for a red reference standard value). In one variation, the color of a composition comprising a polypeptide produced by cells cultured in a culture medium described herein has a reference standard value as described in Table 3. 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 of 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 of polypeptide) having a reference standard color value greater than any 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 reference standard color values applies 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.

In some embodiments, color intensity is determined using a total colorimetric method. See, for example, Vijayasankaran et al., Biotechol. Prog. 29: 1270-1277, 2013, which is incorporated herein by reference. For the total colorimetric method, the CIE system of color measurement is used to derive a quantitative value for the relative color of a sample, as described in Berns et al., Billmeyer and Saltzman's Principles of Color Technology, ^3rd Edition. New York, NY, John Wiley & Sons, Inc., (2000). In brief, after blank correction with water, the absorbance spectrum of the net test sample is measured in the visible light region (380-780 nm) using an HP8453A spectrophotometer (1 cm path cuvette). The absorption spectrum is then converted to the CIE L*a*b* color scale as previously 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). L*a*b* is a three-dimensional color space with roughly uniform spacing in visual perception. The L*a*b* color space can quantify differences in visual judgments of color. For example, two solutions that are visually judged to have very different colors will be further apart in the L*a*b* color space, while two solutions with similar colors will be closer together in the L*a*b* color space. In the three-dimensional L*a*b* color space, the distance between points is calculated as the Euclidean (ΔE) between the points. This allows the measurement of the ΔE between points in the L*a*b* color space and the correlation of this distance with the visual perception judgment of color difference, with a large ΔE representing two solutions of very different colors, and a small ΔE representing two solutions of similar colors. The conversion of the absorption spectrum to the L*a*b* color space requires a limited light source. For example, an artificial flat spectrum in the visible light region can be used as a light source. In some embodiments, the "total color" can represent a 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, the "total color" can represent the overall color of the monoclonal antibody sample tested without distinguishing different hues. The total color can be standardized with the value measured for the reference standard. For example, the color intensity value can then be determined by calculating the ratio of the measured value of the "total color" of the test monoclonal antibody sample to the measured value of the reference monoclonal antibody sample containing a COC reading ≤ B5.

Color intensity can also be measured using the NIFTY (intrinsic fluorescence tool for standardization of yellow/brown proteins) assay. In this assay, the fluorescence of the antibody molecule is used as a surrogate for color because it has been shown that color intensity and fluorescence intensity are well correlated in the protein A pool (R2=0.84). See Vijayasankaran et al., Biotechnol Prog 27:1270-1277 (2013). A higher NIFTY value indicates a higher color intensity, and a lower NIFTY value indicates a lower color intensity. Using a G3000SWXL column (TOSOH), about 50 to 125 μg of monoclonal antibody sample was analyzed by size exclusion chromatography (SEC) at an isocratic flow rate of 0.5 mL/min. The mobile phase for SEC was 0.2 M potassium phosphate, 0.25 M potassium chloride, and a pH of 6.2. The column temperature was controlled at 15 ° C. For example, the SEC eluent can be monitored for UV absorption at 280 nm and fluorescence at 350 nm excitation wavelength and 425 nm emission wavelength. The SEC peak of monoclonal antibody is used for the quantification of color intensity.These wavelengths are selected based on the strong correlation and maximum fluorescence reaction observed with these wavelengths.On UV absorption and fluorescence emission chromatogram, the SEC peak of monoclonal antibody is used Agilent Chemstation software integration.For every kind of monoclonal antibody sample, by the fluorescence peak area of main peak divided by the UV absorption peak area of main peak, determine the fluorescence of standardization, it is corrected fluorescence reaction with antibody mass contribution.The color intensity value is determined subsequently by the ratio of the standardized fluorescence of the monoclonal antibody sample of calculation test and the standardized fluorescence of reference monoclonal antibody sample (for example, containing the sample of COC reading≤B5).Because the sample required for NIFTY is very little, when culture volume is limited, it is useful as the substitute of color.

The NIFTY value can be calculated as follows: F = peak area on the fluorescence chromatogram; U = peak area on the UV absorption chromatogram; i = variable; S = sample; R = reference.

Table 3. Exemplary Reference Standard Values

In another example, 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 a quantitative assay. In some embodiments, an automated method can be used to perform a quantitative assay. In some embodiments, the higher value (e.g., higher numerical value) provided by the quantitative assay represents a higher color intensity, while the lower value (e.g., lower numerical value) represents a lower color intensity.

The color assays detailed herein can be used to assess the color of any solution (e.g., a solution containing a polypeptide), including but not limited to, the polypeptide compositions provided herein.

Compositions and pharmaceutical preparations

The present invention also provides compositions comprising cell culture medium and one or more other components, such as cells or desired polypeptides (e.g., antibodies). Cells comprising nucleic acids encoding target polypeptides (e.g., antibodies) can secrete the polypeptides of the present invention into cell culture medium during cell culture. Therefore, the compositions of the present invention can comprise cells producing polypeptides and cell culture medium as provided herein, wherein the polypeptides are secreted into the cell culture medium. Compositions containing produced polypeptides and cell culture medium as provided herein can also be considered. In some aspects of the present invention, the compositions comprise: (a) cells comprising nucleic acids encoding polypeptides; and (b) cell culture medium as provided herein. In some aspects, the compositions comprise: (a) polypeptides; and (b) cell culture medium as provided herein, wherein the polypeptides are secreted into culture medium by cells comprising nucleic acids encoding polypeptides. In other aspects, the compositions comprise: (a) polypeptides; and (b) cell culture medium as provided herein, wherein the polypeptides are secreted into culture medium by cells comprising nucleic acids encoding polypeptides. In other aspects, the compositions comprise: (a) polypeptides; and (b) cell culture medium as provided herein, wherein the polypeptides are released into culture medium by lysing cells comprising isolated nucleic acids encoding polypeptides. The cells of the compositions can be any cells described herein (e.g., CHO cells), and the culture medium of the compositions can be any culture medium as described herein, such as culture medium comprising one or more compounds described in detail in Table 1 or Table 2. Similarly, the polypeptide of the composition can be any polypeptide described herein, such as an antibody. In some aspects, the composition can have color. In some embodiments, the color is determined, measured, or evaluated by using one or more visual color standards. The visual color standard can be an international or national color standard, for example, 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. Therefore, in some embodiments, the color intensity of a composition comprising (a) a polypeptide; and (b) a cell culture medium provided herein is evaluated. In further embodiments, the polypeptide is isolated and/or purified before evaluating the color intensity. In some embodiments, the color intensity of a composition comprising (a) a polypeptide; and (b) a cell culture medium provided herein is used to predict the color intensity of the final protein composition. For example, the color intensity of a composition comprising a polypeptide and a cell culture medium provided herein is measured using a COC assay as described herein. In some embodiments, the composition of the present invention comprises a polypeptide having a color intensity greater than B3, B4, B5, B6, B7, B8 or B9, wherein the color intensity value is greater than B3, B4, B5, B6, B7, B8 or B9. ... In some aspects, the compositions provided herein comprise 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.

Compositions (e.g., pharmaceutical preparations) of polypeptides (e.g., therapeutic antibodies) produced by any of the methods described herein can be prepared by mixing the polypeptides of desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations used, and include, but are not limited to, buffers, antioxidants, preservatives, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides, and other sugars, chelating agents, sugars, salt-forming counterions, metal complexes (e.g., Zn-protein complexes), and/or nonionic surfactants. 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, with the latter formulations including histidine-acetate buffer. In some embodiments, pharmaceutical preparation is administered to mammals such as people. The pharmaceutical preparation of polypeptide (for example, antibody) can be by any suitable means, including parenteral, intrapulmonary and intranasal administration, and, if desired local treatment, by intralesional administration. Parenteral perfusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Dosage can be by any suitable approach, for example, by injection, such as intravenous or subcutaneous injection, and this part depends on whether administration is short-lived or long-term. Therefore, the preparation containing polypeptide provided herein can be suitable for injection, such as subcutaneous injection to individuality (for example, subcutaneous injection into people). The pharmaceutical preparation for use in vivo is normally sterile. Sterile can easily be achieved by, for example, filtering through a sterile filtration membrane.

In some respects, provided herein are compositions (such as pharmaceutical preparations) comprising for example about 1mg/mL, 10mg/mL, 25mg/mL, 50mg/mL or 75mg/mL concentration, or about 1mg/mL, about 10mg/mL, about 25mg/mL, about 50mg/mL or about 75mg/mL to the polypeptide (such as therapeutic polypeptide) of up to about 100mg/mL concentration. In other respects, provided herein are compositions (such as pharmaceutical preparations) comprising at least about 100mg/mL, 125mg/mL, 150mg/mL, 200mg/mL or 250mg/mL concentration or about 100mg/mL, about 125mg/mL, about 150mg/mL, about 175mg/mL, about 200mg/mL or about 250mg/mL concentration polypeptide (such as therapeutic polypeptide). In some embodiments, provided herein are pharmaceutical preparations comprising a concentration greater than at least about 1 mg/mL, at least about 10 mg/mL, at least about 25 mg/mL, at least about 50 mg/mL or at least about 75 mg/mL of polypeptide, and having a color intensity value greater than B3, B4, B5, B6, B7, B8 or B9 as measured by a COC assay. In some aspects, provided herein are pharmaceutical preparations comprising a concentration greater than at least about 100 mg/mL, at least about 125 mg/mL, at least about 150 mg/mL, or at least about 200 mg/mL of polypeptide, and having a color intensity value greater than B3, B4, B5, B6, B7, B8 or B9 as measured by a COC assay. In some aspects, the color intensity value measured by a COC assay 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 pharmaceutical formulations provided herein comprise a polypeptide at a concentration greater than at least about 1 mg/mL, at least about 10 mg/mL, at least about 25 mg/mL, at least about 50 mg/mL, or at least about 75 mg/mL and have a color intensity value as determined by a colorimetric assay that is less than the color intensity value of a reference solution. For example, the color intensity of a composition (e.g., a pharmaceutical formulation) comprising a polypeptide (e.g., a therapeutic polypeptide) can be reduced by at least 0.1%, or from about 5% to about 50%, compared to a composition comprising the polypeptide produced by cells cultured in a cell culture medium that does not contain one or more of the components of Table 1 or Table 2.

V. Products or Kits

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 culture medium with the kit components). The kit may contain the components provided herein in amounts suitable for supplementing the cell culture medium. In some aspects, the kit comprises one or more components selected from hypotaurine, s-carboxymethylcysteine, anserine, butylated hydroxyanisole, carnosine, lipoic acid, and quercetin hydrate in amounts such that the cell culture medium can be supplemented at the component concentrations provided in Table 1 or Table 2. In some embodiments, the kit comprises one or more of the following: (a) hypotaurine in an amount to provide about 2.0 mM to about 50.0 mM hypotaurine in the cell culture medium; (b) s-carboxymethylcysteine in an amount to provide about 8.0 mM to about 12.0 mM s-carboxymethylcysteine in the cell culture medium; (c) carnosine in an amount to provide about 8.0 mM to about 12.0 mM carnosine in the cell culture medium; (d) anserine in an amount to provide about 3.0 mM to about 5.0 mM carnosine in the cell culture medium. (e) anserine; (e) butylated hydroxyanisole in an amount to provide about 0.025mM to about 0.040mM butylated hydroxyanisole in the cell culture medium; (f) lipoic acid in an amount to provide about 0.040mM to about 0.060mM lipoic acid in the cell culture medium; (g) quercetin hydrate in an amount to provide about 0.010mM to about 0.020mM quercetin hydrate in the cell culture medium; and (h) aminoguanidine in an amount to provide about 0.0003mM to about 10mM aminoguanidine in the cell culture medium. In some aspects, the kit comprises one or more components, wherein the one or more components are hypotaurine or an analog or precursor thereof. In some embodiments, hypotaurine or an analog or precursor thereof is selected from hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulfinic acid and taurine. In some embodiments, a kit for supplementing a cell culture medium with a chemically defined component comprises hypotaurine or an analog or precursor thereof at a concentration of at least about 0.0001 mM, and wherein the hypotaurine or an analog or precursor thereof is selected from the group consisting of hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulfinic acid, and taurine.

In another aspect of the present invention, an article of manufacture is provided that includes a container containing the cell culture medium of the present invention and, optionally, instructions for use. Suitable containers include, for example, bottles and bags. The container can be made of a variety of materials, such as glass or plastic. The container contains the cell culture medium, and a label on or associated with the container can provide instructions for use (e.g., for culturing cells). The article of manufacture can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, and a package insert with instructions for use.

The following examples are provided to illustrate but not to limit the present invention.

Example

Culture media have been identified that can produce protein products (e.g., protein pharmaceutical products) with acceptable quality attributes (e.g., reduced color intensity), particularly when the protein products are present as concentrated solutions (e.g., reaching a concentration of at least about 1 mg/mL to at least about 100 mg/mL). Methods of culturing cells in culture media provided herein and methods of producing polypeptides using the culture media are described herein. The culture media can comprise hypotaurine on the one hand. In some aspects provided herein, the culture media comprises one or more hypotaurine or its analogs or precursors, such as carboxymethylcysteine. Each culture media component can be present at any value provided herein. The culture media can be chemically defined or chemically undefined. The culture media of the present invention can reduce the presence of reactive oxygen species when used in polypeptide production methods compared to polypeptides produced in different culture media. The culture media 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 described herein and pharmaceutical compositions comprising the polypeptide products described in detail herein. In one aspect, the pharmaceutical composition comprises the polypeptide at a concentration of at least or about any of 100 mg/mL, 125 mg/mL, or 150 mg/mL. Methods of producing antibodies and compositions comprising the antibodies are particularly contemplated. The present invention also describes a kit for supplementing cell culture media with chemically defined components.

Example 1: Identification of Antioxidant Compounds Capable of Reducing Color in Antibody Compositions

Compounds that have been reported to react with oxidants were screened based on their ability to reduce the color of protein-containing compositions (Table 4). For the antioxidant screen, a total volume of 40 ml of culture medium was prepared by mixing 1 part basal medium 1 and 0.3 parts feed medium 2, reflecting the representative ratios of culture media used in cell culture conditions (Table 5). A mixture of medium 1 and medium 2 has previously been shown to increase the color intensity of antibody-containing solutions when used to culture antibody-producing cells. The mixture of medium 1 and medium 2 was supplemented with one of 30 antioxidant compounds and spiked with 2 g/L IgG1 monoclonal antibody. The samples were cultured at 37°C with shaking at 250 rpm for five days. Two control samples were included in the screening assay: 1) a 40 mL sample of a mixture of Medium 1 and Medium 2 containing 2 g/L IgG1 monoclonal antibody, incubated at 37°C with shaking at 250 rpm for 5 days in the absence of antioxidants (positive control); and 2) a 40 mL sample of a medium mixture prepared by mixing 1 part Basal Medium 3 with 0.3 parts Feed Medium 4 (Table 5) (this mixture has previously been shown to reduce the color intensity of antibody-containing solutions when used to culture antibody-producing cells), spiked with 2 g/L IgG1 monoclonal antibody, and incubated at 37°C with shaking at 250 rpm for 5 days in the absence of antioxidants (negative control).

Table 4. Representative compounds screened based on color reduction

1X test concentration represents the final concentration in culture medium

After incubation, the monoclonal antibodies were purified using affinity chromatography. The color intensity of the concentrated antibody compositions in the purification pool was determined using an assay in which a higher numerical value represents a higher color intensity and a lower numerical value represents a lower color intensity. The numerical results were standardized relative to a positive control, where the value of the positive control was set to a color intensity change of 0%. Among the 30 antioxidant compounds tested, several compounds, such as gentisic acid, cysteamine, hydrocortisone, and mercaptopropionylglycine, were found to increase the color of the antibody compositions (Figure 1). In comparison, six compounds, such as hypotaurine, anserine, butylated hydroxyanisole, carnosine, lipoic acid, and quercetin hydrate, were found to reduce the color of the antibody compositions (Figure 2). Among the antioxidants that reduced color intensity, hypotaurine showed the greatest effect, reducing the color intensity of the composition containing the antibody by approximately 25%. Taurine, an analog of hypotaurine, also reduced color intensity by approximately 5%.

Table 5. Representative components in the tested media compositions

^a Iron source is ferrous sulfate

^b Iron source is ferric citrate

Example 2: Characterization of antioxidant compounds capable of reducing the color intensity of antibody compositions isolated from antibody-producing cell lines

The ability of hypotaurine to reduce the color intensity of antibody-containing compositions obtained directly from cell culture was evaluated. For these studies, a shake flask cell culture model was utilized, which was found to be representative of larger-scale 2L cell cultures. Briefly, for the shake flask cell culture model, the CHO cells producing the antibodies were seeded with approximately 1.0 × ¹⁰ cells/mL into a 250mL flask containing 100mL basal medium 1 or basal medium 3. For larger-scale 2L cell cultures, the CHO cells producing the antibodies were seeded with approximately 1.0 × ¹⁰ cells/mL into a 2-liter stirred bioreactor (Applikon, Foster City, CA) containing 1L basal medium 1 or basal medium 3. For larger-scale cell growth models, cells were cultured in a fed-batch mode, wherein on days 3, 6, and 9, 100mL of feed medium 2 was added to each liter of cell culture fluid if cultured in basal medium 1, or 100mL of feed medium 4 was added if cultured in basal medium 3 to initiate the production phase. For the shake flask cell culture model, cells were cultured in a fed-batch mode, where on days 3, 6, and 9, 10 mL of feed medium 2 was added per liter of cell culture if cultured in basal medium 1, or 10 mL of feed medium 4 was added if cultured in basal medium 3, to initiate the production phase. Glucose concentration was analyzed daily, and if the glucose concentration dropped below 3 g/L, glucose was replenished from a 500 g/L glucose stock solution to prevent depletion. The reactor was equipped with calibrated dissolved oxygen, pH, and temperature probes. Dissolved oxygen was controlled online by sparging air and/or oxygen. For larger-scale 2 L cell cultures, pH was controlled by adding _CO₂ or _Na₂CO₃ , and antifoaming agents were added to the culture as needed. From day ₀ to day 3, the cell culture was maintained at pH 7.0 and a temperature of 37°C, and then at 35°C after day 3. The cell culture was agitated at 275 rpm and the dissolved oxygen level was maintained at 30% of air saturation. For shake flask cell culture, in 5% _CO2 incubator, culture is placed on a shaker platform and stirred at 150rpm, and the temperature is 37 ℃ from day 0 to day 3 of the cell culture cycle and is converted to 35 ℃ on day 4 until the end of the 14-day cell culture cycle. The osmotic pressure is monitored using an osmometer from Advanced Instruments (Norwood, mA). Nova Bioprofile 400 (Nova Biomedical, Waltham, mA) is also used to measure offline pH and metabolite concentration every day. An automatic cell counter (Beckman Coulter, Fullerton, CA) is used to measure viable cell density (VCC) and cell viability every day. 1mL of cell culture fluid is centrifuged every day to collect cell culture fluid for use in high performance liquid chromatography to determine antibody titers. At the end of the 14th day cell culture duration, the cell culture fluid of all samples is collected by centrifugation. The monoclonal antibodies in the collected cell culture fluid are purified using affinity chromatography. The color intensity of the antibody composition of concentration was measured in the purification pool using the assay method of the higher color intensity and the lower color intensity of the higher numerical value. No matter how the culture medium is used, between larger-scale (2 liters) and shake flask (SF) cell culture model, the growth (Fig. 3 A) and cell viability (Fig. 3 B) measured by VCC are suitable. Antibody production is slightly lower in shake flask cell culture model, and the highest antibody production (Fig. 3 C) is observed in the larger-scale cell culture model hatched in culture medium 1 and culture medium 2. Compared to the antibody composition (with a value of 2.25) obtained from shake flask cell culture composition when cultivated in culture medium 1 and culture medium 2, the color intensity of the antibody composition obtained from shake flask cell culture model when cultivated in culture medium 3 and culture medium 4 is lower, and its value is 1.07. These experiments have established that shake flask model is suitable with 2L cell culture model and is applicable to subsequent experiments.

For experiments with cell culture medium compositions supplemented with the antioxidant hypotaurine, antibody-producing CHO cells were seeded at approximately 1.0×10 ⁶ cells/mL into 250 mL flasks containing 100 mL of basal medium 1. Medium 1 was supplemented with 9.16 mM (100%), 4.58 mM (50%), or 2.29 mM (25%) hypotaurine for cell culture on day 0. Cells were cultured in a fed-batch mode, with 10 mL of feed medium 2 added to each liter of cell culture fluid on days 3, 6, and 9 to initiate the production phase. Additional experimental samples involved the incremental addition of 9.16 mM hypotaurine during cell culture. Specifically, 2.29 mM (25%) hypotaurine was added to basal medium 1 on day 0 of cell culture, and 25% hypotaurine was added to feed medium 2 on days 3, 6, and 9. By culturing cells in medium 1 and 2 without hypotaurine supplementation, including positive controls. By culturing cells in medium 3 and 4 without hypotaurine supplementation, including negative controls. As mentioned above, the concentration of glucose was analyzed every day. If the glucose concentration dropped below 3g/L, glucose was supplemented from a 500g/L glucose stock solution to prevent it from being exhausted. From day 0 to day 3, the cell culture was maintained at a pH of 7.0 and a temperature of 37°C, and then maintained at 35°C after day 3. The cell culture was stirred at 275rpm and the dissolved oxygen level was 30% of air saturation. VCC and cell viability were measured daily using an automated cell counter (Beckman Coulter, Fullerton, CA). 1mL of cell culture fluid was centrifuged every day to collect the cell culture fluid for use in high performance liquid chromatography to determine antibody titers. At the end of the 14th day cell culture duration, the cell culture fluid of all samples was collected by centrifugation. The monoclonal antibodies in the collected cell culture fluid were purified using affinity chromatography. The color intensity of the concentrated antibody compositions was measured in the purification pool using an assay in which a higher numerical value represents a higher color intensity and a lower numerical value represents a lower color intensity. Numerical results were standardized relative to the positive control, where the value of the positive control was set to a color intensity change of 0%. In all cell cultures tested, the growth (Fig. 4A) and cell viability (Fig. 4B) measured by VCC were comparable. In addition, except for the incremental addition of hypotaurine, the cell cultures cultured in the culture medium supplemented with hypotaurine produced the same level of antibody titer (Fig. 4C) as the cell cultures cultured in the culture medium not containing hypotaurine. Utilizing higher concentrations of hypotaurine, it was found that color intensity decreased, with the greatest decrease observed in the culture medium containing 9.16 mM hypotaurine (Fig. 5). This decrease in color intensity was best when hypotaurine was added in a bolus manner on the 1st day rather than when it was added incrementally during the cell culture incubation process. Comparison of the color intensity values obtained from the cell culture experiments and the incubation experiments (see Example 1) demonstrated that the results of the incubation screening experiments (Figure 5, open circles) correlated well with the results from the cell culture experiments (Figure 5, filled circles).

Similar experiments were performed on antibody compositions isolated from cell cultures harvested in basal medium 3 and feed medium 4 to determine whether the color reduction effect of hypotaurine extends to other cell culture media. Briefly, as described above, antibody-producing CHO cells were seeded at approximately 1.0 × ¹⁰ cells/mL into a 250 mL flask containing 100 mL basal medium 3. Medium 3 was supplemented with 12.95 mM (1X), 25.9 mM (2X), or 38.85 mM (3X) hypotaurine for cell culture on day 0. Cells were cultured in a fed-batch mode, with 10 mL of feed medium 4 added to each liter of cell culture fluid on days 3, 6, and 9 for the initial production phase. Positive controls were included by culturing cells in medium 1 and 2 without hypotaurine supplementation. The cultures were placed on a shaking platform and stirred at 150 rpm in a 5% _CO2 incubator with a temperature of 37°C from day 0 to day 3 of the cell culture cycle, and on day 4 the temperature was shifted to 35°C until the end of the cell culture cycle on day 14. Osmolality, offline pH, and metabolite concentrations were measured as described above. VCC and cell viability were measured daily using an automated cell counter (Beckman Coulter, Fullerton, CA). 1 mL of cell culture fluid was centrifuged daily to collect the cell culture fluid for use in high performance liquid chromatography to determine antibody titers. At the end of the cell culture period on day 14, the cell culture fluid of all samples was collected by centrifugation. The monoclonal antibodies in the collected cell culture fluid were purified using affinity chromatography. The color intensity of the concentrated antibody composition was measured in the purification pool using an assay in which a higher numerical value represents a higher color intensity and a lower numerical value represents a lower color intensity. The numerical results were standardized relative to the positive control, in which the value of the positive control was set to a color intensity change of 0%. Higher hypotaurine concentrations were found to reduce color intensity, with the greatest reduction observed in the medium containing 38.85 mM hypotaurine ( FIG. 6 ).

Example 3: Characterization of Hypotaurine Analogs for the Reduction of Color of Antibody Compositions Isolated from Antibody-Producing Cell Lines.

Taurine analogs were tested to assess whether they exhibited a color-reducing effect on antibody-containing compositions. Antibody-producing CHO cells were inoculated at approximately 1.0×10 ⁶ cells/mL into a 2-liter stirred bioreactor (Applikon, Foster City, CA) containing 1 L of basal medium 1 supplemented with 12.95 mM hypotaurine or 10 mM hydroxymethylcysteine (CAS No. 638-23-3). The cells were cultured in a fed-batch mode, with 100 mL of feed medium 2 added to each liter of cell culture fluid on days 3, 6, and 9 for the initial production phase. A positive control was included by culturing cells in medium 1 and 2 without hypotaurine supplementation. Glucose concentrations were analyzed daily and, if the glucose concentration dropped below 2 g/L, glucose was supplemented from a 1.5 g/L glucose stock solution to prevent it from being depleted. The reactor was equipped with calibrated dissolved oxygen, pH, and temperature probes. Dissolved oxygen was controlled online by bubbling air and/or oxygen. pH is controlled by adding CO ₂ or Na ₂ CO ₃ and defoamer is added to the culture as needed. From the 0th to the 3rd day, the cell culture was maintained at a temperature of pH 7.0 and 37 ℃, and then maintained at 35 ℃ after the 3rd day. The cell culture was stirred at 275rpm and the dissolved oxygen level was 30% of air saturation. The osmometer from Advanced Instruments (Norwood, mA) was used to monitor osmotic pressure. Nova Bioprofile 400 (Nova Biomedical, Waltham, mA) was also used to measure offline pH value and metabolite concentration every day. Automatic cell counter (Beckman Coulter, Fullerton, CA) was used to measure VCC and cell viability every day. The cell culture fluid of 1mL was centrifuged every day to collect the cell culture fluid for use in high performance liquid chromatography to determine antibody titer. At the end of the 14th day cell culture duration, when the amount of protein in the culture was about 2-10g/L, the cell culture fluid of all samples was collected by centrifugation. Use protein A affinity chromatography to purify the monoclonal antibody in the collected cell culture fluid.Use Amicon Centricon centrifugal filter unit (Millipore Corporation, Billerica, mA) that protein A pond is concentrated to 150g/L.Use two different determination methods that wherein higher numerical value represents higher color intensity and lower numerical value represents lower color intensity, the color intensity of concentrated antibody composition is measured in concentrated protein A pond.In all cell cultures tested, the growth (Fig. 7 A) and cell viability (Fig. 7 B) measured by VCC are suitable.The cell culture cultivated in the substratum supplemented with hypotaurine or carboxymethylcysteine produces the antibody titer (Fig. 8) of comparable level.Use specific color determination method, find that when the cell of producing antibody is cultivated in the substratum supplemented with hypotaurine and carboxymethylcysteine respectively, the color intensity of the antibody composition separated has reduced by 27% and 13% (Fig. 9 A) respectively. This reduction in color intensity was confirmed by using a secondary color assay, in which approximately 17% and 13% reductions in color intensity were detected for antibody compositions isolated from cells cultured in media supplemented with hypotaurine and carboxymethylcysteine, respectively ( FIG. 9B ).

Example 4: Characterization of the effect of aminoguanidine on the reduction of color of an antibody composition isolated from an antibody-producing cell line.

In order to identify the compound that reduces and works under cell culture conditions in antibody composition, screening test is carried out in cell-free culture medium. Taurine, carnosine and aminoguanidine are selected for screening. These compounds are dissolved in the concentration of 1.2g/L (taurine), 13.6g/L (carnosine) and 27.2g/L (aminoguanidine hydrochloride) in 25mL culture medium. After adjusting pH to the scope of 6.8 to 7.2 and sterilizing filtration (Millipore, Billerica, mA) with Steriflip filter element, solution is hatched in the Falcon tube (BD Biosciences, San Jose, CA) of 50mL equipment TubeSpin cover (TPP Techno Plastic Products AG, Trasadingen, Switzerland). At 37 ℃ and 250rpm, not lucifuge, Chinese hamster ovary celI cells are cultivated 7 days in the cell culture incubator of control humidity, to produce monoclonal antibody.

The monoclonal antibodies in the harvested cell culture fluid (HCCF) and incubation broth were further purified by affinity chromatography. The color intensity of the concentrated antibody composition was determined in the purified pool using an assay where higher values represent higher color intensity and lower values represent lower color intensity.

The relative color intensity of antibodies produced in medium containing taurine, carnosine, or aminoguanidine is shown in Figure 10. The data show that aminoguanidine can reduce the color by approximately 71%, and the relative color intensity value is lower than the negative control in which the antibody was incubated without any glucose.

Claims (24)

1. A method for producing a recombinant polypeptide, comprising the step of culturing cells containing nucleic acids encoding said polypeptide in a cell culture medium, wherein said cell culture medium comprises one or more of components (a)-(d): (a) Taurine; (b) S-carboxymethylcysteine; (c) Butylated hydroxyanisole; and (d) Quercetin hydrate; Furthermore, compared to a composition containing a polypeptide produced by cells cultured in a cell culture medium that does not contain one or more of said components (a)-(d), the cell culture medium containing one or more of said components (a)-(d) reduces the color intensity of the composition containing the polypeptide produced by the cells. 2. The method of claim 1, wherein the cell culture medium containing one or more of components (a)-(d) reduces the color intensity of the composition containing the polypeptides produced by cells cultured in a cell culture medium that does not contain one or more of said components (a)-(d) by at least 0.1%. 3. The method of claim 1, wherein, compared to a composition of polypeptides produced by cells cultured in a cell culture medium containing one or more of said components (a)-(d), the cell culture medium containing one or more of said components (a)-(d) reduces the color intensity of the composition of polypeptides produced by the cells by 5% to 50%. 4. The method of any one of claims 1-3, wherein the cell culture medium comprises one or more of components (a)-(d) in an amount selected from: (a) Taurine at a concentration of at least 0.0001 mM; (b) at a concentration of at least 0.0001 mM of s-carboxymethylcysteine; (c) Butylated hydroxyanisole at a concentration of at least 0.0001 mM; and (d) Quercetin hydrate at a concentration of at least 0.0001 mM. 5. The method of claim 4, wherein the cell culture medium contains taurine at a concentration of 2.0 mM to 50.0 mM. 6. The method of claim 4, wherein the cell culture medium contains 8.0 mM to 12.0 mM of s-carboxymethylcysteine. 7. The method of claim 4, wherein the cell culture medium comprises butylated hydroxyanisole at a concentration of 0.025 mM to 0.040 mM. 8. The method of claim 4, wherein the cell culture medium comprises quercetin hydrate at a concentration of 0.010 mM to 0.020 mM. 9. The method of claim 1, wherein the cell culture medium is a chemically determined cell culture medium. 10. The method of claim 1, wherein the cell culture medium is a chemically uncertain cell culture medium. 11. The method of claim 1, wherein the cell culture medium is a basic cell culture medium. 12. The method of claim 1, wherein the cell culture medium is a fed cell culture medium. 13. The method of claim 1, wherein the cells are in contact with the cell culture medium during the cell growth phase. 14. The method of claim 1, wherein the cells are contacted with the cell culture medium during cell production. 15. The method of claim 1, wherein one or more of said components (a)-(d) are added to the cell culture medium at least one day during the cell culture cycle. 16. The method of claim 1, wherein one or more of said components (a)-(d) are added to the cell culture medium on day 0 of a 14-day cell culture cycle. 17. The method of claim 1, wherein the cell is a mammalian cell. 18. The method of claim 17, wherein the mammalian cells are Chinese hamster ovary (CHO) cells. 19. The method of claim 1, wherein the polypeptide is an antibody or a fragment thereof. 20. The method of claim 19, wherein the antibody is an IgG1 antibody. 21. The method of claim 19 or 20, wherein the antibody is secreted into a cell culture medium. 22. The method of claim 1, further comprising the step of recovering the polypeptide from a cell culture medium containing one or more of said components (a)-(d). 23. The method of claim 22, wherein the composition comprising the recovered polypeptide is a liquid composition or a non-liquid composition. 24. The method of claim 23, wherein the composition comprising the recovered polypeptide is a colorless or slightly colored liquid.