HK40076465B - Method for producing less colored composition containing polypeptide - Google Patents

Description

Method for preparing compositions containing peptides that inhibit staining

Technical Field

This invention provides a method for preparing a composition containing a polypeptide that inhibits staining.

Background Technology

The method of preparing peptides in vitro using recombinant cell cultures is well-known and widely used in industrial-scale production. In the formulation of compositions containing peptides, such as antibodies, and for provision as pharmaceutical preparations, a quality characteristic requires that the color of the formulation be suppressed to an acceptable level, for example, to meet the regulatory requirements for marketability of the product.

Especially in formulations containing antibodies at high concentrations (e.g., above 150 mg/mL), it becomes important to suppress the color of the formulation to an acceptable level due to the increased coloring associated with concentration.

The reason for the coloring of antibody-containing preparations has been reported as the adsorption of antibodies by vitamin _B12 (hereinafter referred to as "VB12") (Non-Patent Literature 1-3, Patent Literature 1). One molecule of cobalt coordinates with one molecule of VB12. VB12 contains cyanocobalamin and hydroxycobalamin ions, and it has been reported that the molecule is colored pink or red by binding to the antibody through the hydroxycobalamin ions (Non-Patent Literature 1-3).

Furthermore, as a culture method aimed at preventing antibody staining, a culture method using a culture medium containing specific amounts of vitamins B2, B6, B9, B12, and cystine has been reported (Patent Document 2). Additionally, a method for preventing staining by preventing the conversion of cyanocobalamin bodies in the culture medium to hydroxycobalamin and the reduction of disulfide bonds in antibodies has been reported (Patent Document 1).

On the other hand, it was reported that VB12 is an essential component of cell culture medium for eukaryotic cells (Non-Patent Document 1) and an essential component of culture for mammalian cells (Patent Document 1).

Existing technical documents

Patent documents

Patent Document 1: WO2018/208743

Patent Document 2: Patent No. 2015-515281

Non-patent literature

Non-Patent Literature 1: MAbs. 2013 Nov 1; 5(6): 974-981

Non-Patent Literature 2: MAbs. 2014 May 1; 6(3): 679-688

Non-patent literature 3: Biotechnology and Bioengineering. 2018; 115: 900-909

Invention Overview

The problem that the invention aims to solve

The objective of this invention is to provide a method that, for polypeptides that are prone to staining, maintains the amount and physical properties of the polypeptides within acceptable limits by modifying a relatively simple preparation method, while simultaneously solving the staining problem.

Methods for solving problems

In order to achieve the above-mentioned problem, the inventors conducted in-depth research and found that by using a culture medium without VB12, the amount of peptide production can be maintained while inhibiting staining, thus completing the present invention.

Therefore, the present invention can be embodied as follows (1) to (17).

(1) A method for preparing a composition containing a polypeptide that inhibits staining, comprising: (a) culturing eukaryotic cells containing nucleic acids encoding polypeptides in a cell culture medium free of vitamin B12; and (b) recovering the composition containing polypeptides from the culture.

The method for preparing the composition described in (2)(1) wherein, in the aforementioned composition, the molar concentration ratio of vitamin B12 to polypeptide is less than 0.26%.

The method for preparing the composition described in (3)(1) or (2) wherein fed-batch culture is performed using an initial culture medium without vitamin B12 and a fed-batch culture medium without vitamin B12.

The method for preparing the composition described in (4)(3) wherein the VCD (viable cell density) in the cell culture medium on the 7th day from the start of fed batch culture is 80×10 ⁵ cells/mL or more.

The method for preparing the composition described in any one of (5)(1) to (4), wherein the eukaryotic cell is a CHO cell.

The method for preparing the composition described in any one of (6)(1) to (5), wherein the aforementioned polypeptide is a polypeptide containing Fc.

The preparation method of the composition described in (7)(6) wherein the aforementioned Fc-containing polypeptide is an antibody.

The method for preparing the composition described in (8), (6) or (7), wherein the aforementioned polypeptide comprises a change of at least one amino acid residue selected from the amino acid residues at positions 214, 234, 238, 250, 264, 307, 311, 330, 343, 428, 434, 436, 438 and 440 of the Fc region according to EU numbers.

The preparation method of the composition described in (9)(6) to (8), wherein the aforementioned polypeptide comprises at least one variation selected from the Fc region according to EU numbers 214R, 234Y, 238D, 250V, 264I, 307P, 311R, 330K, 343R, 428L, 434A, 436T, 438R and 440E.

The preparation method of the composition described in (10)(6) to (9) wherein the aforementioned polypeptide is an antibody containing a heavy chain constant region containing an amino acid sequence of sequence number 1 and a light chain constant region containing an amino acid sequence of sequence number 2.

The method for preparing the composition described in (11)(10) wherein the aforementioned antibody is an IgG1 humanized antibody that binds to latent myostatin.

(12) A method for preparing a composition containing an antibody that inhibits staining, comprising:

a) The step of confirming that the target antibody is an antibody containing a change of at least one amino acid residue selected from the Fc region at positions 214, 234, 238, 250, 264, 307, 311, 330, 343, 428, 434, 436, 438 and 440 according to EU numbers;

b) For antibodies containing the altered amino acid residues, the step of selecting a cell culture medium without vitamin B12;

c) The step of culturing eukaryotic cells containing nucleic acids encoding antibodies with altered amino acid residues using the cell culture medium selected in step b); and

d) The step of recovering the antibody-containing composition from the culture.

The preparation method described in (13)(12) is wherein step b) is the step of selecting an initial culture medium without vitamin B12 and a fed batch culture medium without vitamin B12.

The preparation method described in (14), (12) or (13), wherein the eukaryotic cells mentioned above are CHO cells.

(15) A method for preparing an antibody-containing formulation, comprising: (b) culturing eukaryotic cells containing nucleic acids encoding antibodies in a cell culture medium free of vitamin B12; (c) recovering the antibody-containing composition from the culture; and (d) preparing the resulting composition into a pharmaceutical formulation.

(16) A method for inhibiting antibody staining, comprising: in the preparation of an antibody using a recombinant cell culture, (a) culturing eukaryotic cells containing nucleic acids encoding the antibody in a cell culture medium free of vitamin B12; and (b) recovering the composition containing the antibody from the culture.

The preparation method of the composition described in (17) and (7), the preparation method of the antibody-containing preparation described in (15), or the method of inhibiting antibody staining described in (16), wherein the aforementioned antibody is an anti-IL-8 antibody or an anti-myosin antibody.

Invention Effects

According to the present invention, a method for preparing a composition containing a polypeptide that inhibits staining is provided.

sequence list

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Brief description of the attached diagram

[Figure 1] Shows the number of viable cells as determined by a Vi-CELL system (Beckman Courter, model Vi-CELL XR) for each day of a 14-day culture period in cells that produce the polypeptide (antibody A: antimystifier antibody), with VB+ defined as the medium containing VB12 in the initial culture medium and fed batch culture medium, and VB- defined as the medium without VB12 in the initial culture medium and fed batch culture medium.

[Figure 2] shows the cell viability measured by the Vi-CELL system (Beckman Courter, model Vi-CELL XR) for each day of the 14-day culture period in the culture of cells that produce antibody A, with VB+ as the medium containing VB12 in the initial medium and fed batch medium and VB- as the medium without VB12 in the initial medium and fed batch medium.

[Figure 3] shows that in the culture of cells that produce antibody A, the medium containing VB12 in the initial medium and fed batch medium is VB+, and the medium without VB12 in the initial medium and fed batch medium is VB-. For each day during the 14-day culture period, the cumulative cell count was calculated based on the number of viable cells measured by the Vi-CELL system (Beckman Courter, model Vi-CELL XR) of the automated cell metering device.

[Figure 4] shows the cell proliferation behavior on each day during the culture of cells producing antibody A, with VB12-containing medium (VB+) and VB12-free medium (VB-), and during a passage period of 3 days or 4 days up to 100 days (28 passages), plotted using cell doubling time as the indicator. (Cell doubling time was measured using the Vi-CELL automated cell metering system (Beckman Courte).)

[Figure 5] Shows the number of viable cells as determined by a Vi-CELL automated cell metering system (Beckman Courter, model Vi-CELL XR) for each day of a 14-day culture period in a culture of cells producing a different polypeptide (antibody B: anti-IL-8 antibody) than in Figure 1, with VB+ defined as the medium containing VB12 in the initial culture medium and fed batch culture medium, and VB- defined as the medium without VB12 in the initial culture medium and fed batch culture medium.

[Figure 6] shows the number of viable cells as determined by a Vi-CELL automated cell metering system (Beckman Courter, model Vi-CELL XR) for each day of a 14-day culture period in cells that produce a polypeptide (antibody C: anti-FIXa/FX bispecific antibody) different from those in Figures 1 and 5, with VB+ defined as the medium containing VB12 in the initial culture medium and fed batch culture medium, and VB- defined as the medium without VB12 in the initial culture medium and fed batch culture medium.

[Figure 7] shows the number of viable cells measured by a Vi-CELL automated cell metering system (Beckman Courter, model Vi-CELL XR) for each day of the 14-day culture period in the culture of cells that produce antibody B, with VB+ being the medium containing VB12 in the initial medium and the fed batch medium, and VB- being the medium without VB12 in the initial medium but containing VB12 in the fed batch medium.

[Figure 8] shows the number of viable cells measured by a Vi-CELL automated cell metering system (Beckman Courter, model Vi-CELL XR) for each day of the 14-day culture period in the culture of cells that produce antibody C, with VB+ being the medium containing VB12 in the initial medium and the fed batch medium, and VB- being the medium without VB12 in the initial medium but containing VB12 in the fed batch medium.

[Figure 9] shows the cell viability measured by a Vi-CELL automated cell metering system (Beckman Courter, model Vi-CELL XR) for each day of a 14-day culture period in the culture of cells that produce antibody B, with VB+ defined as the medium containing VB12 in the initial medium and fed batch medium, and VB- defined as the medium without VB12 in the initial medium and fed batch medium.

[Figure 10] shows the cell viability measured by the Vi-CELL system (Beckman Courter, model Vi-CELL XR) for each day of the 14-day culture period in the culture of cells that produce antibody C, with VB+ as the medium containing VB12 in the initial medium and fed batch medium, and VB- as the medium without VB12 in the initial medium and fed batch medium.

[Figure 11] shows the cell viability measured by the Vi-CELL system (Beckman Courter, model Vi-CELL XR) for each day of the 14-day culture period in the culture of cells that produce antibody B, with VB+ as the medium containing VB12 in the initial medium and fed batch medium, and VB- as the medium without VB12 in the initial medium but containing VB12 in the fed batch medium.

[Figure 12] shows the cell viability measured by the Vi-CELL automated cell metering system (Beckman Courter, model Vi-CELL XR) for each day of the 14-day culture period in the culture of cells that produce antibody C, with VB+ as the medium containing VB12 in the initial medium and fed batch medium, and VB- as the medium without VB12 in the initial medium but containing VB12 in the fed batch medium.

[Figure 13] shows that in the culture of cells that produce antibody B, the medium containing VB12 in the initial medium and fed batch medium is VB+, and the medium without VB12 in the initial medium and fed batch medium is VB-. For each day during the 14-day culture period, the cumulative cell count is calculated based on the number of viable cells measured by the Vi-CELL system (Beckman Courter, model Vi-CELL XR) of the automated cell metering device.

[Figure 14] shows that in the culture of cells that produce antibody C, the medium containing VB12 in the initial medium and fed batch medium is VB+, and the medium without VB12 in the initial medium and fed batch medium is VB-. For each day during the 14-day culture period, the cumulative cell count is calculated based on the number of viable cells measured by the Vi-CELL system (Beckman Courter, model Vi-CELL XR) automated cell metering device.

[Figure 15] shows that in the culture of cells that produce antibody B, the medium containing VB12 in the initial medium and the fed batch medium is VB+, and the medium that does not contain VB12 in the initial medium but contains VB12 in the fed batch medium is VB-. For each day during the 14-day culture period, the cumulative cell count is calculated based on the number of viable cells measured by the Vi-CELL automated cell metering system (Beckman Courter, model Vi-CELL XR).

[Figure 16] shows that in the culture of cells that produce antibody C, the medium containing VB12 in the initial medium and the fed batch medium is VB+, and the medium that does not contain VB12 in the initial medium but contains VB12 in the fed batch medium is VB-. For each day during the 14-day culture period, the cumulative cell count is calculated based on the number of viable cells measured by the Vi-CELL automated cell metering system (Beckman Courter, model Vi-CELL XR).

[Figure 17] Shows the cell proliferation behavior on each day of culture for antibody B production, using VB12-containing medium (VB+) and VB12-free medium (VB-), with passages performed in 3-day or 4-day cycles up to 136 days (39 passages), and plotted using cell doubling time as the indicator. (Cell doubling time was measured using the Vi-CELL automated cell metering system (Beckman Courte).)

[Figure 18] shows the cell proliferation behavior on each day during the culture of cells producing antibody C, with VB12-containing medium (VB+) and VB12-free medium (VB-), and passaged in 3-day or 4-day cycles up to 24 days (7 passages), using cell doubling time as the indicator. (Cell doubling time was measured using the Vi-CELL automated cell metering system (Beckman Courte).)

[Figure 19] shows the results of antibody removal using a 5KD cutoff filter and analysis of recovered samples using reversed-phase chromatography (Column: Acclaim ^™ PolarAdvantage II LC) for both potassium cyanide-treated and untreated antibody-containing compositions. Untreated samples are indicated as KCN-, treated samples as KCN+, and the VB12 peak is indicated as STD.

Methods of implementing the invention

The embodiments of the present invention will now be described in detail.

This invention relates to a method for preparing a composition containing a polypeptide that inhibits staining.

VB12

In a narrow sense, VB12 refers to cyanocobalamin, while in a broad sense, it refers to cobalamin, which is a general term for vitamin _B12 . VB12 as a component of culture media is cyanocobalamin. When the components of the culture media are in aqueous solution, they are in a state of equilibrium of cyanocobalamin, hydroxycobalamin, hydrocobalamin, adenosylcobalamin, and methylcobalamin.

In VB12, each molecule binds to one molecule of cobalt. Therefore, in the embodiments of this application, the relative concentration of VB12 relative to the antibody is quantified by quantifying the relative concentration of cobalt molecules relative to the antibody. In this invention, for the culture of eukaryotic cells, initial culture medium and fed-batch culture medium without VB12 can be used. The "VB12-free" culture medium in this invention includes not only the case where the concentration of VB12 in the culture medium is 0 mg/L, but also the case where the culture medium contains VB12 at a level lower than the actual content level as a culture medium additive. Specifically, in the case where the antibody-containing composition (antibody concentration of about 30 mg/mL) is obtained by culturing using this culture medium under the same conditions as in Example 1 of this invention, and the concentration of cobalt molecules contained in VB12 is below the quantitative limit of 20 ppb, it is equivalent to "VB12-free".

eukaryotic cells

Eukaryotic cells refer to cells having a nucleus surrounded by a nuclear membrane. In this invention, "eukaryotic cells containing nucleic acids encoding polypeptides" are host cells that can be used as production systems for the preparation of desired polypeptides. Such cells can be natural cells capable of producing the desired polypeptide, or cells with nucleic acids encoding (or expressing) the desired polypeptide artificially introduced, preferably transformed cells with nucleic acids encoding the desired polypeptide introduced. An example of such transformed cells is a polypeptide-producing strain obtained by introducing exogenous DNA encoding the desired polypeptide into eukaryotic cells using gene recombination technology. Therefore, the term "containing nucleic acids encoding polypeptides" can also be read alternatively as "nucleic acids artificially introduced to encode polypeptides" or "exogenous nucleic acids encoding polypeptides introduced."

Typical examples of eukaryotic cells in this invention are cells suitable as host cells for the production of recombinant proteins, and may be selected from cells derived from insects, pygmy mammals, amphibians, reptiles, or mammals. Preferred examples of eukaryotic cells in this invention are mammalian cells. Mammalian cells are selected from CHO cells, NSO cells, Sp2/O cells, COS cells, HEK cells, BHK cells, PER.C6 ^{(registered trademark)} cells, and hybridoma cells, with CHO cells being more preferred.

Cell culture medium

In this invention, cell culture medium refers to the culture medium used for culturing cells containing nucleic acids encoding polypeptides, and may also be simply referred to as culture medium. Commercially available or known culture media can be used as cell culture media. In addition to the VB12-free culture medium used in this invention, conventional culture media contain VB12 as an essential component necessary for cellular transcriptional activity and nucleic acid synthesis. In embodiments of this invention, a culture medium that maintains the existing composition of the culture medium is used, while excluding, removing, or reducing the VB12 component.

Those skilled in the art can appropriately select the type of culture medium suitable for a specific cell line. For example, well-known or commercially available culture media for animal cell culture may include IMDM (Iscove's Modified Dulbecco Medium), DMEM (Dulbecco's Modified Eagle Medium), Ham's F12 medium, D-MEM/F-12 1:1 Mixture (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), RPMI1640, CHO-S-SFMII (Invitrogen), CHO-SF (Sigma-Aldrich), EX-CELL 301 (JRH Biosciences), CD-CHO (Invitrogen), IS CHO-V (Irvine Scientific), PF-ACF-CHO (Sigma-Aldrich), etc., but are not limited to these, as will be understood by those skilled in the art. Most culture media optimized for cell proliferation and improved yields of antibody and recombinant protein production are well known to those skilled in the art.

For example, examples of culture media that can be used to culture CHO cell lines that produce humanized antibodies (IgG1 antibodies) that bind to the latent myosin used in the examples include F12 medium of IMDM, DMEM or Ham, or combinations thereof.

Cell culture

Cell culture methods are generally classified as batch culture, continuous culture, and fed-batch culture.

Batch culture is a method of cell proliferation in which a small amount of seed culture medium is added to the culture medium without adding new culture medium or draining the culture medium.

The continuous culture method involves continuously adding and draining the culture medium during cultivation. Additionally, the continuous culture method also includes perfusion culture.

Because fed-batch culture falls between batch and continuous culture methods, it is also called semi-batch culture. It involves adding culture medium continuously or sequentially during cultivation, without the continuous draining of culture medium as in continuous culture. The culture medium added during fed-batch culture (hereinafter referred to as the fed-batch medium) does not need to be the same as the culture medium already used in the initial culture (hereinafter referred to as the initial medium). Different media can be added, or only specific components can be added. Alternatively, the fed-batch medium can also be a culture medium with the same composition as the initial medium.

In this invention, any of the following cultivation methods can be applied: batch culture, continuous culture, or fed-batch culture, with fed-batch culture being preferred.

Typically, in the preparation of desired peptides by culturing cells, a seed culture medium containing the cells is added in a certain amount to the initial culture medium, and the cells are then cultured. Furthermore, to increase the yield of the desired peptide, fed-batch culture medium is added to the culture.

Seed medium refers to the medium used to expand the number of cells (working cell bank) necessary for the eventual transfer to the medium used to produce the desired polypeptide (initial medium). Additionally, initial medium generally refers to the medium used to culture cells to produce the desired polypeptide, i.e., the medium used in the initial stage of cell culture. Fed-batch medium generally refers to the medium added to the initial culture medium. Fed-batch medium is sometimes added in multiple installments. Furthermore, fed-batch medium can be added continuously or intermittently.

In this invention, cells are passaged using a seed culture medium. Subsequently, a certain amount of the seed culture medium containing the cells is added to the initial culture medium, and the cells are cultured in the initial culture medium to produce the desired polypeptide. Further, depending on the situation, feed-batch culture medium is added to the culture medium at least once.

Fed-batch culture can be further classified according to the method of feeding. Isorate fed-batch culture is a culture method in which a certain amount of fed-batch culture medium is continuously added to the initial culture medium.

Recovery of peptides, compositions containing peptides, and compositions containing peptides.

The polypeptide used in this invention preferably contains an Fc-containing polypeptide that corresponds to the Fc region of an antibody, and more preferably an antibody.

A composition containing peptides (a composition containing peptides) means a composition containing peptides and other components. In embodiments of the invention, a composition containing peptides can be recovered from a culture obtained through a cell culture step for peptide production. "Recovery" as used herein refers to the recovery of a supernatant (culture supernatant) containing peptides from a culture or culture medium obtained through a cell culture step, or the recovery of a filtrate containing peptides using a filter.

The recovered solution can be adjusted to a composition containing appropriate concentrations of peptides through purification and concentration steps such as affinity column chromatography.

In cases where the polypeptide is an Fc-containing polypeptide or an antibody, the composition containing the polypeptide (the composition containing the polypeptide) can be read alternatively as a composition containing an Fc-containing polypeptide (the composition containing an Fc-containing polypeptide) or a composition containing an antibody (the composition containing an antibody).

Fc area (Fc)

The term Fc region (or simply Fc) encompasses both native Fc regions and variant Fc regions. The boundaries of the Fc region of the immunoglobulin heavy chain are variable, referring to the region composed of the hinge portion or a portion thereof in the antibody molecule, and the CH2 and CH3 domains. The Fc region is not limited to any antibody (IgA, IgD, IgE, IgG, IgM), particularly the Fc region of IgG, but is preferably the Fc region of human IgG (IgG1, IgG2, IgG3, IgG4), and more preferably the Fc region of human IgG1.

Inhibition of coloring

The composition containing a stain-inhibiting polypeptide in this invention refers to a composition comprising a polypeptide that reduces staining caused by VB12 adsorption, specifically a red or pink polypeptide. In embodiments of this invention, for a polypeptide (antibody) composition containing a polypeptide (antibody) with the polypeptide (antibody) concentration adjusted to approximately 30 mg/mL, a colorless evaluation was performed by visual inspection to confirm that staining was inhibited. Furthermore, for the composition containing a stain-inhibiting polypeptide (antibody), the molar ratio [%] of VB12 to polypeptide, quantified according to the conditions of Example 1, is less than 0.26, preferably less than 0.2, more preferably less than 0.1, and even more preferably less than 0.05.

Antibody identification, selection of cell culture medium

In one embodiment of the invention, antibodies that readily produce staining are pre-identified, and a culture step that inhibits staining, differing from the usual culture steps, is selected. In this embodiment, the first step involves confirming that the target polypeptide is a polypeptide containing at least one altered amino acid residue selected from amino acid residues at positions 214, 234, 238, 250, 264, 307, 311, 330, 343, 428, 434, 436, 438, and 440 in the Fc region (using EU numbers). "Confirmation" does not necessarily mean specifying the exact altered position of the polypeptide through analytical methods; it includes pre-identifying that the target polypeptide contains the alteration.

Subsequently, for antibodies confirmed to contain the altered amino acid residues, a different step than the usual culture procedure is selected. That is, while a cell culture medium containing VB12 as an essential component (normal cell culture medium) is used in the usual culture procedure, a cell culture medium without VB12 is used instead of the usual cell culture medium in the culture step for this antibody. Here, the cell culture medium without VB12 can be one whose composition is identical to that of a normal cell culture medium, except that it lacks VB12.

A composition containing a polypeptide is prepared by culturing cells containing nucleic acids encoding the polypeptide in a cell culture medium selected in this way, and by recovering the composition containing the polypeptide from the culture.

Example

The present invention will now be described in detail through specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention.

The following antibodies, cells, and culture media were used in the examples.

Antibody:

As antibody A, an IgG1 humanized antibody containing a heavy chain constant region with amino acid sequence number 1 and a light chain constant region with amino acid sequence number 2 is applied, namely an anti-myosin antibody that binds to latent myosin. This antibody contains alterations in the Fc region according to EU numbers 214R, 234Y, 238D, 250V, 264I, 307P, 311R, 330K, 343R, 428L, 434A, 436T, 438R, and 440E.

As antibody B, use the anti-IL-8 antibody that binds to IL-8 as described in WO/2016/125495 or WO/2017/046994.

As antibody C, the anti-FIX(a)/FX bispecific antibody that binds to both FIX(a) and FX, as described in WO2019065795, is used.

cell:

CHO cells were prepared using a CHO-DXB11 derivative.

Culture medium:

The initial culture medium and fed-batch culture medium used were culture media manufactured by Thermo Fisher Scientific, Fujifilm, and Koden Pharmaceuticals. In this embodiment, as the initial culture medium, an initial culture medium containing VB12 (100% relative VB12 concentration), an initial culture medium with VB12 completely removed and identical in composition except for VB12 (0% relative VB12 concentration), and an initial culture medium obtained by mixing these two media at a 1:1 ratio (50% relative VB12 concentration) were prepared. Furthermore, as the fed-batch culture medium, a fed-batch culture medium containing VB12 (100% relative VB12 concentration) and a fed-batch culture medium with VB12 completely removed and identical in composition except for VB12 (0% relative VB12 concentration) were prepared.

Example 1. Study on the effect of VB12 on antibody staining

Using cells that produce antibody A, samples 1–9 of the initial culture medium and fed-batch culture medium combinations shown in Table 1 were cultured under identical conditions using a 1L–25L culture apparatus via a constant-rate fed-batch method. The culture was carried out for 14 days at a pH range of 6.7–7.2 and a temperature range of 34℃–38℃, with the fed-batch culture medium added on day 3 after the start of culture.

The antibody-containing composition was recovered from the culture medium after 14 days of incubation, purified by affinity column chromatography with Protein A, and the eluent was concentrated and adjusted in samples 1–8 to an antibody concentration of approximately 30 mg/mL (26.7 mg/mL–31.4 mg/mL). Sample 9 was further concentrated to an antibody concentration of over 200 mg/mL.

The staining and VB12 content of the antibody-containing compositions obtained in this way were evaluated. Staining was assessed visually. Some staining (light red) was observed in samples 1, 5, and 7. Furthermore, staining (red) was observed in sample 9. On the other hand, no staining was observed in samples 2–4 and 6–8 (colorless).

Since each VB12 molecule in the antibody-containing composition binds to one cobalt molecule, the molar concentration ratio of cobalt to antibody (cobalt/antibody [%)) was calculated by determining the cobalt concentration in the antibody-containing composition using ICP-MS (Inductively Coupled Plasma-Mass Spectrometry). The content of VB12 (molar concentration ratio of VB12 to antibody (VB12/antibody [%))) was then estimated. These results are shown in Table 1.

[Table 1]

※The cobalt concentration [ppb], cobalt/antibody [%), and VB12/antibody [%) of samples 2-4, 6, and 8 are below the quantitation limit.

Experimental results confirmed that VB12 in the antibody-containing composition was the cause of staining. Furthermore, it was confirmed that inhibiting VB12 in the initial culture medium and fed-batch culture medium could suppress VB12 in the cultured antibody-containing composition.

Example 2. Study on the effect of VB12 in culture medium on cell culture

Cell culture was performed under the same conditions as in Example 1, and the effect of the presence or absence of VB12 in the culture medium on the number of viable cells, cell viability, and cumulative cell count was observed. The results confirmed that culture using a medium without VB12 (VB-) in the initial culture medium and fed-batch culture medium was almost identical in terms of the number of viable cells, cell viability, and cumulative cell count on each day compared to culture using a medium containing VB12 (VB+) in the initial culture medium and fed-batch culture medium. (Figures 1, 2, 3)

Using cells producing antibody B or antibody C, and with the initial culture medium and fed-batch culture medium prepared as shown in Table 1-1 for Sample 10 (VB+) and Sample 11 (VB-), respectively, 1L culture apparatus were used for isorate fed-batch culture under the same conditions. The culture was carried out for 14 days at a pH range of 6.7–7.2 and a temperature range of 34–38°C, with the fed-batch culture medium added on day 3 after the start of culture.

In cell culture under these conditions, the effects of the presence or absence of VB12 in the culture medium on the number of viable cells, cell viability, and cumulative cell count were observed. The results confirmed that culture using a medium without VB12 (VB-) in the initial culture medium and fed-batch culture medium was almost identical in terms of the number of viable cells, cell viability, and cumulative cell count on each day compared to culture using a medium containing VB12 (VB+) in the initial culture medium and fed-batch culture medium (Figures 5, 6, 9, 10, 13, 14).

Using cells producing antibody B or antibody C, and with the initial culture medium and fed-batch culture medium prepared as shown in Table 1-1 for Sample 10 (VB+) and Sample 12 (VB-), respectively, 1L culture apparatus were used for isorate feeding batch culture under the same conditions. The culture was carried out for 14 days at a pH range of 6.7–7.2 and a temperature range of 34–38°C, with the fed-batch culture medium added on day 3 after the start of culture.

In cell culture under these conditions, the effect of the presence or absence of VB12 in the culture medium on the number of viable cells, cell viability, and cumulative cell count was observed. The results confirmed that culture using an initial culture medium without VB12 followed by fed-batch culture medium containing VB12 (VB-) resulted in almost identical viable cell counts, cell viability, and cumulative cell counts on each day compared to culture using an initial culture medium followed by fed-batch culture medium containing VB12 (VB+) (Figures 7, 8, 11, 12, 15, 16).

[Table 1-1]

Example 3. Study on the effect of VB12 in culture medium on antibody production.

Cells were cultured under the same conditions as in Example 1 to observe the effect of the presence or absence of VB12 in the culture medium on antibody production. The results confirmed that the antibody production after 14 days of culture was almost equal between culture in a medium without VB12 (VB-) and culture in a medium containing VB12 (VB+) (Table 2: Antibody production in cultures with VB+ as the control is expressed as 100%).

Cells producing antibody B were also cultured under the conditions listed in Table 1-1 to observe the effect of the presence or absence of VB12 in the culture medium on antibody production. The results confirmed that the antibody production after 14 days of culture was almost equal to that of cultured under the conditions of Sample 10 (initial culture medium and fed-batch culture medium without VB12 (VB-) and Sample 12 (initial culture medium without VB12 and fed-batch culture medium containing VB12 (VB-)). (Tables 2-1 and 2-2: Antibody production in cultures with VB+ as a control is expressed as 100%).

[Table 2]

Production amount (%) Standard error (%) VB+ 100 1.36 VB- 104.6 1.53

[Table 2-1]

Antibody B

VB+ represents sample number 10, and VB- represents sample number 11.

Production (%) VB+ 100 VB- 100.5

[Table 2-2]

Antibody B

VB+ represents sample number 10, and VB- represents sample number 12.

Production amount (%) Standard error (%) VB+ 100 1.709 VB- 100.7 1.861

Cells producing antibody C were also cultured under the conditions listed in Table 1-1 to observe the effect of the presence or absence of VB12 in the culture medium on antibody production. The results confirmed that the antibody production after 14 days of culture was almost equal to that after culture in the initial culture medium and fed-batch culture medium (as in Sample 11) without VB12 (VB-) and the initial culture medium and fed-batch culture medium (as in Sample 12) without VB12 (VB-) compared to that after culture in the initial culture medium and fed-batch culture medium (as in Sample 10) containing VB12 (VB+). (Tables 2-3 and 2-4: Antibody production in cultures with VB+ as the control is expressed as 100%).

[Table 2-3]

Antibody C

VB+ represents sample number 10, and VB- represents sample number 11.

Production amount (%) Standard error (%) VB+ 100 1.027 VB- 106.2 1.558

[Table 2-4]

Antibody C

VB+ represents sample number 10, and VB- represents sample number 12.

Production amount (%) Standard error (%) VB+ 100 1.027 VB- 107.4 1.623

Example 4. Study on the effect of VB12 in culture medium on the physical properties of antibodies

Cell culture, affinity column chromatography, and concentration were performed under the same conditions as in Example 1. The presence or absence of VB12 in the culture medium was observed to affect the physical properties of the antibodies by analyzing the antibodies in the resulting composition. The results confirmed that antibodies obtained from cultures in a medium without VB12 (VB-) in the initial culture medium and fed-batch culture medium had almost equal glycan modifications (Afcosyl, fucose, galactosyl, high-mannose, hybrids), cellular proteins (HCP), cellular DNA (DNA), the proportion of charge variants (acidic, basic), and the proportion of polymers (HMWs) compared to antibodies obtained from cultures in a medium containing VB12 (VB+). (Table 3: Physical properties of antibodies obtained from cultures with VB+ as a control are expressed as 100%)

[Table 3]

Afucosyl fucosyl Galactosyl High mannitol hybrid HCP DNA acidic alkaline HMWs VB+(%) 100 100 100 100 100 100 100 100 100 100 VB-(%) 93.5 100.4 84.0 94.5 88.3 93.8 113.8 91.4 106.8 113.9 VB+ Standard Error (%) 8.2 0.3 3.1 2.3 14.6 4.9 31.4 1.6 2.5 21.8 VB - Standard Error (%) 5.4 0.2 1.7 2.3 6.6 8.9 26.2 1.0 1.3 14.0

Example 5. Study on the effect of VB12 in culture medium on cell passage.

The effects of removing VB12 from the cell passage medium (Thermo Fisher Scientific) were observed. The results showed almost identical proliferation behavior between passage culture in VB12-free medium (VB-) and passage culture in VB12-containing medium (VB+). It was confirmed that passage culture of antibody A-producing cells could be carried out for 100 days (28 passages) (Figure 4), antibody B-producing cells for 136 days (39 passages) (Figure 17), and antibody C-producing cells for 24 days (7 passages) (Figure 18).

Example 6. Study on the possibility of removing VB12 by cation exchange chromatography

Using an initial culture medium containing VB12 and fed-batch culture medium under the same conditions as in Example 1, affinity column chromatography was performed. For the resulting antibody-containing composition, it was investigated whether VB12 could be removed by a cation exchange chromatography step as a binding/elution method. As in Example 1, the concentration of cobalt was determined by ICP-MS as an indicator of the VB12 concentration.

As a result, the cobalt/antibody [%] ratio remained unchanged before passing through the CEX (cation exchange) column and after elution from the CEX column (Table 4). This confirms that the VB12 contained in the antibody-containing composition cannot be removed even with additional chromatographic steps.

[Table 4]

Cobalt/Antibody [%) Passing in front of the column 0.29 After washing 0.31

Example 7. Further study on the effect of VB12 on antibody staining

Further studies were conducted on the adsorption of VB12 and antibody in an antibody-containing composition (antibody concentration of approximately 30 mg/ml, with staining (light red)) obtained by culturing, affinity column chromatography, purification, and concentration under the same conditions as in Example 1.

Potassium cyanide was added to the antibody-containing composition to a concentration of 0.1% (a control was also performed without potassium cyanide), and the mixture was incubated at 37°C for 45 minutes. These samples were then passed through a 5KD cutoff filter, and the recovered antibody samples were analyzed by reversed-phase chromatography. The results showed that no peak of VB12 (cyanocobalamin) was detected in the potassium cyanide-untreated sample (KCN-), while a peak of VB12 (cyanocobalamin) was observed in the potassium cyanide-treated sample (KCN+) (Figure 19, Antibody A). Based on these results, it was confirmed that VB12 is released from the antibody by potassium cyanide treatment, confirming that VB12 affects antibody staining.

Industrial availability

This invention can be used to prepare drugs containing polypeptides.

Claims (6)

1. A method for preparing a composition containing an antibody that inhibits staining, comprising: a) Feed-batch culture of eukaryotic cells containing nucleic acids encoding antibodies in a vitamin B12-free cell culture medium, wherein feed-batch culture is performed using an initial culture medium without vitamin B12 and a feed-batch culture medium without vitamin B12. b) The step of recovering the antibody-containing composition from the culture. The antibody comprises a heavy chain constant region containing an amino acid sequence of sequence number 1 and a light chain constant region containing an amino acid sequence of sequence number 2. 2. The method for preparing the composition according to claim 1, wherein the molar concentration ratio of vitamin B12 to antibody in the composition is less than 0.26%. 3. The method for preparing the composition according to claim 1, wherein the VCD (viable cell density) in the cell culture medium on day 7 from the start of fed-batch culture is 80 × ^10⁵ cells/mL or higher. 4. The method for preparing the composition according to claim 1, wherein the antibody is a humanized IgG1 antibody that binds to latent myosin. 5. A method for preparing an antibody-containing pharmaceutical formulation, comprising preparing an antibody-containing composition by the method according to any one of claims 1 to 4, and preparing the obtained composition into a pharmaceutical formulation. 6. The method according to claim 5, wherein the eukaryotic cell is a CHO cell.