阅读说明：本技术 在抗体制造期间控制粉红色形成的方法 (Method of controlling pink color formation during antibody manufacture ) 是由 杜诚 A·U·博尔万卡尔 余骏昌 谭志俊 于 2018-05-08 设计创作，主要内容包括：本发明是通过防止在制造和收获期间抗体的还原或抑制在制造和收获期间氰钴胺(CN?Cbl)转化为羟钴胺(HO?Cbl)来防止在抗体制造期间产生粉红色的方法。用红光替换细胞培养基制备和储存区域中的白光抑制CN?Cbl向HO?Cbl的转化。在收获时将过氧化物添加到澄清的物料中抑制抗体二硫键的还原。(The present invention is a method of preventing pink color generation during antibody production by preventing reduction of antibodies during production and harvesting or inhibiting the conversion of cyanocobalamin (CN-Cbl) to hydroxycobalamin (HO-Cbl) during production and harvesting. Replacing white light in the cell culture medium preparation and storage area with red light inhibited the conversion of CN-Cbl to HO-Cbl. Peroxide was added to the clarified material at harvest to inhibit reduction of antibody disulfide bonds.)

1. A method of inhibiting pink color formation during antibody manufacture comprising

a) Preventing reduction of disulfide bonds of antibodies, and

b) inhibit the conversion of cyanocobalamin (CN-Cbl) to hydroxycobalamin (HO-Cbl) in the medium.

2. The method of claim 1, wherein the conversion of CN-Cbl to HO-Cbl is inhibited by preventing exposure of the cell culture medium to visible light during one or more times selected from the group consisting of medium preparation, medium storage, antibody production, and antibody harvest.

3. The method of claim 2, wherein only the CN-Cbl is exposed to UVA light during one or more times selected from the group consisting of media preparation, media storage, antibody production, and antibody harvest.

4. The method of claim 2, wherein CN-Cbl is exposed to red light (>6000nm) only during media preparation, media storage, and optionally during antibody production and antibody harvesting.

5. The method of any one of claims 1-4, wherein reduction of antibody disulfide bonds is prevented at harvest by adding hydrogen peroxide to the clarified material.

6. The method of claim 5, wherein the hydrogen peroxide concentration is maintained at ≤ 10 mM.

7. The method of claim 1, wherein the protein is selected from the group consisting of an antibody (Ab), a monoclonal antibody (mAb), a human antibody (HuMAb), a humanized antibody, and a chimeric antibody.

8. A method for inhibiting vitamin B in cell culture production₁₂A method of binding to an antibody comprising

a) Prevent reduction of disulfide bonds of antibodies, or

b) Inhibit the conversion of cyanocobalamin (CN-Cbl) to hydroxycobalamin (HO-Cbl) in the medium.

9. The method of claim 8, wherein the conversion of CN-Cbl to HO-Cbl is inhibited by preventing exposure of the cell culture medium to visible light during one or more times selected from the group consisting of medium preparation, medium storage, antibody production, and antibody harvest.

10. The method according to claim 9, wherein CN-Cbl is exposed to red light (>6000nm) only during culture medium preparation, culture medium storage and optionally during antibody production and antibody harvesting.

11. The method of any one of claims 8, wherein reduction of protein disulfide bonds is prevented at harvest by adding hydrogen peroxide to the clarified material.

12. The method of claim 11, wherein the hydrogen peroxide concentration is maintained at ≤ 10 mM.

13. The method of claim 8, wherein the protein is selected from the group consisting of an antibody (Ab), a monoclonal antibody (mAb), a human antibody (HuMAb), a humanized antibody, and a chimeric antibody.

14. A cell culture method for producing an antibody, comprising:

a) cell culture media were prepared and stored under red light (>600nm),

b) culturing the target antibody producing cells under red light conditions,

c) harvesting the antibody from the cell culture under red light conditions, and

d) the concentration of hydrogen peroxide in the clarified mass at harvest is maintained at 10mM or less.

15. A cell culture method for producing an antibody, comprising:

a) the culture medium was prepared and stored under red light (>600nm),

b) culturing the target antibody-producing cells under red light conditions, and

c) harvesting the antibody of interest from the cell culture under red light conditions.

16. A cell culture method for producing an antibody, comprising:

a) culturing the cells producing the antibody of interest,

b) harvesting the antibody of interest from the cell culture, and

b) the concentration of hydrogen peroxide in the clarified mass at harvest is maintained at 10mM or less.

Background

Monoclonal antibody (mAb) therapeutics have become more popular in the treatment of a variety of human diseases. As macromolecules, mabs have some heterogeneity due to a wide range of post-translational modifications. Successful control of the manufacturing process is critical to ensure product quality and safety of therapeutic proteins as well as consistency from batch to batch. Indeed, ICH Q6B guidelines require product appearance specifications.

Normal antibody production typically had some acceptable color changes (Derfus GE et al, MAbs2014,6(3): 679-. However, color change due to impurities in the process is a problem of lack of process control. One of the major sources of pink/red color in the final drug substance has been identified as vitamin B₁₂mAb complex (Derfus, 2014; Prentice, 2013). Although the nature of the interaction has not yet been elucidated, vitamin B₁₂The attachment to the protein appears to be sufficiently strong to allow co-elution by multiple downstream purification steps including protein a affinity chromatography, low pH viral inactivation, various polishing chromatographies, and ultrafiltration and diafiltration.

Antibodies are typically produced in mammalian cells, such as Chinese Hamster Ovary (CHO) cells, and secreted extracellularly into the cell culture medium. At the end of the cultivation process, the cells are separated from the culture medium during a primary recovery step in which the harvest is clarified using methods such as centrifugation, depth filtration or flocculation. During this process, cells are often subjected to various pressures, including mechanical shear, exposure to a low Dissolved Oxygen (DO) environment, or temperature and pH changes. These stresses cause cell damage, resulting in the release of undesirable intracellular components into the supernatant. These cytosolic components (such as lipids and enzymes) can potentially affect product quality and must be carefully monitored or removed. For example, release of intracellular reducing agents can result in reduction of the disulfide bonds of the antibodies (Mun M. et al Biotechnol Bioeng 2015; 112: 734-.

During the manufacturing process, substantial reduction of the antibody was observed after the harvesting operation and/or protein a chromatography. Various process parameters may have an effect on the extent of antibody reduction. For example, maintaining high levels of Dissolved Oxygen (DO) during harvest is critical to keeping the antibody molecule intact (Mun M.2015; Trexler-Schmidt M, 2010). Mechanical shear forces, which lead to cell lysis and leakage of cellular components into the harvest, also significantly lead to reduction (Kao YH et al Biotechnol Bioeng.2010; 107: 622-. Other process parameters such as harvest retention time (Chung WK et al Biotechnol Bioeng.2017; 114: 1264-.

Control of Low Molecular Weight (LMW) species formed by reduction of antibody disulfide bonds is essential in the manufacturing process in order to ensure antibody product quality. Therefore, several strategies to control disulfide reduction have been proposed in recent years. Chemical inhibitors have been tested to prevent antibody reduction, including pre-and post-harvest treatment with anti-reducing agents such as copper sulfate (Chaderjian WB et al Biotechnol prog.2005; 21: 550-. Although the knowledge and methods surrounding mitigation strategies have increased over the years, the implementation of these methods in production is not without difficulties, such as the introduction of chemical by-products that need to be removed by chromatographic steps, increased processing time and the risk of off-target modifications or damage to the antibody product.

Disclosure of Invention

The present invention is a method of preventing the development of pink color during antibody production by 1) preventing the reduction of antibodies during production and harvesting or 2) inhibiting the conversion of cyanocobalamin (CN-Cbl) to hydroxycobalamin (HO-Cbl) or 3) both, preventing the reduction of antibodies during production and harvesting and inhibiting the conversion of cyanocobalamin to hydroxycobalamin.

In one embodiment of the invention, the conversion of CN-Cbl to HO-Cbl is inhibited by preventing exposure of the culture medium to visible light during one or more times selected from the group consisting of culture medium preparation, culture medium storage, antibody production, and antibody harvest.

In one embodiment of the invention, only the CN-Cbl is exposed to UVA light during one or more times selected from the group consisting of medium preparation, medium storage, antibody production, and antibody harvest.

In one embodiment of the invention, only CN-Cbl is exposed to red light (>6000nm) during culture medium preparation, culture medium storage and optionally during antibody production and antibody harvesting.

In one embodiment of the invention, reduction of the disulfide bonds of the antibodies is prevented at harvest time by adding hydrogen peroxide to the clarified material.

Another embodiment of the invention is the inhibition of vitamin B during manufacture₁₂A method of binding to an antibody comprising 1) preventing reduction of the disulfide bond of the antibody during manufacture and harvest, or 2) inhibiting the conversion of CN-Cbl to HO-Cbl during cell culture medium preparation, medium storage, cell culture process, or antibody harvest.

Another embodiment of the invention is a method of producing an antibody comprising 1) preparing and storing a cell culture medium under red light (>600nm), 2) culturing cells that produce an antibody of interest under red light conditions, 3) harvesting the antibody from the cell culture under red light conditions, and 4) adding hydrogen peroxide to the harvest solution.

Another embodiment of the invention is a method of producing an antibody comprising 1) preparing and storing a cell culture medium under red light (>600nm), 2) culturing cells that produce an antibody of interest under red light conditions, and 3) harvesting the antibody of interest from the cell culture under red light conditions.

Another embodiment of the invention is a method of producing an antibody comprising 1) culturing cells that produce an antibody of interest, 2) harvesting the antibody of interest from the cell culture, and 3) adding hydrogen peroxide to the harvest solution.

Drawings

FIGS. 1A-1D show vitamin B₁₂And spectral absorption of two batches of mAb B. 1A is vitamin B₁₂And the absorbance spectra of two batches of mAb B. 5mg/L vitamin B in PBS₁₂(cyanocobalamin) (blue line), pink batch (red line) and colourless batch (purple line), from 250 to 800 nm. 1B is a rescaled view of (1A) to display detailed information. 1D is a vial of mAb B protein A eluate with a visible pink color.

FIGS. 2A-2D show vitamin B₁₂Attachment to the antibody is a secondary reaction. (2A) Two batches of colorless mAb a (the batch in which no reduced antibody molecules were detected ("good" batch) and another batch with mostly reduced antibody ("bad" batch)) were mixed with CN-Cbl or HO-Cbl. After incubation, excess free vitamin B12 was removed. Samples from left to right, good batches and CN-Cbl; bad batch and CN-Cbl; good lot and HO-Cbl; bad batch and HO-Cbl; and the bad batch was first treated with hydrogen peroxide and then mixed with HO-Cbl. (2B) As shown by CE SDS, hydrogen peroxide can oxidize the reduced antibody to reform the antibody monomer. (2C) Quantification of mAb monomers and various low molecular weight species shown in 2B in hydrogen peroxide treated and untreated mabs. (2D) mAb A batch 2 material in vitamin B₁₂Non-reducing CE-SDS analysis before (blue line) and after (black line) attachment. In combination with vitamin B₁₂Before and after incubation together, the individual peak patterns changed, probably due to oxidation of free thiol groups and re-formation of disulfide bonds in air.

FIGS. 3A-3C show the identification of vitamin B₁₂Coupled mass spectral data. (3A) MS/MS spectra of m/z 664.79 from HO-Cbl and m/z 678.29 from CN-Cbl. (3B) MS/MS spectra of peptide-cobalamin complex L19-cobalamin and L18-19-cobalamin. (3C) Schematic representation of the disulfide bond of the IgG4 antibody. With vitamin B₁₂The bound cysteine residues are labeled blue and disulfide bonds are formedThe cysteine residue in (b) is red for the circle.

Fig. 4A to 4E show the transmission spectrum (4A) of the color filter used in example 1. (4B) CN-Cbl and HO-Cbl in standards; (4C) fresh medium for mAb a; (4D) a medium exposed to red light; and (4E) chromatograms of media exposed to green light separated on RP-UPLC and detected at 360 nm. In (4D and 4E), black, blue and red lines represent exposure energies of 30, 60 and 120 million lux hours, respectively.

Fig. 5A-5C show the free thiol level and Low Molecular Weight (LMW) in CB. (5A) Correlation of free thiol levels of mAb1CB sample with the presence of LMW in the corresponding protein a purified sample (PAVIB). The LMW species of PAVIB were analyzed by NR _ Caliper. Those samples with LMW ≧ 5% are labeled "YES," otherwise labeled "NO. Analysis was performed using JMP software. (5B) Measurement of free thiol levels in CB under different harvest treatments. Clarified harvest material (CB) of the lab scale bioreactor (5-L) containing mAb1 was treated by lowering the pH to 4.8 in the absence or presence of dextran sulfate. CB samples were incubated for 1 hour at room temperature before depth filtration of the samples. After depth filtration, aliquots of each sample were incubated at 37 DEG C for 75 minutes with or without the addition of 1mM NAPDH before measuring the free thiol content. Manufacturing lots with high LMW percentages and representative lots (normal lots) were also included for comparison. (5C) Percentage of intact antibody in the sample of (5B). After protein a purification, intact antibodies were measured by NR _ Caliper.

Fig. 6A-6F show observations of redox indicator dcppip treatment and color change as predictive markers of LMW production. (6A) Structure and reaction of dcppip. (6B) mAb2 purified DS sample with > 95% LMW (3.1 thiol groups per antibody protein) on the left showed no color after treatment with dcppip. The mAb2 sample with more than 98% intact antibody on the right (0.4 thiol groups per antibody protein) showed purple color (in pH 5.5) in the presence of dcppip. (6C) Color change of dcppip in CB of mAb2 under different culture conditions. The DCPIP of the two tubes on the left side becomes completely colorless, which indicates that CB has higher reduction potential; DCPIP in the middle two tubes was blue, indicating no reduction events; and the DCPIP part of the right tube is reduced. (6D) The free thiol concentration is a function of the percentage of mAb2 lysed. 100% cell lysate of mAb2 was mixed with conventional CB to generate a dilution curve of the cell lysate and the free thiol concentration was measured and reported as the average of duplicate samples. The linear regression equation and the R-square were calculated using Excel. (6E) Study design to test the order of color change and amount of free thiol of dcppip. (6F) Images of the DCPIP test results studied in table 7. The upper sample received air before and during the primary recovery. The samples in the lower panel were subjected to a nitrogen purge before and during the primary recovery. The sequence of the test tubes from left to right is the same as the sequence from top to bottom in table 7.

Fig. 7A-7K illustrate the use of hydrogen peroxide to prevent disulfide bond reduction. mAb2 lab scale produced CB was aliquoted into containers containing various concentrations of hydrogen peroxide. The air-free condition inside the container was generated by nitrogen purging, and was left at room temperature for one day. The resulting samples were analyzed directly by non-reducing (7A, 7C, 7E, 7G, 7I) and reducing (7B, 7D, 7F, 7H, 7J) calipers without protein a purification. (7A and 7B) CB with no hydrogen peroxide added was exposed to air as a control. CB with 0mM (7C and 7D), 0.33mM (7E and 7F), 1mM (7G and 7H) and 3mM (7I and 7J) hydrogen peroxide was kept under air-free conditions. (7K) Summary of mAb-fragmented non-reducing Caliper results. The amount of light chain is not included as a fragment due to the presence of excess light chain of this mAb secreted from the host cell. Abbreviations for LMW species: LC, light chain; HC, heavy chain; HL, half antibody with one light chain and one heavy chain; HHL, partial antibody with one light chain and two heavy chains.

Fig. 8A to 8F show evaluation of prevention of disulfide bond reduction using hydrogen peroxide in the worst case. As described in example 2, mAb 2CB tested had 100% cell lysis. The holding conditions with or without air are the same as in fig. 7. (8A) CB samples that were not treated with hydrogen peroxide and maintained in air served as controls. (8B) CB samples that were not treated with hydrogen peroxide and maintained under airless conditions. (8C) 5mM hydrogen peroxide was added to the CB samples prior to being maintained in an airless condition. (8C) 10mM hydrogen peroxide was added to the CB samples prior to being maintained in an airless condition. The resulting CB samples (8A to 8D) were analyzed directly by non-reducing Caliper without protein a purification. (8E) Summary of mAb-fragmented non-reducing Caliper results from unpurified CB. (8F) Summary of intact antibody purity of samples 8A to 8D purified by protein a chromatography and analyzed by non-reducing Caliper.

Fig. 9A-9D show the use of alternative peroxides to prevent reduction of antibody disulfide bonds. In the worst case of mAb 2CB with 100% cell lysis, sodium percarbonate and sodium perborate were used. (9A) CB samples kept in air without any peroxide. (9B) CB samples maintained under airless conditions without any peroxide. (9C) Representative results for 5mM sodium percarbonate treated CB samples. Sodium percarbonate was added before being kept in an air-free condition. Results were from non-reducing calipers (9A to 9C) of unpurified CB. (9D) Summary of sodium percarbonate and sodium perborate treatments. Results were from a non-reducing Caliper of unpurified CB.

Detailed Description

Process control in bioproduct manufacture is critical to ensure product quality and safety of therapeutic proteins and consistency from batch to batch. Knowledge of the mechanisms of color generation during manufacturing is crucial to developing control strategies. The inventors found that the generation of pink color during antibody production is a secondary reaction, depending on the free thiol and hydroxycobalamin (vitamin B) on the reduced antibody₁₂Active form of (ii) of the composition. Both reactants are necessary and neither is sufficient to produce a pink color. The present invention is a method of preventing the development of pink color during antibody production by 1) preventing the reduction of antibodies during production and harvesting or 2) inhibiting the conversion of cyanocobalamin (CN-Cbl) to hydroxocobalamin (HO-Cbl) during production and harvesting. Alternatively, the present invention is a method of preventing pink color during antibody production by 1) preventing reduction of antibodies during production and harvesting and 2) inhibiting the conversion of cyanocobalamin to hydroxycobalamin during production and harvesting。

The inventors found that vitamin B₁₂Is attached to the free thiol group of the cysteines located in heavy chain 134(HC134), light chain 214(LC214), heavy chain 321(HC321), heavy chain 367(HC367), and heavy chain 425(HC 425). These five cysteine residues are distributed between the Fab region and the Fc region. There are two pairs of disulfide bonds in these five cysteine residues, LC214 and HC134 and HC367 and HC425 (fig. 3 c). When these specific disulfide bonds are reduced during manufacture and harvesting, the two cysteine residues in the disulfide pair have equal hydroxycobalamin attachment accessibility.

As used herein, "cyanocobalamin" and "CN-Cbl" are used interchangeably and refer to the vitamin B of the cell culture medium₁₂(VB12) component. Likewise, "hydroxycobalamin" and "HO-Cbl" are used interchangeably and refer to vitamin B in cell culture media₁₂(VB12) that can bind reduced antibodies during manufacture and harvesting.

Antibodies are typically produced in mammalian cells, such as Chinese Hamster Ovary (CHO) cells, and secreted extracellularly into the cell culture medium. At the end of the cell culture process, the cells are separated from the culture medium during a primary recovery step in which the harvest is clarified using methods such as centrifugation, depth filtration or flocculation.

As used herein, "clarified bulk" and "CB" are used interchangeably and refer to the solution collected after a primary recovery step (such as centrifugation, depth filtration or flocculation) for clarifying the cell culture fluid.

As used herein, "cell culture medium preparation," "cell culture medium storage," "cell culture process," "antibody manufacture," "antibody production," and "antibody harvest," "harvest" refer to the many steps required to produce an antibody. Each step may occur at a different location within the manufacturing facility.

Red light is used as a control strategy for pink in a manufacturing environment.

Since the production of pink products requires both hydroxycobalamin and reduced antibodies, controlling either or both factors will prevent the generation of pink.

Photoinduced vitamin B₁₂Transformation can occur during the media preparation, media storage, and cell culture processes. Use of transparent disposable bioprocessing bags in glass bioreactors for media preparation, storage duration, and cell culture duration will increase vitamin B₁₂Exposure and risk of transformation. General control strategies, such as using appropriate containers (e.g., stainless steel containers), covering with additional layers of material, avoiding unnecessary exposure, may provide protection from light. However, the implementation of these strategies is not without challenges and often involves additional costs.