CN110373374B

CN110373374B - Methods and compositions for reducing antibody core fucosylation

Info

Publication number: CN110373374B
Application number: CN201810324841.6A
Authority: CN
Inventors: 张莹; 肖志华
Original assignee: Shanghai Haozhe Information Technology Ltd
Current assignee: Shanghai Haozhe Information Technology Ltd
Priority date: 2018-04-12
Filing date: 2018-04-12
Publication date: 2023-07-07
Anticipated expiration: 2038-04-12
Also published as: CN110373374A; WO2019196697A1

Abstract

The present invention provides small molecule mannose analogs for use in the manufacture of recombinant antibodies having complex N-linked glycans but reduced core fucosylation, which mannose analogs are normally taken up by a host cell (e.g., by active transport or passive diffusion) and inhibit the production of GDP fucose. The method provided by the invention has extremely low price because mannose is adopted as a synthetic substrate; and the method can reduce core fucosylation and simultaneously can not influence the high mannose glycoform proportion of N-linked glycans.

Description

Methods and compositions for reducing antibody core fucosylation

Technical Field

The invention relates to a method for reducing core fucosylation of an antibody and the technical field of used compounds.

Background

Recombinant therapeutic proteins are produced by a number of different methods. One preferred method is to produce recombinant proteins from mammalian host cell lines. Cell lines such as Chinese Hamster Ovary (CHO) cells have been engineered to express therapeutic proteins of interest. The advantages and disadvantages of different cell lines in recombinant proteins vary, including protein characteristics and yields. Monoclonal antibodies or antibody derivatives are one type of recombinant proteins and require a balance of considerations in selecting producer cells for high yield and product consistency.

The Fc fragment functions as an effector by binding the active region to the receptor. N-glycosylation of IgG is located in the consensus sequence of the CH2 region of the Fc fragment (Asn 297-X-Ser/Thr, X can be any amino acid residue other than proline) and is covalently bound to the antibody via an amide bond. Proteins and sugars interact in a peer-to-peer fashion to affect each other's conformation as found by crystal X-diffraction. In addition to Fc fragment glycosylation, 30% of IgG is N-glycosylated in the Fab fragment, with beneficial, detrimental or neutral effects on the efficacy of the antibody molecule. The Fc glycosylated sugar molecule has a complex double-antenna core structure consisting of two pentose molecules, mannose (Man) and N-acetylglucosamine (GlcNAc), and different glycoforms contain different numbers of sugar molecules in addition to the core structure, such as fucose (Fuc), mannose, N-acetylglucosamine, galactose (Gal), bisecting N-acetylglucosamine and sialic acid. Variations in the length, branching pattern and monosaccharide sequence of the sugar chains lead to complexity of glycosylation modification. In addition, the N-glycosylated pentasaccharide core structure is divided into three classes due to various enzymes: high mannose type, heterozygous type and complex type.

The above formula is the structural heterogeneity of the polysaccharide molecule modified by glycosylation at Asn297 of immunoglobulin.

The fucose core oligosaccharide is biosynthesized from fucose residues transferred from GDP-Fuc by a-1, 6-fucosyltransferase in the transport Golgi apparatus. Analysis of the Fc fragment structure with fucose and afucose modifications showed: the electron densities of the Asp280 and Asn297 residues differ between the two; there was also a difference in hydration pattern of the Tyr296 residue. The human IgG expressed by Lec13 mutant cells without fucose modification showed: affinity to fcγriiia is improved by 50-fold and ADCC action is improved by 100-fold. The coreless fucose results in Asn162 in fcγriiia receptor having a higher affinity for Fc glycans and thus a stronger binding. While steric hindrance of fucose may prevent such interactions. This finding has led to interest in engineering cell lines to produce antibodies with reduced core fucosylation.

Methods of engineering cell lines to reduce core fucosylation include gene knockout, gene knock-in, and RNA interference (RNAi). In gene knockout, the gene encoding FUT8 (α1, 6-fucosyltransferase) is inactivated. FUT8 catalyzes the transfer of a fucose residue from GDP-fucose to the Asn-linked (N-linked) GlcNac position 6 of N-glycans. FUT8 is said to be the only enzyme responsible for adding fucose to the N-linked biantennary saccharide Asn 297. Gene knock-in is the addition of a gene encoding an enzyme such as GNTIII or Golgi. Alpha. Mannosidase II. Increasing the level of this enzyme in the cell diverts the monoclonal antibody from the fucosylation pathway (resulting in reduced core fucosylation) and has an elevated amount of bisected N-acetylglucosamine. RNAi also typically targets FUT8 gene expression, resulting in reduced mRNA transcript levels or complete knockout of gene expression.

In addition to engineering cell lines, the use of small molecule inhibitors of enzymes for glycosylation pathways is also included. Inhibitors, such as catheterization, act early on the glycosylation pathway, producing antibodies with immature glycans (e.g., high mannose levels) and low fucosylation levels. Antibodies made by these methods typically lack the complex N-linked glycan structures associated with mature antibodies.

Seattle Gene company in patent CN200980124932.4 discloses a small molecule fucose analogue for the production of recombinant antibodies with complex N-linked glycans but reduced core fucosylation. This patent reduces core fucosylation levels by adding fucose analogs during host cell production, which can reduce core fucosylation of antibodies, but raw material fucosylation is very expensive, and using this method, while reducing core fucosylation, increases the high mannose glycoform fraction of antibody N-linked glycans. IgG molecules containing high mannose residues have a shorter serum half-life than IgG molecules containing a dual antenna core structure, which may result in increased immunogenicity and are not beneficial for drug therapy.

Disclosure of Invention

The object of the present invention is to overcome the above-mentioned drawbacks of the prior art by providing a further method for reducing the core fucosylation of an antibody, which is less costly than the prior art and has less impact on the high mannose glycoform proportion of N-linked glycans.

It is another object of the present invention to provide mannose analogues or biologically acceptable salts or solvates thereof for use in a method of reducing the fucosylation of an antibody core, which can be achieved by the preparation method of the invention.

The invention also provides a mammalian cell culture medium for preparing the antibody for reducing the core fucosylation modification.

The methods and compositions of the present invention are presented in part based on the results described in the examples, which show that culturing host cells expressing an antibody or antibody derivative in the presence of a mannose analog produces an antibody with reduced core fucosylation (i.e., reduced fucosylation of N-acetylglucosamine linked to the reducing end of a complex N-glycoside-linked sugar chain through the N-acetylglucosamine linked to the reducing end of the sugar chain of the Fc region). Such antibodies and antibody derivatives may have improved effector function (ADCC) compared to antibodies or antibody derivatives produced by such host cells cultured in the absence of mannose analogs or fucose analogs.

In the present application

The term "antibody" means: (a) Immunoglobulin polypeptides and immunologically active portions of immunoglobulin polypeptides, i.e., polypeptides of the immunoglobulin family or portions thereof, comprise an antigen binding site for an immunospecific binding domain-specific antigen (e.g., CD 20) and an Fc domain comprising a complex N-glycoside-linked sugar chain, or (b) conservatively substituted derivatives of such immunoglobulin polypeptides or fragments of an immunospecifically binding antigen (e.g., CD 20).

"antibody derivative" means an antibody (including antibody fragments) as defined above, or an Fc domain or Fc region of an antibody comprising a complex N-glycosidically linked sugar chain, modified by covalent binding to a heterologous molecule, e.g., by binding to a heterologous polypeptide (e.g., a ligand binding domain of a heterologous protein), or by glycosylation (except for core fucosylation), deglycosylation (except for non-core fucosylation), acetylation, phosphorylation, or other modifications normally unrelated to the antibody or Fc domain or Fc region.

The term "monoclonal antibody" refers to an antibody derived from a single cell clone, including any eukaryotic or prokaryotic cell clone or phage clone, but is not limited to methods of production thereof. Thus, the term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology.

The term "Fc region" refers to the constant region of an antibody, e.g., C H1-hinge-C H2-C H3 domain, optionally having C H4 domain, or a conservatively substituted derivative of such an Fc region.

The term "Fc domain" refers to a constant region domain of an antibody, e.g., a C H1, hinge, C H2, C H3, or C H4 domain, or a conservatively substituted derivative of such an Fc domain.

An "antigen" is a molecule to which an antibody specifically binds.

The term "specifically binds" refers to an antibody or antibody derivative that binds to its corresponding target antigen in a highly selective manner, but does not interact with various other antigens.

The term "inhibit" refers to the occurrence of a detectable decrease, or complete arrest.

The term "GDP-Fucose" refers to guanosine diphosphate Fucose.

The technical scheme provided by the application is as follows:

mannose analogues of the formula:

wherein R1-R5 are each independently selected from the group consisting of: -OH, -OAc, X, wherein X is F, cl, br or I.

Preferably, R2 is X, and R1, R3, R4 and R5 are each independently selected from the group consisting of-OH and-OAc.

Preferably, R5 is X and R1 to R4 are each independently selected from the group consisting of-OH and-OAc.

The following three mannose analogues or biologically acceptable salts or solvates thereof

A mammalian cell culture medium for producing an antibody that reduces core fucosylation modification comprising an effective amount of a mannose analog as described above, or a biologically acceptable salt or solvate thereof.

Preferably, the volume of the medium is at least 10 liters.

Preferably, one or more mannose analogues or biologically acceptable salts or solvates thereof are added to the medium to maintain an effective concentration thereof.

Preferably, the medium is an animal protein-free medium; the medium is serum-free; the medium is free of added fucose or mannose. More preferably, the medium is free of animal proteins, free of serum, free of added fucose or mannose.

Further, the medium is a powder or a liquid.

A method of reducing core fucosylation modified antibodies, the method comprising the steps of:

1) Culturing a host cell expressing an antibody having an Fc domain in a medium comprising an effective amount of a mannose analog under suitable growth conditions;

2) Isolating the antibody from the cell;

the Fc domain has at least one N-glycoside-linked sugar chain linked to the Fc domain by N-acetylglucosamine at the reducing end thereof;

the mannose analogues are selected from any mannose analogues described in the patent or biologically acceptable salts or solvates thereof; wherein the core fucosylation of the antibody is lower than that of an antibody from a host cell cultured in the absence of the mannose analog.

Preferably, the cell is a recombinant host cell or a hybridoma cell; the recombinant host cell refers to Chinese hamster ovary cells, NS0 or SP2/0.

Preferably, the host cells are cultured in batch, fed-batch, continuous fed-or continuous perfusion medium, or in microcarriers.

Preferably, the culture medium is a mammalian cell culture medium provided in this patent for the preparation of antibodies that reduce core fucosylation modifications.

Preferably, the antibody is an intact antibody, an IgG1, a single chain antibody or a fusion protein comprising an Fc domain.

The mannose analogues may be used in the form of a composition, i.e. a mixture of different mannose analogues as described herein. The mannose analog composition is dissolved in a solvent at a suitable concentration for addition, and the composition is added to a dry powder or liquid medium and then present in a suitable concentration in the medium of the host cell. The solvent includes a biologically acceptable solvate, which means a combination of one or more solvent molecules and a mannose analog. Examples of solvents that form a biologically acceptable solvate include, but are not limited to: water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

The invention has the beneficial effects that:

the ratio of Fucose (Fucose) in N-glycosylation of an Fc segment of an antibody has an important influence on ADCC activity of the antibody. Seattle gene company can inhibit fucosylation with high efficiency by synthesizing fucose analog (patent grant publication No. CN 102076865B), but in practice it was found that inhibition of fucosylation was often accompanied by an increase in high mannose glycation sugar. While high mannose glycoforms of antibodies have a short serum half-life and may result in increased immunogenicity, which should be avoided in the process. Therefore, the inhibition process causes the change of three proportions of fucosylation, nonfucosylation and high mannose saccharification antibodies, which is unfavorable for the regulation and control of precisely controlling the glycoform proportion, thereby affecting the therapeutic properties of the antibodies and antibody derivatives.

The present patent provides mannose analog inhibitors that can act at very low concentrations, while being advantageous for cost optimization because the precursor for synthesizing mannose inhibitors is mannose, which is much cheaper than fucose. Mannose analogues and fucose analogues, particularly halogen substituents, are not metabolized and therefore can be maintained in concentration in the medium for a long period of time, and at lower concentrations, the effective concentration can be maintained.

The data show that the synthesized 2-F-Mannose can inhibit more than 50% of fucosylation at the concentration of 5ppm, completely meets the industrial requirement, and is completely equivalent to the inhibition efficiency of 2-F-Fucose with the same concentration. Similar effects can be achieved by both synthetic 2-Cl-Mannose and 6-F-Mannose. It was demonstrated that mannose analogs and fucose analogs can achieve inhibitory effects in various configurations, reducing fucosylation of antibodies and antibody derivatives.

In summary, the present invention provides small molecule mannose analogs for use in the manufacture of recombinant antibodies having complex N-linked glycans but reduced core fucosylation, which mannose analogs are normally taken up by a host cell (e.g., by active transport or passive diffusion) and inhibit the production of GDP fucose. The method provided by the invention has extremely low price because mannose is adopted as a synthetic substrate; and the method can reduce core fucosylation without greatly influencing the high mannose glycoform proportion of N-linked glycans.

These and other aspects of the invention will be more fully understood from the following detailed description, non-limiting examples of specific embodiments, and the accompanying drawings.

Drawings

FIG. 1 is a graph showing the comparison of culture densities of cells of example 4.

FIG. 2 is a graph showing the comparative culture activity of cells of example 4.

FIG. 3 is a graph showing comparison of cell expression amounts of humanized trastuzumab antibodies in culture supernatants of example 4.

FIG. 4 is a data analysis chart of example 5 capillary electrophoresis showing an electrophoresis chart of glycans from a control humanized trastuzumab.

Figure 5 is a data analysis plot of capillary electrophoresis of example 5 showing an electrophoresis plot of glycans from humanized trastuzumab monoclonal antibodies produced from host cells grown in the presence of 2-fluoro-mannose analogs.

FIG. 6 is a data analysis plot of example 5 capillary electrophoresis showing an electrophoresis plot of glycans from humanized trastuzumab monoclonal antibodies produced from host cells grown in the presence of 2-fluoro-fucose analogs.

FIG. 7 shows the results of Qtof analysis of antibodies, a blank, in which about 95% of the oligosaccharides are core fucosylated.

FIG. 8 is a graph showing Qtof analysis of antibodies expressed in the presence of 5. Mu.M of 2-fluoro-mannose analogues.

FIG. 9 is a graph showing Qtof analysis of antibodies expressed in the presence of 100. Mu.M of 2-fluoro-mannose analogues.

FIG. 10 is a graph showing Qtof analysis of antibodies expressed in the presence of 500. Mu.M of 2-fluoro-mannose analogues.

FIG. 11 is a HNMR spectrum of the product 2-fluoro-mannose analogue of example 1.

FIG. 12 is a HNMR spectrum of the product 2-chloro-mannose analog of example 2.

FIG. 13 is a HNMR spectrum of the product 6-fluoro-mannose analogue of example 3.

Detailed Description

The materials and instruments used in the invention are all conventional products in the technical field which are sold in the market unless specified.

Example 1: synthesis of 2-fluoro-mannose analogues, i.e. 2-deoxy-2-fluoro-1, 3,4, 6-tetra-oxo-acetyl-D-mannose

Example 1.1

D-mannose (50 g,0.278 mol) was added to pyridine (500 mL) under nitrogen atmosphere and dissolved with stirring. Cooled to 0℃and acetic anhydride (500 g,4.09 mol) was added thereto, and the temperature was raised to 25℃to react for 16 hours. Pyridine and acetic anhydride were removed by concentration under reduced pressure, the residue was dissolved in ethyl acetate (500 mL), washed successively with 1mol/L dilute hydrochloric acid (500 mL. Times.2), saturated sodium bicarbonate solution (500 mL) and saturated sodium chloride solution (500 mL), and the organic phase was concentrated under reduced pressure to give 97.5g of an off-white solid in 90% yield.

Example 1.2

The compound prepared in example 1.1 (97.5 g,0.25 mol) was added to dichloromethane (1500 mL) under nitrogen, dissolved with stirring, and cooled to 0 ℃. 40% hydrobromic acid acetic acid solution (400 mL) was slowly added and the reaction was allowed to proceed at 25℃for 3 hours. The mixture was washed with ice water (1500 mL x 2), saturated sodium bicarbonate solution (1500 mL) and saturated sodium chloride solution (1500 mL) in this order, and the organic phase was concentrated under reduced pressure to give 82g of a yellow oil in 80% yield.

Example 1.3

Zinc powder (165 g,2.54 mol), N-methylimidazole (32 mL,0.39 mol) and ethyl acetate (1300 mL) were added to the reaction vessel under nitrogen, the mixture was heated to reflux, the compound (82 g,0.2 mol) prepared in example 1.2 was dissolved in ethyl acetate (325 mL), and the mixture was added dropwise to the above suspension, followed by a reflux reaction for 1 hour. Cooled to 25 degrees and filtered. The filtrate was washed successively with 1mol/L dilute hydrochloric acid (650 mL), saturated sodium bicarbonate solution (650 mL) and saturated sodium chloride solution (650 mL), and the organic phase was concentrated under reduced pressure to give 36.2g of an off-white solid in 66% yield.

Example 1.4

The off-white solid (15 g,0.055 mol) obtained in example 1.3 was dissolved in N, N-dimethylformamide (125 mL) and water (125 mL), cooled to 0℃and 1-chloromethyl-4-fluoro-1, 4-diazabicyclo [2.2.2] octane bis (tetrafluoroboric acid) salt (39 g,0.11 mol) was added in portions and the mixture was heated to 25℃to react for 16 hours. The reaction was extracted with ethyl acetate (250 mL x 2), and the organic phases were combined and washed with water (250 mL) and then with saturated brine (250 mL). Concentration gave 13.6g of a pale yellow oil in 80% yield.

Example 1.5

The pale yellow oil (13.6 g,0.044 mol) prepared in example 1.4 was added to pyridine (140 mL) and dissolved with stirring. Cooled to 0℃and acetic anhydride (70 g,0.69 mol) was added thereto, and the temperature was raised to 25℃to react for 16 hours. Pyridine and acetic anhydride were removed by concentrating under reduced pressure, the residue was dissolved in ethyl acetate (150 mL), washed successively with 1mol/L dilute hydrochloric acid (150 mL x 2), saturated sodium bicarbonate solution (150 mL) and saturated sodium chloride solution (150 mL), the organic phase was concentrated under reduced pressure, and then purified by column chromatography to give 10.2g of an oil, which was dissolved in ethyl acetate (7 mL), petroleum ether (100 mL) was slowly added, and recrystallized to give 7.6g of a white powder with a yield of 49%. m/z (MH+) 351,1H NMR (400 MHz, CDCl) ₃ )，δ6.27(dd，1H)，5.42(t，1H)，5.27(dd，1H)，4.82&4.70 (d, 1H), 4.30 (dd, 1H), 4.27 (dd, 1H), 4.05 (m, 1H), 2.17 (s, 3H), 2.11 (s, 3H), 2.10 (s, 3H), 2.06 (s, 3H). The HNMR spectrum of the final product is shown in FIG. 11.

Example 2: synthesis of 2-chloro-mannose analogues, namely 2-deoxy-2-chloro-1, 3,4, 6-tetra-oxo-acetyl-D-mannose

Example 2.1

The off-white solid (15 g,0.055 mol) of example 1.3 was dissolved in N, N-dimethylformamide (125 mL) and water (125 mL), cooled to 0℃and N-chlorosuccinimide (14.7 g,0.11 mol) was added in portions and the reaction mixture was warmed to 25℃for 16 hours. The reaction was extracted with ethyl acetate (250 mL x 2), and the organic phases were combined and washed with water (250 mL) and then with saturated brine (250 mL). After concentration, 12.7g of a pale yellow oil was obtained in 71% yield.

Example 2.2

The pale yellow oil (12.7 g,0.039 mol) prepared in example 2.1 was added to pyridine (120 mL) and dissolved with stirring. Cooled to 0℃and acetic anhydride (60 g,0.59 mol) was added and the reaction was continued at 25℃for 16 hours. Pyridine and acetic anhydride were removed by concentrating under reduced pressure, the residue was dissolved in ethyl acetate (120 mL), washed successively with 1mol/L dilute hydrochloric acid (120 mL x 2), saturated sodium bicarbonate solution (120 mL) and saturated sodium chloride solution (120 mL), the organic phase was concentrated under reduced pressure, and then purified by column chromatography to give 9.4g of an oil, which was dissolved in ethyl acetate (6 mL), petroleum ether (80 mL) was slowly added, and recrystallized to give 6.6g of a white powder in 46% yield. m/z (MH+) 367,1H NMR (400 MHz, CDCl) ₃ ) Delta 6.24 (s, 1H), 5.48 (t, 1H), 5.37 (dd, 1H), 4.40 (d, 1H), 4.21 (dd, 1H), 4.16 (d, 1H), 4.08 (m, 1H), 2.18 (s, 3H), 2.11 (s, 3H), 2.10 (s, 3H), 2.02 (s, 3H). HNMR spectra are shown in FIG. 12.

Example 3: synthesis of 6-fluoro-mannose analogues, i.e. 6-deoxy-6-fluoro-1, 2,3, 4-tetra-oxo-acetyl-D-mannose

Example 3.1

D-mannose (23 g,0.128 mol) was added to pyridine (200 mL) under nitrogen atmosphere and dissolved with stirring. Cooled to 0℃and triphenylchloromethane (53 g,0.192 mol) was added and the reaction was allowed to proceed at 25℃for 16 hours. Pyridine was removed by concentration under reduced pressure, and the residue was purified by column chromatography to give 48g of an off-white solid in 88% yield.

Example 3.2

The compound (45 g,0.107 mol) prepared in example 3.1 was added to pyridine (500 mL) under nitrogen atmosphere and dissolved with stirring. Cooled to 0℃and acetic anhydride (500 g,4.09 mol) was added thereto, and the temperature was raised to 25℃to react for 16 hours. Pyridine and acetic anhydride were removed by concentration under reduced pressure, the residue was dissolved in ethyl acetate (500 mL), washed successively with 1mol/L dilute hydrochloric acid (500 mL. Times.2), saturated sodium bicarbonate solution (500 mL) and saturated sodium chloride solution (500 mL), and the organic phase was concentrated under reduced pressure to give 58g of an off-white solid in 92% yield.

Example 3.3

The compound (55 g,0.093 mol) in example 3.2 was added to methanol (500 mL), cooled to 0℃and reacted with 2mol/L hydrochloric acid (150 mL) for 4 hours. Concentrating under reduced pressure until no methanol flows out, extracting the residue with ethyl acetate (250 mL x 2), washing the combined organic phases with water (250 mL), saturated sodium bicarbonate (250 mL) and saturated sodium chloride solution (250 mL), and purifying the residue after concentrating under reduced pressure by column chromatography to obtain 29g of white solid with a yield of 90%.

Example 3.4

The compound produced in example 11 (25 g,0.072 mol) and N, N-dimethylaminopyridine (17.5 g,0.144 mol) were dissolved in dry dichloromethane (500 mL), cooled to-25℃and slowly added diethylaminosulfur trifluoride (46 g, 0.284 mol), and the reaction was maintained at this temperature for 2 hours and heated to 25℃for 12 hours. Cooled to 0 ℃, quenched with methanol (5 mL), washed with water (500 mL) and the residue concentrated under reduced pressure was purified by column chromatography to give 15.9g of a white solid in 63% yield. m/z (MH+) 351,1H NMR (400 MHz, CDCl) ₃ )，δ6.12&5.84(s，1H)，5.46&5.32(t，1H)，5.31(t，1H)，5.37&5.16(dd，1H)，4.60(m，1H)，4.48(m，1H)，4.06&3.84 (m, 1H), 2.21 (s, 3H), 2.14 (s, 3H), 2.11 (s, 3H), 2.02 (s, 3H). HNMR spectra are shown in FIG. 13.

Example 4: antibody expression in the presence of 2-fluoro-mannose analogues

To determine the effect of 2-fluoro-mannose analogs on antibody glycosylation, the CHODG44 cell line expressing humanized trastuzumab was expressed at 5X 10 ⁶ Each/mL was incubated in 30mL CHO medium at 37℃under 5% CO2 while shaking in 150mL shake flasks at 125 RPM. Insulin-like growth factor (IGF), 50. Mu.M of 2-fluoro-mannose analog (prepared in example 1) was added to CHO medium. Different cultures were supplemented with 0.1% by volume of the 50mM 2-fluoro-mannose analogue and 0.1% by volume of the 50mM 2-fluoro-fucose on day 3, respectively. On

days

3,5,7,9, 11, feed cultures were performed, respectively. Conditioned medium was collected by passing the medium through a 0.2 μm filter on day 13. 1mL was used for expression level-HPLC detection. The culture density of the cells is shown in FIG. 1, the culture activity of the cells is shown in FIG. 2, and the cell expression level of the humanized trastuzumab in the culture supernatant is shown in FIG. 3.

Conditioned medium was applied to protein a column pre-equilibrated with 1X Phosphate Buffered Saline (PBS), ph7.4 for antibody purification. After washing the column with 20 column volumes of 1 XPBS, the antibody was eluted with 5 column volumes of Immunopure IgG elution buffer. 10% by volume of 1M tris pH8.0 was added to the eluted fraction. Samples were dialyzed overnight against 1 XPBS.

To identify glycosylation patterns in purified trastuzumab antibodies derived from example 4 by LC-MS (Q-Tof) analysis of antibodies produced by expression in the presence of 2-fluoro-mannose analogs, 10 μl of 100mMDTT to 90 μl of 1mg/mL antibody in PBS was added and incubated for 15 min at 37 ℃ to reduce the inter-chain disulfide bonds of the antibodies. This solution (20. Mu.L) was injected into a PLRP-S HPLC column (PL company (Polymer Laboratories; amerst, mass.)) and the following gradient was run: solvent a,0.05% tfa in water; solvent B,0.035% tfa in acetonitrile; the linear gradient was 70-50% A0-12.5 minutes. The HPLC effluent was analyzed by electrospray ionization Q-Tof mass spectrometer (Waters, milford, mass.) with a cone voltage of 35V and a collection m/z of 500-4000. The heavy chain data was deconvolved with the MaxEnt1 function of masslynx4.0.

Example 5: capillary electrophoresis of oligosaccharides

To further characterize the glycans on the antibodies obtained from example 4, capillary electrophoresis was performed. The antibody sample was exchanged into water with buffer. 300 μg of each sample was treated overnight with PNGaseF at 37℃to release oligosaccharides. The supernatant was dried, the oligosaccharides were labeled overnight with APTS (8-aminopyrene-1, 3, 6-trisulfonic acid trisodium salt) in 1M sodium cyanoborohydride/THF at 22 ℃ C.,. The labeled oligosaccharides were diluted with water and analyzed by capillary electrophoresis in an N-CHO coated capillary (BC Co. (Beckman Coulter)) with Beckman Coulter PA-800. Samples were injected at 0.5psi for 8 seconds and separated at 30kV for 15 minutes. The labeled oligosaccharides were detected with laser-induced fluorescence (LFI) at excitation wavelength 488. The emitted fluorescence was detected at 520. Lamda.).

The antibody sample was also treated with beta-galactosidase to remove galactose. The antibody sample was exchanged into water with buffer. 300 μg of each sample was treated overnight with PNGaseF at 37℃to release oligosaccharides. The oligosaccharides were dried, then labeled with APTS in 1M sodium cyanoborohydride/THF at 22℃overnight, the labeled oligosaccharides were diluted with water and analyzed by capillary electrophoresis in an N-CHO coated capillary (BC Co.) using Beckman Coulter PA-800, running in 40mM EAA, 0.2% HPMC, pH 4.5. Samples were injected at 0.5psi for 8 seconds and separated at 30kV for 15 minutes. The labeled oligosaccharides were detected with laser-induced fluorescence (LFI) at excitation wavelength 488. The emitted fluorescence was detected at 520. Lambda..

The analysis of the data for capillary electrophoresis is shown in figures 4-6 of the specification. Referring to fig. 4, an electropherogram of glycans from a control humanized trastuzumab is shown. Description figure 5 shows an electrophoretogram of glycans from humanized trastuzumab antibodies produced from host cells grown in the presence of 2-fluoro-mannose. Description figure 6 shows an electrophoretogram of glycans from humanized trastuzumab antibodies produced from host cells grown in the presence of 2-fluoro-fucose. Figure 4 about 96% of the oligosaccharides are core fucosylated, while less than 2% are non-core fucosylated. Figure 5 about 70% of the oligosaccharides are core fucosylated, while about 23% are non-core fucosylated. Figure 6 about 60% of the oligosaccharides are core fucosylated, while about 18% are non-core fucosylated, about 22% of the oligosaccharides are Man5. Comparing fig. 4 and fig. 5, it was found that the content of core fucosylation G0F was significantly reduced after addition of 2-fluoro-mannose. Comparing fig. 4 and fig. 6, it was found that the content of core fucosylation G0F was significantly reduced after the addition of 2-fluoro-fucose. Comparing fig. 4, 5 and 6, it was found that the content of core fucosylation G0F was significantly reduced after addition of both 2-fluoro-mannose and 2-fluoro-fucose, but that the Man5 ratio in the oligosaccharides was significantly increased after addition of 2-fluoro-fucose, whereas the core fucosylation ratio was reduced in the oligosaccharides without other effects after addition of 2-fluoro-mannose.

Example 6: expression of other antibodies in the presence of 2-F-mannose analogues

To demonstrate the effect on other antibody glycosylation, antibodies were expressed from the following cell lines:

bevacizumab, CHOS cells; anti-CD 20 antibody, CHOK1 cells; and cetuximab, SP2/0 and CHO-K1 cells. Briefly, the cell lines were first cultured at 5X 10 ⁶ Each/mL was incubated in 30mL CHO medium at 37℃under 5% CO2 while shaking in 150mL shake flasks at 125 RPM. Insulin-like growth factor (IGF), 50. Mu.M 2-fluoro-mannose (obtained in example 1) was added to CHO medium. Different cultures were supplemented with 0.1% by volume of 2-fluoro-mannose containing 50mM on day 3. On

days

3,5,7,9, 11, feed cultures were performed, respectively. Conditioned medium was collected by passing the medium through a 0.2 μm filter on day 13. 1mL was used for expression level-HPLC detection.

Antibody purification was performed by applying conditioned medium to protein a column pre-equilibrated with 1X Phosphate Buffered Saline (PBS), ph 7.4. The antibodies were eluted with 5 column volumes of Immunopure IgG elution buffer (Pierce Biotechnology, rockwell, ill.). 10% by volume of 1M tris pH8.0 was added to the eluted fraction. Samples were dialyzed against 1 XPBS overnight.

The Qtof analysis of the antibodies showed similar results to example 4. The content of core fucosylation G0F of the oligosaccharides was significantly reduced relative to the antibody heavy chain produced from the host cells grown in the absence of 2-F-mannose, observed for antibodies obtained from cells grown in the presence of 2-fluoro-fucose.

Example 7: antibody expression in the presence of 2-chloro-mannose analogues

To determine the effect of 2-chloro-mannose on antibody glycosylation, the CHO DG44 cell line expressing humanized trastuzumab was expressed at 5×10 ⁶ Each/mL was incubated in 30mL CHO medium at 37℃under 5% CO2 while shaking in 150mL shake flasks at 125 RPM. Insulin-like growth factor (IGF), 50. Mu.M 2-chloro-mannose (obtained in example 2) was added to CHO medium. Different cultures were supplemented with 0.1% by volume of 2-fluoro-mannose containing 50mM on day 3. On

days

The Qtof analysis of the antibodies showed similar results to example 4. The content of core fucosylation G0F of oligosaccharides was significantly reduced relative to antibodies produced from host cells grown in the absence of 2-chloro-mannose, observed for antibodies obtained from cells grown in the presence of 2-chloro-fucose.

Example 8: antibody expression in the presence of 6-fluoro-mannose analogues

To determine the effect of 6-fluoro-mannose on antibody glycosylation, the CHO DG44 cell line expressing humanized trastuzumab was expressed as 5×10 ⁶ Each/mL was incubated in 30mL CHO medium at 37℃under 5% CO2 while shaking in 150mL shake flasks at 125 RPM. Pancreas addition to CHO MediumIsland-like growth factor (IGF), 50. Mu.M 6-fluoro-mannose (obtained in example 3). Different cultures were supplemented with 0.1% volume of 6-fluoro-mannose containing 50mM on day 3. On

days

The Qtof analysis of the antibodies showed similar results to example 4. The content of core fucosylation G0F of oligosaccharides was significantly reduced relative to antibodies produced from host cells grown in the absence of 6-fluoro-mannose, observed for antibodies obtained from cells grown in the presence of 6-fluoro-fucose.

Example 9: antibody expression in the presence of 2-fluoro-mannose analogues in an effective concentration range

To determine the effective concentration of 2-fluoro-mannose derivatives on antibody glycosylation, the humanized trastuzumab expressing CHO DG44 cell line was expressed at 5×10 ⁶ Each/mL was incubated in 30mL CHO medium at 37℃under 5% CO2 while shaking in 150mL shake flasks at 125 RPM. Insulin-like growth factor (IGF) was added to CHO medium, and 5. Mu.M, 100. Mu.M, and 500. Mu.M of 2-fluoro-mannose (obtained in example 1) were added, respectively. Different cultures were supplemented with different concentrations of 2-fluoro-mannose at day 3. On

days

3,5,7,9, 11, feed cultures were performed, respectively. Conditioned medium was collected by passing the medium through a 0.2 μm filter on day 13.

The Qtof analysis of the antibodies showed the results shown in FIGS. 7-10 of the specification. Figure 7 is a blank, where approximately 95% of the oligosaccharides in the antibody are core fucosylated. FIG. 8 shows the presence of 5. Mu.M of 2-fluoro-mannose expressed antibodies, of which about 70% of the oligosaccharides are core fucosylated. FIG. 9 is a graph showing the presence of 100. Mu.M of 2-fluoro-mannose expressed antibodies, in which about 55% of the oligosaccharides are core fucosylated. FIG. 10 shows the presence of 500. Mu.M of 2-fluoro-mannose expressed antibodies, of which about 15% of the oligosaccharides are core fucosylated.

Example 10: antibody expression in the presence of 2-chloro-mannose analogues in an effective concentration range

To determine the effective concentration of 2-chloro-mannose on antibody glycosylation effect, the humanized trastuzumab expressing CHO DG44 cell line was expressed at 5X 10 ⁶ Each/mL was incubated in 30mL CHO medium at 37℃under 5% CO2 while shaking in 150mL shake flasks at 125 RPM. Insulin-like growth factor (IGF) was added to CHO medium, and 5. Mu.M, 100. Mu.M, 500. Mu.M 2-chloro-mannose was added, respectively (prepared as described in example 1). Different cultures were supplemented with different concentrations of 2-chloro-mannose at day 3. On

days

The Qtof analysis of the antibodies showed similar results to example 9.

Example 11: antibody expression in the presence of 6-fluoro-mannose analogues in the effective concentration range

To determine the effective concentration of 6-fluoro-mannose on antibody glycosylation effect, the humanized trastuzumab expressing CHO DG44 cell line was expressed at 5X 10 ⁶ Each/mL was incubated in 30mL CHO medium at 37℃under 5% CO2 while shaking in 150mL shake flasks at 125 RPM. Insulin addition to CHO MediumLike growth factor (IGF), 5. Mu.M, 100. Mu.M, 500. Mu.M 6-fluoro-mannose (prepared as described in example 1) was added, respectively. Different cultures were supplemented with different concentrations of 6-fluoro-mannose at day 3. On

days

The Qtof analysis of the antibodies showed similar results to example 9.

Example 12: antibody expression in the presence of 2-fluoro-mannose analogues in different media

To demonstrate the effect on antibody glycosylation in the presence of 2-fluoro-mannose in different media, the following media were selected to culture cell lines expressing antibodies:

CD FortiCHO ^TM Medium(Gibco ^TM ) Siemens Feishul technologies;

CD CHO Fusion (Sigma-Aldrich Co.); OPM-CHO CD07 (Shanghai Olympic Biotechnology Co., ltd.). CHO cells expressing humanized trastuzumab were selected. Briefly, the cell lines were first cultured at 5X 10 ⁶ Each/mL was incubated in 30mL CHO medium at 37℃under 5% CO2 while shaking in 150mL shake flasks at 125 RPM. Insulin-like growth factor (IGF), 50. Mu.M 2-fluoro-mannose (prepared as described in example 1) was added to CHO medium. Different cultures were supplemented with 0.1% by volume of 2-fluoro-mannose containing 50mM on day 3. On

days

The scope of the invention is not limited by the specific embodiments described herein. In addition to those described herein, variations of the present invention will become apparent to those skilled in the art upon reading the description and drawings of the present patent. Such variations are intended to fall within the scope of the appended claims. Any step, element, embodiment, feature or aspect of the invention may be used in any combination unless clearly and substantially different from what is described herein. All patent applications, scientific publications, accession numbers, etc. cited in this application are hereby incorporated by reference in their entirety for all purposes as if each were individually set forth.

Claims

1. Use of a mammalian cell culture medium for the preparation of an antibody that reduces core fucosylation modification, characterized in that: the medium is an animal protein free medium and comprises an effective amount of one or any combination of mannose analogs of the formula:

2. use of a mammalian cell culture medium according to claim 1 for the preparation of an antibody that reduces core fucosylation modifications, wherein: the volume of the medium is at least 10 liters.

3. Use of a mammalian cell culture medium according to claim 1 for the preparation of an antibody that reduces core fucosylation modifications, wherein: one or more mannose analogues or biologically acceptable salts thereof are added to the medium to maintain an effective concentration thereof.

4. Use of a mammalian cell culture medium according to claim 1 for the preparation of an antibody that reduces core fucosylation modifications, wherein: the medium is serum-free.

5. Use of a mammalian cell culture medium according to claim 1 for the preparation of an antibody that reduces core fucosylation modifications, wherein: the medium is free of added fucose or mannose.

6. Use of a mammalian cell culture medium according to claim 1 for the preparation of an antibody that reduces core fucosylation modifications, wherein: the culture medium is a powder or a liquid.

7. A method of reducing core fucosylation modified antibodies, the method comprising the steps of:

2) Isolating the antibody from the cell;

the mannose analog is selected from the mannose analog of claim 1, or a biologically acceptable salt thereof, wherein the core fucosylation of the antibody is lower than that of an antibody from a host cell cultured in the absence of the mannose analog; the cell is a recombinant host cell or a hybridoma cell.

8. The method of reducing core fucosylation modified antibody according to claim 7, wherein: the recombinant host cell refers to Chinese hamster ovary cells, NS0 or SP2/0.

9. The method of reducing core fucosylation modified antibody according to claim 7, wherein: the host cells are cultured in batch, fed-batch, continuous fed-or continuous perfusion medium, or in microcarriers.

10. The method of reducing core fucosylation modified antibody according to claim 7, wherein: the culture medium is a mammalian cell culture medium for producing the antibody for reducing core fucosylation modification according to claim 1.

11. The method of reducing core fucosylation modified antibody according to claim 7, wherein: the antibody is an intact antibody, an IgG1, a single chain antibody or a fusion protein comprising an Fc domain.

12. The method of reducing core fucosylation modified antibody according to claim 7, wherein: the mannose analogues are a composition formed by mixing different mannose analogues, and the composition is dissolved in a solvent for addition at a proper concentration; the composition is added to a dry or liquid medium and then present in the medium of the host cell at a suitable concentration.