WO2019196697A1

WO2019196697A1 - Method and composition for reducing core fucosylation of antibody

Info

Publication number: WO2019196697A1
Application number: PCT/CN2019/080902
Authority: WO
Inventors: 张莹; 肖志华
Original assignee: 上海颢哲信息科技有限公司
Priority date: 2018-04-12
Filing date: 2019-04-02
Publication date: 2019-10-17
Also published as: CN110373374A; CN110373374B

Abstract

A small molecule mannose analog for the manufacture of a recombinant antibody having complex N-linked glycans but reduced core fucosylation. Said mannose analog is typically taken in by host cells (e.g. by means of active transport or passive diffusion), inhibiting the production of GDP fucose. As mannose is being used as the synthetic substrate, the cost is extremely low. Moreover, by using said method, the core fucosylation will be reduced while the high mannose glycoform ratio of N-linked glycans will not be affected.

Description

Method and composition for reducing antibody core fucosylation

Technical field

The present invention relates to a method for reducing core fucosylation of antibodies and a technical field of compounds used.

Background technique

Recombinant therapeutic proteins are produced by a number of different methods. A 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 yield. Monoclonal antibodies or antibody derivatives are one type of recombinant protein that requires a balanced consideration of high yields and product consistency when selecting cells for production.

The Fc fragment exerts an effector function by binding the active region to the receptor. The N-glycosylation of IgG is located in the consensus sequence of the CH2 region of the Fc fragment (Asn297-X-Ser/Thr, X may be any amino acid residue other than proline) and is covalently bound to the antibody via an amide bond. It was found by crystal X-ray diffraction that proteins interact with sugars in a peer-to-peer manner to affect each other's conformation. In addition to glycosylation of the Fc fragment, 30% of the IgG is N-glycosylated in the Fab fragment, which has a beneficial, deleterious or neutral effect on the efficacy of the antibody molecule. The Fc glycosylated sugar molecule has a complex double-antenna core structure composed of two pentose molecules, mannose (Man) and N-acetylglucosamine (GlcNAc), which additionally contain, in addition to the core structure. Different numbers of sugar molecules, such as fucose (Fuc), mannose, N-acetylglucosamine, galactose (Gal), aliquots of N-acetylglucosamine and sialic acid. The length of the sugar chain, the bifurcation pattern, and the change in the monosaccharide sequence result in the complexity of glycosylation modification. In addition, due to various enzymes, the N-glycosylated pentasaccharide core structure is divided into three categories: high mannose type, heterozygous type and complex type.

The above formula is a molecular structural heterogeneity structural formula of glycan modified by immunoglobulin Asn297.

The fucose core oligosaccharide is a biosynthesis of a fucose residue transported from GDP-Fuc by a 1,6-fucosyltransferase in a transport Golgi apparatus. The structure analysis of Fc fragments modified with fucose and fucose showed that there were differences in electron densities between Asp280 and Asn297 residues; the hydration patterns of Tyr296 residues also differed. The afucose-modified human IgG expressed by the Lec13 mutant cells showed a 50-fold increase in affinity with FcγRIIIa and a 100-fold increase in ADCC action. The core-free fucose makes Asn162 and Fc glycans in the FcγRIIIa receptor have higher affinity and thus stronger binding. The steric hindrance of fucose blocks this interaction. This finding led to an interest in engineered cell lines to create antibodies with reduced core fucosylation.

Methods for 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-alginyltransferase) is inactivated. FUT8 catalyzes the transfer of fucose residues from GDP-fucose to the 6-position of the Asn-linked (N-linked) GlcNac of the N-glycan. FUT8 is said to be the only enzyme responsible for the addition of fucose to the N-linked biantennary saccharide Asn297. 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 decreased mRNA transcription levels or complete knockdown of gene expression.

In addition to engineered cell lines, small molecule inhibitors using enzymes that act on the glycosylation pathway are also included. Inhibitors, such as catanospermine, act early on the glycosylation pathway to produce antibodies with immature glycans (eg, high levels of mannose) and low fucosylation levels. Antibodies produced by these methods typically lack the complex N-linked glycan structure associated with mature antibodies.

A small molecule fucose analog for the production of recombinant antibodies with complex N-linked glycans but reduced core fucosylation is disclosed in the patent CN200980124932.4 by the Seattle Genetics Corporation. The patent reduces core fucosylation levels by adding fucose analogs during host cell production, which reduces core fucosylation of antibodies, but the price of the raw fucose is very high and is reduced using this method. At the same time as the core fucosylation, the high mannose glycoform ratio of the antibody N-linked glycan is increased. An IgG molecule containing a high mannose residue has a shorter serum half-life than an IgG molecule containing a double antenna type core structure, which may cause an increase in immunogenicity and is not conducive to drug treatment.

Summary of the invention

The object of the present invention is to overcome the above-mentioned drawbacks of the prior art and to provide another method for reducing the core fucosylation of an antibody which is low in cost compared to the prior art and has a high mannose for N-linked glycans. The glycoform ratio has less effect.

Another object of the present invention is to provide a mannose analog or a biologically acceptable salt or solvate thereof for use in a method for reducing core fucosylation of an antibody, which can be achieved by the preparation method of the present invention.

The invention also provides a mammalian cell culture medium for the preparation of antibodies that reduce core fucosylation modifications.

The methods and compositions of the present invention are presented, in part, based on the results described in the Examples, which show that host cells expressing antibodies or antibody derivatives can produce core fucosylation in the presence of a mannose analog. An antibody that reduces (i.e., decreases in fucosylation of N-acetylglucosamine linked to the sugar chain of the N-acetylglucosamine linked to the Fc region by a complex N-glycoside-linked sugar chain). Such antibodies and antibody derivatives may have an enhanced effector function (ADCC) compared to antibodies or antibody derivatives made from such host cells cultured in the absence of a mannose analog or fucose analog.

In this application

The term "antibody" means: (a) an immunologically active portion of an immunoglobulin polypeptide and an immunoglobulin polypeptide, ie, a polypeptide of an immunoglobulin family or a portion thereof, an antigen comprising an immunospecific binding domain specific antigen (eg, CD20). A binding site and an Fc domain comprising a complex N-glycoside-linked sugar chain, or (b) a conservative substitution derivative of such an immunoglobulin polypeptide or a fragment of an immunospecific binding antigen (eg, CD20).

"Antibody derivative" means an antibody (including an antibody fragment) as defined above, or an Fc domain or Fc region of an antibody comprising a complex N-glycosidic linked sugar chain, modified by covalent attachment of a heterologous molecule, eg, by Binding to a heterologous polypeptide (eg, a ligand binding domain of a heterologous protein), or through glycosylation (except for core fucosylation), deglycosylation (except for non-core fucosylation), acetylation , phosphorylation or other modifications that are normally unrelated to the antibody or Fc domain or Fc region.

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

The term "Fc region" denotes a constant region of an antibody, eg, a CH1-strand-CH2-CH3 domain, optionally having a CH4 domain, or a conservatively substituted derivative of such an Fc region.

The term "Fc domain" refers to the constant region domain of an antibody, eg, a CH1, hinge, CH2, CH3 or CH4 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 "inhibiting" refers to a detectable decrease, or complete inhibition.

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

The technical solutions provided by the present application are as follows:

A mannose analog of the formula: or a biologically acceptable salt or solvate thereof:

Wherein R1 to 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 -OH and -OAc.

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

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

Preferably, the medium has a volume of at least 10 liters.

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

Preferably, the medium is an animal protein free medium; the medium is serum free; the medium does not contain added fucose or mannose. More preferably, the medium is free of animal protein, serum free, and contains no added fucose or mannose.

Further, the medium is a powder or a liquid.

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

1) cultivating 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, and the sugar chain is linked to the Fc domain via N-acetylglucosamine at its reducing end;

The mannose analog is selected from any of the above-described mannose analogs or biologically acceptable salts or solvates thereof; wherein the core fucosylation of the antibody is lower than An antibody to a host cell cultured in the mannose analog.

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

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

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

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

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

The beneficial effects of the invention:

The ratio of fucose in the N-glycosylation of the Fc portion of the antibody has an important influence on the ADCC activity of the antibody. The Seattle Gene Company can efficiently inhibit fucosylation by synthesizing fucose analogs (Patent Grant Bulletin No. CN 102076865 B), but it has been found during practice that inhibition of fucosylation is accompanied by an increase in high mannose glycoforms. High-mannose glycosylated antibodies have a short serum half-life and may cause an increase in immunogenicity, which should be avoided in the process. Therefore, the inhibition process causes changes in the three ratios of fucosylation, non-fucosylation and high-mannose antibody, which is not conducive to precise control of the regulation of glycoform ratio, thereby affecting the therapeutic properties of antibodies and antibody derivatives.

This patent provides a mannose analog inhibitor that works at very low concentrations, while the precursor of the synthetic mannose inhibitor is mannose, and the price of mannose is much lower than fucose. , is conducive to cost optimization. Mannose analogs and fucose analogs, especially halogen substitutes, are not suitable for metabolism, so the concentration can be maintained in the medium for a long period of time, and the effective concentration can be maintained at a lower concentration.

The data show that the synthesized 2-F-Mannose can inhibit more than 50% of the fucose glycosylation at a concentration of 5 ppm, fully meeting the industrialization requirements, and is completely equivalent to the inhibition efficiency of the same concentration of 2-F-Fucose. Synthetic 2-Cl-Mannose and 6-F-Mannose can achieve similar effects. It was demonstrated that mannose analogs and fucose analogs can achieve an inhibitory effect in various configurations, reducing fucosylation of antibodies and antibody derivatives.

In summary, the present invention provides small molecule mannose analogs for the manufacture of recombinant antibodies having complex N-linked glycans but reduced core fucosylation, which are typically taken up by host cells (eg, by Active transport or passive diffusion), inhibiting the production of GDP fucose. The method provided by the invention has extremely low price due to the use of mannose for the synthetic substrate; and the method can reduce the core fucosylation without affecting the high mannose glycoform ratio of the N-linked glycan. . Moreover, the mannose analogs provided by the present invention also have potential for tumor treatment.

These and other aspects of the present invention can be more fully understood from the following detailed description of the appended claims.

DRAWINGS

Figure 1 is a graph showing the comparison of the culture density of the cells of Example 4.

Fig. 2 is a graph showing the comparison of the culture viability of the cells of Example 4.

Fig. 3 is a graph showing the comparison of the cell expression levels of the humanized trastuzin monoclonal antibody in the culture supernatant of Example 4.

4 is a data analysis diagram of capillary electrophoresis of Example 5, showing an electrophoretogram of glycans from a control humanized trastuzole monoclonal antibody.

Figure 5 is a data analysis diagram of capillary electrophoresis of Example 5, showing an electropherogram of a glycan from a humanized trastuzole monoclonal antibody, which is a host grown from the presence of a 2-fluoro-mannose analog. Made by cells.

Figure 6 is a data analysis diagram of capillary electrophoresis of Example 5, showing an electropherogram of a glycan from a humanized trastuzole monoclonal antibody grown from the presence of a 2-fluoro-fucose analog Made by host cells.

Figure 7 is the result of Qtof analysis of the antibody, which is a blank control panel in which approximately 95% of the oligosaccharides in the antibody are core fucosylated.

Figure 8 is a Qtof analysis display of antibodies expressed in the presence of 5 μM 2-fluoro-mannose analog.

Figure 9 is a Qtof analysis display of antibodies expressed in the presence of 100 μM 2-fluoro-mannose analog.

Figure 10 is a Qtof analysis display of antibodies expressed in the presence of 500 μM 2-fluoro-mannose analog.

Figure 11 is a H NMR spectrum of the product 2-fluoro-mannose analog of Example 1.

Figure 12 is a H NMR spectrum of the product 2-chloro-mannose analog of Example 2.

Figure 13 is a H NMR spectrum of the product 6-fluoro-mannose analog of Example 3.

detailed description

The materials and instruments used in the present invention are all conventional products commercially available in the art unless otherwise specified.

Example 1: Synthesis of 2-fluoro-mannose analog, 2-deoxy-2-fluoro-1,3,4,6-tetra-oxy-acetyl-D-mannose

Example 1.1

D-mannose (50 g, 0.278 mol) was added to pyridine (500 mL) under nitrogen and dissolved with stirring. After cooling to 0 degree, acetic anhydride (500 g, 4.09 mol) was added, and the mixture was heated to 25 degrees for 16 hours. Concentrated under reduced pressure to remove pyridine and acetic anhydride, and the residue was dissolved with ethyl acetate (500 mL), and then 1 mol/L diluted hydrochloric acid (500mL*2), saturated sodium hydrogen carbonate solution (500mL) and saturated sodium chloride solution (500mL) The organic phase was concentrated under reduced pressure to give 97.5 g,yield of white solid.

Example 1.2

The compound obtained in Example 1.1 (97.5 g, 0.25 mol) was added to dichloromethane (1500 mL) under a nitrogen atmosphere, stirred and dissolved, and cooled to 0. A 40% aqueous solution of hydrobromic acid (400 mL) was slowly added, and the mixture was heated to 25 degrees for 3 hours. The organic layer was washed with EtOAc (EtOAc) EtOAc (EtOAc)

Example 1.3

Under a nitrogen atmosphere, zinc powder (165 g, 2.54 mol), N-methylimidazole (32 mL, 0.39 mol) and ethyl acetate (1300 mL) were added to the reaction mixture, and the mixture was heated to reflux. The compound (82 g, 0.2 mol) was dissolved in ethyl acetate (325 mL), and added dropwise to the suspension, and the mixture was refluxed for 1 hour. Cool to 25 degrees and filter. The filtrate was washed successively with 1 mol/L of dilute hydrochloric acid (650 mL), saturated sodium hydrogen carbonate solution (650 mL) and saturated sodium chloride solution (650 mL), and the organic phase was concentrated under reduced pressure to give 36.2 g of white solid.

Example 1.4

The white-like solid obtained in Example 1.3 (15 g, 0.055 mol) was dissolved in N,N-dimethylformamide (125 mL) and water (125 mL), cooled to 0. 4-fluoro-1,4-diazabicyclo[2.2.2]octane bis(tetrafluoroborate) salt (39 g, 0.11 mol) was heated to 25 ° for 16 hours. The reaction mixture was extracted with ethyl acetate (250 mL*2), and the organic phase was combined and washed with water (250 mL) and brine (250 mL). After concentration, 13.6 g of a pale yellow oil was obtained in a yield of 80%.

Example 1.5

The pale yellow oil (13.6 g, 0.044 mol) obtained in Example 1.4 was added to pyridine (140 mL). After cooling to 0 degree, acetic anhydride (70 g, 0.69 mol) was added, and the mixture was heated to 25 degrees for 16 hours. Concentrated under reduced pressure to remove pyridine and acetic anhydride, and the residue was dissolved with ethyl acetate (150 mL), and then 1 mol/L diluted hydrochloric acid (150mL*2), saturated sodium hydrogen carbonate solution (150mL) and saturated sodium chloride solution (150mL) After washing, the organic phase was concentrated under reduced pressure and purified by EtOAc EtOAcjjjjjjjjjjj . m/z (MH+) 351, 1H NMR (400 MHz, CDCl ₃ ), δ 6.27 (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 Figure 11.

Example 2: Synthesis of 2-chloro-mannose analog, 2-deoxy-2-chloro-1,3,4,6-tetra-oxy-acetyl-D-mannose

Example 2.1

The white-like solid of Example 1.3 (15 g, 0.055 mol) was dissolved in N,N-dimethylformamide (125 mL) and water (125 mL), cooled to 0. The diimide (14.7 g, 0.11 mol) was heated to 25 degrees for 16 hours. The reaction mixture was extracted with ethyl acetate (250 mL*2), and the organic phase was combined and washed with water (250 mL) and brine (250 mL). After concentration, 12.7 g of a pale yellow oil was obtained, yield 71%.

Example 2.2

The pale yellow oil (12.7 g, 0.039 mol) obtained in Example 2.1 was added to pyridine (120 mL). After cooling to 0 degree, acetic anhydride (60 g, 0.59 mol) was added, and the mixture was heated to 25 degrees for 16 hours. Concentrated under reduced pressure to remove pyridine and acetic anhydride, and the residue was dissolved with ethyl acetate (120 mL), and then 1 mol/L diluted hydrochloric acid (120mL*2), saturated sodium hydrogen carbonate solution (120mL) and saturated sodium chloride solution (120mL) After washing, the organic phase was concentrated under reduced pressure. EtOAcjjjjjjjjjjjjj . m/z (MH+) 367, 1H NMR (400 MHz, CDCl ₃ ), δ 6.24 (s, 1H), 5.48 (t, 1H), 5.37 (dd, 1H), 4.40 (d, 1H), 4.21. , 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). The HNMR spectrum is shown in Figure 12.

Example 3: Synthesis of 6-fluoro-mannose analog, 6-deoxy-6-fluoro-1,2,3,4-tetra-oxy-acetyl-D-mannose

Example 3.1

D-mannose (23 g, 0.128 mol) was added to pyridine (200 mL) under nitrogen and dissolved with stirring. After cooling to 0 degree, triphenylchloromethane (53 g, 0.192 mol) was added, and the mixture was heated to 25 degrees for 16 hours. The pyridine was concentrated under reduced pressure.

Example 3.2

The compound obtained in Example 3.1 (45 g, 0.107 mol) was added to pyridine (500 mL) under a nitrogen atmosphere and stirred to dissolve. After cooling to 0 degree, acetic anhydride (500 g, 4.09 mol) was added, and the mixture was heated to 25 degrees for 16 hours. Concentrated under reduced pressure to remove pyridine and acetic anhydride, and the residue was dissolved with ethyl acetate (500 mL), and then 1 mol/L diluted hydrochloric acid (500mL*2), saturated sodium hydrogen carbonate solution (500mL) and saturated sodium chloride solution (500mL) After washing, the organic phase was concentrated under reduced pressure to give white crystals (yield: 58%).

Example 3.3

The compound (55 g, 0.093 mol) in Example 3.2 was added to methanol (500 mL), cooled to 0°, and then reacted with 2 mol/L hydrochloric acid (150 mL) for 4 hours. The organic layer was washed with water (250 mL), and then the organic layer was washed with water (250mL), saturated sodium bicarbonate (250mL) and saturated sodium chloride solution (250mL). The residue after concentration under reduced pressure was purified by column chromatography to yield white solid (yield: 29%).

Example 3.4

The compound prepared 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 After the ethylaminosulfur trifluoride (46 g, 0.285 mol) was kept at this temperature for 2 hours, the temperature was raised to 25 degrees for 12 hours. The mixture was cooled to 0. EtOAc (5 mL) was evaporated. m/z (MH+) 351, 1H NMR (400 MHz, CDCl ₃ ), δ 6.12 & 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). The HNMR spectrum is shown in Figure 13.

Example 4: Antibody expression in the presence of 2-fluoro-mannose analog

To determine the effect of 2-fluoro-mannose analogs on antibody glycosylation, CHO DG44 cell line expressing humanized trastuzin monoclonal antibody was at 5×10 ⁶ cells/mL in 30 mL CHO medium at 37 °C. Incubate under 5% CO2 while shaking at 125 RPM in a 150 mL shake flask. Insulin-like growth factor (IGF), 50 μM 2-fluoro-mannose analog (prepared in Example 1) was added to the CHO medium. On day 3, different cultures were supplemented with 0.1% by volume of 50 mM 2-fluoro-mannose analog and 0.1% by volume of 50 mM 2-fluoro-fucose. On the 3rd, 5th, 7th, 9th, and 11th days, the fed culture was separately performed. The conditioned medium was collected through a 0.2 μm filter in the 13th Angel medium. 1 mL was taken for expression amount-HPLC detection. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the culture density of cells, and Fig. 2 shows the culture activity of cells, and Fig. 3 of the specification shows the cell expression level of humanized trastuzin monoclonal antibody in the culture supernatant.

The conditioned medium was applied to a Protein A column pre-equilibrated with IX phosphate buffered saline (PBS), pH 7.4 for antibody purification. The column was washed with 20 column volumes of IX PBS and the antibody was eluted with 5 column volumes of Immunopure IgG elution buffer. A 10% volume of 1 M Tris pH 8.0 was added to the eluted fraction. The samples were dialyzed against IX PBS overnight.

LC-MS (Q-Tof) analysis of antibodies produced by expression in the presence of 2-fluoro-mannose analogs to identify glycosylation patterns in purified trastuzole monoclonal antibodies from Example 4, adding 10 μL of 100 mMDTT The antibody interchain disulfide bond was reduced by 90 μL of 1 mg/mL antibody in PBS and incubated at 37 ° C for 15 minutes. This solution (20 μL) was injected into a PLRP-S HPLC column (Polymer Laboratories; Amherst, MA) and the following gradient was run: solvent A, 0.05% TFA in water; solvent B, 0.035% TFA in acetonitrile; linear gradient 70-50% A0-12.5 min. The HPLC effluent was analyzed by electrospray ionization Q-Tof mass spectrometer (Waters, Milford, MA) with a cone voltage of 35 V and m/z 500-4000 was collected. The heavy chain data was deconvoluted using MassLynx 4.0's MaxEnt1 function.

Example 5: Capillary Electrophoresis of Oligosaccharides

To further characterize the glycan on the antibody obtained in Example 4, capillary electrophoresis was performed. Antibody samples were exchanged in buffer with water. 300 μg of each sample was treated with PNGaseF at 37 ° C overnight to release oligosaccharides. The cold methanol ((-20 ° C) was added and centrifuged at 14,000 rpm for 10 minutes to remove the protein component in the sample. The supernatant was dried, and the oligosaccharide was APTS (8-aminoindole-1,3,6-trisulphonic acid). The trisodium salt) was labeled overnight in 1 M sodium cyanoborohydride/THF at 22 ° C. The labeled oligosaccharide was diluted with water and used in an N-CHO coated capillary (Beckman Coulter) with Beckman Coulter PA- The sample was analyzed by capillary electrophoresis. The sample was injected at 0.5 psi for 8 seconds and separated at 30 kV for 15 minutes. The labeled oligosaccharide was detected by laser induced fluorescence (LFI) at an excitation wavelength of 488 λ. The emitted fluorescence was detected at 520 λ.

Antibody samples were also treated with beta-galactosidase to remove galactose. Antibody samples were exchanged in buffer with water. 300 μg of each sample was treated with PNGaseF at 37 ° C overnight to release oligosaccharides. The methanol component was removed by adding cold methanol ((-20 ° C) and centrifugation at 14,000 rpm for 10 minutes. The supernatant was dried, resuspended in water and treated with β-galactosidase. The oligosaccharides were dried and then Labeled overnight with APTS in 1 M sodium cyanoborohydride/THF at 22 ° C. The labeled oligosaccharides were diluted with water and analyzed by capillary electrophoresis in a N-CHO coated capillary (BC) using Beckman Coulter PA-800. 40 mM EACA, 0.2% HPMC, pH 4.5. The sample was injected at 0.5 psi for 8 seconds and separated at 30 kV for 15 minutes. The labeled oligosaccharides were detected by laser induced fluorescence (LFI) at an excitation wavelength of 488 λ. The emitted fluorescence was detected at 520 λ.

Data analysis of capillary electrophoresis is shown in Figures 4-6 of the specification. Referring to Figure 4, an electropherogram of glycans from a control humanized trastuzole monoclonal antibody is shown. Figure 5 of the specification shows an electropherogram of a glycan from a humanized trastuzole monoclonal antibody produced from a host cell grown in the presence of 2-fluoro-mannose. Figure 6 of the specification shows an electropherogram of a glycan from a humanized trastuzole monoclonal antibody produced from a host cell grown in the presence of 2-fluoro-fucose. Figure 4 shows that approximately 96% of the oligosaccharides are core fucosylated, while less than 2% are non-core fucosylated. Figure 5 shows that approximately 70% of the oligosaccharides are core fucosylated, while approximately 23% are non-core fucosylated. Figure 6 Approximately 60% of the oligosaccharides are core fucosylated, while approximately 18% are non-core fucosylated, and approximately 22% of the oligosaccharides are Man5. Comparing Fig. 4 with Fig. 5, it was found that the content of core fucosylated G0F was significantly reduced after the addition of 2-fluoro-mannose. Comparing Fig. 4 with Fig. 6, it was found that the content of core fucosylated G0F was significantly reduced after the addition of 2-fluoro-fucose. Comparing Fig. 4, Fig. 5 and Fig. 6, it was found that the content of core fucosylated G0F was significantly reduced after adding 2-fluoro-mannose and 2-fluoro-fucose, but adding 2-fluoro-fucoid After sugar, the proportion of Man5 in oligosaccharides was significantly increased, and after adding 2-fluoro-mannose, the proportion of core fucosylation in oligosaccharides was reduced, and there was no other effect. It can be seen that the 2-fluoro-mannose analog can inhibit the formation of high mannose type during the production of antibody-like glycoproteins.

Example 6: Expression of other antibodies in the presence of 2-F-mannose analogs

To confirm the effect on glycosylation of other antibodies, antibodies were expressed from the following cell lines:

Bevacizumab, CHOS cells; anti-CD20 antibody, CHOK1 cells; and cetuximab, SP2/0 and CHO-K1 cells. Briefly, cell lines first with 5 × 10 ⁶ cells / mL at 37 ℃, 5% CO2 in 30mL CHO cultured medium, while 150mL shake flasks shaking at 125RPM. Insulin-like growth factor (IGF), 50 μM 2-fluoro-mannose (obtained in Example 1) was added to the CHO medium. On day 3, different cultures were supplemented with 0.1% by volume of 50 mM 2-fluoro-mannose. On the 3rd, 5th, 7th, 9th, and 11th days, the fed culture was separately performed. The conditioned medium was collected through a 0.2 μm filter in the 13th Angel medium. 1 mL was taken for expression amount-HPLC detection.

The conditioned medium was applied to a protein column pre-equilibrated with 1X phosphate buffered saline (PBS), pH 7.4 for antibody purification. The antibody was eluted with 5 column volumes of Immunopure IgG Elution Buffer (Pierce Biotech, Rockford, Ill.). A 10% volume of 1 M Tris pH 8.0 was added to the eluted fraction. Samples were dialyzed against 1 x PBS overnight.

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

Example 7: Antibody expression in the presence of 2-chloro-mannose analogs

To determine the effect of 2-chloro-mannose on antibody glycosylation, CHO DG44 cell line expressing humanized trastuzin monoclonal antibody was at 5×10 ⁶ cells/mL in 30 mL CHO medium at 37 ° C, 5 Incubate under % CO2 while shaking at 125 RPM in a 150 mL shake flask. Insulin-like growth factor (IGF), 50 μM 2-chloro-mannose (obtained in Example 2) was added to the CHO medium. On day 3, different cultures were supplemented with 0.1% by volume of 50 mM 2-fluoro-mannose. On the 3rd, 5th, 7th, 9th, and 11th days, the fed culture was separately performed. The conditioned medium was collected through a 0.2 μm filter in the 13th Angel medium. 1 mL was taken for expression amount-HPLC detection.

The Qtof analysis of the antibody showed similar results to Example 4. An antibody obtained from cells grown in the presence of 2-chloro-fucose was observed relative to an antibody produced from a host cell grown in the absence of 2-chloro-mannose, and the core fucosylated G0F of the oligosaccharide was observed. The content is significantly reduced.

Example 8: Antibody expression in the presence of 6-fluoro-mannose analog

To determine the effect of 6-fluoro-mannose on antibody glycosylation, the CHO DG44 cell line expressing the humanized trastuzin monoclonal antibody was at 5 × 10 ⁶ /mL in 30 mL CHO medium at 37 ° C, 5 Incubate under % CO2 while shaking at 125 RPM in a 150 mL shake flask. Insulin-like growth factor (IGF), 50 μM of 6-fluoro-mannose (obtained in Example 3) was added to the CHO medium. On day 3, different cultures were supplemented with 0.1% by volume of 50 mM 6-fluoro-mannose. On the 3rd, 5th, 7th, 9th, and 11th days, the fed culture was separately performed. The conditioned medium was collected through a 0.2 μm filter in the 13th Angel medium. 1 mL was taken for expression amount-HPLC detection.

Conditioned medium was applied to a protein column pre-equilibrated with 1X phosphate buffered saline (PBS), pH 7.4 for antibody purification. The antibody was eluted with 5 column volumes of Immunopure IgG Elution Buffer (Pierce Biotech, Rockford, Ill.). A 10% volume of 1 M Tris pH 8.0 was added to the eluted fraction. Samples were dialyzed against 1 x PBS overnight.

The Qtof analysis of the antibody showed similar results to Example 4. An antibody obtained from cells grown in the presence of 6-fluoro-fucose was observed relative to an antibody produced from a host cell grown in the absence of 6-fluoro-mannose, and the core fucosylated G0 of the oligosaccharide was observed. The content is significantly reduced.

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

To determine the effective concentration of the 2-fluoro-mannose derivative on antibody glycosylation, the CHO DG44 cell line expressing the humanized trastuzin monoclonal antibody was at 5×10 ⁶ cells/mL in 30 mL CHO medium. Incubate at 37 ° C, 5% CO 2 while shaking at 125 RPM in a 150 mL shake flask. Insulin-like growth factor (IGF) was added to CHO medium, and 5 μM, 100 μM, and 500 μM of 2-fluoro-mannose (obtained in Example 1) were added. Different concentrations of 2-fluoro-mannose were supplemented in different cultures on day 3. On the 3rd, 5th, 7th, 9th, and 11th days, the fed culture was separately performed. The conditioned medium was collected through a 0.2 μm filter in the 13th Angel medium.

The Qtof analysis of the antibodies showed the results as shown in Figures 7-10 of the specification. Figure 7 is a blank control where approximately 95% of the oligosaccharides in the antibody are core fucosylated. Figure 8 is an antibody in the presence of 5 [mu]M of 2-fluoro-mannose in which approximately 70% of the oligosaccharides in the antibody are core fucosylated. Figure 9 shows the presence of 100 [mu]M of 2-fluoro-mannose expressed antibodies, with approximately 55% of the oligosaccharides in the antibody being core fucosylated. Figure 10 shows the presence of 500 [mu]M of 2-fluoro-mannose expressed antibody, with approximately 15% of the oligosaccharides in the antibody being core fucosylated.

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

To determine the effective concentration of 2-chloro-mannose on antibody glycosylation, CHO DG44 cell line expressing humanized trastuzin monoclonal antibody was at 5×10 ⁶ cells/mL in 30 mL CHO medium at 37 °C. Incubate under 5% CO2 while shaking at 125 RPM in a 150 mL shake flask. Insulin-like growth factor (IGF) was added to CHO medium, and 5 μM, 100 μM, and 500 μM of 2-chloro-mannose (prepared as described in Example 1) were added, respectively. Different concentrations of 2-chloro-mannose were supplemented in different cultures on day 3. On the 3rd, 5th, 7th, 9th, and 11th days, the fed culture was separately performed. The conditioned medium was collected through a 0.2 μm filter in the 13th Angel medium.

The Qtof analysis of the antibody showed similar results to Example 9.

Example 11: Antibody expression in the presence of an effective concentration range of 6-fluoro-mannose analogs

To determine the effective concentration of 6-fluoro-mannose on antibody glycosylation, CHO DG44 cell line expressing humanized trastuzin monoclonal antibody was at 5×10 ⁶ cells/mL in 30 mL CHO medium at 37 °C. Incubate under 5% CO2 while shaking at 125 RPM in a 150 mL shake flask. Insulin-like growth factor (IGF) was added to CHO medium, and 5 μM, 100 μM, and 500 μM of 6-fluoro-mannose (prepared as described in Example 1) were added, respectively. Different concentrations of 6-fluoro-mannose were supplemented in different cultures on day 3. On the 3rd, 5th, 7th, 9th, and 11th days, the fed culture was separately performed. The conditioned medium was collected through a 0.2 μm filter in the 13th Angel medium.

The Qtof analysis of the antibody showed similar results to Example 9.

Example 12: Antibody expression in the presence of 2-fluoro-mannose analogs in different media

To confirm the effect of 2-fluoro-mannose on the glycosylation of antibodies in different media, the following media culture cell lines were selected to express antibodies:

CD FortiCHO ^TM Medium (Gibco ^TM ) Thermo Fisher Scientific;

CD CHO Fusion (Sigma-Aldrich); OPM-CHO CD07 (Shanghai Aopumai Biotechnology Co., Ltd.). CHO cells expressing a humanized trastuzin monoclonal antibody were selected. Briefly, cell lines first with 5 × 10 ⁶ cells / mL at 37 ℃, 5% CO2 in 30mL CHO cultured medium, while 150mL shake flasks shaking at 125RPM. Insulin-like growth factor (IGF), 50 μM 2-fluoro-mannose (prepared as described in Example 1) was added to CHO medium. On day 3, different cultures were supplemented with 0.1% by volume of 50 mM 2-fluoro-mannose. On the 3rd, 5th, 7th, 9th, and 11th days, the fed culture was separately performed. The conditioned medium was collected through a 0.2 μm filter in the 13th Angel medium. 1 mL was taken for expression amount-HPLC detection.

The scope of the invention is not limited by the specific embodiments described herein. Various modifications of the invention will be apparent to those skilled in the <RTIgt; Such modifications are intended to fall within the scope of the appended claims. Any of the steps, elements, embodiments, features, or aspects of the invention may be used in any combination, unless substantially different from the substance herein. All patent applications, as well as scientific publications, accession numbers, and the like, cited in this application are hereby incorporated by reference in their entirety for all purposes in the the the the the the

Claims

A mannose analog of the formula: or a biologically acceptable salt or solvate thereof:

In the middle

R1-R5 are each independently selected from the group consisting of: -OH, -OAc, X, wherein X is F, Cl, Br or I.
The mannose analog or a biologically acceptable salt or solvate thereof according to claim 1, wherein R 2 is X, and R 1 , R 3 , R 4 and R 5 are each independently selected from the group consisting of -OH and -OAc.
The mannose analog or a biologically acceptable salt or solvate thereof according to claim 1, wherein R5 is X, and R1 to R4 are each independently selected from -OH and -OAc.
The mannose analog or a biologically acceptable salt or solvate thereof according to claim 1, which has the following structural formula
The mannose analog or a biologically acceptable salt or solvate thereof according to claim 1, which has the following structural formula
The mannose analog or a biologically acceptable salt or solvate thereof according to claim 1, which has the following structural formula
A mammalian cell culture medium for preparing an antibody that reduces core fucosylation modification, characterized in that it comprises an effective amount of any of the mannose analogs of claims 1-6 or a biology thereof An acceptable salt or solvate.
The mammalian cell culture medium for producing an antibody for reducing core fucosylation modification according to claim 7, wherein the medium has a volume of at least 10 liters.
The mammalian cell culture medium for preparing an antibody for reducing core fucosylation modification according to claim 7, wherein one or more mannose analogs or A biologically acceptable salt or solvate to maintain its effective concentration.
The mammalian cell culture medium for preparing an antibody for reducing core fucosylation modification according to claim 7, wherein the medium is an animal protein-free medium.
The mammalian cell culture medium for preparing an antibody for reducing core fucosylation modification according to claim 7, wherein the medium is serum-free.
The mammalian cell culture medium for preparing an antibody for reducing core fucosylation modification according to claim 7, wherein the medium contains no added fucose or mannose.
The mammalian cell culture medium for preparing an antibody for reducing core fucosylation modification according to claim 7, wherein the medium is a powder or a liquid.
A method of reducing core fucosylation-modified antibodies, the method comprising the steps of:

1) cultivating 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, and the sugar chain is linked to the Fc domain via N-acetylglucosamine at its reducing end;

The mannose analog is selected from any of the mannose analogs of any of claims 1-6, or a biologically acceptable salt or solvate thereof; wherein the core fucosylation of the antibody is lower than An antibody from a host cell cultured in the absence of a mannose analog.
The method of reducing a core fucosylation-modified antibody according to claim 14, wherein the cell is a recombinant host cell or a hybridoma cell.
The method for reducing a core fucosylation-modified antibody according to claim 15, wherein the recombinant host cell means Chinese hamster ovary cells, NS0 or SP2/0.
The method for reducing a core fucosylation-modified antibody according to claim 14, wherein the host cell is cultured in a batch medium, a fed-batch medium, a continuous feed medium or a continuous perfusion culture. Base culture, or culture with microcarriers.
The method for reducing a core fucosylation-modified antibody according to claim 14, wherein the medium is any one of claims 7-13 for preparing a reduced core fucosylation. Modified mammalian cell culture medium.
The method of reducing a core fucosylation-modified antibody according to claim 14, wherein the antibody is an intact antibody, an IgG1, a single-chain antibody or a fusion protein comprising an Fc domain.
The method of reducing core fucosylation-modified antibodies according to claim 14, wherein said mannose analog is a composition in which different mannose analogs are mixed, said composition being The appropriate concentration is dissolved in a solvent for addition; the composition is added to a dry powder or liquid medium and then present in a suitable concentration in the culture medium of the host cell.
Any of the mannose analogs of claims 1-6 and mixtures thereof are useful for inhibiting the production of high mannose forms during glycoprotein production.