CN110373374A - A kind of method and composition reducing antibody core fucosylation - Google Patents
A kind of method and composition reducing antibody core fucosylation Download PDFInfo
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- CN110373374A CN110373374A CN201810324841.6A CN201810324841A CN110373374A CN 110373374 A CN110373374 A CN 110373374A CN 201810324841 A CN201810324841 A CN 201810324841A CN 110373374 A CN110373374 A CN 110373374A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
Abstract
The present invention provides the small molecule sweet dew sugar analogues for manufacturing the recombinant antibodies that there is complicated N- connection glycan but core fucosylation to reduce, sweet dew sugar analogue usually by host cell intake (such as, pass through active transport or Passive diffusion), inhibit to generate GDP fucose.For method provided by the invention since synthesis substrate uses mannose, price is extremely low；And while reducing core fucosylation using this method, the high mannose sugar-type ratio of N- connection glycan will not be had an impact.
Description
Technical field
The present invention relates to a kind of methods of core fucosylation for reducing antibody and the compound technical used.
Background technique
Recombinating treatment albumen is produced by many distinct methods.A kind of preferred method is from mammalian host cell
System's production recombinant protein.The cell line of engineered such as Chinese hamster ovary (CHO) cell etc is interested to express
Treatment albumen matter.Advantage and disadvantage of the different cell lines in recombinant protein are had nothing in common with each other, including protein characteristic and yield.Monoclonal
Antibody or antibody derivatives are exactly one kind of recombinant protein, need to balance consideration when selection produces cell to high yield and product
Consistency.
Fc segment combines performance effector function with receptor by active region.The N- glycosylation of IgG is located at Fc segment
The area CH2 consensus sequence (Asn297-X-Ser/Thr, X can be the arbitrary amino acid residue except proline), pass through amide
Key and antibody covalent bond.Find that albumen and sugar influence conformation each other with Peer interaction by crystal X diffraction.It removes
The glycosylation of Fc segment is outer, and there are N- glycosylations in Fab segment by 30% IgG, generate to the effect of antibody molecule beneficial, harmful
Or neutral influence.Fc, which glycosylates glycan molecule, has complicated double antenna type nuclear structure, and the structure is by mannose (Man) and N- second
In addition two kinds of pentose molecule compositions of acyl aminoglucose (GlcNAc), different sugar-type also contain different number of sugar in addition to nuclear structure
Son, such as fucose (Fuc), mannose, N-acetyl-glucosamine, galactolipin (Gal), bisection N-acetyl-glucosamine and sialic acid.
The variation of the length, bifurcated pattern and monosaccharide sequence of sugar chain results in glycosylation modified complexity.Additionally, due to various enzymes
Reason makes the glycosylated pentasaccharides nuclear structure of N- be divided into three classes: high mannose type, heterozygous and complexity.
Above formula is immunoglobulin A sn297 glycosylation modified glycan molecule structural heterogeneity structural formula.
Fucose core oligosaccharide is to be turned from GDP-Fuc in transhipment golgiosome by -1,6- fucosyltransferase
The fucosyl residues biosynthesis shipped.Pass through the Fc fragment structure analytical table with fucose and without fucose modification
Bright: the electron density of Asp280 and Asn297 residue has differences between the two；There is also differences for the hydration mode of Tyr296 residue.
The human IgG without fucose modification of Lec13 mutant cell expression is shown: improving 50 times with Fc γ RIIIa affinity, ADCC makees
With 100 times of raising.Coreless fucose makes Asn162 and Fc glycan in Fc γ RIIIa receptor have more high-affinity, thus
In conjunction with stronger.And the steric hindrance of fucose can hinder this interaction.This discovery causes people to engineered cells
System takes up, to manufacture the antibody of core fucosylation reduction.
Engineered cells system includes gene knockout, gene knock-in and RNA dry in the method for reducing core fucosylation
Disturb (RNAi).In gene knockout, the gene of coding FUT8 (α 1,6- algae glycosyl transferase) is inactivated.FUT8 is catalyzed fucose
Residue is transferred to 6 of Asn- connection (N- connection) GlcNac of N- glycan from GDP- fucose.It is said that FUT8 is unique is responsible for
Fucose is added to the enzyme on the double feeler carbohydrate Asn297 of N- connection.Gene knock-in is that codase is added, such as GNTIII or Gao Er
The gene of matrix α mannosidase II.The level for improving the enzyme in cell turns to monoclonal antibody from fucosylation pathway
(core fucosylation is caused to reduce), and the bisection N-acetyl-glucosamine with rise.RNAi generally also targets FUT8
Gene expression causes mRNA transcriptional level to reduce or knock out completely gene expression.
It further include the micromolecular inhibitor using the enzyme for acting on glycosylation approach in addition to engineered cells system.Inhibit
Agent, such as Kate's Northey amine (catanospermine), early stage effect in glycosylation approach, generate have prematurity glycan (for example,
Sweet dew sugar level is high) and low fucosylation level antibody.The antibody as made from these methods is generally deficient of mature antibody
The N- connection glycan structures of relevant complexity.
Seattle Genetics Inc. discloses a kind of for manufacturing the N- with complexity in patent CN200980124932.4
Connect the small molecule fucose analogue for the recombinant antibodies that glycan but core fucosylation reduce.The patent passes through thin in host
Fucose analogue is added in born of the same parents' production process reduces core fucosylation level, and this method can reduce the core rock algae of antibody
While glycosylating, but the price of raw material fucose is very high, and reduce core fucosylation using this method, it can increase
Add the high mannose sugar-type ratio of antibody N- connection glycan.The IgG molecular proportion core knot of type containing double antenna of the residue containing high mannose
The IgG molecule serum half-life of structure is short, is likely to result in the raising of immunogenicity, is unfavorable for drug therapy.
Summary of the invention
The object of the invention is to the drawbacks described above in order to overcome the prior art, provide another reduction antibody core rock algae
Glycosylated method, this method is at low cost compared with prior art, generates shadow to the high mannose sugar-type ratio of N- connection glycan
Sound is smaller.
Another object of the present invention is to provide sweet dew sugar analogue used in the method for reducing antibody core fucosylation
Or its biologically acceptable salt or solvate, it the preparation method can realize through the invention.
The present invention additionally provides a kind of mammalian cell culture simultaneously, and being used to prepare, which reduces core fucosylation, repairs
The antibody of decorations.
The it is proposed of the method for the invention and composition is based partially on described in embodiment as a result, the embodiment is aobvious
Show, the host cell of culture expression antibody or antibody derivatives can generate core fucosylation when there are sweet dew sugar analogue
It reduces (that is, being connected to the N- acetyl of the sugar chain in the area Fc by complexity N- glucosides-connection sugar chain reducing end N-acetyl-glucosamine
The fucosylation of aminoglucose reduces) antibody.With from be not present mannose analog or fucose analogue when cultivate this
The antibody or antibody derivatives of kind of host cell manufacture are compared, and this antibody and antibody derivatives can have the effector function of raising
(ADCC)。
In the application
Term " antibody " indicates: the immunologic competence part of immunoglobulin polypeptides and immunoglobulin polypeptides (a) is exempted from
The polypeptide or part thereof of epidemic disease globulin family includes the antigen binding of immunologic specificity binding domain specific antigen (for example, CD20)
Site and Fc structural domain including complicated N- glucosides-connection sugar chain, or (b) this immunoglobulin polypeptides or immunologic specificity knot
Close the conservative replaces derivative of the segment of antigen (for example, CD20).
" antibody derivatives " indicate antibody (including antibody fragment) as defined above, or connect comprising complicated N- glucosides
The Fc structural domain of the antibody of sugar chain or the area Fc, are modified by covalent bond heterologous molecule, for example, by combining heterologous polypeptide
(for example, ligand binding domains of heterologous protein), or pass through glycosylation (except core fucosylation), deglycosylation
(except non-core fucosylation), acetylation, phosphorylation or under normal circumstances with antibody or Fc structural domain or the area Fc without
Other modifications closed.
Term " monoclonal antibody " refers to clones derived from individual cells, including any eukaryon or procaryotic cell clone or phagocytosis
The antibody of body clone, but its production method is not limited.Therefore, terms used herein " monoclonal antibody " is not limited by hybridoma
The antibody that technology generates.
Term " area Fc " indicates the constant region of antibody, for example, 3 structural domain of C H 1- hinge-C H 2-C H, optionally has
The derivative of the conservative replaces in 4 structural domain of C H or this area Fc.
Term " Fc structural domain " indicates the constant region domain of antibody, for example, C H 1, hinge, C H 2, C H 3 or C H
The conservative replaces derivative of 4 structural domains or this Fc structural domain.
" antigen " is the molecule that antibody specificity combines.
Term " specific binding " refer to antibody or antibody derivatives in a manner of high selectivity with its respective target antigen binding,
But not with various other antigen interactions.
Term " inhibition ", which refers to, occurs detectable decline, or prevent completely.
Term " GDP-Fucose " refers to guanosine diphosphate fucose.
Technical solution provided by the present application is as follows:
Such as the sweet dew sugar analogue of following formula or its biologically acceptable salt or solvate:
R1-R5 is independently selected from formula :-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-OH and-OAc.
Preferably, R5 is X, and R1 to R4 is each independently selected from-OH and-OAc.
Three kinds of following sweet dew sugar analogues or its biologically acceptable salt or solvate
A kind of to be used to prepare the mammalian cell culture for reducing the antibody of core fucosylation modification, it includes have
The above-mentioned sweet dew sugar analogue of effect amount or its biologically acceptable salt or solvate.
Preferably, the volume of the culture medium is at least 10 liters.
Preferably, it is added to one or more kinds of sweet dew sugar analogues in the culture medium or it is biologically subjected to
Salt or solvate to maintain its effective concentration.
Preferably, the culture medium is animal protein-free culture medium；The culture medium is free of serum；The culture medium is free of
The fucose or mannose of addition.It is furthermore preferred that the culture medium animal protein-free, the rock algae without serum, without addition
Sugar or mannose.
Further, the culture medium is powder or liquid.
The method for reducing the antibody of core fucosylation modification, this method comprises the following steps:
1) under the conditions of proper growth, in the culture medium comprising a effective amount of sweet dew sugar analogue, culture expression has
The host cell of the antibody of Fc structural domain；
2) antibody is separated from the cell；
The Fc structural domain has at least one N- glucosides-connection sugar chain, and the sugar chain passes through the N- second of its reducing end
Acyl aminoglucose is connected to the Fc structural domain；
The sweet dew sugar analogue is selected from the above-mentioned any sweet dew sugar analogue of this patent or it is biologically subjected to
Salt or solvate；Wherein the core fucosylation of the antibody is lower than from cultivating when being free of sweet dew sugar analogue
The antibody of host cell.
Preferably, the cell is recombinant host cell or hybridoma；The recombinant host cell refers to China
Hamster ovary cell, NS0 or SP2/0.
Preferably, host cell batch cultivation base, fed-batch culture base, continuous feeding culture medium or continuous filling
Culture medium culture is infused, or with microcarrier culture.
Preferably, the culture medium is used to prepare the anti-of reduction core fucosylation modification for what is provided in this patent
The mammalian cell culture of body.
Preferably, the antibody is complete antibody, IgG1, single-chain antibody or the fusion protein comprising Fc structural domain.
The sweet dew sugar analogue can the application in the form of composition, i.e., different mannose described herein is similar
The form of mixtures of object.The mannose analogue composition is to be dissolved in solvent to be added with suitable concentration, by institute
The composition stated is added in dry powder or fluid nutrient medium, then is present in the culture medium of host cell with suitable concentration.It is described
Solvent include the upper acceptable solvate of biology, solvate indicates one or more solvent molecules and sweet dew sugar analogue
Combination.Form the example of the solvent of the upper acceptable solvate of biology, including but not limited to: water, isopropanol, ethyl alcohol,
Methanol, DMSO, ethyl acetate, acetic acid and ethanol amine.
Beneficial effects of the present invention:
Fucose (Fucose) ratio has important influence to the ADCC activity of antibody in antibody Fc section N glycosylation.It is western refined
Genome company is schemed by synthesis fucose analogue (license notification number CN 102076865B), can efficiently inhibit rock algae
Glycosylation, but find often to inhibit the fucosylated raising along with high mannose sugar-type in practice process.And Gao Ganlu
The antibody serum half-life short for the sugar-type that is saccharified, thereby increases and it is possible to will cause the raising of immunogenicity, this should be avoided in technique.Institute
Caused with the process of inhibition fucosylated, non-fucosylated and three kinds of ratios of high mannose antibody variation is unfavorable for essence
The really regulation of control sugar-type ratio, to influence the therapeutic of antibody and antibody derivatives.
This patent provides mannose analog inhibitors, which can work under extremely low concentration, simultaneously because
The precursor for synthesizing mannose inhibitor is mannose, and the price of mannose is conducive to cost optimization well below fucose.It is sweet
Reveal sugar analogue and fucose analogue especially halogen substituents, should not be metabolized, it is possible to protect in the medium for a long time
Concentration is held, under conditions of low concentration, maintains action concentration.
Data show that the 2-F-Mannose of synthesis can inhibit 50% or more fucose to glycosylate under 5ppm concentration,
Industrialization demand is fully meet, it is completely suitable with the inhibition efficiency of the 2-F-Fucose of same concentrations.The 2-Cl- of synthesis
Mannose, 6-F-Mannose can achieve similar effects.Prove sweet dew sugar analogue and fucose analogue in each structure
It can achieve inhibitory effect in type, reduce the fucosylation of antibody and antibody derivatives.
To sum up, the present invention provides for manufacturing the weight that there is complicated N- connection glycan but core fucosylation to reduce
The small molecule sweet dew sugar analogue of group antibody, sweet dew sugar analogue usually by host cell intake (for example, by active transport or
Passive diffusion), inhibit to generate GDP fucose.For method provided by the invention since synthesis substrate uses mannose, price is extremely low；
And while reducing core fucosylation using this method, the high mannose sugar-type ratio of N- connection glycan will not be generated
Larger impact.
It can more fully be managed by reading described in detail below, particular implementation non-limiting embodiment and attached drawing
Solve the aspects of the invention and other aspects.
Detailed description of the invention
Fig. 1 is the culture density contrast curve chart of 4 cell of embodiment.
Fig. 2 is the culture motility rate contrast curve chart of 4 cell of embodiment.
Fig. 3 is the cell expression quantity contrast curve chart of humanization Trastuzumab in 4 culture supernatant of embodiment.
Fig. 4 is the data analysis chart of 5 Capillary Electrophoresis of embodiment, it is shown that anti-from control humanization toltrazuril monoclonal
The electrophoretogram of the glycan of body.
Fig. 5 is the data analysis chart of 5 Capillary Electrophoresis of embodiment, it is shown that from humanization Trastuzumab
The electrophoretogram of glycan, the antibody are from there are the host cells grown when 2-FM analog to manufacture.
Fig. 6 is the data analysis chart of 5 Capillary Electrophoresis of embodiment, it is shown that from humanization Trastuzumab
The electrophoretogram of glycan, the host cell that the antibody is grown when being from fucose analogue fluoro- there are 2- manufacture.
Fig. 7 be the Qtof of antibody analysis shows that as a result, for blank control figure, about 95% oligosaccharides is core in antibody
Fucosylation.
Fig. 8 is that there are the Qtof of the antibody of 5 μM of 2-FM analog expression analysis shows that figure.
Fig. 9 is that there are the Qtof of the antibody of 100 μM of 2-FM analog expression analysis shows that figure.
Figure 10 is that there are the Qtof of the antibody of 500 μM of 2-FM analog expression analysis shows that figure.
Figure 11 is the product 2-FM analog HNMR spectrogram of embodiment 1.
Figure 12 is the chloro- sweet dew sugar analogue HNMR spectrogram of product 2- of embodiment 2.
Figure 13 is the fluoro- sweet dew sugar analogue HNMR spectrogram of product 6- of embodiment 3.
Specific embodiment
Material used in the present invention and instrument are commercially available conventional products in the art if not otherwise specified.
Embodiment 1: synthesis 2-FM analog, i.e. 2- deoxidation -2- fluoro- 1,3,4,6- tetra--oxy-acetyl-D- are sweet
Dew sugar
Embodiment 1.1
Under nitrogen protection, D-MANNOSE (50g, 0.278mol) is added in pyridine (500mL), stirring and dissolving.It is cooling
It to 0 degree, is added acetic anhydride (500g, 4.09mol), is warming up to 25 degree and reacts 16 hours.It is concentrated under reduced pressure and removes pyridine and acetic anhydride,
Residue is dissolved with ethyl acetate (500mL), successively uses 1mol/L dilute hydrochloric acid (500mL*2), saturated sodium bicarbonate solution
The washing of (500mL) and saturated sodium chloride solution (500mL), organic phase are concentrated under reduced pressure to give off-white powder 97.5g, yield
90%.
Embodiment 1.2
Under nitrogen protection, the compound (97.5g, 0.25mol) being prepared in embodiment 1.1 is added to methylene chloride
In (1500mL), stirring and dissolving is cooled to 0 degree.Be slowly added to 40% hydrobromic acid acetum (400mL), be warming up to 25 degree it is anti-
It answers 3 hours.Successively with ice water (1500mL*2), saturated sodium bicarbonate solution (1500mL) and saturated sodium chloride solution (1500mL)
Washing, organic phase are concentrated under reduced pressure to give yellow oil 82g, yield 80%.
Embodiment 1.3
Under nitrogen protection, zinc powder (165g, 2.54mol), N- methylimidazole (32mL, 0.39mol) are added in a kettle
With ethyl acetate (1300mL), mixture is heated to flow back, the compound (82g, 0.2mol) that will be prepared in embodiment 1.2
It is dissolved into ethyl acetate (325mL), is added drop-wise in aforementioned suspension, maintain the reflux for reaction 1 hour.25 degree are cooled to, filtering.
Filtrate is successively with successively with 1mol/L dilute hydrochloric acid (650mL), saturated sodium bicarbonate solution (650mL) and saturated sodium chloride solution
(650mL) washing, organic phase are concentrated under reduced pressure to give off-white powder 36.2g, yield 66%.
Embodiment 1.4
The off-white powder (15g, 0.055mol) that embodiment 1.3 obtains is dissolved in n,N-Dimethylformamide
In (125mL) and water (125mL), it is cooled to 0 degree, the fluoro- Isosorbide-5-Nitrae-diazabicyclo [2.2.2] of 1- chloromethyl -4- is added in batches
Octane two (tetrafluoro boric acid) salt (39g, 0.11mol) is warming up to 25 degree and reacts 16 hours.Reaction solution ethyl acetate (250mL*
2) it extracts, organic phase is successively washed with water (250mL) and saturated salt solution (250mL) after merging.Faint yellow oil is obtained after concentration
Shape object 13.6g, yield 80%.
Embodiment 1.5
The light yellow oil (13.6g, 0.044mol) being prepared in embodiment 1.4 is added to pyridine (140mL)
In, stirring and dissolving.It is cooled to 0 degree, is added acetic anhydride (70g, 0.69mol), 25 degree is warming up to and reacts 16 hours.Reduced pressure removes
Pyridine and acetic anhydride are removed, residue is dissolved with ethyl acetate (150mL), successively uses 1mol/L dilute hydrochloric acid (150mL*2), saturated carbon
Sour hydrogen sodium solution (150mL) and saturated sodium chloride solution (150mL) washing, after organic phase is concentrated under reduced pressure, by column chromatographic purifying
Grease 10.2g is obtained, is dissolved in ethyl acetate (7mL), petroleum ether (100mL) is slowly added to, is recrystallized to give white powder
Last 7.6g, yield 49%.M/z (MH+) 351,1H NMR (400MHz, CDCl3), 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 spectrogram of final product is shown in attached drawing 11.
Embodiment 2: the synthesis chloro- sweet dew sugar analogue of 2-, i.e. 2- deoxidation -2- chloro- 1,3,4,6- tetra--oxy-acetyl-D- are sweet
Dew sugar
Embodiment 2.1
Off-white powder (15g, 0.055mol) in embodiment 1.3 is dissolved in n,N-Dimethylformamide (125mL)
In water (125mL), it is cooled to 0 degree, N- chlorosuccinimide (14.7g, 0.11mol) is added in batches, is warming up to 25 degree
Reaction 16 hours.Reaction solution is extracted with ethyl acetate (250mL*2), and organic phase is successively eaten with water (250mL) and saturation after merging
Salt water (250mL) washing.Light yellow oil 12.7g, yield 71% are obtained after concentration.
Embodiment 2.2
The light yellow oil (12.7g, 0.039mol) being prepared in embodiment 2.1 is added to pyridine (120mL)
In, stirring and dissolving.It is cooled to 0 degree, is added acetic anhydride (60g, 0.59mol), 25 degree is warming up to and reacts 16 hours.Reduced pressure removes
Pyridine and acetic anhydride are removed, residue is dissolved with ethyl acetate (120mL), successively uses 1mol/L dilute hydrochloric acid (120mL*2), saturated carbon
Sour hydrogen sodium solution (120mL) and saturated sodium chloride solution (120mL) washing, after organic phase is concentrated under reduced pressure, by column chromatographic purifying
Grease 9.4g is obtained, is dissolved in ethyl acetate (6mL), petroleum ether (80mL) is slowly added to, is recrystallized to give white powder
6.6g, yield 46%.M/z (MH+) 367,1H NMR (400MHz, CDCl3), δ 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 spectrogram is shown in attached drawing 12.
Embodiment 3: the synthesis fluoro- sweet dew sugar analogue of 6-, i.e. 6- deoxidation -6- fluoro- 1,2,3,4- tetra--oxy-acetyl-D- are sweet
Dew sugar
Embodiment 3.1
Under nitrogen protection, D-MANNOSE (23g, 0.128mol) is added in pyridine (200mL), stirring and dissolving.It is cooling
It to 0 degree, is added triphenylchloromethane (53g, 0.192mol), is warming up to 25 degree and reacts 16 hours.It is concentrated under reduced pressure and removes pyridine, it is residual
Excess obtains off-white powder 48g, yield 88% by column chromatographic purifying.
Embodiment 3.2
Under nitrogen protection, the compound (45g, 0.107mol) being prepared in embodiment 3.1 is added to pyridine
In (500mL), stirring and dissolving.It is cooled to 0 degree, is added acetic anhydride (500g, 4.09mol), 25 degree is warming up to and reacts 16 hours.Subtract
Pressure concentration removes pyridine and acetic anhydride, and residue is dissolved with ethyl acetate (500mL), successively uses 1mol/L dilute hydrochloric acid (500mL*
2), saturated sodium bicarbonate solution (500mL) and saturated sodium chloride solution (500mL) washing, it is white that organic phase is concentrated under reduced pressure to give class
Color solid 58g, yield 92%.
Embodiment 3.3
Compound (55g, 0.093mol) in embodiment 3.2 is added in methanol (500mL), is cooled to 0 degree, is added
2mol/L hydrochloric acid (150mL) reacts 4 hours.It is concentrated under reduced pressure into no methanol outflow, residue is extracted with ethyl acetate (250mL*2)
It takes, the organic phase washed with water (250mL), saturated sodium bicarbonate (250mL) and saturated sodium chloride solution (250mL) after merging are washed
It washs, the residue after reduced pressure obtains white solid 29g, yield 90% by column chromatographic purifying.
Embodiment 3.4
By the compound (25g, 0.072mol) prepared in embodiment 11 and N, N- dimethylamino naphthyridine (17.5g,
It 0.144mol) is dissolved in dry methylene chloride (500mL), is cooled to -25 degree, is slowly added to diethylin sulfur trifluoride
(46g, 0.285mol) kept the thermotonus 2 as a child afterwards, is warming up to 25 degree and reacts 12 hours.It is cooled to 0 degree, methanol is added
(5mL) quenching reaction, reaction solution are washed with water (500mL), and the residue after reduced pressure obtains white by column chromatographic purifying
Solid 15.9g, yield 63%.M/z (MH+) 351,1H NMR (400MHz, CDCl3), 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).HNMR spectrogram is shown in attached drawing 13.
Embodiment 4: there are antibody expressions when 2-FM analog
To determine that 2-FM analog on the glycosylated influence of antibody, expresses humanization Trastuzumab
CHODG44 cell line with 5 × 106A/mL is cultivated under 37 DEG C, 5%CO2 in 30mL CHO culture medium, while in 150mL
It is vibrated in shaking flask with 125RPM.Insulin-like growth factor (IGF), 50 μM of 2-FM class are added in CHO culture medium
Like object (preparation of embodiment 1 gained).Supplement the fluoro- sweet dew of the 2- containing 50mM of 0.1% volume at the 3rd day in difference culture respectively
The fluoro- fucose of the 2- containing 50mM of sugar analogue and 0.1% volume.At the 3rd, 5,7,9,11 day, feed-batch culture was carried out respectively.?
13rd angel's culture medium is by 0.2 μm of filter come collection condition culture medium.1mL is taken to detect for expression quantity-HPLC.Specification is attached
Fig. 1 shows that the culture density of cell, Figure of description 2 show that the culture motility rate of cell, Figure of description 3 are shown in culture supernatant
The cell expression quantity of humanization Trastuzumab.
Conditioned medium is applied to 1X phosphate buffered saline (PBS), the A albumen column that pH7.4 is pre-equilibrated carries out antibody
Purifying.With the Immunopure IgG elution buffer antibody elution after the 1X PBS column scrubber of 20 column volumes with 5 column volumes.?
The 1Mtris pH8.0 of 10% volume is added in the flow point of elution.Sample 1X PBS dialysed overnight.
LC-MS (Q-Tof) analysis by expressing the antibody generated when there are 2-FM analog is to identify
Glycosylation pattern from the purifying Trastuzumab of embodiment 4, it is anti-to be added 10 μ L100mMDTT to 90 μ L1mg/mL
15 minutes are cultivated in the PBS solution of body and at 37 DEG C to restore antibody interchain disulfide bond.The solution (20 μ L) is injected into PLRP-S
HPLC column (PL company, Massachusetts A Mu Hirst city (Polymer Laboratories；Amherst, MA)), operation with
Gradient: solvent A, 0.05%TFA aqueous solution；The acetonitrile solution of solvent B, 0.035%TFA；Linear gradient is 70-50%A0-
12.5 minutes.HPLC efflux is analyzed with electro-spray ionization Q-Tof mass spectrograph (Waters, Penelope Milford, Massachusetts),
Cone voltage is 35V, collects m/z500-4000.Heavy chain data are deconvoluted with the MaxEnt1 function of MassLynx4.0.
Embodiment 5: the Capillary Electrophoresis of oligosaccharides
To further characterize the feature from glycan on the antibody that embodiment 4 obtains, Capillary Electrophoresis has been carried out.Antibody samples
With buffer-exchanged into water.300 every kind of samples of μ g are with PNGaseF37 DEG C of processing overnight to discharge oligosaccharides.Cold methanol is added
((- 20 DEG C) and 14,000rpm are centrifuged 10 minutes to remove the protein component in sample.Dry, the oligosaccharides APTS by supernatant
(8- amino pyrene -1,3,6- trisulfonic acid trisodium salt) is stayed overnight in 1M sodium cyanoborohydride/THF in 22 DEG C of labels.The oligosaccharides of label
It is diluted with water, and uses Beckman Coulter in the capillary (BC company (Beckman Coulter)) of N-CHO coating
PA-800 passes through capillary electrophoresis analysis.Sample is separated 15 minutes with 0.5psi injection 8 seconds in 30kV.The oligosaccharides of label is used
Laser induced fluorescence (LFI) is with 488 λ of excitation wavelength detection.Transmitting fluorescence is detected in 520 λ.
Also with beta galactosidase processing antibody samples to remove galactolipin.Antibody samples buffer-exchanged is into water.
300 every kind of samples of μ g are with PNGaseF37 DEG C of processing overnight to discharge oligosaccharides.Cold methanol ((- 20 DEG C) and 14,000rpm is added
Centrifugation 10 minutes to remove the protein component in sample.Supernatant is dry, it is resuspended in water and is handled with beta galactosidase.
Oligosaccharides is dry, and then with APTS, 22 DEG C of labels are stayed overnight in 1M sodium cyanoborohydride/THF.The oligosaccharides of label is diluted with water, and
Pass through capillary electrophoresis analysis with Beckman Coulter PA-800 in the capillary (BC company) of N-CHO coating,
It is run in 40mMEACA, 0.2%HPMC, pH4.5.Sample is separated 15 minutes with 0.5psi injection 8 seconds in 30kV.Label
Oligosaccharides is with laser induced fluorescence (LFI) with 488 λ of excitation wavelength detection.Transmitting fluorescence is detected in 520 λ.
The data analysis of Capillary Electrophoresis is shown in Figure of description 4-6.With reference to Fig. 4, it is shown that bent from control humanization
The electrophoretogram of the glycan of appropriate pearl monoclonal antibody.Figure of description 5 shows poly- from humanization Trastuzumab
The electrophoretogram of sugar, the antibody is from there are the host cells grown when 2-FM to manufacture.Figure of description 6 is shown
The electrophoretogram of glycan from humanization Trastuzumab, the growth when antibody is from fucose fluoro- there are 2-
Host cell manufacture.The oligosaccharides of Fig. 4 about 96% is core fucosylation, and is non-core fucose lower than 2%
Base.The oligosaccharides of Fig. 5 about 70% is core fucosylation, and about 23% is non-core fucosylation.Fig. 6
About 60% oligosaccharides is core fucosylation, and about 18% is non-core fucosylation, about 22% widow
Sugar is Man5.By Fig. 4 and Fig. 5, it was found that, after 2-FM is added, the content of core fucosylation G0F obviously drops
It is low.By Fig. 4 and Fig. 6, it was found that, after the fluoro- fucose of 2- is added, the content of core fucosylation G0F is substantially reduced.It will figure
4, Fig. 5 and Fig. 6 it was found that, after 2-FM and the fluoro- fucose of 2- is added, the content of core fucosylation G0F is bright
It is aobvious to reduce, but after the addition fluoro- fucose of 2-, Man5 ratio is significantly raised in oligosaccharides, and after 2-FM is added, remove oligosaccharides
Middle core fucosylation ratio reduces, no other influences.
Embodiment 6: there are the expression of other antibody of 2-F- sweet dew sugar analogue
To confirm the influence to other antibody glycosylations, antibody is expressed from following cell line:
Bevacizumab, CHOS cell；Anti-CD 20 antibodies, CHOK1 cell；And Cetuximab, SP2/0 and CHO-K1 are thin
Born of the same parents.In short, cell line is first with 5 × 106A/mL is cultivated under 37 DEG C, 5%CO2 in 30mL CHO culture medium, while
It is vibrated in 150mL shaking flask with 125RPM.Addition insulin-like growth factor (IGF), 50 μM of 2- are fluoro- sweet in CHO culture medium
Dew sugar (1 gained of embodiment).The 2-FM containing 50mM of 0.1% volume is supplemented at the 3rd day in difference culture.?
3,5,7,9,11 days, feed-batch culture was carried out respectively.In the 13rd angel's culture medium by 0.2 μm of filter come collection condition culture medium.
1mL is taken to detect for expression quantity-HPLC.
Apply conditioned medium on the A albumen column pre-equilibrated with 1X phosphate buffered saline (PBS), pH7.4 and carries out antibody
Purifying.Immunopure IgG elution buffer (the Illinois Luo Ke Ford city Pierre Si biology skill of 5 column volumes of antibody
Art company) elution.The 1Mtris pH8.0 of 10% volume is added in the flow point of elution.Sample 1x PBS dialysed overnight.
The Qtof of antibody analysis shows that result it is similar to Example 4.Relative to from there is no grow when 2-F- mannose
Host cell manufacture heavy chain of antibody, the antibody that the cell grown when observing from fucose fluoro- there are 2- obtains, oligosaccharides
The content of core fucosylation G0F is substantially reduced.
Embodiment 7: antibody expression when sweet dew sugar analogue chloro- there are 2-
To determine that the chloro- mannose of 2- on the glycosylated influence of antibody, expresses the CHO of humanization Trastuzumab
DG44 cell line is with 5 × 106A/mL is cultivated under 37 DEG C, 5%CO2 in 30mL CHO culture medium, while in 150mL shaking flask
In vibrated with 125RPM.Insulin-like growth factor (IGF) is added in CHO culture medium, the chloro- mannose of 50 μM of 2- (is implemented
2 gained of example).The 2-FM containing 50mM of 0.1% volume is supplemented at the 3rd day in difference culture.The 3rd, 5,7,9,11
It, carries out feed-batch culture respectively.In the 13rd angel's culture medium by 0.2 μm of filter come collection condition culture medium.Take 1mL for table
It is detected up to amount-HPLC.
Apply conditioned medium on the A albumen column pre-equilibrated with 1X phosphate buffered saline (PBS), pH7.4 and carries out antibody
Purifying.Immunopure IgG elution buffer (the Illinois Luo Ke Ford city Pierre Si biology skill of 5 column volumes of antibody
Art company) elution.The 1Mtris pH8.0 of 10% volume is added in the flow point of elution.Sample 1x PBS dialysed overnight.
The Qtof of antibody analysis shows that result it is similar to Example 4.Relative to from there is no grow when the chloro- mannose of 2-
Host cell manufacture antibody, the antibody that the cell grown when observing from fucose chloro- there are 2- obtains, the core of oligosaccharides
The content of fucosylation G0F is substantially reduced.
Embodiment 8: antibody expression when sweet dew sugar analogue fluoro- there are 6-
To determine that the fluoro- mannose of 6- on the glycosylated influence of antibody, expresses the CHO of humanization Trastuzumab
DG44 cell line is with 5 × 106A/mL is cultivated under 37 DEG C, 5%CO2 in 30mL CHO culture medium, while in 150mL shaking flask
In vibrated with 125RPM.Insulin-like growth factor (IGF) is added in CHO culture medium, the fluoro- mannose of 50 μM of 6- (is implemented
3 gained of example).The fluoro- mannose of the 6- containing 50mM of 0.1% volume is supplemented at the 3rd day in difference culture.The 3rd, 5,7,9,11
It, carries out feed-batch culture respectively.In the 13rd angel's culture medium by 0.2 μm of filter come collection condition culture medium.Take 1mL for table
It is detected up to amount-HPLC.
Apply conditioned medium on the A albumen column pre-equilibrated with 1X phosphate buffered saline (PBS), pH7.4 and carries out antibody
Purifying.Immunopure IgG elution buffer (the Illinois Luo Ke Ford city Pierre Si biology skill of 5 column volumes of antibody
Art company) elution.The 1Mtris pH8.0 of 10% volume is added in the flow point of elution.Sample 1x PBS dialysed overnight.
The Qtof of antibody analysis shows that result it is similar to Example 4.Relative to from there is no grow when the fluoro- mannose of 6-
Host cell manufacture antibody, the antibody that the cell grown when observing from fucose fluoro- there are 6- obtains, the core of oligosaccharides
The content of fucosylation G0F is substantially reduced.
Embodiment 9: there are antibody expressions when Valid concentration 2-FM analog
To determine that 2-FM derivative to the effective concentration of antibody glycosylation effects, expresses humanization toltrazuril list
The CHO DG44 cell line of clonal antibody is with 5 × 106A/mL is cultivated under 37 DEG C, 5%CO2 in 30mL CHO culture medium, together
When vibrated in 150mL shaking flask with 125RPM.Insulin-like growth factor (IGF) is added in CHO culture medium, adds 5 μ respectively
M, 100 μM, 500 μM of 2-FM (1 gained of embodiment).Various concentration is supplemented respectively in difference culture at the 3rd day
2-FM.At the 3rd, 5,7,9,11 day, feed-batch culture was carried out respectively.The 13rd angel's culture medium by 0.2 μm of filter come
Collection condition culture medium.
Conditioned medium is applied to 1X phosphate buffered saline (PBS), the A albumen column that pH7.4 is pre-equilibrated carries out antibody
Purifying.With the Immunopure IgG elution buffer antibody elution after the 1X PBS column scrubber of 20 column volumes with 5 column volumes.?
The 1Mtris pH8.0 of 10% volume is added in the flow point of elution.Sample 1X PBS dialysed overnight.
The Qtof of antibody analysis shows that result such as Figure of description 7-10.Fig. 7 is blank control, about 95% in antibody
Oligosaccharides be core fucosylation.Fig. 8 is there are the antibody that 5 μM of 2-FM is expressed, about 70% in antibody
Oligosaccharides is core fucosylation.Fig. 9 is there are the antibody that 100 μM of 2-FM is expressed, about 55% in antibody
Oligosaccharides is core fucosylation.Figure 10 is there are the antibody that 500 μM of 2-FM is expressed, about 15% in antibody
Oligosaccharides be core fucosylation.
Embodiment 10: antibody expression when sweet dew sugar analogue chloro- there are Valid concentration 2-
To determine the chloro- mannose of 2- to the effective concentration of antibody glycosylation effects, expression humanization toltrazuril monoclonal is anti-
The CHO DG44 cell line of body is with 5 × 106A/mL is cultivated under 37 DEG C, 5%CO2 in 30mL CHO culture medium, while
It is vibrated in 150mL shaking flask with 125RPM.In CHO culture medium add insulin-like growth factor (IGF), respectively add 5 μM,
100 μM, 500 μM of the chloro- mannose of 2- (prepared by the description of such as embodiment 1).Supplement is different respectively in difference culture at the 3rd day
The chloro- mannose of the 2- of concentration.At the 3rd, 5,7,9,11 day, feed-batch culture was carried out respectively.Pass through 0.2 μm in the 13rd angel's culture medium
Filter carrys out collection condition culture medium.
Conditioned medium is applied to 1X phosphate buffered saline (PBS), the A albumen column that pH7.4 is pre-equilibrated carries out antibody
Purifying.With the Immunopure IgG elution buffer antibody elution after the 1X PBS column scrubber of 20 column volumes with 5 column volumes.?
The 1Mtris pH8.0 of 10% volume is added in the flow point of elution.Sample 1X PBS dialysed overnight.
The Qtof of antibody analysis shows that result it is similar to Example 9.
Embodiment 11: antibody expression when sweet dew sugar analogue fluoro- there are Valid concentration 6-
To determine the fluoro- mannose of 6- to the effective concentration of antibody glycosylation effects, expression humanization toltrazuril monoclonal is anti-
The CHO DG44 cell line of body is with 5 × 106A/mL is cultivated under 37 DEG C, 5%CO2 in 30mL CHO culture medium, while
It is vibrated in 150mL shaking flask with 125RPM.In CHO culture medium add insulin-like growth factor (IGF), respectively add 5 μM,
100 μM, 500 μM of the fluoro- mannose of 6- (prepared by the description of such as embodiment 1).Supplement is different respectively in difference culture at the 3rd day
The fluoro- mannose of the 6- of concentration.At the 3rd, 5,7,9,11 day, feed-batch culture was carried out respectively.Pass through 0.2 μm in the 13rd angel's culture medium
Filter carrys out collection condition culture medium.
Conditioned medium is applied to 1X phosphate buffered saline (PBS), the A albumen column that pH7.4 is pre-equilibrated carries out antibody
Purifying.With the Immunopure IgG elution buffer antibody elution after the 1X PBS column scrubber of 20 column volumes with 5 column volumes.?
The 1Mtris pH8.0 of 10% volume is added in the flow point of elution.Sample 1X PBS dialysed overnight.
The Qtof of antibody analysis shows that result it is similar to Example 9.
Embodiment 12: there is antibody expression when 2-FM analog in different medium
On the glycosylated influence of antibody when there is 2-FM in different medium in fact as evidence, following culture is selected
Base cultured cells system expresses antibody:
CD FortiCHOTMMedium(GibcoTM) Thermo Fischer Scient Inc.；CD CHO Fusion
(Sigma-Aldrich company)；OPM-CHO CD07 (Shanghai Ao Pumai Biotechnology Co., Ltd).Selection expression humanization is bent
The Chinese hamster ovary celI of appropriate pearl monoclonal antibody.In short, cell line is first with 5 × 106A/mL in 30mL CHO culture medium in 37 DEG C,
It cultivates under 5%CO2, while being vibrated in 150mL shaking flask with 125RPM.Insulin-like growth factor is added in CHO culture medium
(IGF), 50 μM of 2-FM (prepared by the description of such as embodiment 1).0.1% volume is supplemented in difference culture at the 3rd day
The 2-FM containing 50mM.At the 3rd, 5,7,9,11 day, feed-batch culture was carried out respectively.Pass through in the 13rd angel's culture medium
0.2 μm of filter carrys out collection condition culture medium.1mL is taken to detect for expression quantity-HPLC.
Apply conditioned medium on the A albumen column pre-equilibrated with 1X phosphate buffered saline (PBS), pH7.4 and carries out antibody
Purifying.Immunopure IgG elution buffer (the Illinois Luo Ke Ford city Pierre Si biology skill of 5 column volumes of antibody
Art company) elution.The 1Mtris pH8.0 of 10% volume is added in the flow point of elution.Sample 1x PBS dialysed overnight.
The Qtof of antibody analysis shows that result it is similar to Example 4.Relative to from there is no grow when 2-F- mannose
Host cell manufacture heavy chain of antibody, the antibody that the cell grown when observing from fucose fluoro- there are 2- obtains, oligosaccharides
The content of core fucosylation G0F is substantially reduced.
Protection scope of the present invention is not limited by specific embodiment described herein.In addition to content described herein, this
Field technical staff would appreciate that various modifications of the invention by reading the description and accompanying drawings of this patent.These deformations should fall into institute
In the range of attached claims.Unless from the content of this paper, obviously essence is different, any step of the invention, element, implementation
Mode, features or aspect can be used in any combination.All patent applications quoted in the application and scientific publications, login
It is number equal to be therefore incorporated herein by reference in their entirety for all purposes, it just look like that each piece document is individually listed like that.
Claims (20)
1. such as the sweet dew sugar analogue or its biologically acceptable salt or solvate of following formula:
In formula
R1-R5 is independently selected from :-OH ,-OAc, X, wherein X is F, Cl, Br or I.
2. sweet dew sugar analogue as described in claim 1 or its biologically acceptable salt or solvate, feature exist
In: R2 is X, and R1, R3, R4 and R5 are each independently selected from-OH and-OAc.
3. sweet dew sugar analogue as described in claim 1 or its biologically acceptable salt or solvate, feature exist
In: R5 is X, and R1 to R4 is each independently selected from-OH and-OAc.
4. sweet dew sugar analogue as described in claim 1 or its biologically acceptable salt or solvate, feature exist
In: have the following structure formula
5. sweet dew sugar analogue as described in claim 1 or its biologically acceptable salt or solvate, feature exist
In: have the following structure formula
6. sweet dew sugar analogue as described in claim 1 or its biologically acceptable salt or solvate, feature exist
In: have the following structure formula
7. a kind of be used to prepare the mammalian cell culture for reducing the antibody of core fucosylation modification, feature exists
In: any sweet dew sugar analogue that it includes a effective amount of as described in claim 1-6 or its biologically acceptable salt or
Solvate.
8. being used to prepare the mammaliancellculture for reducing the antibody of core fucosylation modification as claimed in claim 7
Base, it is characterised in that: the volume of the culture medium is at least 10 liters.
9. being used to prepare the mammaliancellculture for reducing the antibody of core fucosylation modification as claimed in claim 7
Base, it is characterised in that: be added to one or more kinds of sweet dew sugar analogues in the culture medium or it is biologically subjected to
Salt or solvate to maintain its effective concentration.
10. being used to prepare the mammalian cell training for reducing the antibody of core fucosylation modification as claimed in claim 7
Support base, it is characterised in that: the culture medium is animal protein-free culture medium.
11. being used to prepare the mammalian cell training for reducing the antibody of core fucosylation modification as claimed in claim 7
Support base, it is characterised in that: the culture medium is free of serum.
12. being used to prepare the mammalian cell training for reducing the antibody of core fucosylation modification as claimed in claim 7
Support base, it is characterised in that: fucose or mannose of the culture medium without addition.
13. being used to prepare the mammalian cell training for reducing the antibody of core fucosylation modification as claimed in claim 7
Support base, it is characterised in that: the culture medium is powder or liquid.
14. a kind of method for the antibody for reducing core fucosylation modification, this method comprises the following steps:
1) under the conditions of proper growth, in the culture medium comprising a effective amount of sweet dew sugar analogue, culture expression has Fc knot
The host cell of the antibody in structure domain；
2) antibody is separated from the cell；
The Fc structural domain has at least one N- glucosides-connection sugar chain, and the sugar chain passes through the N- acetyl Portugal of its reducing end
Osamine is connected to the Fc structural domain；
The sweet dew sugar analogue is selected from any sweet dew sugar analogue described in claim 1-6 or it is biologically subjected to
Salt or solvate；Wherein the core fucosylation of the antibody is lower than from cultivating when being free of sweet dew sugar analogue
The antibody of host cell.
15. reducing the method for the antibody of core fucosylation modification as claimed in claim 14, it is characterised in that: described thin
Born of the same parents are recombinant host cell or hybridoma.
16. reducing the method for the antibody of core fucosylation modification as claimed in claim 15, it is characterised in that: described
Recombinant host cell refers to Chinese hamster ovary cell, NS0 or SP2/0.
17. reducing the method for the antibody of core fucosylation modification as claimed in claim 14, it is characterised in that: the place
Chief cell batch cultivation base, fed-batch culture base, continuous feeding culture medium or continuous perfusion culture base culture, or with micro- load
Body culture.
18. reducing the method for the antibody of core fucosylation modification as claimed in claim 14, it is characterised in that: described
Culture medium is the mammal of any antibody for being used to prepare reduction core fucosylation modification described in claim 7-13
Cell culture medium.
19. reducing the method for the antibody of core fucosylation modification as claimed in claim 14, it is characterised in that: described anti-
Body is complete antibody, IgG1, single-chain antibody or the fusion protein comprising Fc structural domain.
20. reducing the method for the antibody of core fucosylation modification as claimed in claim 14, it is characterised in that: described
Sweet dew sugar analogue is the composition that different sweet dew sugar analogues is mixed into, and the composition is dissolved in molten with suitable concentration
It is added in agent；It is thin that the composition is present in host again after being added in dry powder or fluid nutrient medium with suitable concentration
In the culture medium of born of the same parents.
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| PCT/CN2019/080902 WO2019196697A1 (en) | 2018-04-12 | 2019-04-02 | Method and composition for reducing core fucosylation of antibody |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102076865A (en) * | 2008-05-02 | 2011-05-25 | 西雅图基因公司 | Methods and compositions for making antibodies and antibody derivatives with reduced core fucosylation |
| CN102216452A (en) * | 2008-09-26 | 2011-10-12 | 尤里卡治疗公司 | Cell lines and proteins with variant glycosylation pattern |
| CN102311986A (en) * | 2000-10-06 | 2012-01-11 | 协和发酵麒麟株式会社 | Produce the cell of antibody compositions |
| WO2016144773A1 (en) * | 2015-03-06 | 2016-09-15 | Abbvie Inc. | Arabinosylated glycoproteins |
-
2018
- 2018-04-12 CN CN201810324841.6A patent/CN110373374B/en active Active
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311986A (en) * | 2000-10-06 | 2012-01-11 | 协和发酵麒麟株式会社 | Produce the cell of antibody compositions |
| CN102076865A (en) * | 2008-05-02 | 2011-05-25 | 西雅图基因公司 | Methods and compositions for making antibodies and antibody derivatives with reduced core fucosylation |
| CN102216452A (en) * | 2008-09-26 | 2011-10-12 | 尤里卡治疗公司 | Cell lines and proteins with variant glycosylation pattern |
| WO2016144773A1 (en) * | 2015-03-06 | 2016-09-15 | Abbvie Inc. | Arabinosylated glycoproteins |
Non-Patent Citations (2)
| Title |
|---|
| IZABELA FOKT等: "D-Glucose- and D-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-D-glucoses and -D-mannoses", 《CARBOHYDRATE RESEARCH》 * |
| ZHILING ZHU等: "Surfaces presenting α-phenyl mannoside derivatives enable formation of stable, high coverage, non-pathogenic Escherichia coli biofilms against pathogen colonization", 《ZHILING ZHU等》 * |
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