CN1242776A - Carbohybrate purification using ultrafiltration, reverse osmosis and nanofiltration - Google Patents

Abstract

The invention provides methods for purifying carbohydrates, including oligosaccharides, nucleotide sugars, and related compounds, by use of ultrafiltration, nanofiltration and/or reverse osmosis. The carbohydrates are purified away from undesired contaminants such as compounds present in reaction mixtures following enzymatic synthesis or degradation of oligosaccharides.

Description

With ultrafiltration, reverse osmosis and nanofiltration Carbohybrate purification

The intersection document of related application

The application is U.S. Provisional Application No.60/028, the part renewal application of 226 (submissions on October 10th, 1996), and it is for referencial use to fit into this paper in this application.

Invention field

The present invention relates to the synthetic of oligosaccharides.Particularly, the present invention relates to improving one's methods of purifying oligosaccharides with ultrafiltration, nanofiltration (nanofiltration) and/or reverse osmosis.

Background of invention

Along with the understanding as the effect of cell surface recognition component improves constantly to carbohydrate, the interest of producing the carbohydrate molecule with definite structure also improves day by day.For example, the compound that comprises oligosaccharides part saliva acidic group lactose (sialyl lactose) has become the interested neutralizing agent (for example referring to U.S. Patent No. 5,330,975) of directed toward bacteria (as vibrio cholerae, intestinal bacteria and Salmonellas) enterotoxin.After deliberation the treatment of saliva acidic group lactose to sacroiliitis and relevant autoimmune disease.Think that especially saliva acidic group lactose suppresses or destroyed the degree that occupies of the last Fc carbohydrate binding site of IgG, thereby prevented the formation (seeing United States Patent (USP) 5,164,374) of immunocomplex.Advised recently treating ulcer, and the purposes of last compound on this performance begun the I clinical trial phase with saliva acidic group-α (2,3) galactoside, saliva acidic group lactose and saliva acidic group lactose amine (lactosamine).See Balkonen etc., FEMS Immunology and Medical Microbiology 7:29 (1993) and BioWorld Today, p.5, April 4 nineteen ninety-five.Another example is, can comprise saliva acidic group Louis part (saliva acidic group Louis in conjunction with existing in such as the white corpuscle of ELAM-1 and GMP 140 acceptors and non-leucocyte cell line ^xWith saliva acidic group Louis ^a) compound.Polley etc., Proc.Natl.Acad.Sci.USA, 88:6224 (1991) and Phillips etc., Science, 250:1130 (1990), other sees USSN08/063,181.

Because to preparing the interest of required carbohydrate structure, urge the deep research that has been used as in being combined at present to glycosyltransferase and at the carbohydrate enzyme.Come the synthetic carbohydrate of enzymatic to have more superiority with glycosyltransferase, because enzyme provides stereoselectivity virtually completely and has been connected specificity (Ito etc., Pure Appl.Chem. than chemical process, 65:753 (1993), United States Patent (USP) 5,352,670 and 5,374,541).Therefore, glycosyltransferase is used for the treatment of in synthetic more and more in many carbohydrates with other purpose and is used as enzymatic catalysts.

The saccharide compound that enzymatic is synthetic or other method makes normally obtains with the form of mixtures of complexity, it not only comprises required compound, also comprises other pollutent such as unreacted sugar, salt, pyruvate salt, phosphoric acid salt, PEP, nucleosides, Nucleotide and protein.The existence of these pollutents is to use in many application of saccharide compound undesirable.Be used for the method for purifying oligosaccharides in the past,, several shortcomings arranged as chromatography (being ion exchange chromatography and size exclusion chromatography).For example, the chromatography purification method is not suitable for large scale purification, thereby has hindered its application in sugared commercialization is produced.And, chromatography purification method expense costliness.Therefore, need a kind of purification process that method therefor is faster, more effective, cost is lower in the past.The present invention has satisfied this demand and other demand.

Background technology

USPN 5,454, and 952 have described a kind of method of removing unwanted impurity in the sugary soln (especially suppress sugared crystalline molasses form ion) that makes up with film.The nanofiltration that this method relates to ultrafiltration and carries out thereafter can be used to improve the recovery of the crystallised sugar in sugarcane or the beet solution according to description.

USPN 5,403, and 604 have described with nanofiltration remove fruit juice sugar from fruit juice, obtain high reservation liquid of sugared content and the lower percolate of sugared content.

USPN 5,254,174 described the employing chromatography and/nanofiltration, desalt and glucose, fructose and sucrose by from the fruit juice that contains synanthrin (inulide) compound or syrup, removing, coming the purifying chemical formula is the synanthrin compound (wherein G is a glucose, and F is a fructose) of GFn.

USPN 4,956,458 described with reverse osmosis come from poly-dextrose (it is a crosslinked dextran polymer at random, and the acid catalysis condensation by glucose forms), to remove, most of bad composition such as anhydroglucose and furtural derivative poly-dextrose.

USPN 4,806, and 244 have described film and the adsorption system with combination, remove vitriol by nanofiltration from water, remove in the percolate nitrate by film by being adsorbed onto ion exchange resin then.

Summary of the invention

The invention provides a kind of from the stock liquid that contains pollutent the method for Carbohybrate purification compound.This method relates to contacts stock liquid under certain condition with nanofiltration membrane or reverse osmosis membrane, film has kept required saccharide compound under this condition, and most of pollutents then pass through film.The invention provides the method for Carbohybrate purification compound, these carbohydrates for example are saliva acidic group lactose; Sialic acid; Lacto-N-neotetraose (LNnT) and GlcNAc β 1; 3Gal β 1,4Glc (LNT-2), NeuAc α (2 → 3) Gal β (1 → 4) (Fuc α 1 → 3) Glc (R ¹) β 1-OR ², R wherein ¹Be OH or NAc; R ²Be hydrogen, alkoxyl group, sugar, oligosaccharides or the aglycone that has a carbon atom at least; And Gal α (1 → 3) Gal β (1 → 4) Glc (R ¹) β-O-R ³, R wherein ¹Be OH or NAc; R ³Be-(CH ₂) _n-COX, X=OH, OR ⁴,-NHNH ₂, R ⁴Be hydrogen, sugar, oligosaccharides or the aglycone that has a carbon atom at least, the integer of n=2 to 18.

Also provide purifying that the method for the saccharide compound of following chemical formula is arranged in addition: NeuAc α (2 → 3) Gal β (1 → 4) GlcN (R ¹) β-OR ², NeuAc α (2 → 3) Gal β (1 → 4) GlcN (R ¹) β (1 → 3) Gal β-OR ², NeuAc α (2 → 3) Gal β (1 → 4) (Fuc α 1 → 3) GlcN (R ¹) β OR ²Or NeuAc α (2 → 3) Gal β (1 → 4) (Fuc α 1 → 3) GlcN (R ¹) β (1 → 3) Gal β-OR ², R wherein ¹Be the alkyl or the acyl group, 5,6,7 of 1-18 carbon atom, 8-tetrahydrochysene-2-naphthoyl amino; Benzamido, 2-naphthoyl amino; 4-aminobenzoic amido; Or 4-nitrobenzoyl amido, R ²Be hydrogen, sugar, oligosaccharides or the aglycone that has a carbon atom at least.

In another example, the invention provides the method for Carbohybrate purification compound from stock liquid (containing the reaction mixture that is used for synthesizing saccharide compound).Synthetic can be enzymatic or chemical process or its combination.Method relates to, contact with ultra-filtration membrane by stock liquid, the albumen tunicle keep and saccharide compound as percolate by film, thereby remove any albumen that exists in the stock liquid.The percolate of ultrafiltration step is contacted with nanofiltration membrane or reverse osmosis membrane under certain condition, and nanofiltration membrane or reverse osmosis membrane can be detained saccharide compound under this condition, and most of unwanted pollutents then pass through film.

Another example of the present invention provides the method for purifying Nucleotide, nucleosides and nucleotide sugar, this method is that the stock liquid that contains Nucleotide or related compound is contacted with nanofiltration membrane or reverse osmosis membrane under certain condition, and film can keep Nucleotide or the most of pollutents of related compound then pass through film under this condition.

The present invention also provides a kind of method of removing one or more pollutents from the solution that contains carbohydrate interested.This method relates to a film contacts side surfaces that makes this solution and semi-permeable membranes, and the repulsion coefficient of this semi-permeable membranes can keep carbohydrate and make pollutent pass through film.According to size and the electric charge of interested carbohydrate with respect to pollutent, used film is selected from ultra-filtration membrane, nanofiltration membrane and reverse osmosis membrane.This film will contain the glycogen feed liquid and be separated into retained part and diafiltration part.If film is higher than pollutent to the repulsion coefficient of sugar, then the Pollutant levels of retained part will be lower than the Pollutant levels in the stock liquid relatively, and the ratio of carbohydrate and unwanted pollutent is higher usually.On the contrary, film to the repulsion coefficient of carbohydrate be lower than to pollutent the time, then the separation of Shi Xianing will be that the concentration of pollutent is lower than stock liquid in the percolate, sugar will be higher than stock liquid with the ratio of pollutent in the percolate.If desired, can contain sugared fraction so that be further purified by the recirculation of film system.

The pollutent example that available the inventive method is removed from the solution that contains required compound includes, but are not limited to, unreacted sugar, mineral ion, pyruvate salt, phosphoric acid salt, phosphoenolpyruvic acid and albumen.

Detailed Description Of The Invention

Definition

Here adopted following abbreviation:

The Arabic glycosyl of Ara=;

The Fru=fructosyl;

The Fuc=fucosido;

The Gal=galactosyl;

GalNAc=N-acetyl galactosyl;

The Glc=glucosyl;

GlcNAc=N-acetyl glucosyl;

The Man=mannose group;

NeuAc=saliva acidic group (N-acetylneuraminic amine base).

Term " carbohydrate " comprises having general formula (CH ₂O) _nChemical compound, it comprises monose, disaccharides, oligosaccharides and polysaccharide.Term used herein " widow " refers to by 2 to about 10 polymerizable moleculars that residue is formed, for example amino acid (oligopeptides), monose (oligosaccharides) and nucleic acid (oligonucleotide).Term " poly-(many) " refers to comprise the polymerizable molecular of about 10 above residues.

Think that oligosaccharides has a reducing end under neutral and a non reducing end, whether reducing end under neutral that no matter should sugar is reducing sugar.According to the name that people accept, think that here the left side of oligosaccharides is a non reducing end, the right side is a reducing end under neutral.

All oligosaccharides as herein described are write like this: the title of non-reducing sugar or abbreviation (as Gal) are thereafter the ring positions of the reducing sugar of glycosidic link (α or β), ring key, participation bonding, are the title or the abbreviation (as GlcNAc) of reducing sugar then.Key between two sugar can be expressed as, for example, and 2,3,2-3 or (2,3).

If unwanted component concentrations is no more than the about 40% of this concentration of component before the purifying behind purifying, claim that then compound is " to have purified basically " in the unwanted component from solution.Preferable, the concentration of unwanted component behind purifying should be less than 20% (weight) of concentration before about purifying, and better is less than about 10%.

Term used herein " pharmaceutically pure " refers to the compound of enough purifying from unwanted pollutent, and this compound is suitable for coming administration as pharmaceutical preparation.Preferable, this compound is purified to does not wish that the amount of pollutent behind purifying is about before the purifying in the stock liquid 5% (weight) of Pollutant levels or still less.Better, the Pollutant levels behind the purifying be about Pollutant levels before the purifying 1% or lower, better is about 0.5% or lower.

" stock liquid " refers to contain any solution of compound to be purified.For example, be used for the reaction mixture of synthesis of oligose and can be used as stock liquid, can be with method of the present invention from wherein being purified into required reaction product.

The invention example

The invention provides with semi-permeable membranes (as reverse osmosis membrane and/or nanofiltration membrane) specificity sugar and oligosaccharide structure are purified to highly purified rapidly effective purification process.This method is specially adapted to isolate required oligosaccharide compound in the reactant from remain in reaction mixture and other pollutent after oligosaccharides synthesizes or degrades.For example, the invention provides and be used for from enzymatic synthetic or enzymatic degradation oligosaccharides, nucleotide sugar, glucose lipid, glycolipid, Nucleotide, nucleosides and other and contain the method for the reaction mixture of sugar compounds oligosaccharides and enzyme and/or other component being separated.Also provide in addition come from stock liquid to remove with ultrafiltration, nanofiltration or reverse osmosis desalt, the method for sugar and other component.By adopting these technology, can produce the sugar that reaches 100% purity basically (as saliva acidic group lactose, SLe ^xWith many other carbohydrates).And, to compare with previously known carbohydrate purification process, purification process of the present invention is more effective rapider, is more suitable for carrying out large scale purification.

Required purifying can be realized in a step usually; Usually need be such as other additional purification steps such as crystallizations.Therefore, the invention provides the one-step method that purifying contains sugar compounds.

According to the present invention, for purifying sugar, select a kind of film, this film is suitable for isolating required sugar from the unwanted component of (treat therefrom be purified into sugar) solution.The purpose of selective membrane is, the molecular weight of film is held back (MWCO), film composition, perviousness and repulsion characteristic are for specific application optimum, that is, make film keep specific molecular and the best performance that allows salt and other (normally less or oppositely charged) molecule pass through.Component i (R _i) reservation percentage ratio by formula R _i=(1-C _Ip/ C _Ir) * 100% provides, wherein C _IpBe the concentration of component i in the percolate, C _IrBe the concentration of component i in the retention, both all represent with weight percentage.The reservation percentage ratio of certain component is also referred to as retention property or film repels coefficient.

For effective separation, selected film is tackled the repulsion of carbohydrate interested than the repulsion ratio that is higher than the need separating compound.If film is higher than second kind of compound for the repulsion ratio of first kind of compound, then lack than second kind of compound by first kind of compound concentrations in the diafiltration solution of film.Otherwise then first kind of compound concentrations is higher than second kind of compound concentrations in the percolate.If film does not repel certain compound, then percolate and this compound concentration in the retained part keep substantially with stock liquid in identical.If certain compound concentration is higher than this compound concentration in the stock liquid in the percolate, also may be that film has negative repulsion ratio to this compound.About " the Membranes and Membrane Sepraration Processes " part among the visible Ullmann ' of the total description s Encyclopedia of Industrial Chemistry (VCH, 1990) of membrane technique; Other sees, Noble and Stern, Membrane Separations Technology:Principles and Applications (Elsevier, 1995).

At first, normally selecting molecular weight to hold back (MWCO, usually relevant with membrane pore size) expectation can keep required compound and allow the film that unwanted compound passes through in the stock liquid.Required MWCO is usually less than the molecular weight of compound to be purified, and usually greater than the molecular weight of the unwanted pollutent that will from the solution that contains compound to be purified, remove.For example, for the compound of purifying molecule amount 200Da, should select MWCO to be lower than the film of about 200Da.For example, MWCO is that the film of 100Da also is suitable material standed for.Can be used for film of the present invention can partly be categorized as ultrafiltration (UF) film, nanofiltration (NF) film according to its MWCO, or reverse osmosis (RO) film, and this depends on required separation.Describe for ease of the present invention, as Pure Water Handbook, Osmonics, determined UF, NF and the RO film of being categorized as among the Inc. (Minnetonka MN).The RO film has less than the nominal MWCO of about 200Da usually and repels most of ions, the NF film nominal MWCO between 150Da to 5kDa that has an appointment usually, the UF film nominal MWCO (these MWCO scopes are with regard to carbohydrate molecule) between 1kDa to 300kDa that has an appointment usually.

When selecting to be applicable to the film of particular separation, the parameter of second consideration is the polymer form of film.Film used in each zone is made with conventional mould material, no matter is inorganic, organic, or inorganic and organic mixing material.Typical inorganic materials comprises glass, pottery, sintering metal, metal etc.The ceramic membrane that is applicable to the UF zone can be according to making described in (for example) U.S. Patent No. 4,692,354 (Asaeda etc.), 4,562,021 (Alary etc.) and other document like that.The organic materials that is applicable to NF and RO zone is polymkeric substance normally, no matter is isotropy or anisotropic, the hole side of fiber (bore side) or shell-side has thin layer or " cortex ".Preferable filamentary material is that polymeric amide, polyphenyl methane amide, polysulfones (comprising sulfonated polysulfones and sulfonated polyethersulfone), polystyrene (comprise and contain cinnamic multipolymer, as acrylonitrile-styrene, butadiene-styrene and styrene-ethylene base benzyl halide multipolymer), polycarbonate, cellulose polymer compound (comprising cellulose ethanoate), polypropylene, polyvinyl chloride, polyethylene terephthalate, polyvinyl alcohol, fluorocarbon etc., as U.S. Patent No. 4,230,463,4,806,244 and 4, described in 259,183.Except polymeric was distinguished layer, NF and RO film also had porousness to support base material usually.

When selecting suitable membrane to form, film surface charge particularly importantly.In required MWCO scope, its surface charge of selected film is fit to the ionic charge of carbohydrate and pollutent.Although the MWCO of certain films is normally constant, the pH that changes stock liquid but can influence the separation performance of film by changing the film surface charge.For example, by reducing the pH of solution, the film that can will have clean surface negative charge under neutral pH is adjusted to has clean neutral charge simply.Another effect of regulator solution pH is the ionic charge of having regulated on pollutent and the carbohydrate interested.Therefore, by selecting suitable membrane polymer class shape and pH, can obtain pollutent and film is the neutral system, thereby pollutent is passed through easily.For example, if pollutent is electronegative under neutral pH, then need usually to reduce the pH of stock liquid so that the pollutent protonization.For example, be about 3, make phosphate anion protonated, make it pass through film, just can remove phosphoric acid salt if the pH of solution is reduced to.As described in example 5 above, pH is reduced to 3.0 from 7.5 can make the GlcNAc percentage ratio by polyamide membrane reduce (for example Osmonics MX07 was reduced to 28% from 70% in 30 minutes), make simultaneously the phosphoric acid salt percentage ratio that passes through from 10% be increased to 46% (see embodiment 6, table 5 about change pH to other compound during by various nanofiltration membrane pass through than other embodiment of influence).For purifying negatively charged ion carbohydrate, pH is usually between about pH1 to 7.Otherwise, if pollutent has positive surface charge, then can be with the pH regulator of stock liquid between about pH7-14.For example improve and contain amino (NH ₃ ⁺) pH of pollutent solution can make and aminoly become neutral, thereby make it pass through film easily.Therefore, the pH that relates to by regulating the solution contact with film of one aspect of the present invention regulates separation efficiency; Can change the ionic charge of pollutent like this, can also influence the surface charge of film, thereby the purifying of required carbohydrate is carried out easily.Certainly, must in the acceptable pH scope of certain films, carry out according to manufacturer's specification sheets, to avoid damage film.

For some application, at first under a pH, mixture is carried out nanofiltration or reverse osmosis, the reservation liquid that will contain sugar interested then is adjusted to different pH, again it is carried out the film purifying that another is taken turns.For example, make and be used for the reaction mixture of sialic acid synthetase base lactose and under pH3.0, filter by Osmonics MX07 film (a kind of MWCO is about the nanofiltration membrane of 500Da) and will keep saliva acidic group lactose, and remove most phosphates in the solution, pyruvate salt, salt and manganese, and some GlcNAc, lactose and sialic acid have been removed.After with pH regulator to 7.4, make its recirculation can remove most of residual phosphoric acid salt, all pyruvate salts, all lactose, some sialic acids and a large amount of remaining manganese by the MX07 film.

If from the mixture that contains the albumen enzyme of synthetic required oligosaccharides or nucleotide sugar (for example be used for) purifying sugar, wish usually to remove the first step of district's albumen as purification step.For less than proteic carbohydrate, by selecting MWCO but (promptly greater than oligosaccharide molecular amount to be purified less than the albumen for the treatment of from solution, to remove or other macromolecular molecular weight, proteic in this case repulsion is than being higher than required carbohydrate) film, can realize this separation.Like this, film can exclusion molecular weight greater than the albumen of MWCO and other macromole, and make sugar pass through film.Otherwise if from less than purifying oligosaccharides or nucleotide sugar the albumen of oligosaccharides or nucleotide sugar, the MWCO of then used film should be greater than molecular weight of albumen less than the molecular weight of oligosaccharides or nucleotide sugar.Usually, protein isolate and the used film of sugar are commonly referred to ultrafiltration (UF) film.The ultra-filtration membrane that is applicable to the inventive method is available from several commercial production merchants, they comprise Millipore Corp. (Bedford, MA), Osmonics, Inc. (Minnetonka, MN), Filmtec (Minneapolis, MN), UOP, Desalination Systems, Advanced MembraneTechnologies and Nitto.

The present invention also provides with nanofiltration (NF) film or reverse osmosis (RO) film and removed the method that desalts with other lower-molecular-weight component from the mixture that contains sugar interested.Nanofiltration membrane is that a class is come isolating film according to molecular weight and ionic charge.With regard to regard to the material size of film, nanofiltration membrane is usually between reverse osmosis membrane and ultra-filtration membrane.Between all chains of swollen polymer network of nanofiltration membrane, micropore or perforate are arranged usually.To the molecular weight cut-off of unionized molecule usually between 100-20000 dalton.For the ion that the same molecular amount is arranged, it will be 0 with the ionic charge of concrete film that film repels (reservation) effect, 1,2,3 and increase gradually, because electric density increases and (for example sees Eriksson, P., " Nanofiltration Extends the Range of Membrane Filtration, " Enviromental Progress, 7:58-59 (1988)).At Chemical Engineering Progress, pp.68-74 (in March, 1994), Rautenbach etc. have also described nanofiltration among Desalintion 77:73 (1990) and the USPN 4,806,244.In a typical application, interested sugar is kept by nanofiltration membrane, and salt and other unwanted component of polluting then can be passed through.The nanofiltration membrane that is used for the inventive method is about 40% to 100% usually for the retention property of sugar interested, and preferable is about 70% to 100%.Be used for the nanofiltration membrane that nanofiltration membrane of the present invention can be any routine, wherein polyamide membrane is specially suitable.Several coml manufacturers sell the nanofiltration membrane that is applicable to the inventive method, comprising Millipore Corp. (Bedford, MA), Osmonics, Inc. (Minnetonka, MN), Filmtec (Minneapolis, MN), UOP, Advanced Membrane Technologies, DesalinationSystems and Nitto.For example, suitable membrane comprises Osmonics MX07, YK, GH (G-10), GE (G-5) and HL film.

Reverse osmosis (RO) film also can make various aqueous solutes by also keeping selected molecule simultaneously.Usually, infiltration refers to that neat liquid (normally water) enters solution (normally sugar or salt and water) by semi-permeable membranes and diluting soln and make two kinds of liquid reach the method for osmotic equilibrium.On the contrary, reverse osmosis is by pressure-actuated membrane process, wherein applies external pressure to the film system, makes water molecules oppositely flow into the pure water compartment of film system from salts solution or sugar soln compartment.The RO film is semi permeable, atresia, and it need the osmotic pressure of dissolved substance be pressed onto on the film in the water to be higher than with aqueous materials with pressure pump.The RO film can be removed low-molecular-weight molecule (＜200 dalton) and ion effectively from water.Preferable, reverse osmosis membrane is about 40% to 100% usually for the retention property of interested sugar, and preferable is about 70% to 100%.Wherein suitable R O film comprises, but be not limited to Filmtec BW-30, Filmtec SW-30, Filmtec SW-30HR, UOP RO film, Desal RO film, Osmonics RO film, Advanced Membrane Technologies RO film and Nitto RO film.Suitable R O film example is Millipore Cat.No.CDRN500 60 (Millipore Corp., Bedford MA).

Be used for film of the present invention and can adopt any known membrane structure.For example, film can be flat, tabular and frame shape, tubulose, spiral wound form, tubular fibre etc.In a preferable example, film is the spiral wound form.Film can adopt any suitable configuration, comprises cross-flow (cross-flow) or filters conformation (depthconfigutation) deeply.Filter in (being suitable for ultrafiltration of the present invention, nanofiltration and reverse osmosis purifying) in " cross-flow ", treat that therefrom " raw material " or the solution of the interested sugar of purifying are flowed through by membrane channel to be parallel to film surface or tangential direction, and be separated into reservation (being also referred to as recirculation or concentrated) liquid and percolate.In order to keep effective membrane filtration, liquid stream should be parallel to the film surface flow under sufficiently high speed, to produce shearing force and/or turbulent flow, removes tunicle and repels and the particle of accumulation.Cross flow filter has three strands of liquid stream-stock liquid streams, percolate stream and debris stream.On the contrary, " terminal type " or " deep layer " filters and only needs two strands of liquid stream-stock liquid streams and filtered solution stream (or percolate).Recirculated liquid or reservation liquid have kept all particles and macromole that tunicle repels, and they can all be recycled in the membrane module that produces recycle stream, or can part remove from system.For example, when coming sugared in the purifying lower-molecular-weight component with method of the present invention, keep in the liquid stream (or be stock liquid for depth type filtration) and contain required sugar, and percolate contains the pollutent that is removed.

Pressure when filtering by adjusting, flow velocity and temperature can further be optimized purification process of the present invention.UF, NF and RO need be increased to pressure greater than environmental stress usually, to overcome the osmotic pressure of the solution that passes film.Can do further optimization by progressively regulating again according to maximum and working pressure recommendation of film manufacturer specification sheets.For example, the recommended pressure of UF usually between about 25-100psi, for NF then between 50-1500psi, for RO between about 100-1500psi.Also can regulate the flow velocity of concentrated solution (stock liquid) and percolate so that required purifying reaches optimum.Can also the manufacturer to the suggestion of concrete film as starting point, optimize purge process by progressively regulating.Concentrated solution (P _c) flow velocity usually between about 1-15 gallon per minute (GPM), better between about 3-7GPM.For percolate, flow velocity is usually between about 0.05-10GPM, and is preferable between about 0.2-1GPM.Temperature when carrying out purifying is also influential to purification effect and speed.For most of occasions, temperature is usually between about 0-100 ℃, and is preferable between about 20-40 ℃.For some films, higher temperature can make membrane pore size increase, thereby provides adjustable another kind of parameter to optimize purifying.

In a preferable example, filter and in film purifying machine, to carry out, this machine can be controlled flow velocity, pressure, temperature automatically and other influences the parameter of purifying.For example, the machine of Osmonics 213T film purifying machine and above-mentioned other company's production is applicable to method of the present invention.

After the use or after perviousness reduced, film was easy to clean.Cleaning can be carried out (if wishing like this) under high slightly temperature, water or caustic solution wash.If liquid stream contains a small amount of enzyme, then can be at low quantity of surfactant (ULTRASIL for example ^) exist down and wash.In addition, also available pre-filter membrane (100-200 μ m) is protected more expensive nanofiltration membrane.If desired, can use other sanitising agent.Film to be cleaned is depended in the selection of purging method, specification sheets that should the reference membrane manufacturer.Clean available following current flushing or back flushing.

Purification process of the present invention can use separately or unite use with other method of Carbohybrate purification.For example, before and after nanofiltration/reverse osmosis, or before and after filtering, but spent ion exchange resin is removed specific ion from the mixed solution that contains sugar interested.Deionizing (as remaining phosphoric acid salt and Nucleotide after first round nanofiltration or the reverse osmosis) if desired, ion exchange resin is special ideal.Under above-mentioned saliva acidic group lactose synthetic situation, this can by (for example) pH be about 3.0 or lower reservation liquid in add anionite-exchange resin (as AG1 X-8, acetate form, BioRad; For example referring to, the products catalogue of other ion exchange resin of BioRad) reduce to required degree until phosphate concn and realize.In this process, acetate discharges, and so also permits after ion-exchange by nanofiltration or reverse osmosis system and carries out once more purifying.For example, can make pH3.0 or lower solution circulated pass through Osmonics MX07 or similar film, very low and stable until the specific conductivity of percolate.Then, make the pH of solution be increased to 7.4, make solution recirculation by identical film, to remove remaining sodium acetate and salt with NaOH.Decationize in a similar manner; For example, in order to remove Mn ²⁺, can add acidic ion exchange resin, as AG50WX8 (H ⁺) (BioRad).

Purification process of the present invention is specially adapted to purifying and synthesizes the oligosaccharides that makes with enzymatic.Enzymatic synthesis method with glycosyltransferase provides the very effective method for preparing oligosaccharides; Use for some, need from the enzyme the enzymatic synthesis reaction mixture and other reactant, be purified into oligosaccharides.The preferred methods of producing many oligosaccharides relates to the glycosyltransferase circulation, and every formation 1 product of moles will produce at least 1 mole of inorganic pyrophosphate, and it carries out in the presence of divalent-metal ion usually.Glycosyltransferase round-robin example is the sialic acid based transferase circulation of adopting one or more enzymes and other reactant.For example referring to, U.S. Patent No. 5,374,541 WO 9425615 A, PCT/US96/04790 and PCT/US96/04824.For example, being used for the reactant of sialic acid synthetase base oligosaccharides can contain sialic acid based transferase, cmp sialic acid synthetic enzyme, sialic acid and sialic acid based transferase acceptor, CTP and soluble divalent metal.Typical α (2,3) sialic acid based transferase refers to that α (2,3) saliva transferring enzyme (sialtransferase) (EC 2.4.99.6) transfers to sialic acid on the non-reduced terminal Gal of Gal β 1 → 3Glc disaccharides or glucosides.See Van den Eijnden etc., J.Biol.Chem., 256:3159 (1981), Weinstein etc., J.Biol.Chem., 257:13845 (1982) and Wen etc., J.Biol.Chem., 267:21011 (1992).Another typical α 2,3-sialic acid based transferase (EC 2.4.99.4) is transferred to sialic acid on the non-reduced terminal Gal of disaccharides or glucosides.See Rearick etc., J.Biol.Chem., 254:4444 (1979) and Gillespie etc., J.Biol.Chem., 267:21004 (1992).Other typical enzyme comprises Gal-β-1,4-GlcNAc α-2,6 sialic acid based transferase (seeing Kurosawa etc., Eur.J.Biochem.219:375-381 (1994)).Reaction mixture also contains the acceptor (acceptor) of sialic acid based transferase, and preferable have a galactosyl unit.Suitable acceptor comprises, for example, and Gal β 1 → 3GalNAc, lactose-N-tetrose, Gal β 1 → 3GlcNAc, Gal β 1 → 3Ara, Gal β 1 → 6GlcNAc, Gal β 1 → 4Glc (lactose), Gal β 1 → 4Glc β 1-OCH ₂CH ₃, Gal β 1 → 4Glc β 1-OCH ₂CH ₂CH ₃, Gal β 1 → 4Glc β 1-OCH ₂C ₆H ₅, Gal β 1 → 4GlcNAc, Gal β 1 → OCH ₃, melibiose, raffinose, stachyose and lacto-N-neotetraose (LNnT).Sialic acid in the reaction mixture not only can comprise sialic acid (5-N-n acetylneuraminic acid n itself; 5-N-acetylaminohydroxyphenylarsonic acid 3,5-dideoxy-D-glycerine-D-semi-lactosi-2-nonulosonic acid; NeuAc also abbreviates AcNeu or NANA sometimes as), but also comprise the sialic acid that 9-replaces, as 9-O-C ₁-C ₆-ethanoyl-NeuAc is as 9-O-lactyl-NeuAc or 9-O-ethanoyl-NeuAc, 9-deoxidation-9-fluoro-NeuAc and 9-nitrine-9-'-deoxy-n euAc.Synthetic and being applied in the International Patent Application WO 9216640 (on October 1st, 1992 is open) in sialylated process of these compounds described to some extent.

In preferable example, reaction medium also can comprise the cmp sialic acid recirculation system, it comprises the phosphodonor of at least 2 moles of every mole of sialic acids, the adenine nucleotide of catalytic amount, phosphoric acid can be transferred to kinases on the nucleoside diphosphate, terminal phosphate can be transferred to nucleoside monophosphate kinase on the CMP from ribonucleoside triphosphote from phosphodonor.For example, appropriate C MP-sialic acid regeneration system rapidly comprises cytidine monophosphate (CMP), ribonucleoside triphosphote (for example adenosine triphosphate (ATP)), phosphodonor (for example phosphoenolpyruvic acid or acetylphosphate), phosphoric acid can be transferred to the kinases (for example pyruvate kinase or E.C. 2.7.2.1) on the nucleoside diphosphate and terminal phosphate can be transferred to nucleoside monophosphate kinase (for example myokinase) on the CMP from ribonucleoside triphosphote from phosphodonor.Aforesaid α (2,3) sialic acid based transferase and cmp sialic acid synthetic enzyme also can formally be regarded the part of cmp sialic acid regeneration system rapidly as.Do not adopt the example of cmp sialic acid recirculation system for those, reaction medium preferably also comprises Phosphoric acid esterase.

Pyruvic acid is a sialic acid based transferase round-robin by product, and it can be used in another reaction, and wherein N-acetylmannosamine (ManNAc) and pyruvic acid react in the presence of NeuAc zymohexase (EC 4.1.3.3) and form sialic acid.In addition, be also advantageous in that the GlcNac isomery becomes the effect of ManAc, the starting raw material that the GlcNAc of available cheapness produces as sialic acid.Like this, sialic acid can be replaced by the NeuAc zymohexase of ManNAc (or GlcNAc) and catalytic amount.Although the NeuAc zymohexase is catalysis backward reaction (NeuAc is to ManNAc and pyruvate salt) also, the NeuAc that produces has irreversibly mixed in the catalytic reaction cycle via CMP-NeuAc of cmp sialic acid synthetic enzyme.In addition, also can make starting raw material ManNAc (for example referring to Simon etc., J.Am.Chem.Soc.110:7159 (1988)) with the method from the GlcNAc chemical conversion known in the art.The enzymic synthesis of the derivative that sialic acid and 9-thereof replace and the application of gained sialic acid in the sialylated reaction scheme of difference are disclosed in the International Patent Application WO 9216640 (on October 1st, 1992 is open) and to include this paper in for referencial use.

When coming the enzymic synthesis oligosaccharides with galactosyltransferase, reaction medium is except galactosyltransferase, donor substrate, acceptor sugar and divalent metal, preferably also contain a donor substrate recirculation system, this system comprises every mole of glucose-1-phosphate of admitting at least 1 mole of sugar, phosphodonor, phosphoric acid can be transferred to kinases on the nucleoside diphosphate, can be formed the pyrophosphorylase of UDP-glucose and the UDP and the UDP-semi-lactosi-4-epimerase of catalytic amount from UTP and glucose-1-phosphate from phosphodonor.Typical galactosyltransferase comprises α (1,3) galactosyltransferase (E.C.No.2.4.1.151, for example see Dabkowski etc., Transplant Proc.25:2921 (1993) and Yamamoto etc., Nature 345:229-233 (1990)) and β (1,4) galactosyltransferase (E.C.No.2.4.1.38).

With the also available the inventive method purifying of other enzyme method synthetic oligosaccharides.For example, this method can be used to the oligosaccharides of generation in the acyclic or part circulating reaction of purifying (for example shift effectively and sugar is covalently bound to cultivate activatory sugar and suitable admittance molecule and glycosyltransferase under the condition of admitting molecule simply).Glycosyltransferase is included in for example U.S. Patent No. 5,180,674 and International Patent Publication No. WO 93/13198 and WO95/02683 in describe those, and (see USPN 5 by the glycosyltransferase of the los locus of eisseria (Neisseria) coding, 545,553), find that they combine or debond with cell surface.The oligosaccharides that available these glycosyltransferases obtain comprises, for example, Gal α (1 → 4) Gal β (1 → 4) Glc, GlcNAc β (1,3) Gal β (1,4) Glc, Gal β (1 → 4) GlcNAc β (1 → 3) Gal β (1 → 4) Glc and GalNAc β (1 → 3) Gal β (1 → 4) GlcNAc β (1 → 3) Gal β (1 → 4) Glc.

The compound of available described method purifying comprises sialic acid and any sugar of sialic acid part is arranged.They comprise saliva acidic group semi-lactosi, comprise saliva acidic group lactose and the compound that following formula is arranged:

NeuAc α (2,3) Gal β (1 → 4) GlcN (R ') β-OR or

NeuAcα(2，3)Galβ(1→4)GlcN(R′)β(1→3)Galβ-OR。

In these chemical formulas, R ' is the alkyl or the acyl group, 5,6,7 of 1-8 carbon atom, 8-tetrahydrochysene-2-naphthoyl amino; Benzamido, 2-naphthoyl amino; 4-aminobenzoic amido; Or 4-nitrobenzoyl amido.R is hydrogen, C ₁-C ₆Alkyl, sugar, oligosaccharides or have the aglycone of a carbon atom at least.Term " aglycone that has a carbon atom at least " refers to group-A-Z, and wherein A represents the alkylidene group of 1-18 carbon atom, and this alkylidene group can be by halogen, sulfydryl, hydroxyl, oxygen, sulphur, amino, imino-or alkoxyl group; Z be hydrogen ,-OH ,-SH ,-NH ₂,-NHR ¹,-N (R ¹) ₂,-CO ₂H ,-CO ₂R ¹,-CONH ₂,-CONHR ¹,-CON (R ¹) ₂,-CON (R ¹) ₂Or-OR ¹, R wherein ¹Independent is the alkyl of 1-5 carbon atom.In addition, R can be

N wherein, m, o=1-18; (CH ₂) _n-R ²R in (wherein n=0-18) ²Be the aromatic ring of various replacements, preferably by one or more alkoxyl groups (preferably methoxyl group or O (CH ₂) _mCH ₃(wherein m=0-18), or its combination) phenyl that replaces.R can also be 3-(3,4, a 5-trimethoxyphenyl) propyl group.

The present invention also can be used to purifying and comprises in conjunction with all cpds of selecting proteic sugar moieties.These select protein binding that following general formula is partly arranged:

R ¹Galβ1，m(Fucα1，n)GlcNR ⁰(R ²) _p-

R wherein ⁰Be (C ₁-C ₈Alkyl) carbonyl, (C ₁-C ₈Alkoxyl group) carbonyl or (C ₂-C ₉Alkenyl oxygen) carbonyl, R ¹It is the oligosaccharides that following formula is arranged

R ³And R ⁴Can be identical or different, can be the C of H, replacement or non-replacement ₁-C ₈Alkyl, hydroxyl-(C ₁-C ₈Alkyl), aryl-(C ₁-C ₈Alkyl) or (C ₁-C ₈Alkoxyl group)-(C ₁-C ₈Alkyl).R ²Can be H, C ₁-C ₈Alkyl, hydroxyl-(C ₁-C ₈Alkyl), aryl (C ₁-C ₈Alkyl), (C ₁-C ₈Alkyl)-and aryl, alkylthio, α 1,2Man, α 1,6GalNAc, β 1,3Gal β 1,4Glc, α 1,2Man-R ⁸, α 1,6GalNAc-R ⁸, and β 1,3Gal-R ⁸R ⁸Can be H, C ₁-C ₈Alkyl, C ₁-C ₈Alkoxyl group, hydroxyl-(C ₁-C ₈Alkyl), aryl-(C ₁-C ₈Alkyl), (C ₁-C ₈Alkyl) aryl or alkylthio.In the formula, m and n are integers, can be 3 or 4; P can be 0 or 1.

Above-mentioned substituting group can be by hydroxyl, hydroxyl (C ₁-C ₄Alkyl), poly-hydroxy (C ₁-C ₄Alkyl), alkanol amido or hydroxyl alkanol amido substituting group replace.Preferable substituting group comprises hydroxyl, poly-hydroxy (C ₃Alkyl), kharophen and glycoloyl amino.Substituting group can have more than one substituting group, and they can be identical or different.

For R ¹Be the example of oligosaccharides, this oligosaccharides is preferably trisaccharide.Preferable trisaccharide comprises NeuAc α 2,3Gal β 1,4GlcNAc β 1,3 or NeuGc α 2,3Gal β 1,4GlcNAc β 1,3.

For R ¹Be the example of following formula,

R ³And R ⁴Should form chemical formula is-R ⁵-or-(R ⁶) _q-O-(R ⁷) _r-a group,

R wherein ⁵Be the C of replacement or non-replacement ₃-C ₇Divalent alkyl, R ₆And R ₇Identical or different, be the C of replacement or non-replacement ₁-C ₆Divalent alkyl.In the formula, q and r are integers, can be identical or different, and can be 0 or 1.The summation of q and r is at least 1.

R ³And R ⁴The better structure of the single group that forms is that chemical formula is-(R ⁶The group of)-O-, wherein R ⁶Be the C of replacement or non-replacement ₃-C ₄Divalent alkyl.For example, R ⁶Formula is-CH ₂-CH ₂-CH ₂-CH ₂-, preferably replace.This group can be used hydroxyl, poly-hydroxy (C ₃Alkyl) and the alkanol amido of replacement or non-replacement, replace as kharophen or hydroxyl kharophen.Structure after the replacement forms monose usually, and preferably sialic acid such as NeuAc or NeuGc are connected to α 2,3 on the Gal residue.

In above-mentioned general formula, m and n are integers, can be 3 or 4.Therefore, in one group of structure, Gal and GlcNAc β 1,4 connect, and Fuc and GlcNAc α 1,3 connect.This formula comprises SLe ^xTetrose.SLe ^xChemical formula be NeuAc α 2,3Gal β 1,4 (Fuc α 1,3) GlcNAc β 1-.This structure is carried the cell selective identification of LECCAM.The SLe of available method purifying of the present invention ^xCompound comprises NeuAc α 2,3Gal β 1,4 (Fuc α 1,3) GlcNAc β-1-Gal-OEt, NeuAc α 2,3Gal β 1,4 (Fuc α 1,3) GlcNAc β 1,4Gal β 1-OEt, and other compound of International Application No. WO 91/19502 description.Other compound of available this method purifying comprises U.S. Patent No. 5,604, the compound of describing in 207 that following formula is arranged

Wherein Z is a hydrogen, C ₁-C ₆Acyl group or

Y is selected from C (O), SO ₂, HNC (O), OC (O) and SC (O);

R ¹Be selected from aryl, the phenyl C of aryl, replacement ₁-C ₃Alkylidene group, wherein said aryl substituent are selected from halogen, trifluoromethyl, nitro, C ₁-C ₁₈Alkyl, C ₁-C ₁₈Alkoxyl group, amino, list-C ₁-C ₁₈Alkylamino, two-C ₁-C ₁₈Alkylamino, benzylamino, C ₁-C ₁₈Alkyl benzyl amino, C ₁-C ₁₈Alkylthio and C ₁-C ₁₈Alkyl carbonylamino, or

R ¹Y is allyloxycarbonyl or chloracetyl;

R ²Be selected from monose (comprising β 1,3Gal-OR, wherein R=H, alkyl, aryl or acyl group), disaccharides, hydrogen, C ₁-C ₁₈Straight chain, side chain or cyclic hydrocarbon group, C ₁-C ₆Alkyl, 3-(3,4, the 5-trimethoxyphenyl) propyl group, C ₁-C ₅Alkylidene group ω-carboxylate salt, ω-trisubstituted silyl C ₂-C ₄Alkylidene group, the trisubstituted silyl of wherein said ω are that three substituent silyls of independence are arranged, and they independently are selected from C ₁-C ₄Alkyl, phenyl,

Or OR ²Form C together ₁-C ₁₈Straight chain, side chain or cyclic hydrocarbon group carbamate;

R ³Be hydrogen or C ₁-C ₆Acyl group;

R ⁴Be hydrogen, C ₁-C ₆Alkyl or benzyl;

R ⁵Be selected from hydrogen, benzyl, methoxy-benzyl, dimethoxy-benzyl and C ₁-C ₆Acyl group; R ⁷Be methyl or hydroxymethyl; With

X is selected from C ₁-C ₆Acyloxy, C ₂-C ₆Hydroxyl acyloxy, hydroxyl, halogen and azido-.

The one group of relevant structure that comprises in the general formula is that Gal and β 1,3 connect, and Fuc and α 1,4 connect.For example, tetrose NeuAc α 2,3Gal β 1,3 (Fuc α 1,4) GlcNAc β 1-is called SLe here ^x, by selecting protein receptor identification.See Berg etc., J.Biol.Chem., 266:14869-14872 (1991).People such as Berg show E-especially and select albumen cDNA cell transformed optionally in conjunction with comprising SLe ^xAt interior neoglycoprotein.

Method of the present invention also can be used for purifying general formula Gal α 1, the oligosaccharide compound of 3Gal-, and it comprises Gal α 1,3Gal β 1,4Glc (R) β-O-R ¹, R wherein ₁Be-(CH ₂) _n-COX, X are OH, OR ₂,-NHNH ₂, R is OH or NAc, R ₂Be hydrogen, sugar, oligosaccharides or the aglycone that has a carbon atom at least, n is the integer of 2-18, and that better is 2-10.For example, but be Gal α 1 with the described step purifying of embodiment 7-8 chemical formula, 3Gal β 1,4GlcNAc β-O-(CH ₂) ₅The compound of-COOH.Comprise lacto-N-neotetraose (LNnT), GlcNAc β 1 in the compound according to purifying of the present invention, 3Gal β 1,4Glc (LNT-2), saliva acidic group (α 2,3)-lactose and saliva acidic group (α 2,6)-lactose.

In the foregoing description, term adopts its standard implication usually.Term used herein " alkyl " refers to have the alkyl of 1-20 carbon atom of side chain or unbranched, saturated or undersaturated, unit price or divalence, the low alkyl group (as methyl, ethyl, n-propyl, butyl, n-hexyl etc.) that comprises 1-8 carbon atom, cycloalkyl (3-7 carbon atom), cycloalkanes methyl (4-8 carbon atom) and aralkyl.Term " alkoxyl group " refers to the alkyl by oxygen link molecule rest part, for example oxyethyl group, methoxyl group or positive propoxy.Term " alkylthio " refers to by all the other alkyl that distribute and be connected of sulphur and molecule.Term " acyl group " refers to remove hydroxyl and the group of deriving and obtaining from organic acid.Example comprises ethanoyl, propionyl, oleoyl, myristoyl.

Term " aryl " refers to for example obtain phenyl from benzene by removing the group that an atom is derived and obtained from aromatic hydrocarbons.Aromatic hydrocarbons can have more than one unsaturated carbocyclic, as naphthyl.

Term " alkoxyl group " refers to the alkyl that is connected with the molecule rest part by oxygen, for example oxyethyl group, methoxyl group or positive propoxy.

Term " alkylthio " refers to the alkyl that is connected with the molecule rest part by sulphur.

The general formula of term " alkanol amido " is-NH-CO-(C ₁-C ₆Alkyl), can be substituted or not replace.If be substituted, substituting group is generally hydroxyl.This term is particularly including two preferable structures, kharophen (NH-CO-CH ₃) and hydroxyl kharophen (NH-CO-CH ₂-OH).

Term " heterogeneous ring compound " refers to have the ring compound of three or more atom, wherein the non-carbon atom of at least one atom (for example being N, O, S, Se, P or As).The example of these compounds comprises furans (furanose form that comprises pentasaccharides is as Fucose), pyrans (the pyranose form that comprises six sugar is as glucose and semi-lactosi), pyrimidine, purine, pyrazine etc.

Method of the present invention not only can be used to the new synthetic carbohydrate of purifying, but but also purifying degraded (as enzyme liberating) product.For example referring to, Sinnott, M.L., among the Chem.Rev.90:1171-1202 (1990) about the example of the enzyme of catalysis oligosaccharides degraded.

The present invention also provides the method for purifying Nucleotide, nucleotide sugar and related compound.But for example use method purifying nucleotide sugar as herein described, as GDP-Fucose, GDP-seminose, CMP-NeuAc, UDP-glucose, UDP-semi-lactosi, UDP-N-acetylgalactosamine etc.This method also is applicable to purifying Nucleotide and is in the Nucleotide (as CMP, CDP, CTP, GMP, GDP, GTP, TMP, TDP, TTP, AMP, ADP, ATP, UMP, UDP, UTP) of various phosphorylation states, and the deoxidation form of these and other Nucleotide.

Provide the following example so that describe, they also do not mean that restriction or define the present invention.

Embodiment

Embodiment 1-5 has illustrated the synthetic of saliva acidic group lactose and the purifying that carries out with nanofiltration and ion-exchange.Put it briefly, N-acetyl-D-mannosamine (ManNAc) is to obtain from N-acetyl-D-glycosamine (GluNAc) under alkaline condition.The short enzyme condensation of ManNAc and Sodium.alpha.-ketopropionate generates sialic acid.With the sialytransferase circulation sialic acid is transformed into saliva acidic group lactose, uses nanofiltration and ion-exchange purification then.Embodiment 6 has illustrated with nanofiltration and has come separation of organic substances and inorganic salt.Embodiment has illustrated the separation performance of polyphenyl methane amide nanofiltration membrane.The separation performance of embodiment 8 explanation polyamide nanofiltration membranes.

Embodiment 1

Sialic synthetic and purifying

Present embodiment has illustrated the method (rather than synthesize or directly adopt sialic acid with expensive ManNAc) of coming sialic acid synthetase with relatively inexpensive substrate GlcNAc.Employing is similar to Simon etc., and the described step of J.Am.Chem.Soc.110:7159 (1988) is transformed into ManNAc with GlcNAc.In brief, GlcNAc (1000g, 4.52 moles) is dissolved in 500 ml waters.With 50% sodium hydroxide (about 115 milliliters) pH is transferred to 12.0.Stirred solution is 7.5 hours under argon gas, cools off in ice bath then, with concentrated hydrochloric acid (about 200 milliliters) pH is transferred to 7.7.Aldol condensation by ManNAc forms sialic acid then.÷

Be subjected to the aldol condensation of N-n acetylneuraminic acid n (Neu5Ac) zymohexase and pyruvic acid mediation for the ManNAc that obtains sialic acid, make to produce in the back.In the 1.5 premium on currency solution that contain about 57 gram (0.258 mole) ManNAc and 193 gram GlcNAc (from the epimerization of base catalysis), add 123.8 gram Sodium.alpha.-ketopropionates (1.125 moles), 1.5 gram bovine serum albumins and 0.75 gram sodium azide.PH is transferred to 7.5, add the 11930U acetylneuraminate aldolase.Solution was cultivated 7 days down at 37 ℃.Aminex HPX87H (BioRad) post (0.004M sulfuric acid, 0.8 ml/min, monitoring A ₂₂₀) on the HPLC analysis revealed, solution contains 0.157M sialic acid (91% ManNAc transforms, 0.235 mole).

Embodiment 2

With sialic acid based transferase circulation sialic acid synthetase base lactose

Add lactose monohydrate (79.2 grams, 0.22 mole), 0.7 gram bovine serum albumin, phosphoenolpyruvic acid one sylvite (37 grams, 0.22 mole) in the sialic acid that embodiment 1 makes, pH transfers to 7.5.Add CMP (2.48 grams, 0.0088 mole), ATP (0.54 gram, 0.0009 mole), pH is transferred to 7.5.Add sodium azide (0.35 gram), and following enzyme: pyruvate kinase (19800U), myokinase (13200U), CMP sialic acid synthetic enzyme (440U) and sialic acid based transferase (165U).Add 66 milliliters of MnCl ₂, water transfers to 2.2 liters with final volume.Reaction is at room temperature carried out.

Measure [Mn with thin-layer chromatography (TLC) monitoring reaction with ion chromatography every day ²⁺].The as shown in table 1 interpolation/regulate:

Table 1

Added 44 milliliters of 1M MnCl on the 2nd day ₂

Added 43 milliliters of 1M MnCl on the 4th day ₂

Added 34.3 milliliters of 1M MnCl on the 6th day ₂, 37 gram PEP; Readjust pH7.5; Pyruvate kinase (19800U), myokinase (13200U), CMP sialic acid synthetic enzyme (440U) and sialic acid based transferase (165U)

The 7th day 31.7 milliliters of 1M MnCl ₂

The 8th day 24.6 milliliters of 1M MnCl ₂

The 9th day 44 milliliters of 1M MnCl ₂

The 10th day 30.8 milliliters of 1M MnCl ₂

The 11st day 31.7 milliliters of 1M MnCl ₂

The 12nd day 24.6 milliliters of 1M MnCl ₂, readjust pH7.5

The 13rd day 440U CMP sialic acid synthetic enzyme, 82.5U sialic acid based transferase

Readjusted pH7.5 on the 14th day

The 16th day 37.7 milliliters of 1M MnCl ₂, 19800U pyruvate kinase, 13200U myokinase

The 17th day 26 gram phosphoenolpyruvic acid, trisodium salt

Record through TLC, saliva acidic group lactose productive rate is about 70-80%.

Embodiment 3

With sialic acid based transferase circulation sialic acid synthetase base lactose

Present embodiment has been described with the circulation of sialic acid based transferase and to manganese ion concentration and has been controlled to produce α-N-n acetylneuraminic acid n (2,3) beta galactose base (1,4) glucose.

In polypropylene container, phosphoenolpyruvic acid trisodium salt (285.4 grams, 1.22 moles) and sialic acid (197 grams, 0.637 mole) are dissolved in 5 premium on currency, regulate pH to 7.1 with 6M sodium hydroxide.The adding Cytidine-5 '-single phosphoric acid (5.14 grams, 15.9 mmoles) and Repone K (7.9 grams, 0.106 mole), readjust pH to 7.45 with 6M sodium hydroxide.Add pyruvate kinase (28000 unit), myokinase (17000 unit), adenosine triphosphate (0.98 gram, 1.6 mmoles), CMP NeuAc synthetic enzyme (1325 unit), α 2,3 sialic acid based transferases (663 unit) and MnCl ₂4H ₂O (52.4 grams, 0.265 mole) also mixes.In dividing, 3.7 ascending parts of gained mixture add lactose (119 grams, 0.348 mole) and sodium azide (1.75 gram).Reaction mixture is kept at room temperature, and monitor with thin-layer chromatography (TLC) and ion chromatography every day.Two days later, the extra enzyme of following adding: pyruvate kinase (38100 unit), myokinase (23700 unit), CMP NeuAc synthetic enzyme (935 unit) and α 2,3 sialic acid based transferases (463 unit).Regularly pH is transferred to 7.5 with 6M sodium hydroxide.Measure manganese ion concentration and interpolation as shown in table 2 below in addition.

Table 2

Fate	????[Mn ++] (measured value, mM)	??Mn ++Loss (from the day before yesterday)	Addition (the milliliter number of 1M, the ultimate density after the adding
				????1	????28	????22.0	No
????2	????23.9	????4.1	No
				????3	????10.7	????13.2	111 milliliters ,+30mM
????4	????1.4	????3.9.3	111 milliliters ,+30mM
				????5	????3.0	????28.4	148 milliliters ,+40mM
????6	????12.9	????30.1	74 milliliters ,+20mM
				????7	????10.0	????22.9	80 milliliters ,+20mM
????8	????12.0	????18.0	80 milliliters ,+20mM
				????9	????24.3	????7.7	No

In the time of the 9th day, TLC shows that reaction is complete substantially.Shown in result in the table, Mn ⁺⁺Loss cause almost that all need add MnCl every day ₂4H ₂O is to keep concentration of metal ions.Mn ion is the required cofactor of at least a enzyme in the circulation of sialic acid based transferase.Yet, the inorganic phosphate salt formation of mn ion and generation the low-down title complex of solubleness.Because in the restriction of solubleness, though the transferring enzyme circulation can be proceeded, speed of response slows down.Replenish because of precipitating the mn ion that loses by adding, can keep speed of response with pyrophosphate salt.Therefore, in the time of in manganese ion concentration maintains the suitableeest scope, sialic acid based transferase reaction cycle can tend to complete.

Embodiment 4

With ion-exchange and reverse osmosis purified salivary acidic group lactose

Present embodiment has been described trisaccharide that embodiment 2 makes verification and the purity after through acetylize and esterification.The 5-acetylaminohydroxyphenylarsonic acid 3 that the sialic acid based transferase is produced down at suitable lactose cofactor (55 gram), 5-dideoxy-α-D-glycerine-D-semi-lactosi-nonulo pyrans glycosyl ester-(2-3)-O-β-D-semi-lactosi pyrans glycosyl-(1-4)-O-β-D-glucose pyranose sodium solution (2 liters) filtration passing through filter paper.The filter membrane that filtrate is held back by the 3000-10000 molecular weight is to remove Deproteinization from required product.Make elutriant suitable device (Cat.No.CDRN500 60, Millipore, Bedford MA) concentrates and desalination on the polyamide reverse osmose membrane in.The retention that will contain product flashes to thick slurry.Also can randomly handle reservation liquid, to remove divalent cation with resin.After filtration, the filtrate of containing required product is substantially free of salt, warp ¹The Hmr spectrography shows, its purity height.In addition, evaporate soup compound twice with pyridine (2 * 200 milliliters).In evaporative flask, add the N in 1.2 liters of pyridines, N-dimethyl aminopyridine (2.2 gram) solution.In 1 hour, add diacetyl oxide (0.83 liter).The gained mixture was at room temperature slowly stirred 24-48 hour.With TLC (methyl alcohol: methylene dichloride is 1: 9) monitoring reaction.After reaction finishes, vacuumize, evaporating solns obtains resistates.

Resistates is dissolved in 1.5 liters of ethyl acetate.Wash this solution with 5% aqueous hydrochloric acid (1.5 liters), use saturated sodium bicarbonate aqueous solution (1.5 liters) washing then, last water (1.5 liters) washing.With anhydrous sodium sulfate drying organic layer and filtration.Filtrate is condensed into the semi-solid state resistates.Mistake-O-ethanoyl lactose trisaccharide (69 gram) is dissolved in the methyl alcohol (350 milliliters), the adding sodium methoxide solution (17.5 milliliters, 25%, in methanol solution), add 3.5 ml waters then.When using Virahol: ammonium hydroxide: water is to carry out TLC at 7: 1: 2 to show when reaction finishes, and adds acetate (2 milliliters) in solution.In solution, add ether (180 milliliters), make the product precipitation.Filter this solid, and be dissolved in (350 milliliters) in the water.In this solution, add charcoal 24 grams, under 60 ℃, be heated to 1 hour.Then this solution is cooled to room temperature and filtration.Evaporated filtrate obtains solid product (34 gram). ¹The H-NMR spectrography shows that this solid is pure saliva acidic group lactose, and it contains the sodium acetate of 11% weight.

Embodiment 5

With nanofiltration purified salivary acidic group lactose

The ultra-filtration membrane that with MWCO is 10kDa filters being similar to embodiment 2 described reaction mixtures, to remove Deproteinization.Record phosphoric acid concentration [PO through following standard phosphorus testing sequence ₄ ^3-] greater than 2.8mM.

With concentrated hydrochloric acid (about 500 milliliters) regulator solution to pH3.0.It was gone up purifying 3-5 hour at Osmonics213T film purifying instrument (film type is MX07) under pH3, remain unchanged until the specific conductivity of percolate.Wash solution from instrument then, the scavenging solution of merging and stock liquid with sodium-hydroxide treatment to pH be 7.4.As described belowly measure Mn with HPLC ²⁺Concentration.The nanofiltration parameter is as follows:

Working pressure: P _f=100psi

Concentrated solution flow velocity: Q _c=5GPM

Percolate flow velocity: Q _f=7GPH

Temperature range: 20-40 ℃

Volume: 5 gallons

Initial percolate specific conductivity is 28.1mS; After the recirculation 5 hours, specific conductivity is reduced to 115 μ S, phosphate concentration [PO ₄ ³⁺] be reduced to 770 μ M, manganese concentration [Mn ²⁺] be 3.4mM.

Regulator solution is further purified about 1 hour to pH7.4 on film purifying instrument (Osmonics, film type are MX07) then, remains unchanged until the specific conductivity of percolate.Then solution is washed out from the film instrument.The nanofiltration parameter is:

Working pressure: P _f=100psi

Concentrated solution flow velocity: Q _c=5GPM

Percolate flow velocity: Q _f=0.3GPH

Temperature range: 20-40 ℃

Volume: 5 gallons

Filter result is as follows:

Specific conductivity: initial percolate specific conductivity: 2.01mS

After the recirculation 5 hours: 93.7 μ S

Phosphate concentration: [PO ₄ ^3-]=410 μ M

Manganese concentration: [Mn ²⁺]=3.0mM

Use AG50WX8 (H then ⁺) resin (BioRad, 1.18 kilograms) handles above-mentioned solution (6Gal), stirs 2 hours until pH=2.0.Filter resin then so that very bright yellow solution to be provided.HPLC monitors has only a small amount of [Mn ²⁺].Use sodium hydroxide (50%w/w) that solution is neutralized to pH7.4 then.Before the plastic resin treatment: [Mn ²⁺]=3mM [PO ₄ ^3-After the μ M plastic resin treatment of]=410: pH=3, [Mn ²⁺]=1.23mM

pH＝2，[Mn ²⁺]＝6.8μM????????[PO ₄ ^3-]＝190μM

With the solution of AG1X8 (acetate form) the more above-mentioned small parts of plastic resin treatment, further to remove phosphoric acid salt.The result is presented in the following table 3:

Table 3

Sample volume (milliliter)	Weight resin (gram)	Churning time (hour)	??[PO 4 3-]μM
				50 milliliters	0.25 gram	????1	????86
50 milliliters	0.5 gram	????1	????41
				50 milliliters	1.0 gram	????1	????30
50 milliliters	2.0 gram	????1	????8

Make solution recirculation with Osmonic film purifying instrument (Osmonic MX07), purification solution is 5 hours under the following conditions:

Working pressure: P _f=100psi

Concentrated solution flow velocity: Q _{C '}=5GPM

Percolate flow velocity: Q _f=0.2GPH

Temperature range: 20-40 ℃

Volume: 5 gallons

The result is as follows:

Percolate specific conductivity: initial percolate specific conductivity: 0.136mS

Separate after 5 hours: 45 μ S

Then, solution concentration to the 3-4 liter, is added gac (J.T.Baker, 180 grams) then.Make suspension be heated to 55 ℃ 2 hours.Remove by filter charcoal then, obtain very bright yellow solution, its freeze-drying solid that is white in color.

The analytical data that has shown the saliva acidic group lactose solution that above-mentioned purifying obtains in the table 4.

Table 4

Measure	The result	Method
			????PO 4 3-Content	330ppm (weight)	The phosphate determination method 1
Nucleotide/nucleosides content	A) do not survey (ABS 280=0.0) b) do not survey	UV (0.1mM, saliva acidic group lactose) 1H-NMR
			????Mn 2+Content	80ppm (weight)	????HPLC 2Record
Saliva acidic group lactose-content	????71％	1H-NMR (with 1,2-isopropylidene D-glucose furanose is as standard)
			Sialic acid content	About 2%	???? 1H-NMR
Lactose-content	Do not survey	???? 1H-NMR
			Acetate content	Do not survey	???? 1H-NMR
N-acetyl-glucosamine content	Do not survey	???? 1H-NMR
			Pyruvate salt content	Do not survey	???? 1H-NMR

¹The phosphatase assay method

With deionized water (775 μ l) dilution unknown sample (100 μ l).Use 100 μ l bimolybdates (be dissolved in 100 μ l 2.5N sulfuric acid make 1.25 gram ammonium molybdates), 25 μ l Fiska Subha Row solution (powder is available from Sigma, and prepares by manufacturer's specification sheets) to handle this solution then.100 ℃ were heated this mixture 7 minutes, and write down the absorbancy under the 810nm then.Compare with the phosphoric acid typical curve, to determine concentration.

²HPLC test determination Mn ²⁺Concentration:

Post: Alltech Universal cation seperation column, 0.46 * 10cm

Monitor: Alltech302 type electric conductivity monitor

Moving phase: 3mM phthalandione, 0.5mM pyridine dicarboxylic acid

Flow velocity: 1.5 ml/min

Post oven temperature: 35 ℃.

Embodiment 6

Nanofiltration separation organism and inorganic salt

Test various nanofiltration membrane separation and go out the ability of various organic compound and inorganic salt in the aqueous solution.Under two different pH, film is tested, can be regulated separation distributions figure by the ionic charge of regulating some compound with proof.The result is as shown in table 5.

The nanofiltration membrane of test is Osmonics, MX07, SX12 and B006 that Inc. (Minnetonka MN) produces, and Osmonics, the DL2540 that DeSalination Systems produces.The MX07 film uses as mode as described in the above-mentioned embodiment 5.The parameter of other film is listed in table 6.

Show to pass through in 5:30 minute the compound percentage ratio of film

Film	??????MX07 a		??????SX12 a		??????B006 a		?????DL2540 a
										??pH7.5	?pH3.0	?pH7.5	?pH3.0	?pH7.5	?pH3.0	??pH7.5	?pH3.0
Compound
									Sodium phosphate	????10	????46	????20	????39	????15	????64	????1.8 b
Manganese	????86		????40	????40	????92	????92
									Sodium.alpha.-ketopropionate	????35	????59	????45	????65	????34	????65
??GlcNAc	????70	????28			????84		????12
									Lactose	????36	????＜5			By
Raffinose	????0	????0	????8		????52
									Sialic acid	????12	????5					????＜1	????1
Sodium	????56
									??CMP	????＜1	????＜1
??PEP	????＜1	????8

Attention: ^aSeparation degree by % during according to 30 minutes

^b20 ℃ and 40 ℃ of tests down

GluNAc:N-acetyl-D-glycosamine

PEP:2-phosphoenolpyruvic acid trisodium salt

CMP: cytidine 5 '-single phosphoric acid

Film MX07, SX12, B006 be available from Osmonics, Inc., DL2540 available from

Osmonics，Desalination?Systems(Escondido，CA)。

Table 6

	????SX12	????B006	????DL2540
				Pressure (Pf) (PSI)	????200	????100	????200
Concentrated solution flow velocity (Q c)(GPM)	????4.5	????4	????4
				Percolate flow velocity (Q f)(GPM)	????0.2	????0.5	????0.6
Temperature range (℃)	????20-40	????20-40	????20-40
				Volume (Gal)	????5	????5	????5

Embodiment 7

The separation performance of polyphenyl methane amide nanofiltration membrane

The evidence that present embodiment is described flat and spiral twine polyphenyl methane amide film (YK, Osmonics) the purifying sugar effectively of form.Test membrane passing through and stick effect under various pH levels to sugar and salt.

Material and method

A. flat and spiral wound form instrumentation and membrane prepare.

(Escondido CA), cleans 4 to 5 times, uses about 1 liter of distilled water at every turn for Osmonics, Desalination Systems at first thoroughly to clean the desalination membrane instrument that the YK film is arranged.Water is poured in the feed chute, and circulate about 1 minute (about 100psi) forwards out water discharge valve to position, emptying counterclockwise.Valve-off after the draining repeats this process 4-5 time.After cleaning, add the water more than 1 liter again.Again make system under the pressure of 150psi, circulate 30 minutes emptying then.To be with the film system to be used for following test then.

After each off-test, wash instrument 3-4 time like that as mentioned above.Make about 1 premium on currency then under 100-150psi the about 15-20 of recirculation minute, and emptying in the instrument.Still remain in the instrument if suspect some test compounds, also can carry out extra simple cleaning after this step sometimes.Should monitor specific conductivity all the time, remove to guarantee all samples.If specific conductivity is still very high, then clean instrument until fundamental surveillance less than pollutent.Most of ionic compounds are easy to be removed, exception be MnCl ₂, it also needs 1 or 2 extra brief cleaning.

B. the test of salt

In order to measure retention property, test the 10mM solution of following salt: MnCl with flat film to various salt ₂, NaH ₂PO ₄, NaC ₃H ₃O ₃, NaOAc, Na ₄P ₂O ₇, Sodium Benzoate, MgSO ₄, NaN ₃, and NaCl.A kind of 1 liter of solution of salt is poured in the feed chute, and recirculation is about 15 minutes under 100psi.At this moment, collect percolate and concentrated solution sample, measure with conductivitimeter.After this, collected sample every 5 minutes, each sample operation is collected three times altogether at least.With the specific conductivity of percolate specific conductivity divided by concentrated solution, the percentage ratio (" passing through percentage ratio ") of the salt of calculating by film.

Behind end of run first, the pH of solution is reduced to pH3.0, if possible, adopt the conjugate acid of the salt of test.When regulating pH, make solution recirculation, to guarantee the solution thorough mixing in the instrument.Repeated test process, and the specific conductivity of mensuration percolate and concentrated solution.With conjugate base pH value of solution is increased to then and is about 7.0, under new pH, rerun once more.Measure the specific conductivity of percolate and concentrated solution once more.

C. Tang test

The steamed bun stuffed with sugar of test is drawn together saliva acidic group lactose, lactose, N-acetyl-glucosamine, NeuAc α 2,3Gal β 1,4 (Fuc α 1,3) GlcNAc β 1,4Gal β 1-OEt (Compound I), Gal α 1,3Gal β 1,4GlcNAc β-O-(CH ₂) ₅-COOH (Compound I I), LNT-2, LNnT, CMP, cytidine and sialic acid.Sugar soln (1 liter) is poured in the feed containers, and recirculation is at least 10 minutes under 100psi.In the time of 10 minutes, get percolate and concentrated solution sample, in the time of 15 minutes, get another sample of percolate again.With TLC naked eyes comparative sample.Any pH regulator adopts HCl and/or NaOH.

The result:

A. flat film

(YK002, on YV+ paper backing, Osmonics) retention property to various salt and sugar is presented in the table 7 flat polyphenyl methane amide nanofiltration membrane.Test is carried out under 25-35 ℃, and the percolate flow velocity is the 2-8 ml/min.

Table 7

Material	Concentration	Pressure (psi)	Pass through % *
						??pH3.0	??pH5 **	????pH7
			????MnCl 2	??10mM	????100				????66	????12	????9.8
????NaH 2PO 4	??10mM	????100				????82	????15	????4.6
			Sodium.alpha.-ketopropionate	??10mM	????100				????80	????36	????9.8
Sodium-chlor	??10mM	????100				????-	????-	????18
			Saliva acidic group lactose ***	??10g/L	????100				????0	????-	????0
Compound I ***	??10g/L	????100				????0	????-	????0
			Compound I I ***	??2g/L	????100				????0	????-	????0
????LNT-2 ***	??0.4g/L	????100				????0	????-	????0
			????LNnT ***	??0.35g/L	????100				????0	????-	????0
Lactose	??10g/L	????100				????0.0	????0.3	????-
			????GlcNAc	??10g/L	????100				????5.9	????-	????3.7
????Na 4P 2O 7	??10mM	????100				????19	????2.0	????1.4
			Sialic acid ***	??10mM	????100				????0	????-	????-
Cytidine ***	??1g/L	????100				????0	????-	Trace
			????CMP ***	??1g/L	????100				????0	????-	????0
Benzylalcohol ***	1.5% (volume)	????100				????-	????-	????100
			????NaN 3	??10mM	????100				????81	????-	????67
????MgSO 4	??10mM	????100				????38	????-	????2.9
			Phenylformic acid	About 0.5g/L	????100				????99	????-	????-
Sodium Benzoate	??2.5％	????100				????-	????-	????42

^*By % is the per-cent of amount of substance in amount of substance and the concentrated solution in the percolate.

^*" pH5 " scope is 4.8-5.6

" pH7 " scope is 6.1-7.4

^{* *}Measure with the TLC naked eyes

B. spiral membrane

Spirrillum polyphenyl methane amide nanofiltration membrane (YK1812CZA; Osmonics) retention property to various salt and sugar is presented in the table 8.Test is carried out under 25-35 ℃, and the percolate flow velocity is 3 milliliters/second.

Table 8

Material	Concentration	Pressure (psi)	Pass through % *
						??pH3.0	??pH5 **	????pH7
			????MnCl 2	??10mM	????100				????50	????-	?40(pH6.2)
????NaH 2PO 4	??10mM	????100				????67	????49	????19
			????NaOAc	??10mM	????100				????-	????81	????65
Sodium.alpha.-ketopropionate	??10mM	????100				????81	????-	????26
			Sodium-chlor	??10mM	????100				????79	????78	????-
Saliva acidic group lactose ***	??10g/L	????100				????0	????-	????0
			Compound I ***	??10g/L	????100				????0	????-	????0
Compound I I ***	??2g/L	????100				????0	????-	????0
			????LNT-2 ***	??0.4g/L	????100				????0	????-	????0
Lactose	??10g/L	????100				????0.59	????-	????2.3
			????GlcNAc	??10g/L	????100				????13	????7.1	????19
????Na 4P 2O 7	??10mM	????100				????65	????-	????5.2
			Sialic acid ***	??10mM	????100				????0	????-	????0
Cytidine ***	??1g/L	????100				About 10	????-	About 5-10
			????CMP ***	??1g/L	????100				Trace	????-	Trace
Sodium Benzoate	About 0.5g/L	????100				????93	????-	????97

^*By % is the per-cent of amount of substance in amount of substance and the concentrated solution in the percolate.

^*" pH5 " scope is 4.5-5.2

" pH7 " scope is 6.6-7.0

" pH3 " scope is 2.8-3.4

^{* *}Measure with the TLC naked eyes

# " trace " refers to that TLC goes up inadequate observation of naked eyes and obtains.

These results show that flat film of YK002 and YK1812CZA spiral membrane have kept saliva acidic group lactose and Compound I and II, LNT-2 and LNnT, and ionic compound is passed through, and make that this film is the good selection of these sugar of purifying.

Embodiment 8

The separation performance of polyamide nanofiltration membrane

Present embodiment has been described the evaluation of polyamide membrane in purifying sugar purposes to several flat and spiral wound forms.Test membrane passing through or stick effect under various pH levels to sugar and salt.

Material and method

A. flat and spiral wound form instrumentation and membrane prepare.

At first thoroughly clean polyamide membrane G-5 (GE is arranged; Osmonics) (Escondido CA), cleans 4 to 5 times desalination membrane instrument, uses about 1 liter of distilled water at every turn for Osmonics, Desalination Systems.Water is poured in the feed chute, and circulate about 1 minute (about 100psi) uses the water discharge valve emptied of water.Valve-off after the emptying repeats this process 4-5 time.After cleaning, add the water more than 1 liter again.Make system's recirculation 30 minutes under the pressure of 150psi, emptying then.Then, band film system has been ready to can be used for following test.

After each test, wash instrument 3-4 time like that as mentioned above.Make about 1 premium on currency then under 100-150psi the about 15-20 of recirculation minute, and emptying in the instrument.Still remain in the instrument if suspect some test compounds, after this also can carry out extra simple cleaning sometimes.All the time monitor specific conductivity, be removed to guarantee all samples.If specific conductivity is still very high, then cleans instrument and do not go out pollutent until basic survey.Most of ionic compounds are easy to be removed, exception be MnCl ₂, it also needs 1 or 2 extra brief cleaning.

B. the test of salt

Test the 10mM solution of following salt: MnCl with flat film ₂, NaH ₂PO ₄, NaC ₃H ₃O ₃And NaCl.A kind of 1 liter of solution of salt is poured in the feed chute, and recirculation is about 15 minutes under 100psi.At this moment, collect percolate and concentrated solution sample, measure with conductivitimeter.After this, collected sample every 5 minutes, each sample operation is collected three times altogether at least.With the specific conductivity of percolate specific conductivity, calculate to such an extent that pass through percentage ratio divided by concentrated solution.Behind end of run, the pH of solution is reduced to 3.0, if possible, adopt the conjugate acid of test salt.When regulating pH, make solution recirculation, to guarantee the solution thorough mixing in the instrument.The repeated test process is collected data as mentioned above.With conjugate base pH is increased to approximately 7.0 then, under new pH, repeats once more.Emptying and cleaning as mentioned above then.

C. Tang test

The steamed bun stuffed with sugar of test is drawn together saliva acidic group lactose, lactose, NeuAc α 2,3Gal β 1,4 (Fuc α 1,3) GlcNAc β 1,4Gal β 1-OEt (Compound I), Gal α 1,3Gal β 1,4GlcNAc β-O-(CH ₂) ₅-COOH (Compound I I), LNT-2, LNnT.Sugar soln (1 liter) is poured in the feed containers, and recirculation is at least 10 minutes under 100psi.In the time of 10 minutes, get percolate and concentrated solution sample, in the time of 15 minutes, get another sample of percolate again.With TLC naked eyes comparative sample.Any pH regulator adopts HCl and/or NaOH.After sugar is tested, it is transferred in the Pyrex flask in order to being used for other film once more.

The result:

A. flat film

((GH, Osmonics) retention property to various salt and sugar is presented in the table 9 G-10 flat polyamide nanofiltration membrane.The A-value of film is 10.0, and it is 62.8 (to use 2000ppmMgSO that tap water sees through percentage ratio ₄At room temperature record).Test is carried out under 25-35 ℃, and the percolate flow velocity is the 5-8 ml/min.

Table 9

Material	Concentration	Pressure (psi)	Pass through % *
						??pH3.0	??pH5 **	???pH7
			????MnCl 2	??10mM	????200				????82.4	????82.4	????84.6
????NaH 2PO 4	??10mM	????200				????33.0	????18.0	????10.5
			Sodium.alpha.-ketopropionate	??10mM	????200				????49.4	?????-	????8.9
Sodium-chlor	??10mM	????200				????-	?????-	????17.8
			Saliva acidic group lactose ***	??10g/L	????200				????＜5	?????-	????＜5
Compound I ***	??10g/L	????200				????-	?????-	????0
			Compound I I ***	??2g/L	????200				????0	?????-	????-
????LNT-2 ***	??0.4g/L	????200				????-	?????-	Trace #
			????LNnT ***	??0.35g/L	????200				????-	?????-	Trace #
Lactose	??10g/L	????200				????2.0	?????-	????4.2

^*By % is the per-cent of amount of substance in amount of substance and the concentrated solution in the percolate.

^*" pH5 " scope is 4.8-5.6

^{* *}Measure with the TLC naked eyes

# " trace " refers to that TLC goes up inadequate observation of naked eyes and obtains.

In another experiment, test A-value is 8.0, tap water through percentage ratio is 38.9 G-10 (GH) polyamide membrane.Test is carried out under 25-35 ℃, and the percolate flow velocity is the 6-8 ml/min.The results are shown in Table 10.

Table 10

Material	Concentration	Pressure (psi)	Pass through % *
						??pH3.0	??pH5 **	????pH7
			????MnCl 2	??10mM	????200				???70.8	????-	????77.7
????NaH 2PO 4	??10mM	????200				???39.4	????32.1	????16.2
			Sodium.alpha.-ketopropionate	??10mM	????200				???60.8	????-	????21.8
Sodium-chlor	??10mM	????200				????-	????-	????14.2
			Saliva acidic group lactose ***	??10g/L	????200				Trace #	????-	Trace #
Compound I ***	??10g/L	????200				????-	????-	????0
			Compound I I ***	??2g/L	????200				Trace #	????-	????-
????LNT-2 ***	??0.4g/L	????200				????-	????-	Trace #
			????LNnT ***	??0.35g/L	????200				????-	????-	Trace #
Lactose	??10g/L	????200				???3.8	????-	?????22.1

^*By % is the per-cent of amount of substance in amount of substance and the concentrated solution in the percolate.

^*" pH5 " scope is 4.8-5.6

^{* *}Measure with the TLC naked eyes

Also tested G-5 (GE) polyamide membrane (the A-value is 3.9, and it is 33.9 that tap water sees through percentage ratio).Test is carried out under 25-35 ℃, and the percolate flow velocity is the 3-5 ml/min.The results are shown in Table 11.

Table 11

Material	Concentration	Pressure (psi)	Pass through % *
						???pH3.0	??pH5 **	???pH7
			????MnCl 2	??10mM	????200				????77.6	????80.1	????81.8
????NaH 2PO 4	??10mM	????200				????30.0	????8.6	????4.8
			Sodium.alpha.-ketopropionate	??10mM	????200				????48.2	?????-	????8.4
Sodium-chlor	??10mM	????200				????-	?????-	????15.0
			Saliva acidic group lactose ***	??10g/L	????200				????0	?????-	????0
Compound I ***	??10g/L	????200				????-	?????-	????0
			Compound I I ***	??2g/L	????200				????0	?????-	????-
????LNT-2 ***	??0.4g/L	????200				????-	?????-	????0
			????LNnT ***	??0.35g/L	????200				????-	?????-	????0
Lactose	??10g/L	????200				????6.3	?????-	????15.1

^*By % is the per-cent of amount of substance in amount of substance and the concentrated solution in the percolate.

^*" pH5 " scope is 4.8-5.6

^{* *}Measure with the TLC naked eyes

HL (Osmonics) polyamide membrane is shown in the table 12 retention property of sugar and salt.Test is carried out under 25-35 ℃, and the percolate flow velocity is the 8-13 ml/min.

Table 12

Material	Concentration	Pressure (psi)	Pass through % *
						??pH3.0	??pH5 **	????pH7
			????MnCl 2	??10mM	????100				????48	????22	????23
????NaH 2PO 4	??10mM	????100				????67	????24	????7.5
			Sodium.alpha.-ketopropionate	??10mM	????100				????76	????29	????16
Sodium-chlor	??10mM	????100				????71	????66	????-
			Saliva acidic group lactose ***	??10g/L	????100				????0	????-	????0
Lactose	??10g/L	????100				????1.9	????4.1	????-

^*By % is the per-cent of amount of substance in amount of substance and the concentrated solution in the percolate.

^*" pH5 " scope is 4.5-5.8

^{* *}Measure with the TLC naked eyes

B. spiral wound form film

Also measured the retention property of spiral wound form shape polyamide membrane to salt and sugar.GH1812CAZ film (Osmonics) is tested under 25-35 ℃, and the percolate flow velocity is the 1.5-2 milliliters/second.The results are shown in table 13.

Table 13

Material	Concentration	Pressure (psi)	Pass through % *
						??pH3.0	??pH5 **	??pH7
			????MnCl 2	??10mM	????100				????93	????94	????-
????NaH 2PO 4	??10mM	????100				????69	????29	????19
			Sodium.alpha.-ketopropionate	??10mM	????100				????68	????-	????42
Sodium-chlor	??10mM	????100				????66	????61	????64
			Saliva acidic group lactose ***	??10g/L	????100				Trace #	????-	Trace #
Compound I ***	??10g/L	????100				????0	????-	????0
			Compound I I ***	??2g/L	????100				????0	????-	????0
????LNT-2 ***	??0.4g/L	????100				Trace #	????-	Trace #
			Lactose	??10g/L	????100				????73	????-	????34
????GlcNAc	??10g/L	????100				????48	????-	????56
			????Na 4P 2O 7	??10mM	????100				????13	????-	????5.7
Sialic acid ***	??10mM	????100				????25-50	????-	????-
			Cytidine ***	??1g/L	????100				????＞50	????-	????＞50
????CMP ***	??1g/L	????100				????＞50	????-	????＞50
			Sodium Benzoate	About 0.5g/L	????100				????90	????-	????-

^*By % is the per-cent of amount of substance in amount of substance and the concentrated solution in the percolate.

^*" pH5 " scope is 4.5-5.6

" pH7 " scope is 6.1-7.4

^{* *}Measure with the TLC naked eyes

# " trace " refers to that TLC goes up inadequate observation of naked eyes and obtains.

GE1812CAZ spiral wound form polyamide membrane (Osmonics) under 25-35 ℃, percolate flow velocity 0.9 milliliters/second that reduces the test gained the results are shown in Table 14.

Table 14

Material	Concentration	Pressure (psi)	Pass through % *
						???pH3.0	??pH5 **	???pH7
			????MnCl 2	??10mM	????100				????90	????94	????-
????NaH 2PO 4	??10mM	????100				????54	????14	????8.7
			????NaOAc	??10mM	????100				????98	????-	????24
Sodium.alpha.-ketopropionate	??10mM	????100				????73	????-	????45
			Sodium-chlor	??10mM	????100				????54	????-	????44
Saliva acidic group lactose ***	??10g/L	????100				????0	????-	????0
			Compound I ***	??10g/L	????100				????0	????-	????0
Compound I I ***	??2g/L	????100				????0	????-	????0
			Lactose	??10g/L	????100				????41	????-	????43
????GlcNAc	??10g/L	????100				????72	????-	????69
			????MgSO 4	??10mM	????100				????50	????37	????-
????Na 4P 2O 7	??10mM	????100				????11	????-	????4.7
			Sialic acid I ***	??10mM	????100				Trace #	????-	Trace #
Cytidine ***	??1g/L	????100				????＞50	????-	????＞50
			????CMP ***	??1g/L	????100				????＞50	????-	????＞50
Sodium Benzoate	About 0.5g/L	????100				????63	????40	????-

^*By % is the per-cent of amount of substance in amount of substance and the concentrated solution in the percolate.

^*" pH5 " scope is 4.8-5.6

^{* *}Measure with the TLC naked eyes

# " trace " refers to that TLC goes up inadequate observation of naked eyes and obtains.

These results show that G-10 (GH) (the A value is 10), G-10 (GH) (the A value is 8) flat film and GH1812CZA spiral wound form film pass through ion, but can not effectively keep saliva acidic group lactose or similar trisaccharide.G-5 (GE) (the A value is 3.9) flat film and GE1812CZA spiral wound form film can keep saliva acidic group lactose, Compound I and II, LNT-2 and LNnT, and ionic compound is passed through.

It is for referencial use that this paper is all included in all publications, patent and the patent application of mentioning in this specification sheets in, and it is for referencial use to include this paper particularly, individually in as each independent publication, patent or patent application.

Foregoing description is descriptive, unrestriction.With reference to after this paper disclosure, those skilled in the art are easy to the present invention is done many variations.For example, many substrates, enzyme and reaction conditions are alternative goes in the glycosyltransferase circulation, and this is a part of the present invention, does not depart from the scope of the present invention.Therefore scope of the present invention should not determined according to foregoing description, but should determine according to claims and four corner of equal value thereof.

Claims (30)

1. method from the stock liquid Carbohybrate purification compound that contains pollutent, this method comprises contacts stock liquid under certain condition with nanofiltration membrane or reverse osmosis membrane, film keeps saccharide compound and makes most of pollutents by film under this condition, and wherein saccharide compound is selected from:

Saliva acidic group lactose;

Sialic acid;

LnNT；

LNT-2；

NeuAc α (2 → 3) Gal β (1 → 4) (Fuc α 1 → 3) Glc (R ¹) β 1-OR ², R wherein ¹Be OH or NAc; R ²Be hydrogen, alkoxyl group, sugar, oligosaccharides or the aglycone that has a carbon atom at least; And

Gal α (1 → 3) Gal β (1 → 4) Glc (R ¹) β-O-R ³, R wherein ¹Be OH or NAc; R ³Be-(CH ₂) _n-COX, X=OH, OR ⁴,-NHNH ₂, R ⁴Be hydrogen, sugar, oligosaccharides or the aglycone that has a carbon atom at least, n is 2 to 18 integer.

2. method according to claim 1, wherein saccharide compound is selected from saliva acidic group lactose, LnNT, LNT-2 and Gal α 1,3Gal β 1,4GlcNAc β 1-O-(CH ₂) ₅-COOH.

3. method according to claim 2, wherein saccharide compound is selected from saliva acidic group (α 2,3) lactoside and saliva acidic group (α 2,6) lactoside.

4. method according to claim 2, wherein film comprises polymeric amide or polyphenyl methane amide.

5. method according to claim 4, wherein film is the film that is selected from YK, GE (G-5) and MX07.

6. method according to claim 1, wherein saccharide compound is NeuAc α 2,3Gal β 1,4 (Fuc α 1,3) GlcNAc β 1,4Gal β 1-OEt.

7. method according to claim 6, wherein film is selected from the film of YK, GE (G-5), GH (G-10) and HL and MX07.

8. method according to claim 1, wherein stock liquid is the enzymatic reaction mixture that is used for synthesizing saccharide compound.

9. method according to claim 1 is wherein by regulating the pH of stock liquid, the ability optimum that can make film keep saccharide compound and pollutent is passed through.

10. method from the stock liquid Carbohybrate purification compound that contains pollutent, this method comprises contacts stock liquid under certain condition with nanofiltration membrane or reverse osmosis membrane, under this condition film keep saccharide compound and most of pollutent by film, wherein saccharide compound has following formula

Wherein Z is hydrogen, C ₁-C ₆Acyl group or

Y is selected from C (O), SO ₂, HNC (O), OC (O) and SC (O);

R ¹Be selected from aryl, the phenyl C of aryl, replacement ₁-C ₃Alkylidene group, wherein said aryl substituent are selected from halogen, trifluoromethyl, nitro, C ₁-C ₁₈Alkyl, C ₁-C ₁₈Alkoxyl group, amino, list-C ₁-C ₁₈Alkylamino, two-C ₁-C ₁₈Alkylamino, benzylamino, C ₁-C ₁₈Alkyl benzyl amino, C ₁-C ₁₈Alkylthio and C ₁-C ₁₈Alkyl carbonylamino, or

R ¹Y is allyloxycarbonyl or chloracetyl;

R ²Be selected from monose, wherein monose comprises β 1,3Gal-OR, and wherein R is H, alkyl, aryl or acyl group, disaccharides, hydrogen, C ₁-C ₁₈Straight chain, side chain or cyclic hydrocarbon group, C ₁-C ₆Alkyl, 3-(3,4, the 5-trimethoxyphenyl) propyl group, C ₁-C ₅Alkylidene group ω-carboxylate salt, ω-trisubstituted silyl C ₂-C ₄Alkylidene group, the trisubstituted silyl of wherein said ω are to have independently to be selected from C ₁-C ₄Three substituent silyls of alkyl, phenyl,

Or OR ²Form C together ₁-C ₁₈Straight chain, side chain or cyclic hydrocarbon group carbamate;

R ³Be hydrogen or C ₁-C ₆Acyl group;

R ⁴Be hydrogen, C ₁-C ₆Alkyl or benzyl;

R ⁵Be selected from hydrogen, benzyl, methoxy-benzyl, dimethoxy-benzyl and C ₁-C ₆Acyl group;

R ⁷Be methyl or methylol; With

X is selected from C ₁-C ₆Acyloxy, C ₂-C ₆Hydroxyl acyloxy, hydroxyl, halogen and azido-.

11. method according to claim 10, wherein the saccharide compound formula is NeuAc α (2 → 3) Gal β (1 → 4) GlcN (R ¹) β-OR ²Or NeuAc α (2 → 3) Gal β (1 → 4) GlcN (R ¹) β (1 → 3) Gal β-OR ², R wherein ¹Be the alkyl or the acyl group, 5,6,7 of 1-18 carbon atom, 8-tetrahydrochysene-2-naphthoyl amino; Benzamido; 2-naphthoyl amino; 4-aminobenzoic amido; Or 4-nitrobenzoyl amido, R ²Be hydrogen, sugar, oligosaccharides or the aglycone that has a carbon atom at least.

12. method according to claim 10, wherein the saccharide compound formula is NeuAc α (2 → 3) Gal β (1 → 4) (Fuc α 1 → 3) GlcN (R ¹) β OR ²Or NeuAc α (2 → 3) Gal β (1 → 4) (Fuc α 1 → 3) GlcN (R ¹) β (1 → 3) Gal β-OR ², R wherein ¹Be the alkyl or the acyl group, 5,6,7 of 1-18 carbon atom, 8-tetrahydrochysene-2-naphthoyl amino; Benzamido; 2-naphthoyl amino; 4-aminobenzoic amido; Or 4-nitrobenzoyl amido, R ²Be hydrogen, sugar, oligosaccharides or the aglycone that has a carbon atom at least.

13. the method for a Carbohybrate purification compound from stock liquid, this stock liquid contain the enzymatic reaction mixture that is used for synthesizing saccharide compound, this method comprises the following steps:

By stock liquid is contacted with ultra-filtration membrane, saccharide compound passes through film as percolate to make the albumen tunicle keep simultaneously, and removes any albumen that exists in the stock liquid; With

The percolate of ultrafiltration step is contacted with nanofiltration membrane or reverse osmosis membrane under certain condition, and nanofiltration membrane or reverse osmosis membrane keep saccharide compound under this condition, and most of unwanted pollutents pass through film simultaneously.

14. the method for a Carbohybrate purification compound from contain the pollutent stock liquid, this method comprises contacts stock liquid under certain condition with nanofiltration membrane or reverse osmosis membrane, film keeps most of pollutents of saccharide compound while by film under this condition, and wherein saccharide compound is Nucleotide or nucleotide sugar.

15. method according to claim 14, wherein nucleotide sugar is selected from GDP-Fucose, GDP-seminose, CMP-NeuAc, UDP-glucose, UDP-semi-lactosi, UDP-N-acetylgalactosamine.

16. method according to claim 14, wherein saccharide compound is Nucleotide or nucleosides.

17. method of from the stock liquid that contains carbohydrate interested, removing pollutent, this method comprises makes stock liquid contact with a side form face of nanofiltration membrane or reverse osmosis membrane, this side form make carbohydrate be retained in the face of the repulsion coefficient of carbohydrate and pollutent and pollutent by film, wherein stock liquid comprises and is used for the enzymatic reaction mixture of synthetic carbohydrate.

18. method according to claim 17, wherein enzymatic reaction comprises the enzymatic degradation of oligosaccharides or polysaccharide.

19. method according to claim 17, wherein film is separated into retained part and diafiltration part with material solution, compares with the Pollutant levels in the stock liquid, and retained part contains the pollutent of low concentration.

20. method according to claim 17, wherein the pH to stock liquid selects to regulate the repulsion coefficient of film to saccharide compound and pollutent, and increase is removed the pollutent in the stock liquid and/or increased the reservation of saccharide compound.

21. method according to claim 20, wherein pollutent is a negatively charged ion, and the pH of stock liquid is between 1 to 7.

22. method according to claim 20, wherein pollutent is a positively charged ion, and the pH of stock liquid is between 7 to 14.

23. method according to claim 20, wherein to make the surperficial net charge of nanofiltration membrane be positive charge to the pH of selected stock liquid.

24. method according to claim 20, wherein to make the surperficial net charge of nanofiltration membrane be negative charge to the pH of selected stock liquid.

25. method according to claim 20, wherein the pH of selected stock liquid makes the surperficial net charge of nanofiltration membrane for neutral.

26. method according to claim 17, wherein carbohydrate is oligosaccharides or polysaccharide.

27. method according to claim 17, wherein carbohydrate is a saliva acidic group sugar.

28. method according to claim 17, wherein pollutent comprises and is selected from nucleotide sugar, unreacted sugar, mineral ion, pyruvate salt, phosphoric acid salt, phosphoenolpyruvic acid, Nucleotide, nucleosides and proteic one or more compounds.

29. method according to claim 17 is wherein removed Deproteinization earlier from mixture before nanofiltration.

30. method according to claim 17, wherein this method also comprises the ion-exchange purification step.