US20050269265A1 - Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration - Google Patents

Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration Download PDF

Info

Publication number
US20050269265A1
US20050269265A1 US11/198,839 US19883905A US2005269265A1 US 20050269265 A1 US20050269265 A1 US 20050269265A1 US 19883905 A US19883905 A US 19883905A US 2005269265 A1 US2005269265 A1 US 2005269265A1
Authority
US
United States
Prior art keywords
membrane
solution
nanofiltration
membranes
permeate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/198,839
Inventor
Shawn DeFrees
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Neose Technologies Inc
Original Assignee
Neose Technologies Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Neose Technologies Inc filed Critical Neose Technologies Inc
Priority to US11/198,839 priority Critical patent/US20050269265A1/en
Publication of US20050269265A1 publication Critical patent/US20050269265A1/en
Abandoned legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B30/00Preparation of starch, degraded or non-chemically modified starch, amylose, or amylopectin
    • CCHEMISTRY; METALLURGY
    • C13SUGAR INDUSTRY
    • C13BPRODUCTION OF SUCROSE; APPARATUS SPECIALLY ADAPTED THEREFOR
    • C13B20/00Purification of sugar juices
    • C13B20/16Purification of sugar juices by physical means, e.g. osmosis or filtration
    • C13B20/165Purification of sugar juices by physical means, e.g. osmosis or filtration using membranes, e.g. osmosis, ultrafiltration
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D61/00Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/02Reverse osmosis; Hyperfiltration ; Nanofiltration
    • B01D61/025Reverse osmosis; Hyperfiltration
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D61/00Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/02Reverse osmosis; Hyperfiltration ; Nanofiltration
    • B01D61/025Reverse osmosis; Hyperfiltration
    • B01D61/026Reverse osmosis; Hyperfiltration comprising multiple reverse osmosis steps
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D61/00Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/02Reverse osmosis; Hyperfiltration ; Nanofiltration
    • B01D61/027Nanofiltration
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D61/00Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/02Reverse osmosis; Hyperfiltration ; Nanofiltration
    • B01D61/029Multistep processes comprising different kinds of membrane processes selected from reverse osmosis, hyperfiltration or nanofiltration
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D61/00Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/02Reverse osmosis; Hyperfiltration ; Nanofiltration
    • B01D61/04Feed pretreatment
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • CCHEMISTRY; METALLURGY
    • C13SUGAR INDUSTRY
    • C13KSACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
    • C13K13/00Sugars not otherwise provided for in this class
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2311/00Details relating to membrane separation process operations and control
    • B01D2311/04Specific process operations in the feed stream; Feed pretreatment
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02PCLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00Technologies relating to chemical industry
    • Y02P20/50Improvements relating to the production of bulk chemicals
    • Y02P20/582Recycling of unreacted starting or intermediate materials

Definitions

  • the present invention relates to the synthesis of oligosaccharides.
  • it relates to improved methods for purifying oligosaccharides using ultrafiltration, nanofiltration and/or reverse osmosis.
  • sialyl lactose has been of interest as neutralizers for enterotoxins from bacteria such as Vibrio cholerae, Escherichia coli, and Salmonella (see, e.g., U.S. Pat. No. 5,330,975).
  • Sialyl lactose has also been investigated for the treatment of arthritis and related autoimmune diseases.
  • sialyl lactose is thought to inhibit or disrupt the degree of occupancy of the Fc carbohydrate binding site on IgG, and thus prevent the formation of immune complexes (see, U.S.
  • glycosyltransferases Because of interest in making desired carbohydrate structures, glycosyltransferases and their role in enzyme-catalyzed synthesis of carbohydrates are presently being extensively studied.
  • the use of glycosyltransferases for enzymatic synthesis of carbohydrate offers advantages over chemical methods due to the virtually complete stereoselectivity and linkage specificity offered by the enzymes (Ito et al., Pure Appl. Chem., 65: 753 (1993) U.S. Pat. Nos. 5,352,670, and 5,374,541). Consequently, glycosyltransferases are increasingly used as enzymatic catalysts in synthesis of a number of carbohydrates used for therapeutic and other purposes.
  • Carbohydrate compounds produced by enzymatic synthesis or by other methods are often obtained in the form of complex mixtures that include not only the desired compound but also contaminants such as unreacted sugars, salts, pyruvate, phosphate, PEP, nucleosides, nucleotides, and proteins, among others. The presence of these contaminants is undesirable for many applications for which the carbohydrate compounds are useful.
  • Previously used methods for purifying oligosaccharides, such as chromatography, i.e., ion exchange and size exclusion chromatography have several disadvantages. For example, chromatographic purification methods are not amenable to large-scale purifications, thus precluding their use for commercial production of saccharides. Moreover, chromatographic purification methods are expensive. Therefore, a need exists for purification methods that are faster, more efficient, and less expensive than previously used methods. The present invention fulfills this and other needs.
  • a method for using a combination of membranes to remove undesirable impurities from a sugar-containing solution, especially molasses-forming ions which inhibit sugar crystallization is described in U.S. Pat. No. 5,454,952.
  • the method which involves ultrafiltration followed by nanofiltration, is described as being useful for improving the recovery of crystalline sugar from sugar cane or sugar beet solutions.
  • U.S. Pat. No. 5,403,604 describes the removal of fruit juice sugars from fruit juice by nanofiltration to obtain a retentate having a high level of sugars and a permeate having a lower level of sugars.
  • U.S. Pat. No. 5,254,174 describes the use of chromatography and/or nanofiltration to purify inulide compounds of formula GF n (where G is glucose and F is fructose) by removing salts and glucose, fructose, and sucrose from a juice or syrup containing the inulide compounds.
  • U.S. Pat. No. 4,956,458 describes the use of reverse osmosis to remove from polydextrose, which is a randomly cross-linked glucan polymer produced through the acid-catalyzed condensation of glucose, most of the off-flavor constituents such as anhydroglucose and furaldehyde derivatives polydextrose.
  • U.S. Pat. No. 4,806,244 describes the use of a combined membrane and sorption system in which sulfate is removed from water by nanofiltration, after which the nitrate, which passed through the membrane, was removed from the permeate by absorption to an ion exchange resin.
  • the present invention provides methods of purifying a carbohydrate compound from a feed solution containing a contaminant.
  • the methods involve contacting the feed solution with a nanofiltration or reverse osmosis membrane under conditions such that the membrane retains the desired carbohydrate compound while a majority of the contaminant passes through the membrane.
  • the invention provides methods for purifying carbohydrate compounds such as sialyl lactosides, sialic acid, lacto-N-neotetraose (LNnT) and GlcNAc ⁇ 1,3Gal ⁇ 1,4Glc (LNT-2), NeuAc ⁇ (2 ⁇ 3)Gal ⁇ (1 ⁇ 4)(Fuc ⁇ 1 ⁇ 3)Glc(R 1 ) ⁇ 1-OR 2 , wherein R 1 is OH or NAc; R 2 is a hydrogen, an alkoxy, a saccharide, an oligosaccharide or an aglycon group having at least one carbon atom; and Gal ⁇ ( ⁇ 13)Gal ⁇ (1 ⁇ 4)Glc(R 1 ) ⁇ -O—R 3 , wherein R 1 is OH or NAc; R 3 is —(CH 2 ) n —COX, with X ⁇ OH, OR 4 , —NHNH 2 , R 4 being a hydrogen, a saccharide, an oligosacc
  • the invention provides methods of purifying a carbohydrate compound from a feed solution comprising a reaction mixture used to synthesize the carbohydrate compound.
  • the synthesis can be enzymatic or chemical, or a combination thereof.
  • the methods involve removing any proteins present in the feed solution by contacting the feed solution with an ultrafiltration membrane so that proteins are retained the membrane while the carbohydrate compound passes through the membrane as a permeate.
  • the permeate from the ultrafiltration step is then contacted with a nanofiltration or reverse osmosis membrane under conditions such that the nanofiltration or reverse osmosis membrane retains the carbohydrate compound while a majority of an undesired contaminant passes through the membrane.
  • Another embodiment of the invention provides methods for purifying nucleotides, nucleosides, and nucleotide sugars by contacting a feed solution containing the nucleotide or related compound with a nanofiltration or reverse osmosis membrane under conditions such that the membrane retains the nucleotide or related compound while a majority of the contaminant passes through the membrane.
  • the present invention also provides methods for removing one or more contaminants from a solution that contains a carbohydrate of interest.
  • the methods involve contacting the solution with a first side of a semipermeable membrane having rejection coefficients so as to retain the carbohydrate while allowing the contaminant to pass through the membrane.
  • the membrane is selected from the group consisting of an ultrafiltration membrane, a nanofiltration membrane, and a reverse osmosis membrane, depending on the size and charge of the carbohydrate of interest relative to those of the contaminants.
  • the membrane separates a feed solution containing a carbohydrate into a retentate portion and a permeate portion.
  • the rejection coefficient of the membrane is greater for the carbohydrate than for the contaminant, the retentate portion will have a lower concentration of the contaminant relative to the contaminant concentration in the feed solution, and generally also a higher ratio of the carbohydrate to the undesired contaminant.
  • a membrane having a rejection coefficient for the carbohydrate that is lesser than that for the contaminant will effect a separation wherein the concentration of the contaminant is lower in the permeate than in the feed solution, and the permeate will have a higher ratio of carbohydrate to contaminant than the feed solution.
  • the fraction containing the carbohydrate can be recycled through the membrane system for further purification.
  • contaminants that can be removed from solutions containing the compound of interest using the methods of the invention include, but are not limited to, unreacted sugars, inorganic ions, pyruvate, phosphate, phosphoenolpyruvate, and proteins.
  • carbohydrate encompasses chemical compounds having the general formula (CH 2 O) n , and includes monosaccharides, disaccharides, oligosaccharides, and polysaccharides.
  • oligo refers to a polymeric molecule consisting of 2 to approximately 10 residues, for example, amino acids (oligopeptide), monosaccharides (oligosaccharide), and nucleic acids (oligonucleotide).
  • poly refers to a polymeric molecule comprising greater than about 10 residues.
  • Oligosaccharides are considered to have a reducing end and a non-reducing end, whether or not the saccharide at the reducing end is in fact a reducing sugar. In accordance with accepted nomenclature, oligosaccharides are depicted herein with the non-reducing end on the left and the reducing end on the right.
  • oligosaccharides described herein are described with the name or abbreviation for the non-reducing saccharide (e.g., Gal), followed by the configuration of the glycosidic bond ( ⁇ or ⁇ ), the ring bond, the ring position of the reducing saccharide involved in the bond, and then the name or abbreviation of the reducing saccharide (e.g., GlcNAc).
  • the linkage between two sugars may be expressed, for example, as 2,3, 2 ⁇ 3, or (2,3).
  • a compound is “substantially purified” from an undesired component in a solution if the concentration of the undesired component after purification is no greater than about 40% of the concentration of the component prior to purification.
  • the post-purification concentration of the undesired component will be less than about 20% by weight, and more preferably less than about 10%, of the pre-purification concentration.
  • pharmaceutically pure refers to a compound that is sufficiently purified from undesired contaminants that the compound is suitable for administration as a pharmaceutical agent.
  • the compound is purified such that the undesired contaminant is present after purification in an amount that is about 5% by weight or less of the pre-purification concentration of the contaminant in the feed solution.
  • the post-purification concentration of the contaminant is about 1% or less of the pre-purification contaminant concentration, and most preferably about 0.5% or less of the pre-purification concentration of contaminant.
  • a “feed solution” refers to any solution that contains a compound to be purified.
  • a reaction mixture used to synthesize an oligosaccharide can be used as a feed solution from which the desired reaction product is purified using the methods of the invention.
  • the present invention provides methods for rapidly and efficiently purifying specific carbohydrate and oligosaccharide structures to a high degree of purity using semipermeable membranes such as reverse osmosis and/or nanofiltration membranes.
  • the methods are particularly useful for separating desired oligosaccharide compounds from reactants and other contaminants that remain in a reaction mixture after synthesis or breakdown of the oligosaccharides.
  • the invention provides methods for separating oligosaccharides from enzymes and/or other components of reaction mixtures used for enzymatic synthesis or enzymatic degradation of oligosaccharides, nucleotide sugars, glycolipids, liposaccharides, nucleotides, nucleosides, and other saccharide-containing compounds.
  • the saccharides e.g., sialyl lactose, SLe x , and many others
  • the purification methods of the invention are more efficient, rapid, and amenable to large-scale purifications than previously known carbohydrate purification methods.
  • the invention provides single-step methods for purifying saccharide-containing compounds.
  • a membrane is selected that is appropriate for separating the desired carbohydrate from the undesired components of the solution from which the carbohydrate is to be purified.
  • the goal in selecting a membrane is to optimize for a particular application the molecular weight cutoff (MWCO), membrane composition, permeability, and rejection characteristics, that is, the membrane's total capacity to retain specific molecules while allowing salts and other, generally smaller or opposite charged molecules, to pass through.
  • MWCO molecular weight cutoff
  • the percent retention of a component is also called the retention characteristic or the membrane rejection coefficient.
  • a membrane For effective separation, a membrane is chosen that has a high rejection ratio for the saccharide of interest relative to the rejection ratio for compounds from which separation is desired. If a membrane has a high rejection ratio for a first compound relative to a second compound, the concentration of the first compound in the permeate solution which passes through the membrane is decreased relative to that of the second compound. Conversely, the concentration of the first compound increases relative to the concentration of the second compound in the retentate. If a membrane does not reject a compound, the concentration of the compound in both the permeate and the reject portions will remain essentially the same as in the feed solution. It is also possible for a membrane to have a negative rejection rate for a compound if the compound's concentration in the permeate becomes greater than the compound's concentration in the feed solution.
  • a membrane having a molecular weight cut-off (MWCO, which is often related to membrane pore size) that is expected to retain the desired compounds while allowing an undesired compound present in the feed stream to pass through the membrane.
  • the desired MWCO is generally less than the molecular weight of the compound being purified, and is typically greater than the molecular weight of the undesired contaminant that is to be removed from the solution containing the compound being purified.
  • MWCO molecular weight cut-off
  • UF ultrafiltration
  • NF nanofiltration
  • RO reverse osmosis
  • RO membranes typically have a nominal MWCO of less than about 200 Da and reject most ions
  • NF membranes generally have a nominal MWCO of between about 150 Da and about 5 kDa
  • UF membranes generally have a nominal MWCO of between about 1 kDa and about 300 kDa (these MWCO ranges assume a saccharide-like molecule).
  • a second parameter that is considered in choosing an appropriate membrane for a particular separation is the polymer type of the membrane.
  • the membranes used in each zone are made of conventional membrane material whether inorganic, organic, or mixed inorganic and organic. Typical inorganic materials include glasses, ceramics, cermets, metals and the like. Ceramic membranes, which are preferred for the UF zone, may be made, for example, as described in U.S. Pat. Nos. 4,692,354 to Asaeda et al, 4,562,021 to Alary et al., and others.
  • the organic materials which are preferred for the NF and RO zones, are typically polymers, whether isotropic, or anisotropic with a thin layer or “skin” on either the bore side or the shell side of the fibers.
  • Preferred materials for fibers are polyamides, polybenzamides, polysulfones (including sulfonated polysulfone and sulfonated polyether sulfone, among others), polystyrenes, including styrene-containing copolymers such as acrylo-nitrile-styrene, butadiene-styrene and styrene-vinylbenzylhalide copolymers, polycarbonates, cellulosic polymers including cellulose acetate, polypropylene, poly(vinyl chloride), poly(ethylene terephthalate), polyvinyl alcohol, fluorocarbons, and the like, such as those disclosed in U.S. Pat. Nos. 4,230,463, 4,806,24
  • a membrane surface charge is selected that has a surface charge that is appropriate for the ionic charge of the carbohydrate and that of the contaminants. While MWCO for a particular membrane is generally invariable, changing the pH of the feed solution can affect separation properties of a membrane by altering the membrane surface charge. For example, a membrane that has a net negative surface charge at neutral pH can be adjusted to have a net neutral charge simply by lowering the pH of the solution. An additional effect of adjusting solution pH is to modulate the ionic charge on the contaminants and on the carbohydrate of interest.
  • a suitable membrane polymer type and pH By choosing a suitable membrane polymer type and pH, one can obtain a system in which both the contaminant and the membrane are neutral, facilitating pass-through of the contaminant. If, for instance, a contaminant is negatively charged at neutral pH, it is often desirable to lower the pH of the feed solution to protonate the contaminant. For example, removal of phosphate is facilitated by lowering the pH of the solution to about 3, which protonates the phosphate anion, allowing passage through a membrane.
  • Example 5 a decrease in pH from 7.5 to 3.0 decreases the percentage of GlcNAc passing through a polyamide membrane such as an Osmonics MX07 in thirty minutes from 70% to 28%, while increasing the pass percentage of phosphate from 10% to 46% (see, Example 6, Table 5 for additional examples of the effect of pH change on passage rate of other compounds through various nanofiltration membranes).
  • the pH will generally between about pH 1 and about pH 7.
  • the pH of the feed solution can be adjusted to between about pH 7 and about pH 14.
  • one aspect of the invention involves modulating a separation by adjusting the pH of a solution in contact with the membrane; this can change the ionic charge of a contaminant and can also affect the surface charge of the membrane, thus facilitating purification if the desired carbohydrate.
  • the manufacturer's instructions must be followed as to acceptable pH range for a particular membrane to avoid damage to the membrane.
  • a mixture is first subjected to nanofiltration or reverse osmosis at one pH, after which the retentate containing the saccharide of interest is adjusted to a different pH and subjected to an additional round of membrane purification.
  • filtration of a reaction mixture used to synthesize sialyl lactose through an Osmonics MX07 membrane (a nanofiltration membrane having a MWCO of about 500 Da) at pH 3.0 will retain the sialyl lactose and remove most phosphate, pyruvate, salt and manganese from the solution, while also removing some of the GlcNAc, lactose, and sialic acid.
  • a saccharide is to be purified from a mixture that contains proteins, such as enzymes used to synthesize a desired oligosaccharide or nucleotide sugar, it is often desirable to remove the proteins as a first step of the purification procedure.
  • proteins such as enzymes used to synthesize a desired oligosaccharide or nucleotide sugar
  • this separation is accomplished by choosing a membrane that has an MWCO which is less than the molecular mass of the protein or other macromolecule to be removed from the solution, but is greater than the molecular mass of the oligosaccharide being purified (i.e., the rejection ratio in this case is higher for the protein than for the desired saccharide).
  • UF membranes that are suitable for use in the methods of the invention are available from several commercial manufacturers, including Millipore Corp. (Bedford, Mass.), Osmonics, Inc. (Minnetonka, Minn.), Filmtec (Minneapolis, Minn.), UOP, Desalination Systems, Advanced Membrane Technologies, and Nitto.
  • the invention also provides methods for removing salts and other low molecular weight components from a mixture containing a saccharide of interest by using a nanofiltration (NF) or a reverse osmosis (RO) membrane.
  • Nanofiltration membranes are a class of membranes for which separation is based both on molecular weight and ionic charge. These membranes typically fall between reverse osmosis and ultrafiltration membranes in terms of the size of species that will pass through the membrane.
  • Nanofiltration membranes typically have micropores or openings between chains in a swollen polymer network. Molecular weight cut-offs for non-ionized molecules are typically in the range from 100-20,000 Daltons.
  • a nanofiltration membrane useful in the methods of the invention will typically have a retention characteristic for the saccharide of interest of from about 40% to about 100%, preferably from about 70% to about 100%.
  • the nanofilter membranes used in the invention can be any one of the conventional nanofilter membranes, with polyamide membranes being particularly suitable.
  • suitable membranes include the Osmonics MX07, YK, GH (G-10), GE (G-5), and HL membranes, among others.
  • RO membranes also allow a variety of aqueous solutes to pass through them while retaining selected molecules.
  • osmosis refers to a process whereby a pure liquid (usually water) passes through a semipermeable membrane into a solution (usually sugar or salt and water) to dilute the solution and achieve osmotic equilibrium between the two liquids.
  • reverse osmosis is a pressure driven membrane process wherein the application of external pressure to the membrane system results in a reverse flux with the water molecules passing from a saline or sugar solution compartment into the pure water compartment of the membrane system.
  • a RO membrane which is semipermeable and non-porous, requires an aqueous feed to be pumped to it at a pressure above the osmotic pressure of the substances dissolved in the water.
  • An RO membrane can effectively remove low molecular weight molecules ( ⁇ 200 Daltons) and also ions from water.
  • the reverse osmosis membrane will have a retention characteristic for the saccharide of interest of from about 40% to about 100%, preferably from about 70% to about 100%.
  • Suitable RO membranes include, but are not limited to, the Filmtec BW-30, Filmtec SW-30, Filmtec SW-30HR, UOP RO membranes, Desal RO membranes, Osmonics RO membranes, Advanced Membrane Technologies RO membranes, and the Nitto RO membranes, among others.
  • a suitable RO membrane is Millipore Cat. No. CDRN500 60 (Millipore Corp., Bedford Mass.).
  • the membranes used in the invention may be employed in any of the known membrane constructions.
  • the membranes can be flat, plate and frame, tubular, spiral wound, hollow fiber, and the like.
  • the membrane is spiral wound.
  • the membranes can be employed in any suitable configuration, including either a cross-flow or a depth configuration.
  • cross-flow which is preferred for ultrafiltration, nanofiltration and reverse osmosis purifications according to the invention
  • the “feed” or solution from which the carbohydrate of interest is to be purified flows through membrane channels, either parallel or tangential to the membrane surface, and is separated into a retentate (also called recycle or concentrate) stream and a permeate stream.
  • retentate also called recycle or concentrate
  • the feed stream should flow, at a sufficiently high velocity, parallel to the membrane surface to create shear forces and/or turbulence to sweep away accumulating particles rejected by the membrane.
  • Cross-flow filtration thus entails the flow of three streams—feed, permeate and retentate.
  • a “dead end” or “depth” filter has only two streams—feed and filtrate (or permeate).
  • the recycle or retentate stream which retains all the particles and large molecules rejected by the membrane, can be entirely recycled to the membrane module in which the recycle stream is generated, or can be partially removed from the system.
  • the methods of the invention are used to purify saccharides from lower molecular weight components, for example, the desired saccharides are contained in the retentate stream (or feed stream, for a depth filter), while the permeate stream contains the removed contaminants.
  • the purification methods of the invention can be further optimized by adjusting the pressure, flow rate, and temperature at which the filtration is carried out.
  • UF, NF, and RO generally require increasing pressures above ambient to overcome the osmotic pressure of the solution being passed through the membrane.
  • the membrane manufacturers' instructions as to maximum and recommended operating pressures can be followed, with further optimization possible by making incremental adjustments.
  • the recommended pressure for UF will generally be between about 25 and about 100 psi, for NF between about 50 psi and about 1500 psi, and for RO between about 100 and about 1500 psi.
  • Flow rates of both the concentrate (feed solution) and the permeate can also be adjusted to optimize the desired purification.
  • Typical flow rates for the concentrate (P c ) will be between about 1 and about 15 gallons per minute (GPM), and more preferably between about 3 and about 7 GPM.
  • flow rates (P f ) of between about 0.05 GPM and about 10 GPM are typical, with flow rates between about 0.2 and about 1 GPM being preferred.
  • the temperature at which the purification is carried out can also influence the efficiency and speed of the purification. Temperatures of between about 0 and about 100° C. are typical, with temperatures between about 20 and 40° C. being preferred for most applications. Higher temperatures can, for some membranes, result in an increase in membrane pore size, thus providing an additional parameter that one can adjust to optimize a purification.
  • the filtration is performed in a membrane purification machine which provides a means for automating control of flow rate, pressure, temperature, and other parameters that can affect purification.
  • a membrane purification machine which provides a means for automating control of flow rate, pressure, temperature, and other parameters that can affect purification.
  • the Osmonics 213T membrane purification machine is suitable for use in the methods of the invention, as are machines manufactured by other companies listed above.
  • the membranes can be readily cleaned either after use or after the permeability of the membrane diminishes. Cleaning can be effected at a slightly elevated temperature if so desired, by rinsing with water or a caustic solution. If the streams contain small amounts of enzyme, rinsing in the presence of small amounts of surfactant, for instance ULTRASIL°, might be useful. Also, one can use prefilters (100-200 ⁇ m) to protect the more expensive nanofiltration membranes. Other cleaning agents can, if desired, be used. The choice of cleaning method will depend on the membrane being cleaned, and the membrane manufacturer's instructions should be consulted. The cleaning can be accomplished with a forward flushing or a backward flushing.
  • the purification methods of the invention can be used alone or in combination with other methods for purifying carbohydrates.
  • an ion exchange resin can be used to remove particular ions from a mixture containing a saccharide of interest, either before or after nanofiltration/reverse osmosis, or both before and after filtration. Ion exchange is particularly desirable if it is desired to remove ions such as phosphate and nucleotides that remain after a first round of nanofiltration or reverse osmosis.
  • this can be accomplished, for example, by adding an anion exchange resin such as AG1X-8 (acetate form, BioRad; see, e.g., BioRad catalog for other ion exchange resins) to a retentate that is at about pH 3.0 or lower until the phosphate concentration is reduced as desired.
  • AG1X-8 acetate form, BioRad; see, e.g., BioRad catalog for other ion exchange resins
  • acetic acid is released, so one may wish to follow the ion exchange with an additional purification through the nanofiltration or reverse osmosis system.
  • one can circulate the pH 3.0 or lower solution through an Osmonics MX07 or similar membrane until the conductivity of the permeate is low and stabilized.
  • the pH of the solution can then be raised to 7.4 with NaOH and the solution recirculated through the same membrane to remove remaining sodium acetate and salt. Cations can be removed in a similar manner; for example, to remove Mn 2+ , an acidic ion exchange resin can be used, such as AG50WX8 (H + ) (BioRad).
  • the purification methods of the invention are particularly useful for purifying oligosaccharides that have been prepared using enzymatic synthesis.
  • Enzymatic synthesis using glycosyltransferases provides a powerful method for preparing oligosaccharides; for some applications it is desirable to purify the oligosaccharide from the enzymes and other reactants in the enzymatic synthesis reaction mixture.
  • Preferred methods for producing many oligosaccharides involve glycosyl transferase cycles, which produce at least one mole of inorganic pyrophosphate for each mole of product formed and are typically carried out in the presence of a divalent metal ion.
  • glycosyltransferase cycles are the sialyltransferase cycles, which use one or more enzymes as well as other reactants. See, e.g., U.S. Pat. No. 5,374,541 WO 9425615 A, PCT/US96/04790, and PCT/US96/04824.
  • a reaction used for synthesis of sialylated oligosaccharides can contain a sialyltransferase, a CMP-sialic acid synthetase, a sialic acid, an acceptor for the sialyltransferase, CTP, and a soluble divalent metal cation.
  • ⁇ (2,3)sialtransferase transfers sialic acid to the non-reducing terminal Gal of a Gal ⁇ 1 ⁇ 3Glc disaccharide or glycoside.
  • ⁇ (2,3)sialtransferase EC 2.4.99.6 transfers sialic acid to the non-reducing terminal Gal of a Gal ⁇ 1 ⁇ 3Glc disaccharide or glycoside.
  • Another exemplary ⁇ 2,3-sialyltransferase (EC 2.4.99.4) transfers sialic acid to the non-reducing terminal Gal of the disaccharide or glycoside. See, Rearick et al., J. Biol. Chem., 254: 4444 (1979) and Gillespie et al., J. Biol. Chem., 267: 21004 (1992). Further exemplary enzymes include Gal- ⁇ -1,4-GlcNAc ⁇ -2,6 sialyltransferase (See, Kurosawa et al. Eur. J. Biochem. 219: 375-381 (1994)).
  • the reaction mixture will also contain an acceptor for the sialyltransferase, preferably having a galactosyl unit.
  • Suitable acceptors include, for example, Gal ⁇ 1 ⁇ 3GalNAc, lacto-N-tetraose, Gal ⁇ 1 ⁇ 3GlcNAc, Gal ⁇ 1 ⁇ 3Ara, Gal ⁇ 1 ⁇ 6GlcNAc, Gal ⁇ 1 ⁇ 4Glc (lactose), Gal ⁇ 1 ⁇ 4Glc ⁇ 1-OCH 2 CH 3 , Gal ⁇ 1 ⁇ 4Glc ⁇ 1-OCH 2 CH 2 CH 3 , Gal ⁇ 1 ⁇ 4Glc ⁇ 1-OCH 2 C 6 H 5 , Gal ⁇ 1 ⁇ 4GlcNAc, Gal ⁇ 1-OCH 3 , melibiose, raffinose, stachyose, and lacto-N-neotetraose (LNnT).
  • LNnT lacto-N-neotetraose
  • the sialic acid present in the reaction mixture can include not only sialic acid itself (5-N-acetylneuraminic acid; 5-N-acetylamino-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid; NeuAc, and sometimes also abbreviated AcNeu or NANA), but also 9-substituted sialic acids such as a 9-O—C 1 -C 6 acyl-NeuAc like 9-O-lactyl-NeuAc or 9-O-acetyl-NeuAc, 9-deoxy-9-fluoro-NeuAc and 9-azido-9-deoxy-NeuAc.
  • the synthesis and use of these compounds in a sialylation procedure is described in international application WO 92/16640, published Oct. 1, 1992.
  • the reaction medium can further comprise a CMP-sialic acid recycling system comprising at least 2 moles of phosphate donor per each mole of sialic acid, and catalytic amounts of an adenine nucleotide, a kinase capable of transferring phosphate from the phosphate donor to nucleoside diphosphates, and a nucleoside monophosphate kinase capable of transferring the terminal phosphate from a nucleoside triphosphate to CMP.
  • a CMP-sialic acid recycling system comprising at least 2 moles of phosphate donor per each mole of sialic acid, and catalytic amounts of an adenine nucleotide, a kinase capable of transferring phosphate from the phosphate donor to nucleoside diphosphates, and a nucleoside monophosphate kinase capable of transferring the terminal phosphate from a nucleoside triphosphate to CMP.
  • a suitable CMP-sialic acid regenerating system comprises cytidine monophosphate (CMP), a nucleoside triphosphate (for example adenosine triphosphate (ATP), a phosphate donor (for example, phosphoenolpyruvate or acetyl phosphate), a kinase (for example, pyruvate kinase or acetate kinase) capable of transferring phosphate from the phosphate donor to nucleoside diphosphates and a nucleoside monophosphate kinase (for example, myokinase) capable of transferring the terminal phosphate from a nucleoside triphosphate to CMP.
  • CMP cytidine monophosphate
  • a nucleoside triphosphate for example adenosine triphosphate (ATP)
  • a phosphate donor for example, phosphoenolpyruvate or acetyl phosphate
  • reaction medium will preferably further comprise a phosphatase.
  • Pyruvate is a byproduct of the sialyltransferase cycle and can be made use of in another reaction in which N-acetylmannosamine (ManNAc) and pyruvate are reacted in the presence of NeuAc aldolase (EC 4.1.3.3) to form sialic acid.
  • ManNAc N-acetylmannosamine
  • NeuAc aldolase EC 4.1.3.3
  • advantage can be taken of the isomerization of GlcNAc to ManNAc, and the less expensive GlcNAc can be used as the starting material for sialic acid generation.
  • the sialic acid can be replaced by ManNAc (or GlcNAc) and a catalytic amount of NeuAc aldolase.
  • NeuAc aldolase also catalyzes the reverse reaction (NeuAc to ManNAc and pyruvate)
  • the produced NeuAc is irreversibly incorporated into the reaction cycle via CMP-NeuAc catalyzed by CMP-sialic acid synthetase.
  • the starting material, ManNAc can also be made by the chemical conversion of GlcNAc using methods known in the art (see, e.g., Simon et al., J. Am. Chem. Soc. 110: 7159 (1988).
  • the reaction medium will preferably contain, in addition to a galactosyltransferase, donor substrate, acceptor sugar and divalent metal cation, a donor substrate recycling system comprising at least 1 mole of glucose-1-phosphate per each mole of acceptor sugar, a phosphate donor, a kinase capable of transferring phosphate from the phosphate donor to nucleoside diphosphates, and a pyrophosphorylase capable of forming UDP-glucose from UTP and glucose-1-phosphate and catalytic amounts of UDP and a UDP-galactose-4-epimerase.
  • a donor substrate recycling system comprising at least 1 mole of glucose-1-phosphate per each mole of acceptor sugar, a phosphate donor, a kinase capable of transferring phosphate from the phosphate donor to nucleoside diphosphates, and a pyrophosphorylase capable of forming UDP-glucose from UTP and glucose-1-phosphat
  • Exemplary galactosyltransferases include ⁇ (1,3) galactosyltransferase (E.C. No. 2.4.1.151, see, e.g., Dabkowski et al., Transplant Proc. 25: 2921 (1993) and Yamamoto et al., Nature 345: 229-233 (1990)) and ⁇ (1,4) galactosyltransferase (E.C. No. 2.4.1.38).
  • Oligosaccharides synthesized by other enzymatic methods can also be purified by the methods of the invention.
  • the methods are useful for purifying oligosaccharides produced in non-cyclic or partially cyclic reactions such as simple incubation of an activated saccharide and an appropriate acceptor molecule with a glycosyltransferase under conditions effective to transfer and covalently bond the saccharide to the acceptor molecule.
  • Glycosyltransferases which include those described in, e.g., U.S. Pat. No. 5,180,674, and International Patent Publication Nos.
  • WO 93/13198 and WO 95/02683 as well the glycosyltransferases encoded by the los locus of Neisseria (see, U.S. Pat. No. 5,545,553), can be bound to a cell surface or unbound.
  • Oligosaccharides that can be obtained using these glycosyltransferases include, for example, Gal ⁇ (1 ⁇ 4)Gal ⁇ (1 ⁇ 4)Glc, GlcNAc ⁇ (1,3)Gal ⁇ (1,4)Glc, Gal ⁇ (1 ⁇ 4)GlcNAc ⁇ (1 ⁇ 3)Gal ⁇ (1 ⁇ 4) Glc, and GalNAc ⁇ (1 ⁇ 3)Gal ⁇ (1 ⁇ 4)GlcNAc ⁇ (1 ⁇ 3) Gal ⁇ (1 ⁇ 4)Glc, among many others.
  • sialic acid and any sugar having a sialic acid moiety include the sialyl galactosides, including the sialyl lactosides, as well as compounds having the formula: NeuAc ⁇ (2 ⁇ 3)Gal ⁇ (1 ⁇ 4)GlcN(R′) ⁇ -OR or NeuAc ⁇ (2 ⁇ 3)Gal ⁇ (1 ⁇ 4)GlcN(R′) ⁇ (1 ⁇ 3)Gal ⁇ -OR
  • R′ is alkyl or acyl from 1-18 carbons, 5,6,7,8-tetrahydro-2-naphthamido; benzamido; 2-naphthamido; 4-aminobenzamido; or 4-nitrobenzamido.
  • R is a hydrogen, a alkyl C 1 -C 6 , a saccharide, an oligosaccharide or an aglycon group having at least one carbon atom.
  • A represents an alkylene group of from 1 to 18 carbon atoms optionally substituted with halogen, thiol, hydroxy, oxygen, sulfur, amino, imino, or alkoxy
  • Z is hydrogen, H, —SH, —NH 2 , R 1 , —N(R 1 ) 2 , —CO 2 H, —CO 2 R 1 , —CONH 2 , —CONHR 1 , —CON(R 1 ) 2 , —CONHNH 2 , or —OR 1 wherein each R 1 is independently alkyl of from 1 to 5 carbon atoms.
  • R can also be 3-(3,4,5-trimethoxyphenyl)propyl.
  • the present invention is also useful for purifying a variety of compounds that comprise selectin-binding carbohydrate moieties.
  • selectin-binding moieties have the general formula: R 1 Gal ⁇ 1,m(Fuc ⁇ 1,n)GlcNR 0 (R 2 ) p —
  • R 0 is (C 1 -C 8 alkyl)carbonyl, (C 1 -C 8 alkoxy)carbonyl, or (C 2 -C 9 alkenyloxy)carbonyl
  • R 1 is an oligosaccharide or a group having the formula
  • R 3 and R 4 may be the same or different and may be H, C 1 -C 8 alkyl, hydroxy-(C 1 -C 8 alkyl), aryl-(C 1 -C 8 alkyl), or (C 1 -C 8 alkoxy)-(C 1 -C 8 alkyl), substituted or unsubstituted.
  • R 2 may be H, C 1 -C 8 alkyl, hydroxy-(C 1 -C 8 alkyl), aryl-(C 1 -C 8 alkyl), (C 1 -C 8 alkyl)-aryl, alkylthio, ⁇ 1,2Man, ⁇ 1,6GalNAc, ⁇ 1,3Gal ⁇ 1,4Glc, ⁇ 1,2Man-R 8 , ⁇ 1,6GalNAc—R 8 , and ⁇ 1,3Gal-R 8 .
  • R 8 may be H, C 1 -C 8 alkyl, C 1 -C 8 alkoxy, hydroxy-(C 1 -C 8 alkyl), aryl-(C 1 -C 8 alkyl), (C 1 -C 8 alkyl)-aryl, or alkylthio.
  • m and n are integers and may be either 3 or 4; p may be zero or 1.
  • substituted groups mentioned above may be substituted by hydroxy, hydroxy(C 1 -C 4 alkyl), polyhydroxy(C 1 -C 4 alkyl), alkanoamido, or hydroxyalknoamido substituents.
  • Preferred substituents include hydroxy, polyhydroxy(C 3 alkyl), acetamido and hydroxyacetamido.
  • a substituted radical may have more than one substitution, which may be the same or different.
  • the oligosaccharide is preferably a trisaccharide.
  • Preferred trisaccharides include NeuAc ⁇ 2,3Gal ⁇ 1,4GlcNAc ⁇ 1,3 or NeuGc ⁇ 2,3Gal ⁇ 1,4GlcNAc ⁇ 1,3.
  • R 1 is the group having the formula R 3 and R 4 preferably form a single radical having the formula —R 5 — or —(R 6 ) q —O—(R 7 ) r — in which R 5 is C 3 -C 7 divalent alkyl, substituted or unsubstituted, R 6 and R 7 are the same or different and are C 1 -C 6 divalent alkyl, substituted or unsubstituted.
  • q and r are integers which may be the same or different and are either zero or 1. The sum of q and r is always at least 1.
  • a more preferred structure for a single radical formed by R 3 and R 4 is one having the formula —(R 6 )—O— in which R 6 is C 3 -C 4 divalent alkyl, substituted or unsubstituted.
  • R 6 may have the formula —CH 2 —CH 2 —CH 2 —CH 2 —, preferably substituted.
  • the radical can be substituted with hydroxy, polyhydroxy(C 3 alkyl), and substituted or unsubstituted alkanoamido groups, such as acetamido or hydroxyacetamido.
  • the substituted structure will typically form a monosaccharide, preferably a sialic acid such as NeuAc or NeuGc linked ⁇ 2,3 to the Gal residue.
  • both m and n are integers and can be either 3 or 4.
  • Gal is linked ⁇ 1,4 and Fuc is linked ⁇ 1,3 to GlcNAc.
  • This formula includes the SLe x tetrasaccharide.
  • SLe x has the formula NeuAc ⁇ 2,3Gal ⁇ 1,4(Fuc ⁇ 1,3)GlcNAc ⁇ 1—. This structure is selectively recognized by LECCAM-bearing cells.
  • Other compounds that one-can purify using the methods include those described in U.S. Pat. No. 5,604,207 having the formula wherein Z is hydrogen, C 1 -C 6 acyl or
  • Y is selected from the group consisting of C(O), SO 2 , HNC(O), OC(O) and SC(O);
  • R 1 is selected from the group consisting of an aryl, a substituted aryl and a phenyl C 1 -C 3 alkylene group, wherein said aryl substitutent is selected from the group consisting of a halo, trifuloromethyl, nitro, C 1 -C 18 alkyl, C 1 -C 18 alkoxy, amino, mono-C 1 -C 18 alkylamino, di-C 1 -C 18 alkylamino, benzylamino, C 1 -C 18 alkylbenzylamino, C 1 -C 18 thioaklyl and C 1 -C 18 alkyl carboxamido groups, or
  • R 1 Y is allyloxycarbonyl or chloroacetyl
  • R 2 is selected from the group consisting of monosaccharide (including ⁇ 1,3Gal-OR, where R ⁇ H, alkyl, aryl or acyl), disaccharide, hydrogen, C 1 -C 18 straight chain, branched chain or cyclic hydrocarbyl, C 1 -C 6 alkyl, 3-(3,4,5-trimethoxyphenyl)propyl, C 1 -C 5 alkylene ⁇ -carboxylate, ⁇ -trisubstituted silyl C 2 -C 4 alkylene wherein said ⁇ -trisubstituted silyl is a silyl group having three substituents independently selected from the group consisting of C 1 -C 4 alkyl, phenyl,
  • R 3 is hydrogen or C 1 -C 6 acyl
  • R 4 is hydrogen, C 1 -C 6 alkyl or benzyl
  • R 5 is selected from the group consisting of hydrogen, benzyl, methoxybenzyl, dimethoxybenzyl and C 1 -C 6 acyl;
  • R 7 is methyl or hydroxymethyl
  • X is selected from the group consisting of C 1 -C 6 acyloxy, C 2 -C 6 hydroxylacyloxy, hydroxy, halo and azido.
  • a related set of structures included in the general formula are those in which Gal is linked ⁇ 1,3 and Fuc is linked ⁇ 1,4.
  • the tetrasaccharide, NeuAc ⁇ 2,3Gal ⁇ 1,3(Fuc ⁇ 1,4)GlcNAc ⁇ 1- termed here SLe a
  • selectin receptors See, Berg et al., J. Biol. Chem., 266: 14869-14872 (1991).
  • Berg et al. showed that cells transformed with E-selectin cDNA selectively bound neoglycoproteins comprising SLe a .
  • compounds that can be purified according to the invention are lacto-N-neotetraose (LNnT), GlcNAc ⁇ 1,3Gal ⁇ 1,4Glc (LNT-2), sialyl( ⁇ 2,3)-lactose, and sialyl( ⁇ 2,6)-lactose.
  • alkyl as used herein means a branched or unbranched, saturated or unsaturated, monovalent or divalent, hydrocarbon radical having from 1 to 20 carbons, including lower alkyls of 1-8 carbons such as methyl, ethyl, n-propyl, butyl, n-hexyl, and the like, cycloalkyls (3-7 carbons), cycloalkylmethyls (4-8 carbons), and arylalkyls.
  • alkoxy refers to alkyl radicals attached to the remainder of the molecule by an oxygen, e.g., ethoxy, methoxy, or n-propoxy.
  • alkylthio refers to alkyl radicals attached to the remainder of the molecule by a sulfur.
  • acyl refers to a radical derived from an organic acid by the removal of the hydroxyl group. Examples include acetyl, propionyl, oleoyl, myristoyl.
  • aryl refers to a radical derived from an aromatic hydrocarbon by the removal of one atom, e.g., phenyl from benzene.
  • the aromatic hydrocarbon may have more than one unsaturated carbon ring, e.g., naphthyl.
  • alkoxy refers to alkyl radicals attached to the remainder of the molecule by an oxygen, e.g., ethoxy, methoxy, or n-propoxy.
  • alkylthio refers to alkyl radicals attached to the remainder of the molecule by a sulfur.
  • alkanoamido has the general formula —NH—CO—(C 1 -C 6 alkyl) and may or may not be substituted. If substituted, the substituent is typically hydroxyl.
  • the term specifically includes two preferred structures, acetamido, —NH—O—CH 3 , and hydroxyacetamido, —NH—CO—CH 2 —OH.
  • heterocyclic compounds refers to ring compounds having three or more atoms in which at least one of the atoms is other than carbon (e.g., N, O, S, Se, P, or As).
  • examples of such compounds include furans (including the furanose form of pentoses, such as fucose), pyrans (including the pyranose form of hexoses, such as glucose and galactose) pyrimidines, purines, pyrazines and the like.
  • the methods of the invention are useful not only for purifying carbohydrates that that are newly synthesized, but also those that are the products of degradation, e.g., enzymatic degradation. See, e.g., Sinnott, M. L., Chem. Rev. 90: 1171-1202 (1990) for examples of enzymes that catalyze degradation of oligosaccharides.
  • the invention also provides methods for purifying nucleotides, nucleotide sugars, and related compounds.
  • a nucleotide sugar such as GDP-fucose, GDP-mannose, CMP-NeuAc, UDP-glucose, UDP-galactose, UDP—N-acetylgalactosamine, and the like, can be purified by the methods described herein.
  • the methods are also useful for purifying nucleotides and nucleotides in various states of phosphorylation (e.g., CMP, CDP, CTP, GMP, GDP, GTP, TMP, TDP, TTP, AMP, ADP, ATP, UMP, UDP, UTP), as well as the deoxy forms of these and other nucleotides.
  • states of phosphorylation e.g., CMP, CDP, CTP, GMP, GDP, GTP, TMP, TDP, TTP, AMP, ADP, ATP, UMP, UDP, UTP
  • Examples 1-5 demonstrate the synthesis of sialyl lactose and its purification using nanofiltration and ion exchange.
  • ManNAc N-acetyl-D-mannosamine
  • GluNAc N-acetyl-D-glucosamine
  • Example 6 demonstrates the separation of organics and inorganic salts by nanofiltration.
  • Example 7 demonstrates the separation characteristics of polybenzamide nanofiltration membranes.
  • Example 8 demonstrates the separation characteristics of polyamide nanofiltration membranes.
  • This example demonstrates a method for synthesizing sialic acid using a relatively inexpensive substrate, GlcNAc, rather than the more expensive ManNAc or sialic acid.
  • the ManNAc produced in the previous step was subjected to aldol condensation mediated by N-acetylneuraminic acid (Neu5Ac) aldolase and pyruvic acid.
  • N-acetylneuraminic acid (Neu5Ac) aldolase N-acetylneuraminic acid (Neu5Ac) aldolase and pyruvic acid.
  • N-acetylneuraminic acid (Neu5Ac) aldolase N-acetylneuraminic acid (Neu5Ac) aldolase and pyruvic acid.
  • N-acetylneuraminic acid (Neu5Ac) aldolase N-acetylneuraminic acid aldolase
  • Example 2 To the sialic acid produced in Example 1 was added lactose monohydrate (79.2 g, 0.22 mol), 0.7 g bovine serum albumin, phosphoenolpyruvate monopotassium salt (37 g, 0.22 mol), and the pH was adjusted to 7.5. CMP (2.84 g, 0.0088 mol), ATP (0.54 g, 0.0009 mol) were added, and the pH readjusted to 7.5. Sodium azide (0.35 g) was added, as were the following enzymes: pyruvate kinase (19,800 U), myokinase (13,200 U), CMP sialic acid synthetase (440 U, and sialyltransferase (165 U). 66 ml of 1M MnCl 2 was added and the final volume adjusted to 2.2 L with water. The reaction was carried out at room temperature.
  • the sialyl lactose yield was approximately 70-80% as determined by TLC.
  • This example illustrates the production of ⁇ -N-acetylneuraminic acid(2,3) ⁇ -galactosyl(1,4)glucose using the sialyl transferase cycle with control of the manganese ion concentration.
  • phosphoenolpyruvate trisodium salt 285.4 g, 1.22 mol
  • sialic acid 197 g, 0.637 mol
  • Cytidine-5′-monophosphate 5.14 g, 15.9 mmol
  • potassium chloride 7.9 g, 0.106 mol
  • Mn ++ Loss of Mn ++ (mL of 1 M, final Day (measured, mM) (from previous day) added conc) 1 28 22.0 none 2 23.9 4.1 none 3 10.7 13.2 111 mL, +30 mM 4 1.4 39.3 111 mL, +30 mM 5 3.0 28.4 148 mL, +40 mM 6 12.9 30.1 74 mL, +20 mM 7 10.0 22.9 80 mL, +20 mM 8 12.0 18.0 80 mL, +20 mM 9 24.3 7.7 none
  • This example illustrates the workup and purification of the trisaccharide produced in Example 2 followed by peracetylation and esterification.
  • the eluate was concentrated and desalted by running it against a polyamide reverse osmosis membrane in a suitable apparatus (Cat. No. CDRN500 60, Millipore, Bedford, Mass.).
  • the retentate containing the product was evaporated to a thick syrup.
  • the retentate can be treated with a chelating resin to remove divalent cations.
  • the filtrate contained the desired product substantially free of salts and in a high state of purity as shown by 1 Hmr spectroscopy. Otherwise the syrup was so evaporated twice with pyridine (2 ⁇ 200 mL).
  • the evaporation flask was charged with a solution of N,N-dimethylaminopyridine (2.2 g) in pyridine (1.2 L).
  • Acetic anhydride (0.83 L) was added during a period of 1 hour. The resulting mixture was left for 24-48 hours rotating slowly at room temperature. The reaction is checked by TLC (methanol:dichloromethane 1:9). Upon complete reaction vacuum is applied and the solution is evaporated to give a residue.
  • a reaction mixture similar to that described in Example 2 was subjected to filtration using an ultrafiltration membrane having a MWCO of 10 kDa to remove the proteins.
  • the phosphate concentration [PO 4 3 ⁇ ] was greater than 2.8 mM.
  • the conductivity of the initial permeate was 28.1 mS; after 5 hours of recirculation, the conductivity had dropped to 115 ⁇ S, the phosphate concentration [PO 4 3 ⁇ ] had decreased to 770 ⁇ M, and the manganese concentration [Mn 2+ ] was 3.4 mM.
  • the unknown sample (100 ⁇ l) was diluted with D.I. water (775 ⁇ l). The solution was then treated with 100 ⁇ l of acid molybdate (prepared by dissolving 1.25 g of ammonium molybdate in 100 ⁇ l of 2.5N H 2 SO 4 ), 25 ⁇ l of Fiska Subha Row Solution (purchased from Sigma as a powder, and prepared according to manufacturer's directions). The mixture was heated at 100° C. for 7 min, the absorption at 810 nm was then recorded. The concentration was determined by comparing the absorption with a phosphate standard curve.
  • the nanofiltration membranes tested were the MX07, SX12, and B006 produced by Osmonics, Inc. (Minnetonka Minn.) and the DL2540 produced by Osmonics, DeSalination Systems.
  • the MX07 membrane was used as described in Example 5 above. Parameters for the remaining membranes were as shown in Table 6.
  • GluNAc N-Acetyl-D-Glucosamine
  • PEP 2-Phosphoenolpyruvate Trisodium Salt
  • CMP Cytidine 5′-monophosphate Membranes MX07, SX12, B006 from Osmonics, Inc., DL2540 from Osmonics, Desalination Systems (Escondido, CA).
  • This Example describes experiments which demonstrate that a polybenzamide membrane (YK, Osmonics) is effective for the purification of sugars, in both flat-sheet and spiral-wound forms.
  • the membrane was tested at varying pH levels for the passage or retention of sugars and salts.
  • a Desal membrane machine (Osmonics, Desalination Systems, Escondido, Calif.) with membrane YK was washed thoroughly by first rinsing the machine 4 to 5 times, each with approximately 1 L of distilled water. The water was poured into the feed tank, circulated for about a minute ( ⁇ 100 psi), and emptied using the drain valve, twisting it counterclockwise to an open position. Thee valve was closed after emptying, and the process was repeated 4 to 5 times. After rinsing, approximately 1 more L of water was added. The system was recirculated at a pressure of 150 psi for 30 min and then was emptied. The system including the membrane was then used in the following experiments.
  • the pH of the solution was then lowered to pH 3.0, when possible, using a conjugate acid of the salt being tested.
  • the solution was recirculated while adjusting the pH to assure that the solution inside the machine was mixed as well.
  • the testing process was repeated, with conductivity of both the permeate and the concentrate being determined.
  • the solution was then brought to a pH of about 7.0 with a conjugate base, and once again the run was repeated at the new pH. Again, conductivity of both the permeate and concentrate was determined.
  • This Example describes the evaluation of several polyamide membranes for use in the purification of sugars, in both flat-sheet and spiral-wound forms.
  • the membranes were tested at varying pH levels for the passage or retention of sugars and salts.
  • a Desal membrane machine (Osmonics, Desalination Systems) with a polyamide membrane G-5 (GE; Osmonics) was washed thoroughly by first rinsing the machine 4 to 5 times, each with approximately 1 L of distilled water. The water was poured into the feed tank, circulated for about a minute ( ⁇ 100 psi), and emptied using the drain valve. The valve was closed after emptying, and the process was repeated 4 to 5 times. After rinsing, approximately one more L of water was added. The system was recirculated at a pressure of 150 psi for 30 min and then was emptied. The system including the membrane was then ready for application testing.
  • GE polyamide membrane G-5
  • the machine was washed with water 3 to 4 times as described above. Then, about 1 L of water was recirculated for about 15-20 minutes at 100-150 psi and the machine was emptied. Occasionally this was followed by an extra brief washing, if some of the compound was suspected to still remain in the apparatus. The conductivity was always checked to make sure that all the sample was removed. If the conductivity remained high, the machine was washed until the contaminants were virtually undetectable. Most of the ionic compounds were removed easily, with the exception of MnCl 2 , which only required 1 or 2 extra short washings.
  • a 10 mM solution of the following salts were tested with the flat sheet membranes: MnCl 2 , NaH 2 PO 4 , NaC 3 H 3 O 3 , and NaCl.
  • a 1 L solution of one of the salts was poured into the feed tank and recirculated at 100 psi for about 15 min.
  • samples of both the permeate and the concentrate were collected and measured using a conductivity meter.
  • the samples were collected every five minutes thereafter, with a total of at least three collections for each sample run. The percentage pass was calculated by dividing the conductivity of the permeate by the conductivity of the concentrate.
  • the pH of the solution was lowered to pH 3.0, when possible, using a conjugate acid of the salt being tested.
  • the solution was recirculated while adjusting the pH to assure that the solution inside the machine was mixed as well.
  • the testing process was repeated, collecting data as before.
  • the solution was brought to a pH of about 7.0 with a conjugate base, and once again the run was repeated at the new pH.
  • the machine was then emptied and rinsed as described above.
  • the retention characteristics for various salts and sugars of a flat sheet polyamide nanofiltration membrane are shown in Table 9.
  • the A-value of the membrane was 10.0, and the percent transmission of tap water was 62.8 (tested using 2000 ppm MgSO 4 at ambient temperature).
  • the experiments were conducted at a temperature of 25-35° C. and a permeate flow rate of 5-8 mL/min.
  • a G-5 (GE) polyamide membrane (A-value: 3.9, percent transmission of tap water: 33.9) was also tested. The experiment was conducted at 25-35° C. and a permeate flow rate of 3-5 mL/min. Results are shown in Table 11.

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

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This is a continuation-in-part of U.S. Provisional Application No. 60/028,226, filed Oct. 10, 1996, the disclosure of which is incorporated by reference.
  • FIELD OF THE INVENTION
  • The present invention relates to the synthesis of oligosaccharides. In particular, it relates to improved methods for purifying oligosaccharides using ultrafiltration, nanofiltration and/or reverse osmosis.
  • BACKGROUND OF THE INVENTION
  • Increased understanding of the role of carbohydrates as recognition elements on the surface of cells has led to increased interest in the production of carbohydrate molecules of defined structure. For instance, compounds comprising the oligosaccharide moiety, sialyl lactose, have been of interest as neutralizers for enterotoxins from bacteria such as Vibrio cholerae, Escherichia coli, and Salmonella (see, e.g., U.S. Pat. No. 5,330,975). Sialyl lactose has also been investigated for the treatment of arthritis and related autoimmune diseases. In particular, sialyl lactose is thought to inhibit or disrupt the degree of occupancy of the Fc carbohydrate binding site on IgG, and thus prevent the formation of immune complexes (see, U.S. Pat. No. 5,164,374). Recently, sialyl-α(2,3)galactosides, sialyl lactose and sialyl lactosamine have been proposed for the treatment of ulcers, and Phase I clinical trials have begun for the use of the former compound in this capacity. See, Balkonen et al., FEMS Immunology and Medical Microbiology 7: 29 (1993) and BioWorld Today, p. 5, Apr. 4, 1995. As another example, compounds comprising the sialyl Lewis ligands, sialyl Lewisx and sialyl Lewisa are present in leukocyte and non-leukocyte cell lines that bind to receptors such as the ELAM-1 and GMP 140 receptors. Polley et al., Proc. Natl. Acad Sci., USA, 88: 6224 (1991) and Phillips et al., Science, 250: 1130 (1990), see, also, U.S. Ser. No. 08/063,181.
  • Because of interest in making desired carbohydrate structures, glycosyltransferases and their role in enzyme-catalyzed synthesis of carbohydrates are presently being extensively studied. The use of glycosyltransferases for enzymatic synthesis of carbohydrate offers advantages over chemical methods due to the virtually complete stereoselectivity and linkage specificity offered by the enzymes (Ito et al., Pure Appl. Chem., 65: 753 (1993) U.S. Pat. Nos. 5,352,670, and 5,374,541). Consequently, glycosyltransferases are increasingly used as enzymatic catalysts in synthesis of a number of carbohydrates used for therapeutic and other purposes.
  • Carbohydrate compounds produced by enzymatic synthesis or by other methods are often obtained in the form of complex mixtures that include not only the desired compound but also contaminants such as unreacted sugars, salts, pyruvate, phosphate, PEP, nucleosides, nucleotides, and proteins, among others. The presence of these contaminants is undesirable for many applications for which the carbohydrate compounds are useful. Previously used methods for purifying oligosaccharides, such as chromatography, i.e., ion exchange and size exclusion chromatography, have several disadvantages. For example, chromatographic purification methods are not amenable to large-scale purifications, thus precluding their use for commercial production of saccharides. Moreover, chromatographic purification methods are expensive. Therefore, a need exists for purification methods that are faster, more efficient, and less expensive than previously used methods. The present invention fulfills this and other needs.
  • BACKGROUND ART
  • A method for using a combination of membranes to remove undesirable impurities from a sugar-containing solution, especially molasses-forming ions which inhibit sugar crystallization is described in U.S. Pat. No. 5,454,952. The method, which involves ultrafiltration followed by nanofiltration, is described as being useful for improving the recovery of crystalline sugar from sugar cane or sugar beet solutions.
  • U.S. Pat. No. 5,403,604 describes the removal of fruit juice sugars from fruit juice by nanofiltration to obtain a retentate having a high level of sugars and a permeate having a lower level of sugars.
  • U.S. Pat. No. 5,254,174 describes the use of chromatography and/or nanofiltration to purify inulide compounds of formula GFn (where G is glucose and F is fructose) by removing salts and glucose, fructose, and sucrose from a juice or syrup containing the inulide compounds.
  • U.S. Pat. No. 4,956,458 describes the use of reverse osmosis to remove from polydextrose, which is a randomly cross-linked glucan polymer produced through the acid-catalyzed condensation of glucose, most of the off-flavor constituents such as anhydroglucose and furaldehyde derivatives polydextrose.
  • U.S. Pat. No. 4,806,244 describes the use of a combined membrane and sorption system in which sulfate is removed from water by nanofiltration, after which the nitrate, which passed through the membrane, was removed from the permeate by absorption to an ion exchange resin.
  • SUMMARY OF THE INVENTION
  • The present invention provides methods of purifying a carbohydrate compound from a feed solution containing a contaminant. The methods involve contacting the feed solution with a nanofiltration or reverse osmosis membrane under conditions such that the membrane retains the desired carbohydrate compound while a majority of the contaminant passes through the membrane. The invention provides methods for purifying carbohydrate compounds such as sialyl lactosides, sialic acid, lacto-N-neotetraose (LNnT) and GlcNAcβ1,3Galβ1,4Glc (LNT-2), NeuAcα(2→3)Galβ(1→4)(Fucα1→3)Glc(R1)β1-OR2, wherein R1 is OH or NAc; R2 is a hydrogen, an alkoxy, a saccharide, an oligosaccharide or an aglycon group having at least one carbon atom; and Galα(→13)Galβ(1→4)Glc(R1)β-O—R3, wherein R1 is OH or NAc; R3 is —(CH2)n—COX, with X═OH, OR4, —NHNH2, R4 being a hydrogen, a saccharide, an oligosaccharide or an aglycon group having at least one carbon atom, and n=an integer from 2 to 18.
  • Also provided are methods for purifying carbohydrate compounds having a formula NeuAcα(2→3)Galβ(1→4)GlcN(R1)β-OR2, NeuAcα(2→3)Galβ(1→4)GlcN(R1)β(1→3)Galβ-OR2, NeuAcα(2→3)Galβ(1→4) (Fucα1→3)GlcN(R1)β-OR2, or NeuAcα(2→3)Galβ(1→4) (Fucα1→3)GlcN(R1)β(1→3)Galβ-OR2, wherein R1 is alkyl or acyl from 1-18 carbons, 5,6,7,8-tetrahydro-2-naphthamido; benzamido; 2-naphthamido; 4-aminobenzamido; or 4-nitrobenzamido, and R2 is a hydrogen, a saccharide, an oligosaccharide or an aglycon group having at least one carbon atom.
  • In another embodiment, the invention provides methods of purifying a carbohydrate compound from a feed solution comprising a reaction mixture used to synthesize the carbohydrate compound. The synthesis can be enzymatic or chemical, or a combination thereof. The methods involve removing any proteins present in the feed solution by contacting the feed solution with an ultrafiltration membrane so that proteins are retained the membrane while the carbohydrate compound passes through the membrane as a permeate. The permeate from the ultrafiltration step is then contacted with a nanofiltration or reverse osmosis membrane under conditions such that the nanofiltration or reverse osmosis membrane retains the carbohydrate compound while a majority of an undesired contaminant passes through the membrane.
  • Another embodiment of the invention provides methods for purifying nucleotides, nucleosides, and nucleotide sugars by contacting a feed solution containing the nucleotide or related compound with a nanofiltration or reverse osmosis membrane under conditions such that the membrane retains the nucleotide or related compound while a majority of the contaminant passes through the membrane.
  • The present invention also provides methods for removing one or more contaminants from a solution that contains a carbohydrate of interest. The methods involve contacting the solution with a first side of a semipermeable membrane having rejection coefficients so as to retain the carbohydrate while allowing the contaminant to pass through the membrane. The membrane is selected from the group consisting of an ultrafiltration membrane, a nanofiltration membrane, and a reverse osmosis membrane, depending on the size and charge of the carbohydrate of interest relative to those of the contaminants. The membrane separates a feed solution containing a carbohydrate into a retentate portion and a permeate portion. If the rejection coefficient of the membrane is greater for the carbohydrate than for the contaminant, the retentate portion will have a lower concentration of the contaminant relative to the contaminant concentration in the feed solution, and generally also a higher ratio of the carbohydrate to the undesired contaminant. Conversely, a membrane having a rejection coefficient for the carbohydrate that is lesser than that for the contaminant will effect a separation wherein the concentration of the contaminant is lower in the permeate than in the feed solution, and the permeate will have a higher ratio of carbohydrate to contaminant than the feed solution. If desired, the fraction containing the carbohydrate can be recycled through the membrane system for further purification.
  • Examples of contaminants that can be removed from solutions containing the compound of interest using the methods of the invention include, but are not limited to, unreacted sugars, inorganic ions, pyruvate, phosphate, phosphoenolpyruvate, and proteins.
  • DESCRIPTION OF THE PREFERRED EMBODIMENTS DEFINITIONS
  • The following abbreviations are used herein:
      • Ara=arabinosyl;
      • Fru=fructosyl;
      • Fuc=fucosyl;
      • Gal=galactosyl;
      • GalNAc=N-acetylgalacto;
      • Glc=glucosyl;
      • GlcNAc=N-acetylgluco;
      • Man=mannosyl; and
      • NeuAc=sialyl (N-acetylneuraminyl).
  • The term “carbohydrate” encompasses chemical compounds having the general formula (CH2O)n, and includes monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term “oligo,” as used herein, refers to a polymeric molecule consisting of 2 to approximately 10 residues, for example, amino acids (oligopeptide), monosaccharides (oligosaccharide), and nucleic acids (oligonucleotide). The term “poly” refers to a polymeric molecule comprising greater than about 10 residues.
  • Oligosaccharides are considered to have a reducing end and a non-reducing end, whether or not the saccharide at the reducing end is in fact a reducing sugar. In accordance with accepted nomenclature, oligosaccharides are depicted herein with the non-reducing end on the left and the reducing end on the right.
  • All oligosaccharides described herein are described with the name or abbreviation for the non-reducing saccharide (e.g., Gal), followed by the configuration of the glycosidic bond (α or β), the ring bond, the ring position of the reducing saccharide involved in the bond, and then the name or abbreviation of the reducing saccharide (e.g., GlcNAc). The linkage between two sugars may be expressed, for example, as 2,3, 2→3, or (2,3).
  • A compound is “substantially purified” from an undesired component in a solution if the concentration of the undesired component after purification is no greater than about 40% of the concentration of the component prior to purification. Preferably, the post-purification concentration of the undesired component will be less than about 20% by weight, and more preferably less than about 10%, of the pre-purification concentration.
  • The term “pharmaceutically pure,” as used herein, refers to a compound that is sufficiently purified from undesired contaminants that the compound is suitable for administration as a pharmaceutical agent. Preferably, the compound is purified such that the undesired contaminant is present after purification in an amount that is about 5% by weight or less of the pre-purification concentration of the contaminant in the feed solution. More preferably, the post-purification concentration of the contaminant is about 1% or less of the pre-purification contaminant concentration, and most preferably about 0.5% or less of the pre-purification concentration of contaminant.
  • A “feed solution” refers to any solution that contains a compound to be purified. For example, a reaction mixture used to synthesize an oligosaccharide can be used as a feed solution from which the desired reaction product is purified using the methods of the invention.
  • Embodiments of the Invention
  • The present invention provides methods for rapidly and efficiently purifying specific carbohydrate and oligosaccharide structures to a high degree of purity using semipermeable membranes such as reverse osmosis and/or nanofiltration membranes. The methods are particularly useful for separating desired oligosaccharide compounds from reactants and other contaminants that remain in a reaction mixture after synthesis or breakdown of the oligosaccharides. For example, the invention provides methods for separating oligosaccharides from enzymes and/or other components of reaction mixtures used for enzymatic synthesis or enzymatic degradation of oligosaccharides, nucleotide sugars, glycolipids, liposaccharides, nucleotides, nucleosides, and other saccharide-containing compounds. Also provided are methods for removing salts, sugars and other components from feed solutions using ultrafiltration, nanofiltration or reverse osmosis. Using these techniques, the saccharides (e.g., sialyl lactose, SLex, and many others) can be produced at up to essentially 100% purity. Moreover, the purification methods of the invention are more efficient, rapid, and amenable to large-scale purifications than previously known carbohydrate purification methods.
  • Often, a desired purification can be effected in a single step; additional purification steps such as crystallization and the like are generally not required. Accordingly, the invention provides single-step methods for purifying saccharide-containing compounds.
  • To purify saccharides according to the invention, a membrane is selected that is appropriate for separating the desired carbohydrate from the undesired components of the solution from which the carbohydrate is to be purified. The goal in selecting a membrane is to optimize for a particular application the molecular weight cutoff (MWCO), membrane composition, permeability, and rejection characteristics, that is, the membrane's total capacity to retain specific molecules while allowing salts and other, generally smaller or opposite charged molecules, to pass through. The percent retention of a component i (Ri) is given by the formula Ri=(1−Cip/Cir)×100%, wherein Cip is the concentration of component i in the permeate and Cir is the concentration of component i in the retentate, both expressed in weight percent. The percent retention of a component is also called the retention characteristic or the membrane rejection coefficient.
  • For effective separation, a membrane is chosen that has a high rejection ratio for the saccharide of interest relative to the rejection ratio for compounds from which separation is desired. If a membrane has a high rejection ratio for a first compound relative to a second compound, the concentration of the first compound in the permeate solution which passes through the membrane is decreased relative to that of the second compound. Conversely, the concentration of the first compound increases relative to the concentration of the second compound in the retentate. If a membrane does not reject a compound, the concentration of the compound in both the permeate and the reject portions will remain essentially the same as in the feed solution. It is also possible for a membrane to have a negative rejection rate for a compound if the compound's concentration in the permeate becomes greater than the compound's concentration in the feed solution. A general review of membrane technology is found in “Membranes and Membrane Separation Processes,” in Ullmann's Encyclopedia of Industrial Chemistry (VCH, 1990); see also, Noble and Stern, Membrane Separations Technology: Principles and Applications (Elsevier, 1995).
  • As a starting point, one will generally choose a membrane having a molecular weight cut-off (MWCO, which is often related to membrane pore size) that is expected to retain the desired compounds while allowing an undesired compound present in the feed stream to pass through the membrane. The desired MWCO is generally less than the molecular weight of the compound being purified, and is typically greater than the molecular weight of the undesired contaminant that is to be removed from the solution containing the compound being purified. For example, to purify a compound having a molecular weight of 200 Da, one would choose a membrane that has a MWCO of less than about 200 Da. A membrane with a MWCO of 100 Da, for example, would also be a suitable candidate. The membranes that find use in the present invention are classified in part on the basis of their MWCO as ultrafiltration (UF) membranes, nanofiltration (NF) membranes, or reverse osmosis (RO) membranes, depending on the desired separation. For purposes of this invention, UF, NF, and RO membranes are classified as defined in the Pure Water Handbook, Osmonics, Inc. (Minnetonka Minn.). RO membranes typically have a nominal MWCO of less than about 200 Da and reject most ions, NF membranes generally have a nominal MWCO of between about 150 Da and about 5 kDa, and UF membranes generally have a nominal MWCO of between about 1 kDa and about 300 kDa (these MWCO ranges assume a saccharide-like molecule).
  • A second parameter that is considered in choosing an appropriate membrane for a particular separation is the polymer type of the membrane. The membranes used in each zone are made of conventional membrane material whether inorganic, organic, or mixed inorganic and organic. Typical inorganic materials include glasses, ceramics, cermets, metals and the like. Ceramic membranes, which are preferred for the UF zone, may be made, for example, as described in U.S. Pat. Nos. 4,692,354 to Asaeda et al, 4,562,021 to Alary et al., and others. The organic materials which are preferred for the NF and RO zones, are typically polymers, whether isotropic, or anisotropic with a thin layer or “skin” on either the bore side or the shell side of the fibers. Preferred materials for fibers are polyamides, polybenzamides, polysulfones (including sulfonated polysulfone and sulfonated polyether sulfone, among others), polystyrenes, including styrene-containing copolymers such as acrylo-nitrile-styrene, butadiene-styrene and styrene-vinylbenzylhalide copolymers, polycarbonates, cellulosic polymers including cellulose acetate, polypropylene, poly(vinyl chloride), poly(ethylene terephthalate), polyvinyl alcohol, fluorocarbons, and the like, such as those disclosed in U.S. Pat. Nos. 4,230,463, 4,806,244, and 4,259,183. The NF and RO membranes often consist of a porous support substrate in addition to the polymeric discrimination layer.
  • Of particular importance in selecting a suitable membrane composition is the membrane surface charge. Within the required MWCO range, a membrane is selected that has a surface charge that is appropriate for the ionic charge of the carbohydrate and that of the contaminants. While MWCO for a particular membrane is generally invariable, changing the pH of the feed solution can affect separation properties of a membrane by altering the membrane surface charge. For example, a membrane that has a net negative surface charge at neutral pH can be adjusted to have a net neutral charge simply by lowering the pH of the solution. An additional effect of adjusting solution pH is to modulate the ionic charge on the contaminants and on the carbohydrate of interest. Therefore, by choosing a suitable membrane polymer type and pH, one can obtain a system in which both the contaminant and the membrane are neutral, facilitating pass-through of the contaminant. If, for instance, a contaminant is negatively charged at neutral pH, it is often desirable to lower the pH of the feed solution to protonate the contaminant. For example, removal of phosphate is facilitated by lowering the pH of the solution to about 3, which protonates the phosphate anion, allowing passage through a membrane. As shown in Example 5, a decrease in pH from 7.5 to 3.0 decreases the percentage of GlcNAc passing through a polyamide membrane such as an Osmonics MX07 in thirty minutes from 70% to 28%, while increasing the pass percentage of phosphate from 10% to 46% (see, Example 6, Table 5 for additional examples of the effect of pH change on passage rate of other compounds through various nanofiltration membranes). For purification of an anionic carbohydrate, the pH will generally between about pH 1 and about pH 7. Conversely, if contaminant has a positive surface charge, the pH of the feed solution can be adjusted to between about pH 7 and about pH 14. For example, increasing the pH of a solution containing a contaminant having an amino group (—NH3 +) will make the amino group neutral, thus facilitating its passage through the membrane. Thus, one aspect of the invention involves modulating a separation by adjusting the pH of a solution in contact with the membrane; this can change the ionic charge of a contaminant and can also affect the surface charge of the membrane, thus facilitating purification if the desired carbohydrate. Of course, the manufacturer's instructions must be followed as to acceptable pH range for a particular membrane to avoid damage to the membrane.
  • For some applications, a mixture is first subjected to nanofiltration or reverse osmosis at one pH, after which the retentate containing the saccharide of interest is adjusted to a different pH and subjected to an additional round of membrane purification. For example, filtration of a reaction mixture used to synthesize sialyl lactose through an Osmonics MX07 membrane (a nanofiltration membrane having a MWCO of about 500 Da) at pH 3.0 will retain the sialyl lactose and remove most phosphate, pyruvate, salt and manganese from the solution, while also removing some of the GlcNAc, lactose, and sialic acid. Further recirculation through the MX07 membrane after adjusting the pH of the retentate to 7.4 will remove most of the remaining phosphate, all of the pyruvate, all of the lactose, some of the sialic acid, and substantial amounts of the remaining manganese.
  • If a saccharide is to be purified from a mixture that contains proteins, such as enzymes used to synthesize a desired oligosaccharide or nucleotide sugar, it is often desirable to remove the proteins as a first step of the purification procedure. For a saccharide that is smaller than the proteins, this separation is accomplished by choosing a membrane that has an MWCO which is less than the molecular mass of the protein or other macromolecule to be removed from the solution, but is greater than the molecular mass of the oligosaccharide being purified (i.e., the rejection ratio in this case is higher for the protein than for the desired saccharide). Proteins and other macromolecules that have a molecular mass greater than the MWCO will thus be rejected by the membrane, while the saccharide will pass through the membrane. Conversely, if an oligosaccharide or nucleotide sugar is to be purified from proteins that are smaller than the oligosaccharide or nucleotide sugar, a membrane is used that has a MWCO that is larger than the molecular mass of the protein but smaller than that of the oligosaccharide or nucleotide sugar. Generally, separation of proteins from carbohydrates will employ membranes that are commonly referred to as ultrafiltration (UF) membranes. UF membranes that are suitable for use in the methods of the invention are available from several commercial manufacturers, including Millipore Corp. (Bedford, Mass.), Osmonics, Inc. (Minnetonka, Minn.), Filmtec (Minneapolis, Minn.), UOP, Desalination Systems, Advanced Membrane Technologies, and Nitto.
  • The invention also provides methods for removing salts and other low molecular weight components from a mixture containing a saccharide of interest by using a nanofiltration (NF) or a reverse osmosis (RO) membrane. Nanofiltration membranes are a class of membranes for which separation is based both on molecular weight and ionic charge. These membranes typically fall between reverse osmosis and ultrafiltration membranes in terms of the size of species that will pass through the membrane. Nanofiltration membranes typically have micropores or openings between chains in a swollen polymer network. Molecular weight cut-offs for non-ionized molecules are typically in the range from 100-20,000 Daltons. For ions of the same molecular weight, membrane rejections (retentions) will increase progressively for ionic charges of 0, 1, 2, 3 etc. for a particular membrane because of increasing charge density (see, e.g., Eriksson, P., “Nanofiltration Extends the Range of Membrane Filtration,” Environmental Progress, 7: 58-59 (1988)). Nanofiltration is also described in Chemical Engineering Progress, pp. 68-74 (March 1994), Rautenbach et al., Desalination 77: 73 (1990), and U.S. Pat. No. 4,806,244). In a typical application, saccharides of interest will be retained by the nanofiltration membrane and contaminating salts and other undesired components will pass through. A nanofiltration membrane useful in the methods of the invention will typically have a retention characteristic for the saccharide of interest of from about 40% to about 100%, preferably from about 70% to about 100%. The nanofilter membranes used in the invention can be any one of the conventional nanofilter membranes, with polyamide membranes being particularly suitable. Several commercial manufacturers, including Millipore Corp. (Bedford, Mass.), Osmonics, Inc. (Minnetonka, Minn.), Filmtec, UOP, Advanced Membrane Technologies, Desalination Systems, and Nitto, among others, distribute nanofiltration membranes that are suitable for use in the methods of the invention. For example, suitable membranes include the Osmonics MX07, YK, GH (G-10), GE (G-5), and HL membranes, among others.
  • Reverse osmosis (RO) membranes also allow a variety of aqueous solutes to pass through them while retaining selected molecules. Generally, osmosis refers to a process whereby a pure liquid (usually water) passes through a semipermeable membrane into a solution (usually sugar or salt and water) to dilute the solution and achieve osmotic equilibrium between the two liquids. In contrast, reverse osmosis is a pressure driven membrane process wherein the application of external pressure to the membrane system results in a reverse flux with the water molecules passing from a saline or sugar solution compartment into the pure water compartment of the membrane system. A RO membrane, which is semipermeable and non-porous, requires an aqueous feed to be pumped to it at a pressure above the osmotic pressure of the substances dissolved in the water. An RO membrane can effectively remove low molecular weight molecules (<200 Daltons) and also ions from water. Preferably, the reverse osmosis membrane will have a retention characteristic for the saccharide of interest of from about 40% to about 100%, preferably from about 70% to about 100%. Suitable RO membranes include, but are not limited to, the Filmtec BW-30, Filmtec SW-30, Filmtec SW-30HR, UOP RO membranes, Desal RO membranes, Osmonics RO membranes, Advanced Membrane Technologies RO membranes, and the Nitto RO membranes, among others. One example of a suitable RO membrane is Millipore Cat. No. CDRN500 60 (Millipore Corp., Bedford Mass.).
  • The membranes used in the invention may be employed in any of the known membrane constructions. For example, the membranes can be flat, plate and frame, tubular, spiral wound, hollow fiber, and the like. In a preferred embodiment, the membrane is spiral wound. The membranes can be employed in any suitable configuration, including either a cross-flow or a depth configuration. In “cross-flow” filtration, which is preferred for ultrafiltration, nanofiltration and reverse osmosis purifications according to the invention, the “feed” or solution from which the carbohydrate of interest is to be purified flows through membrane channels, either parallel or tangential to the membrane surface, and is separated into a retentate (also called recycle or concentrate) stream and a permeate stream. To maintain an efficient membrane, the feed stream should flow, at a sufficiently high velocity, parallel to the membrane surface to create shear forces and/or turbulence to sweep away accumulating particles rejected by the membrane. Cross-flow filtration thus entails the flow of three streams—feed, permeate and retentate. In contrast, a “dead end” or “depth” filter has only two streams—feed and filtrate (or permeate). The recycle or retentate stream, which retains all the particles and large molecules rejected by the membrane, can be entirely recycled to the membrane module in which the recycle stream is generated, or can be partially removed from the system. When the methods of the invention are used to purify saccharides from lower molecular weight components, for example, the desired saccharides are contained in the retentate stream (or feed stream, for a depth filter), while the permeate stream contains the removed contaminants.
  • The purification methods of the invention can be further optimized by adjusting the pressure, flow rate, and temperature at which the filtration is carried out. UF, NF, and RO generally require increasing pressures above ambient to overcome the osmotic pressure of the solution being passed through the membrane. The membrane manufacturers' instructions as to maximum and recommended operating pressures can be followed, with further optimization possible by making incremental adjustments. For example, the recommended pressure for UF will generally be between about 25 and about 100 psi, for NF between about 50 psi and about 1500 psi, and for RO between about 100 and about 1500 psi. Flow rates of both the concentrate (feed solution) and the permeate can also be adjusted to optimize the desired purification. Again, the manufacturers' recommendations for a particular membrane serve as a starting point from which to begin the optimization process by making incremental adjustments. Typical flow rates for the concentrate (Pc) will be between about 1 and about 15 gallons per minute (GPM), and more preferably between about 3 and about 7 GPM. For the permeate, flow rates (Pf) of between about 0.05 GPM and about 10 GPM are typical, with flow rates between about 0.2 and about 1 GPM being preferred. The temperature at which the purification is carried out can also influence the efficiency and speed of the purification. Temperatures of between about 0 and about 100° C. are typical, with temperatures between about 20 and 40° C. being preferred for most applications. Higher temperatures can, for some membranes, result in an increase in membrane pore size, thus providing an additional parameter that one can adjust to optimize a purification.
  • In a preferred embodiment, the filtration is performed in a membrane purification machine which provides a means for automating control of flow rate, pressure, temperature, and other parameters that can affect purification. For example, the Osmonics 213T membrane purification machine is suitable for use in the methods of the invention, as are machines manufactured by other companies listed above.
  • The membranes can be readily cleaned either after use or after the permeability of the membrane diminishes. Cleaning can be effected at a slightly elevated temperature if so desired, by rinsing with water or a caustic solution. If the streams contain small amounts of enzyme, rinsing in the presence of small amounts of surfactant, for instance ULTRASIL°, might be useful. Also, one can use prefilters (100-200 μm) to protect the more expensive nanofiltration membranes. Other cleaning agents can, if desired, be used. The choice of cleaning method will depend on the membrane being cleaned, and the membrane manufacturer's instructions should be consulted. The cleaning can be accomplished with a forward flushing or a backward flushing.
  • The purification methods of the invention can be used alone or in combination with other methods for purifying carbohydrates. For example, an ion exchange resin can be used to remove particular ions from a mixture containing a saccharide of interest, either before or after nanofiltration/reverse osmosis, or both before and after filtration. Ion exchange is particularly desirable if it is desired to remove ions such as phosphate and nucleotides that remain after a first round of nanofiltration or reverse osmosis. In the case of sialyl lactose synthesis as discussed above, this can be accomplished, for example, by adding an anion exchange resin such as AG1X-8 (acetate form, BioRad; see, e.g., BioRad catalog for other ion exchange resins) to a retentate that is at about pH 3.0 or lower until the phosphate concentration is reduced as desired. In this process, acetic acid is released, so one may wish to follow the ion exchange with an additional purification through the nanofiltration or reverse osmosis system. For example, one can circulate the pH 3.0 or lower solution through an Osmonics MX07 or similar membrane until the conductivity of the permeate is low and stabilized. The pH of the solution can then be raised to 7.4 with NaOH and the solution recirculated through the same membrane to remove remaining sodium acetate and salt. Cations can be removed in a similar manner; for example, to remove Mn2+, an acidic ion exchange resin can be used, such as AG50WX8 (H+) (BioRad).
  • The purification methods of the invention are particularly useful for purifying oligosaccharides that have been prepared using enzymatic synthesis. Enzymatic synthesis using glycosyltransferases provides a powerful method for preparing oligosaccharides; for some applications it is desirable to purify the oligosaccharide from the enzymes and other reactants in the enzymatic synthesis reaction mixture. Preferred methods for producing many oligosaccharides involve glycosyl transferase cycles, which produce at least one mole of inorganic pyrophosphate for each mole of product formed and are typically carried out in the presence of a divalent metal ion. Examples of glycosyltransferase cycles are the sialyltransferase cycles, which use one or more enzymes as well as other reactants. See, e.g., U.S. Pat. No. 5,374,541 WO 9425615 A, PCT/US96/04790, and PCT/US96/04824. For example, a reaction used for synthesis of sialylated oligosaccharides can contain a sialyltransferase, a CMP-sialic acid synthetase, a sialic acid, an acceptor for the sialyltransferase, CTP, and a soluble divalent metal cation. An exemplary α2,3)sialyltransferase referred to as α(2,3)sialtransferase (EC 2.4.99.6) transfers sialic acid to the non-reducing terminal Gal of a Galβ1→3Glc disaccharide or glycoside. See, Van den Eijnden et al., J. Biol. Chem., 256: 3159 (1981), Weinstein et al., J. Biol. Chem., 257: 13845 (1982) and Wen et al., J. Biol. Chem., 267: 21011 (1992). Another exemplary α2,3-sialyltransferase (EC 2.4.99.4) transfers sialic acid to the non-reducing terminal Gal of the disaccharide or glycoside. See, Rearick et al., J. Biol. Chem., 254: 4444 (1979) and Gillespie et al., J. Biol. Chem., 267: 21004 (1992). Further exemplary enzymes include Gal-β-1,4-GlcNAc α-2,6 sialyltransferase (See, Kurosawa et al. Eur. J. Biochem. 219: 375-381 (1994)). The reaction mixture will also contain an acceptor for the sialyltransferase, preferably having a galactosyl unit. Suitable acceptors, include, for example, Galβ1→3GalNAc, lacto-N-tetraose, Galβ1→3GlcNAc, Galβ1→3Ara, Galβ1→6GlcNAc, Galβ1→4Glc (lactose), Galβ1→4Glcβ1-OCH2CH3, Galβ1→4Glcβ1-OCH2CH2CH3, Galβ1→4Glcβ1-OCH2C6H5, Galβ1→4GlcNAc, Galβ1-OCH3, melibiose, raffinose, stachyose, and lacto-N-neotetraose (LNnT). The sialic acid present in the reaction mixture can include not only sialic acid itself (5-N-acetylneuraminic acid; 5-N-acetylamino-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid; NeuAc, and sometimes also abbreviated AcNeu or NANA), but also 9-substituted sialic acids such as a 9-O—C1-C6 acyl-NeuAc like 9-O-lactyl-NeuAc or 9-O-acetyl-NeuAc, 9-deoxy-9-fluoro-NeuAc and 9-azido-9-deoxy-NeuAc. The synthesis and use of these compounds in a sialylation procedure is described in international application WO 92/16640, published Oct. 1, 1992.
  • In preferred embodiments the reaction medium can further comprise a CMP-sialic acid recycling system comprising at least 2 moles of phosphate donor per each mole of sialic acid, and catalytic amounts of an adenine nucleotide, a kinase capable of transferring phosphate from the phosphate donor to nucleoside diphosphates, and a nucleoside monophosphate kinase capable of transferring the terminal phosphate from a nucleoside triphosphate to CMP. For example, a suitable CMP-sialic acid regenerating system comprises cytidine monophosphate (CMP), a nucleoside triphosphate (for example adenosine triphosphate (ATP), a phosphate donor (for example, phosphoenolpyruvate or acetyl phosphate), a kinase (for example, pyruvate kinase or acetate kinase) capable of transferring phosphate from the phosphate donor to nucleoside diphosphates and a nucleoside monophosphate kinase (for example, myokinase) capable of transferring the terminal phosphate from a nucleoside triphosphate to CMP. The previously discussed α(2,3)sialyltransferase and CMP-sialic acid synthetase can also be formally viewed as part of the CMP-sialic acid regenerating system. For those embodiments in which a CMP-sialic acid recycling system is not used, the reaction medium will preferably further comprise a phosphatase.
  • Pyruvate is a byproduct of the sialyltransferase cycle and can be made use of in another reaction in which N-acetylmannosamine (ManNAc) and pyruvate are reacted in the presence of NeuAc aldolase (EC 4.1.3.3) to form sialic acid. Alternatively, advantage can be taken of the isomerization of GlcNAc to ManNAc, and the less expensive GlcNAc can be used as the starting material for sialic acid generation. Thus, the sialic acid can be replaced by ManNAc (or GlcNAc) and a catalytic amount of NeuAc aldolase. Although NeuAc aldolase also catalyzes the reverse reaction (NeuAc to ManNAc and pyruvate), the produced NeuAc is irreversibly incorporated into the reaction cycle via CMP-NeuAc catalyzed by CMP-sialic acid synthetase. In addition, the starting material, ManNAc, can also be made by the chemical conversion of GlcNAc using methods known in the art (see, e.g., Simon et al., J. Am. Chem. Soc. 110: 7159 (1988). The enzymatic synthesis of sialic acid and its 9-substituted derivatives and the use of a resulting sialic acid in a different sialylating reaction scheme is disclosed in International application WO 92/16640, published on Oct. 1, 1992, and incorporated herein by reference.
  • When a galactosyltransferase is used for enzymatic synthesis of an oligosaccharide, the reaction medium will preferably contain, in addition to a galactosyltransferase, donor substrate, acceptor sugar and divalent metal cation, a donor substrate recycling system comprising at least 1 mole of glucose-1-phosphate per each mole of acceptor sugar, a phosphate donor, a kinase capable of transferring phosphate from the phosphate donor to nucleoside diphosphates, and a pyrophosphorylase capable of forming UDP-glucose from UTP and glucose-1-phosphate and catalytic amounts of UDP and a UDP-galactose-4-epimerase. Exemplary galactosyltransferases include α(1,3) galactosyltransferase (E.C. No. 2.4.1.151, see, e.g., Dabkowski et al., Transplant Proc. 25: 2921 (1993) and Yamamoto et al., Nature 345: 229-233 (1990)) and β(1,4) galactosyltransferase (E.C. No. 2.4.1.38).
  • Oligosaccharides synthesized by other enzymatic methods can also be purified by the methods of the invention. For example, the methods are useful for purifying oligosaccharides produced in non-cyclic or partially cyclic reactions such as simple incubation of an activated saccharide and an appropriate acceptor molecule with a glycosyltransferase under conditions effective to transfer and covalently bond the saccharide to the acceptor molecule. Glycosyltransferases, which include those described in, e.g., U.S. Pat. No. 5,180,674, and International Patent Publication Nos. WO 93/13198 and WO 95/02683, as well the glycosyltransferases encoded by the los locus of Neisseria (see, U.S. Pat. No. 5,545,553), can be bound to a cell surface or unbound. Oligosaccharides that can be obtained using these glycosyltransferases include, for example, Galα(1→4)Galβ(1→4)Glc, GlcNAcβ(1,3)Galβ(1,4)Glc, Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4) Glc, and GalNAcβ(1→3)Galβ(1→4)GlcNAcβ(1→3) Galβ(1→4)Glc, among many others.
  • Among the compounds that one can purify using the described methods are sialic acid and any sugar having a sialic acid moiety. These include the sialyl galactosides, including the sialyl lactosides, as well as compounds having the formula:
    NeuAcα(2→3)Galβ(1→4)GlcN(R′)β-OR or
    NeuAcα(2→3)Galβ(1→4)GlcN(R′)β(1→3)Galβ-OR
  • In these formulae, R′ is alkyl or acyl from 1-18 carbons, 5,6,7,8-tetrahydro-2-naphthamido; benzamido; 2-naphthamido; 4-aminobenzamido; or 4-nitrobenzamido. R is a hydrogen, a alkyl C1-C6, a saccharide, an oligosaccharide or an aglycon group having at least one carbon atom. The term “aglycon group having at least one carbon atom” refers to a group-A-Z, in which A represents an alkylene group of from 1 to 18 carbon atoms optionally substituted with halogen, thiol, hydroxy, oxygen, sulfur, amino, imino, or alkoxy; and Z is hydrogen, H, —SH, —NH2, R1, —N(R1)2, —CO2H, —CO2R1, —CONH2, —CONHR1, —CON(R1)2, —CONHNH2, or —OR1 wherein each R1 is independently alkyl of from 1 to 5 carbon atoms. In addition, R can be
    Figure US20050269265A1-20051208-C00001

    where n,m,o=1-18; (CH2)n—R2 (in which n=0-18), wherein R2 is a variously substituted aromatic ring, preferably, a phenyl group, being substituted with one or more alkoxy groups, preferably methoxy or O(CH2)mCH3, (in which m=0-18), or a combination thereof. R can also be 3-(3,4,5-trimethoxyphenyl)propyl.
  • The present invention is also useful for purifying a variety of compounds that comprise selectin-binding carbohydrate moieties. These selectin-binding moieties have the general formula:
    R1Galβ1,m(Fucα1,n)GlcNR0(R2)p
  • in which R0 is (C1-C8 alkyl)carbonyl, (C1-C8 alkoxy)carbonyl, or (C2-C9 alkenyloxy)carbonyl, R1 is an oligosaccharide or a group having the formula
    Figure US20050269265A1-20051208-C00002
  • R3 and R4 may be the same or different and may be H, C1-C8 alkyl, hydroxy-(C1-C8 alkyl), aryl-(C1-C8 alkyl), or (C1-C8 alkoxy)-(C1-C8 alkyl), substituted or unsubstituted. R2 may be H, C1-C8 alkyl, hydroxy-(C1-C8 alkyl), aryl-(C1-C8 alkyl), (C1-C8 alkyl)-aryl, alkylthio, α1,2Man, α1,6GalNAc, β1,3Galβ1,4Glc, α1,2Man-R8, α1,6GalNAc—R8, and β1,3Gal-R8. R8 may be H, C1-C8 alkyl, C1-C8 alkoxy, hydroxy-(C1-C8 alkyl), aryl-(C1-C8 alkyl), (C1-C8 alkyl)-aryl, or alkylthio. In the formula, m and n are integers and may be either 3 or 4; p may be zero or 1.
  • The substituted groups mentioned above may be substituted by hydroxy, hydroxy(C1-C4 alkyl), polyhydroxy(C1-C4 alkyl), alkanoamido, or hydroxyalknoamido substituents. Preferred substituents include hydroxy, polyhydroxy(C3 alkyl), acetamido and hydroxyacetamido. A substituted radical may have more than one substitution, which may be the same or different.
  • For embodiments in which R1 is an oligosaccharide, the oligosaccharide is preferably a trisaccharide. Preferred trisaccharides include NeuAcα2,3Galβ1,4GlcNAcβ1,3 or NeuGcα2,3Galβ1,4GlcNAcβ1,3.
  • For embodiments in which R1 is the group having the formula
    Figure US20050269265A1-20051208-C00003

    R3 and R4 preferably form a single radical having the formula
    —R5— or —(R6)q—O—(R7)r
    in which R5 is C3-C7 divalent alkyl, substituted or unsubstituted, R6 and R7 are the same or different and are C1-C6 divalent alkyl, substituted or unsubstituted. In the formula, q and r are integers which may be the same or different and are either zero or 1. The sum of q and r is always at least 1.
  • A more preferred structure for a single radical formed by R3 and R4 is one having the formula
    —(R6)—O—
    in which R6 is C3-C4 divalent alkyl, substituted or unsubstituted. For instance, R6 may have the formula —CH2—CH2—CH2—CH2—, preferably substituted. The radical can be substituted with hydroxy, polyhydroxy(C3 alkyl), and substituted or unsubstituted alkanoamido groups, such as acetamido or hydroxyacetamido. The substituted structure will typically form a monosaccharide, preferably a sialic acid such as NeuAc or NeuGc linked α2,3 to the Gal residue.
  • In the general formula, above, both m and n are integers and can be either 3 or 4. Thus, in one set of structures Gal is linked β1,4 and Fuc is linked α1,3 to GlcNAc. This formula includes the SLex tetrasaccharide. SLex has the formula NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1—. This structure is selectively recognized by LECCAM-bearing cells. SLex compounds that can be purified using the methods of the invention include NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1-Gal-OEt, NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,4Galβ1-OEt, and others that are described in international application WO 91/19502. Other compounds that one-can purify using the methods include those described in U.S. Pat. No. 5,604,207 having the formula
    Figure US20050269265A1-20051208-C00004

    wherein Z is hydrogen, C1-C6 acyl or
    Figure US20050269265A1-20051208-C00005
  • Y is selected from the group consisting of C(O), SO2, HNC(O), OC(O) and SC(O);
  • R1 is selected from the group consisting of an aryl, a substituted aryl and a phenyl C1-C3 alkylene group, wherein said aryl substitutent is selected from the group consisting of a halo, trifuloromethyl, nitro, C1-C18 alkyl, C1-C18 alkoxy, amino, mono-C1-C18 alkylamino, di-C1-C18 alkylamino, benzylamino, C1-C18 alkylbenzylamino, C1-C18 thioaklyl and C1-C18 alkyl carboxamido groups, or
  • R1Y is allyloxycarbonyl or chloroacetyl;
  • R2 is selected from the group consisting of monosaccharide (including β1,3Gal-OR, where R═H, alkyl, aryl or acyl), disaccharide, hydrogen, C1-C18 straight chain, branched chain or cyclic hydrocarbyl, C1-C6 alkyl, 3-(3,4,5-trimethoxyphenyl)propyl, C1-C5 alkylene ω-carboxylate, ω-trisubstituted silyl C2-C4 alkylene wherein said ω-trisubstituted silyl is a silyl group having three substituents independently selected from the group consisting of C1-C4 alkyl, phenyl,
  • or OR2 together form a C1-C18 straight chain, branched chain or cyclic hydrocarbyl carbamate;
  • R3 is hydrogen or C1-C6 acyl;
  • R4 is hydrogen, C1-C6 alkyl or benzyl;
  • R5 is selected from the group consisting of hydrogen, benzyl, methoxybenzyl, dimethoxybenzyl and C1-C6 acyl;
  • R7 is methyl or hydroxymethyl; and
  • X is selected from the group consisting of C1-C6 acyloxy, C2-C6 hydroxylacyloxy, hydroxy, halo and azido.
  • A related set of structures included in the general formula are those in which Gal is linked β1,3 and Fuc is linked α1,4. For instance, the tetrasaccharide, NeuAcα2,3Galβ1,3(Fucα1,4)GlcNAcβ1-, termed here SLea, is recognized by selectin receptors. See, Berg et al., J. Biol. Chem., 266: 14869-14872 (1991). In particular, Berg et al. showed that cells transformed with E-selectin cDNA selectively bound neoglycoproteins comprising SLea.
  • The methods of the invention are also useful for purifying oligosaccharide compounds having the general formula Galα1,3Gal—, including Galα1,3Galβ1,4Glc(R)β—O—R1, wherein R1 is —(CH2)n—COX, with X═OH, OR2, —NHNH2, R═OH or NAc, and R2 is a hydrogen, a saccharide, an oligosaccharide or an aglycon group having at least one carbon atom, and n=an integer from 2 to 18, more preferably from 2 to 10. For example, one can purify a compound having the formula Galα1,3Galβ1,4GlcNAcβ-O—(CH2)5—COOH using procedures such as those described in Examples 7-8. Also among the compounds that can be purified according to the invention are lacto-N-neotetraose (LNnT), GlcNAcβ1,3Galβ1,4Glc (LNT-2), sialyl(α2,3)-lactose, and sialyl(α2,6)-lactose.
  • In the above descriptions, the terms are generally used according to their standard meanings. The term “alkyl” as used herein means a branched or unbranched, saturated or unsaturated, monovalent or divalent, hydrocarbon radical having from 1 to 20 carbons, including lower alkyls of 1-8 carbons such as methyl, ethyl, n-propyl, butyl, n-hexyl, and the like, cycloalkyls (3-7 carbons), cycloalkylmethyls (4-8 carbons), and arylalkyls. The term “alkoxy” refers to alkyl radicals attached to the remainder of the molecule by an oxygen, e.g., ethoxy, methoxy, or n-propoxy. The term “alkylthio” refers to alkyl radicals attached to the remainder of the molecule by a sulfur. The term of “acyl” refers to a radical derived from an organic acid by the removal of the hydroxyl group. Examples include acetyl, propionyl, oleoyl, myristoyl.
  • The term “aryl” refers to a radical derived from an aromatic hydrocarbon by the removal of one atom, e.g., phenyl from benzene. The aromatic hydrocarbon may have more than one unsaturated carbon ring, e.g., naphthyl.
  • The term “alkoxy” refers to alkyl radicals attached to the remainder of the molecule by an oxygen, e.g., ethoxy, methoxy, or n-propoxy.
  • The term “alkylthio” refers to alkyl radicals attached to the remainder of the molecule by a sulfur.
  • An “alkanoamido” radical has the general formula —NH—CO—(C1-C6 alkyl) and may or may not be substituted. If substituted, the substituent is typically hydroxyl. The term specifically includes two preferred structures, acetamido, —NH—O—CH3, and hydroxyacetamido, —NH—CO—CH2—OH.
  • The term “heterocyclic compounds” refers to ring compounds having three or more atoms in which at least one of the atoms is other than carbon (e.g., N, O, S, Se, P, or As). Examples of such compounds include furans (including the furanose form of pentoses, such as fucose), pyrans (including the pyranose form of hexoses, such as glucose and galactose) pyrimidines, purines, pyrazines and the like.
  • The methods of the invention are useful not only for purifying carbohydrates that that are newly synthesized, but also those that are the products of degradation, e.g., enzymatic degradation. See, e.g., Sinnott, M. L., Chem. Rev. 90: 1171-1202 (1990) for examples of enzymes that catalyze degradation of oligosaccharides.
  • The invention also provides methods for purifying nucleotides, nucleotide sugars, and related compounds. For example, a nucleotide sugar such as GDP-fucose, GDP-mannose, CMP-NeuAc, UDP-glucose, UDP-galactose, UDP—N-acetylgalactosamine, and the like, can be purified by the methods described herein. The methods are also useful for purifying nucleotides and nucleotides in various states of phosphorylation (e.g., CMP, CDP, CTP, GMP, GDP, GTP, TMP, TDP, TTP, AMP, ADP, ATP, UMP, UDP, UTP), as well as the deoxy forms of these and other nucleotides.
  • The following examples are offered solely for the purposes of illustration, and are intended neither to limit nor to define the invention.
  • EXAMPLES
  • Examples 1-5 demonstrate the synthesis of sialyl lactose and its purification using nanofiltration and ion exchange. In summary, N-acetyl-D-mannosamine (ManNAc) was generated from N-acetyl-D-glucosamine (GluNAc) under basic conditions. The ManNAc was condensed with sodium pyruvate to produce sialic acid enzymatically. The sialyltransferase cycle was used to convert the sialic acid into sialyl lactose, which was then purified by nanofiltration and ionic exchange. Example 6 demonstrates the separation of organics and inorganic salts by nanofiltration. Example 7 demonstrates the separation characteristics of polybenzamide nanofiltration membranes. Example 8 demonstrates the separation characteristics of polyamide nanofiltration membranes.
  • Example 1 Synthesis and Purification of Sialic Acid
  • This example demonstrates a method for synthesizing sialic acid using a relatively inexpensive substrate, GlcNAc, rather than the more expensive ManNAc or sialic acid. A procedure similar to that described in Simon et al., J. Am. Chem. Soc. 110: 7159 (1988), was used to convert GlcNAc to ManNAc. Briefly, GlcNAc (1000 g, 4.52 mole) was dissolved in water (500 ml). The pH was adjusted to 12.0 with 50% NaOH (˜115 ml). The solution was stirred under argon for 7.5 hours, then cooled in an ice bath and the pH was adjusted to 7.7 with concentrated HCl (˜200 ml). Sialic acid was then produced by aldol condensation of ManNAc.
  • To obtain sialic acid, the ManNAc produced in the previous step was subjected to aldol condensation mediated by N-acetylneuraminic acid (Neu5Ac) aldolase and pyruvic acid. To a 1.5 L aqueous solution containing approximately 57 g (0.258 mol) ManNAc and 193 g GlcNAc from base-catalyzed epimerization was added 123.8 g sodium pyruvate (1.125 mole), 1.5 g bovine serum albumin, and 0.75 g sodium azide. The pH was adjusted to 7.5 and 11,930 U of sialic acid aldolase was added. The solution was incubated at 37° C. for 7 days. HPLC analysis on an Aminex HPX87H (BioRad) column (0.004 M H2SO4, 0.8 ml/min, monitor A220) revealed that the solution contained 0.157 M sialic acid (91% conversion of ManNAc, 0.235 mol).
  • Example 2 Synthesis of Sialyl Lactose Using Sialyltransferase Cycle
  • To the sialic acid produced in Example 1 was added lactose monohydrate (79.2 g, 0.22 mol), 0.7 g bovine serum albumin, phosphoenolpyruvate monopotassium salt (37 g, 0.22 mol), and the pH was adjusted to 7.5. CMP (2.84 g, 0.0088 mol), ATP (0.54 g, 0.0009 mol) were added, and the pH readjusted to 7.5. Sodium azide (0.35 g) was added, as were the following enzymes: pyruvate kinase (19,800 U), myokinase (13,200 U), CMP sialic acid synthetase (440 U, and sialyltransferase (165 U). 66 ml of 1M MnCl2 was added and the final volume adjusted to 2.2 L with water. The reaction was carried out at room temperature.
  • The reaction was monitored daily by thin layer chromatography (TLC) and [Mn2+] was determined by ion chromatography. Additions/adjustments were made as shown in Table 1:
    TABLE 1
    Day 2   44 ml 1M MnCl2 added
    Day 4   43 ml 1M MnCl2 added
    Day 6 added 34.3 ml 1M MnCl2, 37 g PEP; pH readjusted to 7.5;
    pyruvate kinase (19,800 U), myokinase (13,200 U), CMP
    sialic acid synthetase (440 U), and sialyltransferase (165 U)
    Day 7 31.7 ml 1M MnCl2
    Day 8 24.6 ml 1M MnCl2
    Day 9   44 ml 1M MnCl2
    Day 10 30.8 ml 1M MnCl2
    Day 11 31.7 ml 1M MnCl2
    Day 12 24.6 ml 1M MnCl2, pH readjusted to 7.5
    Day 13  440 U CMP sialic acid synthetase, 82.5 U sialyltransferase
    Day 14 pH readjusted to 7.5
    Day 16 37.7 ml 1 M MnCl2, 19,800 U pyruvate kinase, 13,200 U
    myokinase
    Day 17   26 g phosphenolpyruvate, trisodium salt
  • The sialyl lactose yield was approximately 70-80% as determined by TLC.
  • Example 3 Synthesis of Sialyl Lactose Using Sialyltransferase Cycle
  • This example illustrates the production of α-N-acetylneuraminic acid(2,3)β-galactosyl(1,4)glucose using the sialyl transferase cycle with control of the manganese ion concentration.
  • In a polypropylene vessel, phosphoenolpyruvate trisodium salt (285.4 g, 1.22 mol) and sialic acid (197 g, 0.637 mol) were dissolved in 5 L of water and the pH was adjusted to 7.1 with 6 M NaOH. Cytidine-5′-monophosphate (5.14 g, 15.9 mmol) and potassium chloride (7.9 g, 0.106 mol) were added and the pH was re-adjusted to 7.45 with 6 M NaOH. Pyruvate kinase (28,000 units), myokinase (17,000 units), adenosine triphosphate (0.98 g, 1.6 mmol), CMP NeuAc synthetase (1325 units), α2,3 sialyltransferase (663 units) and MnCl4H2O (52.4 g, 0.265 mol) were added and mixed. To a 3.7 L portion of the resulting mixture was added lactose (119 g, 0.348 mol) and sodium azide (1.75 g). The reaction mixture was kept at room temperature and monitored daily by thin layer chromatography (tlc) and ion chromatography. After two days, additional enzymes were added as follows: pyruvate kinase (38,100 units), myokinase (23,700 units), CMP NeuAc synthetase (935 units), and α2,3 sialyltransferase (463 units). The pH was periodically adjusted to 7.5 with 6 M NaOH. Additionally, the manganese ion concentration was measured and supplemented as shown in Table 2 below.
    TABLE 2
    Amount Supplemented
    [Mn++] Loss of Mn++ (mL of 1 M, final
    Day (measured, mM) (from previous day) added conc)
    1 28 22.0 none
    2 23.9 4.1 none
    3 10.7 13.2 111 mL, +30 mM
    4 1.4 39.3 111 mL, +30 mM
    5 3.0 28.4 148 mL, +40 mM
    6 12.9 30.1  74 mL, +20 mM
    7 10.0 22.9  80 mL, +20 mM
    8 12.0 18.0  80 mL, +20 mM
    9 24.3 7.7 none
  • On day 9, the reaction was essentially complete by tlc. As the results in the table indicate, the depletion of Mn++ resulted in additional amounts of MnC4H2O being added almost daily to maintain the metal ion concentration. Manganese ion is a required cofactor for at least one enzyme in the sialyl transferase cycle. However, the manganese ion inorganic phosphate produced form a complex of very low solubility. Because of this limited solubility, the transferase cycle can continue to proceed, but at reduced reaction rates. By supplementing the manganese ions which are lost by precipitation with pyrophosphate, the rate of reaction can be maintained. Thus, when manganese ion concentration is maintained in an optimal range, the sialyl transferase reaction cycle can be driven to completion.
  • Example 4 Purification of Sialyllactose Using Ion Exchange and Reverse Osmosis
  • This example illustrates the workup and purification of the trisaccharide produced in Example 2 followed by peracetylation and esterification. A solution (2L) of sodium 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-nonulopyranosylonate-(2-3)-O-β-D-galactopyranosyl-(1-4)-O-β-D-glucopyranose produced from the action of a sialyl transferase in the presence of the appropriate cofactors on lactose (55 g) was filtered through paper. The filtrate was run through a membrane with a 3000 or 10,000 molecular weight cut off to remove protein from the desired product. The eluate was concentrated and desalted by running it against a polyamide reverse osmosis membrane in a suitable apparatus (Cat. No. CDRN500 60, Millipore, Bedford, Mass.). The retentate containing the product was evaporated to a thick syrup. Optionally the retentate can be treated with a chelating resin to remove divalent cations. After filtration the filtrate contained the desired product substantially free of salts and in a high state of purity as shown by 1Hmr spectroscopy. Otherwise the syrup was so evaporated twice with pyridine (2×200 mL). The evaporation flask was charged with a solution of N,N-dimethylaminopyridine (2.2 g) in pyridine (1.2 L). Acetic anhydride (0.83 L) was added during a period of 1 hour. The resulting mixture was left for 24-48 hours rotating slowly at room temperature. The reaction is checked by TLC (methanol:dichloromethane 1:9). Upon complete reaction vacuum is applied and the solution is evaporated to give a residue.
  • The residue was dissolved in ethyl acetate (1.5 L). This solution was washed with 5% aqueous hydrochloric acid (1.5 L) followed by saturated aqueous sodium bicarbonate (1.5 L) and finally water (1.5 L). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to a semi-solid residue. The per-O-acetylated lactone trisaccharide (69 g) was dissolved in methanol (350 mL) and a sodium methoxide solution (17.5 mL, 25% solution in methanol) was added followed by water (3.5 mL). When TLC developed with isopropanol:ammonium hydroxide:water 7:1:2 showed the reaction to be complete acetic acid (2 mL) was added to the solution. Ethyl ether (180 mL) was added to the solution to precipitate the product. This solid was filtered and dissolved in water (350 mL). Charcoal (24 g) was added to this solution and heated to 60° C. for one hour. This solution was allowed to cool to ambient temperature and filtered. Evaporation of the filtrate gave the solid product (34 g). 1H-NMR spectroscopy showed this solid to be pure sialyl lactose containing 11% sodium acetate weight by weight.
  • Example 5 Purification of Sialyl Lactose Using Nanofiltration
  • A reaction mixture similar to that described in Example 2 was subjected to filtration using an ultrafiltration membrane having a MWCO of 10 kDa to remove the proteins. The phosphate concentration [PO4 3−], as determined by a standard phosphorus assay procedure described below, was greater than 2.8 mM.
  • The solution was adjusted with concentrated HCl (˜500 ml) to pH=3.0. It was then purified on the Osmonics 213T membrane purification machine (membrane type MX07) at pH=3 for 5 hours until the conductivity of the permeate solution remained unchanged. The solution was then rinsed from the machine and the combined rinse and feed solution treated with NaOH until pH 7.4. The Mn2+ concentration was measured by HPLC, as described below. The nanofiltration parameters were as follows:
    Operation pressure: Pf = 100 psi
    Concentrate Flow Rate: Qc = 5 GPM
    Permeate Flow Rate: Qf = 7 GPH
    Temperature range: 20-40° C.
    Volume: 5 Gallons
  • The conductivity of the initial permeate was 28.1 mS; after 5 hours of recirculation, the conductivity had dropped to 115 μS, the phosphate concentration [PO4 3−] had decreased to 770 μM, and the manganese concentration [Mn2+] was 3.4 mM.
  • The solution was then adjusted to pH 7.4 and further purified on the membrane purification machine (Osmonics, membrane type MX07) for about 1 hour until the conductivity of the permeate solution remained unchanged. The solution was then rinsed out from the membrane machine. The nanofiltration parameters were:
    Operation Pressure: Pf = 100 psi
    Concentrate Flow Rate: Qc = 5 GPM
    Permeate Flow Rate: Qf = 0.3 GPM
    Temperature Range: 20-40° C.
    Volume: 5 Gallon
  • The results of the filtration were as follows:
    Conductivity: initial permeate conductivity: 2.01 mS
    after 5 hours recirculation: 93.7 μS
    Phosphate Concentration: [P04 3−] = 410 μM
    Manganese Concentration: [Mn2+] = 3.0 mM
  • The above solution (6 Gal) was then treated with AG50WX8 (H+) resin (BioRad, 1.18 Kg) and stirred for 2 hours until pH=2.0. The resin was then filtered to provide a very light yellow solution. Only minimal amount of [Mn2+] was detected by HPLC. The solution was then neutralized with NaOH (50% w/w) to a pH of 7.4.
    Before resin [Mn2+] = 3 mM; [PO4 3−] = 410 μM
    treatment:
    After resin pH = 3, [Mn2+] = 1.23 mM; [PO4 3−] = 190 μM
    treatment: pH = 2, [Mn2+] = 6.8 μM;
  • Some small portions of the above solution were treated with AG1X8 (acetate form) resin to further remove the phosphate. The results are shown in Table 3 below:
    TABLE 3
    Sample Weight of Stirring
    Volume (ml) resin (g) Time (hour) [PO4 3−] μM)
    50 ml 0.25 g  1 86
    50 ml 0.5 g 1 41
    50 ml 1.0 g 1 30
    50 ml 2.0 g 1 8
  • The solution was further purified by recirculation of the solution using an Osmonic membrane purification machine (Osmonic MX07) for 5 hours under the following conditions:
    Operation pressure: Pf = 100 psi
    Concentrate Flow Rate: Qc, = 5 GPM
    Permeate Flow Rate: Qf = 0.2 GPM
    Temperature range: 20-40° C.
    Volume: 5 Gallon
    Results were as follows:
    Permeate Conductivity: initial permeate conductivity: 0.136 mS
    after 5 hours' separation: 45 μS
  • The solution was then concentrated to 3-4 L, after which activated charcoal (J. T. Baker, 180 g) was added. The suspension was heated at 55° C. for 2 hours. Charcoal was then removed by filtration to yield a very light yellow solution, which was lyophilized to a white solid.
  • Analysis data for the sialyl lactose solution purified as described above are shown in Table 4.
    TABLE 4
    Assay Result Method
    PO4 3− content 330 ppm (by weight) Phosphate assay1
    Nucleotide/ a)Not detected UV (0.1 mM,
    nucleoside content (ABS280 = 0.0) sialylactose)
    b)Not detected 1H-NMR
    Mn2+ content 80 ppm (by weight) Determined by HPLC2
    Sialyl lactose 71% 1H-NMR (1,2-
    content isopropylidene D-glucose
    furanose was used as a
    standard
    Sialic acid content ˜2% 1H-NMR
    Lactose content Not detectable 1H-NMR
    Acetate content Not detectable 1H-NMR
    N-acetyl glucosamine Not detectable 1H-NMR
    content
    Pyruvate content Not detectable 1H-NMR

    1Phosphate Assay Method
  • The unknown sample (100 μl) was diluted with D.I. water (775 μl). The solution was then treated with 100 μl of acid molybdate (prepared by dissolving 1.25 g of ammonium molybdate in 100 μl of 2.5N H2SO4), 25 μl of Fiska Subha Row Solution (purchased from Sigma as a powder, and prepared according to manufacturer's directions). The mixture was heated at 100° C. for 7 min, the absorption at 810 nm was then recorded. The concentration was determined by comparing the absorption with a phosphate standard curve.
    2HPLC Assay for the determination of Mn2+ concentration:
    Column: Alltech Universal Cation column, 0.46 × 10 cm
    Detector: Alltech model 320 conductivity detector
    Mobile phase: 3 mM phthalic acid, 0.5 mM dipicolinic acid
    Flow rate: 1.5 ml/min
    Column oven 35° C.
    temperature:
  • Example 6 Separation of Organics and Inorganic Salts by Nanofiltration
  • Various nanofiltration membranes were tested for ability to separate various organic compounds and inorganic salts from an aqueous solution. The membranes were tested at two different pHs to demonstrate that by adjusting the ionic charge of certain compounds, the separation profile can be modulated. Results are shown in Table 5.
  • The nanofiltration membranes tested were the MX07, SX12, and B006 produced by Osmonics, Inc. (Minnetonka Minn.) and the DL2540 produced by Osmonics, DeSalination Systems. The MX07 membrane was used as described in Example 5 above. Parameters for the remaining membranes were as shown in Table 6.
    TABLE 5
    Percentage of Compound Passing Through Membrane in 30 Minutes
    Membrane
    MX07a SX12a B006a DL2540a
    Compound pH 7.5 pH 3.0 pH 7.5 pH 3.0 pH 7.5 pH 3.0 pH 7.5 pH 3.0
    Sodium 10 46 20 39 15 64 1.8b
    Phosphate
    Manganese 86 40 40 92 92
    Sodium 35 59 45 65 34 65
    Pyruvate
    GlcNAc 70 28 84 12
    Lactose 36 <5 pass
    Raffinose 0 0 8 52
    Sialic Acid 12 5 <1 1
    Sodium 56
    CMP <1 <1
    PEP <1 8

    Notes:

    aPass % based upon separation time 30 mins.

    bTemp. tested at 20° C. and 40° C.

    GluNAc: N-Acetyl-D-Glucosamine

    PEP: 2-Phosphoenolpyruvate Trisodium Salt

    CMP: Cytidine 5′-monophosphate

    Membranes MX07, SX12, B006 from Osmonics, Inc., DL2540 from Osmonics, Desalination Systems (Escondido, CA).
  • TABLE 6
    SX12 B006 DL2540
    Pressure (Pf) (PSI) 200 100 200
    Concentrate Flow Rate (Qc) 4.5 4 4
    (GPM)
    Permeate Flow Rate (Qf) 0.2 0.5 0.6
    (GPM)
    Temperature Range (° C.) 20-40 20-40 20-40
    Volume (Gal) 5 5 5
  • Example 7 Separation Characteristics of Polybenzamide Nanofiltration Membranes
  • This Example describes experiments which demonstrate that a polybenzamide membrane (YK, Osmonics) is effective for the purification of sugars, in both flat-sheet and spiral-wound forms. The membrane was tested at varying pH levels for the passage or retention of sugars and salts.
  • Materials and Methods
  • A. Flat Sheet and Spiral Wound Machine Operations and Membrane Preparation
  • A Desal membrane machine (Osmonics, Desalination Systems, Escondido, Calif.) with membrane YK was washed thoroughly by first rinsing the machine 4 to 5 times, each with approximately 1 L of distilled water. The water was poured into the feed tank, circulated for about a minute (˜100 psi), and emptied using the drain valve, twisting it counterclockwise to an open position. Thee valve was closed after emptying, and the process was repeated 4 to 5 times. After rinsing, approximately 1 more L of water was added. The system was recirculated at a pressure of 150 psi for 30 min and then was emptied. The system including the membrane was then used in the following experiments.
  • After the completion of each experiment, the machine was washed with water 3 to 4 times as described above. Then, about IL of water was recirculated for about 15-20 minutes at 100-150 psi and emptied from the machine. Occasionally this was followed by an extra brief washing, if some of the test compound was suspected to still remain in the apparatus. The conductivity was always checked to make sure that all the sample was removed. If the conductivity remained high, the machine was washed until the contaminants were virtually undetectable. Most of the ionic compounds were removed easily, with the exception of MnCl2, which only required 1 or 2 extra short washings.
  • B. Testing of Salts
  • To determine the retention characteristics of various salts, 10 mM solutions of the following salts were tested with the flat sheet membranes: MnCl2, NaH2PO4, NaC3H3O3, NaOAc, Na4P2O7, sodium benzoate, MgSO4, NaN3, and NaCl. A 1 L solution of one of the salts was poured into the feed tank and recirculated at 100 psi for about 15 min. At this point, samples of both the permeate and the concentrate were collected and measured using a conductivity meter. The samples were collected every five minutes thereafter, with a total of at least three collections for each sample run. The percentage of salt passing through the membrane (the “percentage pass”) was calculated by dividing the conductivity of the permeate by the conductivity of the concentrate.
  • After the first run was completed, the pH of the solution was then lowered to pH 3.0, when possible, using a conjugate acid of the salt being tested. The solution was recirculated while adjusting the pH to assure that the solution inside the machine was mixed as well. The testing process was repeated, with conductivity of both the permeate and the concentrate being determined. The solution was then brought to a pH of about 7.0 with a conjugate base, and once again the run was repeated at the new pH. Again, conductivity of both the permeate and concentrate was determined.
  • C. Testing of Sugars
  • Sugars that were tested included sialyl lactose, lactose, N-acetyl glucosamine, NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,4Galβ1-OEt (Compound I), Galα1,3Galβ1,4GlcNAcβ-O—(CH2)5—COOH (Compound II), LNT-2, LNnT, CMP, cytidine, and sialic acid. A sugar solution (1 L) was poured into the feed container and recirculated at 100 psi for at least 10 minutes. Samples of the permeate and concentrate were taken at 10 min, and another sample of the permeate was taken at 15 min. The samples were compared visually by TLC. Any pH adjustments that were made were by using HCl and/or NaOH.
  • Results:
  • A. Flat Sheet Membrane
  • The retention characteristics for various salts and sugars of a flat sheet polybenzamide nanofiltration membrane (YK 002 on YV+ paper backing (Osmonics) are shown in Table 7. The experiments were conducted at a temperature of 25-35° C. and a permeate flow rate of 2-8 mL/min.
    TABLE 7
    Pressure % Pass*
    Material Concentration (psi) pH 3.0 pH 5** pH 7
    MnCl2 10 mM 100 66 12 9.8
    NaH2PO4 10 mM 100 82 15 4.6
    NaPyruvate 10 mM 100 80 36 9.8
    NaCl 10 mM 100 18
    Sialyl lactose*** 10 g/L 100 0 0
    Compound I*** 10 g/L 100 0 0
    Compound II*** 2 g/L 100 0 0
    LNT-2*** .4 g/L 100 0 0
    LNnT*** .35 g/L 100 0 0
    Lactose 10 g/L 100 0.0 0.3
    GlcNAc 10 g/L 100 5.9 3.7
    Na4P207 10 mM 100 19 2.0 1.4
    Sialic Acid*** 10 mM 100 0
    Cytidine*** 1 g/L 100 0 trace
    CMP*** 1 g/L 100 0 0
    Benzyl Alcohol*** 1.5% vol 100 100
    NaN3 10 mM 100 81 67
    MgSO4 10 mM 100 38 2.9
    Benzoic acid ˜0.5 g/L 100 99
    Na Benzoate 2.5% 100 42

    *% Pass is the percent ratio of the amount of material in the permeate to the amount of material in the concentrate.

    **“pH 5” ranges from 4.8 to 5.6 “pH 7” ranges from 6.1 to 7.4

    ***Determined visually from TLC
  • B. Spiral Wound Membrane
  • The retention characteristics for various salts and sugars of a spiral wound polybenzamide nanofiltration membrane (YK1812CZA; Osmonics) are shown in Table 8. The experiments were conducted at a temperature of 25-35° C. and a permeate flow rate of 3 mL/sec.
    TABLE 8
    Pressure % Pass*
    Material Concentration (psi) pH 3** pH 5** pH 7**
    MnCl2 10 mM 100 50 40
    (pH
    6.2)
    NaH2PO4 10 mM 100 67 49 19
    NaOAc 10 mM 100 81 65
    NaPyruvate 10 mM 100 81 26
    NaCl 10 mM 100 79 78
    Sialyl lactose*** 10 g/L 100 0 0
    Compound I*** 10 g/L 100 0 0
    Compound II*** 2 g/L 100 0 0
    LNT-2*** 0.4 g/L 100 0 0
    Lactose 10 g/L 100 0.59 2.3
    GlcNAc 10 g/L 100 13 7.1 19
    Na4P2O7 10 mM 100 65 5.2
    Sialic Acid*** 10 mM 100 0 0
    Cytidine*** 1 g/L 100 ˜10 ˜5-10
    CMP*** 1 g/L 100 trace trace
    Sodium Benzoate ˜0.5 g/L 100 93 97

    *% Pass is the percent ratio of the amount of material in the permeate to the amount of material in the concentrate.

    **“pH 5” ranged from 4.5 to 5.2 “pH 7” ranged from 6.6 to 7.0 “pH 3” ranged from 2.8 to 3.4

    ***Determined visually from TLC

    # “trace” is defined as barely detectable by TLC as seen by eye.
  • These results indicate that the YK002 flat sheet membrane and the YK1812CZA spiral wound membrane retained sialyl lactose as well as Compounds I and II, LNT-2, and LNnT, while allowing ionic compounds to pass, making this membrane type a good choice for purification of such saccharides.
  • Example 8 Separation Characteristics of Polyamide Nanofiltration Membranes
  • This Example describes the evaluation of several polyamide membranes for use in the purification of sugars, in both flat-sheet and spiral-wound forms. The membranes were tested at varying pH levels for the passage or retention of sugars and salts.
  • Materials and Methods
  • A. Flat Sheet and Spiral Wound Machine Operations and Membrane Preparation:
  • A Desal membrane machine (Osmonics, Desalination Systems) with a polyamide membrane G-5 (GE; Osmonics) was washed thoroughly by first rinsing the machine 4 to 5 times, each with approximately 1 L of distilled water. The water was poured into the feed tank, circulated for about a minute (˜100 psi), and emptied using the drain valve. The valve was closed after emptying, and the process was repeated 4 to 5 times. After rinsing, approximately one more L of water was added. The system was recirculated at a pressure of 150 psi for 30 min and then was emptied. The system including the membrane was then ready for application testing.
  • After each experiment, the machine was washed with water 3 to 4 times as described above. Then, about 1 L of water was recirculated for about 15-20 minutes at 100-150 psi and the machine was emptied. Occasionally this was followed by an extra brief washing, if some of the compound was suspected to still remain in the apparatus. The conductivity was always checked to make sure that all the sample was removed. If the conductivity remained high, the machine was washed until the contaminants were virtually undetectable. Most of the ionic compounds were removed easily, with the exception of MnCl2, which only required 1 or 2 extra short washings.
  • B. Testing of Salts
  • A 10 mM solution of the following salts were tested with the flat sheet membranes: MnCl2, NaH2PO4, NaC3H3O3, and NaCl. A 1 L solution of one of the salts was poured into the feed tank and recirculated at 100 psi for about 15 min. At this point, samples of both the permeate and the concentrate were collected and measured using a conductivity meter. The samples were collected every five minutes thereafter, with a total of at least three collections for each sample run. The percentage pass was calculated by dividing the conductivity of the permeate by the conductivity of the concentrate. After the run was completed, the pH of the solution was lowered to pH 3.0, when possible, using a conjugate acid of the salt being tested. The solution was recirculated while adjusting the pH to assure that the solution inside the machine was mixed as well. The testing process was repeated, collecting data as before. Then the solution was brought to a pH of about 7.0 with a conjugate base, and once again the run was repeated at the new pH. The machine was then emptied and rinsed as described above.
  • C. Testing of Sugars
  • The sugars that were tested included sialyl lactose, lactose, NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,4Galβ1-OEt (Compound I), Galβ1,3Galβ1,4GlcNAcβ-O—(CH2)5—COOH (Compound II), LNT-2, and LNnT. A sugar solution (1 L) was poured into the feed container and recirculated at 100 psi for at least 10 minutes. Samples of the permeate and concentrate were taken at 10 min, and another sample of the permeate was taken at 15 min. The samples were compared visually by TLC. Any pH adjustments that were made were by using HCl and/or NaOH. After the sugar had been tested, it was transferred into a Pyrex flask to be reused for other membranes.
  • Results
  • A. Flat Sheet Membrane
  • The retention characteristics for various salts and sugars of a flat sheet polyamide nanofiltration membrane (G-10 (GH; Osmonics) are shown in Table 9. The A-value of the membrane was 10.0, and the percent transmission of tap water was 62.8 (tested using 2000 ppm MgSO4 at ambient temperature). The experiments were conducted at a temperature of 25-35° C. and a permeate flow rate of 5-8 mL/min.
    TABLE 9
    Pressure % Pass*
    Material Concentration (psi) pH 3 pH 5** pH 7
    MnCl2 10 mM 200 82.4 82.4 84.6
    NaH2PO4 10 mM 200 33.0 18.0 10.5
    NaPyruvate 10 mM 200 49.4 8.9
    NaCl 10 mM 200 17.8
    Sialyl lactose*** 10 g/L 200 <5 <5
    Compound I*** 10 g/L 200 0
    Compound II*** 2 g/L 200 0
    LNT-2*** 0.4 g/L 200 trace#
    LNnT*** 0.35 g/L 200 trace#
    Lactose 10 g/L 200 2.0 4.2

    *% Pass is the percent ratio of the amount of material in the permeate to the amount of material in the concentrate.

    **“pH 5” ranged from 4.8 to 5.6

    ***Determined visually from TLC

    #“Trace” is defined as barely visible with TLC
  • In another experiment, a G-10 (GH) polyamide membrane with an A-value of 8.0 and a percent transmission of tap water of 38.9 was tested. The experiment was conducted at 25-35° C. and a permeate flow rate of 6-8 mL/min. The results are shown in Table 10.
    TABLE 10
    Pressure % Pass*
    Material Concentration (psi) pH 3 pH 5** pH 7
    MnCl2 10 mM 200 70.8 77.7
    NaH2PO4 10 mM 200 39.4 32.1 16.2
    NaPyruvate 10 mM 200 60.8 21.8
    NaCl 10 mM 200 14.2
    Sialyl lactose*** 10 g/L 200 trace# trace#
    Compound I*** 10 g/L 200 0
    Compound II*** 2 g/L 200 trace#
    LNT-2*** .4 g/L 200 trace#
    LNnT*** 0.35 g/L 200 trace#
    Lactose 10 g/L 200  3.8 22.1

    *% Pass is the percent ratio of the amount of material in the permeate to the amount of material in the concentrate.

    **“pH 5” ranged from 4.8 to 5.6

    ***Determined visually from TLC
  • A G-5 (GE) polyamide membrane (A-value: 3.9, percent transmission of tap water: 33.9) was also tested. The experiment was conducted at 25-35° C. and a permeate flow rate of 3-5 mL/min. Results are shown in Table 11.
    TABLE 11
    Pressure % Pass*
    Material Concentration (psi) pH 3 pH 5** pH 7
    MnCl2 10 mM 200 77.6 80.1 81.8
    NaH2PO4 10 mM 200 30.0  8.6 4.8
    NaPyruvate 10 mM 200 48.2 8.4
    NaCl 10 mM 200 15.0
    Sialyl lactose*** 10 g/L 200 0 0
    Compound I*** 10 g/L 200 0
    Compound II*** 2 g/L 200 0
    LNT-2*** 0.4 g/L 200 0
    LNnT*** 0.35 g/L 200 0
    Lactose 10 g/L 200  6.3 15.1

    *% Pass is the percent ratio of the amount of material in the permeate to the amount of material in the concentrate.

    **“pH 5” ranged from 4.8 to 5.6

    ***Determined visually from TLC
  • The sugar and salt retention characteristics of an HL (Osmonics) polyamide membrane are shown in Table 12. The experiments were carried out at 25-35° C. and a permeate flow rate of 8-13 mL/min.
    TABLE 12
    Pressure % Pass*
    Material Concentration (psi) pH 3 pH 5** pH 7
    MnCl2 10 mM 100 48 22 23
    NaH2PO4 10 mM 100 67 24 7.5
    NaPyruvate 10 mM 100 76 29 16
    NaCl 10 mM 100 71 66
    Sialyl lactose*** 10 g/L 100 0 0
    Lactose 10 g/L 100 1.9 4.1

    *% Pass is the percent ratio of the amount of material in the permeate to the amount of material in the concentrate.

    **“pH 5” ranged from 4.5 to 5.8

    ***Determined visually from TLC
  • B. Spiral Wound Membrane
  • The characteristics of sugar and salt retention on several spiral wound polyamide membranes were also determined. A GH1812CZA membrane (Osmonics) was tested at a temperature of 25-35° C. and a permeate flow rate of 1.5-2 mL/sec. Results are shown in Table 13.
    TABLE 13
    Pressure % Pass*
    Material Concentration (psi) pH 3 pH 5** pH 7
    MnCl2 10 mM 100 93 94
    NaH2PO4 10 mM 100 69 29 19
    NaPyruvate 10 mM 100 68 42
    NaCl 10 mM 100 66 61 64
    Sialyl lactose*** 10 g/L 100 trace# trace#
    Compound I*** 10 g/L 100 0 0
    Compound II*** 2 g/L 100 0 0
    LNT-2*** 0.4 g/L 100 trace# trace#
    Lactose 10 g/L 100 73 34
    GlcNAc 10 g/L 100 48 56
    Na4P2O7 10 mM 100 13 5.7
    Sialic Acid*** 10 mM 100 25-50
    Cytidine*** 1 g/L 100 >50 >50
    CMP*** 1 g/L 100 >50 >50
    Benzoic Acid ˜0.5 g/L 100 90

    *% Pass is the percent ratio of the amount of material in the permeate to the amount of material in the concentrate.

    **“pH 5” ranged from 4.5 to 5.6 “pH 7” ranged from 6.1 to 7.4

    ***Determined visually from TLC

    #“trace” is defined as barely detectable on TLC by eye
  • Results obtained for a GE1812CZA spiral wound polyamide membrane (Osmonics) tested at 25-35° C. and a decreased permeate flow rate of 0.9 mL/sec are shown in Table 14.
    TABLE 14
    Pressure % Pass*
    Material Concentration (psi) pH 3 pH 5** pH 7
    MnCl2 10 mM 100 90 94
    NaH2PO4 10 mM 100 54 14 8.7
    NaOAc 10 mM 100 98 24
    NaPyruvate 10 mM 100 73 45
    NaCl 10 mM 100 54 44
    Sialyl lactose*** 10 g/L 100 0 0
    Compound I*** 10 g/L 100 0 0
    Compound II*** 2 g/L 100 0 0
    Lactose 10 g/L 100 41 43
    GlcNAc 10 g/L 100 72 69
    MgSO4 10 mM 100 50 37
    Na4P2O7 10 mM 100 11 4.7
    Sialic Acid*** 10 mM 100 trace# trace#
    Cytidine*** 1 g/L 100 >50 >50
    CMP*** 1 g/L 100 >50 >50
    Benzoic Acid ˜0.5 g/L 100 63 40

    *% Pass is the percent ratio of the amount of material in the permeate to the amount of material in the concentrate.

    **pH 5″ ranged from 4.8 to 5.6

    ***Determined visually from TLC

    #“Trace” is defined as barely visible with TLC
  • These results demonstrate that the G-10 (GH) (A value=10) and the G-10 (GH) (A value=8) flat sheet membranes and the GH1812CZA spiral wound membrane allowed ions to pass but did not efficiently retain sialyl lactose or similar trisaccharides. The G-5 (GE) (A-value=3.9) flat sheet membrane and the GE1812CZA spiral wound membrane retained sialyl lactose as well as Compounds I and II, LNT-2, and LNnT, while allowing ionic compounds to pass.
  • All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.
  • The above description is illustrative and not restrictive. Many variations of the invention will become apparent to those of skill in the art upon review of this disclosure. Merely by way of example a number of substrates, enzymes, and reaction conditions can be substituted into the glycosyl transferase cycles as part of the present invention without departing from the scope of the invention. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.

Claims (2)

1.-30. (canceled)
31. A method of removing an unsialylated carbohydrate contaminant from a mixture comprising said contaminant and a sialyl galactoside which is a member selected from sialyl lactosides; glycolipids; liposaccharides;
NeuAcα2,3 Galβ1,4(Fucα1,3)GlcNAcβ1,4Gal-OEt;
NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAcβ1,4Galβ1-OEt; and
NeuAcα2,3Galβ1,3-Y, in which Y is a saccharide or an oligosaccharide, said method comprising, contacting said mixture with a nanofiltration or reverse osmosis membrane at a pH of from about 1 to about 7, for a length of time sufficient to allow said unsialylated carbohydrate contaminant to pass through said membrane.
US11/198,839 1996-10-10 2005-08-04 Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration Abandoned US20050269265A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US11/198,839 US20050269265A1 (en) 1996-10-10 2005-08-04 Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US2822696P 1996-10-10 1996-10-10
US08/947,775 US6454946B1 (en) 1996-10-10 1997-10-09 Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration
US10/104,609 US6936173B2 (en) 1996-10-10 2002-03-22 Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration
US11/198,839 US20050269265A1 (en) 1996-10-10 2005-08-04 Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US10/104,609 Continuation US6936173B2 (en) 1996-10-10 2002-03-22 Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration

Publications (1)

Publication Number Publication Date
US20050269265A1 true US20050269265A1 (en) 2005-12-08

Family

ID=21842245

Family Applications (4)

Application Number Title Priority Date Filing Date
US08/947,775 Expired - Fee Related US6454946B1 (en) 1996-10-10 1997-10-09 Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration
US10/104,609 Expired - Lifetime US6936173B2 (en) 1996-10-10 2002-03-22 Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration
US10/184,211 Abandoned US20030029799A1 (en) 1996-10-10 2002-06-27 Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration
US11/198,839 Abandoned US20050269265A1 (en) 1996-10-10 2005-08-04 Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration

Family Applications Before (3)

Application Number Title Priority Date Filing Date
US08/947,775 Expired - Fee Related US6454946B1 (en) 1996-10-10 1997-10-09 Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration
US10/104,609 Expired - Lifetime US6936173B2 (en) 1996-10-10 2002-03-22 Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration
US10/184,211 Abandoned US20030029799A1 (en) 1996-10-10 2002-06-27 Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration

Country Status (13)

Country Link
US (4) US6454946B1 (en)
EP (1) EP0931097B1 (en)
JP (1) JP2001502005A (en)
KR (1) KR100490507B1 (en)
AT (1) ATE304546T1 (en)
AU (1) AU735695B2 (en)
CA (1) CA2268168C (en)
DE (1) DE69734205T2 (en)
DK (1) DK0931097T3 (en)
HU (1) HUP0001634A2 (en)
IL (2) IL129363A (en)
NZ (1) NZ335203A (en)
WO (1) WO1998015581A1 (en)

Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060070953A1 (en) * 2004-08-06 2006-04-06 Luigi Villanova Process for the recovery of tyrosol and hydroxytyrosol from oil mill wastewaters and catalytic oxidation method in order to convert tyrosol in hydroxytyrosol
US20090014386A1 (en) * 2005-10-28 2009-01-15 Danisco A/S Separation Process
WO2008154639A3 (en) * 2007-06-12 2009-12-30 Novo Nordisk A/S Improved process for the production of nucleotide sugars
US20100006495A1 (en) * 2008-07-09 2010-01-14 Eltron Research And Development, Inc. Semipermeable polymers and method for producing same
US7795210B2 (en) 2001-10-10 2010-09-14 Novo Nordisk A/S Protein remodeling methods and proteins/peptides produced by the methods
US7803777B2 (en) 2003-03-14 2010-09-28 Biogenerix Ag Branched water-soluble polymers and their conjugates
US7842661B2 (en) 2003-11-24 2010-11-30 Novo Nordisk A/S Glycopegylated erythropoietin formulations
US7932364B2 (en) 2003-05-09 2011-04-26 Novo Nordisk A/S Compositions and methods for the preparation of human growth hormone glycosylation mutants
US7956032B2 (en) 2003-12-03 2011-06-07 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
US8008252B2 (en) 2001-10-10 2011-08-30 Novo Nordisk A/S Factor VII: remodeling and glycoconjugation of Factor VII
US8053410B2 (en) 2002-06-21 2011-11-08 Novo Nordisk Health Care A/G Pegylated factor VII glycoforms
US8063015B2 (en) 2003-04-09 2011-11-22 Novo Nordisk A/S Glycopegylation methods and proteins/peptides produced by the methods
US8076292B2 (en) 2001-10-10 2011-12-13 Novo Nordisk A/S Factor VIII: remodeling and glycoconjugation of factor VIII
US8207112B2 (en) 2007-08-29 2012-06-26 Biogenerix Ag Liquid formulation of G-CSF conjugate
US8268967B2 (en) 2004-09-10 2012-09-18 Novo Nordisk A/S Glycopegylated interferon α
US8361961B2 (en) 2004-01-08 2013-01-29 Biogenerix Ag O-linked glycosylation of peptides
US8404809B2 (en) 2005-05-25 2013-03-26 Novo Nordisk A/S Glycopegylated factor IX
US8633157B2 (en) 2003-11-24 2014-01-21 Novo Nordisk A/S Glycopegylated erythropoietin
US8632770B2 (en) 2003-12-03 2014-01-21 Novo Nordisk A/S Glycopegylated factor IX
US8716240B2 (en) 2001-10-10 2014-05-06 Novo Nordisk A/S Erythropoietin: remodeling and glycoconjugation of erythropoietin
US8716239B2 (en) 2001-10-10 2014-05-06 Novo Nordisk A/S Granulocyte colony stimulating factor: remodeling and glycoconjugation G-CSF
US8791070B2 (en) 2003-04-09 2014-07-29 Novo Nordisk A/S Glycopegylated factor IX
US8791066B2 (en) 2004-07-13 2014-07-29 Novo Nordisk A/S Branched PEG remodeling and glycosylation of glucagon-like peptide-1 [GLP-1]
US8841439B2 (en) 2005-11-03 2014-09-23 Novo Nordisk A/S Nucleotide sugar purification using membranes
US8911967B2 (en) 2005-08-19 2014-12-16 Novo Nordisk A/S One pot desialylation and glycopegylation of therapeutic peptides
US8916360B2 (en) 2003-11-24 2014-12-23 Novo Nordisk A/S Glycopegylated erythropoietin
US8969532B2 (en) 2006-10-03 2015-03-03 Novo Nordisk A/S Methods for the purification of polypeptide conjugates comprising polyalkylene oxide using hydrophobic interaction chromatography
US9005625B2 (en) 2003-07-25 2015-04-14 Novo Nordisk A/S Antibody toxin conjugates
US9029331B2 (en) 2005-01-10 2015-05-12 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
US9050304B2 (en) 2007-04-03 2015-06-09 Ratiopharm Gmbh Methods of treatment using glycopegylated G-CSF
US9150848B2 (en) 2008-02-27 2015-10-06 Novo Nordisk A/S Conjugated factor VIII molecules
US9187532B2 (en) 2006-07-21 2015-11-17 Novo Nordisk A/S Glycosylation of peptides via O-linked glycosylation sequences
US9187546B2 (en) 2005-04-08 2015-11-17 Novo Nordisk A/S Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
US9200049B2 (en) 2004-10-29 2015-12-01 Novo Nordisk A/S Remodeling and glycopegylation of fibroblast growth factor (FGF)
US11198702B2 (en) 2016-02-04 2021-12-14 Industrial Technology Research Institute Method for separating hydrolyzed product of biomass
US11206851B2 (en) * 2017-01-17 2021-12-28 Zea 10, LLC Process for producing protein concentrate or isolate and cellulosic thermochemical feedstock from brewers spent grains

Families Citing this family (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ335203A (en) * 1996-10-10 2000-10-27 Neose Technologies Inc Carbohydrate, purification using ultrafiltration, reverse osmosis and nanofiltration
EP1903114A3 (en) 1997-12-01 2008-07-23 Neose Technologies, Inc. Enzymatic synthesis of gangliosides
CN1086391C (en) * 1998-12-12 2002-06-19 中国科学院新疆化学研究所 Purification method of oligosaccharide
CA2627846C (en) * 1999-03-23 2011-01-04 Oji Paper Co., Ltd. Process for bleaching lignocellulose pulp
US6942754B2 (en) 1999-03-23 2005-09-13 Oji Paper Co., Ltd. Process for producing xylooligosaccharide from lignocellulose pulp
US6824646B2 (en) 1999-03-23 2004-11-30 Oji Paper Co., Ltd. Process for oxygen bleaching and enzyme treating lignocellulosic pulp with liquid treatment and recovery
US20030075506A1 (en) * 2000-04-14 2003-04-24 Tudhope Bryan R Apparatus and method for isolating and/or eliminating solutes from a solution
US7465398B1 (en) 2000-09-29 2008-12-16 Delta-T Corporation Method for filtering and recovering solids from potato process water
CA2431399C (en) * 2000-12-13 2009-04-28 Institut D'optique Theorique Et Appliquee Method for characterising a surface, and device therefor
US6736903B2 (en) * 2001-04-23 2004-05-18 Sunshine Raisin Corporation Method and apparatus for producing a work product
KR20020085699A (en) * 2001-05-09 2002-11-16 박영규 Rotational plate-styled membrane separation method and apparatus of protein
WO2003031464A2 (en) 2001-10-10 2003-04-17 Neose Technologies, Inc. Remodeling and glycoconjugation of peptides
DK2279753T3 (en) 2001-10-10 2015-11-23 Novo Nordisk As The conversion of peptides and glycokonjugering
US6648978B2 (en) 2001-10-15 2003-11-18 A. E. Staley Manufacturing Co. Membrane filtration for thickening and starch washing in corn wet milling
GB0229423D0 (en) 2002-12-18 2003-01-22 Avecia Ltd Process
AU2004220087A1 (en) 2003-03-06 2004-09-23 Seneb Biosciences, Inc. Methods and compositions for the enzymatic synthesis of gangliosides
JP2005147788A (en) * 2003-11-13 2005-06-09 Inst Nuclear Energy Research Rocaec Boric acid purification and reutilization system, and method, using reverse osmosis separation method
US8075780B2 (en) * 2003-11-24 2011-12-13 Millipore Corporation Purification and concentration of synthetic biological molecules
US7404926B2 (en) * 2003-12-30 2008-07-29 Rhoades Frank G Water treatment system
FR2848877B1 (en) * 2004-01-28 2012-04-20 Applexion Ste Nouvelle De Rech S Et D Applic Ind D Echangeurs D Ions Applexion PROCESS FOR THE NANOFILTRATION PURIFICATION OF A SUGAR-AQUEOUS SOLUTION CONTAINING MONOVALENT AND VERSATILE ANIONS AND CATIONS
NZ548893A (en) 2004-02-04 2009-09-25 Biogernerix Ag Methods of refolding mammalian glycosyltransferases
US20050287515A1 (en) * 2004-06-24 2005-12-29 Reinhold Deppisch Removal of bacterial DNA from therapeutic fluid formulations
EP1869184B1 (en) 2005-04-11 2011-09-28 National Research Council Of Canada Identification of a beta-1,3-n-acetylgalactosaminyltransferase (cgte) from campylobacter jejuni lio87
US8771991B2 (en) 2005-08-11 2014-07-08 National Research Council Of Canada SOAT polypeptide reaction mixture
DK1951060T3 (en) * 2005-11-04 2018-01-29 Arla Foods Amba CONCENTRATE derived from a milk product enriched with naturally occurring sialyl lactose and a process for its preparation
PT2068907T (en) 2006-10-04 2018-01-15 Novo Nordisk As Glycerol linked pegylated sugars and glycopeptides
KR100888513B1 (en) * 2006-12-15 2009-03-12 주식회사 진켐 Novel N-Acetylglucosamine-2-Epimerase and Method for Producing CMP-neuraminic acid Using the Same
DK176760B1 (en) 2007-10-03 2009-06-29 Arla Foods Amba Process for producing lactose-free milk
KR101768561B1 (en) * 2008-12-09 2017-08-16 도레이 카부시키가이샤 Method for producing sugar liquid
US20120035120A1 (en) 2009-03-25 2012-02-09 Seneb Biosciences, Inc. Glycolipids as treatment for disease
JP5641598B2 (en) * 2009-09-29 2014-12-17 雪印メグミルク株式会社 Separation method of sialyl lactose material
DE102010053749B4 (en) * 2010-12-08 2015-02-19 Airbus Defence and Space GmbH Device for identifying biotic particles
MY182178A (en) * 2011-09-01 2021-01-18 Chugai Pharmaceutical Co Ltd Method for preparing a composition comprising highly concentrated antibodies by ultrafiltration
EP2596852A1 (en) * 2011-11-28 2013-05-29 Annikki GmbH Method for the regeneration of an aqueous solution containing lignin
CN102532208B (en) * 2012-01-06 2014-10-29 南京工业大学 Method for continuously separating sialic acid
ITMI20121184A1 (en) * 2012-07-05 2014-01-06 Altergon Italia Srl PROCESS FOR INDUSTRIAL PRODUCTION OF SODIUM IALURONATE (HANA) HIGHLY PURIFIED WITH CONTROLLED MOLECULAR WEIGHT
EP2857410A1 (en) 2013-10-04 2015-04-08 Jennewein Biotechnologie GmbH Process for purification of 2´-fucosyllactose using simulated moving bed chromatography
DK2896628T3 (en) 2014-01-20 2019-01-14 Jennewein Biotechnologie Gmbh Process for Effective Purification of Neutral Milk Oligosaccharides (HMOs) from Microbial Fermentation
DK3169696T3 (en) 2014-07-18 2019-08-05 Corbion Biotech Inc PROCEDURE FOR EXTRACTION OF SOLUBLE PROTEINS FROM MICROALGE BIOMASS
GB201414213D0 (en) * 2014-08-11 2014-09-24 Imp Innovations Ltd Filtration process
US10899782B2 (en) 2016-03-07 2021-01-26 Glycom A/S Separation of oligosaccharides from fermentation broth
WO2017182965A1 (en) * 2016-04-19 2017-10-26 Glycom A/S Separation of oligosaccharides from fermentation broth
US11160298B1 (en) * 2016-05-20 2021-11-02 Thermolife International, Llc Methods of producing decolorized beet products and compositions produced therefrom
WO2018020473A1 (en) 2016-07-28 2018-02-01 Fonterra Co-Operative Group Limited Dairy product and process
WO2019003135A1 (en) * 2017-06-30 2019-01-03 Glycom A/S Purification of oligosaccharides
EP3450443A1 (en) * 2017-08-29 2019-03-06 Jennewein Biotechnologie GmbH Process for purifying sialylated oligosaccharides
WO2019209559A1 (en) 2018-04-23 2019-10-31 Danisco Us Inc Synthesis of glucan comprising beta-1,3 glycosidic linkages with phosphorylase enzymes
WO2019215073A1 (en) * 2018-05-07 2019-11-14 Jennewein Biotechnologie Gmbh A SIMPLE METHOD FOR THE PURIFICATION OF LACTO-N-NEOTETRAOSE (LNnT) FROM CARBOHYDRATES OBTAINED BY MICROBIAL FERMENTATION
CN112203519B (en) 2018-06-01 2023-09-15 科汉森母乳低聚糖股份有限公司 Simple method for purifying sialyllactose
EP3686210A1 (en) * 2019-01-24 2020-07-29 DuPont Nutrition Biosciences ApS Process for purifying a human milk oligosaccharide and related compositions
KR20230151745A (en) 2022-04-26 2023-11-02 (주)에코샤인 Ultrafiltration separation and purification equipment capable of continuous production

Citations (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4230463A (en) * 1977-09-13 1980-10-28 Monsanto Company Multicomponent membranes for gas separations
US4259183A (en) * 1978-11-07 1981-03-31 Midwest Research Institute Reverse osmosis membrane
US4562021A (en) * 1983-09-06 1985-12-31 Ceraver, S.A. Method of manufacturing a medium for microfiltration, for ultrafiltration, or for reverse osmosis
US4692354A (en) * 1986-08-12 1987-09-08 Masashi Asaeda Method for preparing ceramic membrane for separation of condensed component
US4741823A (en) * 1986-12-23 1988-05-03 Osmonics, Inc. Flow control manifold for cross-flow membrane system
US4806244A (en) * 1986-07-15 1989-02-21 The Dow Chemical Company Combined membrane and sorption process for selective ion removal
US4839037A (en) * 1987-03-09 1989-06-13 Osmonics, Inc. Tapered, spirally wound filter cartridge and method of making same
US4956458A (en) * 1988-05-13 1990-09-11 Warner-Lambert Company Purification of polydextrose by reverse osmosis
US5164374A (en) * 1990-12-17 1992-11-17 Monsanto Company Use of oligosaccharides for treatment of arthritis
US5233033A (en) * 1990-09-04 1993-08-03 Taiyo Kagaku Co., Ltd. Method for production of sialic acid
US5254174A (en) * 1989-09-22 1993-10-19 Danisco A/S Method for preparing a mixture of saccharides
US5330975A (en) * 1989-02-07 1994-07-19 Snow Brand Milk Products Co., Ltd. Bacterial toxin neutralizer
US5352670A (en) * 1991-06-10 1994-10-04 Alberta Research Council Methods for the enzymatic synthesis of alpha-sialylated oligosaccharide glycosides
US5403604A (en) * 1991-10-15 1995-04-04 The Nutrasweet Company Sugar separation from juices and product thereof
US5454952A (en) * 1990-11-09 1995-10-03 Applied Membrand Systems Pty Ltd. Method and apparatus for fractionation of sugar containing solution
US5501797A (en) * 1993-08-30 1996-03-26 Holland Sweetener Company V.O.F. Process for recovery of raw materials in the aspartame preparation process
US5676980A (en) * 1995-09-29 1997-10-14 Continental General Tire, Inc. Center split segmented mold for curing pneumatic tires
US5874541A (en) * 1992-08-21 1999-02-23 Vrije Universiteit Immunoglobulins devoid of light chains
US6030815A (en) * 1995-04-11 2000-02-29 Neose Technologies, Inc. Enzymatic synthesis of oligosaccharides
US6288222B1 (en) * 2000-02-16 2001-09-11 Neose Technologies, Inc. Method of filtration of a dairy stream
US6454946B1 (en) * 1996-10-10 2002-09-24 Neose Technologies, Inc. Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03133387A (en) * 1989-10-19 1991-06-06 Nichiden Kagaku Kk Production of maltooligosaccharide
JP2802654B2 (en) * 1989-10-30 1998-09-24 雪印乳業株式会社 A method for recovering oligosaccharide-bound sialic acids from alkaline washing waste liquid of anion exchange resin generated during desalting of whey
CA2038485A1 (en) * 1990-03-23 1991-09-24 Donald K. Hadden Nanofiltration process for making dextrose
JPH04108395A (en) * 1990-08-28 1992-04-09 Higeta Shoyu Kk Production of galactosamino-oligosaccharide
JPH05168490A (en) * 1991-12-20 1993-07-02 Fushimi Seiyakushiyo:Kk Production of physilogically active pectin oligomer and its use
JPH0686684A (en) * 1992-05-26 1994-03-29 Monsanto Co Synthesis of sialo conjugation
US5374541A (en) 1993-05-04 1994-12-20 The Scripps Research Institute Combined use of β-galactosidase and sialyltransferase coupled with in situ regeneration of CMP-sialic acid for one pot synthesis of oligosaccharides
CN1129953A (en) 1993-07-15 1996-08-28 尼奥斯药品公司 Synthesis of saccharic composition
AU8129394A (en) 1993-10-28 1995-05-22 Valeriy S. Maisotsenko Method of determining working media motion and designing flow structures for same
WO1996008459A1 (en) 1994-09-12 1996-03-21 Aeci Limited Process for recovering citric acid
US5728554A (en) * 1995-04-11 1998-03-17 Cytel Corporation Enzymatic synthesis of glycosidic linkages
US5876980A (en) 1995-04-11 1999-03-02 Cytel Corporation Enzymatic synthesis of oligosaccharides

Patent Citations (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4230463A (en) * 1977-09-13 1980-10-28 Monsanto Company Multicomponent membranes for gas separations
US4259183A (en) * 1978-11-07 1981-03-31 Midwest Research Institute Reverse osmosis membrane
US4562021A (en) * 1983-09-06 1985-12-31 Ceraver, S.A. Method of manufacturing a medium for microfiltration, for ultrafiltration, or for reverse osmosis
US4806244A (en) * 1986-07-15 1989-02-21 The Dow Chemical Company Combined membrane and sorption process for selective ion removal
US4692354A (en) * 1986-08-12 1987-09-08 Masashi Asaeda Method for preparing ceramic membrane for separation of condensed component
US4741823A (en) * 1986-12-23 1988-05-03 Osmonics, Inc. Flow control manifold for cross-flow membrane system
US4839037A (en) * 1987-03-09 1989-06-13 Osmonics, Inc. Tapered, spirally wound filter cartridge and method of making same
US4956458A (en) * 1988-05-13 1990-09-11 Warner-Lambert Company Purification of polydextrose by reverse osmosis
US5330975A (en) * 1989-02-07 1994-07-19 Snow Brand Milk Products Co., Ltd. Bacterial toxin neutralizer
US5254174A (en) * 1989-09-22 1993-10-19 Danisco A/S Method for preparing a mixture of saccharides
US5233033A (en) * 1990-09-04 1993-08-03 Taiyo Kagaku Co., Ltd. Method for production of sialic acid
US5454952A (en) * 1990-11-09 1995-10-03 Applied Membrand Systems Pty Ltd. Method and apparatus for fractionation of sugar containing solution
US5164374A (en) * 1990-12-17 1992-11-17 Monsanto Company Use of oligosaccharides for treatment of arthritis
US5352670A (en) * 1991-06-10 1994-10-04 Alberta Research Council Methods for the enzymatic synthesis of alpha-sialylated oligosaccharide glycosides
US5403604A (en) * 1991-10-15 1995-04-04 The Nutrasweet Company Sugar separation from juices and product thereof
US5874541A (en) * 1992-08-21 1999-02-23 Vrije Universiteit Immunoglobulins devoid of light chains
US5501797A (en) * 1993-08-30 1996-03-26 Holland Sweetener Company V.O.F. Process for recovery of raw materials in the aspartame preparation process
US6030815A (en) * 1995-04-11 2000-02-29 Neose Technologies, Inc. Enzymatic synthesis of oligosaccharides
US5676980A (en) * 1995-09-29 1997-10-14 Continental General Tire, Inc. Center split segmented mold for curing pneumatic tires
US6454946B1 (en) * 1996-10-10 2002-09-24 Neose Technologies, Inc. Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration
US6936173B2 (en) * 1996-10-10 2005-08-30 Neose Technologies, Inc. Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration
US6288222B1 (en) * 2000-02-16 2001-09-11 Neose Technologies, Inc. Method of filtration of a dairy stream

Cited By (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8716240B2 (en) 2001-10-10 2014-05-06 Novo Nordisk A/S Erythropoietin: remodeling and glycoconjugation of erythropoietin
US8008252B2 (en) 2001-10-10 2011-08-30 Novo Nordisk A/S Factor VII: remodeling and glycoconjugation of Factor VII
US8076292B2 (en) 2001-10-10 2011-12-13 Novo Nordisk A/S Factor VIII: remodeling and glycoconjugation of factor VIII
US8716239B2 (en) 2001-10-10 2014-05-06 Novo Nordisk A/S Granulocyte colony stimulating factor: remodeling and glycoconjugation G-CSF
US7795210B2 (en) 2001-10-10 2010-09-14 Novo Nordisk A/S Protein remodeling methods and proteins/peptides produced by the methods
US8053410B2 (en) 2002-06-21 2011-11-08 Novo Nordisk Health Care A/G Pegylated factor VII glycoforms
US8247381B2 (en) 2003-03-14 2012-08-21 Biogenerix Ag Branched water-soluble polymers and their conjugates
US7803777B2 (en) 2003-03-14 2010-09-28 Biogenerix Ag Branched water-soluble polymers and their conjugates
US8791070B2 (en) 2003-04-09 2014-07-29 Novo Nordisk A/S Glycopegylated factor IX
US8853161B2 (en) 2003-04-09 2014-10-07 Novo Nordisk A/S Glycopegylation methods and proteins/peptides produced by the methods
US8063015B2 (en) 2003-04-09 2011-11-22 Novo Nordisk A/S Glycopegylation methods and proteins/peptides produced by the methods
US7932364B2 (en) 2003-05-09 2011-04-26 Novo Nordisk A/S Compositions and methods for the preparation of human growth hormone glycosylation mutants
US9005625B2 (en) 2003-07-25 2015-04-14 Novo Nordisk A/S Antibody toxin conjugates
US8916360B2 (en) 2003-11-24 2014-12-23 Novo Nordisk A/S Glycopegylated erythropoietin
US7842661B2 (en) 2003-11-24 2010-11-30 Novo Nordisk A/S Glycopegylated erythropoietin formulations
US8633157B2 (en) 2003-11-24 2014-01-21 Novo Nordisk A/S Glycopegylated erythropoietin
US8632770B2 (en) 2003-12-03 2014-01-21 Novo Nordisk A/S Glycopegylated factor IX
US7956032B2 (en) 2003-12-03 2011-06-07 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
US8361961B2 (en) 2004-01-08 2013-01-29 Biogenerix Ag O-linked glycosylation of peptides
US8791066B2 (en) 2004-07-13 2014-07-29 Novo Nordisk A/S Branched PEG remodeling and glycosylation of glucagon-like peptide-1 [GLP-1]
US20060070953A1 (en) * 2004-08-06 2006-04-06 Luigi Villanova Process for the recovery of tyrosol and hydroxytyrosol from oil mill wastewaters and catalytic oxidation method in order to convert tyrosol in hydroxytyrosol
US7427358B2 (en) * 2004-08-06 2008-09-23 Lachifarma S.R.L. Laboratorio Chimico Farmaceutico Salentino Process for the recovery of tyrosol and hydroxytyrosol from oil mill wastewaters and catalytic oxidation method in order to convert tyrosol in hydroxytyrosol
US8268967B2 (en) 2004-09-10 2012-09-18 Novo Nordisk A/S Glycopegylated interferon α
US9200049B2 (en) 2004-10-29 2015-12-01 Novo Nordisk A/S Remodeling and glycopegylation of fibroblast growth factor (FGF)
US10874714B2 (en) 2004-10-29 2020-12-29 89Bio Ltd. Method of treating fibroblast growth factor 21 (FGF-21) deficiency
US9029331B2 (en) 2005-01-10 2015-05-12 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
US9187546B2 (en) 2005-04-08 2015-11-17 Novo Nordisk A/S Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
US8404809B2 (en) 2005-05-25 2013-03-26 Novo Nordisk A/S Glycopegylated factor IX
US8911967B2 (en) 2005-08-19 2014-12-16 Novo Nordisk A/S One pot desialylation and glycopegylation of therapeutic peptides
US20090014386A1 (en) * 2005-10-28 2009-01-15 Danisco A/S Separation Process
US8613858B2 (en) * 2005-10-28 2013-12-24 Dupont Nutrition Biosciences Aps Separation process
US8841439B2 (en) 2005-11-03 2014-09-23 Novo Nordisk A/S Nucleotide sugar purification using membranes
US9187532B2 (en) 2006-07-21 2015-11-17 Novo Nordisk A/S Glycosylation of peptides via O-linked glycosylation sequences
US8969532B2 (en) 2006-10-03 2015-03-03 Novo Nordisk A/S Methods for the purification of polypeptide conjugates comprising polyalkylene oxide using hydrophobic interaction chromatography
US9050304B2 (en) 2007-04-03 2015-06-09 Ratiopharm Gmbh Methods of treatment using glycopegylated G-CSF
WO2008154639A3 (en) * 2007-06-12 2009-12-30 Novo Nordisk A/S Improved process for the production of nucleotide sugars
US9493499B2 (en) 2007-06-12 2016-11-15 Novo Nordisk A/S Process for the production of purified cytidinemonophosphate-sialic acid-polyalkylene oxide (CMP-SA-PEG) as modified nucleotide sugars via anion exchange chromatography
US8207112B2 (en) 2007-08-29 2012-06-26 Biogenerix Ag Liquid formulation of G-CSF conjugate
US9150848B2 (en) 2008-02-27 2015-10-06 Novo Nordisk A/S Conjugated factor VIII molecules
US20100006495A1 (en) * 2008-07-09 2010-01-14 Eltron Research And Development, Inc. Semipermeable polymers and method for producing same
US8147735B2 (en) 2008-07-09 2012-04-03 Eltron Research & Development, Inc. Semipermeable polymers and method for producing same
US11198702B2 (en) 2016-02-04 2021-12-14 Industrial Technology Research Institute Method for separating hydrolyzed product of biomass
US11206851B2 (en) * 2017-01-17 2021-12-28 Zea 10, LLC Process for producing protein concentrate or isolate and cellulosic thermochemical feedstock from brewers spent grains

Also Published As

Publication number Publication date
JP2001502005A (en) 2001-02-13
EP0931097A4 (en) 2000-01-12
KR20000049057A (en) 2000-07-25
DE69734205D1 (en) 2005-10-20
US20020148791A1 (en) 2002-10-17
IL149275A (en) 2007-07-04
DE69734205T2 (en) 2006-07-06
HUP0001634A2 (en) 2000-09-28
KR100490507B1 (en) 2005-05-19
EP0931097A1 (en) 1999-07-28
US20030029799A1 (en) 2003-02-13
IL129363A (en) 2003-02-12
WO1998015581A1 (en) 1998-04-16
NZ335203A (en) 2000-10-27
EP0931097B1 (en) 2005-09-14
CA2268168A1 (en) 1998-04-16
ATE304546T1 (en) 2005-09-15
AU735695B2 (en) 2001-07-12
AU5081698A (en) 1998-05-05
IL129363A0 (en) 2000-02-17
US6936173B2 (en) 2005-08-30
US6454946B1 (en) 2002-09-24
DK0931097T3 (en) 2006-01-16
CA2268168C (en) 2008-04-29

Similar Documents

Publication Publication Date Title
US6936173B2 (en) Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration
US20090048440A1 (en) Nucleotide Sugar Purification Using Membranes
JP5876649B2 (en) Improved process for producing nucleotide sugars
US6030815A (en) Enzymatic synthesis of oligosaccharides
EP3714059A1 (en) Process for the purification of l-fucose from a fermentation broth
US20220340942A1 (en) Separation of neutral oligosaccharides from fermentation broth
CN1242776A (en) Carbohybrate purification using ultrafiltration, reverse osmosis and nanofiltration
MXPA99003318A (en) Carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration
WO2022263426A1 (en) Separation of human milk oligosaccharides from a fermentation broth
BE1029436B1 (en) SEPARATION OF BREAST MILK OLIGOSACCHARIDES FROM A FERMENTATION BROTH
DK181124B1 (en) Separation of neutral human milk oligosaccharides from a fermentation broth
WO2023242184A1 (en) Separation of human milk oligosaccharides from a fermentation broth
DK202200566A1 (en) Separation of human milk oligosaccharides from a fermentation broth
JP2024500676A (en) Biomass removal by centrifugation
WO2022263406A1 (en) Separation of human milk oligosaccharides from a fermentation broth
BE1029435A1 (en) SEPARATION OF BREAST MILK OLIGOSACCHARIDES FROM A FERMENTATION BROTH
WO2022072333A1 (en) Process for purifying a human milk oligosaccharide and related compositions
Morelli Pressure Driven Membrane Technology for Food and Biotechnology Industry
JPH0377896A (en) Preparation of highly pure oligosaccharide from saccharide mixture

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION