Carbohydrate purification using ultrafiltration, reverse...

Details Carbohydrate purification using ultrafiltration, reverse... Carbohydrate purification using ultrafiltration, reverse...

1997-10-09

2002-09-24

Wilson, James O. (Department: 1623)

Liquid purification or separation

Processes

Liquid/liquid solvent or colloidal extraction or diffusing...

C210S651000, C210S652000, C210S649000, C127S034000, C127S042000, C127S053000

Reexamination Certificate

active

06454946

ABSTRACT:

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. 5,164,374). Recently, sialyl-&agr;(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 Bio World Today, p. 5, Apr. 4, 1995. As another example, compounds comprising the sialyl Lewis ligands, sialyl Lewis
x
and sialyl Lewis
a
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 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 trough 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 GlcNAc&bgr;1,3Gal&bgr;1,4Glc (LNT-2), NeuAc&agr;(2→3)Gal&bgr;(1→4)(Fuc
&agr;
1→3)Glc(R
1
) &bgr;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&agr;(1→3)Gal&bgr;(1&Dgr;4)Glc(R
1
) &bgr;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 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 NeuAc&agr;(2→3)Gal&bgr;(1→4)GlcN(R
1
)&bgr;—OR
2
, NeuAc&agr;(2→3)Gal&bgr;(1→4)GlcN(R
1
)&bgr;(1→3)Gal&bgr;—OR
2
, NeuAc&agr;(2→3)Gal&bgr;(1→4) (Fuc
&agr;
1→3)GlcN(R
1
)&bgr;—OR
2
, or NeuAc&agr;(2→3)Gal&bgr;(1→4) (Fuc&agr;1→3)GlcN(R
1
)&bgr;(1→3)Gal&bgr;—OR
2
, wherein R
1
is alkyl or acyl from 1-18 carbons, 5,6,7,8-tetrahydro-2-naphthamido; benzamido; 2-naphthamido; 4-aminobenzamido; or 4-nitrobenzamido, and R
2
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 throug

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