U.S. patent application number 11/198839 was filed with the patent office on 2005-12-08 for carbohydrate purification using ultrafiltration, reverse osmosis and nanofiltration.
This patent application is currently assigned to Neose Technologies. Invention is credited to DeFrees, Shawn.
Application Number | 20050269265 11/198839 |
Document ID | / |
Family ID | 21842245 |
Filed Date | 2005-12-08 |
United States Patent
Application |
20050269265 |
Kind Code |
A1 |
DeFrees, Shawn |
December 8, 2005 |
Carbohydrate purification using ultrafiltration, reverse osmosis
and nanofiltration
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.
Inventors: |
DeFrees, Shawn; (North
Wales, PA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS LLP (SF)
2 PALO ALTO SQUARE
PALO ALTO
CA
94306
US
|
Assignee: |
Neose Technologies
Horsham
PA
|
Family ID: |
21842245 |
Appl. No.: |
11/198839 |
Filed: |
August 4, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11198839 |
Aug 4, 2005 |
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10104609 |
Mar 22, 2002 |
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6936173 |
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10104609 |
Mar 22, 2002 |
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08947775 |
Oct 9, 1997 |
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6454946 |
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60028226 |
Oct 10, 1996 |
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Current U.S.
Class: |
210/639 ; 127/34;
210/651; 210/652 |
Current CPC
Class: |
B01D 61/04 20130101;
B01D 61/027 20130101; B01D 2311/04 20130101; C13K 13/00 20130101;
C08B 37/0003 20130101; B01D 61/022 20130101; Y02P 20/582 20151101;
C13B 20/165 20130101; B01D 61/025 20130101; B01D 2311/04 20130101;
B01D 2311/18 20130101; B01D 2311/04 20130101; B01D 2311/12
20130101; B01D 2311/18 20130101 |
Class at
Publication: |
210/639 ;
210/651; 210/652; 127/034 |
International
Class: |
B01D 061/16 |
Claims
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; NeuAc.alpha.2,3
Gal.beta.1,4(Fuc.alpha.1,3)GlcNAc.beta.1,4Gal-OEt;
NeuAc.alpha.2,3Gal.beta.1,4(Fuc.alpha.1,3)GlcNAc.beta.1,4Gal.beta.1-OEt;
and NeuAc.alpha.2,3Gal.beta.1,3-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.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] 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
[0002] 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
[0003] 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-.alpha.(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 Lewis.sup.x and sialyl Lewis.sup.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.
[0004] 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.
[0005] 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
[0006] 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.
[0007] 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.
[0008] U.S. Pat. No. 5,254,174 describes the use of chromatography
and/or nanofiltration to purify inulide compounds of formula
GF.sub.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.
[0009] 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.
[0010] 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
[0011] 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.beta.1,3Gal.beta.1,4Glc (LNT-2),
NeuAc.alpha.(2.fwdarw.3)Gal.beta.(1.fwdarw.4)(Fuc.alpha.1.fwdarw.3)Glc(R.-
sup.1).beta.1-OR.sup.2, wherein R.sup.1 is OH or NAc; R.sup.2 is a
hydrogen, an alkoxy, a saccharide, an oligosaccharide or an aglycon
group having at least one carbon atom; and
Gal.alpha.(.fwdarw.13)Gal.beta.(1.fw-
darw.4)Glc(R.sup.1).beta.-O--R.sup.3, wherein R.sup.1 is OH or NAc;
R.sup.3 is --(CH.sub.2).sub.n--COX, with X.dbd.OH, OR.sup.4,
--NHNH.sub.2, R.sup.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.
[0012] Also provided are methods for purifying carbohydrate
compounds having a formula
NeuAc.alpha.(2.fwdarw.3)Gal.beta.(1.fwdarw.4)GlcN(R.sup.-
1).beta.-OR.sup.2,
NeuAc.alpha.(2.fwdarw.3)Gal.beta.(1.fwdarw.4)GlcN(R.sup-
.1).beta.(1.fwdarw.3)Gal.beta.-OR.sup.2,
NeuAc.alpha.(2.fwdarw.3)Gal.beta.- (1.fwdarw.4)
(Fuc.alpha.1.fwdarw.3)GlcN(R.sup.1).beta.-OR.sup.2, or
NeuAc.alpha.(2.fwdarw.3)Gal.beta.(1.fwdarw.4)
(Fuc.alpha.1.fwdarw.3)GlcN(-
R.sup.1).beta.(1.fwdarw.3)Gal.beta.-OR.sup.2, wherein R.sup.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.sup.2 is a hydrogen, a saccharide, an oligosaccharide or an
aglycon group having at least one carbon atom.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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
[0017] The following abbreviations are used herein:
[0018] Ara=arabinosyl;
[0019] Fru=fructosyl;
[0020] Fuc=fucosyl;
[0021] Gal=galactosyl;
[0022] GalNAc=N-acetylgalacto;
[0023] Glc=glucosyl;
[0024] GlcNAc=N-acetylgluco;
[0025] Man=mannosyl; and
[0026] NeuAc=sialyl (N-acetylneuraminyl).
[0027] The term "carbohydrate" encompasses chemical compounds
having the general formula (CH.sub.2O).sub.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.
[0028] 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.
[0029] 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 (.alpha. or
.beta.), 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.fwdarw.3, or
(2,3).
[0030] 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.
[0031] 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.
[0032] 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
[0033] 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, SLe.sup.x,
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.
[0034] 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.
[0035] 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 (R.sub.i) is given by the
formula R.sub.i=(1-C.sub.ip/C.sub.ir).times.100%, wherein C.sub.ip
is the concentration of component i in the permeate and C.sub.ir 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.
[0036] 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).
[0037] 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).
[0038] 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.
[0039] 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 (--NH.sub.3.sup.+)
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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.).
[0044] 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.
[0045] 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 (P.sub.c) 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 (P.sub.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.degree. C. are
typical, with temperatures between about 20 and 40.degree. 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.
[0046] 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.
[0047] 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.degree., might be useful.
Also, one can use prefilters (100-200 .mu.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.
[0048] 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 Mn.sup.2+, an acidic ion
exchange resin can be used, such as AG50WX8 (H.sup.+) (BioRad).
[0049] 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 .alpha.2,3)sialyltransferase referred to as
.alpha.(2,3)sialtransferase (EC 2.4.99.6) transfers sialic acid to
the non-reducing terminal Gal of a Gal.beta.1.fwdarw.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 .alpha.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-.beta.-1,4-GlcNAc
.alpha.-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.beta.1.fwdarw.3GalNAc, lacto-N-tetraose,
Gal.beta.1.fwdarw.3GlcNAc, Gal.beta.1.fwdarw.3Ara,
Gal.beta.1.fwdarw.6GlcNAc, Gal.beta.1.fwdarw.4Glc (lactose),
Gal.beta.1.fwdarw.4Glc.beta.1-OCH.sub.2CH.sub.3,
Gal.beta.1.fwdarw.4Glc.b- eta.1-OCH.sub.2CH.sub.2CH.sub.3,
Gal.beta.1.fwdarw.4Glc.beta.1-OCH.sub.2C.- sub.6H.sub.5,
Gal.beta.1.fwdarw.4GlcNAc, Gal.beta.1-OCH.sub.3, 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-galac- to-2-nonulosonic
acid; NeuAc, and sometimes also abbreviated AcNeu or NANA), but
also 9-substituted sialic acids such as a 9-O--C.sub.1-C.sub.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.
[0050] 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 .alpha.(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.
[0051] 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.
[0052] 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
.alpha.(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 .beta.(1,4)
galactosyltransferase (E.C. No. 2.4.1.38).
[0053] 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,
Gal.alpha.(1.fwdarw.4)Gal.beta.(1.fwdarw.4)Glc,
GlcNAc.beta.(1,3)Gal.beta.(1,4)Glc,
Gal.beta.(1.fwdarw.4)GlcNAc.beta.(1.f-
wdarw.3)Gal.beta.(1.fwdarw.4) Glc, and
GalNAc.beta.(1.fwdarw.3)Gal.beta.(1-
.fwdarw.4)GlcNAc.beta.(1.fwdarw.3) Gal.beta.(1.fwdarw.4)Glc, among
many others.
[0054] 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:
NeuAc.alpha.(2.fwdarw.3)Gal.beta.(1.fwdarw.4)GlcN(R').beta.-OR
or
NeuAc.alpha.(2.fwdarw.3)Gal.beta.(1.fwdarw.4)GlcN(R').beta.(1.fwdarw.3)Gal-
.beta.-OR
[0055] 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
C.sub.1-C.sub.6, 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,
--NH.sub.2, R.sup.1, --N(R.sup.1).sub.2, --CO.sub.2H,
--CO.sub.2R.sup.1, --CONH.sub.2, --CONHR.sup.1,
--CON(R.sup.1).sub.2, --CONHNH.sub.2, or --OR.sup.1 wherein each
R.sup.1 is independently alkyl of from 1 to 5 carbon atoms. In
addition, R can be 1
[0056] where n,m,o=1-18; (CH.sub.2).sub.n--R.sup.2 (in which
n=0-18), wherein R.sup.2 is a variously substituted aromatic ring,
preferably, a phenyl group, being substituted with one or more
alkoxy groups, preferably methoxy or O(CH.sub.2).sub.mCH.sub.3, (in
which m=0-18), or a combination thereof. R can also be
3-(3,4,5-trimethoxyphenyl)propyl.
[0057] 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:
R.sup.1Gal.beta.1,m(Fuc.alpha.1,n)GlcNR.sup.0(R.sup.2).sub.p--
[0058] in which R.sup.0 is (C.sub.1-C.sub.8 alkyl)carbonyl,
(C.sub.1-C.sub.8 alkoxy)carbonyl, or (C.sub.2-C.sub.9
alkenyloxy)carbonyl, R.sup.1 is an oligosaccharide or a group
having the formula 2
[0059] R.sup.3 and R.sup.4 may be the same or different and may be
H, C.sub.1-C.sub.8 alkyl, hydroxy-(C.sub.1-C.sub.8 alkyl),
aryl-(C.sub.1-C.sub.8 alkyl), or (C.sub.1-C.sub.8
alkoxy)-(C.sub.1-C.sub.- 8 alkyl), substituted or unsubstituted.
R.sup.2 may be H, C.sub.1-C.sub.8 alkyl, hydroxy-(C.sub.1-C.sub.8
alkyl), aryl-(C.sub.1-C.sub.8 alkyl), (C.sub.1-C.sub.8 alkyl)-aryl,
alkylthio, .alpha.1,2Man, .alpha.1,6GalNAc,
.beta.1,3Gal.beta.1,4Glc, .alpha.1,2Man-R.sup.8,
.alpha.1,6GalNAc--R.sup.- 8, and .beta.1,3Gal-R.sup.8. R.sup.8 may
be H, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkoxy,
hydroxy-(C.sub.1-C.sub.8 alkyl), aryl-(C.sub.1-C.sub.8 alkyl),
(C.sub.1-C.sub.8 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.
[0060] The substituted groups mentioned above may be substituted by
hydroxy, hydroxy(C.sub.1-C.sub.4 alkyl),
polyhydroxy(C.sub.1-C.sub.4 alkyl), alkanoamido, or
hydroxyalknoamido substituents. Preferred substituents include
hydroxy, polyhydroxy(C.sub.3 alkyl), acetamido and
hydroxyacetamido. A substituted radical may have more than one
substitution, which may be the same or different.
[0061] For embodiments in which R.sup.1 is an oligosaccharide, the
oligosaccharide is preferably a trisaccharide. Preferred
trisaccharides include NeuAc.alpha.2,3Gal.beta.1,4GlcNAc.beta.1,3
or NeuGc.alpha.2,3Gal.beta.1,4GlcNAc.beta.1,3.
[0062] For embodiments in which R.sup.1 is the group having the
formula 3
[0063] R.sup.3 and R.sup.4 preferably form a single radical having
the formula
--R.sup.5-- or --(R.sup.6).sub.q--O--(R.sup.7).sub.r--
[0064] in which R.sup.5 is C.sub.3-C.sub.7 divalent alkyl,
substituted or unsubstituted, R.sup.6 and R.sup.7 are the same or
different and are C.sub.1-C.sub.6 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.
[0065] A more preferred structure for a single radical formed by
R.sup.3 and R.sup.4 is one having the formula
--(R.sup.6)--O--
[0066] in which R.sup.6 is C.sub.3-C.sub.4 divalent alkyl,
substituted or unsubstituted. For instance, R.sup.6 may have the
formula --CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--, preferably
substituted. The radical can be substituted with hydroxy,
polyhydroxy(C.sub.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 .alpha.2,3
to the Gal residue.
[0067] 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
.beta.1,4 and Fuc is linked .alpha.1,3 to GlcNAc. This formula
includes the SLe.sup.x tetrasaccharide. SLe.sup.x has the formula
NeuAc.alpha.2,3Gal.beta.1,4(Fu- c.alpha.1,3)GlcNAc.beta.1--. This
structure is selectively recognized by LECCAM-bearing cells.
SLe.sup.x compounds that can be purified using the methods of the
invention include NeuAc.alpha.2,3Gal.beta.1,4(Fuc.alpha.1,-
3)GlcNAc.beta.1-Gal-OEt,
NeuAc.alpha.2,3Gal.beta.1,4(Fuc.alpha.1,3)GlcNAc.-
beta.1,4Gal.beta.1-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 4
[0068] wherein Z is hydrogen, C.sub.1-C.sub.6 acyl or 5
[0069] Y is selected from the group consisting of C(O), SO.sub.2,
HNC(O), OC(O) and SC(O);
[0070] R.sup.1 is selected from the group consisting of an aryl, a
substituted aryl and a phenyl C.sub.1-C.sub.3 alkylene group,
wherein said aryl substitutent is selected from the group
consisting of a halo, trifuloromethyl, nitro, C.sub.1-C.sub.18
alkyl, C.sub.1-C.sub.18 alkoxy, amino, mono-C.sub.1-C.sub.18
alkylamino, di-C.sub.1-C.sub.18 alkylamino, benzylamino,
C.sub.1-C.sub.18 alkylbenzylamino, C.sub.1-C.sub.18 thioaklyl and
C.sub.1-C.sub.18 alkyl carboxamido groups, or
[0071] R.sup.1Y is allyloxycarbonyl or chloroacetyl;
[0072] R.sup.2 is selected from the group consisting of
monosaccharide (including .beta.1,3Gal-OR, where R.dbd.H, alkyl,
aryl or acyl), disaccharide, hydrogen, C.sub.1-C.sub.18 straight
chain, branched chain or cyclic hydrocarbyl, C.sub.1-C.sub.6 alkyl,
3-(3,4,5-trimethoxyphenyl)p- ropyl, C.sub.1-C.sub.5 alkylene
.omega.-carboxylate, .omega.-trisubstituted silyl C.sub.2-C.sub.4
alkylene wherein said .omega.-trisubstituted silyl is a silyl group
having three substituents independently selected from the group
consisting of C.sub.1-C.sub.4 alkyl, phenyl,
[0073] or OR.sup.2 together form a C.sub.1-C.sub.18 straight chain,
branched chain or cyclic hydrocarbyl carbamate;
[0074] R.sup.3 is hydrogen or C.sub.1-C.sub.6 acyl;
[0075] R.sup.4 is hydrogen, C.sub.1-C.sub.6 alkyl or benzyl;
[0076] R.sup.5 is selected from the group consisting of hydrogen,
benzyl, methoxybenzyl, dimethoxybenzyl and C.sub.1-C.sub.6
acyl;
[0077] R.sup.7 is methyl or hydroxymethyl; and
[0078] X is selected from the group consisting of C.sub.1-C.sub.6
acyloxy, C.sub.2-C.sub.6 hydroxylacyloxy, hydroxy, halo and
azido.
[0079] A related set of structures included in the general formula
are those in which Gal is linked .beta.1,3 and Fuc is linked
.alpha.1,4. For instance, the tetrasaccharide,
NeuAc.alpha.2,3Gal.beta.1,3(Fuc.alpha.1,4)- GlcNAc.beta.1-, termed
here SLe.sup.a, 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 SLe.sup.a.
[0080] The methods of the invention are also useful for purifying
oligosaccharide compounds having the general formula
Gal.alpha.1,3Gal--, including
Gal.alpha.1,3Gal.beta.1,4Glc(R).beta.--O--R.sup.1, wherein R.sup.1
is --(CH.sub.2).sub.n--COX, with X.dbd.OH, OR.sup.2, --NHNH.sub.2,
R.dbd.OH or NAc, and R.sup.2 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
Gal.alpha.1,3Gal.beta.1,4Glc- NAc.beta.-O--(CH.sub.2).sub.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), GlcNAc.beta.1,3Gal.beta.1,4Glc
(LNT-2), sialyl(.alpha.2,3)-lactose, and
sialyl(.alpha.2,6)-lactose.
[0081] 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.
[0082] 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.
[0083] The term "alkoxy" refers to alkyl radicals attached to the
remainder of the molecule by an oxygen, e.g., ethoxy, methoxy, or
n-propoxy.
[0084] The term "alkylthio" refers to alkyl radicals attached to
the remainder of the molecule by a sulfur.
[0085] An "alkanoamido" radical has the general formula
--NH--CO--(C.sub.1-C.sub.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.sub.3, and hydroxyacetamido,
--NH--CO--CH.sub.2--OH.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] The following examples are offered solely for the purposes
of illustration, and are intended neither to limit nor to define
the invention.
EXAMPLES
[0090] 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
[0091] 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 (.about.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 (.about.200 ml). Sialic acid was then
produced by aldol condensation of ManNAc.
[0092] 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.degree. C. for 7 days. HPLC analysis on an Aminex HPX87H
(BioRad) column (0.004 M H.sub.2SO.sub.4, 0.8 ml/min, monitor
A.sub.220) 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
[0093] 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.sub.2 was added
and the final volume adjusted to 2.2 L with water. The reaction was
carried out at room temperature.
[0094] The reaction was monitored daily by thin layer
chromatography (TLC) and [Mn.sup.2+] was determined by ion
chromatography. Additions/adjustments were made as shown in Table
1:
1TABLE 1 Day 2 44 ml 1M MnCl.sub.2 added Day 4 43 ml 1M MnCl.sub.2
added Day 6 added 34.3 ml 1M MnCl.sub.2, 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 MnCl.sub.2 Day 8 24.6 ml 1M MnCl.sub.2 Day 9 44 ml 1M
MnCl.sub.2 Day 10 30.8 ml 1M MnCl.sub.2 Day 11 31.7 ml 1M
MnCl.sub.2 Day 12 24.6 ml 1M MnCl.sub.2, 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 MnCl.sub.2, 19,800 U
pyruvate kinase, 13,200 U myokinase Day 17 26 g phosphenolpyruvate,
trisodium salt
[0095] The sialyl lactose yield was approximately 70-80% as
determined by TLC.
Example 3
Synthesis of Sialyl Lactose Using Sialyltransferase Cycle
[0096] This example illustrates the production of
.alpha.-N-acetylneuramin- ic acid(2,3).beta.-galactosyl(1,4)glucose
using the sialyl transferase cycle with control of the manganese
ion concentration.
[0097] 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), .alpha.2,3 sialyltransferase (663
units) and MnCl.sub.2.degree.4H.sub.2O (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 .alpha.2,3 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.
2TABLE 2 Amount Supplemented [Mn.sup.++] Loss of Mn.sup.++ (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
[0098] On day 9, the reaction was essentially complete by tlc. As
the results in the table indicate, the depletion of Mn.sup.++
resulted in additional amounts of MnC.sub.2.degree.4H.sub.2O 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
[0099] 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-.alpha.-
-D-glycero-D-galacto-nonulopyranosylonate-(2-3)-O-.beta.-D-galactopyranosy-
l-(1-4)-O-.beta.-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 .sup.1Hmr
spectroscopy. Otherwise the syrup was so evaporated twice with
pyridine (2.times.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.
[0100] 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.degree. 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).
.sup.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
[0101] 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.sub.4.sup.3-], as determined by a standard
phosphorus assay procedure described below, was greater than 2.8
mM.
[0102] The solution was adjusted with concentrated HCl (.about.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 Mn.sup.2+
concentration was measured by HPLC, as described below. The
nanofiltration parameters were as follows:
3 Operation pressure: P.sub.f = 100 psi Concentrate Flow Rate:
Q.sub.c = 5 GPM Permeate Flow Rate: Q.sub.f = 7 GPH Temperature
range: 20-40.degree. C. Volume: 5 Gallons
[0103] The conductivity of the initial permeate was 28.1 mS; after
5 hours of recirculation, the conductivity had dropped to 115
.mu.S, the phosphate concentration [PO.sub.4.sup.3-] had decreased
to 770 .mu.M, and the manganese concentration [Mn.sup.2+] was 3.4
mM.
[0104] 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:
4 Operation Pressure: P.sub.f = 100 psi Concentrate Flow Rate:
Q.sub.c = 5 GPM Permeate Flow Rate: Q.sub.f = 0.3 GPM Temperature
Range: 20-40.degree. C. Volume: 5 Gallon
[0105] The results of the filtration were as follows:
5 Conductivity: initial permeate conductivity: 2.01 mS after 5
hours recirculation: 93.7 .mu.S Phosphate Concentration:
[P0.sub.4.sup.3-] = 410 .mu.M Manganese Concentration: [Mn.sup.2+]
= 3.0 mM
[0106] The above solution (6 Gal) was then treated with AG50WX8
(H.sup.+) 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.
6 Before resin [Mn.sup.2+] = 3 mM; [PO.sub.4.sup.3-] = 410 .mu.M
treatment: After resin pH = 3, [Mn.sup.2+] = 1.23 mM;
[PO.sub.4.sup.3-] = 190 .mu.M treatment: pH = 2, [Mn.sup.2+] = 6.8
.mu.M;
[0107] 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:
7 TABLE 3 Sample Weight of Stirring Volume (ml) resin (g) Time
(hour) [PO.sub.4.sup.3-] .mu.M) 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
[0108] 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:
8 Operation pressure: P.sub.f = 100 psi Concentrate Flow Rate:
Q.sub.c, = 5 GPM Permeate Flow Rate: Q.sub.f = 0.2 GPM Temperature
range: 20-40.degree. C. Volume: 5 Gallon Results were as follows:
Permeate Conductivity: initial permeate conductivity: 0.136 mS
after 5 hours' separation: 45 .mu.S
[0109] 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.degree. C. for 2 hours. Charcoal was then removed
by filtration to yield a very light yellow solution, which was
lyophilized to a white solid.
[0110] Analysis data for the sialyl lactose solution purified as
described above are shown in Table 4.
9TABLE 4 Assay Result Method PO.sub.4.sup.3- content 330 ppm (by
weight) Phosphate assay.sup.1 Nucleotide/ a)Not detected UV (0.1
mM, nucleoside content (ABS.sub.280 = 0.0) sialylactose) b)Not
detected .sup.1H-NMR Mn.sup.2+ content 80 ppm (by weight)
Determined by HPLC.sup.2 Sialyl lactose 71% .sup.1H-NMR (1,2-
content isopropylidene D-glucose furanose was used as a standard
Sialic acid content .about.2% .sup.1H-NMR Lactose content Not
detectable .sup.1H-NMR Acetate content Not detectable .sup.1H-NMR
N-acetyl glucosamine Not detectable .sup.1H-NMR content Pyruvate
content Not detectable .sup.1H-NMR .sup.1Phosphate Assay Method
[0111] The unknown sample (100 .mu.l) was diluted with D.I. water
(775 .mu.l). The solution was then treated with 100 .mu.l of acid
molybdate (prepared by dissolving 1.25 g of ammonium molybdate in
100 .mu.l of 2.5N H.sub.2SO.sub.4), 25 .mu.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.degree. 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.
10 .sup.2HPLC Assay for the determination of Mn.sup.2+
concentration: Column: Alltech Universal Cation column, 0.46
.times. 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.degree. C. temperature:
Example 6
Separation of Organics and Inorganic Salts by Nanofiltration
[0112] 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.
[0113] 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.
11TABLE 5 Percentage of Compound Passing Through Membrane in 30
Minutes Membrane MX07.sup.a SX12.sup.a B006.sup.a DL2540.sup.a
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 1.8.sup.b 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: .sup.aPass % based upon
separation time 30 mins. .sup.bTemp. tested at 20.degree. C. and
40.degree. 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).
[0114]
12 TABLE 6 SX12 B006 DL2540 Pressure (P.sub.f) (PSI) 200 100 200
Concentrate Flow Rate (Q.sub.c) 4.5 4 4 (GPM) Permeate Flow Rate
(Q.sub.f) 0.2 0.5 0.6 (GPM) Temperature Range (.degree. C.) 20-40
20-40 20-40 Volume (Gal) 5 5 5
Example 7
Separation Characteristics of Polybenzamide Nanofiltration
Membranes
[0115] 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.
[0116] Materials and Methods
[0117] A. Flat Sheet and Spiral Wound Machine Operations and
Membrane Preparation
[0118] 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 (.about.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.
[0119] 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 MnCl.sub.2, which only required 1 or
2 extra short washings.
[0120] B. Testing of Salts
[0121] To determine the retention characteristics of various salts,
10 mM solutions of the following salts were tested with the flat
sheet membranes: MnCl.sub.2, NaH.sub.2PO.sub.4,
NaC.sub.3H.sub.3O.sub.3, NaOAc, Na.sub.4P.sub.2O.sub.7, sodium
benzoate, MgSO.sub.4, NaN.sub.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. 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.
[0122] 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.
[0123] C. Testing of Sugars
[0124] Sugars that were tested included sialyl lactose, lactose,
N-acetyl glucosamine,
NeuAc.alpha.2,3Gal.beta.1,4(Fuc.alpha.1,3)GlcNAc.beta.1,4Gal-
.beta.1-OEt (Compound I),
Gal.alpha.1,3Gal.beta.1,4GlcNAc.beta.-O--(CH.sub- .2).sub.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.
[0125] Results:
[0126] A. Flat Sheet Membrane
[0127] 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.degree. C. and a permeate
flow rate of 2-8 mL/min.
13 TABLE 7 Pressure % Pass* Material Concentration (psi) pH 3.0 pH
5** pH 7 MnCl.sub.2 10 mM 100 66 12 9.8 NaH.sub.2PO.sub.4 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 Na.sub.4P.sub.20.sub.7 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 NaN.sub.3
10 mM 100 81 -- 67 MgSO.sub.4 10 mM 100 38 -- 2.9 Benzoic acid
.about.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
[0128] B. Spiral Wound Membrane
[0129] 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.degree. C. and a permeate flow rate of 3
mL/sec.
14 TABLE 8 Pressure % Pass* Material Concentration (psi) pH 3** pH
5** pH 7** MnCl.sub.2 10 mM 100 50 -- 40 (pH 6.2) NaH.sub.2PO.sub.4
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 Na.sub.4P.sub.2O.sub.7 10 mM 100 65 -- 5.2
Sialic Acid*** 10 mM 100 0 -- 0 Cytidine*** 1 g/L 100 .about.10 --
.about.5-10 CMP*** 1 g/L 100 trace -- trace Sodium Benzoate
.about.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 .sup.# "trace" is defined as barely detectable by TLC as seen
by eye.
[0130] 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
[0131] 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.
[0132] Materials and Methods
[0133] A. Flat Sheet and Spiral Wound Machine Operations and
Membrane Preparation:
[0134] 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 (.about.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.
[0135] 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 MnCl.sub.2, which only required 1 or 2 extra short
washings.
[0136] B. Testing of Salts
[0137] A 10 mM solution of the following salts were tested with the
flat sheet membranes: MnCl.sub.2, NaH.sub.2PO.sub.4,
NaC.sub.3H.sub.3O.sub.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. 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.
[0138] C. Testing of Sugars
[0139] The sugars that were tested included sialyl lactose,
lactose,
NeuAc.alpha.2,3Gal.beta.1,4(Fuc.alpha.1,3)GlcNAc.beta.1,4Gal.beta.1-OEt
(Compound I),
Gal.beta.1,3Gal.beta.1,4GlcNAc.beta.-O--(CH.sub.2).sub.5--C- OOH
(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.
[0140] Results
[0141] A. Flat Sheet Membrane
[0142] 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 MgSO.sub.4 at ambient temperature). The experiments
were conducted at a temperature of 25-35.degree. C. and a permeate
flow rate of 5-8 mL/min.
15 TABLE 9 Pressure % Pass* Material Concentration (psi) pH 3 pH
5** pH 7 MnCl.sub.2 10 mM 200 82.4 82.4 84.6 NaH.sub.2PO.sub.4 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.sup.# LNnT*** 0.35 g/L 200 -- --
trace.sup.# 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 .sup.#"Trace" is defined as barely
visible with TLC
[0143] 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.degree. C. and a
permeate flow rate of 6-8 mL/min. The results are shown in Table
10.
16 TABLE 10 Pressure % Pass* Material Concentration (psi) pH 3 pH
5** pH 7 MnCl.sub.2 10 mM 200 70.8 -- 77.7 NaH.sub.2PO.sub.4 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.sup.# -- trace.sup.#
Compound I*** 10 g/L 200 -- -- 0 Compound II*** 2 g/L 200
trace.sup.# -- -- LNT-2*** .4 g/L 200 -- -- trace.sup.# LNnT***
0.35 g/L 200 -- -- trace.sup.# 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
[0144] 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.degree. C. and a permeate flow rate of 3-5
mL/min. Results are shown in Table 11.
17 TABLE 11 Pressure % Pass* Material Concentration (psi) pH 3 pH
5** pH 7 MnCl.sub.2 10 mM 200 77.6 80.1 81.8 NaH.sub.2PO.sub.4 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
[0145] 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.degree. C. and a permeate
flow rate of 8-13 mL/min.
18 TABLE 12 Pressure % Pass* Material Concentration (psi) pH 3 pH
5** pH 7 MnCl.sub.2 10 mM 100 48 22 23 NaH.sub.2PO.sub.4 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
[0146] B. Spiral Wound Membrane
[0147] 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.degree. C.
and a permeate flow rate of 1.5-2 mL/sec. Results are shown in
Table 13.
19 TABLE 13 Pressure % Pass* Material Concentration (psi) pH 3 pH
5** pH 7 MnCl.sub.2 10 mM 100 93 94 -- NaH.sub.2PO.sub.4 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.sup.# -- trace.sup.# Compound
I*** 10 g/L 100 0 -- 0 Compound II*** 2 g/L 100 0 -- 0 LNT-2*** 0.4
g/L 100 trace.sup.# -- trace.sup.# Lactose 10 g/L 100 73 -- 34
GlcNAc 10 g/L 100 48 -- 56 Na.sub.4P.sub.2O.sub.7 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
.about.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 .sup.#"trace" is defined as
barely detectable on TLC by eye
[0148] Results obtained for a GE1812CZA spiral wound polyamide
membrane (Osmonics) tested at 25-35.degree. C. and a decreased
permeate flow rate of 0.9 mL/sec are shown in Table 14.
20 TABLE 14 Pressure % Pass* Material Concentration (psi) pH 3 pH
5** pH 7 MnCl.sub.2 10 mM 100 90 94 -- NaH.sub.2PO.sub.4 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 MgSO.sub.4
10 mM 100 50 37 -- Na.sub.4P.sub.2O.sub.7 10 mM 100 11 -- 4.7
Sialic Acid*** 10 mM 100 trace.sup.# -- trace.sup.# Cytidine*** 1
g/L 100 >50 -- >50 CMP*** 1 g/L 100 >50 -- >50 Benzoic
Acid .about.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 .sup.#"Trace" is defined as barely visible with
TLC
[0149] 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.
[0150] 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.
[0151] 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.
* * * * *