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Title:
PREPARATION OF SUGAR-NUCLEOTIDES
Document Type and Number:
WIPO Patent Application WO/2018/115309
Kind Code:
A1
Abstract:
The present invention relates to methods for preparation of glyco-conjugated proteins and in particular to processes for preparation of sugar-nucleotides and modified sugar-nucleotides.

Inventors:
AMANN FRANZ (CH)
Application Number:
EP2017/084141
Publication Date:
June 28, 2018
Filing Date:
December 21, 2017
Export Citation:
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Assignee:
NOVO NORDISK AS (DK)
CARBOGEN AMCIS AG (CH)
International Classes:
C07H1/06; C07H19/02; C07H19/10
Domestic Patent References:
WO2008154639A22008-12-18
WO2007056191A22007-05-18
WO2006127910A22006-11-30
WO2008154639A22008-12-18
WO2008060780A22008-05-22
Foreign References:
US20120083600A12012-04-05
US20120083600A12012-04-05
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Claims:
A method for purifying or preparing a sugar-nucleotide using an anion exchange media.

The method according to claim 1 , wherein the sugar-nucleotide is obtained from a protected sugar-nucleotide.

The method according to claim 1 or claim 2, where in the sugar-nucleotide consists of a sugar moiety and a nucleotide moiety.

The method according to claim 3, where in the sugar moiety is a sialic acid, such as SA- Glycine

The method according to any of the previous claims 3-4, where in the nucleotide moiety comprises a mono-phosphate moiety, such as CMP.

The method according to any of the previous claims, where in the sugar-nucleotide is CMP-SA-Glycine (GSC).

The method according to any of the previous claims, where in the protected sugar- nucleotide is N-protected GSC.

The method according to any of the previous claims, wherein the anion exchange media is a strong anion exchange resin with quaternary ammonium groups.

The method according to any of the previous claims 1-8, comprising the steps of

a. providing a protected sugar-nucleotide,

b. immobilizing the protected sugar-nucleotide using an anion exchange media c. optionally releasing the protected sugar-nucleotide from the media

d. removing the protection group from the protected sugar-nucleotide and thereby e. obtaining the sugar-nucleotide and

f. optionally releasing the sugar-nucleotide from the media.

10. The method according to any of the previous claims 1-8, comprising the steps of

a. providing protected sugar-nucleotide b. removing the protection group from the protected sugar-nucleotide and thereby c. obtaining a sugar-nucleotide,

d. immobilizing the sugar-nucleotide using an anion exchange media and e. optionally releasing the sugar-nucleotide from the media.

1 1 . A method for preparing a modified sugar-nucleotide comprising the steps of

a. providing a property modifying agent

b. reacting said modifying agent with a sugar-nucleotide obtained according to any of the claims 1-10

c. obtaining a modified sugar-nucleotide.

12. A method for preparing a modified glyco-protein comprising the steps of:

a. providing a glyco-protein

b. reacting said glyco-protein with a modified sugar-nucleotide obtained according to claim 1 1 and thereby

c. obtaining a modified glyco-protein.

13. An immobilized form of a protected sugar-nucleotide or a sugar-nucleotide.

14. An immobilized protected sugar-nucleotide or an immobilized sugar-nucleotide.

15. A salt comprising an anion exchange media and a protected sugar-nucleotide or a sugar- nucleotide.

Description:
PREPARATION OF SUGAR-NUCLEOTIDES

TECHNICAL FIELD OF THE INVENTION

The invention relates to a method for preparing sugar-nucleotide molecules. Such molecules are useful as intermediates in the preparation of modified proteins.

BACKGROUND

The production of protracted therapeutic products is of great interest in the medical industry. A specific example of such technology is glyco-conjugation, such as described in WO2008154639, US2012083600 and WO2008060780 wherein the preparation of 5-N- modified CMP-NAN, such as pegylated CMP-SA (also termed CMP-SA- PEG, PEG-GSC and PSC) is described.

A critical step in the preparation of glyco-conjugates is the production of the intermediate Cytidine 5'-monophospho-/V-glycyl-neuraminic acid or Glycyl Sialic acid Cytidine monophosphate (GSC) to which the protractor is linked for subsequent attachment to the glycan group of the target protein.

In US2012083600 the GSC intermediate is obtained by removal of an Fmoc group using dimethylamine, followed by a filtering step to remove a precipitate and subsequently freeze dried to yield a white powder of CMP-glycyl-sialic acid (GSC).

Much interest and effort have a been used to provide efficient methods for producing and purification of the modified CMP-NAN (CMP-SA-PEG ) used in the glyco-pegylation of the protein of interest, while less emphasis has be put into the production of the limiting GSC intermediate.

It is of general interest in the industry to optimize processes used in the production of medical compounds. Certain intermediates, that are used as starting material for subsequent process steps are preferably prepared by an efficient process leading to a product suitable for storage and later use.

SUMMARY

The present invention relates to a method for preparing sugar-nucleotides using an anion exchange media. The association of sugar-nucleotides to an anion exchange media is here referred to as immobilization. And the invention thus relates to immobilization of sugar- nucleotides or precursor molecules of sugar-nucleotides, such as protected sugar- nucleotides. The methods described herein are useful in the preparation of sugar-nucleotides which are intermediate products used in the preparation of modified glyco-proteins as the sugar of the sugar-nucleotide forms the link between the glycan of the protein and the modifying group. Prior to the glyco-conjugation step the sugar-nucleotide is itself modified by linkage of the modifying group to the sugar-nucleotide. In order for this step to be efficient the purity and reactivity of the sugar-nucleotide employed is critical. The present invention allows preparation of such sugar-nucleotide where any protection group is removed and the resulting sugar-nucleotide is highly pure. The finding that the sugar-nucleotide (and the precursor, the protected sugar-nucleotide) can be efficiently captured by an anion exchange media also allows for storage of the intermediates. In one embodiment the invention relates to a process for preparation of Cytidine 5'- monophospho-/V-glycyl-neuraminic acid (GSC) used in the preparation of glyco-conjugated products. The inventors have demonstrated that Cytidine 5'-monophospho-/V-glycyl- neuraminic acid (GSC) can be efficiently prepared using a resin and that both the precursor, the N-protected GSC and GSC can be stored in an immobilised form (Example 3).

This enables the inventors to produce the intermediate (GSC) with increased purity and stability and simplify subsequent use of the intermediate as no or only modest additional purification steps are required. The GSC produced may subsequently be used in the preparation of modified GSC, such as PEG-GSC which in turn can be use in the preparation of modified glyco-proteins, such as pegylated glyco-proteins.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 shows the overall synthesis of a PEG-glycyl-sialic acid cytidine-5 ' -monophosphate (PSC) exemplified using a branched 40K PEG. Cytidine 5'-monophospho-N-glycyl- neuraminic acid (GSC) is reacted with a para-nitrophenol linked PEG (PEG-NP) yielding PEG-glycyl-sialic acid cytidine-5 ' -monophosphate.

Fig. 2 shows the process of generating Cytidine 5'-monophospho-/V-glycyl-neuraminic acid (GSC) starting from D-mannosamine. The last step (step 3) is removal of the protection group from the N-protected GSC, wherein the protecting group is Fmoc yielding GSC.

Fig.3 shows the structure of GSC detached from the resin as characterised by nuclear magnetic resonance (NMR) including proton and carbon chemical shifts. Fig.4 illustrates the process of example 4, leading to the production of GSC immobilized on the resin. The intermediate /V-protected GSC is captured on the resin in step 2 and the protection group removed in step 3 providing resin bound GSC. DESCRIPTION

The present invention relates to process improvements useful in the preparation of compounds including a modified sugar-nucleotide. The invention relates to the finding that intermediates used in the synthesis of modified sugar-nucleotides, exemplified by CMP-SA- PEG used in the preparation of glyco-PEGylated proteins, may be efficiently bound to an anion exchange media. This enables production of the GSC intermediate in a highly efficient manner both with respect to yield and purity. It furthermore enables a practical "holding step" as the GSC intermediate can be stored in immobilized form bound to the anion resin for later use.

The inventors have found that sugars-nucleotides both in protected and de- protected form can be bound to an anion exchange media and furthermore that the de- protection step can be performed while the sugar-nucleotides are associated with the anion exchange media.

The present invention in an aspect relates to a method for preparing a sugar- nucleotide from protected sugar-nucleotide using a resin as will be described in further details here below.

Sugar moiety

According to the invention the sugar moiety of the sugar-nucleotide can be any glycosyl moiety or glycosyl-mimetic moiety including mono- and oligo-saccharides. Almost all sugars have the formula C n H2nO n (n is between 3 and 7). Both glucose and fructose has the molecular formula C 6 H 12 0 6 while they differ in structure as glucoseusually forms a 6 member ring (pyranose) and fructose usually a 5 member ring structure (furanose).

In one embodiment the sugar moiety comprise a monosaccharide, such as a 5- or 6- member ring, comprising one oxygen atom and 4 and 5 carbons, respectively.

Exemplary sugar moieties include glucose, mannose, fructose, galactose, sialic acid (SA), /V-Acetylglucosamine (GlcNAc) or /V-Acetylgalactosamin (GalNAc).

The family of sialic acid includes a series of nine-carbon sugars acids including neuramic acid and N- or O-substituted derivatives thereof. In most situations the 5-amino group comprises an acetyl or a glycolyl group while hydroxyl substituents may vary.

In one embodiment the sugar moiety is a sialic acid. In one embodiment the sialic acid is /V-acetyl-neuraminic acid (2-keto-5-acetamido- 3,5-dideoxy-D-glycero-D-galactononulopyranos-1 -onic acid (often abbreviated as Neu5Ac, NeuAc, NAN or NANA).

In a further embodiment the sialic acid is /V-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which the /V-acetyl group of NeuAc is hydroxylated.

In yet a further embodiment the sialic acid is 2-keto-3-deoxy-nonulosonic acid (KDN). Further included are 9-substituted sialic acids such as a 9-0-C1-C6 acyl-Neu5Ac like 9-0-lactyl-Neu5Ac or 9-0-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-0- azido-9-deoxy- Neu5Ac.

A sialic acid sugar moiety can be prepared from D-mannosamine, Fmoc protected glycine -succinimidyl ester and pyruvate as described in example 3 Step 1 .

Substituted sugar

The sugar moiety may be described as a substituted sugar where the 5 or 6 member ring is substituted at one or more sites.

In one embodiment the linkage to the property modifying group, optionally via a linker, occurs via R 3 , R 4 , R 5 , R 5 or R 6 . In a further embodiment the linkage to the property modifying group, is via R , R or R b .

In a preferred embodiment the sugar moiety is of formula I wherein

R 1 provides the linkage to the nucleotide,

R 2 is -H, -CH 2 OH, -COOH or -OH, R 3 represent -H or-OH,

R 4 represent -H or-OH,

R 5 provides the linkage to the property modifying group optionally via a linker and R 6 and R 6 ' independently represent -H, unsubstituted alkyl, -OR 9 , NHC(0)R'° wherein d is 0 or 1 and R 9 and R lo are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl or sialic acid.

In one embodiment linkage to the nucleotide moiety is through -O- or -N- and R 1 is thus -O- or -N-.

In a preferred embodiment the linkage is directly to an -O- of a phosphate group of the nucleotide providing a -0-P(0)(0-)(0-Nucleotide) group linked to C-1 in the R 1 position of formula I.

In an exemplary embodiment, R6 and R6', together with the carbon to which they are attached constitutes the side chain of the carbohydrate.

In one embodiment the side chain attached at C-5 is -CH(-OH)-CH(-OH)-CH 2 -OH .

In one embodiment R 1 provides a negative charge. The negative charge may be provided by a group that is deprotonated in water forming the conjugate base, such as O- P(0)(OH)(OR) that becomes - " , 0-P(0)(0-)(OR).

In one embodiment R 2 provides a negative charge, such as a group that is deprotonated in water forming the conjugate base, such as -COOH that becomes COO-. Substituent R5 at C-4 provides the linkage to the property modifying group. Linkage to a property modifying group may be obtained by various routes. A frequently used technology involves a primary amine (sugar moiety) and a carbonyl derivative (property modifying group) which can be reacted to form an amide bond.

In an embodiment C-4 is substituted with a linker moiety comprising an amine. In one embodiment R5 of formula I comprises a linker moiety comprising a primary amine.

In one embodiment R5 is - NH-C(=0)-CH 2 -NH-X, wherein X is a proton or a protecting group that is absent when the linker is connected with the property modifying group.

In one embodiment X of the unreacted substituted sugar may thus be a protecting group such as Fmoc. In one embodiment the unreacted sugar moiety includes a protected primary amine which is also referred to as an N-protected sugar moiety.

In one embodiment the sugar moiety is a sialic acid comprising a protection group at a primary amino group.

As mentioned above the amino group may be located at a distance from C-4, such as by R5 comprising a glycyl group.

In one embodiment the sugar moiety is a sialic acid. In one embodiment the unreacted sugar moiety is CMP-SA-Glycine-Fmoc (FSC).

In the preparation of the modified sugar-nucleotides the protection group present on the intermediate sugar-moiety is removed prior to formation of the modified sugar-nucleotide as exemplified in Example 3 step 3 and Figure 2 step 3.

The examples herein illustrate the N-protected sugar moiety by SA-Glycyl-Fmoc. In one embodiment the sugar moiety is an N-protected sugar moiety. In one embodiment the N-protected sugar moiety is SA-Glycine-Fmoc.

Nucleotide moiety

According to the invention a sugar-nucleotide or a protected sugar-nucleotide as described herein includes a nucleoside moiety covalently linked to a phosphate moiety.

The phosphate moiety may be selected from monophosphate, diphosphate, triphosphate and polyphosphate moieties.

In one embodiment the nucleotide moiety has a mono-phosphate group.

The nucleoside moiety of the sugar-nucleotide can be any nucleoside or

deoxynucleoside, including adenosine, guanosine, 5-methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxyuridine, and deoxycytidine.

Additional nucleoside moieties are described herein.

The methods of the invention are useful for producing sugar-nucleotides, in which the nucleotide is linked to a mono/di/tri/poly-phosphate. Accordingly, exemplary nucleotides of the sugar-nucleotide include CMP, CDP, CTP, AMP, cAMP, ADP, ATP, UMP, UDP, UTP, GMP, cGMP, GDP, GTP, TMP, TDP and TTP as well as the deoxy forms of these and other nucleotides. Sugar-nucleotide

A sugar-nucleotide according to the invention includes a sugar moiety covalently linked to a nucleotide moiety and both have been described herein above. The term sugar-nucleotide also includes salt forms thereof, such as sodium and di- sodium salts. The term sugar-nucleotide according to the invention does not include sugar- nucleotides comprising further functional groups, such as a property modifying group as such molecules are herein described as modified sugar-nucleotides.

The present invention also relates a special salt form where the sugar-nucleotide is reversibly bound to a resin, such as an anion exchange media, which provides the positive charge for the ionic interaction. This form of the sugar-nucleotide is referred to as

immobilized sugar-nucleotide.

The sugar-nucleotide may be described as protected where a leaving group is attached to the sugar moiety, such as to C-4 via a linker (see above).

The linkage between the sugar and nucleotide moieties will usually be through a phosphate group of the nucleotide and C-1 of the sugar moiety (of formula I) such as through R1 as described above.

In one embodiment the sugar-nucleotide is Cytidine 5'-monophospho-N-glycyl- neuraminic acid also referred to as Cytidine 5'-monophospho-N-glycyl-sialic acid, Glycyl-SA- CMP (GSC), CMP-SA-Glycine or CMP-glycyl-Sialic Acid.

The sugar-nucleotides are usually prepared as a protected intermediate as described for the sugar moiety above which are de-protected prior to covalent linkage to the modifying group.

The preparation of a protected sugar-nucleotide is exemplified in Example 3 step 2

In one embodiment the protected sugar-nucleotide is an N-protected sugar- nucleotide. In one embodiment the protected sugar-nucleotide comprises Fmoc-glycyl- neuraminic acid in combination with a cytidine nucleotide moiety.

In one embodiment the protected sugar-nucleotide is Cytidine 5'-monophospho-N- Fmoc-glycyl-neuraminic acid also referred to as N-protected GSC or Fmoc-protected GSC.

Method for preparing a sugar-nucleotide

Various processes can be used for preparing a sugar-nucleotide which can be used as an intermediate in the preparation of a modified therapeutic molecule.

The inventors of the present invention have found that a resin can be used in the process and more particular in the process where the protected sugar-nucleotide is de- protected and the obtained sugar-nucleotide is reacted with the modifying agent to obtain the modified sugar-nucleotide. The binding of a sugar-nucleotide both in the protected and de- protected forms enables an efficient process which allows easy and effective purification of the intermediates by washing.

The present invention in an aspect relates to a method for purifying or preparing a sugar-nucleotide using a resin. The method in an embodiment involves binding of the protected or de-protected sugar-nucleotide to a resin. The de-protected sugar-nucleotide is in general simply referred to as a sugar-nucleotide. In an embodiment the method is for preparing a sugar-nucleotide from a protected sugar-nucleotide. As described herein above the sugar-nucleotide comprise a sugar moiety and a nucleotide moiety and the invention thus relates to various embodiments wherein the sugar moiety and the nucleotide moiety are as described herein above. Non-limiting examples are methods where the nucleotide moiety is CMP and/or the sugar moiety is a substituted sugar moiety, such as a Salic Acid (SA). In one embodiment the protected sugar-moiety is N-protected. In one embodiment the sugar moiety is an N-protected Salic acid.

In one embodiment the sugar moiety is a sialic acid.

In one embodiment the nucleotide moiety is a cytidine

In one embodiment the protected sugar-nucleotide is N-protected GSC.

In one embodiment the sugar-nucleotide is GSC.

In one embodiment the invention relates to a method for preparing GSC from N- protected GSC using an anion exchange media.

In one embodiment the resin is an anion exchange media. Multiple examples of such materials are available and include products from many manufactures such as the examples listed here below.

GE Healthcare Applied Biosystems: Tosohaas

Q-Sepharose FF Poras HQ 10 and 20um self Toyopearl DEAE 650S, M and

Q-Sepharase BB pack C

Q-Sepharose XL Poras HQ 20 and 50um bulk Super Q 650

Q-Sepharose HP media QAE 550C

10 Mini Q Poras PI 20 and 50um

MonoQ Poras D 50um

MonoP

MerckKGgA: Pall Corporation: DEAE Sepharose FF Fractogel DMAE DEAEHyperD Source 15Q FractoPrep DEAE Q Ceramic Hyper D

Source 30Q Fractoprep TMAE Mustang Q membrane Capto Q Fractogel EMD DEAE absorber

ANX Sepharose 4 FF Fractogel EMD TMAE

sub)

Streamline DEAE

Streamline QXL

In one embodiment the anion exchange resin comprises tertiary amines. In one embodiment the anion exchange resin comprise mixed bed resins including blends of anion exchange resins. In one embodiment the anion exchange media comprises a quaternary ammonium functional group. In on embodiment the anion exchange media is an anion exchange resin, selected from Smopex-103, Amberlite ® IRA 958 and Q Sepharose.

In a method according to the invention a sugar-nucleotide is prepared using the steps of

a. providing a protected sugar-nucleotide

b. immobilizing the protected sugar-nucleotide using an anion exchange media c. removing the protection group from the protected sugar-nucleotide and thereby obtaining the sugar-nucleotide.

In a method according to the invention a sugar-nucleotide is prepared using the steps of

a. providing a protected sugar-nucleotide

b. removing the protection group from the protected sugar-nucleotide

c. immobilizing the sugar-nucleotide using an anion exchange media and thereby obtaining the sugar-nucleotide.

As described above the key finding by the inventors relates to the usage of an anion-exchange media in the process of preparing the sugar-nucleotide as both the protected and de-protected sugar-nucleotide is capable of associating with the anion- exchange media. This enables easy handling and an effective process. It follows that the anion-exchange media can be used in one or more steps of the process. It may be used for capturing the protected sugar-nucleotide where by the protected sugar-nucleotide is immobilized on the anion-exchange media. The process may include a step of purification where unbound material is removed leading to a purification of the protected sugar- nucleotide. In a further step the protection group is removed from the protected sugar- nucleotide providing de-protected sugar-nucleotide e.g. the sugar-nucleotide. In this process the protected sugar-nucleotide or de-protected sugar-nucleotide may be associated with the anion-exchange media to various degrees.

In one embodiment the protected sugar-nucleotide and/or de-protected sugar- nucleotide is immobilized on the media throughout the process.

In one embodiment only the protected sugar-nucleotide is immobilized and released from the anion-exchange media before removal of the protection group.

In one embodiment the protected sugar-nucleotide and the sugar-nucleotide are both immobilized and the obtained sugar-nucleotide released from the anion-exchange media after removal of the protection group.

As is apparent from the above various sugar-nucleotide may be obtained according to the invention. The examples herein focus on obtaining GSC from N-protected GSC although it is apparent the same method may be applied to alternative sugar-nucleotides.

The inventors found that the method allowed a highly efficient process with a high yield and provides GSC in a higher purity compared to existing methods (US2012/0083600).

In one embodiment the invention relates to a method for preparing GSC, wherein the method comprises the steps of:

a. providing an N-protected GSC

b. immobilizing or purifying the N-protected GSC using an anion resin

c. obtaining GSC by removing the protecting group from the N-protected GSC.

In one embodiment the invention relates to a method for preparing GSC, wherein the method comprises the steps of:

a. providing N-protected GSC

b. obtaining GSC by removing the protecting group from N-protected GSC and d. immobilizing or purifying GSC using a resin.

In one embodiment the invention relates to a method for preparing GSC, wherein the method comprises the steps of:

a. providing N-protected GSC

b. immobilizing N-protected GSC using a resin

c. optionally releasing N-protected GSC from the resin

d. obtaining GSC by removal of the protecting group from the N-protected GSC.

In one embodiment the invention relates to a method for preparing GSC, wherein the method comprises the steps of:

a. providing N-protected GSC

b. obtaining GSC by removal of the protecting group from N-protected GSC and c. immobilizing GSC using a resin

d. and optionally releasing GSC from the resin.

The method can as described above be performed including one or more steps involving the anion exchange media. Association of the protected sugar-nucleotide and/or the final sugar-nucleotide allows for intermediate purification and storage as the media can easily be washed and dried. Such additional method steps may be included in the method of the invention.

In one embodiment the protected sugar-nucleotide, such as N-protected GSC is immobilized to the anion exchange media and washed. The immobilized protected sugar- nucleotide may be washed with water and/or THF. A further step of drying may be included, which may in an embodiment be performed in a flow of nitrogen. Such drying steps allow storage of the protected sugar-nucleotide. In a preferred embodiment the protected sugar- nucleotide is N-protected GSC, such as is CMP-SA-Glycine-Fmoc (or FSC).

The method according to the invention includes a step of removal of the protection group from the protected sugar-nucleotide to obtain the sugar-nucleotide of interest.

The skilled person will know that the method for removal of the protection group depends on the specific molecule in question. The skilled person will likewise know which method to use based on general knowledge in the art. The present application has illustrated an example of such removal in Example 3 step 3.

In one embodiment where the protection group is a Fmoc group as described herein above. In a further such embodiment the Fmoc group is attached via a nitrogen of a sialic acid and the sugar-nucleotide referred to as N-protected.

In one embodiment the protection group is removed under alkaline condition.

In one embodiment the protection group is removed using pipiridine.

In one embodiment the protection group is removed using piperidine in a water and ethanol mixture.

The handling of immobilized sugar-nucleotides (protected and de-protected) may be performed in various ways. It may be preferred that association of the sugar-nucleotide with the anion exchange media takes place in a column where the relevant sugar-nucleotide preparation is loaded onto a column comprising the relevant media. As described above the media associated sugar-nucleotide may then be easily washed.

Alternatively the anion exchange media including the sugar-nucleotide may be transferred to more stable environment, such as an inert reactor for one or more method steps. In one embodiment the removal of the protection groups takes place in an inert reactor. By transferring the reaction mixture (including the media and now de-protected sugar-nucleotide) to a column allows further processing including washing and drying of the sugar-nucleotide. Such method steps are also described in Example 3 step to with GSC as an example.

In one embodiment the sugar-nucleotide is immobilized using an anion exchange media such as a resin. In one embodiment the immobilized sugar-nucleotides, such as the immobilized GSC is washed. In on embodiment the immobilized sugar-nucleotides, such as the immobilized GSC is washed with water, ethanol and/or THF. In on embodiment the immobilized sugar-nucleotides, such as the immobilized GSC is washed water, ethanol and THF. In on embodiment the immobilized sugar-nucleotides, such as the immobilized GSC is dried. In on embodiment the immobilized sugar-nucleotides, such as the immobilized GSC is dried in a flow of nitrogen. In on embodiment the immobilized sugar-nucleotides, such as the immobilized GSC is dried under reduced pressure. In on embodiment the immobilized sugar-nucleotides, such as the immobilized GSC is pre-dried in a flow of nitrogen and dried under reduced pressure. Immobilized sugar-nucleotide

The method described herein involves a step of immobilizing a sugar-nucleotide or protected sugar-nucleotide which is found to be very beneficial in the process of preparing and for storing of such sugar-nucleotides. The sugar-nucleotide is associated with the anion exchange media via ionic bonds. As seen in figure 3, the interaction between the sugar- nucleotide GSC and the media involves the -COO " of the sugar ring, corresponding to R2 of the substituted sugar-moiety described herein, and a phosphate group from the nucleotide. As described above this may be considered a special salt form of the sugar-nucleotide.

An aspect of the invention relates to an immobilized form of a sugar-nucleotide or protected sugar-nucleotide and may simply be described as an immobilized sugar-nucleotide or an immobilized protected sugar-nucleotide. Alternatively the immobilized sugar-nucleotide or immobilized protected sugar-nucleotide may be described as a salt comprising an anion exchange media and the sugar-nucleotide or the protected sugar-nucleotide.

In further such embodiments the sugar-nucleotide is negatively charged.

The sugar-nucleotide in the salt or in the immobilized form is as the sugar-nucleotide defined herein above and the sugar-nucleotide thus includes a sugar moiety as well as a nucleotide moiety. In one embodiment the sugar moiety has a R1 group selected from a monophosphate, diphosphate, triphosphate or polyphosphate group, such as a -O- P(0)(OR)(0-nucleotide). In one embodiment the nucleotide moiety has monophosphate group. As described the sugar-nucleotide by be in the protected for or in the de-protected form.

In one embodiment the immobilized sugar-nucleotide is GSC or N-protected GSC.

In one embodiment the immobilized sugar-nucleotide is reversibly bound to the anion exchange media, such as a resin. In one embodiment the interaction is by ionic bonds. In further embodiments the immobilized sugar-nucleotide is bound to an anion exchange resin, such as a strong anion exchange resin. In one embodiment the immobilized sugar- nucleotide is bond to an anion exchange media selected from Smopex-103, Amberlite IRA 958 and Q Sepharose, such as Q Sepharose FF. Modified sugar-nucleotide

The sugar-nucleotide produced according to the invention may subsequently be used in the preparation of a modified sugar-nucleotide.

An aspect of the invention relates to a method for preparing a modified sugar- nucleotide comprising the steps of;

a. providing a property modifying agent

b. reacting said modifying agent with a sugar-nucleotide obtained as described

herein

c. obtaining a modified sugar-nucleotide.

A modifying agent may be an agent capable of reacting with the sugar-nucleotide transferring a property modifying group to the sugar-nucleotide providing a modified sugar- nucleotide comprising said property modifying group.

The term property modifying group refers to the functionality of the group when transferred to a protein or polypeptide

The property modifying group may comprise any group including various types of molecules that provides a desired property. Classical examples of property modifying groups are half-life extending groups, such as PEG, Albumin binders and Fc domains. Half-life extending groups may also be described as protractors as they provide a protracting functionality to the protein or polypeptide.

In one embodiment the property modifying group is a polymeric modifying group, such as 40K PEG, 30K PEG, 20K PEG or 10K PEG.

In a preferred embodiment, a polymeric modifying group (e.g., linear or branched PEG moiety) is covalently attached to the sugar moiety of the sugar-nucleotide, optionally via a linker moiety. As seen from the Examples herein the polymeric modifying group can in one embodiment be covalently attached to the sugar moiety. In one embodiment the property modifying group is attached via a primary amine. The linkage is in one embodiment through a de-protected nitrogen of the sugar moiety.

In one embodiment the invention provides a method for preparing a modified sugar- nucleotide, wherein the modified sugar-nucleotide comprise a sugar-moiety and a nucleotide moiety as described herein above. With reference to the example a method to preparing modified GSC is provided, while it is clear that the method of the invention is not limited to this example. In one embodiment the invention provides a method for preparing a modified GSC (GSC-M), wherein GSC is Cytidine 5'-monophospho-N-glycyl-neuraminic acid and M is a property modifying group comprising the steps of:

a. providing a property modifying agent; M-LG, wherein M is a property modifying group and LG is a leaving group,

b. providing GSC obtained according to the method of the invention

c. reacting said M-LG and GSC

d. obtaining GSC-M.

In one embodiment the sugar-nucleotide, i.e. GSC, is provided in solution or as immobilized GSC. In on embodiment the sugar-nucleotide is provided in isolated form, such as a lyophilised powder. As described herein above GSC may be obtained by use of anion exchange resin at any point in the process, and thus provided for later use either in solution e.g. released from the resin or immobilized on the anion exchange media.

In one embodiment the property modifying agent is described by M-LG, wherein M is a property modifying group and LG is a leaving group. In one embodiment the property modifying agent is capable of reacting with a primary amine. A suitable leaving group is para-nitrophenyl (NP).

The reaction of the property modifying agent and the sugar-nucleotide may be performed according to common general knowledge and varied by the skilled person as required.

In one embodiment the reaction of the property modifying agent and the sugar- nucleotide occurs in a phosphate buffer. In one embodiment the reaction of the property modifying agent and the sugar-nucleotide occurs in THF. In one embodiment the reaction of the property modifying agent and the sugar-nucleotide occurs in a mixture of phosphate buffer and THF.

In one embodiment the reaction of the property modifying agent and the sugar- nucleotide occurs at a pH of 7.5-8.5.

In one embodiment the reaction of the property modifying agent and the sugar- nucleotide is performed with a reaction temperature of 15-30 °C. In one embodiment the reaction time for the reaction of the property modifying agent and the sugar-nucleotide is less than 120 hours. The reaction may be followed and stopped when completed or close to completion as decided by the skilled artisan.

In one embodiment the reaction of the property modifying agent and the sugar- nucleotide is performed with an excess of the sugar-nucleotide. In order to minimize waste of material it is preferred that the ratio of GSC/M-NP does not exceed 5. In one embodiment the ratio of GSC/M-NP is 1 -2.5.

In a further embodiment, wherein the sugar-nucleotide is provide in immobilized form such as in the form of a salt with an anion exchange media, the modified sugar- nucleotide may also be obtained in immobilized form. To obtain the modified sugar- nucleotide in soluble form, the modified sugar-nucleotide may be release from the media.

In such embodiments the method may include a further step of releasing the modified sugar-nucleotide from the anion exchange media. In an embodiment, following the examples herein, the method of the invention comprises a step where GSC-M is released from the resin. In such embodiments the modified sugar-nucleotide is released by changing the pH or the salt concentration. In a further such embodiment the modified sugar-nucleotide is released using aqueous sodium hydrogen carbonate, such as a 10-100 mM sodium hydrogen carbonate solution. The modified sugar-nucleotide is thus obtained as di-sodium salt in solution.

In one embodiment GSC-M is released from the resin with aqueous sodium hydrogen carbonate. In one embodiment GSC-M is released from the resin with 10-100 mM sodium hydrogen carbonate, such as 20 mM. In one embodiment GSC-M is released from the resin and the resin is removed. To separate the released modified sugar-nucleotide from the anion exchange media a filtering step is further applied. In one embodiment GSC-M is released from the resin and the resin is removed by filtering.

In order to preserve the modified sugar nucleotide a cooling step may further be included, wherein the modified sugar nucleotide is cooled to 2-8°C.

As described above the property modifying group, M, may be a PEG, which includes such as 40K PEG, 30K PEG, 20K PEG or 10K PEG.

In one embodiment the obtained modified sugar-nucleotide is PEG-GSC. In one embodiment the modified sugar-nucleotide is 40K PEG-GSC. In one embodiment the modified sugar-nucleotide is 40K PEG-GSC with a branched PEG. In one embodiment the modified sugar-nucleotide is 40K PEG-GSC with a branched PEG comprising two 20K PEG'S. The obtained modified sugar-nucleotide may be further purified based on comment general knowledge using standard purification technologies. In one embodiment the modified sugar-nucleotide is purified using ultra- and dia-filtration.

Modified glycoprotein

A modified sugar-nucleotide may be used in the preparation of a modified glycoprotein, where the property modifying group is attached to the protein of interest via glyco- conjugation. Methods for preparing modified glyco-proteins are well-known in the art and the skilled artisan will be able to follow such methods using the sugar-nucleotide and modified sugar-nucleotide prepared according to the invention. The skilled person will also know how to adapt the process when needed, such as to make a selection of a transferase and performing needed trimming of sialic acids to obtain the desired product.

Accordingly, the present invention relates to a method for preparing a modified glyco-protein, wherein the method comprises the steps of:

a. providing a glyco-protein

b. reacting said protein with a modified sugar-nucleotide obtained according to the invention,

c. obtaining a modified glycoprotein.

In one embodiment a NANA aldolase and a CNS enzyme is used.

In one embodiment the glycoprotein is a Factor protein, such as Factor VII, Factor.

VIII or Factor IX

In one embodiment the invention relates to a method for preparing PEG-FVII, PEG- FVIII or PEG-FIX. While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended embodiments are intended to cover all such modifications and changes as fall within the true spirit of the invention. EMBODIMENTS

1. A method for purifying or preparing a sugar-nucleotide using an anion exchange media. 2. The method according to embodiment 1 , wherein the sugar-nucleotide is obtained from a protected sugar-nucleotide.

3. The method according to embodiment 1 , where in the sugar-nucleotide consists of a sugar moiety and a nucleotide moiety.

4. The method according to embodiment 3, where in the sugar moiety is a substituted

sugar.

5. The method according to embodiment 3, where in the sugar moiety is a sialic acid.

6. The method according to embodiment 3, where in the nucleotide moiety comprises a mono-phosphate moiety.

7. The method according to embodiment 1 , where in the sugar-nucleotide is CMP-SA- Glycine (GSC).

8. The method according to embodiment 2, where in the protected sugar-nucleotide is N- protected GSC. 9. The method according to embodiment 1 , wherein the method is for preparing GSC from N-protected GSC using a resin.

10. The method according to embodiment 1 , wherein the anion exchange media is a resin. 1 1 . The method according to embodiment 1 , wherein the anion exchange resin is a strong anion exchange resin.

12. The method according to embodiment 1 , wherein the anion exchange resin is a

quartenary ammonium (Q) exchange resin. 13. The method according to embodiment 1 , wherein the anion exchange resin is an anion exchange resin, selected from Smopex-103, Amberlite IRA 958 and Q Sepharose.

14. The method according to any of the embodiment 1-13, comprising the steps of

a. providing a protected sugar-nucleotide,

b. immobilizing the protected sugar-nucleotide using an anion exchange media c. optionally releasing the protected sugar-nucleotide from the media

d. removing the protection group from the protected sugar-nucleotide and thereby e. obtaining the sugar-nucleotide and

f. optionally releasing the sugar-nucleotide from the media.

15. The method according to embodiment 14, comprising the steps of

a. providing N-protected GSC ,

b. immobilizing N-protected GSC using an anion exchange media,

c. optionally releasing N-protected GSC from the media,

d. removing the protection group from the N-protected GSC and thereby e. obtaining GSC and

f. optionally releasing GSC from the media. 16. The method according to any of the embodiments 1 -13, comprising the steps of

a. providing protected sugar-nucleotide

b. removing the protection group from the protected sugar-nucleotide and thereby c. obtaining a sugar-nucleotide,

d. immobilizing the sugar-nucleotide using an anion exchange media and e. optionally releasing the sugar-nucleotide from the media.

17. The method according to embodiment 16, comprising the steps of

a. providing N-protected GSC

b. removing the protection group from said N-protected GSC and thereby c. obtaining GSC

d. immobilizing GSC using an anion exchange media and

e. optionally releasing GSC from the media

18. The method according to any of the previous embodiments, wherein N-protected GSC immobilized. 19. The method according to any of the previous embodiments, wherein GSC is immobilized.

20. The method according to embodiment 18, wherein the immobilized N-protected GSC is washed with water and/or THF.

21 . The method according to embodiment 18, wherein the immobilized N-protected GSC is washed with water and THF. 22. The method according to embodiment 18, wherein the immobilized N-protected GSC is dried.

23. The method according to embodiment 18, wherein the immobilized N-protected GSC is dried in a flow of nitrogen.

24. The method according to any of the previous embodiment 2-23, wherein the protected sugar-nucleotide comprise a protection group, such as Fmoc.

25. The method according to any of the embodiment 18-23, wherein the N-protected GSC is CMP-SA-Glycine-Fmoc (FSC).

26. The method according to any of the previous embodiment 2-25, wherein the protection group is removed under alkaline condition. 27. The method according to any of the previous embodiment 2-25, wherein the protection group is removed using an amine.

28. The method according to any of the previous embodiment 2-25, wherein the protecting group is removed using piperidine.

29. The method according to any of the previous embodiment 2-25, wherein the protecting group is removed using piperidine in a water and ethanol mixture.

30. The method according to any of the previous embodiment 19-29, wherein the

immobilized GSC is washed. The method according to any of the previous embodiment 19-29, wherein the immobilized GSC is washed with water, ethanol and THF. The method according to any of the previous embodiment 19-29, wherein the

immobilized GSC is dried. The method according to any of the previous embodiment 19-29, wherein the

immobilized GSC is dried in a flow of nitrogen. The method according to any of the previous embodiment 19-29, wherein the

immobilized GSC is dried under reduced pressure. The method according to any of the previous embodiment 19-29, wherein the

immobilized GSC is pre-dried in a flow of nitrogen and dried under reduced pressure. An immobilized form of a protected sugar-nucleotide or a sugar-nucleotide. An immobilized protected sugar-nucleotide or an immobilized sugar-nucleotide. A salt comprising an anion exchange media and a protected sugar-nucleotide or a sugar- nucleotide. The salt, the immobilized protected sugar-nucleotide or the immobilized sugar-nucleotide according to any of the embodiments 36-38, wherein the sugar-nucleotide is negatively charged. The salt, the immobilized protected sugar-nucleotide or the immobilized sugar-nucleotide according to any of the embodiments 36-38, wherein the protected sugar-nucleotide or the sugar-nucleotide is as defined in any of the above embodiments 3-8.

The salt, the immobilized protected sugar-nucleotide or the immobilized sugar-nucleotide according to any of the embodiments 36-40 comprises N-protected GSC or GSC. 42. The salt, the immobilized protected sugar-nucleotide or the immobilized sugar-nucleotide according to any of the embodiments 36-41 , wherein N-protected GSC or GSC is reversibly bound to a resin. 43. The salt, the immobilized protected sugar-nucleotide or the immobilized sugar-nucleotide according to any of the embodiments 36-42, wherein N-protected GSC or GSC is bound to the anion exchange media by ionic interactions.

44. The salt, the immobilized protected sugar-nucleotide or the immobilized sugar-nucleotide according to any of the embodiments 36-43, wherein N-protected GSC or GSC is immobilized on an anion exchange resin.

45. The salt, the immobilized protected sugar-nucleotide or the immobilized sugar-nucleotide according to any of the embodiments 36-44, wherein N-protected GSC or GSC is immobilized on a strong anion exchange resin.

46. The salt, the immobilized protected sugar-nucleotide or the immobilized sugar-nucleotide according to any of the embodiments 36-45, wherein N-protected GSC or GSC s immobilized on a strong anion exchange resin selected from Smopex-103, Amberlite IRA 958 and Q Sepharose.

47. The salt, the immobilized protected sugar-nucleotide or the immobilized sugar-nucleotide according to any of the embodiments 36-46, wherein N-protected GSC or GSC is immobilized on Q Sepharose.

48. A method for preparing a modified sugar-nucleotide

a. providing a property modifying agent

b. reacting said modifying agent with a sugar-nucleotide obtained according to any of the embodiment 1 -35 and

c. obtaining a modified sugar-nucleotide.

49. The method according to embodiment 48, wherein the sugar-nucleotide is as defined in any of the above embodiments 3-7. 50. A method for preparing modified CMP-SA-Glycine (GSC-M), wherein GSC is CMP-SA- Glycine and M is a property modifying group, comprising the steps of:

a. providing a property modifying agent, M-LG, wherein M is a property modifying group and LG is a leaving group,

b. providing GSC obtained according to any of the embodiments 1 -35.

c. reacting said property modifying agent with GSC and thereby

d. obtaining GSC-M.

51 . The method according to embodiment 50, wherein GSC is immobilized on a resin, in solution or in an isolated form.

52. The method according to embodiment 50 or 51 , wherein the leaving group is para- nitrophenyl (NP). 53. The method according to any of the embodiments 50-52, wherein the reaction occurs in a mixture of phosphate buffer and THF.

54. The method according to any of the embodiments 50-53, wherein the reaction

temperature is 15-30 °C.

55. The method according to any of the embodiments 50-54, wherein GSC and M-NP are reacted in excess of GSC.

56. The method according to any of the embodiments 50-55, wherein GSC and M-NP are reacted in excess of GSC wherein the ratio of GSC/M-NP is 1-2.5.

57. The method according to any of the embodiments 50-56, wherein the reaction time is less than 120 hours. 58. The method according to any of the embodiments 50-57, wherein pH of the reaction buffer is 7.5-8.5.

59. The method according to any of the embodiments 50-58, wherein GSC-M is released from the resin. 60. The method according to any of the embodiments 50-58, wherein GSC-M is released from the resin by changing the pH or by changing the salt concentration.

61 . The method according to any of the embodiments 50-58, wherein GSC-M is released from the resin with aqueous sodium hydrogen carbonate.

62. The method according to any of the embodiments 50-58, wherein GSC-M is released from the resin with 10-100 mM sodium hydrogen carbonate, such as 20 mM. 63. The method according to any of the embodiments 50-62, wherein GSC-M is released from the resin and the resin is removed.

64. The method according to any of the embodiments 50-62, wherein GSC-M is released from the resin and the resin is removed by filtering.

65. The method according to any of the embodiments 50-64, wherein GSC-M is cooled (2- 8°C).

66. The method according to any of the embodiments 50-65, wherein the property modifying moiety M is a protractor.

67. The method according to any of the embodiments 50-65, wherein the property modifying moiety M is a half-life extending group. 68. The method according to any of the embodiments 50-67, wherein M is a PEG.

69. The method according to any of the embodiments 50-68, wherein M is 40K PEG, 30K PEG, 20K PEG or 10K PEG. 70. The method according to any of the embodiments 50-67, wherein M is heperosan (HEP).

71 . The method according to any of the embodiments 50-69, wherein GSC-M is PEG-GSC.

72. The method according to any of the embodiments 50-69, wherein GSC-M is a 40K PEG- GSC 73. The method according to any of the embodiments 50-69, wherein GSC-M is a 40K PEG- GSC with a branched PEG. 74. The method according to any of the embodiments 50-69, wherein GSC-M is a 40K PEG- GSC with a branched PEG comprising two 20K PEG'S.

75. A method for purifying PEG-GSC comprising the steps of

a. providing a PEG-GSC solution

b. performing ultra- and diafiltration

c. obtaining purified PEG-GSC.

76. The method according to embodiment 75, wherein the ultra- and diafiltration is performed using a membrane with a molecular weight cut-off below the molecular weight of PEG-GSC obtaining a purified PEG-GSC solution.

77. The method according to embodiment 75, wherein the ultra- and diafiltration is performed using a 5-40 kDa membrane. 78. The method according to embodiment 75, wherein the ultra- and diafiltration is performed using a 10-20 kDa membrane.

79. The method according to any of the embodiments 75-78, wherein the PEG-GSC solution is obtained according to any of the embodiments 50-74.

80. The method according to any of the embodiments 75-79, wherein the method comprise an anion exchange chromatography step.

81 . The method according to any of the embodiments 75-80, wherein the method comprises a second ultra- and diafiltration.

82. The method according to any of the embodiments 75-81 , wherein the purified PEG- GSC solution is filtered. 83. The method according to any of the embodiments 75-81 , wherein the purified PEG- GSC solution is filtered using a 0.2 μηη filter.

84. The method according to any of the embodiments 82-83, wherein the filtered PEG- GSC solution is lyophilized.

85. The method according to any of the embodiments 82-83, wherein the filtered PEG- GSC solution is pre-frozen and lyophilized. 86. The method according to any of the embodiment 84-85, wherein the lyophilized PEG-GSC is stored at 15-25°C.

87. The method according to any of the embodiments 75-86, wherein PEG is 40K PEG, 30K PEG, 20K PEG or 10K PEG

88. The method according to any embodiments 75-87, wherein PEG is 40K and PEG- GSC has a branched PEG.

89. The method according to any embodiments 75-88, wherein PEG is 40K and PEG- GSC has a branched PEG of two 20K PEG'S.

90. A method for preparing a modified glyco-protein wherein the method comprises the steps of:

a. providing a glyco-protein

b. reacting said glyco-protein with GSC-M obtained according to any of the

embodiments 50-74 and optionally purified according to any of the embodiments 75- 89,

c. obtaining a modified protein. 91 . The method according to embodiment 90, wherein the glyco-protein is at therapeutic protein.

92. The method according to embodiment 90, wherein the glyco-protein is a Factor protein. 93. The method according to embodiment 90, wherein the glyco-protein is selected from the group of Factor VII, Factor VIII, Factor IX and Factor X.

94. The method according to embodiment 90, therein the modified protein is PEG-FVII, PEG-FVIII or PEG-FIX.

METHODS AND EXAMPLES

List of Abbreviations

The protracting entity PEG-GSC (Chem.1 ) is produces by the reaction scheme depicted in Figure 1. A PEG-NP (NOF Corporation (Japan)) is reacted with GSC yielding PEG-GSC. The process may be performed as described in US 2012/083600. Chem.1 :

Example 2: General method for preparation of GSC

The production of GSC is prepared according to the general reaction scheme shown in Figure 2 yielding GSC. The last step in the process is the removal of the protection group Fmoc which is required to ensure reactivity with PEG-NP or alternative moieties for conjugation/derivation via a propyl-p-Nitro-phenyl Carbonate (NP) group.

Example 3: Method for preparation of immobilized cvtidine monophosphate-glycyl- sialic acid (GSC) Step 1 : Synthesis of crude Fmoc-glycyl-mannosamine (FGM)

D-mannosamine is coupled with a slight excess of Fmoc-Gly-OSu in the presence of sodium hydrogen carbonate in a water and ethanol mixture at ambient temperature. An inert reactor is charged with D-mannosamine (50 g), Fmoc-glycine-O-succinimide (1 10 g), sodium bicarbonate (24 g), water (0.28 L) and ethanol (1 .1 L). The reaction mixture is stirred at 20°C.

The conversion is monitored by RP-HPLC and upon completion of reaction, toluene

(2.4 L) and water (2.0 L) is added. The aqueous phase is washed with toluene (2x1.5 L) and concentrated under reduced pressure (approximately 0.9 L distilled off at 40-50°C). Sodium chloride (200 g) is added followed by 2-methyl-tetrahydrofurane (2.5 L)..

The organic phase is dried with magnesium sulphate (100 g) and filtered. The filter cake is washed with 2-methyl-tetrahydrofurane (200 ml_). The combined filtrate is

concentrated under reduced pressure at 40-50°C to an oil or a foam.

Step 2: Synthesis of Cytidine monophosphate-Fmoc-glycyl-sialic acid (FSC) and

immobilization on Q Sepharose FF (FSC/Q)

In a one-pot reaction, FGM, excess sodium pyruvate and a slight excess of CTP are enzymatically converted to FSC. Two process enzymes are used: NANA aldolase (CDX-001 from Codexis, Inc) and CNS enzyme (CMP-NAN synthetase) (Center for Biocatalysis and Bioprocessing, University of Iowa). NANA aldolase adds pyruvate to the FGM generating FGS. The excess pyruvate shifts the equilibrium towards FGS. The CNS enzyme transfers CMP (from CTP) to FGS yielding FSC. Manganese (II) is used as a cofactor for the CNS enzyme.

An inert reactor is charged with FGM (51 .8 g) and water (2 L). Aqueous NaOH (1 M) is added keeping the pH at 7.5 and the temperature is increased to 30°C. Manganese chloride (1 1.5 g), CTP disodium salt (86.5 g) and sodium pyruvate (1 12 g) added to the reactor. A solution of NANA Aldolase (16.1 g) in water (0.44 L) is added followed by an aqueous solution of CNS enzyme (1.15 L, approximately 400000 U/L). Water (1 L) is then added to flush remaining reagents into the reactor and the enzymatic reaction is stirred at 30°C. The progress of the reaction is monitored by RP-HPLC and upon completion of reaction, the process enzymes are removed by ultra- and diafiltration (UF/DF using a membrane with a MWCO = 10 kDa).

The permeate containing FSC is purified by RP-HPLC (water - methanol). Product fractions with a purity≥98% are combined and loaded onto an anion exchange resin (Q Sepharose FF, approximately 2.5 L) generating FSC immobilised on Q Sepharose (FSC/Q).

The FSC/Q is washed with water (1.25 L) and THF (2.5 L) and dried in a flow of nitrogen.

Step 3: Synthesis of Cytidine monophosphate-glycyl-sialic acid (GSC) immobilised on Q

Sepharose FF (GSC/Q)

The Fmoc protecting group of FSC/Q is removed by treating the FSC/Q with piperidine in a water and ethanol mixture generating the product GSC, which remains immobilised on Q Sepharose FF (GSC/Q).

FSC/Q is transferred from the column to an inert reactor (approximately 2.5 L).

Ethanol (3 L), water (0.34 L) and piperidine (85 ml.) are added and the reaction mixture is stirred at 20°C. The reaction is followed by HPLC and upon completion of the reaction the product (GSC immobilised on Q Sepharose (GSC/Q)), is transferred to a column and washed with 1 :30 v/v water - ethanol (3 x 3.5 L) and THF (2.5 L). .

The resin is pre-dried in a flow of nitrogen and dried under reduced pressure generating GSC/Q as a dry solid. The solid GSC/Q has a purity≥95%, and can be stored at -20°C for at least 24 months.

Characterisation

GSC/Q consists of GSC bound to Q Sepharose FF resin. GSC detached from the resin has been characterised by nuclear magnetic resonance (NMR) and the structure is depicted in Figure 3, including proton and carbon chemical shifts.

Example 4: Method for producing 5-N -derivatives of cvtidine monophosphate-qlvcyl- sialic acid exemplified by PEG-GSC

The overall process is shown in figure 4. Step A: Coupling of GSC and 40K PEG-NP

Excess GSC/Q (1 .4 equivalents of GSC) is PEGylated with 40K PEG-NP in a mixture of 0.3 M phosphate buffer pH 8 and THF in equal volumes yielding the 40K PSC-. The conversion is monitored by reverse phase HPLC (RP-HPLC) and upon completion of reaction the mixture is cooled. Dilute aqueous sodium hydrogen carbonate (20 mM) is added to the reaction mixture, releasing the partially bound product 40K PSC from the resin. The resin is removed by filtering, washed with aqueous sodium hydrogen carbonate (20 mM) and with water The combined wash and the filtrate are mixed and contain the crude 40K PSC product. The solution is cooled on an ice bath and stored a 2-8°C until purification in step B.

Step B: Purification of 40K PSC

The purification of 40K PSC consists of UF/DF, AIEX, a second UF/DF and filtration with a 0.2 μηη filter.

First ultra- and diafiltration

The crude 40K PSC solution from step A is concentrated by ultrafiltration using a membrane with a molecular weight cut-off = 5 kDa to approximately 5% w/v and then diafiltered with 9 diavolumes of water. 40K PSC and molecules with a molecular weight greater than 5 kDa are retained and small molecules and salts are removed in the permeate.

Anion exchange chromatography

The aqueous solution of 40K PSC (retentate from the first UF/DF) is loaded onto an equilibrated anion exchange resin (Q Sepharose Fast Flow) which is washed with 75 μΜ NaHCOs. Then 40K PSC is eluted with 20 mM NaHC0 3 . Product fractions are cooled, combined and filtered.

Second ultra- and diafiltration

The 40K PSC eluted from AIEX is concentrated by ultrafiltration to approximately 5% w/v and then diafiltered with 9 diavolumes of water using a membrane with a molecular weight cut-off = 5 kDa, retaining molecules with a molecular weight greater than 5 kDa. The 40K PSC retentate from the second UF/DF is filtered with a 0.2 μηη filter into lyophilisation trays and pre-frozen at < - 50°C.The product is lyophilized affording 40K PSC.