Title of Invention

"A PROCESS FOR ROMING A DERIVATIVE OF A SIALIC ACID COMPOUND"

Abstract

A process for forming a derivative of a sialic acid compound in which a starting compound comprising a terminal sialic acid unit is subjected to a preliminary intermediate - forming step comprising the sequential steps of oxidation and reduction or vice versa in which a group selected from a primary amine group, a secondary amine group and a hydrazine is formed on the terminal sialic acid unit, followed by a reaction step in which the intermediate is reacted with a bifunctional reagent, of formula I in which R is H or sulphonyl; R1 is a linker group; and X is a functional group, whereby the ester group is cleaved and the amine or hydrazine group of the intermediate is acylated by -CO-R1-X to form the derivative.

Full Text

The present invention relates to derivatives of sialic acid compounds, preferably polysaccharides which have terminal or intrachain sialic acid units. Preferably the polysaccharide consists only of sialic acid units, for instance linked alpha-2,8, 2,9 to one another. The products are useful for conjugation to substrates such as peptides, proteins, drugs, drug delivery systems, viruses, cells, microbes, synthetic polymers etc. The reaction involves conjugation of an N-hydroxysuccinimide (NHS) group containing reagent with either an amino or hydrazide functional sialic acid derivative. Polysialic acids (PSAs) are naturally occurring unbranched polymers of sialic acid produced in certain bacterial strains and in mammals in certain cells [Roth et. al., 1993]. They can be produced in various degrees of polymerisation: from n = about 80 or more sialic acid residues down to n = 2 by either limited acid hydrolysis, digestion with neuraminidases or by fractionation of the natural, bacterial or cell derived forms of the polymer. The composition of different PSAs also varies such that there are homopolymeric forms i.e. the alpha-2,8-linked PSA comprising the capsular polysaccharide of E. coli strain K1 and of the group-B meningococci, which is also found on the embryonic form of the neuronal cell adhesion molecule (N-CAM). Heteropolymeric forms also exist, such as the alternating alpha-2,8 alpha-2,9 PSA of E. coli strain K92 and the group C polysaccharides of N. meningitidis. In addition, sialic acid may also be found in alternating copolymers with monomers other than sialic acid such as group W135 or group Y of N. meningitidis. PSAs have important biological functions including the evasion of the immune and complement systems by pathogenic bacteria and the regulation of glial adhesiveness of immature neurons during foetal development (wherein the polymer has an anti-adhesive function) [Muhlenhoff et. al., 1998; Rutishauser, 1989; Troy, 1990, 1992; Cho and Troy, 1994], although there are no known receptors for PSAs in mammals. The alpha-2,8-linked PSA of E. coli strain K1 is also known as 'colominic acid' and is used (in various lengths) to exemplify the present invention. The alpha-2,8 linked form of PSA, among bacterial polysaccharides, is uniquely non-immunogenic (el iciting neither T-cell or antibody responses in mammalian subjects) even when conjugated to immunogenic carrier protein, which may reflect its existence as a mammalian (as well as a bacterial) polymer. Shorter forms of the polymer (up to n=4) are found on cell-surface gangliosides, which are widely distributed in the body, and are believed to effectively impose and maintain immunological tolerance to PSA. In recent years, the biological properties of PSAs, particularly those of the alpha-2,8 linked homopolymeric PSA, have been exploited to modify the pharmacokinetic properties of protein and low molecular weight drug molecules [Gregoriadis, 2001; Jain et. al., 2003; US-A-5,846,951; WO-A-0187922]. PSA derivatisation of a number of therapeutic proteins including cataiase and asparaginase [Femandes and Gregoriadis, 1996 and 1997] gives rise to dramatic improvements in circulation half-life, its stability and also allows such proteins to be used in the face of pre-existing antibodies raised as an undesirable (and sometimes inevitable) consequence of prior exposure to the therapeutic protein [Femandes and Gregoriadis, 2001]. In many respects, the modified properties of polysialylated proteins are comparable to proteins derivatised with polyethylene glycol (PEG). For example, in each case, half-lives are increased, and proteins and peptides are more stable to proteolytic digestion, but retention of biological activity appears to be greater with PSA than with PEG [Hreczuk-Hirst et. al., 2002]. Also, there are questions about the use of PEG with therapeutic agents that have to be administered • chronically, as PEG is only very slowly biodegradable [Beranova et.al., 2000] and both high and low molecular weight forms tend to accumulate in the tissues [Bendele, et. al., 1998; Convers, et. al., 1997]. PEGylated proteins have been found to generate anti PEG antibodies that could also influence the residence time of the conjugate in the blood circulation [Cheng et. a)., 1990]. Despite the established history of PEG as a parenterally administered polymer conjugated to therapeutics, a better understanding of its immunotoxicology, pharmacology and metabolism will be required [Hunter and Moghimi, 2002; Brocchini, 2003]. Likewise there are concerns about the utility of PEG in therapeutic agents that require high dosages, (and hence ultimately high dosages of PEG), since accumulation of PEG may lead to toxicity. The alpha 2,8 linked PS A therefore offers an attractive alternative to PEG, being an immunologically 'invisible' biodegradable polymer which is naturally part of the human body, and that can degrade, via tissue neuraminidases, to sialic acid, a non-toxic saccharide. Our group has described, in previous scientific papers and in granted patents, the utility of natural PSAs in improving the pharmacokinetic properties of protein therapeutics [Gregoriadis, 2001; Femandes and Gregoriadis, 1996,1997, 2001; Gregoriadis et. al., 1993, 1998, 2000; Hreczuk-Hirst et. al., 2002; Mital, 2004; Jain et. al., 2003, 2004; US-A- 05846,951; WO-A-0187922]. Now, we describe new derivatives of PSAs, which allow new compositions and methods of production of PSA-derivatised proteins (and other forms of therapeutic agents). These new materials and methods are particularly suitable for the production of PSA-derivatised therapeutic agents intended for use in humans and animals, where the chemical and molecular definition of drug entities is of major importance because of the safety requirements of medical ethics and of the regulatory authorities (e.g. FDA, EMEA). Methods have been described previously for the attachment of polysaccharides to therapeutic agents such as proteins [Jennings and Lugowski, 1981; US-A-5,846,951; WO-A-0187922}. Some of these methods depend upon chemical derivatisation of the 'non-reducing' end of the polymer to create a protein-reactive aldehyde moiety (Fig. 1). The reducing end of PSA (and other polysaccharides) is only weakly reactive with proteins under the mild conditions necessary to preserve protein conformation and the chemical integrity of PSA during conjugation. The sialic acid unit, at the non-reducing terminal of PSA which contains a vicinal diol, can be readily (and selectively) oxidised with periodate to yield a mono-aldehyde derivative. This derivative is much more reactive towards proteins and comprises of a suitably reactive element for the attachment of proteins via reductive amination and other chemistries. We have described this previously in US-A-5,846,951and WO-A-0187922. The reaction is illustrated in Fig. 1 in which: a) shows the oxidation of CA (alpha-2,8 linked PSA from E. coif) with sodium periodate to form a protein-reactive aldehyde at the nonreducing end of the terminal sialic acid and b) shows the reaction of the aldehyde with a primary amine group of a protein followed by the selective reduction of the SchifFs base with sodium cyanoborohydride (NaCNBH3) to form a stable irreversible covalent bond with the protein amino group. In PCT/GB04/03488 we describe polysaccharide derivatives which have a sulfhydryl-reactive group introduced via a terminal sialic acid unit. This unit is usually introduced by derivatisation of a sialic acid unit at the non-reducing end of the polysaccharide. The sulfhydryl reactive group is preferably a maleimido group. The reaction to introduce this group may involve the reaction of a heterobifunctional reagent having a sulfhydrylreactive group at one end and a group such as a hydrazide or an ester at the other end, with an aldehyde or amine group on the sialic acid derived terminal unit of the polysaccharide. The product is useful for site specific derivatisation of proteins, e.g. at Cys units or introduced sulfhydryl groups. Although the various methods that have been described to attach PSAs to therapeutic agents [US-A-5,846,951; WO-A-0187922J, are theoretically useful, achievement of acceptable yields of conjugate via reaction of proteins with the non-reducing end (aldehyde form) of the PSA requires reaction times that are not conducive to protein stability at higher temperature (e.g. interferon alpha-2b). Secondly, reactant concentrations (i.e. polymer excess) are required that may be unattainable or uneconomical. In the invention there is .provided a new process for forming derivatives of a sialic acid compound in which a starting compound comprising a terminal sialic acid unit is subjected to a preliminary intermediate - forming step, in which a group selected from a primary amine group, a secondary amine group and a hydrazine is formed on the terminal sialic acid unit, followed by a reaction step in which the intermediate is reacted with a bifunctional reagent in which R is H or sulphonyl; R1 is a linker group; and X is a functional group, whereby the;ester group is cleaved and the amine or hydrazine group of the intermediate is acylated by -CO-R1-X to form the derivative. In a first embodiment the starting compound has a terminal sialic acid unit joined to another moiety via its 2-carbon atom i.e. as a non-reducing terminal unit, and in which the preliminary step involves oxidation of the C-7, C-8 diol group of the sialic acid to form an aldehyde group followed by reductive amination with H2NR4, in which R4 is H or lower alkyl, or acid addition salt thereof to form the intermediate. This preliminary step is shown in Figure 3. In this first embodiment the starting compound has the following in which R2 is the said other moiety and is selected from a mono-, di-, oligoor poly-saccharide group, a protein or peptide, a lipid , a drug and a drug delivery system (such as a liposome) and in which the amide derivative product has the following formula: COOH in in which X, R1 and R4 are the same groups as in the respective starting compounds and R3 is the same as R2 or is the product of the reaction thereof in the steps of oxidation, reductive amination and reaction with reagent I. The formation of a compound according to this embodiment is shown in Figure 6, wherein the reagent I is a bis-NHS crosslinker. In a second embodiment the starting compound has a reducing terminal sialic acid, joined to another moiety via its 8-carbon atom, and in which the preliminary step involves a ketal ring-opening reduction step whereby a group having vicinal diols is formed followed by a selective oxidation step in which the vicinal diol group is oxidised to an aldehyde group, followed by reductive amination with H2NR4 or acid addition salt to form the intermediate. In this embodiment the starting compound has the following formula .1 COOH iv HO in which R5 is the said other moiety and is selected from a saccharide group an oligo- or poly-saccharide group, an alkyl group, an acyl group, a lipid, a drug delivery system, and in which the amide product has the following in which R1, X and R4 are the same groups as in the respective starting compounds and R6 is the same as R5 or is the product of the reaction thereof in the steps of reduction, oxidation, amination and reaction with reagent I. The formation of a compound of formula V is shown in Figure 2. In a third embodiment the starting compound has a terminal sialic acid unit joined to another moiety via its 2-carbon atom (i.e. as a non-reducing terminal unit), and in which the preliminary step involves oxidation of the 7, C-8-diol group of the sialic acid to form an aldehyde group followed by reaction with hydrazine and reduction to form the intermediate. In this embodiment in which the starting compound has the following formula: HO- i i •R2 II HO in which R2 is the said other moiety and is selected from a mono-, di-, oligoor poly-saccharide group, a protein or peptide, a lipid, a drug or a drug delivery system and in which the product derivative has the following formula vm 8 in which X and R1 are the same as in the respective starting materials and R3 is the same as R2 or is the product of the reaction thereof in the steps of oxidation, reaction with hydrazine, reduction and reaction with reagent I, In a fourth embodiment the starting compound has a reducing end terminal sialic acid, joined to another moiety via its 8-carbon atom, and in which the preliminary step involves a ketal ring-opening reduction step whereby a group having vicinal diols is formed followed by a selective oxidation step in which the vicinal diol group is oxidised to an aldehyde group, followed by reaction with hydrazine and reduction to form the intermediate. In this embodiment in which the starting compound has the following formula rv in which R5 is the said other moiety and is selected from a mono-, di-, oligoand poly-saccharide group, an alkyl group, an acyl group, a lipid and a drug delivery system, and in which the product derivative has the following formula HQ in which X, R1 are same groups as in the respective starting compounds and in which R6 is the same as R5 or is the product of the reaction thereof in the steps of reduction, oxidation, reaction with hydrazine, reduction and reaction with reagent I. An example of a reaction scheme which produces compounds of formula IX is shown in Figure 5, wherein the bifunctional reagent I is bis-NHS. In the process it is generally important that the intermediate is isolated substantially from the product mixture of the preliminary step prior to being contacted with the reagent of formula I. This is because the reagents used in the preliminary step(s) may inactivate the reagent of formula I. In addition, where the preliminary step involves sequential steps of oxidation and reduction or vice versa the oxidising agents or reducing agents of the first step should be inactivated before adding the reagent for the subsequent step. In the process of the invention, it is convenient for the reaction between the intermediate and the reagent of formula I to be conducted in an aprotic solvent, preferably comprising a small amount of a protic solvent. Minimising the level of protic solvent present in the reaction avoids premature deactivation of the NHS group of the reagent of formula I. In general aprotic solvents are found to damage biological molecules. It is surprising that the use of dimethylsulphoxide DMSO, specifically to solubilise PSAs, results in good levels of conjugation to NHS reagents, without excess levels of deactivation of the NHS groups prior to reaction, and allows recovery of the derivative from the product mixture. Preferably therefore the aprotic solvent is DMSO. The reagent of formula 1 is generally used in an amount which is in stoichiometric excess for reaction with the intermediate, and is preferably present in an amount at least twice, more preferably at least five times the amount for stoichiometric reaction with the intermediate. In one embodiment of the reagent of formula I, X is a group 10 in which R has the same definition as above. In an alternative embodiment X is a group selected from the group consisting of vinylsulphone, N-maleimido, N-iodoacetamido, orthopyridyl disulfide, protected hydroxyl, protected amino, and azido. The reagent of formula I is preferably selected from: N-(a-maleimidoacetoxy)succinimide ester, (AMAS), N-(p-maleimidopropyloxy)succinimide ester, (BMPS), N-(^-maleimidocapryloxy)succinimide ester, (EMCS), or its sulfo analog, N-(Y-maleimidobutyryloxy)succinimide ester, (GMBS), or its sulfo analog, succinimidyl-4-(NH7ialeimidomethyl)-cyclohexane-1 -carboxy-(6- amidocaproate), (LC-SMCC), m-maleimido benzoyl-N-hydroxysuccinimide ester (MBS), or, its sulfo analog, succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxyate) (SMCC) or its sulfo analog, succinimidyl-4-(p-maleimido phenyl) butyrate (SMPB) or its sulfo analog, succinimidyl-6-(|3-maleimido-propionamido) hexanoate (SMPH), N-(k-maleimidoundecanoyloxy) sulfosuccinimide-ester(sulfo-KMUS), succinimidyl 6-[3-2(2-pyridyldithio)-propionamido]hexanoate(LCSPDP) or its sulfo analog, 4-succinimidyloxycarbonyl-methyl-a-(2-pyridyldithio) toluene (SMPT) or its sulfo-LC analog, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl [4-vinylsulfonyl) benzoate (SVSB), succinimidyl 3-(bromoacetamido)propionate (SBAP), and N-succinimidyliodoacetate (SI A) and N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB) or its sulfo analog. Another category of heterobifunctional reagents of formula I have photoreactive groups as X, such as azide groups. Examples of such reagents are: N-5-Azido-2-nitrobenzoyloxysuccinimide water insoluble (ANB-NOS), N-Hydroxysuccinimidyl-4-azidosalicylic acid water insoluble, noncleavable (NHS-ASA), N-Succinimidyl (4-azidophenyl)-1,3'-dithiopropionate (SADP), Sulfosuccinimidyl2-(7-azido-4-methyl-coumarin-3-acetamido) ethyl- 1,3'-dithiopropionate (SAED), Sulfosuccinimidyl 2-(m-azido-o-nitro-benzamido)ethyl-1,3'- dithiopropionate (SAND), A/-Succinimidyl6-(4'-azido-21-nitro-phenylamino)hexanoate (SANPAH), Sulfosuccinimidyl 2-(p-azido-o-salicylamido)ethyl-1,3'- dithiopropionate (SASD), Sulfosuccinimidyl-(perfluoroazidobenzamido) ethyl-1,3'- dithiopropionate (SFAD), and A/-Hydroxysulfosuccinimidyl-4-azidobenzoate (Sulfo-HSAB). The reagent of formula I may be selected from bis[2- succinimidyloxycarbonyl-oxy)ethyl]sulfone (BSOCOES) and its sulfo analog, bis(sulfosuccinimidyl)suberate) (BS3), disuccinimidyl glutarate (DSG), dithiobis (succinimidyl propionate) (DSP), disuccinimidyl suberate (DSS), disuccinimidyl tartrate (DST) or its sulfo analog, 3,3'-dithiobis (Sulfosuccinimidyl propionate) (DTSSP), and ethylene glycol bis(succinimidyl succinate) (EGS) and its sulfo analog. The group R1 is a difunctional organic radical. Preferably, R1 is selected from the group consisting of alkanediyl, arylene, alkarylene, heteroarylene and alkylheteroarylene, any of which may substituted and/or interrupted by carbonyl, ester, sulfide, ether, amide and/or amine linkages. Particularly preferred is C3-C6 alkanediyl. Most preferably, R1 corresponds to the appropriate portion of one of the preferred reagents I listed above. The substituent group may be chosen from those listed for R1 above, or alternatively may be an amino acid side chain. In the process preferably the product derivative is isolated substantially completely from any excess reagent. Reaction conditions for the reactions generally used may also be used here, for instance with reference to Hermanson, (1995). More preferably, the product amide derivative is isolated substantially completely from the product mixture. Such isolation and recovery may involve a drying step preferably carried out under reduced pressure and most preferably a freeze-drying step. Thus reactive sialic acid derivatives useful for subsequent reaction with biologically useful compounds may be made available in a stable form. The invention is illustrated further in the accompanying examples and Figures. the following is a brief description of the drawings: Figure 1a is a reaction scheme showing the prior art activation of the non-reducing sialic acid terminal unit; Figure 1b is a reaction scheme showing the prior art reductive amination of the aldehyde moiety of reaction scheme 1a using a proteinamine moiety; Figure 2 shows the preparation of reducing and derivatised NHS colominic acid (when non-reducing end has no vicinal diol); Figure 3 shows the preparation of reducing end derivatised NH2-CA colominic acid (vicinal diol removed at non-reducing end); 13 Figure 4 shows the general scheme for preparation of CA-NHSprotein conjugation; Figure 5 shows the preparation of CA-protein conjugates via NHS on reducing end; Figure 6 shows preparation of non-reducing end derivatised CA; Figure 7 shows the preparation of CA-protein conjugates using bis(sulfosuccinimidyl) suberate (BS3) on non-reducing end; Figure 8 shows the schematic reprsentation of CA-protein conjugation using the crosslinker DSG; Figure 9 shows the HPLC of the CA-GH conjugation reactions; Figure 10 shows the sodium dodecyl sulphate (SDS)-polyacrylamide gel eletrophoresis (PAGE) of CA-NHS-GH conjugates (CA 35kDa); Figure 11 shows native PAGE of unreacted CAs; Figure 12 shows the SDS-PAGE of CAM-p-gal and CAI-p-gal conjugates; Figure 13 shows the SDS-PAGE analysis of the CAH-NHS reactions; and Figure 14 shows the size exclusion chromatography analysis of the CAH-NHS reactions. Examples Materials Sodium meta-periodate and molecular weight markers were obtained from Sigma Chemical Laboratory, UK. The CAs used, linear alpha-(2,8)- linked £. coli K1 PSAs (22.7 kDa average, polydispersity (p.d.) 1.34; 39kDa p.d. 1.4; 11kDa, p.d. 1.27) were from Camida, Ireland. Other materials included 2,4 dinitrophenyl hydrazine (Aldrich Chemical Company, UK), dialysis tubing (3.5kDa and 10kDa cut off limits (Medicell International Limited, UK); Sepharose SP HiTrap, PD-10 columns (Pharmacia, UK); XK50 column (Amersham Biosciences, UK); Sepharose Q FF (Amersham Biosciences); Tris-glycine polyacrylamide gels (4-20% and 16%), Trisglycine sodium dodecylsulphate running buffer and loading buffer (Novex, 14 UK). Deionised water was obtained from an Elgastat Option 4 water purification unit (Elga Limited, UK). All reagents used were of analytical grade. A plate reader (Dynex Technologies, UK) was used for spectrophotometric determinations in protein or CA assays. Methods Protein and CA determination Quantitative estimation of CAs, as sialic acid, was carried out by the resorcinol method [Svennerholm 1957] as described elsewhere [Gregoriadis et. al., 1993; Fernandes and Gregoriadis, 1996,1997]. GH was measured by the bicinchoninic acid (BCA) colorimetric method. Reference Example 1: Fractionation of CA by IEC (CA, 22.7 kDa, pd 1.34) An XK50 column was packed with 900 ml Sepharose Q FFand equilibrated with 3 column volumes of wash buffer (20mM triethanolamine; pH 7.4) at a flow rate of 50ml/min. CA (25 grams in 200 ml wash buffer) was loaded on column at 50 ml/min via a syringe port. This was followed by washing the column with 1.5 column volumes (1350ml) of washing buffer. The bound CA was eluted with 1.5 column volumes of different elution buffers (Triethanolamine buffer, 20 mM pH 7.4, with OmM to 475mM NaCI in 25 mM NaCI steps) and finally with 1000mM NaCI in the same buffer to remove all residual CA and other residues (if any). The samples were concentrated to 20 ml by high pressure ultra filtration over a 5kDa membrane (Vivascience, UK). These samples were buffer exchanged into deionised water by repeated ultra filtration at 4°C. The samples were analysed for average molecular weight and other parameters by GP) and native PAGE (stained with alcian blue). Narrow fractions of CA produced using above procedure were oxidised with sodium periodate and analysed by GPC and native PAGE for gross alteration to the polymer. Reference Example 2: Activation of CA Freshly prepared 0.02 M sodium metaperiodate (NalO4; 6 fold molar excess over CA) solution was mixed with CA at 20°C and the reaction 15 mixture was stirred magnetically for 15 min in the dark (as shown in the first step of Figure 3). The oxidised CA was precipitated with 70% (final concentration) ethanol and by centrifuging the mixture at SOOOg for 20 minutes. The supernatant was removed and the pellet was dissolved in a minimum quantity of deionised water. The CA was again precipitated with 70% ethanol and then centrifuged at 12,000 g. The pellet was dissolved in a minimum quantitiy of water, lyophilized and stored at -20°C until further use. Reference Example 3: Determination of the oxidation state of CA and derivatives Quantitative estimation of the degree of CA oxidation was carried out with 2,4 dinitrophenylhydrazine (2,4-DNPH), which yields sparingly soluble 2,4 dinitrophenyl-hydrazones on interaction with carbonyl compounds. Non-oxidised (CA) and oxidised CA (CAO) (5mg each) were added to the 2,4-DNPH reagent (1.0ml), the solutions were shaken and then allowed to stand at 37°C until a crystalline precipitate was observed [Shriner et. al., 1980]. The degree (quantitative) of CA oxidation was measured with a method [Park and Johnson, 1949] based on the reduction of ferricyanide ions in alkaline solution to ferric ferrocyanide (Persian blue), which is then measured at 630nm. In this instance, glucose was used as a standard. Reference Example 4a: Preparation of amino coiominic acid (CA-NH2) CAO produced as in Reference Example 2 at 10-100 mg/ml was dissolved in 2 ml of deionised water with a 300-fold molar excess of NH4CI, in a 50 ml tube and then NaCNBH4 (5 M stock in 1 N NaOH(aq)), was added at a final concentration of 5 mg/ml (Figure 4, first step). The mixture was incubated at room temperature for 5 days. A control reaction was also set up with CA instead of CAO. Product coiominic acid amine derivative was precipitated by the addition of 5 ml icecold ethanol. The precipitate was recovered by centrifugation at 4000 rpm, 30 minutes, room temperature in a benchtop centrifuge. The pellet was retained and resuspended in 2 ml of deionised water, then precipitated again with 5 ml of ice-cold ethanol in a 10 ml ultracentrifuge tube. The precipitate was collected by centrifugation at 30,000 rpm for 30 minutes at room temperature. The pellet was again resuspended in 2 ml of deionised water and freeze-dried. Reference Example 4b: Assay for amine content The TNBS (picrylsulphonic acid, i.e. 2, 4, 6-tri-nitro-benzene sulphonic acid) assay was used to determine the amount of amino groups present in the product [Satake et. al., 1960]. In the well of a microtitre plate TNBS (0.5 ul of 15 mM TNBS) was added to 90 ul of 0.1 M borate buffer pH 9.5. To this was added 10 ul of a 50 mg/ml solution of CA-amide the plate was allowed to stand for 20 minutes at room temperature, before reading the absorbance at 405nm. Glycine was used as a standard, at a concentration range of 0.1 to 1mM. TNBS trinitrophenylates primary amine groups. The TNP adduct of the amine is detected. Testing the product purified with a double cold-ethanol precipitation using the TNBS assay showed close to 90 % conversion. Example 1: Preparation of CA-NHS CA-NH2 (35 kDa) (15-20 mg) synthesised in Reference Example 4a above was dissolved in 0.15M PBS (350 jiL, pH 7.2) and then either 50 or 75 molar equivalents of BS3 in PBS (150 nL, PH 7.2) was added. The mixture was vortexed for 5 seconds and then reacted for 30 minutes at 20°C. This is shown generally in Figure 4, second step, for a homobifunctional cross-linker and more specifically in Figure 7 for BS3. The CA-NHS product was purified by PD-10 column using PBS as eluent (pH 7.2) and used immediately for site-specific conjugation to the NH2 groups in proteins and peptides. Determination of the CA concentration from the PD 10 fractions was achieved by analysing the sialic acid content using the resorcinol assay. The NHS content on the CA polymer was measured by UV spectroscopy by analysing the CA and NHS reaction solution at 260nm and also by thin layer chromatography with visualization at 254nm. CA-NH2 (35 kDa) (15-20 mg) synthesised in Example 1 above was either dissolved in the minimum amount of water (50-65 u.L) to which was added DMSO (300-285 ul) or in >95% DMSO (350 nL) with the aid of heat (100-125°C). 75 molar equivalents of DSG in DMSO (150 L) was added to the CA-NH2 solution, vortexed for 5 seconds and then reacted for 30 minutes at 20°C (Figure 8). The CA-NHS product was purified either with dioxane precipitation (x2) or by PD-10 column using PBS as eluent (pH 7.2) and used immediately for site-specific conjugation to the NH2 groups in proteins and peptides. As before determination of the CA concentration from the PD- 10 fractions was measured using the resorcinol assay. The NHS content on the CA polymer was measured by UV spectroscopy (260nm) and by thin layer chromatography (254nm). Example 2: Preparation of CA-NHS-protein conjugates (using BS3 and DSG) GH in sodium bicarbonate (pH 7.4) was covalently linked to CA-NHS (35kDa), from reference example 4b with an excess of BS3. The reaction was performed in 0.15 M PBS (pH 7.2; 1.5ml) using a molar ratio of 25:1 or 50:1 of CA-NHS:GH for a period of 30 minutes at 20°C. Polysialylated GH was characterised by SDS-PAGE and the conjugation yield determined by FPLC-size exclusion chromatography. Controls included subjecting the native protein to the conjugation procedure using BS3 in the absence of any CA-NHS. CA-NH2 was also subjected to the conjugation procedure using BS3 in the absence of native GH. GH in sodium bicarbonate (pH 7.4) was covalently linked to CA-NHS (35kDa), which was prepared as discussed in example 4b using an excess of DSG. The reaction was performed in 0.15 M PBS (pH 7.2; 1.5ml) using a molar ratio of 50:1 of CA-NHS:GH for a period of 30 minutes at 20°C. Polysialylated GH was characterised by SDS-PAGE and the conjugation yield determined by HPLC-size exclusion chromatography. Controls included subjecting the native protein to the conjugation procedure using DSG in the absence of any CA-NHS. CA-GH conjugates were dissolved in ammonium bicarbonate buffer (0.2M; pH7) and were chromatographed on superose 6 column with detection by UV index (Agilent, 10/50 system, UK). Samples (1mg/ml) were filtered over 0.45um nylon membrane 175 ul injected and run at 0.25cm/min with ammonium bicarbonate buffer as the mobile phase (Fig. 9). SDS-PAGE (MiniGel, Vertical Gel Unit, model VGT1, power supply model Consort E132; VWR, UK) was employed to detect changes in the molecular size of GH upon polysialylation. SDS-PAGE of GH and its conjugates (with CA-NHS) of 0 (control) and 30 minutes samples from the reaction mixtures as well as a process control (non oxidised CA), was carried out using a 4-20% polyacrylamide gel. The samples were calibrated against a wide range of molecular weight markers (Figs. 10 and 11). RESULTS CA and its derivatives (22.7kDa) were successfully fractionated into various narrow species with a polydispersity less than 1.1 with m.w. averages of up to 46 kDa with different % of populations. Table 2 shows the results of separating the 22.7kDa material. (Table Removed) This process was scalable from 1 ml to 900 ml of matrix with the fractionation profile almost identical at each scale (not all results shown). [The fractionation of larger polymer (CA, 39kDa, pd 1.4) produced species up to 90kDa. This process can successfully be used for the fractionation of even large batches of the polymer. The results show that the ion exchange fractions are narrowly dispersed. This is consistent with the GPCdata.] All narrow fractions were successfully oxidised with 20mM periodate and samples taken from different stages of the production process and analysed by GPC and native PAGE showed no change in the molecular weight and polydispersity. Quantitative measurement of the oxidation state of CA was performed by ferricyanide ion reduction in alkaline solution to ferrocyanide (Prussian Blue) [Park and Johnson, 1949] using glucose as a standard. The oxidized CA was found to have a nearly 100 mol % of apparent aldehyde content as compared to native polymer. The results of quantitative assay of CA intermediates in the oxidation process using ferricyanide were consistent with the results of qualitative tests performed with 2,4 dinitrophenylhydrazine which gave a faint yellow precipitate with the native CA, and intense orange colour with the aldehyde containing forms of the polymer, resulting in an intense orange precipitate after ten minutes of reaction at room temperature. The amination of the polymer was found to be 85% and the CA-NHS was positive for NHS. Further, the thiol content of the polymer was found to be 60% The integrity of the internal alpha-2,8 linked NeuSAc residues post periodate and borohydride treatment was analysed by GPC and the chromatographs obtained for the oxidised (CAO), amino CA (CA-NH2), CANHS materials were compared with that of native CA. It was found (Fig. 9) that all CAs exhibit almost identical elution profiles, with no evidence that the various steps give rise to significant fragmentation or crosslinking (in case of CA-NHS) of the polymer chain. The small peaks are indicative of buffer salts. Formation of the CA-GH conjugates was analysed by SEC-HPLC and SDS-PAGE. For the conjugation reaction with DSG the SDS-PAGE showed that there was no free GH remaining and that the conjugation reaction had gone to completion. This was confirmed by SEC-HPLC, whereby the CA-GH conjugates were eluted before the expected elution time of the free GH (a peak for free GH was not observed). On the other hand, analysis by SDSPAGE of the conjugation reaction of CA-NH2 to GH using BS3showed the presence of free GH, which was confirmed by SEC-HPLC with an elution peak around 70 minutes for the free protein. In addition, the SEC-HPLC enable the degree of conjugation to be determined at 53%. The results (Fig. 10) show that in the conjugate lanes there are shifts in the bands which typically indicates an increase in mass indicative of a polysialylated-GH in comparison to GH. Further, GH conjugates were separated into different species by SEC-HPLC. 3.1 Synthesis To 40 mg colominic acid amine (85 mol % amine) as (described in Reference Example 2) dissolved in 1 ml of PBS pH 7.4 was added 5 mg of N-succinimidyl iodoacetate (SIA). The mixture was left to react for 1h at 25°C in the dark, after which excess SIA was removed by gel filtration over a 5ml Hightrap™ Desalting column(AP Bioscience) eluted with PBS. 0.5 ml fractions were collected from the column and samples from each fraction tested for colominic acid content (resorcinol assay) and reactivity with cysteine indicating Iodide (Ellman's Assay). Fractions positive for both iodide and CA were pooled. 3.2 Conjugation of CAI to (l-galactosidase To E.co// B-galactosidase (5.0 mg, 4.3 xlO"8 mol) in 1ml PBS 15 mg CAI was added (6.59 x10-7 mol, 15 molar equiv). The tube was sealed wrapped in foil and the reaction was allowed to proceed at room temperature for 1 h whilst gently mixing. The resulting conjugate was analysed by SDS page and then purified according to accepted protocols to remove free CAI. Samples were assayed for polymer and protein content as outlined above. Control reactions were carried out with CA as a negative control. All samples were analysed for ft-gal activity as described below in section 3.3. 3.3 Assay for Enzyme Activity Standards from 60 ng/ml to 3.75 ng/ml of fresh (i-galactosidase were prepared in PBS. Sample of CAM-B-gal were diluted to 60 ug/ml in the same buffer. Enzyme activity of the conjugates was measured as follows: In a microtitre plate, to 100 \i\ of sample or standard was added 100 nl of Allin- One IJ-gal substrate (Pierce). The plate was incubated at 37°C for 30 min and absorbance read at 405nm. A calibration curve was prepared from the standards and the activity of the samples calculated from the equation for the linear regression of the curve. 3.4 Conclusions Fractions 3-6 were positive for both polymer and iodoacetate and were pooled. The SDS page (4-12 % Bis/Tris gel; Figure 12) showed an increase in apparent molecular mass for samples incubated with the iodoacetamide derivative but not with control polymer. From the protein and polymer assays the conjugation ratio was determined to be 1.63 CA1:1 li-gal. fc-gal activity was calculated to be 100. 9 % for the conjugated sample, compared to the free enzyme. Example 4: Preparation of colominic acid hydrazide (CAH) 4.1 Synthesis 50mg of oxidised colominic acid (19kDa) was reacted with 2.6mg of hydrazine (liquid) in 400ul of 20mM sodium acetate buffer, pH 5.5, for 2h at 25°C. The colominic acid was then precipitated with 70% ethanol. The precipitate was redissolved in 350ul phosphate buffer saline, pH 7.4 and NaCNBH3 was added to 5mg/ml. The mixture was allowed to react for 4h at 25°C, then frozen overnight. NaCNBH3 and reaction by products were removed by gel permeation chromatography on a PD10 column packed with Sephadex G25, using 0.15M NH4HCO3 as the mobile phase. The fractions (0.5ml each) were analysed by the TNBS assay (specific to amino groups; described earlier). Fractions 6, 7, 8 and 9 (the void volume fractions) had a strong singal, well above the background. The background was high due to the presence of the NH/ ions. Fractions 6, 7, 8 and 9 also contained colominic acid. These four fractions, were freeze dried to recover the CAhydrazide (CAH). 4.2 Preparation of colominic acid NHS (CA-NHS) and colominic acid-protein conjugates 10mg of 19kDa CA-hydrazide were reacted with 9mg of BS3 in 400ul of PBS (pH 7.4) for 30 minutes at room temperature. The reaction mixture was applied to a PD-10 column packed with Sephadex G25 collecting 0.5ml fractions. 0.1 mg of BSA was added to each fraction between 5 and 9. After 2 hours at room temperatures the fractions reacted with BSA. These samples were analysed by SDS-PAGE and SEC HPLC. These fractions have little colominic acid. The colominic acid rich fractions (6 and 7) have a protein streak in addition to the bands present in the other samples and BSA, which is clear evidence of conjugation (Fig. 13). 23 The HPLC chromatogram of fraction 6 shows that there is a big shift in the retention time for conjugate as compared to free protein confirming conjugation (Fig. 14a and b). The BSA used contains impurities. The BSA peak is at 56 minutes (Fig. 14a). In addition to peak at 56 minutes, there are larger species which are conjugates. There is a large peak at 80 minutes, which is the NHS released from the CA-NHS as it reacts with the protein. This cannot be free BS3 as the CAM was passed through a gel permeation chromatography column, which will have removed. This strongly suggests that an NHS ester group was created on the CA molecule (Fig. 14b). References Bendele, A., Seely, J., Richey, C., Sennello, G., Shopp, G., Renal tubular vacuolation in animals treated with polyethylene-glycol conjugated proteins, Toxicological sciences, 42 (1998) 152-157. Beranova, M., Wasserbauer, R., Vancurova, D., Stifter, M., Ocenaskova, J., Mora, M., Biomaterials, 11 (2000) 521-524. Brocchini, S., Polymers in medicine: a game of chess. Drug Discovery Today, 8, (2003) 111-112. Garlsson, J., Drevin, H. And Axen, R., Biochem Journal, 173, (1978), 723-737. Cheng T, Wu, M., Wu.P., Chern, J, Roffer, SR., Accelerated clearance of polyethylene glycol modified proteins by anti-polyethylene glycol IgM. Bioconjugate chemistry, 10 (1999) 520-528. Cho, J.W. and Troy, F. A., PSA engineering: Synthesis of polysialylated neoglycosphingolipid by using the polytransferase from neuroinvasive E.coli K1, Proceedings of National Academic Sciences, USA, 91(1994)11427-11431. Convers, C. D. , Lejeune, L., Shum, K. , Gilbert, C., Shorr, R.G.L, Physiological effect of polyethylene glycol conjugation on stroma-free bovine hemoglobin in the conscious dog after partial exchange transfusion, Artificial organ, 21 (1997) 369-378. Dyer, J.R., Use of periodate oxidation in biochemical analysis, Methods of Biochemical Analysis, 3 (1956) 111-152. Fernandes, A.I., Gregoriadis, GM Polysialylated asparaginase: preparation, activity and pharmacokinetics, Biochimica et Biophysica Acta, 1341(1997)26-34. Fernandes, A.I., Gregoriadis, G., Synthesis, characterization and properties of polysialylated catalase, Biochimica et Biophysica Acta, 1293 (1996)92-96. Fernandes, A.I., Gregoriadis, G., The effect of polysialylation on the immunogenicity and antigenicity of asparaginase: implications in its pharmacokinetics, International Journal of Pharmaceutics, 217 (2001) 224. Fleury, P., Lange, J., Sur I'oxydation des acides alcools et des sucrespar I'acid periodique, Comptes Rendus Academic Sciences, 195 (1932)1395-1397. Gregoriadis, G., Drug and vaccine delivery systems, in: PharmaTech, World Markets Research Centre Limited, London (2001) 172-176. Gregoriadis, G., Fernandes, A., McCormack, B., Mital, M., Zhang, X, Polysialic acids: Potential for long circulating drug, protein, liposome and other microparticle constructs, in Gregoriadis, G and McCormack, B (Eds), Targeting of Drugs, Stealth Therapeutic Systems, Plenum Press, New York (1998)193-205. Gregoriadis, G., Fernandes, A., Mital, M., McCormack, B., Polysialic acids: potential in improving the stability and pharmacokinetics of proteins and other therapeutics, Cellular and Molecular Life Sciences, 57 (2000) 1964-1969. Gregoriadis, G., McCormack, B., Wang, Z., Lifely, R., Polysialic acids: potential in drug delivery, FEBS Letters, 315 (1993) 271-276. Hermanson, G. T., Bioconjugate techniques, Acadamic press, London, 1995. reczuk-Hirst, D., Jain, S., Genkin, D., Laing, P., Gregoriadis, G., Preparation and properties of polysialylated interferon-a-2b, AAPS Annual Meeting, 2002, Toronto, Canada, M1056 Hunter, A. C, Moghimi, S. M., Therapeutic synthetic polymers: a game of Russian Roulette. Drug Discovery Today, 7 (2002) 998-1001. Jain, S., Hirst, D. H., McCormack, B., Mital, M., Epenetos, A, Laing, P., Gregoriadis, G., Polysialylated insulin: synthesis, characterization and biological activity in vivo, Biochemica et. Biophysics Acta, 1622 (2003) 42-49. Jain, S., Hirst, D.H., Laing, P., Gregoriadis, G., Polysialylation: The natural way to improve the stability and pharmacokinetics of protein and peptide drugs, Drug Delivery Systems and Sciences, 4(2) (2004) 3-9. Jennings, H. J., Lugowski, C., Immunogenicity of groups A, B, and C meningococal polysaccharide tetanus toxoid conjugates, Journal of Immunology, 127(1981)1011-1018. Lifely, R., Gilbert, AS., Moreno, C.C., Sialicacid polysaccharide antigen of Neisseria meningitidis and Escherichia coli: esterification between adjacent residues, Carbohydrate Research, 94 (1981) 193-203. Mital, M., Polysialic acids: a role for optimization of peptide and protein therapeutics, Ph.D. Thesis, University of London, 2004. Muflenhoff, M., Ectehardt, M., Gerardy-Schohn, R., Polysialic acid: three-dimensional structure, biosynthesis and function, Current opinions in Structural Biology, 8 (1998) 558-564. Park, J.T., Johnson, M.J., A submicrodetermination of glucose, Journal of Biological Chemistry, 181 (1949) 149-151. Roth, J., Rutishauser, U., Troy, F.A. (Eds.), Polysialic acid: from microbes to man, BirkhSuser Verlag, Basel, Advances in Life Sciences, 1993. Rutishauser, U., Polysialic acid as regulator of cell interactions in: R.U. Morgoles andR.K. Margalis (eds.), Neurobiology of Glycoconjugates, pp 367-382, Plenum Press, New York, 1989. Satake, K., et. al., J. Biochem., 47, 654, 1960. Shriner, R. L, Fuson, R.D.C., Curtin, D.Y., Morill, T.C., The Systematic Identification of Organic Compounds, 6th ed., Wiley, New York, 1980. Svennerholm, L., Quantitative estimation of sialicacid II: A colorimetric resorcinol-hydrochloric acid method, Biochimca et Biophysica Acta, 24 (1957)604-611. Troy, F. A. Polysialylation of neural cell adhesion molecules, Trends in Glycoscience and Glycotechnology, 2 (1990) 430-449. Troy, F.A., Polysialylation: From bacteria to brain, Glycobiology, 2 (1992)1-23. We Claim- 1. A process for forming a derivative of a sialic acid compound in which a starting compound comprising a terminal sialic acid unit is subjected to a preliminary intermediate - forming step comprising the sequential steps of oxidation and reduction or vice versa in which a group selected from a primary amine group, a secondary amine group and a hydrazine is formed on the terminal sialic acid unit, wherein the terminal sialic acid unit is joined to another moiety via its 2-carbon atom or via its 8-carbon atom, followed by a reaction step in which the intermediate is reacted with a bifunctional reagent, of formula I (Formula Removed) in which R is H or sulphonyl; R1 is a linker group; and X is a functional group, whereby the ester group is cleaved and the amine or hydrazine group of the intermediate is acylated by -CO-R1-X to form the derivative. 2. A process as claimed in claim 1 in which the starting compound has a terminal sialic acid unit joined to another moiety via its 2-carbon atom, and in which the preliminary step involves oxidation of the 7, 8-diol group of the sialic acid to form an aldehyde group followed by reductive amination with H2NR4 in which R4 is H or lower alkyl, or acid addition salt thereof to form the intermediate. 3. A process as claimed in claim 2 in which the starting compound has the following formula: (Formula Removed) in which R2 is the said other moiety and is selected from a mono-, di-, oligo- or poly-saccharide group, a protein or peptide, a lipid , a drug or a drug delivery system and in which the amide derivative product has the following formula: (Formula Removed) in which X, R1 and R4 are the same groups as in the respective starting compounds and R3 is the same as R2 or is the product of the reaction thereof in the steps of oxidation, reductive amination and reaction with reagent I. 4. A process as claimed in claim 1 in which the starting compound has a reducing terminal sialic acid, joined to another moiety via its 8-carbon atom, and in which the preliminary step involves a ketal ring-opening reduction step whereby a group having vicinal diols is formed followed by a selective oxidation step in which the vicinal diol group is oxidised to an aldehyde group, followed by reductive amination with H2NR4 in which R4 is H or lower alkyl, or acid addition salt to form the intermediate. 5. A process as claimed in claim 4 in which the starting compound has the following formula (Formula Removed) in which R5 is the said other moiety and is selected from a saccharide group an oligo- or poly¬saccharide group, an alkyl group, an acyl group, a lipid and a drug delivery system, and in which the amide product has the following formula (Formula Removed) in which R1, X and R4 are the same groups as in the respective starting compounds and R6 is the same as R5 or is the product of the reaction thereof in the steps of reduction, oxidation, amination and reaction with reagent I. 6. A process as claimed in claim 1 in which the starting compound has a terminal sialic acid unit joined to another moiety via its 2-carbon atom, and in which the preliminary step involves oxidation of the 7, 8-diol group of the sialic acid to form an aldehyde group followed by reaction with hydrazine and reduction to form the intermediate. 7. A process as claimed in claim 6 in which the starting compound has the following formula: (Formula Removed) in which R2 is the said other moiety and is selected from a mono-, di-, oligo- or poly-saccharide group, a protein or peptide, a lipid, a drug or a drug delivery system and in which the product derivative has the following formula (Formula Removed) in which X and R1 are the same as in the respective starting materials and R3 is the same as R2 or is the product of the reaction thereof in the steps of oxidation, reaction with hydrazine, reduction and reaction with reagent I. 8. A process as claimed in claim 1 in which the starting compound has a reducing terminal sialic acid, joined to another moiety via its 8-carbon atom, and in which the preliminary step involves a ketal ring-opening reduction step whereby a group having vicinal diols is formed followed by a selective oxidation step in which the vicinal diol group is oxidised to an aldehyde group, followed by reaction with hydrazine and reduction to form a the intermediate. 9. A process as claimed in claim 8 in which the starting compound has the following formula (Formula Removed) in which R5 is the said other moiety and is selected from a mono-, di-, oligo- or poly-saccharide group, an alkyl group, an acyl group, a lipid and a drug delivery system, and in which the product derivative has the following formula (Formula Removed) in which X, R1 are same groups as in the respective starting compounds and in which R6 is the same as R5 or is the product of the reaction thereof in the steps of reduction, oxidation, reaction with hydrazine, reduction and reaction with reagent I. 10. A process as claimed in any of claims 1 to 9 in which the intermediate is isolated substantially from the product mixture of the preliminary step prior to being contacted with the reagent of formula I. 11. A process as claimed in any of claims 1 to 10 in which the reaction between the intermediate and the reagent of general formula I is conducted in an aprotic solvent, preferably comprising a small amount of a protic solvent. 12. A process as claimed in claim 11 in which the aprotic solvent is dimethylsulfoxide, and the protic solvent is water. 13. A process as claimed in any of claims 1 to 12 in which the reagent of formula I is present in stoichiometric excess for reaction with the intermediate, preferably present in an amount of at least twice, more preferably present in an amount of at least five times the amount for stoichiometric reaction with the intermediate. 14. A process as claimed in any of claims 1 to 13 in which X is a group (Formula Removed) in which R has the same definition as in claim 1. 15. A process as claimed in any of claims 1 to 14 in which the reagent of formula I is selected from the group consisting of bis[2-succinimidyloxycarbonyl-oxy)ethyl]sulfone (BSOCOES) and its sulfo analog, bis(sulfosuccinimidyl)suberate) (BS3), disuccinimidyl glutarate (DSG), dithiobis (succinimidyl propionate) (DSP), disuccinimidyl suberate (DSS), disuccinimidyl tartrate (DST) or its sulfo analog, 3,3'-dithiobis (sulfosuccinimidyl propionate) (DTSSP), and ethylene glycol bis(succinimidyl succinate) (EGS) and its sulfo analog. 16. A process as claimed in any of claims 1 to 13 in which X is a functional group selected from vinylsulphone, N-maleimido, N-iodoacetamido, orthopyridyl disulfide, protected hydroxyl, protected amino, adazido. 17. A process as claimed in claim 16 in which the reagent is selected from the group consisting of N-(a-maleimidoacetoxy)succinimide ester, (AMAS), N-((3-maleimidopropyloxy)succinimide ester, (BMPS), N-(ε;-maleimidocapryloxy)succinimide ester, (EMCS), or its sulfo analog, N-(Y-maleimidobutyryloxy)succinimide ester, (GMBS), or its sulfo analog, succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate), (LC- SMCC), m-maleimido benzoyl-N-hydroxysuccinimide ester (MBS), or, its sulfo analog, succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxyate) (SMCC) or its sulfo analog, succinimidyl-4-(p-maleimido phenyl) butyrate (SMPB) or its sulfo analog, succinimidyl-6-((3-maleimido-propionamido) hexanoate (SMPH), N-(k-maleimidoundecanoyloxy) sulfosuccinimide-ester(sulfo-KMUS), succinimidyl 6-[3-2(2-pyridyldithio)-propionamido]hexanoate (LC-SPDP) or its sulfo analog, 4-succinimidyloxycarbonyl-methyl-a-(2-pyridyldithio) toluene (SMPT) or its sulfo-LC analog, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl [4-vinylsulfonyl) benzoate (SVSB), succinimidyl 3-(bromoacetamido)propionate (SBAP), and N-succinimidyliodoacetate (SIA) and N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB) or its sulfo analog. 18. A process as claimed in any preceding claim in which R1 is selected from the group consisting of alkanediyl, arylene, alkarylene, heteroarylene and alkylheteroarylene, any of which may be substituted and/or interrupted by carbonyl, ester, sulfide, ether, amide and/or amine linkages. 19. A process as claimed in claim 18 in which R1 is C3-C6 alkanediyl. 20. A process as claimed in any preceding claim in which the product derivative is isolated substantially completely from any excess reagent. 21. A process as claimed in claim 20 in which the product amide or hydrazide derivative is isolated substantially completely from the product mixture. 22. A process as claimed in claim 21 in which the product recovery is finished with a step of drying at reduced pressure to remove solvent. 23. A process as claimed in any preceding claim wherein the starting compound is a polysaccharide, wherein the polysaccharide has terminal sialic acid units and consists of sialic acid units. 24. A derivative of a sialic acid compound, of formula III or formula VIM, prepared by a process as claimed in claim 1 (Formula Removed) ih which X is a functional group selected from the group consisting of N-hydroxysuccinimide (NHS)-esters, vinyl sulfone, N-maleimido, N-iodoacetamido, orthopyridyl disulfide, hydroxy, protected hydroxyl, amino, protected amino, carboxyl, protected carboxyl, or azido; R1 is a linker; R4 is hydrogen or d.4 akyl; and R3 is a mono-, di-, oligo- or poly-saccharide, a protein, a peptide, a lipid, a drug or a drug delivery system. 25. A derivative of a sialic acid compound, of formula V or formula IX, prepared by a process as claimed in claim 1 (Formula Removed) in which X is a functional group selected from the group consisting of N-hydroxysuccinimide (NHS)-esters, vinyl sulfone, N-maleimido, N-iodoacetamido, orthopyridyl disulfide, hydroxy, protected hydroxyl, amino, protected amino, carboxyl, protected carboxyl, or azido; R1 is a linker; R4 is hydrogen or C1-4 akyl; and R6 is a mono-, di-, oligo- or polysaccharide group. 26. A compound as claimed in claim 24 or 25 in which R1 is selected from the group consisting of alkanediyl, arylene, alkarylene, heteroarylene and alkylheteroarylene, any of which may be interrupted by carbonyl, ester, sulfide, ether, amide and/or amine linkages. 27. A compound as claimed in claim 26 in which R1 is C3-C6 alkanediyl. 28. A compound as claimed in any of claims 24 to 27 in which R3 or R6 as the case may be is an oligo or poly-saccharide, wherein the oligo- or polysaccharide is an oligo- or polysialic acid.

Documents:

1100-DELNP-2007-Abstract-(02-12-2011).pdf

1100-delnp-2007-abstract.pdf

1100-delnp-2007-Claims-(06-04-2010).pdf

1100-DELNP-2007-Claims-(19-07-2011).pdf

1100-delnp-2007-Claims-(20-12-2011).pdf

1100-delnp-2007-claims.pdf

1100-DELNP-2007-Correspondence Others-(02-12-2011).pdf

1100-DELNP-2007-Correspondence Others-(05-12-2011).pdf

1100-DELNP-2007-Correspondence Others-(19-07-2011)..pdf

1100-delnp-2007-Correspondence Others-(19-07-2011).pdf

1100-DELNP-2007-Correspondence Others-(23-11-2011).pdf

1100-delnp-2007-Correspondence-Others-(06-04-2010).pdf

1100-delnp-2007-Correspondence-Others-(20-12-2011).pdf

1100-delnp-2007-correspondence-others-1.pdf

1100-DELNP-2007-Correspondence-Others.pdf

1100-delnp-2007-correspondence-po.pdf

1100-DELNP-2007-Description (Complete)-(02-12-2011).pdf

1100-delnp-2007-description (complete).pdf

1100-DELNP-2007-Drawings-(02-12-2011).pdf

1100-delnp-2007-drawings.pdf

1100-DELNP-2007-Form-1-(02-12-2011).pdf

1100-delnp-2007-form-1.pdf

1100-DELNP-2007-Form-13-(19-07-2011).pdf

1100-delnp-2007-form-18.pdf

1100-DELNP-2007-Form-2-(02-12-2011).pdf

1100-delnp-2007-form-2.pdf

1100-DELNP-2007-Form-3-(02-12-2011).pdf

1100-delnp-2007-Form-3-(19-07-2011).pdf

1100-DELNP-2007-Form-3.pdf

1100-delnp-2007-form-5.pdf

1100-delnp-2007-GPA-(20-12-2011).pdf

1100-delnp-2007-gpa.pdf

1100-delnp-2007-pct-101.pdf

1100-delnp-2007-pct-237.pdf

1100-delnp-2007-pct-306.pdf

1100-delnp-2007-pct-308.pdf

1100-delnp-2007-pct-311.pdf

1100-delnp-2007-pct-notification.pdf

1100-delnp-2007-Petition-137-(19-07-2011).pdf