Activated sialic acid derivatives for protein derivatisation and conjugation

Abstract

Derivatives of PSAs are synthesized, in which a reducing and/or non-reducing end terminal sialic acid unit is transformed into a N-hydroxysuccinimide (NHS) group. The derivatives may be reacted with substrates, for instance substrates containing amine or hydrazine groups, to form non-cross-linked/crosslinked polysialylated compounds. The substrates may, for instance, be therapeutically useful drugs, peptides or proteins or drug delivery systems.

Claims

1. A process of making an activated polysialic acid (PSA) molecule comprising a terminal sialic acid unit at the reducing end, the process comprising: a) reducing the terminal sialic acid unit at the reducing end to form a vicinal diol group; b) selectively oxidizing the vicinal diol group of step (a) to form an aldehyde group; c) aminating the aldehyde group of step (b) to form an amine group; and d) reacting the amine group of step (c) with a bifunctional reagent comprising two amine-reactive functional groups to form an activated PSA molecule, the activated PSA molecule being monofunctional and comprising an amine-reactive group at the reducing end.

2. The process of claim 1, wherein the PSA molecules consisting substantially only of units of sialic acid alpha-2,8 PSA molecules and/or alpha-2,9 linked PSA molecules.

3. The process of claim 1, wherein the PSA molecule comprises at least 5 sialic acid units.

4. The process of claim 3, wherein the PSA molecule comprises at least 10 sialic acid units.

5. The process of claim 4, wherein the PSA molecule comprises at least 50 sialic acid units.

6. The process of claim 1, wherein in step (a) the reducing agent used is a hydride or hydrogen with catalysts.

7. The process of claim 6, wherein the hydride is an alkali metal hydride.

8. The process of claim 1, wherein in step (a) the reducing agent used is, an alkali metal cyanoborohydride, L-ascorbic acid, sodium metabisulphite, or triacetoxyborohydride.

9. The process of claim 1, wherein in step (a) the terminal sialic acid unit at the reducing end is joined to an adjacent unit through its 8 carbon atom and the vicinal diol group formed is a 6,7-diol group.

10. The process of claim 9, wherein in step (b) the 6,7-diol group is oxidized to form an aldehyde group at carbon atom 7.

11. The process of claim 1, wherein in step (a) the terminal sialic acid unit at the reducing end is joined to an adjacent unit through its 9 carbon atom and the vicinal diol group formed is a 7,8-diol group.

12. The process of claim 11, wherein in step (b) the 7,8-diol group is oxidized to form an aldehyde group at carbon atom 8.

13. The process of claim 1, wherein in step (b) the selective oxidation is performed using an enzymatic oxidation process or a chemical oxidation process.

14. The process of claim 1, wherein in step (b) the selective oxidation is carried out using perruthenate, or periodate.

15. The process of claim 1, wherein each of the two amine-reactive functional groups comprises an unsubstituted succinimidyl group or a substituted succinimidyl group.

16. The process of claim 15, wherein the substituted succinimidyl group is an ester of N-hydroxysuccinimide.

17. The process of claim 1, wherein the bifunctional reagent is selected from the group consisting of bis[2-succinimidyloxycarbonyl-oxy)ethyl]sulfone (BSOCOES) or a sulfo analog thereof, bis(sulfosuccinimidyl)suberate) (BS.sup.3), disuccinimidyl glutarate (DSG), dithiobis(succinimidyl propionate) (DSP), disuccinimidyl suberate (DSS), disuccinimidyl tartrate (DST) or a sulfo analog thereof, 3,3-dithiobis(sulfosuccinimidyl propionate) (DTSSP), and ethylene glycol bis(succinimidyl succinate) (EGS) or a sulfo analog thereof.

18. The process of claim 1, wherein the activated PSA molecule formed in step (d) is general formula I or II ##STR00005## wherein R.sup.1 is H or sulfonyl; R.sup.2 is a linking group; A is NR.sup.5, NR.sup.5N R.sup.6, O or SR wherein R.sup.5 and R.sup.6 are independently selected from H, C.sub.1-4 alkyl and aryl; SylO is a sialyl group; n is 1-100; R.sup.3 is H, a monosialic acid group, a disialic acid group, an oligosialic acid group or a polysialic acid group; and R.sup.4 is H, a monosialic acid group, a disialic acid group, an oligosialic acid group, a polysialic acid group, an alkyl group or an acyl group.

19. The process of claim 18, wherein R.sup.2 is alkanediyl, arylene, alkarylene, heteroarylene, or alkyl-heteroarylene, any of which is optionally interrupted by either thioester, ester, amine or amide linkages, and in which A is NR.sup.5 or a linker group joined to the rest of the molecule through a group NR.sup.5.

20. The process of claim 18, wherein A is NR.sup.5, wherein R.sup.5 is H.

21. The process of claim 18, wherein R.sup.2 comprises an alkane-diyl group together with a carbonyl to which A is attached.

22. The process of claim 21, wherein R.sup.2 is C.sub.pH.sub.2pCO.sub.3 wherein p is 2-12.

23. The process of claim 18, wherein R.sup.2 comprises an alkanediyl group with an alkane carbon atom attached to A.

24. The process of claim 18, wherein R.sup.2 comprises a mid-chain ester, an amide, ether, a thioether and/or a 1-thio-N-succinimidyl amine linkage.

25. The process of claim 18, wherein R.sup.2 comprises an alkyleneoxyalkylene or an alkyleneoligooxyalkylene.

26. The process of claim 18, wherein ##STR00006## is a member selected from the group consisting of ##STR00007##

27. The process of claim 1, wherein when the PSA molecule comprises at the non-reducing end a terminal sialic acid having a vicinal diol group, the process further comprises: e) selectively oxidizing the vicinal diol group of the non-reducing terminal sialic acid unit to form an aldehyde group; f) aminating the aldehyde group of step (e) to form an amine group; and g) reacting the amine group of step (f) with a bifunctional reagent comprising two amine-reactive functional groups to form the activated PSA molecule, the activated PSA molecule being bifunctional and comprising an amine-reactive group at the reducing end and an amine-reactive group at the non-reducing end.

28. The process of claim 27, wherein in step (e) the non-reducing terminal sialic acid unit is joined to an adjacent unit through its 8 carbon atom and a 7,8-diol group and the aldehyde formed is an aldehyde group at carbon atom 7.

29. The process of claim 27, wherein in step (e) the non-reducing terminal sialic acid unit is joined to an adjacent unit through its 9 carbon atom and a 7,8-diol group and the aldehyde formed is an aldehyde group at carbon atom 8.

30. The process of claim 27, wherein in step (e) the selective oxidation is performed using an enzymatic oxidation process or a chemical oxidation process.

31. The process of claim 27, wherein in step (e) the selective oxidation is carried out using perruthenate, or periodate.

32. The process of claim 27, wherein each of the two amine-reactive functional groups comprises an unsubstituted succinimidyl group or a substituted succinimidyl group.

33. The process of claim 32, wherein the substituted succinimidyl group is an ester of N-hydroxysuccinimide.

34. The process of claim 27, wherein the bifunctional reagent is selected from the group consisting of bis[2-succinimidyloxycarbonyl-oxy)ethyl]sulfone (BSOCOES) or a sulfo analog thereof, bis(sulfosuccinimidyl)suberate) (BS.sup.3), disuccinimidyl glutarate (DSG), dithiobis(succinimidyl propionate) (DSP), disuccinimidyl suberate (DSS), disuccinimidyl tartrate (DST) or a sulfo analog thereof, 3,3-dithiobis(sulfosuccinimidyl propionate) (DTSSP), or ethylene glycol bis(succinimidyl succinate) (EGS) or a sulfo analog thereof.

35. The process of claim 27, wherein the activated PSA molecule formed in step (d) is general formula Ill ##STR00008## wherein R.sup.1 is H or sulfonyl; R.sup.2 is a linking group; A is NR.sup.5, NR.sup.5N R.sup.6, O or SR wherein R.sup.5 and R.sup.6 are independently selected from H, C.sub.1-4 alkyl and aryl; SylO is a sialyl group; and m is 0-100.

36. The process of claim 35, wherein R.sup.2 is alkanediyl, arylene, alkarylene, heteroarylene, or alkyl-heteroarylene, any of which is optionally interrupted by either thioester, ester, amine or amide linkages, and in which A is NR.sup.5 or a linker group joined to the rest of the molecule through a group NR.sup.5.

37. The process of claim 35, wherein A is NR.sup.5, wherein R.sup.5 is H.

38. The process of claim 35, wherein R.sup.2 comprises an alkane-diyl group together with a carbonyl to which A is attached.

39. The process of claim 38, wherein R.sup.2 is C.sub.pH.sub.2pCO.sub.3 wherein p is 2-12.

40. The process of claim 35, wherein R.sup.2 comprises an alkanediyl group with an alkane carbon atom attached to A.

41. The process of claim 35, wherein R.sup.2 comprises a mid-chain ester, an amide, ether, a thioether and/or a 1-thio-N-succinimidyl amine linkage.

42. The process of claim 35, wherein R.sup.2 comprises an alkyleneoxyalkylene or an alkyleneoligooxyalkylene.

43. The process of claim 35, wherein ##STR00009## is a member selected from the group consisting of ##STR00010##

44. A method of conjugating a biological molecule to an activated PSA molecule, the method comprising conjugating an activated PSA molecule of claim 1 by reacting with a biological molecule comprising an amine group.

45. The method of claim 44, wherein the biological molecule is selected from the group consisting of a protein, a peptide, a drug, a lipid, a liposome, a microbe, a cell or component thereof, a synthetic polymer or a synthetic copolymer.

46. The method of claim 45, wherein the protein is selected from the group consisting of a cytokine, an enzyme, a hormone, an antibody or a fragment thereof.

47. A method of conjugating a biological molecule to an activated PSA molecule, the method comprising conjugating an activated PSA molecule of claim 27 by reacting with a biological molecule comprising an amine group.

48. The method of claim 47, wherein the biological molecule is selected from the group consisting of a protein, a peptide, a drug, a lipid, a liposome, a microbe, a cell or component thereof, a synthetic polymer or a synthetic copolymer.

49. The method of claim 48, wherein the protein is selected from the group consisting of a cytokine, an enzyme, a hormone, an antibody, or a fragment thereof.

Description

(1) The following is a brief description of the drawings.

(2) FIG. 1a is a reaction scheme showing the prior art activation of the non-reducing sialic acid terminal unit;

(3) FIG. 1b is a reaction scheme showing the prior art reductive amination of the aldehyde moiety of the product of reaction scheme 1a using a protein-amine moiety;

(4) FIG. 2 represents schematically the potential by-products of the side reactions;

(5) FIG. 3 is a reaction scheme showing the tautomerism between the ketal and ring-closed forms of the reducing terminal sialic acid unit of a PSA; In solution, the terminal sialic acid residue at the reducing end of polysialic acid exists in a tautomeric equilibrium. The ketal form, although in low abundance in the equilibrium mixture (Jennings and Lugowski, 1981) is weakly reactive with protein amine groups, and can give rise to covalent adducts with proteins in the presence of sodium cyanoborohydride, although at a rate and to an extent that are not practically useful.

(6) FIG. 4 shows the preparation of reducing end derivatised NHS colominic acid (when non-reducing end has no vicinal diol)

(7) FIG. 5 shows the preparation of reducing end derivatised NH.sub.2-CA colominic acid (vicinal diol removed at non-reducing end)

(8) FIG. 6 shows the general scheme for preparation of CA-NHS-protein conjugation

(9) FIG. 7a shows the preparation of derivatised thiol colominic acid (CA-SH at non-reducing end)

(10) FIG. 7b shows schematic representation of CA-protein conjugation via CA-SH using NHS-maleimide

(11) FIG. 7c shows the preparation of CA-protein conjugates via CA-SH using NHS-maleimide (AMAS)

(12) FIG. 8a shows the preparation of CA-protein conjugates via NHS on reducing end

(13) FIG. 8b shows capping of reducing end of polysialic acid

(14) FIG. 8c shows preparation of non-reducing end derivatised CA

(15) FIG. 9 shows the preparation of CA-protein conjugates using bis(sulfosuccinimidyl)suberate (BS.sup.3) on non-reducing end

(16) FIG. 10 shows the schematic representation of CA-protein conjugation using the crosslinker DSG

(17) FIGS. 11a and 11b shows gel permeation chromatography (GPC) chromatograms for CAs separated as in example 5.

(18) FIG. 12 shows size exclusion HPLC on CA-NHS-growth hormone (GH) protein conjugation reactions (CA 35 kDa)

(19) FIG. 13 shows the sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) of CA-NHS-GH conjugates (CA 35 kDa)

(20) FIG. 14 shows native PAGE of unreacted and reacted CAs

(21) FIG. 15 shows the SDS-PAGE analysis of the CAH-NHS reactions as in example 10

(22) FIG. 16 shows the HPLC chromatogram of action 6 from FIG. 15.

EXAMPLES

(23) Materials

(24) Sodium meta-periodate and molecular weight markers were obtained from Sigma Chemical Laboratory, UK. The CAs used, linear alpha-(2,8)-linked E. coli K1 PSAs (22.7 kDa average, polydispersity (p.d.) 1.34; 39 kDa p.d. 1.4; 11 kDa, p.d. 1.27) were from Camida, Ireland. Other materials included 2,4 dinitrophenyl hydrazine (Aldrich Chemical Company, UK), dialysis tubing (3.5 kDa and 10 kDa cut off limits (Medicell International Limited, UK); Sepharose SP HiTrap, PD-10 (Pharmacia, UK); XK50 column and Sepharose Q FF (Amersham Biosciences UK); Tris-glycine polyacrylamide gels (4-20% and 16%), Tris-glycine sodium dodecylsulphate running buffer and loading buffer (Novex, 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.

(25) Methods

(26) Protein and CA Determination

(27) Quantitative estimation of CAS, as sialic add, 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 add (BCA) colorimetric method.

Example 1Fractionation of CA by IEC (CA, 22.7 KDa, pd 1.34) [Reference]

(28) An XK50 column was packed with 900 ml Sepharose Q FF and equilibrated with 3 column volumes of wash buffer (20 mM triethanolamine; pH 7.4) at a flow rate of 50 ml/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 (1350 ml) of washing buffer.

(29) The bound CA was eluted with 1.5 column volumes of different elution buffers (Triethanolamine buffer, 20 mM pH 7.4, with 0 mM to 475 mM NaCl in 25 mM NaCl steps) and finally with 1000 mM NaCl in the same buffer to remove all residual CA and other residues (if any).

(30) The samples were concentrated to 20 ml by high pressure ultra filtration over a 5 kDa 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 GPC) (as reported in example 5) and native PAGE (stained with alcian blue; example 8). 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.

Example 2: Activation of CA [Reference]

(31) Freshly prepared 0.02 M sodium metaperiodate (NaIO.sub.4; 6 fold molar excess over CA) solution was mixed with CA at 20 C. and the reaction mixture was stirred magnetically for 15 min in the dark. The oxidised CA was precipitated with 70% (final concentration) ethanol and by centrifuging the mixture at 3000 g 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 quantity of water, lyophilized and stored at 20 C. until further use.

Example 3: Determination of the Oxidation State of CA and Derivatives [Reference]

(32) 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) (5 mg each) were added to the 2,4-DNPH reagent (1.0 ml), 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 630 nm. In this instance, glucose was used as a standard.

Example 4a: Preparation of Amino Colominic Acid (CA-NH2) [Reference]

(33) CAO at 10-100 mg/ml was dissolved in 2 ml of deionised water with a 300-fold molar excess of NH.sub.4Cl, in a 50 ml tube and then NaCNBH.sub.4 (5 M stock in 1 N NaOH(aq), was added at a final concentration of 5 mg/ml. The mixture was incubated at room temperature for 5 days. A control reaction was also set up with colominic acid instead of CAO. Product colominic acid amine derivative was precipitated by the addition of 5 ml ice-cold 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.

Example 4b: Assay for Amine Content

(34) 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].

(35) In the well of a microtitre plate TNBS (0.5 l of 15 mM TNBS) was added to 90 l of 0.1 M borate buffer pH 9.5. To this was added 10 l of a 50 mg/ml solution of CA-amine. The plate was allowed to stand for 20 minutes at room temperature, before reading the absorbance at 405 nm. Glycine was used as a standard, at a concentration range of 0.1 to 1 mM. TNBS trinitrophenylates primary amine groups. The TNP adduct of the amine is detected.

(36) Testing the product purified with a double cold-ethanol precipitation using the TNBS assay showed close to 85% conversion.

Example 4c: Preparation of Colominic AcidSH

(37) Oxidised CA was derivatised with cystamine by reductive amination as described in example 4a, except using a 100-fold molar excess of cystamine instead of NH.sub.4Cl.

(38) Before purifying the product, it was treated with 50 mM DTT at 37 C. for 1 h. The reduced product was purified by double ethanol precipitation and size exclusion chromatography on sepharose G25.

(39) In another example, CANH.sub.2 prepared as in example 4a, is dissolved in 10 mM PBS with 1 mM EDTA pH 8.0. A 50-fold molar excess of 2-iminothiolane is added and the reaction allowed to proceed for 1 h at 25 C. Unreacted 2-iminothiolate is removed by gel filtration on a sephadex G25 column equilibrated with the reaction buffer.

(40) Thiol content is estimated using the Ellman's assay. Briefly 150 of sample are mixed with 150 l of 0.1M phosphate, 1 mM EDTA, pH8 containing 0.08 mg/ml 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and allowed to react for 30 minutes at room temperature and read at 405 nm. The product is suitable for reaction according to the schemes in FIGS. 7b and 7c.

(41) Further, the thiol content of the polymer was found to be 60%.

Example 4d: Preparation of CA-NHS

(42) CA-NH.sub.2 (35 kDa) (15-20 mg) synthesised in Reference Example 4a above was dissolved in 0.15M PBS (3504 pH 7.2) and then either 50 or 75 molar equivalents of BS.sup.3 in PBS (150 PH 7.2) was added. The mixture was vortexed for 5 seconds and then reacted for 30 minutes at 20 C. 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 NH.sub.2 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 260 nm and also by thin layer chromatography with visualization at 254 nm.

(43) CA-NH.sub.2 (35 kDa) (15-20 mg) synthesised in Example 4a above was either dissolved in the minimum amount of water (50-65 0) to which was added DMSO (300-235 L) or in >95% DMSO (350 with the aid of heat (100-125 C.). 75 molar equivalents of DSG in DMSO (150 L) was added to the CA-NH, solution, vortexed for 5 seconds and then reacted for 30 minutes at 20 C. The CA-NHS product was purified either with dioxane precipitation (2) or by PD-10 column using PBS as eluent (pH 7.2) and used immediately for site-specific conjugation to the NH.sub.2 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 (260 nm) and by thin layer chromatography (254 nm).

Example 5: Gel Permeation Chromatography of CA Samples [Reference]

(44) CA (35 kDa) samples were dissolved in NaNO.sub.3 (0.2M), CH.sub.3CN (10%; 5 mg/ml) and were chromatographed on 2GMPW.sub.XL columns with detection by refractive index (GPC system: VE1121 GPC solvent pump, VE3580 R1 detector and collation with Trisec 3 software (Viscotek Europe Ltd). Samples (5 mg/ml) were filtered through 0.45 m nylon membrane and run at 0.7 cm/min with 0.2M NaNO.sub.3 and CH.sub.3CN (10%) as the mobile phase (FIG. 11).

Example 6: Preparation of CA-NHS-Protein Conjugates (Using BS3 and DSG)

(45) GH in sodium bicarbonate (23 mg/ml, pH 7.4) was covalently linked to CA-NHS (35 kDa), from example 4b using an excess of BS.sup.3. The reaction was performed in 0.15 M PBS (pH 7.2; 1.5 ml) 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 HPLC-size exclusion chromatography. Controls included subjecting the native protein to the conjugation procedure using BS.sup.3 in the absence of any CA-NHS. CA-NH.sub.2 was also subjected to the conjugation procedure using BS.sup.3 in the absence of native GH.

(46) GH in sodium bicarbonate (pH 7.4) was covalently linked to CA-NHS (35 kDa), 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; 1.5 ml) 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.

Example 7: HPLC-SEC of CA-NHS-GH Conjugates

(47) 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 (1 mg/ml) were filtered over 0.45 m nylon membrane 175 l injected and run at 0.25 cm/min with ammonium bicarbonate buffer as the mobile phase (FIG. 12).

Example 8: SDS and Native PAGE of CAs and CA-GH Conjugates

(48) SDS-PAGE (MiniGel, Vertical Gel Unit, model VGT 1, 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) at 0 minutes (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. 13 and 14).

(49) Results

(50) CA (22.7 kDa) and its derivatives 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 22.7 kDa material.

(51) TABLE-US-00001 TABLE 2 Ion exchange chromatography of CA22.7 (pd 1.3) Elution buffers (in 20 mM Triethanolamine buffer + mM NaCl, pH 7.4) M.W. Pd % Population 325 mM 12586 1.091 77.4% 350 mM 20884 1.037 3.2% 375 mM 25542 1.014 5.0% 400 mM 28408 1.024 4.4% 425 mM* 7.4% 450 mM 43760 1.032 2.3% 475 mM 42921 1.096 0.2% *Not done

(52) 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).

(53) The fractionation of larger polymer (CA, 39 kDa, pd 1.4) produced species up to 90 kDa. This process can successfully be used for the fractionation of even large batches of the polymer. The results show that the on exchange fractions are narrowly dispersed. This is consistent with the GPC data.

(54) All narrow fractions were successfully oxidised with 20 mM periodate and samples taken from different stages of the production process and analysed by GPC and native PAGE, which showed no change in the molecular weight and polydispersity.

(55) CA, a PSA, is a linear alpha-2,8-linked homopolymer of N-acetylneuraminic acid (Neu5Ac) residues (FIG. 1a).

(56) Quantitative measurement of the oxidation state of CA was performed by ferricyanide on 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 DNPH 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.

(57) The integrity of the internal alpha-2,8 linked Neu5Ac residues post periodate and borohydride treatment was analysed by GPC and the chromatographs obtained for the oxidised (CAO), amino CA (CA-NH.sub.2), CA-NHS materials were compared with that of native CA. It was found (FIG. 12) 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.

(58) 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 SDS-PAGE of the conjugation reaction of CA-NH.sub.2 to GH using BS.sup.3 showed 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%.

(59) The results (FIG. 13) 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.

Example 9: Preparation of Colominic Acid Hydrazide (CAH) [Reference]

(60) 50 mg of oxidised colominic acid (19 kDa) was reacted with 2.6 mg of hydrazine (liquid) in 400 l of 20 mM sodium acetate buffer, pH 5.5, for 2 h at 25 C. The colominic acid was then precipitated with 70% ethanol. The precipitate was redissolved in 350 l phosphate buffer saline, pH 7.4 and NaCNBH.sub.3 was added to 5 mg/ml. The mixture was allowed to react for 4 h at 25 C., then frozen overnight. NaCNBH.sub.3 and reaction by products were removed by gel permeation chromatography on a PD10 column packed with Sephadex G25, using 0.15M NH.sub.4HCO.sub.3 as the mobile phase. The fractions (0.5 ml 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 signal, well above the background. The background was high due to the presence of the NH.sub.3 ions. Fractions 6, 7, 8 and 9 also contained colominic acid. These four fractions, were freeze dried to recover the CA-hydrazide (CAH).

Example 10: Preparation of Colominic Acid NHS (CA-NHS) and Colominic Acid-Protein Conjugates

(61) 10 mg of 19 kDa CA-hydrazide were reacted with 9 mg of BS.sup.3 in 400 l 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 0.5 ml 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.

(62) 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. 15).

(63) 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 (FIGS. 16a and b).

(64) The BSA used contains impurities. The BSA peak is at 56 minutes (FIG. 16a).

(65) 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 BS as the CAH was passed through a gel permeation chromatography column, which will have removed it. This strongly suggests that an NHS ester group was created on the CA molecule (FIG. 16b).

REFERENCES

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