Meningococcus serogroup X conjugate

Abstract

The invention provides a conjugate of a Neisseria meningitidis serogroup X capsular polysaccharide and a carrier molecule. The conjugate is typically made by (a) oxidising a primary hydroxyl group in the capsular polysaccharide, to give an oxidised polysaccharide with an aldehyde group; and (b) coupling the oxidised polysaccharide to a carrier molecule via the aldehyde group, thereby giving the conjugate. The conjugate may be part of an immunogenic composition. This composition may comprise one or more further antigens, particularly capsular polysaccharides from serogroups A, W135, C and Y and conjugated forms thereof. The composition may be in an aqueous formulation. The composition is useful as a vaccine, e.g. for raising an immune response in a mammal. The invention also provides processes for making the conjugate.

Claims

1. A composition comprising (a) a conjugate of a Neisseria meningitidis serogroup X capsular polysaccharide and a carrier protein wherein the conjugation of the capsular polysaccharide to the carrier protein is direct, wherein the carrier protein is conjugated to oxidized primary hydroxyl groups of 1-10% of the residues in the capsular polysaccharide, and (b) a conjugate of a Neisseria meningitidis serogroup A capsular polysaccharide and a carrier protein; wherein the composition comprises less than 50% free saccharide released from the conjugate of the Neisseria meningitidis serogroup X capsular polysaccharide after 28 days at 37 degrees centigrade.

2. The composition of claim 1, wherein the composition is immunogenic.

3. The immunogenic composition of claim 2, wherein the immunogenic composition is a vaccine.

4. The composition of claim 1 further comprising one or more additional antigens.

5. The composition of claim 4, wherein the one or more antigens are selected from the capsular saccharides of Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup W135 and Neisseria meningitidis serogroup Y.

6. The composition of claim 1, wherein the composition is in an aqueous formulation.

7. The composition of claim 1, wherein the carrier protein is selected from a diphtheria toxoid, a tetanus toxoid, CRM197 and Haemophilus influenzae protein D.

8. A method of raising an immune response to Neisseria meningitidis serogroup X and Neisseria meningitidis serogroup A capsular polysaccharides in a mammal by administering an immunologically effective amount of the composition of claim 1 to the mammal.

9. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier, wherein the composition is in an aqueous formulation.

10. The pharmaceutical composition of claim 9 further comprising one or more additional antigens.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the repeat unit of the serogroup X capsular polysaccharide.

(2) FIG. 2 shows an ultra performance liquid chromatogram for native and hydrolysed serogroup X capsular polysaccharide.

(3) FIG. 3 shows a scheme for the conjugation of a serogroup X capsular polysaccharide to CRM197 by TEMPO oxidation followed by reductive amination, and an SDS PAGE analysis of the resultant conjugate.

(4) FIG. 4 shows a chromatogram of conjugation mixture run on a SEPHACRYL S300 column with phosphate buffered saline.

(5) FIG. 5 shows a scheme for the conjugation of a serogroup X capsular polysaccharide to CRM197 via SIDEA linker, and an SDS PAGE analysis of the resultant conjugate.

(6) FIG. 6 shows a scheme for the conjugation of a serogroup X capsular polysaccharide to CRM197 via STDEA linker using a different method, and an SDS PAGE analysis of the resultant conjugate.

(7) FIG. 7 shows an SDS PAGE analysis of a serogroup X capsular polysaccharide-CRM197 conjugate made using a different linker.

(8) FIG. 8 shows IgG antibody titres against serogroup X capsular polysaccharide and serum bactericidal antibody titres against serogroup X following immunisation with a variety of N. meningitidis conjugates.

(9) FIG. 9 shows TgG antibody titres against serogroup A capsular polysaccharide and serum bactericidal antibody titres against serogroup A from the same experiment.

(10) FIG. 10 shows IgG antibody titres against serogroup C capsular polysaccharide and serum bactericidal antibody titres against serogroup C from the same experiment.

(11) FIG. 11 shows IgG antibody titres against serogroup W135 capsular polysaccharide and serum bactericidal antibody titres against serogroup W135 from the same experiment.

(12) FIG. 12 shows IgG antibody titres against serogroup Y capsular polysaccharide and serum bactericidal antibody titres against serogroup Y from the same experiment.

(13) FIG. 13 shows 2D 1H-31P HMBC NMR spectrum recorded at 400 MHz and 250.1 C. on MenA (a) and MenX (b) oligosaccharide generated by acidic hydrolysis. Peaks assignments are labelled.

(14) FIG. 14 shows a) avDP and b) pH as a function of time collected for MenA and MenX capsular polysaccharides and C) O-acetyl status for McnA capsular polysaccharide only at 37 C. and 45 C.

(15) FIG. 15 shows profiles of avDP as a function of time, collected for the stability study at 37 C. and 45 C. for a) MenA and b) MenX capsular polysaccharide at time points (a) 0, (b) 7, (c) 10, (d) 14, (e) 21 days at 45 C. and at (f) 7, (g) 14, (h) 21, (i) 28 days at 37 C. In addition a profile of experimentally degraded MenX capsular polysaccharide, obtained by acidic treatment (sodium acetate pH 4.0, at 80 C. for 4 hrs), is shown in b) (1).

(16) FIG. 16 shows IgG antibody titres against serogroup X capsular polysaccharide and serum bactericidal antibody titres against serogroup X following immunisation with a variety of N. meningitidis conjugates.

(17) FIG. 17 shows a scheme for the conjugation of a serogroup X capsular polysaccharide to CRM197 using a further method, and an SDS PAGE analysis of the resultant conjugate.

(18) FIG. 18 shows IgG antibody titres against serogroup A capsular polysaccharide and serum bactericidal antibody titres against serogroup A following immunisation with a variety of N. meningitidis conjugates.

(19) FIG. 19 shows TgG antibody titres against serogroup C capsular polysaccharide and serum bactericidal antibody titres against serogroup C following immunisation with a variety of N. meningitidis conjugates.

(20) FIG. 20 shows IgG antibody titres against serogroup W135 capsular polysaccharide and serum bactericidal antibody titres against serogroup W135 following immunisation with a variety of N. meningitidis conjugates.

(21) FIG. 21 shows IgG antibody titres against serogroup Y capsular polysaccharide following immunisation with a variety of N. meningitidis conjugates.

(22) FIG. 22 shows high affinity IgG antibody titres against serogroup X capsular polysaccharide following immunisation with a variety of N. meningitidis conjugates.

MODES FOR CARRYING OUT THE INVENTION

(23) Bacterial Growth for Serogroup X Capsular Polysaccharide Production

(24) In order to identify optimal bacterial growth conditions for production and release of serogroup X capsular polysaccharide in the supernatant, three different media were tested using the MenX 5967 (ST 750) strain. Different growths were performed in flasks and monitored by Proton Nuclear Magnetic Resonance spectroscopy (1H NMR). Culture supernatants were analyzed by NMR sequence with a diffusion filter to cut off signals deriving from lower molecular weight (MW) species and highlight the signals of higher MW serogroup X capsular polysaccharide. Further analysis of the corresponding pellets by 1H High-Resolution Magic Angle Spinning NMR (HR-MAS NMR) in solid state did not show serogroup X capsular polysaccharide signals, indicating that the majority of the polysaccharide was released in the supernatant (considering the limit of detection of this assay, the maximum amount of polysaccharide remaining on the bacteria should be of the starting amount). Similar results were obtained with the three media.

(25) In addition to the NMR methodology, a more accurate method for serogroup X capsular polysaccharide quantification in the clarified culture broth was developed (see below) using High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). As shown in the table below, medium #3 resulted in the highest amount of polysaccharide. It was therefore selected for a higher scale (18 L) fermentation which yielded 356 g/mL of serogroup X capsular polysaccharide in the supernatant.

(26) MenX 5967 (ST 750) Strain Growth in Different Media and Relative Polysaccharide Production

(27) TABLE-US-00001 Growth OD Saccharide g saccharide/ medium* (600 nm) (g/mL) OD #1 2 22.55 13.3 #2 6 42.73 7.1 #3 2.8 62.6 22.4 *1. modified Catlin v.6: casaminoacids 10 g/L, NaCl 5.8 g/L, glucose 10 g/L, K.sub.2HPO.sub.4 4 g/L, NH.sub.4Cl 1 g/L, K.sub.2SO.sub.4 1 g/L, MgCl.sub.26H.sub.2O 0.4 g/L, CaCl.sub.22H.sub.2O 0.03 g/L, Fe(III) citrate 0.5 mg/L, pH 7.2; 2. MCDM1: glucose 10 g/L, soy peptone 15 g/L, NaCl 5.80 g/L, K.sub.2SO.sub.4 1 g/L, K.sub.2HPO.sub.4 4 g/L, L-glutamic acid 5 g/L, L-arginine 0.3 g/L, L-serine 0.5 g/L, L-cysteine 0.23 g/L, MgCl.sub.2 0.19 g/L, CaCl.sub.2 0.021 g/L, FeSO.sub.4 0.002 g/L; 3. modified Frantz: L-Glutamic acid 1.6 g/L, Na.sub.2HPO.sub.42H.sub.2O 15.5 g/L, KCl 0.09 g/L, NH.sub.4Cl 1.25 g/L, pH 7.6, supplemented with: glucose 50 g/L, MgSO.sub.47H.sub.2O 30 g/L, 25 g/L ultrafiltered yeast extract, L-cysteine 1.5 g/L.
Purification of Serogroup X Capsular Polysaccharide

(28) The process for purifying serogroup X capsular polysaccharide was purified by a method adapted from reference 235.

(29) Conjugate Production and Characterisation

(30) Conjugates were made using polysaccharides of different chain lengths and different conjugation chemistries.

(31) Conjugation by Oxidation and Reductive Amination (Method A):

(32) Purified serogroup X capsular polysaccharide was hydrolysed in 50 mM sodium acetate at pH 4.7, 100 C. for 1 hour (FIG. 2). The average degree of polymerisation of the resulting oligosaccharide was determined to be 80, corresponding to a molecular weight of 25-30 kDa, by NMR. The polysaccharide depolymerization was monitored in process by Ultra Performance Liquid Chromatography-Size Exclusion Chromatography (UPLC-SEC) and phosphorus (.sub.31P) NMR spectroscopy, and it was quenched by neutralization when the desired avDP was reached. The buffer was exchanged with tetrabutyl ammonium bromide to allow dissolution of the saccharide in dimethylformamide solvent. The saccharide was then oxidized with TEMPO (0.06eq relative to the MenX repeating subunit), NaHCO.sub.3 (9 eq relative to the MenX repeating subunit), TCC (2 eq relative to the MenX repeating subunit) at 0 C. overnight. This oxidation generates an aldehyde group at the C-6 position of individual subunits (FIG. 3). The oxidised saccharide was purified by precipitation with acetone/NaCl and gel filtration using a SEPHADEX G15 column. The saccharide was quantified using HPAEC-PAD and its structural identity confirmed using NMR. There were approximately 4.5 oxidized groups per chain, corresponding to a degree of oxidation of approximately 6% along the circa 80 residue chain. The molecular weight distribution of the oxidized saccharide was measured by UPLC-SEC.

(33) The aldehyde group was used for conjugation to carrier protein CRM197 by reductive amination (FIG. 3). Briefly, the saccharide was mixed with 10 mg/ml CRM197 at a 4:1 w/w ratio and NaBH.sub.3CN at a 1:1 w/w ratio in a NaPi 10 mM pH 7.2 buffer. The mixture was left for 72 hours with slow stirring at 37 C. Conjugates were purified on a SEPHACRYL S300 column with phosphate buffered saline and fractions collected into pools (FIG. 4). Conjugation was verified by SDS PAGE (FIG. 3). The properties of the purified conjugates (pool 1) are given below:

(34) TABLE-US-00002 Total mass Total mass MenX/ MenX CRM197 MenX/ MenX CRM197 MenX CRM197 CRM197 yield yield CRM197 Kd EU/g (g/mL) (g/mL) (mg) (mg) (w/w) (%) (%) (mol/mol) (SEC) (LAL) 104.8 151.3 1.26 1.82 0.69 12.6 72.6 1.7 0.7 1.6

(35) Conjugates were also made by this method in which the polysaccharides were not hydrolysed with sodium acetate and therefore had a native average degree of polymerisation. Further conjugates were made containing polysaccharides with an average degree of polymerisation of 130.

(36) Conjugates were also made by this method in which the carrier protein was tetanus toxoid (TT) or SEQ ID NO: 9 (SEQ9). The polysaccharides in these conjugates had an average degree of polymerisation of 130. Other characteristics of these conjugates are given below:

(37) TABLE-US-00003 MenX/ MenX/ MenX Carrier carrier carrier EU/g Carrier (g/mL) (g/mL) (w/w) (mol/mol) (LAL) TT 197.6 660.80 0.3 0.4 18.81 SEQ9 159.7 757.70 0.21 0.3 6.57
Conjugation by Reductive Amination Followed by Reaction with SIDEA Linker (Method B):

(38) Purified serogroup X capsular polysaccharide was hydrolysed in 50 mM sodium acetate at pH 4.7, 100 C. for 2 hours (FIG. 2). The average degree of polymerisation of the resulting oligosaccharide was determined to be 15, corresponding to a molecular weight of 5 kDa, by NMR. The saccharide was then solubilised at 5 mg/ml in 5 mM sodium acetate buffer at pH 6.5 with 300 mg/ml NH.sub.4OAc and 49 mg/ml NaBH.sub.3CN for 5 days at 37 C. This step resulted in reductive amination of the terminal aldehyde group to generate a primary amine group (FIG. 5). The reaction mixture was then purified by tangential flow filtration with a 200 cm.sup.2 HYDROSART (cellulose) 2 kDa-cut off membrane against 1M NaCl and water. The primary amine group was then used for activation with SIDEA and subsequent conjugation to carrier protein CRM197 (FIG. 5). Briefly, the modified saccharide was dissolved in DMSO/water at 9:1 (v/v) with NEt.sub.3 (at a molar ratio of NEt.sub.3:total NH.sub.2 groups of 5:1) at a mol SIDEA:total mol NH.sub.2 groups of 12:1 for 3 hours at room temperature. The reaction mixture was then purified by precipitation with 90% dioxane (v/v). The SIDEA-modified saccharide was then reacted with 25 mg/ml CRM197 at a ratio of 13:1 (molar ratio active ester groups:CRM197) in a 25 mM NaPi buffer at pH 7.2. The mixture was left for 5 hours with slow stirring at room temperature. The conjugates were purified by precipitation with (NH.sub.4).sub.2SO.sub.4. Conjugation was verified by SDS PAGE (FIG. 5). The properties of one lot of these conjugates are given below:

(39) TABLE-US-00004 Total mass Total mass MenX/ MenX/ MenX MenX CRM197 MenX CRM197 CRM197 CRM197 yield Kd EU/g (g/mL) (g/mL) (mg) (mg) (w/w) (mol/mol) (%) (SEC) (LAL) 167.3 514.4 0.17 0.51 0.33 4.22 12.6 0.32 0.8
Conjugation by Reduction, Oxidation and Reductive Amination Followed by Reaction with SIDEA Linker (Method C):

(40) Purified serogroup X capsular saccharide was reacted at 15 mg/ml in 10 mM NaPi buffer at pH 8 with NaBH.sub.4 (12 eq relative to the molecular weight of MenX, solid) for 1.5 hours at room temperature. This step resulted in reduction of the saccharide. The reduced saccharide was then reacted at 6-8 mg/ml in 10 mM NaPi buffer at pH 7.2 with NaIO.sub.4 (10 eq relative to the molecular weight of MenX, solid) for 1.5 hours at room temperature. The combined effect of these two steps is the generation of an aldehyde group at the reducing terminus of the saccharide (FIG. 6). The modified saccharide is then subjected to reductive amination to provide a primary amine group that can be used for activation with SIDEA and subsequent conjugation to carrier protein CRM197 (FIG. 6). Briefly, the modified saccharide solubilised at 4-5 mg/ml in 10 mM NaPi buffer at pH 7 with 300 mg/ml NH.sub.4OAc and 49 mg/ml NaBH.sub.3CN for 5 days at 37 C. The modified saccharide was then dissolved in DMSO/water at 9:1 (v/v) with NEt.sub.3 (at a molar ratio of NEt.sub.3:total NH.sub.2 groups of 5:1) at a mol SIDEA:total mol NH.sub.2 groups of 12:1 for 3 hours at room temperature. The reaction mixture was then purified by precipitation with 80% acetone (v/v). The resulting SIDEA-modified saccharide was then reacted with 25 mg/ml CRM197 at a ratio of 13:1 (molar ratio active ester groups:CRM197) in a 100 mM NaPi buffer at pH 7.2. The mixture was left overnight with slow stirring at room temperature. The conjugates were purified by precipitation with (NH.sub.4).sub.2SO.sub.4. Conjugation was verified by SDS PAGE (FIG. 6). The properties of one lot of these conjugates are given below:

(41) TABLE-US-00005 MenX/ MenX/ Free MenX CRM197 CRM197 CRM197 saccharide EU/g (g/mL) (g/mL) (w/w) (mol/mol) (%) (LAL) 129.60 628.70 0.21 2.1 <6 0.01
Conjugation Via Alternative Linker (Method D):

(42) The purified serogroup X capsular polysaccharide with an average degree of polymerisation of 15, as described above, was also conjugated to CRM197 using a different linker according to the method of FIG. 7 in U.S. 61/534,751. Conjugation was verified by SDS PAGE (FIG. 7 herein).

(43) Conjugation by Reduction, Oxidation and Reductive Amination with the Carrier (Method E):

(44) Purified serogroup X capsular saccharide was reacted at 15 mg/ml in 10 mM NaPi buffer at pH 8 with NaBH.sub.4 (12 eq relative to the molecular weight of MenX, solid) for 2 hours at room temperature. This step resulted in reduction of the saccharide. The reduced saccharide was then reacted at 6-8 mg/ml in 10 mM NaPi buffer at pH 7.2 with NaIO.sub.4 (10 eq relative to the molecular weight of MenX, solid) for 1.5 hours at room temperature. The combined effect of these two steps is the generation of an aldehyde group at the reducing terminus of the saccharide (FIG. 17). The modified saccharide is then subjected to reductive amination with carrier protein CRM197. Briefly, the modified saccharide at 2 mg/ml was dissolved in 300 mM NaPi buffer at pH 8 (at a weight ratio of saccharide:CRM197 of 8:1) and NaBH.sub.3CN (at a weight ratio of saccharide:NaBH.sub.3CN of 4:1) for 4 days at 37 C. Conjugation was verified by SDS PAGE (FIG. 17).

(45) Immunisation Study (1)

(46) General Assay Protocol:

(47) Balb/c mice were immunized by subcutaneous injection according to the schedule described below. The injection volume was 200 l and the injection contained alum phosphate adjuvant (120 g per dose). Injections were carried out on days 1, 14 and 28, with bleeds taken at day 0 (for preimmune sera), 28 (post second immunisation sera) and 42 (post third sera).

(48) TABLE-US-00006 Grp Mice per group Immunogen Antigen dose 1 8 PBS 2 16 MenX-CRM197 (method A) .sup.1 g 3 16 MenX-CRM197 (method D) .sup.1 g 4 16 MenX-CRM197 (method B) .sup.1 g 5 16 MenX-CRM197 (method A) + MenACWY 1 g + 2, 1, 1, 1 g 6 16 MenX-CRM197 (method D) + MenACWY 1 g + 2, 1, 1, 1 g 7 16 MenX-CRM197 (method B) + MenACWY 1 g + 2, 1, 1, 1 g 8 16 MenACWY 2, 1, 1, 1 g 9 16 MenX-CRM197 (method D) .sup.0.1 g MenACWY = mixture of MenA-CRM197, MenC-CRM197, MenW135-CRM197 and MenY-CRM197 prepared according to ref. 10.

(49) The post third immunisation IgG antibody titre against serogroup X capsular polysaccharide and serum bactericidal antibody titre against serogroup X strain Z9615 are shown in FIG. 8. The serogroup X conjugates were immunogenic and induced bactericidal antibodies. The response was not diminished when the dose was reduced ten-fold (to 0.1 g). Responses were slightly reduced when the conjugates were combined with conjugates derived from serogroups A, C, W135 and Y, but still well above controls. Accordingly, immune interference between these conjugates and the serogroup X conjugates appears to be relatively small.

(50) The post third immunisation IgG antibody titres against serogroups A, C, W135 and Y capsular polysaccharides and serum bactericidal antibody titres against these serogroups (using strains F8238, 11, 240070 and 860800 respectively) were also measured for groups 5, 6, 7 and 8. Results are shown in FIGS. 9-12. The responses to the serogroup A, C, W135 and Y conjugates were generally not diminished when combined with the serogroup X conjugates. Once again, these results suggest that there is little immune interference between these conjugates and the serogroup X conjugates.

(51) Anti-serogroup X capsular polysaccharide IgM ELISA units were found to be low for all the conjugates, as expected for conjugate vaccines due to effective isotype switching from IgM to IgG.

(52) A modified ELISA was used to measure higher avidity IgG antibodies only (FIG. 22). The modified ELISA uses a chaotropic salt to select and detect higher avidity IgG antibodies only. Anti-serogroup X capsular polysaccharide IgG ELISA units were low for all the conjugates both after the second and the third dose compared to the units by standard ELISA, but a statistically significant booster effect was observed after the third dose for all the conjugates (P from 0.0006 to <0.0001).

(53) The following table summarises the rabbit complement serum bactericidal antibody titres against the various strains from the pooled post third immunisation sera.

(54) TABLE-US-00007 Antigen Dose MenX MenA MenC MenW MenY Antigen Name (g) Z9615 F8238 11 240070 860800 PBS + AlumPhosphatc <4 <16 <16 <16 32 MenX-CRM197 (A) 1 4096 <16 <16 <16 32 McnX-CRM197 (D) 1 4096 n/a n/a n/a n/a MenX-CRM197 (B) 1 4096 n/a n/a n/a n/a MenX-CRM197 (A) + 1 + 2, 1, 256 4096 4096 512 1024 McnACWY 1, 1 MenX-CRM197 (D) + 1 + 2, 1, 1024 2048 4096 1024 1024 MenACWY 1, 1 McnX-CRM197 (B) + 1 + 2, 1, 1024 2048 4096 1024 1024 MenACWY 1, 1 MenACWY 2, 1, 1, <4 2048 8192 1024 2048 1 McnX-CRM197 (D) 0.1 4096 n/a n/a n/a n/a
Immunisation Study (2)
General Assay Protocol:

(55) Balb/c mice were immunized by subcutaneous injection according to the schedule described below. The injection volume was 200 l and the injection contained alum phosphate adjuvant.

(56) TABLE-US-00008 Group Mice per group Immunogen Antigen dose 1 1-8 PBS 1 g 2 9-16 MenX-CRM197 (method A, native avDP) 1 g 3 17-24 MenX-CRM197 (method A, 80 avDP) 1 g 4 25-32 MenX-CRM197 (method A, 130 avDP) 1 g 5 33-40 MenX-TT (method A, 130 avDP) 1 g 6 41-48 MenX-SEQ9 (method A, 130 avDP) 1 g 7 49-56 MenX-CRM197 (method A, native avDP) + 1 g + 2, 1, 1, 1 g MenACWY 8 57-64 MenX-CRM197 (method A, 80 avDP) + 1 g + 2, 1, 1, 1 g MenACWY 9 65-72 MenX-CRM197 (method A, 130 avDP) + 1 g + 2, 1, 1, 1 g MenACWY 10 73-80 MenX-TT (method A, 130 avDP) + 1 g + 2, 1, 1, 1 g MenACWY 11 81-88 MenX-SEQ9 (method A, 130 avDP) + 1 g + 2, 1, 1, 1 g MenACWY MenACWY - mixture of MenA-CRM197, MenC-CRM197, MenW135-CRM197 and MenY-CRM197 prepared according to ref. 10.

(57) The post third immunisation IgG antibody titre against serogroup X capsular polysaccharide and serum bactericidal antibody titre against serogroup X strain Z9615 are shown in FIG. 16. The serogroup X conjugates were immunogenic and induced bactericidal antibodies. Responses were slightly reduced when the MenX-CRM197 conjugates were combined with conjugates derived from serogroups A, C, W135 and Y, but still well above controls. In contrast, little or no reduction was seen when MenX-TT or MenX-SEQ9 conjugates were combined with these conjugates. Accordingly, the use of a different carrier protein for the MenX polysaccharide may help to reduce any immune interference between the serogroup X conjugate and these conjugates.

(58) The post third immunisation IgG antibody titre against serogroup A capsular polysaccharide and serum bactericidal antibody titre against serogroup A strain F8238 when the MenX conjugates were combined with conjugates derived from serogroups A, C, W135 and Y are shown in FIG. 18. Corresponding data for serogroups C, W135 and Y are shown in FIGS. 19-21.

(59) Stability Study (1)

(60) Materials:

(61) Purified MenA and MenX polysaccharides were obtained according to the method of ref. 10. The purity of the polysaccharide preparation was assessed by estimation of residual protein and nucleic acids contents, which were lower than 1% w/w of saccharide.

(62) NMR Analyses:

(63) 1H, 13C and 31P NMR experiments were recorded on Bruker Avance III 400 MHz spectrometer, equipped with a high precision temperature controller, and using 5-mm broadband probe (Bruker). For data acquisition and processing, TOPSPIN version 2.6 software (Bruker) was used. 1H NMR spectra were collected at 250.1.degree. C. with 32 k data points over a 10 ppm spectral width, accumulating 128 scans. The spectra were weighted with 0.2 Hz line broadening and Fourier-transformed. The transmitter was set at the water frequency which was used as the reference signal (4.79 ppm). 13C NMR spectra were recorded at 100.6 MHz and 370.1 C., with 32 k data points over a 200 ppm spectral width, accumulating 4 k scans. The spectra were weighted with 0.2 Hz line broadening and Fourier-transformed. The transmitter was set at the acetone frequency which was used as the reference signal (30.89 ppm). 31P NMR spectra were recorded at 161.9 MHz at 250.1 C., with 32 k data points over a 20 ppm spectral width, accumulating approximately 1 k of scans. The spectra were weighted with 3.0 Hz line broadening and Fourier-transformed. 85% phosphoric acid in deuterium oxide was used as an external standard (0 ppm). All the 1H and 31P NMR spectra were obtained in quantitative manner using a total recycle time to ensure a full recovery of each signal (5.times. Longitudinal Relaxation Time T1). To confirm the degradation mechanism of MenA and MenX capsular polysaccharides and consequently to assign the 31P NMR peaks, bidimensional 1H-31P Heteronuclear Multiple-Bond Correlation (HMBC) experiments were acquired on MenA and MenX oligosaccharide samples, previously generated by acidic hydrolysis in 50 mM sodium acetate pH 4.8 (saccharide concentration of .about.10 mg/mL) at 73 C. for 2.5 hrs and pH 4.0 at 80 C. for 5.5 hrs (saccharide concentration of 2.5 mg/mL) respectively. The average degree of polymerization (avDP) of MenA and MenX oligosaccharides was 12 and 10 respectively, as estimated by 31P NMR analysis (see paragraph Stability experiments below). These NMR analytical samples were prepared by solubilizing approximately 10 mg of dried saccharide in 0.75 mL of deuterium oxide (99.9% atom D-Aldrich), with a standard pulse-program. 4096 and 512 data points were collected in F2 and F1 dimension respectively. 64 scans were accumulated prior to Fourier transformation to yield a digital resolution of 0.2 Hz and 5.0 Hz per point in F2 and F1 respectively.

(64) HPLC Analyses:

(65) HPLC analyses were conducted using CARBOPAC PA200 column (4 mm.times.250 mm; DIONEX) with guard column (4 mm50 mm; DIONEX) connected to an ICS 3000 DIONEX system equipped with a Pulsed Amperometric Detector. 100 mM NaOH+10 mM sodium nitrate buffer was used for column equilibration and a three-step gradient with increasing amount of sodium nitrate (100 mM NaOH+10 mM, 250 mM, 500 mM sodium nitrate for 80, 15 and 3 min respectively) was used for elution. A flow rate of 0.4 mL/min was used for the entire run of 120 min. 20 L samples were injected at a concentration of approximately 1 mg/mL. The effluent was monitored using an electrochemical detector in the pulse amperometric mode with a gold working electrode and an Ag/AgCl reference electrode. A quadruple-potential waveform for carbohydrates was applied. The resulting chromatographic data were processed using Chromeleon software 6.8 (DIONEX).

(66) Stability Experiments:

(67) MenA and MenX polysaccharide solutions at a concentration of approximately 1 mg/mL in 100 mM potassium phosphate buffer pH 7.0 prepared with deuterated water were incubated at 37 C. and 45 C. respectively. At different time points, samples were withdrawn and analysed by NMR and HPLC. pH was also monitored at each time point. The avDP of MenA and MenX was monitored for polysaccharide stability. avDP values were calculated by the integration of 31P NMR spectra and expressed as [(Pde/Pme)+1], where Pde is molar concentration of the phosphodiester in chain groups and Pme the molar concentration of phosphomonoester end groups. The HPLC profiles were also evaluated semi-quantitatively in order to confirm the more accurate stability evaluation collected by 31P NMR assay

(68) Degradation Mechanism of MenA and MenX Polysaccharides

(69) The NMR .sup.1H-.sup.31P HMBC data on the MenA oligosaccharide, generated by mild acidic hydrolysis, are reported in FIG. 13(a). Due to the presence of O-acetyl groups at C.sub.3 and C.sub.4 of mannosamine residues, several spin systems were detected and assigned: (i) proton at C1 of 3- or 4-O-acetylated residues (H.sub.1-P.sub.de).sup.3/4OAc; (ii) proton at C.sub.1 of de-O-acetylated residues (H.sub.1-P.sub.de).sup.deOac; (iii) proton at C.sub.3 and C.sub.4 geminal of O-acetyl groups (H.sub.3/H.sub.4-P.sub.de).sup.3/4OAc; (iv) proton at C.sub.2 of 3-O-acetylated residues (H.sub.2-P.sub.de).sup.3OAc; (v) proton at C.sub.2 of 4-O-acetylated residues (H.sub.2-P.sub.de).sup.4OAc; (vi) proton at C.sub.2 of de-O-acetylated residues (H.sub.2-P.sub.de).sup.deOAc; (vii) protons at C.sub.5 and C.sub.6 of 3- or 4-O-acetylated residues (H.sub.5/6-P.sub.de).sup.3/4OAc; (viii) protons at C.sub.3, C.sub.4, C.sub.5 and C.sub.6 of de-O-acetylated residues (H.sub.3/4/5/6-P.sub.de).sup.deOAc. The attachment of phosphate at C.sub.6, confirmed by the cross peaks of phosphomonoester to proton at C.sub.6 of 3- or 4-O-acetylated residues (H.sub.6-P.sub.me).sup.3/4OAc and to proton at C.sub.6 of de-O-acetylated residues (H.sub.6-P.sub.me).sup.deOAc, indicated that during hydrolysis the phosphodiester bond is cleaved leaving a phosphate group attached to the non-reducing terminus, which is consistent with the lower stability of the phosphate-C.sub.1 linkage. Because no other .sup.1H-.sup.31P scalar correlation was detected, no phosphate migration involving free hydroxyl groups at C4 or C3 occurred during hydrolysis. .sup.1H-.sup.31P HMBC on the MenX oligosaccharides (FIG. 13(b)) also indicated that the phosphate-C.sub.1 linkage is less stable and in this case the non-reducing terminus has a phosphate group attached at C.sub.4: the monoester phosphate shows cross-correlation only with proton at C.sub.4. Also for MenX, no phosphate migration involving free hydroxyl groups at C3 or C6 occurred during hydrolysis. All the .sup.31P spin systems were assigned, the phosphodiester and phosphomonoester signals at 1.40 and 4.65 ppm respectively. The proton NMR profile was assigned also by collecting the .sup.31P-decoupled spectrum which reduces the peaks structure due to this scalar coupling. All the spectra assignments were in agreement with the published results mainly based on .sup.13C NMR analysis (ref. 30). 13C NMR chemical shifts of MenX capsular polysaccharide were in agreement with published data (ref. 14), as shown in Table 1 below:

(70) TABLE-US-00009 TABLE 1 13C NMR chemical shifts of MenX capsular polysaccharide. C.sub.1 C.sub.2 C.sub.3 C.sub.4 C.sub.5 C.sub.6 CH.sub.3.sup.NAc CO.sup.NAc Chemical 95.2 54.8 71.1 75.1 73.2 61.3 23.2 175.6 shift (ppm)
Thermal Stability of MenA and MenX Polysaccharides.

(71) Degradation of MenA and MenX capsular polysaccharides, as the consequence of hydrolysis at phosphodiester bonds, results in fragments of lower avDP which expose newly-formed phosphomonoester end groups. In NMR experiments, these phosphomonoester groups generate a .sup.31P resonance signal at higher fields than that originated by the internal phosphodiester groups thus allowing the avDP calculation as described in Stability experiments above. The variation of avDP during storage is an indicator of the polysaccharide stability and so the avDP of samples of MenA and MenX capsular polysaccharides, taken at different time points during exposure at 37 C. and 45 C., was measured by .sup.31P NMR (Table 2 and FIG. 14(a)):

(72) TABLE-US-00010 TABLE 2 avDP estimated by 31P NMR analysis and pH values detected on MenA and MenX samples at different time points and temperatures of 37 C. and 45 C. O-acetylation status of MenA capsular polysaccharide, expressed as mol O-Acetyl groups per mol of repeating unit, is also reported. Temper- MenA PS ature Time OAc MenX PS ( C.) (days) pH avDP (mol/mol) pH avDP 37 0 6.97 >100 0.932 6.96 >100 7 6.91 88.1 0.916 6.91 >100 14 6.91 68.2 0.916 6.89 >100 21 7.00 46.1 0.897 6.93 >100 28 6.96 22.9 0.883 6.91 >100 45 0 6.97 >100 0.932 6.96 >100 7 6.95 22.4 0.891 6.94 >100 10 6.93 15.1 0.886 6.86 >100 14 6.91 10.5 0.851 6.85 >100 21 6.90 5.1 0.826 6.87 >100

(73) At the sample concentration used, the sensitivity of the technique did not allow measurement of the avDP at time zero for both polysaccharides, when the avDP is higher than 100. For each time point sample the pH was maintained in the range of 7.00.1 (FIG. 14(b)).

(74) At 37 C. McnA capsular polysaccharide degraded to an avDP of 22.9 after 28 days of incubation, while at 45 C. degradation was accelerated with an avDP of 5.1 after 21 days. Under the same conditions, MenX capsular polysaccharide did not show degradation (avDP>100 for all time points at both incubation temperatures; based on the assay sensitivity, 100 is the maximum avDP value detectable). HPLC profiles of MenA capsular polysaccharide incubated at 37 C. and 45 C. (FIG. 15) progressively showed increased intensity peaks of shorter oligosaccharides indicating depolymerisation of chains. In comparison, HPLC profiles collected on all MenX samples remain practically unmodified with a broad peak due to long chain polysaccharides at approximately 87 min which additionally demonstrates the higher stability of this carbohydrate. .sup.1H NMR analysis confirmed that the incubation of MenA and MenX capsular polysaccharides at 37 C. and 45 C. did not alter the structure of the polysaccharide repeating units. Only a limited decreasing of O-acetylation level (O-acetyl groups are present in MenA capsular polysaccharide only), from 0.932 to 0.883 and 0.826 mol/mol repeating unit at 37 C. and 45 C. was respectively observed (Table 2 and FIG. 14(c)). Taken together, these NMR and HPLC data confirm the higher stability of MenX as compared to MenA capsular polysaccharide in aqueous solution.

(75) Stability Study (2)

(76) Materials:

(77) MenX-CRM197 conjugates were prepared according to methods A, B and C above.

(78) The conjugates prepared according to method A contained polysaccharides with an average degree of polymerisation of 100. Other characteristics of this conjugate lot are given below:

(79) TABLE-US-00011 MenX/ MenX/ Free MenX CRM197 CRM197 CRM197 saccharide (g/mL) (g/mL) (w/w) (mol/mol) (%) 477.3 1378 0.35 0.7 <2.3

(80) The conjugates prepared according to method B had the following characteristics:

(81) TABLE-US-00012 MenX MenX/CRM197 Free saccharide (g) CRM197 (mg) (w/w) (%) 383 1.71 0.22 5.8

(82) The conjugates prepared according to method C contained polysaccharides with an average degree of polymerisation of 19. Other characteristics of this conjugate lot are given below:

(83) TABLE-US-00013 MenX/ MenX/ Free MenX CRM197 CRM197 CRM197 saccharide (g/mL) (g/mL) (w/w) (mol/mol) (%) 129.6 628.7 0.21 2.1 <6

(84) Accelerated stability studies were performed to provide preliminary information on the stability of these conjugates. Stability studies of were performed at 37 C. for 28 days, the time points for measurement were every 7 days (0, 7, 14, 21, 28 days). Samples were monitored by measuring the free saccharide released from the conjugates. The separation of free saccharide was performed by SPE-C4 cartridge using as elution buffer ACN 10-20%+TFA 0.05%. The total and free saccharide was quantified by HPAEC-PAD analysis, allowing a % free saccharide to be calculated. Values for the three lots of conjugate are given below:

(85) TABLE-US-00014 % free saccharide Time Method A Method B Method C (days) conjugates conjugates conjugates 0 <2.3 5.8 <2.1 7 4.2 29.9 24.9 14 6.2 51.2 31.5 21 8.7 48.7 34.8 28 10.0 56.1 42.8

(86) The conjugates made using method A were more stable than the conjugates made by methods B and C.

(87) Analytical Study

(88) Materials:

(89) MenX polysaccharide was produced by bacterial growth of the Neisseria meningitidis X5967 strain (ST 750) and purified by a method adapted from reference 235. The purity of the polysaccharide preparation was assessed by estimation of residual protein and nucleic acid content using colorimetric assays (both were present at <1% w/w of saccharide), and endotoxin content using the LAL assay (<10 EU/g of saccharide). Sodium acetate salt (Thermo Scientific Dionex), Sodium hydroxide 50% solution (J. T. Baker), Trifluoroacetic acid (Sigma), Water MilliQ grade (Millipore) were of pro analysis quality.

(90) General Methods:

(91) Total phosphorus content was measured according to the method of reference 24. Reactions were monitored by thin-layer chromatography (TLC) on Silica Gel 60 F254 (Sigma Aldrich); after exam under UV light, compounds were visualized by heating with 10% (v/v) ethanolic H.sub.2SO.sub.4. Column chromatography was performed using pre-packed silica cartridges REDISEP (Teledyne-Isco, 0.040-0.063 nm). Unless otherwise specified, a gradient 0.fwdarw.100% of the elution mixture was applied in a COMBIFLASH Rf (Teledyne-Isco) instrument.

(92) .sup.1H, .sup.13C and .sup.31P NMR experiments were recorded on Bruker Avance III 400 MHz spectrometer, equipped with a high precision temperature controller, and using 5-mm broadband probe (Bruker). For data acquisition and processing, TOPSPIN version 2.6 software (Bruker) was used.

(93) .sub.1H NMR spectra were collected at 250.1 C. with 32 k data points over a 10 ppm spectral width. The spectra were weighted with 0.2 Hz line broadening and Fourier transformed. Chemical shift values were reported in ppm, relative to internal Me.sub.4Si (0.00 ppm, CDCl.sub.3) or the solvent signal (4.79 ppm, D2O). .sup.13C NMR spectra were recorded at 100.6 MHz and 370.1 C., with 32 k data points over a 200 ppm spectral width. The spectra were weighted with 0.2 Hz line broadening and Fourier-transformed. Chemical shift values were reported in ppm relative to the signal of CDCl.sub.3 (77.0 ppm, CDCl.sub.3).

(94) .sup.31P NMR spectra were recorded at 161.9 MHz at 250.1 C., with 32 k data points over a 20 ppm spectral width. The spectra were weighted with 3.0 Hz line broadening and Fourier-transformed. 85% phosphoric acid in deuterium oxide was used as an external standard (0 ppm).

(95) Exact masses were measured by electron spray ionization cut-off spectroscopy, using a Q-Tof micro Macromass (Waters) instrument. Optical rotation was measured with a P-2000 Jasco polarimeter. Benzyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido--D-glucopyranoside 3. The starting material 2 (ref. 236) (1.8 g, 3.1 mmol) was dissolved in acetonitrile (200 ml) under nitrogen, and treated with trimethylamineborane (1.4 g, 18.4 mmol) and BF.sub.3.Et.sub.2O (2.6 ml, 18.4 mmol) at 0 C. After stirring for 1 h at 0 C., the mixture was allowed to reach ambient temperature, at which time the reaction was complete (TLC, 7:3 cyclohexane-EtOAc). MeOH (3 ml) and triethylamine (3 ml) were added, and the mixture was concentrated. The residue was partitioned with aq NaHCO.sub.3, and combined organic layers were concentrated and purified on silica gel (cyclohexane-EtOAc) to afford 1.5 g of product 3 (83%). [].sub.D.sup.24=+1.9 (c 0.5, CHCl.sub.3). .sup.1H NMR (CDCl.sub.3, 400 MHz): =7.80-6.95 (m, 19H, Ph), 5.15 (d, 1H, J.sub.1,2 8.0 Hz, H-1), 4.78, 4.47 (2 d, 2H, .sup.2J 12.2 Hz, CH.sub.2Ph), 4.72, 4.51 (2 d, 2H, .sup.2J 12.0 Hz, CH.sub.2Ph), 4.67, 4.59 (2 d, 2H, .sup.2J 12.0 Hz, CH.sub.2Ph), 4.26-4.18 (m, 2H, H-2,3), 3.87-3.88 (m, 3H, H-4,6), 3.66-3.62 (m, 1H, H-5), 2.89 (d, 1H, J.sub.2,OH 2.3 Hz, OH-4). .sup.13C NMR (CDCl.sub.3, 100 MHz): =167.81 (CO), 138.15, 137.59, 137.10, 133.67, 131.61, 128.12, 127.91, 127.86, 127.81, 127.58, 127.40 (Ar), 97.35 (C-1), 78.49 (C-3), 74.37, 74.24 (CH.sub.2Ph), 73.78 (C-5), 73.45 (C-4), 70.80 (CH.sub.2Ph), 70.69 (C-6), 55.37 (C-2). ESI HR-MS (C.sub.35H.sub.33NO.sub.7): m/z=([M+Na].sup.+ found 597.2547; calc 597.2601); ([M+Na].sup.+ found 618.1895; calc 618.1894).

Benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy--D-glucopyranoside 4

(96) A mixture of N-phthalimido compound 3 (1 g, 1.7 mmol) in EtOH (20 ml), containing 1.2 ml of ethylenediamine, was refluxed overnight. After TLC (toluene-EtOAc 4:1) showed the reaction was complete, the mixture was concentrated and re-dissolved in 4:1 EtOH-Ac.sub.2O (25 ml). The mixture was stirred for 3 h, then concentrated. Chromatography of the residue (cyclohexane-EtOAc) gave 740 mg of monosaccharide 4, whose NMR data were identical with those recently reported in literature [237].

Benzyl 2-acetamido-3,6-di-O-benzyl-4-(1,5-dihydro-3-oxo-3.SUP.5.-3H-2,4,3-benzodioxaphosphepin-3-yl)--D-glucopyranoside 5

(97) N,N-diethyl-1,5-dihydro-3H-2,3,4-benzodioxaphosphepin-3-amine (717 mg, 3 mmol) was added to a solution of the monosaccharide (500 mg, 1 mmol) in CH.sub.2Cl.sub.2 (9 ml) and 0.45 M 1H-tetrazole in acetonitrile (9 ml) at 0 C. After 10 min the iced bath was removed and stirring was continued. After stirring further 3 h the reaction went to completion (TLC, 1:1 toluene-EtOAc). The mixture was cooled to 20 C. and m-CPBA was added. After 20 min some aq NaHCO.sub.3 was added to quench it. The mixture was diluted with CH.sub.2Cl.sub.2 and extracted in a separatory funnel with aq NaHCO.sub.3. Combined organic layers were concentrated and the residue was purified on silica gel (cyclohexane-EtOAc) to furnish 630 mg of product (92%). White crystals from EtOAc, m.p. 159-160 C.=[].sub.D.sup.24=+34.7 (c 0.1, CHCl.sub.3). .sup.1H NMR (CDCl.sub.3, 400 MHz): =7.41-7.12 (m, 18H, Ph), 5.90 (d, 1H, J.sub.1,2 7.6 Hz, H-1), 5.17-5.12 (m, 2H, 2CHPh), 5.00-4.78 (m, 4H, 4CHPh), 4.65-4.58 (m, 5H, 4CHPh, H-4), 4.32 (t, 1H, J 9.0 Hz, H-3), 3.89 (d, 1H, J.sub.6a,5 9.0 Hz, H-6a), 3.76-3.69 (m, 2H, H-5,6b), 3.46-3.42 (m, 1H, H-2), 1.80 (s, 3H, CH.sub.3CO). .sup.13C NMR (CDCl.sub.3, 100 MHz): =170.61 (CO), 138.26, 137.36, 134.98, 128.94, 128.35, 127.95, 127.98, 127.80, 127.71, 127.57, 127.50 (Ar), 98.85 (C-1), 78.71 (C-3), 76.72 (C-4), 73.97 (C-5), 73.76, 73.42, 70.09 (CH.sub.2Ph), 69.04 (C-6), 68.30, 60.25 (CH.sub.2Ph), 56.95 (C-2), 23.40 (CH.sub.3CO). .sup.31H NMR (CDCl.sub.3, 162 MHz): =0.32. ESI HR-MS (C.sub.37H.sub.40NO.sub.9P): m/z=([M+H] found 674.2476; calc 674.2519).

2-Acetamido-2-deoxy--D-glucopyranosyl Phosphate 6

(98) The protected monosaccharide 5 (100 mg, 0.15 mmol) was dissolved in MeOH (10 ml) and hydrogenated over 10% Pd/C (30 mg). The mixture was stirred for 1 d, then it was filtered through a celite pad. The solvent was evaporated and the recovered crude material was purified on a C-18 Isolute SPE cartridge. Fractions containing the sugar were freeze-dried to give 42 mg of foamy product 6 (95%), whose NMR data were in agreement with those reported in literature [238].

(99) High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) for MenX Quantification:

(100) MenX samples were treated with TFA at a final concentration of 2 M diluted to a total volume of 600 L in the range 0.5-8 g/mL. Samples were heated at 100 C. for 2.5 hours in a closed screw-cap test tube, then chilled at 2-8 C. for about 30 minutes, added 700 L NaOH 2 M and filtered with 0.45 m ACRODISC (PALL) filters before analysis. A pure preparation of MenX PS or the synthetic monomer 4-GlcNAc-4P, titered through the colorimetric method for total phosphorus content, were used for building the calibration curve, set up with standards in the range of 0.5-8 g/mL. HPAEC-PAD was performed with a DIONEX ICS3000 equipped with a CarboPac PA1 column (4250 mm; DIONEX) coupled with PA1 guard column (450 mm; DIONEX). Samples were run with a flow rate of 1 mL/min, using a gradient in 10 minutes from 100 mM to 500 mM AcONa in 100 mM NaOH. The effluent was monitored using an electrochemical detector in the pulse amperometric mode with a gold working electrode and an Ag/AgCl reference electrode. A quadruple-potential waveform for carbohydrates was applied. The resulting chromatographic data were processed using CHROMELION software 6.8.

(101) Acid Hydrolysis of MenX Polysaccharide and GlcNAc-4P and NMR Characterization:

(102) A large scale acid hydrolysis was conducted on MenX polysaccharide and on the synthetic monomer (10 mg). Both the samples were dissolved in 2 mL 2 M TFA and hydrolysed at 100 C. for 2.5 hours. The samples were dried and exchanged with D.sub.2O for three times before analysis.

(103) Selection of Hydrolysis Conditions:

(104) To identify the optimal conditions for MenX hydrolysis able to completely release the monomer subunits and minimize their degradation, different reaction times for the hydrolysis of MenX polysaccharide (from 1 to 6 hours) performing the hydrolysis in 2 M TFA at 100 C. were explored. A pure preparation of MenX polysaccharide, titered through the colorimetric method for total phosphorus content, was used at two different concentrations (0.5 and 2 g/mL). One prevalent peak was detected by HPAEC-PAD analysis. The area of the peak increased over time, with a maximum among two and three hours, before decreasing for longer times. Eventually 2.5 hours was selected as optimal time of hydrolysis

(105) The linearity of the method was verified in the range 0.5-8 g/mL (R.sup.2=99.807). The method was successfully applied to purification process intermediates, including fermentation broths, and in order to determine the accuracy of the method a recovery study was conducted. Known amounts of polysaccharide were added to standard samples that were subjected to the analysis. The recovery was calculated based on the difference between the total concentration determined for the spiked samples and the concentration found in the un-spiked samples. The mean recovery ranged from 98 to 102%, indicating a high grade of accuracy. Repeatability inter-analysis was performed by analyzing the same sample four times with a CV of 1% and a corresponding average CV of 0.5%.

(106) Synthesis of 4P-GlcNAc:

(107) As shown in Scheme 1, the synthesis of target compound 6 commenced from the regioselective ring opening of protected GlcN 2 (92% yield), which was prepared from galactosamine hydrochloride as described in reference 236. Removal of the N-phthalimido protection by means of ethylenediamine, followed by selective N-acetylation provided known compound 4 in 87% yield [237]. Reaction of 4 with N,N-diethyl-1,5-dihydro-3H-2,3,4-benzodioxaphosphepin-3-amine and 1H-tetrazole and subsequent oxidation with m-chloroperbenzoic acid (m-CPBA) enabled the phosphate group introduction in significantly higher yield than previously reported with other methods [238] and furnished crystalline shelf stable compound 5 (m. p. 159-160 C.). A phosphomonoester peak at 0.32 ppm in the .sup.31P NMR spectrum, which correlated with the H-4 signal at 4.58 ppm and two couples of CH systems (5.13, 4.98 and 5.14, 4.99 ppm respectively) in the .sup.1H-.sup.31P HMBC NMR spectrum allowed to assess the structure of 5. Finally hydrogenolysis over 10% PdC provided the target 4P-GlcNAc 6 in excellent yield (95%) respect to 50% yield attained when unprotected phosphate was present. NMR data of the final product were in good agreement with those reported in literature [239].

(108) ##STR00020##
NMR Characterization of the Products Formed by Acid Hydrolysis of MenX Polysaccharide or 4P-GlcNAc:

(109) MenX polysaccharide and the synthetic monomer 6 were hydrolyzed at larger scale according to the procedure optimized for the HPAEC-PAD analysis in order to confirm the structure of the resulting species by NMR analysis. In both cases 4P-GlcNH was assessed as the prevalent species.

(110) .sup.1H NMR spectrum of 4P-GlcNAc 6 showed the / anomeric peaks at 5.19 and 4.72 ppm respectively, and the proton signals in the range 4.00-3.69 ppm. H-2 and H-2 were assigned at 3.91 ppm and 3.72 ppm signals by homo-nuclear COSY NMR correlation. One single peak for phosphate monoester at 0.58 ppm was detected at the .sup.31P NMR.

(111) After hydrolysis of both the standard 6 and the native MenX PS, .sup.1H NMR analysis of the attained 4P-GlcN showed two major anomeric signals corresponding at / mixtures in the ratio of 5.5:4.5 and 6.7:3.3 at 5.40 and 4.92 ppm, respectively. Remaining ring proton signals fell between 4.08 and 3.44 ppm, while H-2 (dd, J 3.7 and 10.3 Hz, at 3.91 ppm) and H-2 (dd, J 8.5 and 10.5 Hz, at 3.06 ppm) were shifted up-field due to the loss of the acetyl group. Furthermore, no N-acetyl CH.sub.3 signals were detected indicating that hydrolysis resulted in total de-N-acetylation.

(112) Bidimensional .sup.1H-.sup.31P HMBC NMR evidenced two overlapping cross peaks, assigned to the phosphate monoester signals at 0.68 and 0.14 ppm of the .sup.31P NMR spectrum, correlating with H-4 and H-4 at 3.94 and 3.96 ppm, respectively, in the .sup.1H NMR.

(113) Use of 4P-GlcNAc as Standard for MenX Quantification by HPAEC-PAD:

(114) The synthetic monomer 6 was quantified by the colorimetric method for total phosphorus content and then used for building a calibration curve (in the range 0.5-8 g/mL) in comparison to the native MenX polysaccharide. After subjecting the synthetic monomer and the native polysaccharide to the same hydrolysis conditions optimized for MenX polysaccharide samples, the same peak was detected by HPAEC-PAD and the curves obtained perfectly overlapped. The concentration of unknown samples and intermediates of the polysaccharide purification process was consistent independently from the curve used for the quantification (the difference in saccharide concentration values was <2% for all the tested samples). Mixtures of hydrolyzed MenX polysaccharide and synthetic monomer were also analyzed by HPAEC-PAD on a CARBOPAC PA1 column, eluting with 10 mM sodium hydroxide, to verify the eventual formation of GlcN in the hydrolysis conditions used [26]. Formation of GlcN was less than 5% in moles both for native MenX and synthetic monomer samples.

(115) We also verified the possibility to use the commercially available glucosamine-6-phosphate (6P-GlcN) as standard for the analysis, using the same hydrolysis conditions optimized for MenX. The resulting calibration curve overlapped those obtained with native MenX and its synthetic monomer, but the elution time of the resulting peak detected by HPAEC-PAD was different (8.97 min against 9.88 mM for MenX), demonstrating that utilization of 4P-GlcNAc is more straightforward.

(116) This method for MenX polysaccharide quantification is a crucial analytical tool for monitoring the saccharide content of purification process intermediates and a final conjugate vaccine. In addition to allowing the process yield to be calculated, the quantification allows calculation of the saccharide/protein ratio of the conjugate and the % of free saccharide, both of which are important parameters for verifying the quality and consistency of a final vaccine formulation.

(117) The use of a synthetic monomer means that there is no need for the standardization of a batch of polysaccharide for the analysis. The overall method is rapid, permits detection of very low concentrations of sugar (0.5 g/mL of polysaccharide), with minimal sample clean-up and has been verified to work well for the characterization of purification process intermediates, including fermentation broths. The method may be suitable for the quantification of intermediates of conjugation processes and for the characterization of the final vaccine formulations.

(118) It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

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