Novel Polysaccharide-Protein Conjugates And Process To Obtain Thereof

FIELD OF THE INVENTION

The present invention relates to novel polysaccharide-protein conjugates and process to obtain thereof. More particularly, the present invention relates to polysaccharide - protein conjugates produced using carbamate chemistry capable of being used in production of monovalent vaccine or multivalent combination vaccines as well as a diagnostic tool. More specifically the present invention relates to Neisseria meningitidis serogroup X, A, C, Y and W135 polysaccharide-carrier protein conjugate using carbamate chemistry and the process to obtain the same.

BACKGROUND OF THE INVENTION

N. meningitidis (meningococcus) is an aerobic gram-negative bacterium that has been serologically classified mainly into 13 serogroups A, B, Q D, 29E, H, I, K, L, W135, X, Y and Z. The grouping system is based on the capsular polysaccharides of the organism. WHO official website mentions that N. meningitidis is o ne of the most common causes of bacterial meningitis in the world and the only bacterium capable of generating large epidemics of meningitis. Explosive epidemics with incidence rates of up to 1000 cases per 100,000 inhabitants have been reported, particularly in sub-Saharan Africa. N. meningitidis is transmitted by aerosol or direct contact with respiratory secretions of patients or healthy human carriers. As a rule, endemic disease occurs primarily in children and adolescents, with highest attack

l rates in infants aged 3-12 months, whereas in epidemics older children and young adults may be more involved. However, the rapid progression of meningococcal disease frequently results in death within 1-2 days after onset. N. meningitidis infections can be prevented by vaccination.

Haemophilus influenzae type b is a Gram-negative bacterium that causes meningitis and acute respiratory infections, mainly in children. The WHO official website mentions that in both developed and developing countries, it is an important cause of non-epidemic meningitis in young children, and is frequently associated with severe neurological squeal, even if antibiotics are given promptly.

Haemophilus influenzae infection is transmitted by droplets from infected (but not necessarily symptomatic) people. H. influenzae type b infections can be prevented by vaccination.

Majority of meningococcal diseases are caused by 6 serogroups viz., A, B, C, Y, W135 and X. Historically, the majority of the cases in the meningitis belt are caused by serogroup A meningococci. Serogroup C meningococci were responsible for outbreaks in the meningitis belt in the 1980s, and a new outbreak was also experienced in Niger in 2015, while serogroup W (formerly W-135) has emerged as a cause of epidemic meningitis since 2000. Serogroup Y prevalence is minimal in Africa; however, the serogroup is more evident in meningitis cases in South America. According to one NCBI publication, Serogroup X meningococci have previously been considered a rare cause of sporadic meningitis, but during 2006-2010, outbreaks of serogroup X meningitis occurred in Niger, Uganda, Kenya, Togo and Burkina Faso, the latter with at least 1300 cases of serogroup X meningitis among the 6732 reported cases.

According to another NCBI publication, in Togo during 2006-2009, serogroup X meningococci accounted for 16% of the 702 confirmed bacterial meningitis cases. Kozah district experienced a serogroup X meningococci outbreak in March 2007 with a serogroup X meningococci seasonal cumulative incidence of 33/100,000. In Burkina Faso during 2007- 2010, serogroup X meningococci accounted for 7% of the 778 confirmed bacterial meningitis cases, with an increase from 2009 to 2010 (4% to 35% of all confirmed cases, respectively). In 2010, serogroup X meningococci epidemics occurred in northern and central regions of Burkina Faso; the highest district cumulative incidence of serogroup X meningococci was estimated as 130/100,000 during March- April. Based on the above facts, a pentavalent ACYWX pol saccharide-protein conjugate vaccine could offer broader coverage against meningococcal disease except serogroup B. Immunization is the only rational approach to control the meningococcal disease. Currently, various monovalent or multivalent vaccines including serogroup A, C, Y and W135 polysaccharide conjugates are licensed for sale in market, but no licensed vaccine is available against serogroup X meningococci. The serogroup X polysaccharide protein conjugates will be the new addition to multivalent meningococcal conjugate vaccine development in case of public health need.

Several studies suggest that the size of saccharide moiety can influence the immunogenicity of conjugate vaccines. Initial studies on the immunogenicity of dextran-protein conjugates found that dextran of low molecular weight conjugated to chicken serum albumin, induced strong anti-dextran responses in mice, while increasing the dextran size resulted in reduced immunogenicity. Laferriere et al. found little influence of the carbohydrate chain length on the immunogenicity of pneumococcal conjugate vaccines in mice. These studies suggest that there is no clear correlation between polysaccharide chain length and immunogenicity of conjugate vaccine. However, Rana et al. found it important, to establish the optimum saccharide chain length for developing immunogenic Hib polysaccharide conjugate vaccine.

The reaction of the polysaccharides with the CDI has been shown in several literature references. The Carbonyldiimidazole, a particularly preferred reagent, reacts with the hydro xyl groups to form imidazolylurethanes of the polysaccharide, and aryl chloroformates, including, for example, nitrophenyl- chloroformate, producing mixed carbonates of the polysaccharide. In each case, the resulting activated polysaccharide is very susceptible to nucleophilic reagents, such as amines, and is thereby transformed into the respective urethanes. For any conjugation chemistry the polysaccharide structure plays a very significant role. The chemistry of choice for conjugating any polysaccharide to a carrier protein depends upon the available functional groups on that polysaccharide. While some polysaccharides contain chemical groups which can be conveniently utilized for conjugation e.g. amines, carboxyls or aldehydes, many ones require activation and derivatization before they can be coupled to proteins. As evident in the literature the polysaccharide protein conjugate vaccines can be made with or without a linker, wherein the linker can be a part of polysaccharide or can be attached to a carrier protein.

The existing state of art discloses vaccines for the treatment of various serogroups including Neisseria meningitidis serogroup A, B, C, W and Y such as the US patent application no US2015/ 0044253 which discloses the immunogenic composition comprising a saccharide fragment obtained from H. influenzae serotype B (Hib), N. meningitidis serogroup A, B, C, W, Y conjugated with a carrier protein. However, in the US2015/ 0044253, the carbamate linker is linked directly onto amino groups of the carrier protein which results into low conjugation efficiency.

WO2004/ 019992 also discloses modified capsular saccharide, and saccharide protein conjugates for N. meningitidis serogroup A obtained by reductive amination, however, the application talks about the serogroup A only.

There are a fe disclosures available on N. meningitidis serogroup X such as WO2013/ 174832 which discloses conjugate of N. meningitidis serogroup X obtained by reductive amination conjugation chemistry.

The major drawback in the existing state of art disclosures is that none of the prior arts using carbamate chemistry disclose the dissolution of polysaccharide into organic solvent to facilitate the conjugation process. The conjugates presently available suffer from stability issues. More specifically, the stable and commercially viable conjugates for N. meningitidis serogroup X are not available. OBTECT OF THE INVENTION

In order to obviate the drawbacks in the existing state of art, the main object of present invention is to provide novel polysaccharide-protein conjugates.

Another object of the present invention is to provide stable polysaccharide - protein conjugates capable of being used for the preparation of novel conjugate vaccines. Yet another object of the present invention is to provide a N. meningitidis serogroup X polysaccharide-carrier protein conjugate to expand higher coverage of the meningococcal disease incidence prevented by currently licensed vaccines. Yet another object of the present invention is to provide a process to obtain said novel polysaccharide-protein conjugates.

Yet another object of the present invention is to provide a process to obtain said novel polysaccharide-protein conjugates using carbamate chemistry.

Yet another object of the present invention is to provide a process of rapid solubilization of the polysaccharide in non-aqueous aprotic solvents.

Yet another object of the present invention is to carry out the conjugation reaction in broader pH range.

Yet another object of the present invention is to complete the conjugation process in very short time span with high yields. Yet another object of the present invention is to obtain novel polysaccharide-protein conjugates with high immunogenicity capable of being used in vaccine and as diagnostic tool.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides novel polysaccharide (PS)- protein conjugates and process to obtain thereof. More particularly, the present invention relates to obtain the polysaccharide - protein conjugates by carbamate chemistry for the preparation of novel conjugate vaccines. More specifically the present invention relates to N. meningitidis serogroup X, A, C, Y and W135 polysaccharide-carrier protein conjugate using carbamate chemistry and the process to obtain the same. The present invention relates to the process of conjugation wherein capsular polysaccharide is degraded to smaller sizes suitable for conjugation with carrier protein to obtain conjugates with higher antigenicity. The carrier protein is selected from TT (tetanus toxoid), DT (Diphtheria toxoid), CRM197 (non-toxic mutant of DT), OMPV (Outer membrane protein vesicles) or other suitable carrier protein. The polysaccharide fragment is obtained from group of gram negative bacteria, including but not limited to Haemophilus influenzae type b (Hib), Neisseria meningitidis (Men), Streptococcus pneumoniae.

The process of conjugation of Neisseria meningitidis capsular polysaccharide, more preferably Men A, C, Y, W or X capsular polysaccharide is degraded. Such degradation is carried out by using several techniques available in the prior art. More preferably, the degradation is carried out by probe sonication and/ or ultra-sonication at a lab scale and by micro fluidizer at larger scales. The native N. meningitidis capsular polysaccharide is degraded in the size range of distribution coefficient of 0.38+0.06 kD as determined by gel-permeation chromatography (GPC) on high performance liquid chromatography (HPLC). After the degradation process, said degraded polysaccharide is dissolved in strong electrolytic salts for counter ion exchange, including but not limited to Lithium halides, Lithium chloride and Quaternary ammonium halide hydrates. The solution is allowed for proper mixing for 60 ± 30 minutes. Moisture is removed from the resultant capsular polysaccharide by any drying technique such as rotary-evaporation. The dried capsular polysaccharide is then dissolved in the non-aqueous aprotic solvents including but not limited to anhydrous Dimethyl sulfoxide (DMSO), Dimethyl formamide (DMF), N-Methyl pyrrolidone (NMP) or Dimethyl Acetamide. Said degraded and dried capsular polysaccharide in the non-aqueous aprotic solvent is reacted with 5-50 molar excess concentration of moisture free activating agents including but not limited to Ν,Ν'-Carbonyl Di imidazole (CDI), Ν,Ν'-Di- succinimidyl carbonate (DSC) or N,N'-Di-succinimidyl oxalate (DSO) and the mixture is kept in the presence of 4-dimethylaminopyridine (DMAP) or pyridine for pre-determined time to complete the reaction.

The resultant activated capsular polysaccharide is purified by precipitating the polysaccharide in an excess of low polarity solvent including but not limited to ethyl acetate, dichloromethane, n-butyl acetate or their mixture in different proportions, followed by dissolving the precipitated polysaccharide in an aqueous buffer saline to obtain purified activated capsular polysaccharide.

The purified activated capsular polysaccharide so obtained is dissolved in aqueous buffer with an activated carrier protein. The activated carrier protein may be Carbohydrazide labelled or ADH labelled or Hydrazine labelled.

The solution of purified activated capsular polysaccharide and activated carrier protein is allowed for mixing at room temperature for predetermined period of time, preferably 15 ± 5 hours at different pH range depending on whether the said polysaccharide is CDI activated, DSC activated or DSO activated. The pH range for CDI activated polysaccharide is from 8-10, while the pH range for DSC activated or DSO activated polysaccharide is from 6-9. During this reaction the amine of the hydrazide moiety on the protein and -N- containing aromatic residue of carbamate or carbonate moiety on the activated polysaccharide reacts and results in the formation of polysaccharide-protein conjugate with stable carbamate linkage -O- (C=O)-N-. Hence the final conjugate with the formula PS-L1-L2-CR is produced, wherein PS is the polysaccharide, LI is a carbamate linkage, L2 is a hydrazide linkage and CR is the carrier protein. The purified conjugates are obtained by the purification process including but not limited to ultrafiltration, ammonium sulphate precipitation, gel permeation chromatography.

The present invention also discloses the attaching of one linker preferably carbamate to the polysaccharide and second linker preferably hydrazine to the carrier protein, followed by reaction of activated polysaccharide and activated protein having separate linkers to generate a conjugate.

The present improved process of conjugation completes in shorter time span and yields conjugates of more stable covalent bond at wide working range of pH. The novel polysaccharide-protein conjugate of the present invention displays more stability, high yield and high immunogenicity.

The present invention also discloses the method of dissolution of the polysaccharide in the non-aqueous aprotic solvent which facilitates conjugation reaction between the polysaccharide and the carbamate forming agent to complete the conjugation process in shorter time span.

The most significant outcome of the improved process of present invention is to provide novel polysaccharide-protein conjugates capable of being used in vaccine or as diagnostic tool. The said novel polysaccharide- protein conjugate elicit specific and homologous immune response and is therefore useful in the production of monovalent vaccine or multivalent combination vaccines as well as a diagnostic tool.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the EDC (carbodiimide) cross-linking reaction scheme (R-

NH ₂ is either Hydrazine (H2N-NH2) or Adipic Acid Dihydrazide

(ADH) (NH ₂ NHCO(CH2)4CONHNH ₂ ).

Figure 2 depicts the monomer unit of Men X capsular polysaccharide- chemical structure.

Figure 3 depicts the HPLC-SEC profile showing the elution volume on

TSKgel PWXL5000 - PWXL4000 column at a flow rate of lml/ min showing Void volume, Total volume and Sample elution volume.

Figure 4 depicts the HPLC-SEC profile comparison of native MenX polysaccharide with sized polysaccharides.

Figure 5 depicts the HPLC-SEC profile comparison of hydrazine derivatized TT with native TT.

Figure 6 depicts the Chromatogram showing purification of hydrazide derivatized TT on sephadex G25.

Figure 7 depicts the Complete Carbamate Conjugation Scheme.

Figure 8 depicts the HPLC-SEC profile comparison of hydrazine derivatized TT with crude Men X Conjugate.

DETAILED DESCRIPTION OF THE INVENTION WITH ILLUSTRATIONS AND EXAMPLES

The present invention provides novel polysaccharide-protein conjugates and process to obtain thereof. More particularly, the present invention relates to obtain the chemically stable polysaccharide - protein conjugates by carbamate chemistry. The polysaccharide fragment is obtained from group of gram negative bacteria, including but not limited to Haemophilus influenzae type b (Hib), Neisseria meningitidis (Men), Streptococcus pneumoniae, more preferably, N. meningitidis serogroups Men A, C, Y, W and preferably Men X. The present invention also relates to obtain novel polysaccharide-protein conjugates with high immunogenicity capable of being used in vaccine individually or in combinations or as diagnostic tool.

The present invention also relates to the process of conjugation wherein said capsular polysaccharide is degraded to smaller sizes suitable for conjugation with carrier protein to obtain conjugates with higher antigenicity.

The present invention provides a process of conjugation using the CDI for hydroxyl group activation to form a polysaccharide-protein conjugates by a carbamate chemistry.

The carrier protein is selected from TT (tetanus toxoid), DT (Diphtheria toxoid), CRM197 (non-toxic mutant of DT), OMPV (Outer membrane protein vesicles) or other suitable carrier protein.

Said capsular polysaccharide is degraded by using several techniques available in the prior art. More preferably, the degradation is carried out by probe sonication and/ or ultra-sonication at a lab scale and by micro fluidizer at larger scales. The native Neisseria meningitidis capsular polysaccharide is degraded in the size range of distribution coefficient of 0.38+0.06 kD. The size of polysaccharide is determined by gel-permeation chromatography (GPC) on high performance liquid chromatography (HPLC).

After the degradation process, said degraded polysaccharide is dissolved in strong electrolytic salts for counter ion exchange, including but not limited to Lithium halides, Lithium chloride and Quaternary ammonium halide hydrates. The solution is allowed for proper mixing for 60±30 minutes. Moisture is removed from the resultant capsular polysaccharide by any drying technique such as rotary-evaporation. The dried capsular polysaccharide is then dissolved in the non-aqueous aprotic solvents including but not limited to anhydrous Dimethyl sulfoxide (DMSO), Dimethyl formamide (DMF), N-Methyl pyrrolidone (NMP) or Dimethyl Acetamide. Said degraded and dried capsular polysaccharide in the nonaqueous aprotic solvent is reacted with 5-50 molar excess concentration of moisture free activating agents including but not limited to Ν,Ν'- Carbonyl Di imidazole (CDI), Ν,Ν'-Di-succinimidyl carbonate (DSC) or Ν,Ν'-Di-succinimidyl oxalate (DSO) and the mixture is kept in the presence of 4-dimethylaminopyridine (DMAP) or pyridine for predetermined time to complete the reaction. The Men PS has free hydroxyl group in its repeating unit, which when reacted to Carbonyl-diimidazole (COT) forms an intermediate imidazolyl carbamate and imidazole byproduct.

The resultant activated capsular polysaccharide is purified by precipitating the polysaccharide in an excess of low polarity solvent including but not limited to ethyl acetate, dichloromethane, n-butyl acetate or their mixture in different proportions, followed by dissolving the precipitated polysaccharide in an aqueous buffer saline to obtain purified activated capsular polysaccharide. The carrier protein is reacted with water soluble carbodiimide EDC (N-(3- Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride) in the presence of Hydrazine or ADH to yield stable imide bonds with extending terminal hydrazide groups. The EDC reacts with available carboxylate groups to form an intermediate, highly reactive, o-acylisourea. This active ester may further react with nucleophiles such as hydrazide to yield a stable final product (Fig 1). The EDC cross-linking scheme (R-NH2) is either hydrazine or ADH. Carbonyls react with hydrazides and amines at pH 5-7. The hydrolysis of EDC is a competing reaction during coupling and is dependent on temperature, pH and buffer composition. The 4-Morpholinoethanesulfonic acid (MES) is an effective carbodiimide reaction buffer. Phosphate buffers reduce the reaction efficiency of the EDC, but increasing the amount of EDC can compensate for the reduced efficiency. Tris, glycine and acetate buffers may not be used as conjugation buffers.

The hydrazides labeling on TT is determined by TNBS assay; and the protein concentration is determined by Lowry's assay. The degree of derivatization is calculated by dividing the moles of hydrazides generated by moles of protein.

The purified activated capsular polysaccharide so obtained is dissolved in aqueous buffer with an activated carrier protein. The activated carrier protein may be Carbohydrazide labelled or ADH labelled or Hydrazine labelled.

The solution of purified activated capsular polysaccharide and activated carrier protein is allowed for mixing at room temperature for 15 ± 5 hours at different pH range depending on whether the said polysaccharide is CDI activated, DSC activated or DSO activated. The pH range for CDI activated polysaccharide is from 8-10, while the pH range for DSC activated or DSO activated polysaccharide is from 6-9. During this reaction the amine of the hydrazide moiety on the protein and -N- containing aromatic residue of carbamate or carbonate moiety on the activated polysaccharide reacts and results in the formation of polysaccharide-protein conjugate with stable carbamate linkage -O- (OO)-N-. Hence the final conjugate with the formula PS-L1-L2-CR is produced, wherein PS is the polysaccharide, LI is a carbamate linkage, L2 is a hydrazide linkage and CR is the carrier protein. The purified conjugates are obtained by the purification process including but not limited to ultrafiltration, ammonium sulphate precipitation, gel permeation chromatography.

The conjugates prepared using carbamate chemistry has been tested to determine polysaccharide/ protein ratio, amount of free polysaccharide in the conjugates and molecular size distribution. In this invention various optimization experiments have been conducted to attain the polysaccharide/ protein ratio in the range from 0.30 to 1.0 and the conjugation yields of upto 60% with an average of 30±5 % . In one preferred embodiment, the Men PS derived from bacterial fermentation is purified by the downstream purification process and analyzed after purification for all the critical quality parameters. The chemical structure of Men PS consists of repeating units with free hydroxyl group(s) e.g. MenX PS (Fig. 2) consists of repeat unit having a phosphate backbone and N-acetylation at position 3. The hydroxyl groups at position no. 4 and 7 act as reactive functional groups for conjugation with carbamate inducing chemicals like CDI. The monomer repeat units are called as monosaccharides and the long chain formed by a covalent bond in-between monosaccharide units, constitutes the Polysaccharide. The monosaccharide type is specific for specific serogroup (Table 1).

Tablel: Chemical structure of capsular polysaccharides of different N. meningitidis serogroups Group Repeating unit

X →4)-D-GlcpNAc-a-(l→OP0 ₃ →

A →6)-D-ManpNAc(3/40Ac)-a-(l→OP0 ₃ →

C →9)-D-Neup5Ac(7/80Ac)-a-(2→

Y →4)-D-Neup5Ac(7/90Ac)-a-(2→6)-D-Glc-a-(l→

W →4)-D-Neup5Ac(7/90Ac)-a-(2→6)-D-Gal-a-(l→

The native Men PS have size range of distribution coefficient of 0.38+0.06 kD as determined by SEC on HPLC using pullulan standards. The Carbamate chemistry of the present invention and the polysaccharide structure contributes for the formation of stable polysaccharide -protein conjugates to be used as a vaccine candidate either alone or in combination.

The content of degraded polysaccharide have been determined by physico- chemical assays e.g. phosphorus assay for MenX and MenA, and have been found to be similar to that of the initial polysaccharide content.

Different experiments have been conducted to optimize and attain the distribution coefficient to an optimum range of 0.38+0.06 kD. The variable conditions have been used for he conditions varies for different batches depending upon the initial molecular size and structure of individual polysaccharide.

In one preferred embodiment, MenX Polysaccharide is degraded to smaller sizes suitable for conjugation with carrier protein to obtain conjugates with high antigenicity. Tetanus toxoid (TT) is used as carrier protein. Said degraded MenX polysaccharide is dissolved in Lithium chloride. The solution is allowed for proper mixing for a duration of 60 ± 30 minutes. Moisture is removed from the resultant capsular polysaccharide by any known drying technique such as rotary- evaporation. The degraded and dried capsular polysaccharide is then dissolved in anhydrous Dimethyl sulfoxide (DMSO). The dried capsular polysaccharide in the non-aqueous aprotic solvent is reacted with 5-50 molar excess preferably 30 molar excess concentration of activating agent Ν,Ν'-Carbonyl Di imidazole (CDI), and the mixture is kept for mixing for a period of 2 hrs to 3 hrs to complete the reaction. The pH of the reaction mixture is maintained at 7-10 preferably at 9.0. The resultant activated MenX polysaccharide is purified by precipitating the polysaccharide in an excess of low polarity solvent ethyl acetate followed by dissolving the precipitated polysaccharide in an aqueous buffer saline to obtain purified activated capsular polysaccharide.

In an another preferred embodiment the linker is hydrazine or its derivative, attached to the carrier protein. The sized active polysaccharide and the derivatized carrier protein are mixed in a proportion of 0.5:1 to 1:0.5 w/ w more preferably 1:1 w/ w in a buffer of pH 7.0-10.0 preferably 0.1M Sodium carbonate,01M NaCl, pH 9.0. During this reaction the amine of the hydrazide moiety on the protein and -N- containing aromatic residue of carbamate or carbonate moiety on the activated polysaccharide reacts and results in the formation of polysaccharide- protein conjugate with v e r y stable carbamate linkage -O- (OO)-N-. The said conjugate is purified by known techniques and analyzed for total polysaccharide content, protein content, free polysaccharide, polysaccharide-protein ratio and conjugation yield. The purified conjugates are stored 2 to 8°C. The conjugates of the present invention are stable when exposed to high temperature conditions of 37°C. Men conjugates of the present invention show high antigenicity and high immunogenicity as revealed by serum bactericidal assay (SBA) and various immunogenic assays such as ELISA.

NON-LIMITING EXAMPLES

Example V. Sizing of the Men X Polysaccharide:

200mg of Men X Polysaccharide is taken in a concentration at lOmg/ ml in glass beaker on ice bath. Run the sonicator at 20% amplitude for a time of 3 hrs. The size of the degraded Polysaccharide is determined in terms of distribution coefficient (Kd) by running on HP-GPC (Table 2) using refractive index (RI) detector. Samples are eluted in 0.1 M NaN03 at pH 7.2 in isocratic mode on TSK gel G5000 PWXL + G4000 PWXL column in series. Elution volumes are measured in terms of retention time. Void volume (V o) of the column is calculated from the high molecular weight dextran injection retention time and the total volume (V t) of the column is determined by sodium azide injection retention time respectively. Kd is calculated from the formulae Kd=(Ve-Vo/Vt-Vo). Ve is the retention time (RT) of elution volume of the sample (Fig. 3). The data recorded using RI detector shows the shift in peak towards right suggesting depolymerisation of native polysaccharides (Fig. 4).

Table 2: Results showing size distribution of meningococcal polysaccharide serogroup X by HP-SEC on TSK gel G5000 PWXL + G4000 PWXL column. polvsji i h doUv .or (Vo = l ( ⁾ .7»m i n iiriilc Vl= 22.S0 l m m)

492kDa 200mg/20ml 3.0hrs 15.36 5 min 0.384 Kd

570kDa 200mg/20ml 4.5hrs 15.322 min 0.380 Kd

Example 2: Polysaccharide activation with CDI

200 mg of sized polysaccharide and LiCl are mixed with slow stirring at RT for 1 hr and thereafter has been dried on rotavapour at 40°C. Then redissolved in 15 ml of dry DMSO and keep mixing for 2 hrs. 30X molar of Carbonyl di-imidazole (CDI) to polysaccharide is added then. pH has been adjusted to 9.0 by adding Triethylamine (TEA). Slow stirring done at RT for 2 hours and cool the sample in ice to stop the reaction. Added Ethyl acetate and removed the supernatant from the top. Again added Ethyl acetate and removed the supernatant from the top. Dry on high vacuum for 30 minutes.

Example 3: TT activation

250 mg TT is concentrated using 50 kDa molecular weight cut off (MWCO) centrifugal filter to make final 10 ml. The 2.75ml of Hydrazine monohydrate from stock 5 M or 2.5gm of ADH, equivalent to 10X by weight of TT is added to 10 ml of reaction buffer i.e 0.15 M MES buffer containing 0.2 M NaCL pH 5.75 and added to TT above. To this mixture, an equal amount of EDAC (250mg) in 1ml of reaction buffer to make final concentration of approximate 30mM. The pH of the reaction mixture adjusted to 5.85 and final volume of the reaction mixture adjusted to 25 ml. The concentration of TT in reaction mixture is 10 mg/ml. The reaction mixture kept for slow stirring for 1.0 hour in ice bath. The derivatized TT is further purified and analyzed for degree of activation and SEC-HPLC to monitor peak profile. HPLC-SEC profile comparison of Hydrazine activated TT with native TT is shown in Fig 5 wherein the samples have been run on chromatography column in isocratic mode and the data have been recorded using PDA detector.

Example 4: Purification of activated TT

Purification is one of the most important steps in the process and in TT activation. We need to be sure to get rid of any un-reacted hydrazine or ADH to maximum possible. The purification is done by Sephadex G-25 desalting method. Desalt the reaction mixture on sephadex G25 column against 50 mM phosphate buffer containing 75 mM NaCL pH 7.5 to purify the derivatized protein. Chromatography column equilibrated with 50 mM phosphate buffer containing 75 mM NaCL pH 7.5, followed by sample loading and elution at 110 cm/hr. Eluted fractions of 10 ml each collected and fractions corresponding to the peak at 280 nM on UV (Fig. 6) are pooled and concentrated by 50 kDa MWCO Amikon membrane. Selected fractions having TT are concentrated to such a volume to have TT concentration approximately 30-50 mg/ml (considering approximately 75% recovery of activated TT after desalting). Final activated TT is analyzed for protein concentration by Lowry assay and hydrazide labeling by TNBS assay. Both the values used for the calculation of degree of activation (DO A) of TT. Activated TT is run on HPLC at a concentration of 1 mg/ml using PWXL 5000 - PWXL 4000 columns in series to check the peak profile for its integrity.

The process of present invention activates the hydroxyl groups of the different polysaccharides preferably MenX PS with different activating agents preferably carbonyl di-imidazole to make it reactive towards the carrier protein to form a stable PS-TT conjugate (Fig 7). Example 5: Conjugation reaction of activated MenX and derivatized TT

Derivatized TT has been added to the activated Men X PS directly in 3ml of 0.1M Sodium carbonate, 0.1M NaCl buffer at pH 9.0 solution and maintains the pH at 9.0. Activated Polysaccharide and derivatized TT are mixed in a proportion of 1:1 w/w for the conjugation reaction. Formation of conjugate is confirmed in three hours of time only, but the crude conjugate mixture is kept mixing overnight at room temperature. The reaction is then quenched by using a 5-10 molar excess of amine-containing reagent such as glycine.

The course of conjugation is monitored by SEC-HPLC analysis with change in the retention time of the activated TT and peak shift towards the left. HPLC-SEC profile of the conjugate depicts that conjugation reaction is completed to maximum within three to four hours. The SEC-HPLC profiles of native and activated TT and the conjugate indicates that upon activation, the size of activated TT remains unchanged from the native TT, suggesting that little or no aggregation occurs. The samples have been run on chromatography column at the flow rate of 1.0 ml/ min using 0.1M sodium nitrate, pH 7.2 buffer in isocratic mode and the data have been recorded using PDA detector. After conjugation, high molecular weight peak appears to the left of derivatized TT peak, indicating the formation of PS- TT conjugates (Fig. 8). Example 6: Removal of unreacted (free) Polysaccharide from crude conjugate by Ammonium sulphate precipitation

Further, crude conjugate is purified by protein precipitation method to remove free or unreacted polysaccharide. Purification is carried out by slowly adding solid ammonium sulphate to the reaction mixture until conjugate is precipitated out of the solution. The precipitates are separated by centrifugation at 5000 x g for 45 minutes. The supernatant is discarded and the precipitates re-dissolved in 30ml of 50 mM MES buffer containing 100 mM Nad, pH 6.5.

Example 7: Purification of polysaccharide-protein conjugate by Tangential flow filtration (TFF).

The conjugate sample after ammonium sulphate purification is further purified by using 300 kDa MWCO cassettes from Pall. This step ensures the removal of free protein if any from the conjugate and to separate free PS further from the conjugated PS. The conjugate is diafiltered with 20 X volume of MES buffer, pH 6.5 and finally concentrated to a volume of 35 ml.

Hence the removal of residual reagents used during the entire conjugation process is ensured at four different steps, first for PS: at the time of activated PS precipitation step, second for carrier protein: at the GPC purification step, third for whole conjugation process: by Ammonium sulphate precipitation and fourth: by 300kDa diafilteration step. It is further filtered by 0.2μ filters finally and stored at 2-8°C.

Example 8: Characterization of MenX-TT conjugate:

The purified conjugate is analyzed for total polysaccharide content by phosphorous assay for MenX-TT conjugates. Protein content is determined by Lowry's assay. The free polysaccharide determined by precipitating with sodium deoxycholate method and supernatant is analyzed by respective colorimetric assay. Different assays to determine the various residuals are conducted before using it further for formulation. The purified conjugates are stored at 2 - 8 °C.

The Conjugates of the present invention when analyzed for various 5 Quality parameters (Table 3) and found to give polysaccharide/ protein ratio in the range of 0.3 - 0.7 (w/w). Amount of free polysaccharide is different for different conjugates, however, over all free polysaccharide has been less than 10% in purified conjugates. Conjugation yield has also varied for different conjugates from 18% to 43% . In this invention, various 0 conjugation scales are tried from 20mg to 230mg.

Table 3: Characterization of different Men X-TT conjugate lots

Example 9: Characterization of Men A, C, Y, W conjugates obtained by carbamate chemistry

In a similar way to MenX, the serogroups Men A, C, Y & W conjugates were prepared by using the present invention. All the conjugates were 5 characterized for various Quality parameters and found to give the desired results (Table 4).

Table 4: Characterization of different Men-TT conjugates

0 Example 10: Stability of MenX-TT conjugate:

The Conjugates of the present invention are exposed to high temperature conditions of 37°C for 28 days. The samples have been taken out on day 7,14,21 & 28 to monitor the free polysaccharide generated. The stability of the Men X conjugates, prepared by the defined Carbamate chemistry in 5 this embodiment, proved the process applicability to generate extreme stable conjugates (Table 5). Table 5: Stability data for Men X-TT conjugate lot, prepared by the chemistry of present invention

Example 11: Immunogenicity of MenX Conjugates

Groups of 8 female BALB/c mice of 5-9 weeks have been immunized on days 0, 14 and 28 with two different lots of MenX conjugated PS antigen formulated in normal saline (Table 6) at 1 μg dose level. All immunizations are performed by administering 200 μΐ of vaccine dilution via subcutaneous route. Normal saline alone is used for negative control group. Sera is collected at 7-14 days after 2 and 3-doses. Specific anti-PS IgG antibody titers are estimated by ELISA for post 2 and 3-dose.

The maximum IgG titers for MenX conjugates are achieved after two boosters. It is observed that for MenX the increase in titer value after 3 doses, in comparison to negative control is approximately 100 fold in Men X conjugate lot 1 and 150 fold in Men X conjugate lot 2 (Table 6).

Table 6: Geometric mean for IgG titer by ELISA post 2- and 3-dose (+,- 95% Confidence Interval for post 3-dose) for MenX-TT formulations in mouse model, after immunization with of antigen on day 0, 14 and 28.

Example 12: Serum Bactericidal Assay (SBA) for MenX Conjugates

Equal volume from each of the sera sample belonging to a group of mice are pooled together to make group sera pools for testing by serum bactericidal assay. The assay is performed as follows:

Streak the N. meningitidis serogroup X target strain for single colony isolation and incubate overnight at 37°C with 5% CO2 on a Sheep blood agar plate. The strain is subcultured by spreading cells over the entire surface of another Sheep Blood agar plate and then incubated for fresh growth at 37°C with 5% CO2. Bacteria are resuspended in ~ 5 ml of bactericidal assay buffer. Suspension OD ₆ 5o is being adjusted equivalent colony count of approximately lx 10 ⁵ cfu/ ml. The sera are diluted serially 2-fold and assay buffer is added in control wells. ΙΟμΙ of the working solution of bacteria is added to every well. 10 μΐ of heat inactivated (kept at 56°C for 30 min.) complement is added to all inactive complement control wells and 10 μΐ of said inactivated complement is added to sera containing wells and active complement control wells. Shake the plates and incubate the plates for 1 hour at 37°C.

After incubation, spot ΙΟμΤ from each wells on slanted blood agar plate. Incubate all agar plates for overnight at 37°C with 5% CO2. Count the number of colonies on each spot of the plates. The highest serum dilution showing >50% killing of bacteria as compared to complement control is considered as the SB A titre of that serum sample.

The SBA data shows a negligible response from the vehicle immunizations after 3 doses, whereas both the Men X conjugate lots under test showed significantly high SBA titres as compared to the vehicle control indicating the vaccine to be effective in-vivo in the mouse model (Table 7).

Table 7: Geometric mean titer for SBA post 2- and 3-doses (+,- 95% Confidence Interval for post 3-doses) for MenX-TT formulations in mouse model, after immunization with of antigen on day 0, 14 and 28.