FORM-2
THE PATENT ACT,1970
(39 OF 1970)
AND
THE PATENT RULES, 2003
(As Amended)
COMPLETE SPECIFICATION (See section 10; rule 13)
“VACCINE COMPOSITION COMPRISING NEISSERIA MENINGITIDIS POLYSACCHARIDE-PROTEIN CONJUGATES”
SERUM INSTITUTE OF INDIA PVT LTD., a corporation organized and existing under the laws of India, of212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028, Maharashtra, India.
The following specification particularly describes the invention and the manner in which it is to be performed:

FIELD OF INVENTION
The invention relates to vaccine compositions for prophylaxis against gram negative human pathogen – meningococci. The invention specifically relates to immunogenic compositions against Neisseria meningitidis(meningococcus).
BACKGROUND OF THE INVENTION
The background information herein below relates to the present disclosure but is not necessarily prior art.
Neisseria meningitidis(meningococcus) is a Gram negative human pathogen. It colonizes the pharynx, causing meningitis and, occasionally, septicemia in the absence of meningitis. It is closely related to N. gonorrhoeae, although one feature that clearly differentiates meningococcus is the presence of a polysaccharide capsule that is present in all pathogenic meningococci.Based on the organism's capsular polysaccharide, twelve serogroups of N. meningitidishave been identified (A, B, C, H, I, K, L, 29E, W135, X, Y and Z).
Serogroup A ('MenA') is most common cause of epidemic disease in sub-Saharan Africa. Serogroups B & C are responsible for the majority of cases in developed countries, with the remaining cases being caused by serogroups W135 & Y.
The capsular polysaccharideshave been used for vaccination. An injectable tetravalent vaccine of capsular polysaccharides from serogroups A, C, Y & W135 has been known for many years and is licensed for human use. Although effective in adolescents and adults, it induces a poor immune response and short duration of protection and cannot be used in infants.To overcome the relatively low immunogenicity of polysaccharides, thepolysaccharide in vaccines are conjugated to protein carriers to increase immunogenicity and provide long-term protection in young children. Many meningococcal conjugate vaccines are already approved and marketed throughout the world. Examples of such vaccines, known as "Neisseria meningitidis conjugates" are monovalent meningococcal A conjugate (MenAfriVac™),monovalent meningococcal C conjugate (Meningitec™) and quadrivalent A C Y W meningococcal

conjugates(Menveo™ &Menactra™)Menjugate™, NeisVac-C™.Existing conjugate vaccines based on meningococcal polysaccharide are based on two-dose regimen and the minimum relative vaccine efficacy of a single-dose is found to be lessthantwo-dose regimenwith the same amount of vaccine. In the face of limited supply, a one-dose schedule should provide an equal immunogenic protection as two doses in case of an epidemic. Further, the attempts to produce a multivalent meningococcal conjugate vaccine that exhibits equal immunogenicity with respect to each polysaccharide have been unsuccessful.
The capsular polysaccharides of serogroup B, C, Y, and W135 meningococci are composed of sialic acid derivatives. Serogroup B and C meningococci express (α 2-8)-and(α 2-9 239)-linked polysialic acid, respectively, while alternating sequences of D-glucose or D-galactose and sialic acid are expressed by serogroup Y and W135 N. meningitidis.
In contrast,the capsule of serogroup A meningococci is composed of(α 1-6 )-linked N-acetylmannosamine 6-phosphate , while N. meningitidis serogroup X synthesizes capsular polymers of( α 1-4)-linked N-acetylglucosamine 1-phosphate.Refer Yih-Ling Tzeng et al;Genetic Basis for Biosynthesis of the ( 134)-Linked N-Acetyl-D-Glucosamine 1-Phosphate Capsule of Neisseria meningitidis Serogroup X; Infection And Immunity, Dec. 2003, p. 6712–6720 ;Vol. 71, No. 12.
Overall charge densities significantly contribute towards immunological properties on the capsular polysaccharide. Overly crowded placement of immunological functional groups such as O acetyl group can considerably alter the antibody binding properties of the polysaccharide. Thus, trimming the O acetyl groups by controlled de-O-acetylation close to the specification limit is one way to improve the product quality. Little is known about the O-acetylation status of serogroup C meningococci.
According to WHO Technical Report Series on Meningococcal Conjugate Vaccine, following specification should be met by isolated polysaccharides:

Table 1

Control Parameter WHO Specification

Men-C Men-W Men-Y
Polysaccharide content (PS) >3 mg/ml >3 mg/ml >3 mg/ml
Sialic Acid Content >80% >56% >56%
O Acetyl Content >1.5mM/gm of PS >0.3mM/gm of PS >0.3mM/gm of PS
Molecular distribution Size 75% above 0.5kD 80% above 80% above 0.5kD 0.5kD
The existing meningococcal conjugate vaccines are based on A C Y W135 polysaccharides.The increase in incidence of MenX disease in African Meningitis Belt in the last 5 years warrants development and introduction of a MenX polysaccharide conjugate vaccine in selected areas of the region to prevent and control future epidemics.Though has been reported earlier.Inspite of availability of comprehensive seroprevalence and structural data for meningococcal X, a commercially viable conjugate vaccine including X polysaccharide is yet to be developed due to extremely limited success on purification,conjugation and formulation stability aspects for the same. This provides an additional challenge for successfully addressing and controlling various parameters, especially when employing a scalable conjugation process for the large-scale manufacture of Neisseria meningitidis conjugates containing Neisseria meningitidis X polysaccharide.
Thus, a need exists for a meningococcal conjugatevaccine based on A C Y W135 & X polysaccharides capable of conferring broad, long-lived protection against meningococcal disease in children and adults at risk for meningococcal infection wherein each antigen providing equal immunogenic protection in a single dose.

OBJECTS
An object of the present disclosure is to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
It is therefore an object of the present disclosure to provide a meningococcal conjugate vaccine based on A C Y W135 & X polysaccharidescharacterized by a substantially improved immunogenicity and safety wherein, a single dose of the said vaccine composition devoid of any adjuvant is sufficient to elicit the required T-dependent immune response against Neisseria meningitidis serogroup A, C, Y, W135 and X in a human subject 2 years of age or below.
Yet another object of the present disclosure is to provide a meningococcal conjugate vaccine based on A C Y W135 & X polysaccharideswherein, 60-95% of the sialic acid residues in the serogroup C meningococcal capsular saccharide are O-acetylated and the average molecular size of the serogroup C meningococcal capsular saccharide molecule ranges in between 75 kDa to 150 kDathat could be administered either intramuscular (IM) or intradermal (ID) or subcutaneous (SC).
Another object of the present disclosure to provide a meningococcal conjugate vaccine based on A C Y W135 & X polysaccharidesin combination with adjuvants.
Yet another object of the present disclosure is to provide a meningococcal conjugate vaccine based on A C Y W135 & X polysaccharides suitable for treating or preventing meningococcal infection, or to prevent, ameliorate, or delay the onset or progression of the clinical manifestations thereof.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY OF THE INVENTION
The present invention relates to a vaccine composition. The vaccine composition comprises:

a) a Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to a tetanus toxoid carrier protein as the vaccine antigen;
b) a Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to a CRM197 carrier protein as the vaccine antigen;
c) a Neisseria meningitidis serogroup Y (MenY) capsular saccharide conjugated to a CRM197 carrier protein as the vaccine antigen;
d) a Neisseria meningitidis serogroup W135 capsular saccharide conjugated to a CRM197 carrier protein as the vaccine antigen; and
e) a Neisseria meningitidis serogroup X (MenX) capsular saccharide conjugated to a tetanus toxoid carrier protein as the vaccine antigen.
The Neisseria meningitidis serogroup C (MenC) capsular saccharide is sized by High Pressure Cell Disruption and has an average molecular size in the range of 75 kDa to 150 kDa. Further, 60-95% of the sialic acid residues in the Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to CRM197 are O-acetylated. The vaccine composition in an embodiment is devoid of an adjuvant. A single dose of the vaccine composition is sufficient to elicit the required T-dependent immune response against Neisseria meningitidis serogroup A, C, Y, W135 and X in a human subject 2 years of age or below.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING
The present disclosure will now be described with the help of the accompanying drawing, in which:
Figure 1: Overlay of conjugation reaction when Men X polysaccharide(215 kDa) conjugated to Hydrazine derivatized TT;
Figure 2: Purified Conjugate when Men X polysaccharide(215 kDa) conjugated to Hydrazine derivatized TT;
Figure 3: Overlay of conjugation reaction when Men X polysaccharide(326 kDa) conjugated to ADH derivatized TT;

Figure 4: Purified Conjugate when Men X polysaccharide(326 kDa) conjugated to ADH derivatized TT;
Figure 5: Overlay of conjugation reaction when Men X polysaccharide(120 kDa)conjugated to ADH derivatized TT;
Figure 6: Purified Conjugate when Men X polysaccharide(120 kDa) conjugated to ADH derivatized TT;
Figure 7:Chromatogram of Native Meningococcal X polysaccharide;
Figure 8:Overlay of conjugation reaction when Men X polysaccharide(510 kDa) activated with ADH and conjugated to purified TT;
Figure 9:Purified Conjugate when Men X polysaccharide(510 kDa) activated with ADH and conjugated to purified TT;
Figure 10:Overlay of conjugation reaction when Men X polysaccharide(250 kDa) activated with ADH and conjugated to purified TT;
Figure 11:Purified Conjugate when Men X polysaccharide(250 kDa) activated with ADH and conjugated to purified TT;
Figure 12: NMR spectra of Men-C polysaccharide;
Figure 13: NMR spectra of Men-Y polysaccharide;
Figure 14: NMR spectra of Men-W polysaccharide;
Figure 15: NMR spectra of Men-A polysaccharide; and
Figure 16: NMR spectra of Men-X polysaccharide.
DETAILED DESCRIPTIONOF THE INVENTION
The present invention provides vaccine composition. The vaccine composition comprises Neisseria meningitidispolysaccharide-proteinconjugates. Typically, the Neisseria

meningitidisserotypes can be Neisseria meningitidisSerotype A, Neisseria meningitidisSerotype C, Neisseria meningitidisSerotype Y, Neisseria meningitidisSerotype W135, and Neisseria meningitidisSerotype X.
Composition I comprises (a)a conjugate of (i) the capsular saccharide of serogroup A N meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) capsular saccharide of serogroup C N meningitidis and (ii) tetanus toxoid; (c) a conjugate of (i) capsular saccharide of serogroup Y N. meningitidis and (ii) diphtheria toxoid;(d) a conjugate of (i) capsular saccharide of serogroup W135 N.meningitidis and (ii) tetanus toxoid; and (d) a conjugate of (i) capsular saccharide of serogroup X N meningitidis and (ii) tetanus toxoid.
Composition II comprises (a)a conjugate of (i) the capsular saccharide of serogroup A N meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) capsular saccharide of serogroup C N meningitidis and (ii)CRM197; (c) a conjugate of (i) capsular saccharide of serogroup Y N. meningitidis and (ii) tetanus toxoid;(d) a conjugate of (i) capsular saccharide of serogroup W135 N.meningitidis and (ii)CRM197; and (d) a conjugate of (i) capsular saccharide of serogroup X N meningitidis and (ii)CRM197.
Composition III comprises (a)a conjugate of (i) the capsular saccharide of serogroup A N meningitidis and (ii)CRM 197; (b) a conjugate of (i) capsular saccharide of serogroup C N meningitidis and (ii)CRM197; (c) a conjugate of (i) capsular saccharide of serogroup Y N. meningitidis and (ii) tetanus toxoid;(d) a conjugate of (i) capsular saccharide of serogroup W135 N.meningitidis and (ii)CRM197; and (d) a conjugate of (i) capsular saccharide of serogroup X N meningitidis and (ii)tetanus toxoid characterized in that conjugates containing tetanus toxoid as carrier protein are found to enhance immunogenicity of conjugates containing CRM 197 as carrier protein.
composition IV comprises (a)a conjugate of (i) the capsular saccharide of serogroup A N meningitidis and (ii)tetanus toxoid; (b) a conjugate of (i) capsular saccharide of serogroup C N meningitidis and (ii)CRM197; (c) a conjugate of (i) capsular saccharide of serogroup Y N. meningitidis and (ii) CRM197;(d) a conjugate of (i) capsular saccharide of

serogroup W135 N.meningitidis and (ii)CRM197; and (d) a conjugate of (i) capsular saccharide of serogroup X N meningitidis and (ii)tetanus toxoid.
Composition V comprises (a)a conjugate of (i) the capsular saccharide of serogroup A N.meningitidis and (ii)CRM 197; (b) a conjugate of (i) capsular saccharide of serogroup C N.meningitidis and (ii)CRM197; (c) a conjugate of (i) capsular saccharide of serogroup Y N.meningitidis and (ii) CRM197;(d) a conjugate of (i) capsular saccharide of serogroup W135 N.meningitidis and (ii)CRM197; and (d) a conjugate of (i) capsular saccharide of serogroup X N.meningitidis and (ii)`tetanus toxoid.
Accordingly in a first embodiment, the composition can comprise of serogroup A,C,Y,W135 and X saccharide at an amount of 0.5-10µg,0.5-5µg or 0.5-2µg per 0.5 ml dose.
Another aspect of first embodiment is that said composition can comprise of 10 µg of serogroup A saccharide, 5µg of serogroup C saccharide, 5µg of serogroup W135 saccharide, 5µg of serogroup Y saccharide and 5µg of serogroup X saccharide.
Alternatively said multivalent immunogenic composition can comprise of 5 µg of serogroup A saccharide, 5µg of serogroup C saccharide, 5µg of serogroup W135 saccharide, 5µg of serogroup Y saccharide and 5µg of serogroup X saccharide.
Accordingly in a second embodiment,said one or more N.meningitidis saccharide conjugates can optionally be adsorbed onto aluminium hydroxide, aluminium phosphate or a mixture of both or unadsorbed onto adjuvant.
One aspect of second embodiment is that aluminium salt adjuvant can be added at an amount of 20-300µg,20-200µg,25-150µg of Al+++ per 0.5 ml dose.
Another aspect of second embodiment is that aluminium salt adjuvant can be added at an amount of 25-125µg of Al+++ per 0.5 ml.
Yet alternatively the said one or more N.meningitidis saccharide conjugates are not adsorbed onto any adjuvant.

A third embodiment of the instant invention is that said composition can comprise of a preservative selected from thiomersal and 2-phenoxyethanol.
One aspect of third embodiment is that said can further comprise of sodium phosphate, sodium chloride or combination thereof.
A fourth embodiment of the instant invention is that said multivalent immunogenic composition can be in a buffered liquid form or in a lyophilized form.
One aspect of fourth embodiment is that said lyophilized immunogenic composition can comprise of a stabilizer combination selected from a) 2 to 5% (w/v) Trehalose, 0.25 to 0.75% sodium citrate;b) 2 to 5% (w/v) Sucrose and 0.25 to 0.75% sodium citrate;c) 2 to 5% (w/v) Sucrose,2 to 5% (w/v) Lactose and 0.25 to 0.75% sodium citrate;and d) 2 to 5% (w/v) Trehalose ,2 to 5% (w/v) Lactose and 0.25 to 0.75% sodium citrate.
Another aspect of the fourth embodiment is that said lyophilized immunogenic composition can further comprise a buffer selected from Tris and phosphate.
Yet preferably the lyophilized multivalent immunogenic composition can comprise of 5 µg of serogroup A N. meningitidis saccharide conjugated to tetanus toxoid, 5µg of serogroup C N. meningitidis saccharide conjugated to CRM197, 5µg of serogroup W135 N. meningitidis saccharide conjugated to CRM197, 5µg of serogroup Y N. meningitidis saccharide conjugated to CRM197, 5µg of serogroup X N. meningitidis saccharide conjugated to tetanus toxoid, 2.42 mg Sucrose, 0.40 mg sodium citrate, and 0.098 mg TRIS per 0.5 ml dose.
Accordingly in a fifth embodiment,said polysaccharides A C W and X can be mechanically sized with simultaneous de-o-acetylation to have an average molecular weight between 70-800 kDa, ,preferably between70-200 kDa, andmost preferably between 70-150 kDa. Mechanical sizing methods include homogenization, sonication, microfluidization and high pressure cell disruption.
Yet the preferred sizing method include high pressure cell disruption (at pressures as high as 40000 psi).

In one aspect of fifth embodiment, 60-95% of the sialic acid residues in the Neisseria meningitidis serogroup C (MenC) capsular saccharide are O-acetylated.Overall charge densities significantly contribute towards immunological properties on the capsular polysaccharide. Overly crowded placement of immunological functional groups such as O acetyl group can considerably alter the antibody binding properties of the polysaccharide. Thus, trimming the O acetyl groups by controlled de-O-acetylation close to the specification limit is one way to improve the product quality.
In another aspect of fifth embodiment, said polysaccharide Y can be sized to have an average molecular weight between 80-120 kDa, by a method selected from acid hydrolysis, alkaline degradation, oxidation by periodate, ozonolysis, enzymatic hydrolysis, sonication , electron beam fragmentation.Preferably, chemical sizing is by using sodium acetate at a temperature from 60 to 80oC.
In a sixth embodiment,each of the N. meningitidis saccharides is conjugated to the carrier protein via a hetero or homo-bifunctional linker with cyanylation conjugation chemistry.
In one aspect of sixth embodiment,said sized polysaccharide is activated by utilizing a cyanylation reagent selected from but not limited to 1-cyano-4- (dimethylamino)-pyridiniumtetrafluoroborate ('CDAP'), p-nitrophenylcyanate and N-cyanotriethylammoniumtetrafluoroborate ('CTEA').In a preferred conjugation process, cyanylating reagent is other than CDAP and can be selected from a group of 1-cyano- 4-pyrrolidinopyridiniumtetrafluoroborate (CPPT), 1- cyano- imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), or 2- cyanopyridazine -3(2H)one (2-CPO), or a functional derivative or modification thereof.
In anotheraspect of sixth embodiment,said activated polysaccharide or carrier protein,particularly polysaccharide is reacted with hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide,a mixture thereof,preferably with adipic acid dihydrazide.
Hydrazide groups can be introduced into proteins through the carboxyl groups of aspartic acid and glutamic acid residues on the protein using a carbodiimide reaction, for example, by reaction with hydrazine, carbohydrazide, succinyldihydrazide, adipic acid

dihydrazide, hydrazine chloride (e.g., hydrazine dihydrochloride) or any other dihydrazides in the presence of EDC. EDC is employed as a catalyst to activate and modify the protein reactant with hydrazine or the dihydrazide. Any water-soluble carbodiimide including EDC can be used as a catalyst. EDC-catalyzed proteins generally have a tendency to polymerize and precipitate. See Schneerson et al., Infect. Immun. 1986, 52:519-528; Shafer et al., Vaccine 2000; 18(13): 1273-1281; and Inman et al., Biochemistry 1969; 8:4074-4082.
In a seventh embodiment,said multivalent meningococcal polysaccharide protein conjugate composition contains polysaccharides from A, B, C, H, I, K, L, 29E, W135, Y and Z conjugated individually to two or more different types of carrier proteins. The capsular saccharides are chosen from meningococcal serogroups A, C, W135 Y and X, such that the compositions include saccharides from 1, 2, 3, 4,or 5 of these five serogroups. Specific compositions comprise saccharides from: serogroups A & X; serogroups X & W135; serogroups X & Y; serogroups C & X;serogroups A Y & X; serogroups C, X & W135; serogroups X, Y & W135; serogroups A,C & X;serogroups Y,C & X;serogroups A,W & X;serogroups Y &W135 & C & X;serogroups Y & W135 & A & X;serogroups C & W135 & A & X;serogroups Y & C & A & X; serogroups A & C & Y & W135 & X.Compositions including at least serogroup X are preferred , and compositions including saccharides from all five serogroups are most preferred.
In an aspect of seventh embodiment, said carrier protein can be selected from a group of but not limited to CRM 197,diphtheria toxoid,tetanus toxoid, pertussis toxoid, E. coli LT, E: coli ST, and exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA),pneumococcal surface proteins BVH-3 and BVH-11, protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA) and purified protein derivative of tuberculin (PPD).Preferably, combinations of carrier proteins to be utilized comprise tetanus toxoid & diphtheria toxoid,CRM197 & tetanus toxoid.

In another aspect of third embodiment, conjugation reaction utilizes linkers selected from the group consisting of adipic acid dihydrazide, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine and p-nitrophenylethyl amine.
After conjugation,conjugates can be purified from unreacted protein and polysaccharide by any standard techniques including, inter alia, size exclusion chromatography, density gradient centrifugation,ultrafiltration, hydrophobic interaction chromatography or ammonium sulfate fractionation. See, e.g., P. W. Anderson, et. al. (1986). J. Immunol. 137: 1181-1186. See also H. J. Jennings and C. Lugowski (1981) J. Immunol. 127: 1011-1018.
In an eighth embodiment, said immunogenic composition of the instant invention can further comprise of an additional non-meningococcal polysaccharide protein conjugate,wherein said polysaccharides and oligosaccharides for use can be selected from but not limited to pneumococcal polysaccharides of serogroups 1, 2, 3, 4,5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F; Haemophilusinfluenzae type b polysaccharide polyribosylribitol phosphate, group B streptococcal polysaccharides of serotypes III and V and Salmonella typhi Vi polysaccharide. Other polysaccharides of pneumococcal and group B streptococcal serotypes are also suitable for use herein, as are other T-independent polysaccharide and oligosaccharide antigens, for example, polysaccharides or oligosaccharides derived from group A streptococcus, Staphylococci, Enterococci, Klebsiella pneumoniae, E.coli, Pseudomonasaeruginosa, and Bacillusanthracis. While bacterial polysaccharides and oligosaccharides are particularly preferred, gram (-) bacterial lipopolysaccharides and lipooligosaccharides and their polysaccharide and oligosaccharide derivatives, and viral polysaccharides and oligosaccharides can also be employed.
Compositions of the invention may be presented and packaged in various ways.The compositions may be presented in vials, or they may be presented in ready-filled syringes. The syringes may be supplied with or without needles. A syringe will include a single dose of the composition, whereas a vial may include a single dose or multiple doses. Injectable compositions will usually be liquid solutions or suspensions.

Alternatively, they may be presented in solid form (e.g. freeze-dried) for solution or suspension in liquid vehicles prior to injection.
The present invention is further described in light of the following examples which is set forth for illustration purpose only and not to be construed for limiting the scope of the invention.
Examples:
The molecular size of the polysaccharides and the polysaccharide-protein conjugatesused in the present invention were determined using Size Exclusion Chromatography (SE-HPLC). The unit of the molecular size is given in kilo Daltons (kDa or kD).
Example 1:
Thedetail of the biological material used during experimentation is as follows:
Polysaccharides were isolated from:
Table 1

Name of the Organism Strain Designation Source of Strain
Neisseria meningitidis A M1027 SynCoBiopartners ( Netherlands)
Neisseria meningitidis C C11(60E) CBER/FDA, USA
Neisseria meningitidis W S877 CBER/FDA, USA
Neisseria meningitidis Y M10659 CDC, USA
Neisseria meningitidis X M8210 CBER/FDA, USA
Unconjugated carrier protein, i.e. CRM197 or TT. CRM197 was derived from Recombinant Strain CS463-003 (MB101) of Pseudomonas fluorescens from Pfenex USA. TT was derived from Clostridium tetani (Harvard No 49205) obtained from CRI, National Control Authority, Kasauli, Himachal Pradesh, India. CRI got this strain from NVI, Netherland.

Example 2:
Purification of capsular polysaccharides (Downstream process of N. Meningitidis
Serogroup C)
Protocol:
1) IsolatedCrude polysaccharide obtainedwas taken in a vessel.
2) Crude polysaccharide was 3-6 fold concentrated by TFF using 100 kDa cassette.
3) Sodium Dodecyl Sulphate was added to this inactivated harvest to a final concentration of 1% and stirred at room temperature for 2 hour;
4) Sodium Hydroxide was added to a final concentration of 08 - 20mM and adjusted to pH 10.5 with constant stirring at room temperature for 1 hour;
5) Solution obtained from above step was neutralized by addition of Acetic acid.
6) To this neutralized solution, ethanol was added to a final concentration of 33% and incubated for couple of hours with constant stirring.
7) Solution obtained from above step was centrifuged and supernatant was collected.
8) Ethanol concentration was increased to 40%.
9) 0.1M KCl was added to the supernatant and incubated at 2 – 8 °C for not less than 3 hour.
10) Solution obtained from above step was subjected to centrifugation and supernatant was collected. Polysaccharide was precipitated by increasing final concentration of ethanol to 65%.
11) Polysaccharide pellet was dissolved in 1M NaCl. The supernatant was passed through a 0.2µ filter and retentate was diafiltered using 25mM Tris-HCl buffer through 100 kDa tangential flow filtration, to obtain purified polysaccharide (Stage I).
12) Addition of CTAB to a final concentration of 2%; and incubated at RT for 1 hour.

13) Solution obtained from above step was centrifuged and precipitate was collected and dissolved in 96% Ethanol. The dissolved mixture was further subjected to centrifugation for removal of undissolved residues.
14) To supernatant obtained from above step, NaCl was added to a final concentration of 0.2M. The precipitated PS is dissolved in 1M NaCl followed by PS precipitation using 65% alcohol.
15) Solution obtained from above step was extensively diafiltered against 0.5M NaCl, followed by WFI using 100 kDa tangential flow filtration and passed through 0.2µ filter and stored at -20°C as final bulk.
Example 3: Polysaccharide Fermentation & Purification
As an initial step of removal of impurities (proteins, nucleic acids and endotoxins) an anionic detergent sodium deoxy-cholate (DOC) is usually employed. DOC is an animal origin bile-detergent with a core steroid structure. Due to its bulky structure it has limited solubilizing strength and less denaturing ability on biological macromolecules. This drawback results in inconsistency of purification at higher scale. Additionally, high consumption of ethanol and inclusion of redundant steps (carbon filtration) makes the process tedious, time consuming and costly. As an alternative SDS was introduced as replacement for DOC, owing to its amphipathic nature and higher aggregation number SDS has superior denaturing and solubilizing ability for impurities such as the proteins and also disrupts endotoxins irreversibly to its monomeric units. Moreover, as these polysaccharides are highly anionic and hydrophilic SDS specifically targets only the impurities and completely non-invasive on structural integrity of the polysaccharides. Thus, markedly improving the impurity profile (Significantly lower protein impurities), consistency in quality of the Polysaccharide, specific Polysaccharide molecular size range (350-370 kDa) which directly has impact on the conjugation efficiency and thereafter quality of the conjugate product.
Also, Overall charge densities significantly contribute towards immunological properties on the capsular polysaccharide. Overly crowded placement of immunological functional groups such as O acetyl group can considerably alter the antibody binding properties of

the polysaccharide. Thus, trimming the O acetyl groups by controlled de-acetylation close to the specification limit is one way to improve the product quality. This was carried out using very small concentrations of alkali agent for a specified time and observing the subsequent NMR of the de acetylated product.
Table 3

Sample description Parameters Neisseria meningitides serogroup C
Polysaccharide Fermentation and
Purification (350L batches Results)

540N18001 540N18002 540N18003
Conc.crude PS
(100 kDa Conc. harvest) Volume (L) 50 50 50

PS Conc. (mg/mL) 6.87 8.19 7.27

Total PS (g) 343.5 409.5 363.5

Protein impurity (mg/mL) 5.47 3.9 5.99

Protein impurity (%) 79.62154294 47.61904762 82.39339752

NA impurity (mg/mL) 0.29 0.33 0.4

NA impurity (%) 4.22125182 4.029304029 5.502063274

*Purified Polysaccharide Volume (L) 30 30 30

PS Conc. (mg/mL) 7.47 7.22 7.3

Total PS (g) 224.1 216.6 219

Protein Conc. (mg/mL) 0.004 Non detectable Non detectable

Protein percentage (%) 0.053547523

NA impurity (mg/mL) 0.007 0.0024 0.0029

NA impurity (%) 0.093708166 0.033240997 0.039726027

Molecular size (kDa) 366 355 357

Endotoxin (EU/µg of PS) < 10 < 10 < 10

Step recovery (%) 96.92906574 80.34124629 88.02250804

Final recovery (%) 65.24017467 52.89377289 60.24759285
*Purified polysaccharide is further subjected to sizing/fragmentation

Example 4 Polysaccharide sizing Table 4
Men C Conjugate Related -

Menactra (EP 2345422 Serum Institute’s pipeline
Parameter A1)(Quadrivalent Pentavalent Polysaccharide

Conjugate vaccine) Conjugate Vaccine (ACWYX-
(ACWY-DT) TT,CRM197)NmCV-5
Polysaccharide (PS) Chemical method: Size Mechanical method: Size
sizing method reduction by 1% H2O2 in reduction using a high pressure
presence of acetate buffer. homogenizer
Average PS size used Average size: 10-25kDa Average size: 75-150kDa
for conjugation (measured by SE-HPLC), <100kDa (measured by MALS)
Table 5

Product development Stage: Phase-2
Parameter 541N17001* 541N17002
Average PS Size used for conjugation 107kDa (81 kD by MALS) 109kDa (88 kD by MALS)
O-Acetyl in conjugates 90%-95% 90%-95%
Example 5: Polysaccharide – protein conjugation process design Men C Conjugate Related -Table 6

Parameter Menactra (EP 2345422
A1)(Quadrivalent Conjugate
vaccine) (ACWY-DT) Serum Institute’s pipeline Pentavalent Polysaccharide Conjugate Vaccine (ACWYX-TT,CRM197)NmCV-5
Carrier Protein Diphtheria Toxoid (DT) CRM197
Conjugation carbodiimide Combination of

Chemistry cyanylation(cyanylation) and carbodiimide chemistry
Derivatization Polysaccharide is derivatized by adipic acid dihydrazide linker by carbodiimide and ADH-PS is further conjugated to DT by carbodiimide. Protein (CRM197) is derivatized by carbodiimide chemistry and linked to sized polysaccharide by cyanylationchemistry.
Conjugation Conditions Both derivatization and conjugation take place in acidic pH 4.9-5.1. CRM derivatization takes place in acidic conditions pH (6.3 to 6.7) where as protein-polysaccharide coupling takes place at alkaline pH (9.5-10.5).
O-acetylation level in conjugate Not known 65-95%
Example 6:
Purification of capsular polysaccharides (Downstream process of N. meningitidis Serogroup W & Y)
Protocol:
1) Isolated Crude polysaccharide obtained was taken in a vessel.
2) Crude polysaccharide was 3-6 fold concentrated by TFF using 100 kDa cassette.
3) Sodium Dodecyl Sulphate was added to this inactivated harvest to a final concentration of 1% and stirred at room temperature for 2 hour;
4) Sodium Hydroxide was added to a final concentration of 08 - 20mM and adjusted to pH 10.5 with constant stirring at room temperature for 1 hour;
5) Solution obtained from above step was neutralized by addition of Acetic acid.
6) To this neutralized solution, ethanol was added to a final concentration of 33% and incubated for couple of hours with constant stirring.
7) Solution obtained from above step was centrifuged and supernatant was collected.

8) 0.1M KCl was added to the supernatant and incubated at 2-8 °C for ~8 hours.
9) Solution obtained from above step was subjected to centrifugation and supernatant was collected.
10) The supernatant was passed through a 0.2µ filter and retentate was diafiltered using 25mM Tris-HCl buffer through 100 kDa tangential flow filtration, to obtain purified polysaccharide (Stage I).
11) Addition of CTAB to a final concentration of 2%; and incubated at RT for 1 hour.
12) Solution obtained from above step was centrifuged and precipitate was collected and dissolved in 96% Ethanol. The dissolved mixture was further subjected to centrifugation for removal of undissolved residues.
13) To supernatant obtained from above step, NaCl was added to a final concentration of 0.25M. The precipitated PS is dissolved in 1M NaCl followed by PS precipitation using 65% alcohol.
14) Solution obtained from above step was extensively diafiltered against WFI using 100 kDa tangential flow filtration and passed through 0.2µ filter and stored at -20°C as final bulk (Stage II).
Example 7:
Preparation of Meningococcal X polysaccharide
a) Fermentation and Purification of Meningococcal X Polysaccharide
Meningococcal X polysaccharides are obtained from N.meningitidis strains (8210 & 9601) by utilizing a suitable fermentation medium in a continuous fed-batch fermentation mode under optimal fermentorconditions.Further Meningococcal X capsular polysaccharides are typically prepared by a process comprising the steps of CTAB based precipitation, Ethanol(96%)treatment followed by depth filtration, carbon filtration, CaCl2 precipitation, Ethanol (96%)treatment and ultrafiltration.

b) Sizing of Meningococcal X Polysaccharide
Purified Meningococcal X polysaccharides were subjected to 1-2 passes of mechanical sizing(Constant systems cell disruptor)in WFI at a pressure of about 30-40 kpsi.
Example 8:
Conjugation of Meningococcal X polysaccharide to carrier protein.
a) Meningococcal X Polysaccharide of varying average molecular weight conjugated to hydrazine derivatized tetanus toxoid(TT)
Firstly homogenized Polysaccharide of X (Strain 9601), Average molecular weight 215kDa on SEC HPLC, (30 mg/ml) 45 mg was activated with 90 mg CDAP (dissolved 100mg/ml in acetonitrile), pH of mixture was adjusted to 9.5 with 1M NaOH.Then after 3 min hydrazine activated TT (30mg/ml in 1M NaCl) 67.5 was added to the reaction. The reaction was monitored on HPLC and continued upto 18 hrs. After 18 hrs reaction was quenched by addition of glycine and crude conjugate was purified by diafiltration 300kDa TFF membrane in Tris 10mM pH 7.2. Shodex columns SB-804 HQ and SB-805 HQ were used sequentially with PBS as mobile phase at 1 ml/min flow rate. Polysaccharide concentration and protein concentration were determined by phosphorous assay and modified Lowry assay respectively.
Secondly Polysaccharide of X (Strain 8210), average molecular weight 326 kDa on SEC HPLC, (24 mg/ml in 2M NaCl) 60 mg was activated with 150 mg CPPT (dissolved 114mg/ml in acetonitrile)and pH of mixture was adjusted to 9.5 with 2.5M NaOH.Then after 3 min ADH activated TT (37mg/ml in 2M NaCl) 37.5mg was added to the reaction and the reaction was monitored on HPLC and continued upto 5 hrs. After 5 hrs reaction was quenched by addition of glycine and crude conjugate was purified by diafiltration with 500kDa TFF membrane in 10mM PBS followed by Tris 10mM pH 7.2.
Further Polysaccharide of X (Strain 8210), having average molecular weight 120 kDa on SEC HPLC, (20 mg/ml in 1M NaCl) 200 mg was activated with 400 mg CPIP (dissolved 114mg/ml in acetonitrile), and pH of mixture was adjusted to 9.5 with 1M NaOH.Then

after 3 min ADH activated TT (30mg/ml) 150 mg was added to the reaction. The reaction was monitored on HPLC and continued upto 4 hrs. After 4 hrs reaction was quenched by addition of glycine and crude conjugate was purified by diafiltration with 300kDa TFF membrane in 10mM PBS followed by Tris 10mM pH 7.2.
b) Meningococcal X Polysaccharide of varying average molecular weight activated with ADH and conjugated to purified non-activated tetanus toxoid(TT)
Firstly Polysaccharide of X (Strain 8210) having average molecular weight 510kDa on SEC HPLC (27 mg/ml in 2M NaCl) 200 mg was activated with 400 mg CPPT (dissolved 114mg/ml in acetonitrile) and pH of mixture was adjusted to 9.5 with 2.5M NaOH. Then after 3 min,ADH 1.5g (100mg/ml in carbonate buffer) was added and reaction was continued upto 4 hrs. After 4 hrs glycine was added and reaction mixture was diafiltered on 8kDa TFF membrane. Further ADH-Men X polysaccharide was concentrated. To 44 mg of this(7.5mg/ml), purified TT (37.5 mg/ml in 0.9% NaCl) and MES pH 6.0 buffer were added so that final buffer strength of MES was 100mM, followed by addition of 37.5 mg EDAC (dissolved in 100mM MES, pH 6.0). The reaction was continued for 4 hrs and monitored on HPLC. Unbound polysaccharide was removed by Gel filtration Chromatography using Toyopearl HW65 resin on Akta Chromatography System(GE Amersham).The fractions were collected and pooled based on peak profile and saccharide-protein ratio.
Secondly,Polysaccharide of X (Strain 8210)having average molecular weight 250kDa on SEC HPLC was concentrated to 18 mg/ml in 2M NaCl. A quantity of 200 mg was activated with 296 mg CPPT (dissolved 114mg/ml in acetonitrile) and pH of mixture was adjusted to 9.5 with 2.5M NaOH. Then after 3 min ADH 1.12g (100mg/ml in carbonate buffer) was added and reaction was continued upto 4 hrs. After 4 hrs glycine was added and reaction mixture was diafiltered on 8kDa TFF membrane. ADH-Men X polysaccharide was then concentrated.Further to 200 mg of this (7.5mg/ml), purified TT (36.7 mg/ml in 0.9% NaCl) and MES pH 6.0 buffer were added so that final buffer strength of MES was 100mM, followed by addition of 200 mg EDAC (dissolved in 100mM MES, pH 6.0). The reaction was continued for 4 hrs and monitored on HPLC.

The crude conjugate was purified by diafiltration 500kDa TFF membrane in 10mM PBS followed by Tris 10mM pH 7.2.
Table 7Meningococcal X polysaccharide-protein Conjugation

Avg Mw of
Meningococcal X
polysaccharide(kDa) ADH activation Saccharide/
Protein
ratio Polysaccharide Titer (mg/ml) Protein
Titer (mg/ml)
215 TT (ADH activated) 0.23 0.211 0.921
326 TT (ADH activated) 0.57 0.25 0.435
120 Meningococcal X polysaccharide (ADH activated); TT non-activated 0.59 0.20 0.34
510 Meningococcal X polysaccharide (ADH activated); TT non-activated 0.53 0.180 0.34
250 Meningococcal X polysaccharide (ADH activated); TT non-activated 0.43 0.130 0.30
Above data indicates that final conjugate yield of about 20 to 30% can be obtained by utilizing i) Meningococcal X polysaccharide ofAvg Mw of about 100 to 200 kDa,ii)ADH activated Meningococcal X polysaccharide iii) non-activated TT iv)saccharide:protein ratio between 0.5 to 2 during conjugation reaction v) CPPT as cyanylation reagent and vi) conjugation reaction incubation at 2 to 8oC vi)saccharide:protein ratio between 0.2 to 0.6 in final conjugate.
Example 9:
Conjugation of Meningococcal A,C,Y,W135 polysaccharide to carrier protein
CRM197.

Purified Meningococcal polysaccharides A C Y W135 having average Mw between 70 kDato 200 kDa were conjugated to CRM197 in a saccharide:protein ratio of less than 1 by utilizing a suitable cyanylation reagent(CDAP or CPPT).The conjugates were further purified by diafiltration on 300kDa TFF with 50 volumes of 10mM PBS and 50 volume of 10mM Tris.
Example 10:
Structural integrity of the isolated polysaccharides (PS)
Refer Figure 12 – 16 for NMR spectra.
Structural integrity of the isolated polysaccharides of N. meningitidis serogroup A, C, W, Y & X was verified using Nuclear Magnetic Resonance (NMR) spectra recorded were comparable and confirmed their identity as partially O-acetylated Meningococcal polysaccharide of serogroup A, C, W, &Y; and N-acetylated polysaccharide of serogroupX.
Example 11:
Lyophilization& Formulation of Men A C Y W135 & X conjugate containing two different carrier proteins
Lyophilized formulations containing N.meningitidis conjugates, sodium citrate and Tris buffer in various combinations with trehalose,sucrose and lactose were prepared wherein free polysaccharide content was within limits and moisture content was less than 2%.Said stabilizer combination was selected from a) 2 to 5% (w/v) Trehalose, 0.25 to 0.75% sodium citrate; b) 2 to 5% (w/v) Sucrose and 0.25 to 0.75% sodium citrate; c) 2 to 5% (w/v) Sucrose,2 to 5% (w/v) Lactose and 0.25 to 0.75% sodium citrate;and d) 2 to 5% (w/v) Trehalose,2 to 5% (w/v) Lactose and 0.25 to 0.75% sodium citrate.

Table 8
Liquid formulation containing Monovalent Men X—tetanus toxoid conjugate

Formulation Code Strain Composition Amount per 0.5ml
IRCXLT1 9601 Men X-TT conjugate + Saline + Thiomersal 0.5μg
IRCXLTA1 9601 Men X-TT conjugate + Saline + Thiomersal + AlPO4 0.5μg with 125μg
Al+++
IRCXNT1 9601 Men X-TT conjugate+Saline + Thiomersal 1μ
IRCXNAT1 9601 Men X-TT conjugate+Saline + Thiomersal+AlPO4 1μg with 125μg
Al+++
IRCX2LT1 8210 Men X-TT conjugate+Saline + Thiomersal 0.5μg
IRCX2LAT1 8210 Men X-TT conjugate+Saline + Thiomersal 0.5μg with 125μg
Al+++
IRCX2NT1 8210 Men X-TT conjugate+Saline + Thiomersal 1μg
IRCX2NATA1 8210 Men X-TT conjugate+Saline + Thiomersal 1μg with 125μg Al+++
Table 9
Multivalent Liquid formulation containing Men X—tetanus toxoid conjugate

Composition amount per 0.5ml (μ) |
Formulation Strain A-CR C-CR Y-CRM W-CRM X-TT Excipients
M 197 M19 7 197 197
IRCPT1 9601 1 1 1 1 1 1 1 1 1 1 Sodium Chloride and Thiomersal
IRCPTA1 9601




Sodium Chloride Thiomersal +125μg Al+++
IRCP2T1 8210 111 1 1 Sodium

1 1 1 1 1 Chloride and Thiomersal
IRCP2TA1 8210




Sodium Chloride Thiomersal + 125μg Al+++
Table 10
Multivalent Liquid formulation containing Men X—TT conjugate (Strain 8210)

Composition amount per 0.5ml (μ)
Formulation Code A-CR M 197
1
1 C-CR M 197
1
1 Y-CRM 197 W-CRM 197 X-TT Excipients
IRCP4T1

1 1 1 Sodium Chloride and Thiomersal
IRCP4TA1

1 1 1 Sodium
Chloride
Thiomersal
+25μg Al+++
Table 11
Multivalent Liquid formulation containing Men X—TT conjugate (Strain 8210)

Formulation Code Composition amount per 0.5ml (μ) Excipients

A-CR M19
7
1
1 C-CRM 197
1 1 Y-TT
1 1 W-CRM 197 X-TT

IRCP5T1


1 1 Sodium Chloride and Thiomersal
IRCP5TA1


1 1 Sodium Chloride Thiomersal + 25μg Al +++

Table 12
Lyophilized multivalent formulation containing Men X—TT conjugate (Strain 8210)

Composition amount per Vial
Formulation Code A-CR M19
7
25
25 25 C-CRM 197
25
25 25 Y-TT
25
25 25 W-CRM 197 X-TT Excipients Quantity in a vial
IRCLPS3Sc


25 25 Tris 0.6 mg, Sucrose 15mg, Sodium citrate 2.5 mg
IRCLPT3Sc


25 25 Tris, Trehalose 15mg, Sodium citrate 2.5 mg
IRCLPS3L2Sc


25 25 Tris, Sucrose 15mg, Lactose 10mg, Sodium citrate 2.5 mg
Example 12:
Biological activity of Meningococcal monovalent and multivalent conjugate composition containing Men X saccharide conjugate
Each formulation was immunized into six female Swiss Albino Mice of 16-20 g body weight. Mice were immunized subcutaneously on Day 0, 14 and 28. Each mouse was bled after 1 & 2 week post second immunization (Day 21 and Day 35).
Titration of antibody was done by bead based assay and SBA. Pre-immunization serum samples from all six mice were mixed to prepare a single pool serum for each formulation from study 4 onwards and also postimmunization serum sample from six mice all belonging to Swiss Albino strain for each formulation were mixed to prepare pool1, 2 & 3 using serum from Mouse1+2, 3+4 and 5+6, respectively. Each of the pools was analyzed for total IgG titers (Multiplexed bead based assay) and functional antibody titers (SBA).

Table 13

Formulation Code Ig G SBA

Day 21 Day 28 Day 35 Day 21 Day 28 Day 35
128 323 406 406
IRCXLT1 504 800 12800 8 20

IRCXLTA1 12800 32254 51200 256 323

IRCXNT1 2540 2540 8063 81 51

IRCXNAT1 6400 12800 32254 203 323

Table 14

Formulation Code Ig G SBA

Day 28 Day 35 Day 28 Day 35
IRCX2LT1 79 3200 2 20
IRCX2LAT1 4032 40637 20 128
IRCX2NT1 1270 51200 5 256
IRCX2NATA1 16127 51200 5 256
Table 15

Formulati on Code Men A Men C

Ig G SBA Ig G SBA

Day 21
200
2031 9 Day 28
200
2560 0 Day 35
2016
3225 4 Day 21
2 13 Day 28
2 13 Da y 35
13 51 Day 21
635
1612 7 Day 28
800
1612 7 Day 35 Da
y
21 Da
y
28 Day 35
IRCPT1







8063 2 2 16
IRCPTA1







20319 25 32 102

Table 16

Men W135 Men Y

Ig G SBA Ig G SBA
Formulation Code

Day 21 Day 28 Day 35 Day 21 Day 28 Day 35 Day 21 Day 28 Day 35 Day 21 Day 28 Day 35
IRCPT1 3200 3200 16127 20 20 81 252 200 1270 2 16 5
IRCPTA1 1600 2016 12800 40 3 25 504 635 1270 10 6 8
Table 17

Formulation Code MenX

Day 21
1600 5080 Ig G Day 28
2016 10159 Day 35
32254 51200 Day 21
64 81 SBA
Day
28
40 102 Day 35
256 406
IRCPT1

IRCPTA1

Table 18

Formulation Code Men A Men C MenW

Ig G SBA Ig G SBA Ig G SBA

Day 28
79 3200 Day 35 Day 28
2 5 Day 35
5 16 Day 28
400 3200 Day 35 Day 28
3 10 Day 35
20 25 Day 28
800 3200 Day 35 Day 28
6 2 Day 35
81 20
IRCP2T1
252


6400


10159

IRCP2TA1
10159


25600


32254

Table 19

Formulation Code Men Y Men X

Ig G
Day Day
28 35
504 5080 1600 10159 SBA
Day Day
28 35
25 64 3 81 Ig G
Day Day
28 35
2540 20319 6400 32254 SBA
Day Day
28 35
16 81 40 102
IRCP2T1

IRCP2TA1

Table 20

Formulation Code Men A Men C Men W135 Men Y Men X

SBA SBA SBA SBA SBA

Day 35 Day 35 Day 35 Day 35 Day 35
IRCP4T1 13 10 20 8 512
IRCP4TA1 16 25 6 5 512
IRCP5T1 13 25 10 256 406
IRCP5TA1 203 203 102 1625 512
Table 21

Formulation Code MenA Men C Men W135

Ig G SBA Ig G SBA Ig G SBA

Day 28
635
160
0
200
160
0
317
635 Day 35
403
2 640
0 127
0 640
0 127
0 127
0 Da
y
28 Da y 35 Day 28
320
0 403
2 317
320
0
800
508 0 Day 35
6400
2560
0 2540
1015
9 1600
1015 9 Da
y
28 Da y 35 Day 28
320
0 403
2 201
6 254
0 160
0 320
0 Day 35
1015
9 1280
0 4032
1612
7 4032
8063 Da y 28 Da
y
35
IRCLPS3Sc

2 5

2 25

6 40
IRCLPS3DSc + AlPO4

3 25

20 323

13 64
IRCLPT3Sc

2 6

4 25

10 64
IRCLPT3Sc+ AlPO4

5 51

25 102

20 102
IRCLPS3L2Sc

2 2

5 13

64 203
IRCLPS3L2Sc + AlPO4

5 8

13 64

8 25

Table 22

Formulation Code Men Y Men X

Ig Day 28
800 2540
504 504
504 635 G
Day 35
1008 16127
4032 4032
2016 1600 SBA
Day Day
28 35
85 25 323
6 32 4 256
10 162 58 Ig G
Day Day
28 35
1008 16127 3200 51200
252 12800 1270 16127
317 12800 400 2016 SBA
Day Day
28 35
32 323 40 406
16 323 8 256
10 406 64 102
IRCLPS3Sc

IRCLPS3DSc+ AlPO4

IRCLPT3Sc

IRCLPT3Sc+ AlPO4

IRCLPS3L2Sc

IRCLPS3L2Sc+ AlPO4

Above mice immunogenicity data indicates that liquid and lyophilized compositions of monovalent X-tetanus toxoid conjugate and multivalent conjugates containing X-tetanus toxoid conjugate are found to be immunogenic.Further monovalent liquid composition containing 1μg of X-tetanus toxoid conjugate,sodiumchloride,thiomersal and 125 μgAl+++ gives optimal immunogenic response. Also liquid multivalent composition of 0.5 ml containing A-CRM197,C-CRM197,Y-tetanus toxoid,W-CRM197 and X-tetanus toxoid conjugates with 1μg each of all 5 saccharides,sodiumchloride,thiomersal and 25μg Al +++ gives optimal immunogenic response.Thus in this pentavalent conjugate composition, conjugates containing tetanus toxoid as carrier protein are found to enhance immunogenicity of conjugates containing CRM 197 as carrier protein.
Example 13: MCV5 Conjugate formulation
Table 23
Men C Conjugate Related -

Menactra Serum Institute’s
(EP 2345422 pipeline Pentavalent
Parameter A1)(Quadrivalent Polysaccharide
Conjugate vaccine) Conjugate Vaccine
(ACWY-DT) (ACWYX-

TT,CRM197)NmCV-5
Presentation (Final Formulation) Liquid form Freeze-Dried
Formulation buffer Phosphate and Sodium chloride Tris buffer with Sucrose, sodium citrate stabilizer
Dose (Men C) 4µg 5µg
Example 14:NmCV-5 Phase II Clinical Study plan/design, Study assessment.
A Phase 2, randomized (2:2:1), controlled, observer-blind, study was conducted in 375 healthy children 12 to 16 months of age conducted at Centre for Vaccine Development (CVD), Bamako, Mali. The study was approved by ethics committees in Mali & Baltimore, USA as well as the national regulatory agency (NRA) of Mali before initiation.
Phase 2 study: 5 Dose presentation Table 24

Adjuvanted Arm
(Lyophilized product+ Adjuvanted
Diluent) Non-Adjuvanted Arm
(Lyophilized product+ Saline Diluent)
Upon reconstitution with adjuvanted diluent, each 0.5 ml dose contains Upon reconstitution with saline diluent, each 0.5 ml dose contains
Men A-TT Conjugate 5 µg Men A-TT Conjugate 5 µg
Men C-CRM197 conjugate 5 µg Men C-CRM197 conjugate 5 µg
Men Y-CRM197 conjugate 5 µg Men Y-CRM197 conjugate 5 µg
Men W-CRM197 conjugate 5 µg Men W-CRM197 conjugate 5 µg
Men X-TT Conjugate 5 µg Men X-TT Conjugate 5 µg
Sucrose 2.42 mg Sucrose 2.42 mg
Sodium citrate (Tribasic dehydrate) 0.40 mg Sodium citrate (Tribasic dehydrate) 0.40 mg
Tris Buffer 0.098 mg Tris Buffer 0.098 mg
Al+++ (in form of Aluminum phosphate) 125 µg 0.9% Sodium Chloride Qs to 0.5ml
0.9% Sodium Chloride Qs to 0.5ml

The objectives of the study were: Primary Objective:
1. To assess the reactogenicity of non-adjuvanted and adjuvanted formulations of NmCV-5 in comparison with the licensed MenACWY-D vaccine, as measured by the percentage of subjects with at least one severe solicited AE reported within 7 days after any study vaccination
Secondary Immunogenicity Objectives:
1. To assess immunogenicity of non-adjuvanted formulation of NmCV-5 in comparison with MenACWY-D vaccine, as measured by rSBA against serogroups A, C, W, Y and X at 1 month after the second vaccination.
2. To assess immunogenicity of adjuvanted formulation of NmCV-5 in comparison with non-adjuvanted formulation of NmCV-5, as measured by rSBA against serogroups A, C, W, Y and X, at 1 month after the second vaccination.
3. To assess immune response elicited by non-adjuvanted and adjuvanted formulations of NmCV-5 and MenACWY-D vaccine at 1 and 3 months after the first vaccination.
Secondary Safety Objective:
1. To evaluate the safety and reactogenicity of adjuvanted and non-adjuvanted formulations of NmCV-5 in healthy children, when compared to MenACWY-D
Prior to written informed consent, subject’s parents/guardians were asked to sign a pre-screening agreement as early as 9 months of age of the child in order to delay the EPI MenAfriVac dose until the child was at least 12 months of age. To be part of the study children should not have received any meningococcal vaccines.
Parents or legal guardians provided written informed consent for their children to be in the study. Eligible children were randomly assigned to one of the following three study groups:

•NmCV-5_non-adjuvanted group: 150 subjects received the non-adjuvanted
formulation of NmCV-5 at Visit Day 0 and Visit Day 84. •NmCV-5_adjuvanted group: 150 subjects received the adjuvanted formulation of
NmCV-5 at Visit Day 0 and Visit Day 84. •ACWY-D group: 75 subjects received the licensed MenACWY-D (Menactra)
vaccine at Visit Day 0 and Visit Day 84
The study staffs involved in safety assessment of the vaccines including principal investigators were unaware of the treatment group to which a child was assigned. Only the pharmacist and vaccinators knew this assignment.
The total planned duration of follow up for each subject was 168 days (~6 months) after first vaccine dose.
Table 25: Study design

Solicited reactions (viz. tenderness, swelling/induration, irritability, drowsiness, decrease eating, vomiting, and fever) were collected for 7 days post each vaccine dose via daily home visits by study staff. Unsolicited AEs were collected for 28 days after each study vaccination whereas serious AEs were collected throughout the study period of 6 months.
The four blood samples (~5 mL each) collected at Visits 1 (Day 0), 3 (Day 28), 4 (Day 84) and 6 (Day 112) were evaluated for serum bactericidal activity using baby rabbit

complement (rSBA) against all five serogroups of N meningitidis viz. A, C, Y, W & X. The results of rSBA analysis done at Days 0 & 28 are presently available.
The study endpoints were as below:
Primary reactogenicity endpoint:
• Percentage of subjects with at least one severe solicited AE within 7 days after any
study vaccination (Days 0-6 and Days 84-90)
Secondary immunogenicity endpoints:
• Percentage of subjects with rSBAtiter ≥ 8 against serogroups A, C, W, Y and X at Visits Day 0, Day 28, Day 84 and Day 112.
• Percentage of subjects with rSBAtiter ≥ 128 against serogroups A, C, W, Y and X at Visits Day 0, Day 28, Day 84 and Day 112.
• Percentage of subjects with fourfold rise in rSBAtiters against serogroups A, C, W, Y and X at Visits Day 28 and Day 112.
(For subjects with a pre-vaccination rSBAtiter< 8, a post-vaccination titer of ≥ 32; For subjects with a pre-vaccination rSBAtiter ≥ 8, an increase in rSBAtiter of at least 4 times the pre-vaccination titer)
• rSBA GMT for serogroups A, C, W, Y and X at Visits Day 0, Day 28, Day 84 and
Day 112.
Secondary safety endpoints:
• Solicited local and systemic AEs reported during the 7 days after each vaccination (Days 0-6 and Days 84-90);
• Unsolicited AEs reported during 28 days after each vaccination (Days 0-27 and Days 84-111);
• AEs leading to premature withdrawal during the entire study period;
• SAEs reported during the entire study period

Addition of alum as adjuvant to the vaccines has known to accentuate their immune response. With a similar underlying hypothesis, results from this study were also used to interpret difference, if any in safety and immune response of the adjuvanted& non adjuvanted formulations of NmCV-5. The interim analysis results helped to make a choice between the two formulations for future clinical development.
An independent Data and Safety Monitoring Board (DSMB) was set up for periodic review of safety data during the study conduct.
1. Phase II Resultsfor NmCV-5 -
An interim analysis (IA) of the Day 28 post dose 1 safety data and immunogenicity (rSBA) data of all the participants has been completed. Individual subject listings were not generated for the IA and any access to subject-level information about study groups was masked. The study blind was broken only at the treatment (i.e., vaccine) group level for presenting only the immunogenicity results, but there was no subject level unblinding. In case of safety data unlike the immunogenicity results, the treatment (i.e., vaccine) per se was not be identified and the groups were labelled as Vaccine 1, 2, and 3. Moreover, no actual numbers were calculated, neither numerators nor denominators, and only percentages were calculated for each treatment arm.
Out of the 375 vaccinated children, 6 did not provide a Day 28 post dose blood sample for rSBA testing. They are not included in the below presented interim analysis results.
a) Post vaccination rSBA titre of 1:128 or higher is generally considered as immune correlate for long term protection afforded by vaccines against N meningitidis. In the present study more than 97 % of the participants had rSBA titres of ≥ 1:128 against all five serogroups at 28 days post vaccination in both the NmCV-5 groups. These numbers were comparatively lower in the four common serogroups among Menactra recipients. The data is presented in table 1 below.

Table 26: Percentage of subjects with rSBA titres ≥ 1:128 at 28 days after the first dose

Serogro Non-adjuvanted NmCV-5 Adjuvanted NmCV-5 Menactra®
N=74 n (%) [95% CI]
up N=147 n (%) [95% CI] N=148 n (%) [95% CI]

A 147 (100.0)( 148 (100.0)( 73 ( 98.6)( 92.7,100.0)

97.5,100.0) 97.5,100.0)
C 145 ( 98.6)( 95.2, 144 ( 97.3)( 93.2, 40 ( 54.1)( 42.1, 65.7)

99.8) 99.3)
W 145 ( 98.6)( 95.2, 145 ( 98.0)( 94.2, 67 ( 90.5)( 81.5, 96.1)

99.8) 99.6)
X 147 (100.0)( 147 ( 99.3)( 15 ( 20.3)( 11.8, 31.2)

97.5,100.0) 96.3,100.0)
Y 143 ( 97.3)( 93.2, 147 ( 99.3)( 65 ( 87.8)( 78.2, 94.3)

99.3) 96.3,100.0)
b) Similar to rSBA ≥ 1:128 results, the percentage of subjects with four fold rise in rSBA titers at Day 28 from baseline in both the NmCV-5 treatment groups was 97 % or higher for all five serogroups. The numbers and percentages for all the treatment groups are presented in table 2 below.
Table 27: Percentage of subjects with seroconversion (Four fold rise in rSBAtiters as defined under immunogenicity endpoint above) at 28 days after the first dose from the baseline (Visit 1; Day 0)

Non-adjuvanted Adjuvanted Menactra®
N=74 n (%) [95% CI]
Serogrou NmCV-5 NmCV-5

p N=147 n (%) [95% CI] N=148 n (%) [95% CI]

A 146 ( 99.3)( 96.3,100.0) 148 (100.0)( 97.5,100.0) 72 ( 97.3)( 90.6, 99.7)
C 144 ( 98.0)( 94.2, 99.6) 145 ( 98.0)( 94.2, 99.6) 52 ( 70.3)( 58.5, 80.3)
W 143 ( 97.3)( 93.2, 99.3) 144 ( 97.3)( 93.2, 99.3) 67 ( 90.5)( 81.5, 96.1)
X 147 (100.0)( 97.5,100.0) 146 ( 98.6)( 95.2, 99.8) 12 ( 16.2)( 8.7, 26.6)
Y 143 ( 97.3)( 93.2, 99.3) 146 ( 98.6)( 95.2, 99.8) 64 ( 86.5)( 76.5, 93.3)
c) The geometric mean titres (GMTs) of rSBA at 28 days post first vaccine dose were higher among the two NmCV-5 treatment arms as compared to Menactra treatment arms; however these were similar in the NmCV-5 treatment arms. The ratio of GMTs between non adjuvanted and adjuvanted NmCV-5 was close to 1 for all the five serogroups (Table 4)
Table 28: rSBA Geometric mean titres (GMT) at 28 days after the first dose

Serogrou p Non-adjuvanted
NmCV-5
N=147
n (%) [95% CI] Adjuvanted NmCV-5
N=148
n (%) [95% CI] Menactra®
N=74 n (%) [95% CI]
A 7704.9(6458.9,919 1.3) 7286.9(6163.9,8614.4) 3872.1(2867.3,522 9.2)
C 1146.7(935.5,1405. 6) 1058.1(849.9,1317.3) 70.9(41.8,120.4)
W 6563.6(4918.6,875 8.7) 5476.0(4030.0,7440.9) 1135.1(629.2,2047. 8)

X 7596.7(6481.5,890 3.8) 7965.0(6584.9,9634.3) 6.7(3.7,12.0)
Y 2415.5(1883.2,309 8.2) 2951.1(2449.2,3555.8) 629.2(361.9,1093.7 )
Table 29: The ratio of rSBA GMTs between the treatment groups at 28 days after the first dose

Serogrou p Non-adjuvanted
NmCV-5 vs
Adjuvanted NmCV-5
(95% CI)** Non-adjuvanted
NmCV-5 vs
MenACWY-D
(95% CI)** Adjuvanted NmCV-5 vs MenACWY-D (95% CI)**
A 1.06 (0.83,1.35) 1.99 (1.44,2.75) 1.88 (1.37,2.58)
C 1.08 (0.80,1.46) 16.16 (10.12,25.81) 14.91 (9.22,24.14)
W 1.20 (0.79,1.82) 5.78 (3.24,10.32) 4.82 (2.65,8.77)
X 0.95 (0.75,1.22) 1134.56 (713.26,1804.69) 1189.56 (731.40,1934.73)
Y 0.82 (0.60,1.12) 3.84 (2.28,6.47) 4.69 (2.94,7.48)
d) There was no difference in the safety outcomes for all three vaccines.
RESULT: The results clearly indicate that single dose of both the NmCV-5 formulations has rSBA titres ≥ 1:128 against all five serogroups to > 97 % participants at 28 Days post dose. The cut-off 1:128 is a generally acceptable correlate for long term protection against Nisseirameningitidis. The single dose of NmCV-5 is clearly sufficient in providing this protection. On the other hand, Menactra’s immune response is lower for 3 of the 4 common serogroups (with the exception of A) in comparison to NmCV-5. It is to be noted that Menactra is recommended in a 2 dose series for this age group. Further, there was no significant difference in the GMTs of the two formulations of NmCV-5 (with or without adjuvant) at 28 days post single vaccine dose.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

We Claim:
1. A vaccine composition comprising:
(a) a Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to a tetanus toxoid carrier protein as the vaccine antigen;
(b) a Neisseria meningitidis serogroup C (MenC) capsular saccharide sized by High Pressure Cell Disruption and having average molecular size in the range of 75 kDa to 150 kDa conjugated to a CRM197 carrier protein as the vaccine antigen with 60-95% of the sialic acid residues in the Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to CRM197 being O-acetylated;
(c) a Neisseria meningitidis serogroup Y (MenY) capsular saccharide conjugated to a CRM197 carrier protein as the vaccine antigen;
(d) a Neisseria meningitidis serogroup W135 capsular saccharide conjugated to a CRM197 carrier protein as the vaccine antigen; and
(e) a Neisseria meningitidis serogroup X (MenX) capsular saccharide conjugated to a tetanus toxoid carrier protein as the vaccine antigen;
wherein, a single dose of said vaccine composition is sufficient to elicit the required T-dependent immune response against Neisseria meningitidis serogroup A, C, Y, W135 and X in a human subject.
2. The vaccine composition as claimed in claim 1, wherein the vaccine composition is formulated for administration to a human subject 2 years of age or below according to a one dose regimen consisting of a single dose of said vaccine composition.
3. The vaccine composition as claimed in claim 1, wherein the vaccine composition is formulated for administration to a human subject 2 years of age or below according to a two dose regimens consisting of a first dose and a second dose.

4. The vaccine composition as claimed in claim 3, wherein the second dose is to be administered 2 months after the first dose.
5. The vaccine composition as claimed in claim 3, wherein the second dose is to be administered 3 months after the first dose.
6. The vaccine composition as claimed in claim 3, wherein the second dose is to be administered 6 months after the first dose.
7. The vaccine composition as claimed in claim 1, wherein a single dose comprises of no more than 10µg of the vaccine antigen.
8. The vaccine composition as claimed in claim 1, wherein a single dose comprises of no more than 5µg of the vaccine antigen.
9. The vaccine composition as claimed in claim 1, wherein the composition comprises of an adjuvant.
10. The vaccine composition as claimed in claim 1, wherein the composition is devoid of an adjuvant.
11. The vaccine composition as claimed in claim 1, wherein the composition further comprises of a pharmaceutically acceptable excipient selected from the group of sugars, polyols, surfactants, polymers, salts, amino acids, and pH modifiers.
12. The vaccine composition as claimed in claim 1, wherein the composition further comprises of a buffer selected from Tris and phosphate buffer.
13. The vaccine composition as claimed in claim 1, wherein each of Neisseria meningitidis serogroup A (MenA) capsular saccharide, Neisseria meningitidis serogroup Y (MenY) capsular saccharide, Neisseria meningitidis serogroup W135 capsular saccharide and Neisseria meningitidis serogroup X (MenX) capsular

saccharide has an average molecular size between 70 kDa to 800 kDa, preferably between 70 kDa and 200 kDa.
14. The vaccine composition as claimed in claim 1, wherein the composition is lyophilized (freeze-dried).
15. The vaccine composition as claimed in claim 1, wherein the composition further comprises of a preservative selected from the group of 2-phenoxyethanol, Benzethonium chloride (Phemerol), Phenol, m-cresol, Thiomersal, Formaldehyde, methyl and propyl parabens, benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chlor-m-cresol, or a combination thereof.
16. The vaccine composition as claimed in claim 1, wherein the composition is free of preservative.
17. A method of inducing an immune response against a Neisseria meningitidis serogroup A, a Neisseria meningitidis serogroup C, a Neisseria meningitidis serogroup W135, a Neisseria meningitidis serogroup Y and/or a Neisseria meningitidis serogroup X strain in a human subject 2 years of age or below comprising administering of a single dose of the vaccine composition as claimed in any one of claims 1-16 by intramuscular, intravenous, subcutaneous, transcutaneous or intradermal.
18. A kit comprising a first container containing a vaccine composition as claimed in any one of the claims 1-16 and a second container containing an aqueous solution for the reconstitution of the lyophilized (freeze-dried) vaccine composition.
19. A kit comprising a first container containing a vaccine composition as claimed in any one of the claims 1-16 and a second container containing an aqueous solution and adjuvant for the reconstitution of the lyophilized (freeze-dried) vaccine composition.

20. A kit comprising a first container containing a vaccine composition as claimed in any one of the claims 1-16, a second container containing an additional vaccine antigen and a third container containing an aqueous solution for the reconstitution of the lyophilized (freeze-dried) vaccine composition.
21. The kit as claimed in claim 20, wherein the second container comprises of Neisseria meningitidis serogroup B polysaccharide or protein as the vaccine antigen.
22. The kit as claimed in any one of the claims 18-20, wherein the aqueous solution comprises of sodium chloride as dilution medium or buffer for the reconstitution of the lyophilized (freeze-dried) vaccine composition.
23. A vaccine composition comprising Neisseria meningitidis polysaccharide-protein conjugates, said composition comprising:

(a) a Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to a tetanus toxoid carrier protein as the vaccine antigen;
(b) a Neisseria meningitidis serogroup C (MenC) capsular saccharide sized by High Pressure Cell Disruption and having average molecular size in the range of 75 kDa to 150 kDa conjugated to a CRM197 carrier protein as the vaccine antigen with 60-95% of the sialic acid residues in the Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to CRM197 being O-acetylated;
(c) a Neisseria meningitidis serogroup Y (MenY) capsular saccharide conjugated to a CRM197 carrier protein as the vaccine antigen;
(d) a Neisseria meningitidis serogroup W135 capsular saccharide conjugated to a CRM197 carrier protein as the vaccine antigen; and
(e) a Neisseria meningitidis serogroup X (MenX) capsular saccharide conjugated to a tetanus toxoid carrier protein as the vaccine antigen;
wherein, a single dose of said vaccine composition devoid of any adjuvant is sufficient to elicit the required T-dependent immune response against Neisseria

meningitidis serogroup A, C, Y, W135 and X in a human subject 2 years of age or below.
24. A process for preparing a serogroup C meningococcal capsular saccharide conjugated to a CRM197 carrier protein comprising all or either of the following steps:
(1) Isolating and purifying serogroup C meningococcal capsular saccharide;
(2) Subjecting the purified serogroup C meningococcal capsular saccharide to high pressure cell disruption having pressure value of 40000 psi and 5 to 10 passes;
(3) Subjecting the serogroup C meningococcal capsular saccharide and CRM197 carrier protein to a conjugation chemistry rendering them reactive towards the other;
(4) Forming a covalent bond between the serogroup C meningococcal capsular saccharide and CRM197 carrier protein; and
(5) Purifying the resulting serogroup C meningococcal capsular saccharide conjugated to a CRM197 carrier protein;
wherein, 60-95% of the sialic acid residues in the serogroup C meningococcal capsular saccharide conjugated to a CRM197 are O-acetylated and the average molecular size of the conjugate serogroup C meningococcal capsular saccharide-CRM197 molecule ranges between 300 kDa to 1500 kDa, preferably between 300 kDa to 800 kDa.