FORM-2
THE PATENTS ACT, 1970
(39 OF 1970)
AND
THE PATENTS RULES, 2003
(As Amended)
COMPLETE SPECIFICATION
(See section 10; rule 13)
"IMMUNOGENIC COMPOSITION"
Serum Institute of India Ltd., an Institute organized and existing under the laws of India, of 212/2, Off Soli Poonawalia Road, Hadapsar, Pune 411 028 Maharashtra India.
The following specification particularly describes the nature of this invention and the manner in which it is to be performed:

BACKGROUND OF THE INVENTION
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. meningitidis have 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 vaccine utilizing the polysaccharide (PS)alone have relatively low immunogenicity. To overcome the relatively low immunogenicity of polysaccharide, PS 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).
As well as being used for classification, the capsular polysaccharide has 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 i n infants.Mencevax ACWY™ and Menomune™ both contain 50ug of each purified polysaccharide once reconstituted from their lyophiiised forms. The capsular saccharides of serogroups A, C, W135 & Y have also been combined in the form of conjugates to give tetravalent vaccines e.g.the unadjuvanted Menactra™ product. Also conjugated serogroup A polysaccharide have been approved for human use as MenAfriVac™, serogroup C oligosaccharides have been approved for human use as Menjugate™ , Meningitec™ and NeisVac-C™.
N. meningitidis serogroup X strains were first described in
the 1960s and have been isolated from a few cases of
invasive meningococcal diseases in North America, Europe,
Australia, and China. Outbreaks of N. meningitidis
serogroup X strains have been reported in Niger, western
Kenya, and northern Ghana. N. meningitidis serogroup X
strains were reported to be very efficient in colon.i zatlon
among military recruits in the United Kingdom.Refer
Abdullah Kilic et al /Neisseria meningitidis Serogroup X
Sequence Type 767 in Turkey;Journal Of Clinical
Microbiology, Nov. 2010, p. 4340 4341;Vol. 48, No. 11
It was reported that repeated mass vaccination in many African countries might have contributed to colonization by and meningococcal diseases due to serogroup X strains and might result in a changed profile of meningococcal disease

Refer Gagneux, S. P et al ;Prospective study of a serogroup X Neisseria meningitidis outbreak in northern Ghana.J. Infect. Dis. 18 5:618-62 6;2002.
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(a 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 ( a 1-4)-linked N-acetylglucosamine 1-phosphate.Refer Yin-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
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 [1,4] 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.
The present invention arises from the surprising discovery that it is possible to prepare a monovalent or multivalent immunogenic composition based on conjugates of meningococcal polysaccharide from serogroup X by utilizing a scalable and efficient conjugation process.
SUMMARY OF THE INVENTION
The present invention relates to N.meningitidis X saccharide-carrier protein conjugates prepared by a conjugation reaction comprising of i)sizing of polysaccharide il)CPPT based activation of sized polysaccharide having average molecular weight between 100-150 Kda, at a pH between 9 to 9.5 iii)ADH addition after a duration of about 2 to 5 minutes followed by incubation period of 4-20 hrs and iv)reacting ADH activated polysaccharide with purified non-activated carrier protein in a ratio between 0.75-1.5 in presence of MES buffer and EDAC followed by incubation period of 3 ~4 hrs,characterized in that the conjugation reaction is carried at 2~8°C resulting in a conjugate yield from 20% to about 30% and having saccharide to protein ratio from 0.2 to about 0.6 in final conjugate.
Alternatively,N.meningitidis X saccharide-carrier protein conjugates can also be prepared by a conjugation reaction

comprising of i) sizing of polysaccharide .ii) CPPT based activation of sized polysaccharide having average molecular weight between 100-150 Kda, at a pH between 9 to 9.5 iii)addition of ADH activated carrier protein in a saccharide:protein ratio between 0.5 - 2 after 2-3 minutes followed by incubation period of 2 to 20 hrs characterized in that the conjugation reaction is carried at 22°C to 25°C resulting in a conjugate yield from 5% to about 10%.
Accordingly,the instant invention relates to multivalent meningococcal polysaccharide protein conjugate composition comprising capsular saccharide from serogroups X and atleast one capsular saccharide from A, C, W135 and Y wherein,i)polysaccharides A C W135 X are sized mechanically whereas polysaccharide Y is sized chemically,ii)all saccharide are conjugated to carrier protein via a linker with a cyanylation conjugation chemistry iii)all saccharide to protein ratios in final conjugates are between 0.2 - 0.6 and iv) atleast two different carrier proteins selected from the group consisting of TT, DT and CRM197 are utilized.
DESCRIPTION OF THE DRAWINGS
Figure 1: Overlay of conjugation reaction when Men X Ps (215 KDa) conjugated to Hydrazine derivatized TT
Figure 2: Purified Conjugate when Men X Ps (215 KDa) conjugated to Hydrazine derivatized TT
Figure 3: Overlay of conjugation reaction when Men X Ps(32 6 KDa) conjugated to ADH derivatized TT

Figure 4: Purified Conjugate when Men X Ps(326 KDa) conjugated to ADH derivatized TT
Figure 5: Overlay of conjugation reaction when Men X Ps (120 KDa)conjugated to ADH derivatized TT
Figure 6: Purified Conjugate when Men X Ps(120 KDa) conjugated to ADH derivatized TT
Figure 7:Chromatogram of Native Meningococcal X polysaccharide.
Figure 8:0verlay of conjugation reaction when Men X Ps(510 KDa) activated with ADH and conjugated to purified TT.
Figure 9:Purified Conjugate when Men X Ps(510 KDa) activated with ADH and conjugated to purified TT.
Figure 10:0verlay of conjugation reaction when Men X Ps(250 KDa) activated with ADH and conjugated to purified TT.
Figure 11:Purified Conjugate when Men X Ps(250 KDa) activated with ADH and conjugated to purified TT.
DETAILED DESCRIPTION OF THE INVENTION
"Multivalent immunogenic compositions" refer to :
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 (11)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 charachterized 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)CRMl97; (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 ug of serogroup A saccharide, 5ug of serogroup C saccharide, 5μg oi: serogroup W135 saccharide, 5ug of serogroup Y saccharide and 5pg of serogroup X saccharide.
Alternatively said multivalent immunogenic composition can comprise of 5 ug of serogroup A saccharide, 5pg of serogroup C saccharide, 5μg of serogroup W135 saccharide,

5μg of serogroup Y saccharide and 5ug 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 20-300ug,20~200ug,25-150ug 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 A1+++ per 0.5 ml.
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 0.7 5% 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 51 (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.
Accordingly in a fifth embodiment,said polysaccharides A C W and X can be mechanically sized to have an average molecular weight between 100-600 Kda,100-400 Kda,preferably 100-200 Kda,most preferably 100-150 Kda. Mechanical sizing methods like homogenization, microfluidization and high pressure cell disruption are preferred.
In another aspect of fifth embodiment, said polysaccharide Y can be sized to have an average molecular weight between 90-110 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 80°C.
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 l-cyano-4- fdimethylamino)-pyridinium tetrafluoroborate ('CDAP'), p-nitrophenylcyanate and N-cyanotriethylammonium tetrafluoroborate ('CTEA').In a preferred conjugation process, cyanylating reagent is other

than CDAP and can be selected from a group of 1-cyano- 4~ pyrrolidinopyridinium tetrafluoroborate (CPPT), 1- cyano-imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), or 2-cyanopyridazlne -3(2H)one (2-CPO), or a functional derivative or modification thereof.
In another aspect 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, succinyl dihydrazide, 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. EDO 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 BVI-1-3 and BVH-11 , p r o t e c t .1 ve a n 11 g e n (PA) o f B a c i 11 u s a n t h r a c i s 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: 1181-118 6. 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-menlngococca.l 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; Haemophilus influenzae type b polysacchar.i de polyribosyl ribitol 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. coll, Pseudomonas aeruginosa, and Bacillus anthracis. 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.
Examples: Example 1:
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 suit ab1e fermentation mediurn i n a continuous fed-batch fermentation mode under optimal. fermentor conditions.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 , ca rbon fi1trat1on , CaC12 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 30-4 0 kpsi.
Example 2:
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 215kD on SEC HPLC, (30 mg/ml) 4 5 mg was activated with 90 mg CDAP (dissolved lOOmg/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 300kD 'IFF membrane in Tris lOmM 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 kD on SEC HPLC, (24 mg/ml in 2M NaCl) 60 mg was activated with 150 mg CPPT (dissolved 114mg/mi 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. 500kD TF'F membrane in 10mM PBS followed by Tris lOmM pH 7.2.
Further Polysaccharide of X (Strain 8210), having average molecular weight 120 kD on SEC HPLC, (20 mg/ml in 1.M NaCl) 200 mg was activated with 4 00 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 300kD TFF membrane in lOmM PBS followed by Tris lOmM 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 510kD on SEC HPLC (27 mg/ml in 2M NaCl} 200 mg was activated with 4 00 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 (lOOmg/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 8kD TFF membrane. Further ADH-Men X
polysaccharide was concentrated. To 44 mg of
this(7.5mg/ml), purified TT (37.5 mg/mi in 0.9% NaCl) and MES pH 6.0 buffer were added so that final buffer strength of MES was lOOmM, followed by addition of 37.5 mg EDAC (dissolved in lOOmM 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 250kD 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 (lOOmg/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 8kD TFF membrane. ADH-Men X polysaccharide was then concentrated.Further to 200 mg of this (7.Smg/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 lOOmM, followed by addition of 200 mg EDAC (dissolved in lOOmM MES, pH 6.0). The reaction was continued for 4 h r s and monitored on HPLC. The crude conjugate was purified by diafiltration 500kD TFF membrane in lOmM PBS followed by Tris lOmM pll 7.2.
Table 1 Meningococcal X polysaccharide-protein Conjugation

Avg Mw of
Meningococcal X
polysaccharide(KDa) ADH activation Saccharide/
Protein ratio Polysaccharide Titer (mg/ml) Protei
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 of Avg 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 8°C vi)saccharide:protein ratio between 0.2 to 0.6 in final conjugate.
Example 3:
Conjugation of Meningococcal A,C,Y,W135 polysaccharide to
carrier protein CRM197.

Purified Meningococcal polysaccharides A C Y W135 having average Mw between 100 to 200 were conjugated to CRM197 in a saccharide:protein ratio of less than 1 by utilizing a suitable cyanylation reagent(CDAP or CPPTJ.The conjugates were further purified by diafiltration on 300kD TFF with 50 volumes of lOmM PBS and 50 volume of lOmM Tris.
Example 4:
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 2
Liquid formulation containing Monovalent Men X—tetanus toxoid conjugate

Formulation Strain Composition Amount per
Code 0.5ml
IRCXLT1 9601 Men X-TT conjugate + Saline + Thiomersal 0.5μg

IRCXLTA1 9601 Men X-TT conjugate 0.Sμg with
+ Saline + Thiomersal + A1P04 12Sμg Al+H
IRCXNT1 9601 Men X-TT
conjugate+Saline +
Thiomersal lμ
IRCXNAT1 9601 Men X-TT lμg with
conjugate+Saline + Thiomersal+AlP04 125μg Al+++
1RCX2LT1 8210 Men X-TT
conjugate+Saline + Thiomersal 0.5μg
IRCX2LAT1 8210 Men X-TT 0.5μg with
conjugate+Saline + 125μg Al'''
8210 Thiomersal
1RCX2NT1
Men X-TT lug
conjugate+Saline + Thiomersal
IRCX2NATA1 8210 Men X-TT lug with
conjugate+Saline + Thiomersal 12 5μg Al+++
Table 3
Multivalent Liquid formulation containing Men X—tetanus toxoid conjugate

Composition amount per
Formulation Code Strai
n 0.5ml (μ) Excipxents

A- c- Y- W-CRM X -TT

CRM CRM CHM 197
9601 197
1 197
1 197
IRCPT1


1 1 1 Sodium
Chloride
and
Thiomersal
1RCPTA1 9601 1 1 1 1 1 Sodium Chloride Thiomersal +125ug
IRCP2T1 8210 1 1 1 1 1 Sodium Chloride and Thiomersal

IRCP2TA1
8210 1 1 1 1 1 Sodium Chloride Thiomersal + 125μg Al+++
Table 4
Multivalent Liquid formulation containing Men X—TT conjugate (Strain 8210)

Formulation Code Compositi on amount per 0 .5ml (u)
X- Excipients

A- C- Y-

CRM CRM CRM CRM TT
197 197 197 197
IRCP4T1 1 1 1 1 1 Sodium Chloride and Thiomersal
1RCP4TA1 1 1 1 1 1 1 Sodium Chloride Thiomersal + 25μg A1++++
Table 5
Multivalent Liquid formulation containing Men X—TT conjugate (Strain 8210)

Composition amount per 0.5ml
(μ)
Formulation Code A- C- Y- W- X~ Excipients
CRM 197 CRM 197
1 TT CRM 197 TT
IRCP5T1 1
1 1 1 Sodium Chloride
and Thiomersal
IRCP5TA1 1 1 1 1 1 Sodium Chloride Thiomersal + 2 5pg
Al+++

Table 6
Lyophilized multivalent formulation containing Men X—TT conjugate {Strain 8210)

Formulation Code Composition amount per Vial

A-
CRM C-
CRM TT W-CRM X~
TT Excipients
Quantity in a
vial
197 197 197
IRCLPS3SC 25 25 25 25 25 Tris 0.6 mg, Sucrose 15mg, Sodium citrate 2 . 5 mg
IRCLPT3SC 25 25 25 25 2 5 Tris, Trehalose 15mg, Sodium citrate 2.5 mg
1RCLPS3L2SC 25 25 25 25 25 Tris, Sucrose 15mg, Lactose lOmg, Sodium citrate 2.5 mg
Example 5:
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 pooll, 2 & 3 using serum. from Mousel+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 7

Formulation Code Ig G SBA

Day
21 Day 28
800 Day 35
128 00 Day 21 Day 28 Day 35
IRCXLT1 504

8 20 128
1RCXLTA1 12800 32254 51200 256 323 323
IRCXNT1 2540 2540 8063 81 51 406
IRCXNAT1 6400 12800 32254 203 323 406
Table 8

Formulation Code Ig G SBA

Day 28 Day 35 Day 28 Day 35
IRCX2LT1 7 9 4032 3200 40637 2 20 20
XRCX2LAT1


128
IRCX2NT1 1270 51200 5 256
XRCX2NATA1 16127 51200 5 256
Table 9

Men A Men C

1g G SBA Ig G SBA
Formula
tion

Day Day Day Day Day Da Day Day Day Da Da Day
28 35 21 28 y
35 21 28 35 y
21 y
28 35
IRCPT1 200 200 201 6 2 2 13 635 800 8063 2 2 16

IRCPTA1 203 19 256 00 322
54 13 13 51 161 27 161 27 2031 9 25 32 102
Table 10

Formulation Code Men W135 Men Y

Ig G SBA Ig G SBA

Day 21 Day 28 Day
35 Day 21 Day 28 Day 35 Day 21 Day 28 Day 35 Day 21 Day 28 Day 35
1RCPT1 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 11

Formulation Code Men X

Ig G SBA

Day 21 Day
28 Day 35 Day 21 Day 28 Day 35
IRCPT1 IRCPTA1 1600 2016 10159 32254 64 40 256

5080
51200 81 102 406

Table 12

Formulation Code Men A Men C Men W

Ig G SBA Ig G SBA Ig G SBA

Day 28 Day 35 Day 28 Day 35 Day 28 Day 35 Day 28 Day 35 Day 28 Day 35 Day 28 Day 35
IRCP2T1 79 252 2 5 400 6400 3 20 800 1015 9 6 81
IRCP2TA1 3200 10159 5 16 3200 25600 10 25 3200 32254 2 20
Table 13

Formulation Code Men y Men X

Ig G SBA Ig G SBA

Day 28 Day 35 Day 28 Day 35 Day 28 Day 35 Day 28 Day 35
1RCP2T1 504 5080 2 5 64 2 54 0 2 0319 16 81
IRCP2TA1 1600 1015 9 3 81 6400 32254 40 102
Table 14

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

SBA SBA Day 35 SBA SBA SBA

Day 35
Day 35 Day 35 Day 35
IRCP4T1 13 10 20 8 512 512
IRCP4TA1 16 25 6 5

IRCP5T1 13 25 10 256 406
1RCP5TA1 203 203 102 162 5 512
Table 15

Ig Men A Men C Men W135
Formulation
G SBA Ig G SBA Ig G SBA
Code Day 28 Day 35 Day 28 Day 35 Day 28 Day 35 Day 28 Day 35 Day 28 Day 35 Day 28 Day 35
IRCLPS3SC 635 4032 2 5 32 0 0 6400 2 25 3200 10159 6 40
IRCLPS3DSC+ AlPO4 1600 6400 3 2 5 6 4 032 317 25600 20 323 4032 12800 13 64
64
102
IRCLPT3SC 200 160 0 12 7 0 2

2540 4 25 2016 4032 10

IRCLPT3SC+ AlPO4
6400 5 51 32 0 0 1015 9 2 5 102 2540 __ 16127 20

IRCLPS3L2SC 317 12 7 0 2 2 800 1600 5 13
4032 64 203
IRCLPS3L2SC+ A1P04 635 12 7 0 5 8 5080 10159 13 64 3200 8 063 8 2 5
Table 16

Formulation Code Men Y Men X

Ig G SBA Ig G | SBA

Day 28 Eay 35 Day 28 Day
35
5
.323
3 2 Day 28
1008 3200
2 52 Day
35
_________
512 00
12800 Day 28
3 2
1 6
Day 35
323
4 0 6
3 2 3 2 5 6
406
IRCLPS3SC
IRCLPS3DSCH-
A1P04 80 0 1008 8

2 540 1612 7 4032 2 5
6

IRCLPT3SC 504

IRCLPT3SC+ AlPO4 504 4032 4 2 56
162 _ _____ 127 0 1612 7

IRCLPS3L2SC 504 2016 10
317 12 800 10

IRCLPS3L2SC+ A1P04 635 1600 5
400 2 016 64 102
Above mice immunogenicity data indicates that 1iquid and lyophilized compositions of monovalent X-tetanus toxoid con j ugate and multivalent conjugates con taining X-tetanus toxoid conjugate are found to be immunogemc.Further monovalent liquid composition containing lug of X-tetanus toxoid conjugate,sodium chloride,tbiomersal and 125 ]ig Al''' 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 lug each of a 11 5 saccharides,sodium chloride,thiomer sal and 25μg Al+++ gives optimal immunogenic response. Thus in this pentavalent: conjugatecomposition , conjugates containing te1tanus toxoid a s carrier protein are found to enhance immunogenicity of conjugates containing CRM 197 a s carrier protein.
In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it shouId be recognized that the i 1 lustrated 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.

Day 28 Day 35 Day 28 Day 35 Day 28 Day 35 Day 28 Day 35
IRCLPS3SC 8 0 0 1008 8 2 5 5___
3 23 1008 32 00 1612 7
51200 32
4 0
16 3 2 3
IRCLPS3DSC+
A IPO 4 2 54 0 16127




4 0 6
IRCLPT3SC 504 4 03 2 6
4 3 2 2 52 12 8 0 6
3 2 3
IRCLPT3SC+
A1P04
IRCLPS3L2SC
IRCLPS3L2SC+
A1P04 504
504
63 5 4 03 2
2 56
8 12 7 0
317
400 16127
12 8 00
2016 8
10
64 2 5 6

2 016 1600 10
5



40 6
102
Above mice immunogenic!ty data indicates that 1iquid and lyophilized compositions ol monovalent X-tetanus toxoid conjugate and multivalent conjugates containing X-tetanus toxoid conjugate are found to be immunogenic Further monovalent liquid composition containing lug of X-tetanus toxoid conjugate,sodium chloride,thiomersal and 125 ug Alf'' gives optimal immunogenic response. Also j. iquid mu Univalent composition of 0.5 ml containing A-CRM197,C-CRM197,Y-tetanus toxoid,W-CRM197 and X-tetanus toxoid conjugates with lug each of all 5 saccharides , sodium chloride, thiomersal and 25μg Al''' gives optimal immunogenic response.Thus in this pentavalent conjugate composit ion, conjugates containing tetanus toxoid as carrier protein are found to enhance immunogenicity of conjugates containing CRM 197 as carrier protein .
Inview of the many possib1e embodiments to which the
principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examp1es of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the irivention 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. An immunogenic composition comprising a N.meningitidis X
Po1ysaccharide - protein conjugate , wherein said capsu1ar X
sacchar ide i s der ived f rom N . meningitidi s X strain se1ected
from 8210 and 9601.
2 . An immunogenic composition of c1aim 1,wherein at 1east one
additional saccharide conjugate(s) comprises a N. meningitidis
capsular saccharide derived from serogroups A, B, C, W135 and
Y.
3.An immunogenic composition of claim 1, wherein the .N . meningitidis X strain is selected from 8210,9601,9592,9554 and 2526.
4. An immunogenic composition of claim 2, wherein the composition comprises each of capsu1ar po1ysaccharide of N . meningitidis serogroups A, C, W--135, Y & X by utilizi ng at least 2 different carrier proteins for conjugating all 5 polysaccharides.
5 . An immunogenic composition of claim 1 o i 4 , where i n each N. meningitidis saccharide (s) are conjugated to a carrier protein selected from the group consisting of TT, DT, CRM197 , fragment C oi TT, protein D, OHPC and pneumolysin.
6. An immunogenic composition of claim 5, wherein each N. meningitidis saccharide(s) are conjugated to a carrier protein selected from the group consisting of TT, DT and CUM 197.
7. An immunogenic composition of claim 6, wherein each N. meningitidis saccharide is conjugated to a carrier protein selected f .roni the group consi sting of TT and f)T.
8. An immunogenic composition of claim 4,wherein the
composition 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 men ing i t i d is and (i i ) t.e t.anus toxo i d; (c ) a conjugate of (i) capsular saccharide of serogroup Y N. meningi t idis and. ( ii ) diphtheria t:oxoid; (d) a con juga te of ( i ) capsu1ar saccharide of serogroup W13 5 N.meningitidis and ( ii) tetanus toxoid; and (d) a conjugate of (i) capsular saccharide of serogroup X N meningitidis and (ii) tetanus toxoid.
9.An immunogenic composition of claim 4.,wherein the composition comprises {a)ci conjugate of (i) the capsular saccharide of serogroup A N meningitidis and (ii)CRJi'i 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 JN. meningitidis and {.i :i ) tetanus toxoid; (d) a conjugate of (i) capsular saccharide of: serogroup W135 N.meningitidis and (ii)CRMl97; and (d) a conjugate of (i) capsular saccharide of serogroup X N meningitidis and (ii)tetanus toxoid; charachterized in that conjugates containing tetanus toxoid as carrier protein are found to enhance immunogenicity of conjugates containing CRM 197 as carrier protein .
10 .An. immunogenic composition of claim '1 ,wherein the composition comprises (a) a conjugate of (i) the capsular' saccharide of serogroup A N meningitidis and (:i J ) tetanus toxoid; (b) a conjugate of (i) capsular saccharide of serogroup C N meningitidis and (ii)CRM.19 7; (c) a conjugate of (i) capsular saccharide of serogroup Y N. meningi tidis and (ii) tetanus toxoid;(d) a conjugate of (i) capsular saccharide of serogroup W13 5 N . meni ngi t i di s and (ii ) CKM 197 ; and (d) a conjugate of ( i ) capsu 1 ar saccbaride of serogroilp X N meningitidis and (ii)CRMl97.
11. An immunogenic composition of claim 4,wherein the composition comprises (a)a conjugate of (1) the capsular saccharide of serogroup A N meningitidis and (ii)tetanus toxoid; (b) a conjugate of ( i ) capsu.1 ar saccharide of serogroup C N meningitidis and ( ii) CRM19 7 ; (c) a conjugate o f

(i ) capsular saccharide of serogroup Y N. meningcitidis 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.
12 . An immunogenic composition of claim 4,wherein the composition comprises (a) a conjugate of ( i ) the capsu1ar saccharide of serogroup A N meningitidis and (ii)CRM 197; (b) a conjugate of (i} capsu1ar sacchar ide of serogroup C N meningitidis and (ii)CRM197; (c) a conjugate of (i) capsular saccharide of serogroup Y N. meningitidis and (ii) CRM.1.97; (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 (i i)tetanus toxoid.
13. An immunogenic composition of claim 4 ,wherein each capsular polysaccharide has an average size of between 100 and 60 0 KDa.
14. An immunogenic composition of claim 13 wherein each capsular polysaccharide has an average size of between 100 and 300 KDa.
15. An immunogenic composition of claim 14 ,wherein each capsular polysaccharide has an average size of: between 100 and 200 KDa.
16 . An immunogenic composition of claim 4 , wherein at Least 3 N. meningitidis polysaccharides from serog roups A,C, W135 and X post-sizing have an average size of between 3.00 and 150 KDa.
17. An immunogenic composition of claim 16 ,wherein the sizing is by using high pressure cell disruption system.

18. An irmmmogenic composition of claim 4 wherein N.meningitidis polysaccharide from serogroups Y post-chemical sizing have an average size of between 90 and 110 KDa.
19.An immunogenic composition of claim 18 ,wherein the chemical sizing is by using sodium acetate at a temperature from 6 0 to 80°C.
20.An immunogenic composition of claim 4, wherein each of the N. meningitidis saccharides is conjugated to the carrier protein via a hetero or bomo-bifunctional linker with cyanylation conjugation chemistry.
21. An immunogenic composition of claim 20, wherein the linker is ADH.
22. An immunogenic composition, of any preceding claim, wherein said cyanylation reagent is selected from a group or 1-cyano-4-pyrroiidlnopyri di n;i urn tetraf1 uoroborate (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.
23. An immunogenic composition of claim 20, wherein the N.meningitidis X saccharide- tetanus toxoid conj ugates are prepared by a conjugation reaction comprising of i)sizing of polysaccharide ii)CPPT based activat ion oi sized polysaccharide having average molecular weight between. 100-150 Kda, at a pH between 9 to 9. 5 iii) ADH addition after a durat ion of about 2 to 3 minutes tol lowed by incuba t: :i on peri od of A - 2 0 hrs , i v ) d i a f; i 1 t ra t i on I: o remove nnreae ted ADH and v) reacting ADH activated polysaccharide with pur:ff ied non-activated carrier protein in a. ratio between 0.75 -•- 1.5 in presence of MES buffer and EDAC followed by incubation period of 3 -4 hrs , character ized in tha t t.he conjugat:.ion react ion is carr i ed at a. t: empera.t.ure be tween 2 - 8 °C and ra t i o o f s accha r i de to protein in final conjugate is between 0.2 to 0.6.

24. An immunogenic composition of claim 20, wherein the N . me n i n g i t i d i s X s a c c h a r i d e - t e t a n u s t o x o i d c o i i j u g a t e s a r e? prepared by a conjugation reaction comprising of i)siting of polysaccharide ii) CPPT based activation of sized po1ysaccharide having average mo1ecu1ar weight betwteen 100-150 Kda, at a pH between 9 to 9.5 iiijaddition of ADli activated carrier protein in a saccharide:protein ratio between 0.5 - 2 after 2-3 minutes followed by incubation period of 2 to 20 hrs characterized in that the conjugation reaction is carried at 22°C to about 2 5°C and ratio of saccharide to protein in final conjugate is between 0.2 to 0.6.
25. An immunogenic composition of claim 1 or 2, comprising serogroup A, C, Y, W13 5 and X saccharide at an amount of 0 . 5-10/ig , 0 . 5-5/^g or 0 . 5-2/ig per 0 . 5 ml dose .
26. An immunogenic composition of claim 25, comprising 10 /.