FORM-2
THE PATENTS ACT, 1970 (39 OF 1970)
AND
THE PATENTS RULES, 2003
(As Amended)
COMPLETE SPECIFICATION
(See section 10; rule 13)
"Multivalent immunogenic conjugates and preparation thereof
Serum Institute of India Ltd., an Institute organized and existing under the laws of India, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411028 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
Bacterial polysaccharides (PSs) are T-independent antigens inducing short-term immunity in older children and adults, but frequently not in young infants. PSs are incapable of binding to the major histocompatibility complex molecules, which is required for antigen presentation to and stimulation of T-helper lymphocytes. PSs are able to stimulate B lymphocytes for antibody production without the help of T- helper lymphocytes. As a result of the T-independent stimulation of the B lymphocytes, there is a lack of memory induction following immunization by these antigens. T-independent polysaccharide antigens can be converted to T- dependent antigens by covalent attachment of the polysaccharides to protein molecules. B cells that bind the polysaccharide component of the conjugate vaccine can be activated by helper T cells specific for peptides that are a part of the conjugated carrier protein. The T- helper response to the carrier protein serves to augment the antibody production to the polysaccharide. PS- conjugate vaccines are polysaccharide-protein hybrids formed by the covalent attachment of a protein to a PS. Chemical modification of the PS prior to attachment is typically required because most native bacterial PSs cannot be chemically linked to a protein without first undergoing some chemical modification ("activation").
Attachment to the protein renders the PSs to have an

access to the immune property of a number of T cell epitopes of the protein. These T cell epitopes interact with CD4 helper T cells, greatly facilitating an antibody response to the attached polysaccharide. The T helper cell-dependent response to a conjugate results in both serum IgG antibodies and immune memory, even in infants, such as infants less than two years age. Additionally, the immunogeniclity of the PS-conjugate, in contrast to the native PS, is less dependent on the size of the conjugated PS. Accordingly, conjugates prepared with either PS or oligosaccharides can have similar immunogenicity.
There are many conjugation reactions that have been employed for covalently linking polysaccharidesto proteins. Three of the more commonly employed methods include: 1) reductive amination, wherein the aldehyde or ketone group on one component of the reaction reacts with the amino or hydrazide group on the other component, and the C=N double bond formed is subsequently reduced to C-N single bond by a reducing agent; 2) cyanylation conjugation, wherein the polysaccharide is activated either by cyanogens bromide (CNBr) or by l-cyano-4-dimethylammoniumpyridinium tetrafluoroborate (CDAP) to introduce a cyanate group to the hydroxyl group, which forms a covalent bond to the amino or hydra zide group upon addition of the protein component; and 3) a carbodiimide reaction, wherein carbodiimide activates the carboxyl group on one component of the conjugation

reaction, and the activated carbonyl group reacts with the amino or hydrazide group on the other component.
These reactions are also frequently employed to activate the components of the conjugate prior to the conjugation reaction. Vaccines that contain protein covalently linked to carbohydrate have proven remarkably successful in inducing an immune response to the carbohydrate moiety. Examples of such vaccines, known as "conjugates" are available for Haemophilus influenzae type b (e.g., ActHib, Hiberix) , Neisseria meningitidis types A C W and Y (e.g., Menactra) and S. pneumoniae (e.g., Prevnar, Synflorix).
Conventionally monovalent conjugation is performed by linking a single polysaccharide to a protein.Further these methods for synthesis and manufacturing of polysaccharide-protein conjugate vaccines typically employ conjugation reactions with low efficiency (typically about 20%),which means that up to 80% of the added activated polysaccharide is lost. In addition, a chromatographic process for purification of the conjugates from unconjugated PS is typically required.
WO 2007/109129(Robert Lee et all discloses simultaneous conjugation methods by using hydrazide chemistry in reductive amination and cyanylation conjugation reactions to overcome certain drawbacks of conventional methods for

synthesizing cost effective conjugate vaccines.The synthetic methods disclosed by Robert Lee et al utilize the characteristic chemical property of hydrazide groups on one reactant to react with aldehyde groups or cyanate esters on the other reactant with an improved conjugate yield of about 60%.Here CDAP was utilized as cyanylating reagent.
Despite the recent efforts of Lee et al, which have been dedicated to the simultaneous conjugation of polysaccharides to protein ,still challenges exist with respect to following aspects of simultaneous conjugation for obtaining bivalent conjugates such as a) optimal conjugation efficiency, b) equivalent polysaccharide to protein ratio in a bivalent conjugate c) atleast equivalent immunogenicity of said bivalent conjugates in comparison to monovalent conjugates.
The inventors of the instant invention have surprisingly found that bivalent conjugates with optimal conjugation efficiency and minimum free polysaccharide can be obtained by utilizing a novel conjugation reaction,wherein the degree of protection of such multivalent vaccine mixture containing bivalent polysaccharide-protein conjugate is equivalent to that obtained using a multivalent vaccine mixture of single polysaccharides linked to a carrier protein.
SUMMARY OF THE INVENTION

The present invention provides a complex multivalent conjugate vaccine ,particularly polysaccharide-protein bivalent conjugates and methods of preparing said multivalent immunogenic conjugate vaccine.
In particular embodiments, the invention provides bivalent polysaccharide-protein conjugates with atleast 70% conjugation efficiency and free polysaccharide less than 15% can be obtained by utilizing a novel conjugation reaction,wherein the degree of protection of such multivalent vaccine mixture containing bivalent polysaccharide-protein conjugate is equivalent to that obtained using a multivalent vaccine mixture of single polysaccharides linked to a carrier protein.
In another aspect,the invention provides a multivalent polysaccharide protein conjugate composition,particularly a pentavalent meningococcal polysaccharide protein conjugate composition.
In additional embodiments, said pentavalent meningococcal polysaccharide protein conjugate composition contains two bivalent conjugates and a monovalent conjugate,wherein the bacterial capsular polysaccharides are Neisseria meningitidis capsular polysaccharide selected from the group consisting of A, B,C D, X, Y, Z, 29E an W-135; and the carrier protein is a tetanus toxoid, diphtheria

toxoid or CRM-197.
In a specific embodiment, there is described a method for making a multivalent immunogenic polysaccharide protein conjugate , comprising:
a) Activating a first Neisseria meningitidis bacterial
capsular polysaccharide type with CPPT at a temperature
of 24°C and pH of 9.0 for 4 mins with constant stirring
at 300 rpm;
b) Addition of total hydrazide activated CRM 197 to step
a)
c)Incubating said reaction mixture for 40 minutes.
d)Activating a second Neisseria meningitidis bacterial
capsular polysaccharide type with a cyanylating reagent
at a temperature of 24°C and pH of 9.0 for 4 mins with
constant stirring at 300 rpm ; e)Addition of activated second Neisseria meningitidis
bacterial capsular polysaccharide obtained in step
(d)to the reaction mixture of step (c); f)Incubating reaction mixture of step (e) at a
temperature of 24°C and pH of 9.0 for 4 to 6hrs;and g)Subjecting the mixture to quenching by utilizing glycine at 4°C followed by overnight storage at 2-8°C.
Another aspect of the instant invention is that the said cyanylating reagent is other than CDAP ,preferably 1-cyano- 4- pyrrolidinopyridinium tetrafluoroborate (CPPT)

or a functional derivative or modification thereof as described in WO201108470.
Yet another aspect of present invention is that said carrier protein is activated with hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide or a mixture thereof under conditions sufficient to produce a solution of at least one hydrazide-activated protein.
In another embodiment of the invention,said bivalent
conjugation reaction utilizes a
polysaccharide:cyanylating reagent: protein ratio from 0.5:0.8:0.8 to about 1:2:2.
In additional embodiments, polysaccharide to protein ratio for said bivalent conjugation reaction is in the range from 1:1 to 1:5.
Yet another embodiment of the present invention is that the molecular weight of bivalent polysaccharide-protein conjugate ranges from 500 KDa to about 1500 KDa.
DESCRIPTION OF THE DRAWINGS
Fig 1: Men A Native polysaccharide by SEC-HPLC
Fig 2:Depolymerized Men A polysaccharide by SEC-HPLC
Fig 3:Men C Native polysaccharide by SEC-HPLC
Fig 4:Depolymerized Men C polysaccharide by SEC-HPLC
Fig 5 (a) & (b) :Men A-C polysaccharide conjugate with

hydrazine linked carrier protein CRM 197 by SEC-HPLC
Fig 6:Standards for Meningococcal polysaccharides A and C
by HPAEC-PAD
Fig7:Quantification of polysaccharides in Bivalent
Meningococcal A-C conjugate by HPAEC-PAD
Fig 8:Men Y Native polysaccharide by SEC-HPLC
Fig 9:Depolymerized Men Y polysaccharide by SEC-HPLC
Fig 10:Men W135 Native polysaccharide by SEC-HPLC
Fig ll:Depolymerized Men W135 polysaccharide by SEC-HPLC
Fig 12 (a) & (b):Men Y-W135 polysaccharide conjugate with
hydrazine linked carrier protein CRM 197 by SEC-HPLC
Fig 13:Standards for Meningococcal polysaccharides W and
Y by HPAEC-PAD
Fig 14:Quantification of polysaccharides in Bivalent
Meningococcal Y-W conjugate by HPAEC-PAD
Fig 15: Comparison of bivalent A C conjugates and
monovalent conjugates in presence of adjuvant
Fig 16: Comparison of bivalent Y W135 conjugates and
monovalent conjugates without adjuvant
DETAILED DESCRIPTION
Definitions
A "bacterial capsular polysaccharide" is a polysaccharide that is the predominant carbohydrate present in a capsule of a bacteria. The term includes functional derivatives or variants of the polysaccharides. This includes, without limitation, polysaccharides selected from

meningococcal serogroups A, B, C D, X, Y, Z, 2 9E an W-135 saccharides comprising a plurality of repeating units, including, but not limited to polysaccharides having 50 or more repeat units, and oligosaccharides having 50 or less repeating units.
The term "carrier", "carrier protein", or "carrier polypeptide" are used interchangeably to refer to a polypeptide moiety to which the polysaccharide antigens are covalently linked. A carrier protein is often immunogenic and therefore also contributes to the "valency" of the vaccine. Linkage to the carrier protein typically increases the antigenicity of the conjugated carbohydrate molecules. The carrier protein may be from the same target organism as the polysaccharides linked to it or may be from a different organism. The 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).

"Linked" "joined" or "conjugated" refer to covalent linkage of a carbohydrate to the carrier protein. The covalent linkage can be direct or indirect, e.g., linked through a spacer molecule.
"Immunogenic conjugate or composition" is a term used herein to mean a composition useful for stimulating or eliciting a specific immune response (or immunogenic response) in a vertebrate. In some embodiments, the immunogenic response is protective or provides protective immunity, in that it enables the vertebrate animal to better resist infection or disease progression from the organism against which the immunogenic composition is directed. One specific example of a type of immunogenic composition is a vaccine.
A "bivalent conjugate" is a polysaccharide protein
conjugate wherein two different types of capsular
polysaccharides are conjugated to the same carrier
protein molecule of the protein carrier.
In a specific embodiment, there is described a method for
making a multivalent immunogenic polysaccharide protein
conjugate , comprising:
a) Activating a first bacterial capsular polysaccharide
type with a cyanylating agent at a temperature between
20 to 25°C and pH between 8.0-10.0 for 2 to 5mins with

constant stirring at 200-300 rpm;
b) Addition of total carrier protein with or without
linker to step a)
c)Incubating said reaction mixture for a duration
between 30 to about 60 minutes.
d) Activating a second bacterial capsular polysaccharide
type with a cyanylating agent at a temperature between
20 to 25°C and pH between 8.0-10.0 for 2 to 5 mins;
e)Addition of activated second bacterial capsular
polysaccharide obtained in step (d)to the reaction
mixture of step (c);
f) Incubating reaction mixture of step (e) at 20 to 25°C
for 4 to 6hrs;and
g)Subjecting the mixture to quenching by utilizing
glycine at 4°C followed by overnight storage at 2-8°C
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; meningococcal polysaccharides of serotypes , Haemophilus influenzae 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, 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.
The polysaccharides and oligosaccharides can be subjected
to one or more processing steps prior to activation, for
example, purification, functionalization,
depolymerization using mild oxidative conditions, deacetylation, and the like.
After completion of any pre-processing steps, the polysaccharide or oligosaccharide is subjected to an "activation" step. The term "activation" refers to a chemical treatment of the polysaccharide to provide chemical groups capable of reacting with the protein. In a particularly preferred embodiment, activation involves functionalization of the polysaccharide or oligosaccharide with aldehyde groups, ketone groups or cyanate groups that are reacted with hydrazide groups on a functionalized protein. Alternatively, the polysaccharide or oligosaccharide can be functionalized with' hydrazide groups that are reacted with aldehyde

groups or ketone groups on a functionalized protein.
The carrier proteins are preferably proteins that are non-toxic and non-reactogenic and obtainable in sufficient amount and purity that are amenable to the conjugation methods of preferred embodiments;fragments of the native toxins or toxoids, which contain at least one T- cell epitope, are also useful, as are outer membrane protein complexes, as well as certain analogs, fragments, and/or analog fragments of the various proteins listed above The proteins can be obtained from natural sources, can be produced by recombinant technology, or by synthetic methods as are known in the art. Analogs can be obtained by various means, for example, certain amino acids can be substituted for other amino acids in a protein without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules.
Any suitable functionalization reaction can be employed to activate the protein with hydrazide groups. Conventional methods for preparing hydrazide-modified proteins include EDC catalysis and a two-step process using N-succinimidyl iodoacetate and thiol hydrazide through lysine e-amino groups of the protein. See King et al. , Biochemistry 1986; 25:5774-5779. Modified protein prepared by EDC catalysis typically needs to be

fractionated in order for it to be suitable for use in conjugation, and the two-step process is tedious. Accordingly, it is generally not preferred to employ such methods for preparing the hydrazide-modified protein. However, in certain embodiments such methods can be acceptable or even desirable.
Preferably, hydrazide groups are 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. EDOcatalyzed 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.
Conjugates can be prepared by cyanylation conjugation chemistry wherein the 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-

cyanopyridazine -3(2H)one (2-CPO).
The said bivalent conjugation reaction of present invention can employ a polysaccharide:cyanylating reagent: protein ratio selected from 0.5:0.8:0.8, 0.8:1.5:1.5 , 1:0.75:0.75, 1:0.8:1 , 1:1:1 , 1:1.5:1.5 , 1:1:2 ,1:2:1 and 1:2:2.
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.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and . modifications may be made thereto without departing from the spirit or scope of the appended claims.

Examples: Example 1:
Depolymerization of N. meningitidis serogroups A, C, Y and W135 purified capsular polysaccharides
N. meningitides serogroup purified polysaccharide in WFI was depolymerized by homogenization separately at a concentration of 5.0+2.0 gm/1. The size of the depolymerized polysaccharide was above lOOkDa but less than 200kDa.The depolymerized polysaccharide solution was concentrated to 20± 5.0 gms per liter by connecting the reaction tank to an ultra filtration unit equipped with 10,000 MWCO PES cartridges. The depolymerized polysaccharides were stored at -20 °C until the next process step. The molecular size of the depolymerized polysaccharide was determined by passage through a gel filtration chromatography column (Shodex 805/804). The quantity of polysaccharide for serogroup A was determined by estimating phosphorus content (Refer Chen, P.S 1956 Anal. Chem., 28 11, pp 1756-1758) and for serogroups C, Y and W135 by sialic acid content (Refer Svennerholm, L.1955 Biochimica Biophysics Acta 24, pp604-611).Further the O-acetyl content was determined (Refer Hesterin, S. 1949 Journal of Biological Chemistry 180, pp249).Also structural integrity of the depolymerized polysaccharide was determined by 1H and 13C NMR. The purity of the depolymerized polysaccharide content was determined by measuring the LAL (endotoxin) content.
Example 2:
Derivatization of Carrier protein CRM 197 using Hydrazine Hydrochloride
CRM 197 (carrier protein)in phosphate buffer solution was diafiltered and concentrated against 20 volumes of 0.8 5% physiological saline to 5-6gm/l by connecting the reaction tank to an ultra filtration unit equipped with 5000 MWCO PES cartridges. To this solution 1M solution of 2- (N-morpholino) ethanesulfonic acid (MES) was added to

achieve a final concentration of 4.0-5.0 gm/1. 5M hydrazine hydrochloride was later added to this protein solution to achieve a reaction concentration of 4.0- 5.0 gm/l.lM EDC in WFI was added to this mixture and pH was adjusted to 7.0+ 0.2 using sterile hydrochloric acid and sterile 2% sodium hydroxide to achieve a reaction concentration of 3.0-4.0 gm/1 for NLT 4hour at 23±1.0°C and the solution was neutralized with 2% sodium hydroxide.Activated CRM 197 was diafiltered and concentrated to NLT 35 volumes of phosphate buffer to 10-20 gm/1 by connecting the reaction tank to an ultra filtration unit equipped with 5000 MWCO PES cartridges. The protein content was measured by BCA assay. The hydrazine content was measured by 2, 4, 6-trinitrobenzenesulphonic acid (Refer Synder, S.L. and Sobocinski, P.Z.1975 Analytical Biochemistry 64, pp282-288) .
Example 3:
Bivalent conjugation of polysaccharides Men A and Men C to hydrazine linked carrier protein CRM 197 using Cyanylating agent 1 cyano-4-pyrrolino pyridine tetrafluoroborate (CPPT)
Materials used in this preparation include polysaccharide from Neisseria meningitidis serogroups A and C, sterile CRM 197 protein, hydrazine-HCl, EDAC, CPPT, 2M Glycine, sterile IN sodium hydroxide, l0mM Tris buffer pH 7.0-7.2 and 2M Sodium Chloride.
The average ratio of each polysaccharide to derivatized carrier protein is 1:1 to about 1:5 (w/w),preferably, 1 : ( 2+1.25) (w/w).
The saccharide conjugates rely on the activation of the saccharides with CPPT to form cyanate ester. The average ratio of each polysaccharide to CPPT is about 1:1 to about 1:5(w/w),preferably, 1 :( 2±0.5) (w/w).
Initially the Men C saccharide in 2M sodium chloride was CPPT activated for 3±1 mins at a temperature of 23 + 1.0°C and at pH 9.5+0.2 under constant stirring at 200-300rpm

and then conjugated to the hydrazine hydrochloride derivatized CRM 197 catalyzed by using carbodiimide EDC. Hydrazine linked CRM 197 at a concentration of 20+5 mg/ml was added w/w ratio required for both Men C and Men A polysaccharides to the activated Men C polysaccharide. After a time interval of 30-60 mins the second CPPT activated Men A saccharide was added to the reaction mix of Men C - hydrazine linked CRM 197 at a temperature of 23±1.0°C and at pH 9.5±0.2 under constant stirring at 200-300rpm. The conjugation reaction was monitored by SEC-HPLC for 3-4 hours. Conjugation efficiency was monitored to be greater than 90% in terms of protein conversion; the reaction was then quenched with 2M glycine at a temperature of 23+1.0°C and at pH 8.5+0.5 followed by overnight incubation at 2-8 °C.
Example 4:
Bivalent conjugation of polysaccharides Men Y and Men W135 to hydrazine linked carrier protein CRM 197 using Cyanylating agent 1 cyano-4-pyrrolino pyridine tetrafluoroborate (CPPT)
Materials used in this preparation include polysaccharide from Neisseria meningitides serogroups Y and W, sterile CRM 197 protein, hydrazine -HC1, EDAC, CPPT, 2M Glycine, sterile IN sodium hydroxide, l0mM Tris buffer pH 7.0-7.2 and 2M Sodium Chloride.
The average ratio of each polysaccharide to derivatized carrier protein is 1:1 to about 1:5(w/w),preferably, 1 : ( 2+1.2) (w/w).
The saccharide conjugates rely on the activation of the saccharides with CPPT to form cyanate ester.The average ratio of each polysaccharide to CPPT is about 1:1 to about 1:5(w/w),preferably, 1 :( 2+1.0) (w/w).
Initially the Men Y saccharide in 2M sodium chloride was CPPT activated for 3+1 mins at a temperature of 23+1.0°C and at pH 9.5+0.2 under constant stirring at 200-300rpm and then conjugated to the hydrazine derivatized CRM 197 catalyzed by using carbodiimide(EDC). Hydrazine linked

CRM 197 at a concentration of 20±5 mg/ml was added w/w ratio required for both Men Y and Men W polysaccharides to the activated Men Y polysaccharide. After a time interval of 30-60 mins the second CPPT activated Men W saccharide was added to the reaction mix of Men Y -hydrazine linked CRM 197 at a temperature of 23+1.0°C and at pH 9.5+0.2 under constant stirring at 200-3OOrpm. The conjugation reaction was monitored by SEC-HPLC for 3-4 hours. Conjugation efficiency was monitored to be greater than 90% in terms of protein conversion, the reaction was then quenched with 2M glycine at a temperature of 23±1.0°C and at pH 8.5±0.5 followed by overnight incubation at 2-8 °C.
Example 5:
Purification of bivalent conjugates (Men A/C-CRM 197 & Men Y/W135-CRM 197)
The conjugate was purified by ultra filtration by connecting the reaction tank to an ultra filtration unit equipped with 300,000 MWCO PES cartridges using in l0mM Tris buffer pH 7.0+0.5 followed by 0.2 micron filtration.
Example 6:
Estimation of Polysaccharide content in bivalent conjugates by HPAEC-PAD
This HPAEC-PAD method is based on measurement of monosaccharides meningitidis serogroup released by Trifluoro acid hydrolysis (TFA) i.e. glucose (serogroup Y saccharide), galactose (serogroup W135 saccharide), sialic acid (serogroup C saccharide), and mannosamine-6-P (serogroup A saccharide) respectively.
High performance Anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is an improved chromatographic technique developed to separates carbohydrates coupled with pulsed amperometric detection (PAD) . It permits direct quantification of monosaccharides.
Calculation for total Ps mg/ml:

(Sample Area nC - Intercept) X Dilution Fold
Slope Calculation for % Free Ps:
Free Ps (mg/ml) X 100
Total Ps (mg/ml)
Example 7:
Calculation of conjugation efficiency in relation to polysaccharide concentration and Ps: CPPT: Pr ratio by SEC-HPLC
Table 1:
MenA-C bivalent conjugate:

Polysaccharide Men Ratio Protein conversion
A-C Ps: CPPT: Pr by SEC-HPLC
For bivalent
conjugation
15mg/ml Ps conc.
Men A 1:0.75:0.75 >60%
Men C 1:0.75:0.75
Men A 1:1:1 >60%
Men C 1:1:1
Men A 1:1.5:1.5 >70%
Men C 1:1.5:1.5
Men A 1:2:2 >70%
Men C 1:2:2
Men A 1:2:2 >80%
Men C 1:1:2
Men A 1:2:2 >90%
Men C 1:2:1
Table 2:
MenY-W135 bivalent conjugate

Polysaccharide Men Ratio Protein conversion
Y-W135 Ps: CPPT: Pr by SEC-HPLC
For bivalent
conjugation

15mg/ml Ps conc.

Men Y 1:2:2 >60%
Men W 1:2:2
Men Y 1:2:2 >60%
Men W 2:2.5:2.5
Men Y 1:1.5:1.5 >70%
Men W 1:2:2
Men Y 1:2:2 >70%
Men W 1:1:1
Men Y 1:1:1 >80%
Men W 1:1:1
Men Y 1:0.8:1 >90%
Men W 1:0.8:1
Table 3:
MenA-C bivalent conjugate

Polysaccharide Men Ratio Protein conversion
A-C Ps: CPPT: Pr by SEC-HPLC
For bivalent
conjugation
lOmg/ml Ps conc.
Men A 1:0.75:0.75 >50%
Men C 1:0.75:0.75
Men A 1:1:1 >50%
Men C 1:1:1
Men A 1:1.5:1.5 >60%
Men C 1:1.5:1.5
Men A 1:2:2 >60%
Men C 1:2:2
Men A 1:2:2 >70%
Men C 1:1:2
Men A 1:2:2 >70%
Men C 1:2:1
Table 4:
MenY-Wl35 bivalent conjugate:

Polysaccharide Men Ratio Protein conversion
Y-W135 Ps: CPPT: Pr by SEC-HPLC
For bivalent
conjugation
lOmg/ml Ps conc.
Men Y 1:2:2 >50%

Men W
1:2:2
Men Y 1:2:2 >50%
Men W 1:1.5:1.5
Men Y 1:1.5:1.5 >60%
Men W 1:2:2
Men Y 1:2:2 >60%
Men W 1:1:1
Men Y 1:1:1 >70%
Men W 1:1:1
Men Y 1:0.8:1 >70%
Men W 1:0.8:1
Example 8:
A) Confirmation of bivalency of MenA- C -CRM197 bivalent conjugate
The presence of bivalent conjugate molecules in the MenA+C bivalent conjugate was confirmed by sandwich bead based assay i.e. to anlayze whether MenA & MenC Ps both are conjugated to single protein carrier(CRM197)or not.
Assay Conditions
1) The MenA-C bivalent conjugate was diluted 1:100, 1:600
& 1:3600 & added in the wells of the filter plate
containing anti-MenA MAb coupled beads.
2) Also, MenA monovalent Bulk conjugate was added as
negative control in the similar dilutions.
3) Rabbit anti-MenC pooled serum (secondary antibody) was added in 2 different dilutions as 1:500 & 1:2000 dilutions to find out optimized dilution.
4) Anti-rabbit IgG-phycoerythrene conjugate was added to each well to detect the reaction.
Table 5:


Assay Inference
There was no major difference in the FI values with blank wells and monovalent conjugate sample, indicating the absence of bivalent molecules in the monovalent conjugate sample.
The FI with bivalent conjugate are significantly higher than the FI of the blank & monovalent conjugate, hence there is presence of the bivalent molecules (MenA/CRM-97/MenC) in the bivalent conjugate.
As no dilution pattern is seen upon diluting antigen in the conjugate samples (which may be because of the saturation level) , there is a need to repeat the assay with higher conjugate sample dilutions as 1:5000, 1:20000 & 1:80000 in combination of only 1:500 dilution of secondary antibody.
B) Confirmation of bivalency of MenY - W135 -CRM197 bivalent conjugate
The presence of bivalent conjugate molecules in the Men W135+C bivalent conjugate was confirmed by sandwich bead based assay i.e. to analyze whether MenW135 & MenY Ps both are conjugated to single protein carrier(CRM197)or not.

Assay Conditions
1) The MenW135-Y bivalent conjugate was diluted 1:5000,
1:20000 & 1:80000 & added in the wells of the filter
plate containing anti-Meny" MAb coupled beads.
2) Also, MenY of 0.72mg/ml monovalent conjugate was added as negative control in the similar dilutions.
3) The assay utilized mouse anti-MenW135 IgM MAbs (NIBSC) in 1:500 dilution.
Table 6:

The MFI values in monovalent conjugates were similar/slightly higher than blank MFI while the INJFI with bivalent conjugate sample were significantly higher than both i.e. blank & monovalent conjugate sample & hence it confirmed the presence of bivalent conjugate molecules in the MenWl35+Y bivalent conjugate.
The above experiment confirmed the presence of bivalent conjugate molecules in both A-C and W135-Y conjugates as the reaction(FI) with bivalent conjugate bulks is significantly higher than that of monovalent conjugate bulks and also that the blank well fluorescence is similar to that of monovalent conjugates,
Example 8:
Comparative Immunogenicity profile study of bivalent

polysaccharide protein conjugates with corresponding monovalent polysaccharide protein conjugates.
The monovalent and benevolent meningococcal conjugates were injected subcutaneously in 6 mice per formulation. The sera were tested for presence of anti-meningococcal IgGs by a multiplexed bead based assay and the functional antibodies were tested by serum bactericidal (SBAs) antibody titration method against various serogroups. Briefly the sera samples were separated from blood collected 7 days after 3 doses of the vaccine and antibody levels tested. The bead based assay consisted of reacting polystyrene beads already coupled to meningococcal polysaccharide of interest with different dilutions of the sera samples. The secondary antibody coupled to a fluorochrome called Phycoerythrin was added to the wells and reading of the plate was taken using Bioplex 200 system and serogroup specific antibody titers were calculated. The SBAs were performed as per standardized protocol including incubation of bacteria with sera dilutions and rabbit complement and the functional antibody titers were calculated from the highest sera dilution showing 50% or more killing of the bacteria.
The results(Fig 15 & Fig 16) indicate that the monovalent and bivalent conjugates gave rise to equivalent average antibody titers against various meningococcal serogroups with or without the presence of aluminium phosphate as adjuvant.
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 process for preparing a bivalent polysaccharide-

protein conjugate, comprising;
a) Activating a first bacterial capsular polysaccharide
type with a cyanylating agent at a temperature between
20 to 25°C and pH between 8.0-10.0 for 2 to 5mins with
constant stirring at 200-300 rpm;
b) Addition of total carrier protein with or without
linker to step a);
c)Incubating said reaction mixture for a duration
between 30 to about 60 minutes;
d) Activating a second bacterial capsular
polysaccharide type with a cyanylating agent at a
temperature between 20 to 25°c and pH between 8.0-10.0
for 2 to 5 mins;
e)Addition of activated second bacterial capsular
polysaccharide obtained is step (d)to the reaction
mixture of step (c) ;
f)Incubating reaction mixture of step (e) at 20 to 25°C
for 4 to 6hrs;
g)Subjecting the mixture to quenching by utilizing
glycine at 4°C followed by overnight storage at 2-8°C ;
and
wherein the first and second bacterial capsular
polysaccharides are different and are selected from
Neisseria meningitidis group consisting of A, B,C D, X,
Y, Z, 29E and W-135.
2. A bivalent polysaccharide-protein conjugate of claim 1,

wherein the said first and second polysaccharide combination for bivalent conjugate is selected from the group consisting of A/C, A/Y, A/W 135, A/X, C/Y,C/W 135,C/X ,Y/W 135 ,Y/X and W 135/X.
3. A bivalent polysaccharide-protein conjugate of claim 1, wherein the carrier protein is selected from the group consisting of: TT, DT, CRM197, fragment C of TT, protein D of H. influenzae, pneumococcal PhtD, and pneumococcal Pneumolysin,
4 .A bivalent polysaccharide-protein conjugate of claim 1,wherein prior to reacting the protein and polysaccharide together,the polysaccharide is activated by reacting with a cyanylating reagent in a ratio from
1:1 to about 1:5.
5. A bivalent polysaccharide-protein conjugate of claim 1,wherein prior to reacting the protein and polysaccharide together,the polysaccharide is activated using cyanylating reagents selected from a group consisting of 1-cyano- 4- pyrrolidinopyridinium tetrafluoroborate (CPPT), 1- cyano- imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), or 2- cyanopyridazine 3(2H}one (2-CPO).
6. A bivalent polysaccharide-protein conjugate of claim 1,wherein prior to reacting the protein and

polysaccharide together,the carrier protein is activated with hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide or a mixture.
7. A bivalent polysaccharide-protein conjugate of claim 1, wherein ratio of polysaccharide:cyanylating reagent:protein in reaction mixture is selected from 0.5:0.8:0.8, 0.8:1.5:1.5 , 1:0.75:0.75, 1:0.8:1 , 1:1:1 , 1:1.5:1.5 , 1:1:2 ,1:2:1 and 1:2:2.
8. A bivalent polysaccharide-protein conjugate of claim 1,wherein the ratio of polysaccharide to protein in reaction mixture is from 1:1 to about 1:5.
9. A bivalent polysaccharide-protein conjugate of claim 1,wherein the conjugation efficiency is atleast 70%.
10. A bivalent polysaccharide-protein conjugate of claim
1,wherein the free polysaccharide concentration is less
than 15%.
11. A bivalent polysaccharide-protein conjugate of claim 1 ,wherein the size of bivalent conjugate is between 500 KDa and 1500 KDa.
12. A process for preparing a bivalent polysaccharide-protein conjugate, comprising:
a) Activating a first Neisseria meningitidis bacterial

capsular polysaccharide type with CPPT at a temperature of 24°C and pH of 9.0 for 4 mins with constant stirring at 300 rpm;
b) Addition of total hydrazide activated CRM 197 to step a);
c)Incubating said reaction mixture for 40 minutes; d)Activating a second Neisseria meningitidis bacterial capsular polysaccharide type with CPPT at a temperature of 24°C and pH of 9.0 for 4 mins with constant stirring at 300 rpm ;
e)Addition of activated second Neisseria meningitidis bacterial capsular polysaccharide obtained in step (d)to the reaction mixture of step (c);
f)Incubating reaction mixture of step (e) at a temperature of 24°C and pH of 9.0 for 4 to 6hrs; g)Subjecting the mixture to quenching by utilizing glycine at 4°C followed by overnight storage at 2-8°C; and wherein the first and second bacterial capsular polysaccharides are different and are selected from Neisseria meningitidis group consisting of A, B,C D, X, Y, Z, 29E and W-135.
13. A bivalent polysaccharide-protein conjugate of claim
12,
wherein the first bacterial polysaccharide is from
Neisseria meningitidis C and second bacterial capsular
polysaccharide is from Neisseria meningitidis A.

14. A bivalent polysaccharide-protein conjugate of claim
12,
wherein the first bacterial polysaccharide is from
Neisseria meningitidis Y and second bacterial capsular
polysaccharide is from Neisseria meningitidis W 135.
15 .A bivalent polysaccharide-protein conjugate of claim 12,wherein prior to reacting the protein and polysaccharide together,the polysaccharide is activated by reacting with CPPT in a ratio from about 1:1 to about 1:5,preferably about 1:2.
16. A bivalent polysaccharide-protein conjugate of claim 12,wherein prior to reacting the protein and polysaccharide together,the carrier protein is activated with hydrazine hydrochloride.
17. A bivalent polysaccharide-protein conjugate of claim 12, wherein ratio of Neisseria meningitidis polysaccharide:CPPT:CRM197 in reaction mixture is from 0.5:0.8:0.8 to about 1:2:2,preferably selected from 1:0.8:1 ,1:2:1 and 1:2:2.
18. A bivalent polysaccharide-protein conjugate of claim 1,wherein the ratio of Neisseria meningitidis polysaccharide to CRM197 in reaction mixture is from about 1:1 to about 1:5,preferably 1:2.

19. A bivalent polysaccharide-protein conjugate of claim 12,wherein the conjugation efficiency is greater than 70%.
20. A bivalent polysaccharide-protein conjugate of claim 12,wherein the free polysaccharide concentration is less than 15%.
21. A bivalent polysaccharide-protein conjugate of claim 12 ,wherein the size of bivalent conjugate is between 500 KDa and 1500 Kda,preferably between 800 KDa and 1200 KDa.

22. A multivalent polysaccharide protein conjugate vaccine comprising atleast one bivalent polysaccharide protein conjugate prepared according to claim 1 or 12.
23. A multivalent polysaccharide protein conjugate vaccine comprising atleast one bivalent polysaccharide protein conjugate prepared according to claim 1 or 12 and atleast one monovalent polysaccharide protein conjugate .
24. A multivalent polysaccharide protein conjugate vaccine comprising atleast two bivalent polysaccharide protein conjugates prepared according to claim 1 or 12 and atleast one monovalent polysaccharide protein conjugate .

25. A multivalent polysaccharide protein conjugate
vaccine comprising atleast one bivalent polysaccharide
protein conjugate prepared according to claim 1 or 12
further comprising one or more pharmaceutically
acceptable diluents, carriers, adjuvants, and/or buffers.
26. A multivalent polysaccharide protein conjugate vaccine
comprising atleast one bivalent polysaccharide protein
conjugate prepared according to claim 1 or 12 having
equivalent or improved immunogenicity as compared to the
monovalent polysaccharide protein conjugates.