FORM-2
THE PATENTS ACT, 1970
(39 OF 1970)
AND
THE PATENTS RULES, 2003
(As Amended)
COMPLETE SPECIFICATION
{See section 10; rule 13)
"NOVEL METHOD FOR STABILIZATION OF POLYSACCHARIDE-PROTEIN CONSTRUCTS"
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:
Vaccines are widely used for the prevention and/or therapy of many different diseases.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 conjugate vaccines are already approved and marketed throughout the world.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).
The polysaccharide component of conjugate vaccines undergoes gradual depolymerization .This causes reduction in molecular size of the polysaccharide component, i.e. an increase in free polysaccharide.Hence tests should be conducted to ensure stability of product. Polysaccharide-carrier protein conjugates are known to release free polysaccharide after conjugation while further processing, lyophilization or storage in liquid as well as solid formulations. Clinical protection is conferred only by polysaccharide that is covalently bound to the carrier protein. Accordingly vaccines demonstrating. adequate immunogenicity and thermostability contain amounts of unbound polysaccharide ranging from less than 10% up to 40% of the total polysaccharide, refer WHO TRS,pg 47-48,No.897,2000.

George R .Siber et al(2008) discusses that bulk polysaccharide protein conjugates should be tested for identity,any residual reagents or active groups,including capping molecules.Most importantly bulk conjugates should be monitored over a period of time for percentage of unconjugated polysaccharide to ensure vaccine stability during further processing steps.Refer George.Siber et al;"Pneumococcal vaccines:impact of conjugate vaccine";ASM.
US 7754218(Contorni et al) provides compositions to improve the stability of vaccine formulations comprising meningococcal polysaccharide protein conjugates,aluminium salt,histidine buffer,sodium phosphate buffer & polysorbate 80 ,resulting in improved pH stability,adjuvant adsorption and reduction in antigen hydrolysis.However Contorni et al discusses stability of formulation based conjugates and not bulk conjugates.
EP 2457590 discusses that a particular stability problem of many different classes of molecules, ranging from small molecules to complex supramolecular systems, is cleavage of a bond between two conjugated parts of the molecule or the system.Examples of such undesirable processes include cleavage of a polysaccharide moiety from a carrier protein in a number of polysaccharide-based vaccines (e.g. Neisseria meningitidis,Streptococcus pneumoniae & Haemophilus influenzae b vaccines). Acid or base hydrolysis is typically the mechanism of such degradation processes.Hydrolysis is a chemical reaction during which a water molecule is split into hydrogen and hydroxide ions which go on to participate in cleavage of a particular covalent bond. Hydrolysis requires the presence of water

and is known to be a pH-dependent process. However, proton transfer from molecules can also be involved in the mechanism of hydrolytic cleavage.EP 245759 0 discloses that a)less than 11 % increase on free Hib PRP could be observed after 3 weeks in the low ionic strength histidine-based formulation containing 1, 2-propanediol. & b)less than 5% increase of free Hib PRP can be observed at 40°C for 3 weeks in formulation containing displaced buffer based on combination of Tris(aminomethyl)hydroxymethane (10 mM) and sodium lactate(10 mM).
Despite advances in the area of conjugate vaccine formulation stability, there remains a distinct need for stabilizing polysaccharide-protein conjugate bulks. Methods for preparing stabilized polysaccharide protein conjugate formulations are well known in the art,but none of the prior art stabilizers impart the desired enhanced sustained level of stability to bulk polysaccharide protein conjugates.
The inventors have surprisingly found a novel method for stabilizing a polysaccharide-protein bulk conjugate particularly pneumococcal polysaccharide-protein bulk conjugates.
Summary of the Invention
The present invention is based on a surprising discovery that stability of polysaccharide-protein bulk conjugate at 2-8°C and 25°C can be significantly improved by utilizing a stabilizer mixture of sodium salt and amino acid to the bulk conjugate resulting in i)minimum free polysaccharide

content post- storage and ii)minimum change in average molecular weight of polysaccharide post-storage.
Description of Invention
The term "bulk conjugate" is used herein for a conjugate prepared from a single lot or a pool of lots of polysaccharide and a single lot or a pool of lots of carrier protein and conjugated at the same time. This is the parent material from which the final bulk is prepared.
In a first embodiment ,said polysaccharide protein conjugate bulk can be stable for atleast 3 months at 2-8°C, atleast 2 months at 25°C, wherein the free polysaccharide level is minimum,preferably less than 5%.
In a second embodiment, said composition contains a sodium salt selected from a group of sodium phosphate and sodium chloride wherein the concentration of sodium salt can be in the range between 1 mM to 160mM,preferably 154mM.
In a third embodiment of the present invention ,said amino acid is histidine at a concentration between ImM and 2 00 M. The concentration is preferably at least 1 mM (e.g. at most 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM etc.) . More preferably the concentration of histidine in the composition is between 10 mM and 2 0 mM .
In an aspect of the third embodiment, said histidine may be ionized within the composition of the invention.

Yet another aspect of the third embodiment is that said histidine may be added to the composition in the form of amino acid itself or in the form of a salt. A typical histidine salt is the monohydrochloride monohydrate.
In a fourth embodiment,said polysaccharide-protein conjugate bulk of instant invention can contain monovalent or multivalent conjugates,obtained by conventional and simultaneous conjugation respectively.
In a fifth embodiment said polysaccharides and oligosaccharides for use can be selected from but not limited to meningococcal polysaccharides of serogroups A, B,C D, X, Y, Z, 29E ,W-13 5; 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.

In a sixth 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).
In a yet another aspect, the Streptococcus pneumoniae saccharide can be conjugated to the carrier protein via a linker, for instance a bifunctional linker. A possible linker is ADH. Other linkers include B-propionamido (WO 00/10599), nitrophenyl-ethylamine (Geyer et al (1979) Med. Microbiol. Immunol. 165; 171-288), haloalkyl halides (U.S. Pat. No. 4,057,685), glycosidic linkages (U.S. Pat. No. 4,673,574, U.S. Pat. No. 4,808,700), hexane diamine and 6-aminocaproic acid (U.S. Pat. No. 4,459,286).
Further the saccharide conjugates present in the immunogenic compositions of the invention may be prepared by any known coupling technique. The conjugation method may rely on activation of the saccharide with l-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated saccharide may thus be coupled

directly or via a spacer (linker) group to an amino group on the carrier protein.
Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many are described in WO 98/42721. Conjugation may involve a carbonyl linker which may be formed by reaction of a free hydroxyl group of the saccharide with CDI (Bethell et al J. Biol. Chem. 1979, 254; 2572-4, Hearn et al J. Chromatogr. 1981. 218; 509-18) followed by reaction of with a protein to form a carbamate 1inkage. This may involve reduction of the anomeric terminus to a primary hydroxyl group, optional protection/deprotection of the primary hydroxyl group' reaction of the primary hydroxyl group withCDI to form a CDI carbamate intermediate and coupling the CDI carbamate intermediate with an amino group on a protein.
Alternatively the conjugates can also be prepared by direct reductive amination methods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat. No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188, EP-208375 and EP-0-477508.
A further method involves the coupling of a cyanogen bromide (or CDAP) activated saccharide derivatized with adipic acid dihydrazide (ADH) to the protein carrier by Carbodiimide condensation (Chu C. et al Infect. Immunity, 1983 245 256), for example using EDAC.
In an embodiment, a hydroxyl group (preferably an activated hydroxyl group for example a hydroxyl group activated to

make a cyanate ester [e.g. with CDAP]) on a saccharide is linked to an amino or carboxylic group on a protein either directly or indirectly (through a linker}. Where a linker is present, a hydroxy1 group on a saccharide is preferably linked to an amino group on a linker, for example by using CDAP conjugation. A further amino group in the linker for example ADH) may be conjugated to a carboxylic acid group on a protein, for example by using carbodiimide chemistry, for example by using EDAC. In an embodiment, the pneumococcal capsular saccharide(s) is conjugated to the linker first before the linker is conjugated to the carrier protein. Alternatively the linker may be conjugated to the carrier before conjugation to the saccharide.
In another aspect ,a combination of techniques may also be used, with some saccharide-protein conjugates being prepared by CDAP, and some by reductive amination.
Examples:
Example 1
Stability study of Pneumococcal Polysaccharide Protein Conjugates (serotype 14 polysaccharide conjugated to CRMl97)with respect to Mw(Average Molecular weight) as well as free Polysaccharide level.
Type 14-CRM197 Pneumococcal conjugates(prepared by CDAP chemistry) were taken and three aliquots in equal proportion were then diafiltered against different buffers on lOOkDa.
Sample A (Control) : 2 0mM PBS buffer 7.2 , Sample is only diluted with same buffer 0.5 mg/ml.
Sample B : Diafiltered against 0.9% NaCl pH 7.0 & Concentration of 0.5mg/ml was made with. 0.9% NaCl ,pH 7.0.

Sample C : Diafiltered against 0.9% NaCl with 20mM Histidine pH 7.0 & concentration was made 0. 5mg/ml with 0.9% NaCl.
All these samples were kept at 2-8°C & 25°C for stability purpose. 5ml aliquot samples were withdrawn at an interval of 1 month,2 month ,3 months and 4 months respectively.
Table 1
2-8°C Stability of Streptococcus pneumoniae serotype 14 polysaccharide- CRM 197 conjugate

Sample Actual Mw & ] Free 14 serotype Ps values during stability
Initial (0 days) 30 days 90 days 120 days
Mw Free
PS Mw Free Ps Mw Free Ps Mw Free Ps
14A 940 3.98 872 4.7 809 5.73 656 5.53
14B 978 3.50 887 4.56 556 11.96 544 15.05
14C 976 3.25 871 4.25 881 4.25 726 4.15
Mw: Average Molecular Weight estimated by HPLC
Table 2
25°C Stability of Streptococcus pneumoniae serotype 14 polysaccharide- CRM 197 conjugate

Sample Actual Mw & Free 14 serotype Ps values during
stability
Initial (0 days) 30 days 90 days 120 days
Mw Free
PS Mw Free Ps Mw Free Ps Mw Free Ps
14A 940 3.98 860 4.5 845 5.36 650 5.10
14B 978 3.50 877 4.36 536 10.9 512 14.12
14C 949 0.51 940 4.25 935 4.25 932 4.15
Mw: Average Molecular Weight estimated by HPLC

Thus, the data presented in Table 1 and Table 2 demonstrate that the Streptococcus pneumoniae serotype 14 polysaccharide-CRMl97 conjugate bulk "Sample C " comprising 20mM Histidine and 154 mM NaCl were significantly stabilized at 2-8°C and 25°C,wherein the free polysaccharide level was found to be less than 5% with a minimum change in average molecular weight post-storage. whereas the comparator samples(Sample A & Sample B) stability data at 2-8°C and 25°C indicates that the free polysaccharide level was found to be greater than 5% with a significant reduction in average molecular weight post-storage.
Sample C formulation was further considered for bulk conjugate stability studies for other pneumococcal serotype polysaccharide-protein conjugate bulk.
Example 2
Stability study of Pneumococcal Polysaccharide Protein Conjugates (polysaccharides of serotypes 5, 6A,6B, 7F,9V,19A,19F & 23F individually conjugated to CRM197) with respect to Mw(Average Molecular weight) as well as free polysaccharide level.
All the above conjugates{prepared by CDAP conjugation chemistry) were diafiltered against 0.9% NaCl with 20mM Histidine pH 7.0 & concentration was made 0. 5mg/ml with 0.9% NaCl.
Further the said samples were kept at 2-8°C & 25°C for stability purpose. 5ml aliquot samples were withdrawn at an interval of 1 month,2 month or 3 months.

Table 3
2-8°C Stability of Streptococcus pneumoniae serotype
polysaccharide- CRM 197 conjugates

Serotype %Free Polysaccharide Mw

Initial 3M 6M Initial 3M 6M
Type-5 2.76 3.10 8.50 3530 3981 3614
Type-6A 3.19 5.32 7.28 2662 2314 2044
Type-6B 0.49 0.89 0.34 6673 5865 6986
Type-7F 0.25 1.18 0.93 6525 5817 6310
Type-9V ND ND ND 6495 6563 7236
Type-19A 0.61 0.59 0.40 8210 7760 7865
Type-19F ND ND 0.82 6393 6257 6707
Type-23F 0.44 0.79 1.44 5936 5481 6058
Mw: Average Molecular Weight estimated by MALLS
Table 4
25°C Stability of Streptococcus pneumoniae serotype
polysaccharide- CRM 197 conjugates

Serotype % Free Saccharide Mw

Initial 1M 2M 3M initial 1M 2M 3M
Type-5 3.1 5.7 7.3 8.9 3981 3361 3798 3838
Type-GA 5.32 14.88 17-42 23.72 2314 2000 1725 1236
Type-6B 0.89 0.73 0.52 0.87 5865 5786 6142 5110
Type-7F 1.16 1.15 0.78 1.13 5817 5982 6417 5923
Type-9V ND ND ND ND 6563 6724 7235 6993
Type-19A 0.59 4.93 6.33 10.21 7760 6626 6843 5928
Type-19F ND 0.85 1.74 1.87 6257 6302 6020 6127
Type-23F 0.79 0.80 1.24 1.94 5481 6052 5988 6216
Mw: Average Molecular Weight estimated by MALLS

Thus, the data presented in Table 3 and Table 4 demonstrate that the Streptococcus pneumoniae serotype (5, 6A , 6B, 7F ,9V,19A , 19F & 23F) polysaccharide-CRMl97 conjugate bulks comprising 2OmM His tidine and 154 mM NaC1 were significantly stabilized at 2-8°C and 25°C,wherein the free polysaccharide level was found to be less than 10% with a minimum change in average molecular weight post-storage.
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 formulation which stabilizes a polysaccharide-carrier protein bulk conjugate, the formulation comprising
(i) atleast one sodium salt;
(ii) atleast one amino acid having pKa of 6.5;
(iii) one or more polysaccharide-carrier protein conjugates comprising one or more pneumococcal polysaccharides;and
wherein the free polysaccharide content is less than 5% when bulk conjugates are stored at a temperature between' 2 to 2 5°C for atleast 2 months.

2. The formulation of claim 1, wherein the sodium salt is sodium phosphate or sodium chloride at a final concentration of 1 mM to 160 mM.
3. The formulation of claim 2,wherein the sodium salt is sodium chloride at a final concentration of 154 mM.
4. The formulation of claim 1,wherein the amino acid is selected from group of histidine,glycine and lysine at a final concentration of 1 mM to 20 mM.
5. The formulation of claim 4,wherein the amino acid is histidine at a final concentration of 20 mM.
6. The formulation of claim 1 , wherein said capsular polysaccharide is of a serotype selected from the group consisting of 1 , 2, 3, 4, 5,6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F.
7. The formulation of claim 6 , wherein said capsular
polysaccharide is of a serotype selected from the group
consisting of 1 , 4, 5, 6A, 6B, 9V, 14,19A , 19F and 23F.
8. The formulation of claim 1, wherein the carrier protein
is selected from the group of CRM197,diphtheria
toxoid,tetanus toxoid, Haemophilus influenzae protein D,
outer membrane complex c (OMPC),E. coli heat labile toxoid
(LT) , pneumolysin, pneumococcal surface protein A (PspA)
and pneumococcal adhesin protein (PsaA).

9. The formulation of claim 8, wherein the carrier protein
is CRM197.
10. The formulation of claim 1, wherein the formulation
further comprises an adjuvant.
11.The formulation of claim 10, wherein the adjuvant is an aluminium-based adjuvant.
12.The formulation of claim 11, wherein the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide.
13. The formulation of claim 12,wherein the adjuvant is aluminum phosphate.