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 BACTERIAL CAPSULAR POLYOSES"
Serum Institute of India Ltd., an Institute organised and existing under the laws of India, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028, Maharashtra, India,
The following specification particularly describes the invention and the manner in which it is to be performed:

Title
Novel method for stabilization of bacterial capsular polyoses
Background of the invention
Streptococcus pneumoniae is a leading cause of meningitis, pneumonia, and severe invasive disease in infants and young children throughout the world. The multivalent pneumococcal polysaccharide vaccines have been licensed for many years and have proved valuable in preventing pneumococcal disease in elderly adults and high-risk patients. However, infants and young children respond poorly to most pneumococcal polysaccharides. The 7-valent pneumococcal conjugate vaccine (7vPnC, Prevnar®) was the first of its kind demonstrated to be highly immunogenic and effective against invasive disease and otitis media in infants and young children. This vaccine is now approved in many countries around the world. Prevnar contains the capsular polysaccharides from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, each conjugated to a carrier protein designated CRM 197. Prevnar covers aooroximatelv 80-90%, 60-80%, and 40-80% of invasive pneumococcal disease (IPD) in the US, Europe, and other regions of the world, respectively.Surveillance data gathered in the years following Prevnar's introduction has clearly demonstrated a reduction of invasive pneumococcal disease in US infants as expected.
Surveillance of IPD conducted in US infants prior to the introduction of Prevnar demonstrated that a significant portion of disease due to serogroups 6 and 19 was due to the 6A (approximately one-third) and 19A (approximately one-fourth) serotypes . Pneumococcal invasive disease surveillance conducted in the US after licensure of Prevnar suggests that a large burden of disease is still attributable to serotypes 6A and 19A. Moreover, these two serotypes account for more cases of invasive disease than serotypes 1 , 3, 5, and 7F combined (8.2 vs. 3.3 cases/100,000 children 2 years and under). In addition,

serotypes 6A and 19A are associated with high rates of antibiotic resistance . While it is possible that serogroup cross-protection will result in a decline of serotype 6A and 19A disease as more children are immunized, there is evidence that suggested there will be a limit to the decline, and a significant burden of disease due to these serotypes will remain.
Given the relative burden and importance of invasive pneumococcal disease due to serotypes 1 , 3, 5, 6A, 7F and 19A,these serotypes were subsequently added to obtain Prevnar 13®(Wyeth) (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) formulation to increase coverage for invasive disease to >90% in the US and Europe, and as high as 70%-80% in Asia and Latin America.
Many of the pneumococcal capsular polysaccharides are sufficiently stable such that the integrity of the molecules are maintained throughout the process of conjugation of the polysaccharides. However Serotypes 6A, 6B, 19F particularly 19A are much more prone to degradation across conjugation process than other S. pneumoniae serotypes due to the presence of phosphodiester linkages between its repeating units^This provides an additional challenge for successfully addressing and controlling various parameters when scaling-up conjugation processes for the large-scale manufacture of conjugates containing such labile polysaccharides.
Capsular polysaccharides from serotypes 6B, 6A, 10A, 17F, 19A, 19F and 20 contain a phosphodiester bond that connects the repeating units in these polysaccharides (also referred -to as backbone phosphodiester bonds), and polysaccharides from serotypes 11A, 15B, 18C, and 23F contain a phosphodiester bond that links a side chain to their repeating units.The relative order of backbone phosphodiester bond instability due to base hydrolysis was 19A > 10A > 19F > 6B > 17F .Thus pneumococcal polysaccharide 6A, 19A was found to be the more labile, wherein the rate of hydrolysis increased at higher pH and

in the presence of divalent cation.Refer Pujar,Biopolymers 2005:77(6):378-9.
EP 0017322 claims addition of an amphoteric substance, for example glycine, alanine, lysine, valine and other amino acids; albumin, gelatin, peptone, casein and protein hydrolysates and other proteins to type-19 capsular polysaccharide before the lyophilization step, preferably at concentrations of 0.01 to 25.0% (V/V) resulting in a) stabilization of the labile type 19 polysaccharide during lyophilization and b) enhanced solubility of the polysaccharides thereby making them amenable to homogenization.Here the mechanism of the protection of the type 19 polysaccharide was unclear, however it was postulated that if the polysaccharide is being enzymatically degraded by residual autolytic enzymes, it is possible that the ionic interaction of the amphoteric substance stereo- chemically prevents the phosphodiesterase from attaching to the active site.Otherwise if the molecule were degraded because it contained labile bonds that cleaved under the stress of lyophilization (with water molecules being removed), it is possible that the amphoteric substance, by interacting with the labile bond, makes it more resistant to the action of lyophilization.
Synflorix™ (GSK)provides protection against 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F as well as cross protection against 19A & 6A.However such cross protection is found to provide insufficient protection against serotypes 19A.
Further Prevnar 13™(Wyeth) is the only vaccine approved that contains conjugates of polysaccharides from serotypes 6A, 6B, 19A, 19F in addition to 1, 3, 4, 5, 7F, 9V, 14, 18C and 23F.Thus Prevnar 13™ was expected to significantly expand coverage beyond that of Prevnar 7™(Wyeth) , and provide coverage for 6A and 19A that is not dependent on the limitations of serogroup cross-protection as observed in Synflorix™ (GSK) .However Prevnar 13™ conjugates containing 19 A & 19F polysaccharides are found to be less immunogenic as compared to remaining 11 conjugates in said

vaccine composition and thus could fail to provide adequate protection against both serotypes.Thus attempts to produce a multivalent pneumococcal conjugate vaccine that exhibits significant immunogenicity with respect to serotypes 19A as well as 19F have been unsuccessful.
Given the limitations of Prevnar 13™(Wyeth)& Synf lorix™ (GSK) with respect to immunogenicity of 19 A and 19F there remains a distinct need for stabilizing said polysaccharides across sizing, activation and subsequent conjugation processes respectively inorder to improve immunogenicity and stability of such conjugates.
Summary of Invention
The inventors have surprisingly found that low immunogenicity and stability of conjugates containing labile polysaccharides,particularly pneumococcal polysaccharide type 19A & 19F prepared by cyanylation conjugation chemistry can be attributed to a) substantial phosphodiester linkage damage during activation mediated by a cyanylation agent by-product(DMAP) subsequently resulting in polysaccharide-polysaccharide aggregation and greater than 15 % hydrolysis and b)use of sized polysaccharide having average molecular weight above 200KDa resulting in increase in free polysaccharide.
More specifically inventors have found that conjugates of 19A & 19F having improved immunogenicity and stability can be prepared by i) utilizing sized polysaccharide having average molecular weight between 130 KDa & 190 KDa , ii) minimizing 4-dimethylamino-pyridine mediated degradation of sized polysaccharide during activation and preventing subsequent polysaccharide-polysaccharide aggregation by using a polysaccharide to CDAP ratio of 1:1 for 19A & 1:0.8 for 19F at 22°C with a period of activation of 4 min for 19A and 10 min for 19F and iii) using a polysaccharide to protein ratio of 1:1.
Accordingly inventors have found that a conjugate of 19A and having improved immunogenicity and stability can be

prepared by i) utilizing sized polysaccharide having average molecular weight between 130 KDa & 190 KDa , ii) minimizing 4-dimethylamino-pyridine mediated degradation of sized polysaccharide during activation and preventing subsequent polysaccharide-polysaccharide aggregation by using a polysaccharide to CDAP ratio of 1:1 at 22°C with a period of activation of 4 min and iii) using a polysaccharide to protein ratio of 1:1.
Accordingly inventors have found that a conjugate of 19F and having improved immunogenicity and stability can be prepared by i) utilizing sized polysaccharide having average molecular weight between 130 KDa & 180 KDa , ii) minimizing 4-dimethylamino-pyridine mediated degradation of sized polysaccharide during activation and preventing subsequent polysaccharide-polysaccharide aggregation by using a polysaccharide to CDAP ratio of 1:0.8 at 22°C with a period of activation of 9 to 10 min and iii) using a polysaccharide to protein ratio of 1:1.
The modified ratios of polysaccharide to CDAP(1:1 for 19A and 1:0.8 for 19F)can a) prevent degradation and aggregation for both 19A and 19F PnPs b)as well as make process devoid of any diafiltration step generally used for removal of small molecules(including DMAP) , thereby resulting in minimum loss of conjugate.
The immunogenic composition of containing 19F conjugate prepared by improved conjugation method shows immunogenicity (in terms of values of GMT antibody titre and opsonophagocytic assay) for 19F was found to be i) better than that induced by Prevnar 13® vaccine ii) not significantly inferior to that induced by Synflori.x® vaccine.
The immunogenic composition of containing 19A conjugate
prepared by improved conjugation method shows
immunogenicity (in terms of values of GMT antibody titre
and opsonophagocytic assay) for 19A was found to be i)
more than cross-protection value induced by Synflorix®

vaccine ii) not significantly inferior to that induced by Prevnar 13® vaccine.
Detailed description
Present disclosure provides methods for preparing polysaccharide-protein conjugates containing 19A & 19F, wherein free polysaccharide content can be less than 1.7 % in final conjugate as well as when stored at 2 to 8 °C for atleast 3 months.
An important aspect of the instant invention is that conjugation of polysaccharide to carrier protein can be carried out using an improved CDAP conjugation method to minimize 4-dimethylamino-pyridine mediated degradation of sized polysaccharide during activation and also to prevent subsequent polysaccharide-polysaccharide aggregation, wherein such conjugation method modifications can involve i) using a polysaccharide to CDAP ratio of 1:1 at 22°C with a period of activation of 4 min and using a polysaccharide to protein ratio of 1:1 for 19A and iii) using a polysaccharide to CDAP ratio of 1:0.8 at 22°C with a period of activation of 9 to 10 min and a polysaccharide to protein ratio of 1:1 for 19F.
Although this conjugation process is described for use with the S. pneumoniae serotype 19A and 19 F polysaccharides, this process may also be used for serotypes that are structurally similar such as 6A and 6B, which also contain phosphodiester linkages between their repeat units.
The average polysaccharide size (e.g. M w)after mechanical sizing can be between 130 and 190KDa for 19A and between 130-180KDa for 19F.
The carrier protein can be selected from a group of but not limited to CRM 197,diphtheria toxoid,tetanus toxoid, fragment C of tetanus toxoid, pertussis toxoid,protein D of H. influenzae, 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 PhtD, 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),particularly CRM197.
Multivalent vaccine formulations containing 19A and 19F conjugates can comprise of i) atleast one pneumococcal conjugate selected from 19A,19F,6A,6B prepared by conjugation process of instant invention and ii) atleast one pneumococcal conjugate selected from 1, 2, 3, 4,5,7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C,22F, 23F, 33F and can be used to protect or treat a human susceptible to pneumococcal infection, by means of administering the vaccine via a systemic or mucosal route. These administrations can include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral/alimentary, respiratory or genitourinary tracts. In one embodiment, intranasal administration is used for the treatment of pneumonia or otitis media (as nasopharyngeal carriage of pneumococci can be more effectively prevented, thus attenuating infection at its earliest stage).
The present invention further provides a vaccine containing the immunogenic compositions of the invention and a pharmaceutically acceptable excipient.
The vaccines of the present invention may be stored in solution or lyophilized. wherein the lyophilized vaccine composition of the instant invention can be given as 1,5 or 10 dose formulation.
The vaccines of the present invention may be adjuvanted, particularly when intended for use in an elderly population

but also for use in infant populations. Suitable adjuvants include an aluminum salt such as aluminum hydroxide gel or aluminum phosphate or alum, but may also be a salt of calcium, magnesium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatized saccharides, or polyphosphazenes.
The vaccine composition of instant invention can comprise of a step of adding aluminium salt adjuvant at an amount of 100-125ug of Al+++per 0.5 ml.
Description of Figures:
Fig.l: 31P NMR spectrum for 19A native polysaccharide
Fig.2: 31P NMR spectrum for 19A sized polysaccharide at different conditions
Fig 3: SEC-HP-RI profile of sized 19A PnPs (178 kDa) before DMAP treatment
Fig. 4: SEC-HP-RI profile of sized 19A PnPs (70 kDa) after 24 h (A) and 14.5 kDa after 72 h (B) DMAP treatment-degradation profiles
Fig.5: SEC-HP-RI profiles of sized 19A PnPs (178 kDa; A) and time-dependent aggregation of CDAP-mediated activated polysaccharides (5B, 5C & 5D) , 5E (conjugate without 10 kDa DF) ,5F (conjugate with 10 kDa DF),5G-sized 19A/5H-Activated 19A(modified Ps:CDAP ratio 1:1 for 19A)conjugation method without using 10 kDa diafiltration step.
Fig. 6: SEC-HP-RI profiles of sized 19F PnPs (157 kDa; A) and time-dependent aggregation of CDAP-mediated activated polysaccharides (B) , 6C/6D(modified Ps:CDAP ratio 1:1 for 19F)conjugation method without using 10 kDa diafiltration step

Fig.7: Overlay of 31P NMR spectrum of the sized/modified 19A PnPs and 19A conjugate(Conjugate prepared by CDAP conjugation method)
Fig. 8: 31P NMR spectra of sized/modified 19F polysaccharide and conjugate of 19F(Conjugate prepared by CDAP conjugation method)
Fig. 9: 31P NMR spectra of sized/modified 19A polysaccharide and conjugate of 19A (Conjugate prepared by improved /modified CDAP conjugation method)
Fig. 10: 31P NMR spectra of sized/modified 19F polysaccharide and conjugate of 19F (Conjugate prepared by improved /modified CDAP conjugation method)
Examples: Example 1 Sizing of Pneumococcal polysaccharides(PnPs):19A & 19F
A homogenizer (Microfluidics) apparatus was used to reduce the molecular weight of the polysaccharide before the activation step.For 19A size reduction was done at 24-28 Kpsi, whereas for 19F size reduction was done at 26-30 Kpsi wherein the number of passes were about 1 to 3.The size polysaccharide was diafiltered and concentrated followed by 0.22 u filtration.The sized polysaccharide was then subjected to HPSEC-RI for estimation of average molecular weight.
Example 2
Study of effect of "Average Molecular Weight of sized polysaccharide subjected to activation by CDAP" on "free polysaccharide content in final conjugate/on storage": Table 1: Effect of Size of PnPs 19A during activation on % free PnPs 19A.

Sr.No. Size of % free 19A % free 19A % free 19A
Initial PnPs at the PnPs in PnPs in
modified 19A time of conjugates conjugates

PnPs (kDa) release of conjugates after
months
2-8°C two
at after
months
2-8°C 3 at
1 150-170 1.5 1.62 1.7
2 210-240 1.7 2.5 __
3 260-290 2 4.55 9.75
4 310-340 2.4 7.87
5 540 3.77 11.56
Results: It was observed that at the time of release all conjugates containing PnPsl9A & PnPsl9F were having less than 3% free polysaccharide. However with time, the free polysaccharide in polysaccharide-CRMi97 conjugates was found to increase with respect to its initial % free PnPs.
Example 3
Study of effect of sizing reaction conditions on stability
of PnPs 19A
The sized 19A PnPs was found to be stabilized at a pH ranging from 4.8 -7 in WFI as well as sodium acetate buffer at pH 6.25 ± 0.5 and the integrity of sized 19A PnPs at different conditions were checked by 1H proton as well as 31P NMR analysis.Refer Figures 1 & 2.
Example 4
I) Degradation of sized PnPs (19A,19F,6A and 6B)in presence
of DMAP:
Sized PnPs in reaction solution was treated with DMAP in a ratio of 1:1.5 and checked for its degradation profile by SEC-HP-RI. Results:
It was observed that only 19A PnPs undergoes degradation in presence of DMAP whereas other phosphodiester containing PnPs (19F, 6A & 6B) remain intact. Refer Figures 3 (without DMAP) & 4 which show the DMAP-mediated degradation of PnPs. To minimize such degradation of 19A , activated PnPs was subjected to 10 KDa diafiltration using 2M NaCl to remove the DMAP formed from reaction solution before conjugation with CRM197.

II) Degradation and aggregation of sized PnPs (19A,19F,6A
and 6B)in presence of CDAP:
Sized PnPs in reaction solution was treated with CDAP in the ratio of 1:1.5 during activation.It was observed that 50% DMAP is generated as by-product (measured by RP-HPLC)which leads to degradation of PnPs as well as aggregation between activated PnPs after certain time of activation (refer Table 2). Degradation and aggregation was checked by ,SEC-HP-RI profile. Refer Figures 5A (without CDAP), 5B, 5C & 5D (for 19A) & 6A (without CDAP) 6B (for 19F) which show the CDAP-mediated aggregation & degradation of PnPs.
III) Prevention of degradation and aggregation of sized
PnPs (19A and 19F) having PnPs: CDAP of 1:1.5 by employing
diafiltration step:
To minimize such degradation and aggregation of 19A, activated PnPs was subjected to 10 KDa diafiltration using 2M NaCl to remove the DMAP formed from reaction solution before conjugation with CRM197. Refer Figures 5E (conjugate without 10 kDa DF) & 5F (conjugate with 10 kDa DF) which shows the SEC-HP-RI profile of conjugate using 10 kDa-diafiltered activated PnPs and CRM197.However diafiltration step was adversely affecting conjugate yield resulting in a 30%-40% decrease in overall yield.
IV) Prevention of degradation and aggregation of sized PnPs
(19A and 19F) by reducing ratio of PnPs:CDAP to 1:1 (19A)
and 1:0.8 (19F) without employing diafiltration step
Sized PnPs in reaction solution was treated with CDAP in the ratios of 1:1 and 1:0.8 for 19A and 19F respectively and checked for its degradation and aggregation profile by SEC-HP-RI. Results:
It was observed that modified Ps:CDAP ratios(1:1 for 19A and 1:0.8 for 19F)were found to prevent degradation and aggregation for both 19A and 19F PnPs. Refer Figures 5 (G & H for 19A) & 6 (C & D for 19F) .An additional advantage of

using modified ratios was that it was devoid of a 10 kDa
diafiltration step thus ensuring minimum loss in overall
yield.
Example 5:
Factors responsible for polysaccharide-polysaccharide cross
linking & aggregation
Table 2:Effect of "duration of CDAP activation" on
formation of "polysaccharide-polysaccharide aggregates"

Mw of Activated
Serotypes sized/modified PnPs (kDa) modified PnPs (kDa) [SEC-HP- Effects
[SEC-HP-RI] RI]
6A 432 435 No aggregation between activated polysaccharides
6B 131 131 No aggregation between activated polysaccharides
254 (after 20 Aggregation
min) between activated polysaccharides

487 (after 60 Aggregation
19A 178 min) between activated polysaccharides

1173 (after Aggregation
120 min) between activated polysaccharides
19F 157 205 (after 30 Aggregation
min) between activated polysaccharides
Results:
It was observed that in case of serotype 19 A, when " duration of CDAP activation" was more than 10 min,it was resulting in cross linking of activated polysaccharide to activated polysaccharide ultimately leading to formation of "polysaccharide-polysaccharide aggregates" .
Further in case of serotype 19 F, when " duration of CDAP activation" was more than 20 min,it was resulting in cross

linking of activated polysaccharide to activated polysaccharide ultimately leading to formation of "polysaccharide-polysaccharide aggregates" .Further the duration of conjugation reaction was found to be more than the duration required for cross linking of activated polysaccharide to activated polysaccharide thereby resulting in formation of aggregates.Refer Figures 5 & 6.
However for other serotypes like 6APnPs and 6BPnPs, such cross linking was not observed.
Example 6
Role of "Ps:CDAP:CRM ratio" in preventing formation of
"polysaccharide-polysaccharide aggregates" and thereby on
"immunogenicity"
For 19A when the ratios of Ps:CDAP:CRM was 1:1.5:1.5,30% hydrolysis of phosphodiester linkage(degradation into phosphomonoester) was confirmed by NMR such that the immunogenicity of conjugate was also found to be lower than Prevnar 13 , a licensed pneumococcal conjugate vaccine. Refer Figure 7.
For 19F when the ratios of Ps:CDAP:CRM was 1:1.5:1.5,18% hydrolysis of phosphodiester linkage(degradation into phosphomonoester) was confirmed by NMR such that the immunogenicity of conjugate was also found to be lower than Prevnar 13 , a licensed pneumococcal conjugate vaccine. Refer Figure 8.
Results:
It was found that for 19A & 19F , such aggregation between activated polysaccharides before conjugation with CRM197 and subsequent degradation can be prevented by utilizing an advantageous(polysaccharide specific)Ps:CDAP: CRM ratio.
Thus when ratio of Ps: CDAP: CRM ' was 1:1:1 then no hydrolysis of phosphodiester linkage was detected in 19A

conjugate and the rabbit immunogenicity was comparable with Prevnar 13.
Further when ratio of Ps: CDAP: CRM was 1:0.8:1 alongwith an "duration of CDAP activation" of about 9 to 10 mins, then no hydrolysis of phosphodiester linkage was detected in 19F conjugate and rabbit immunogenicity was better than Prevnar 13.Refer Figures 9 & 10.
Example 7
Preparation of conjugates: PnPsl9A & PnPsl9F
Conjugation of polysaccharide to carrier protein was carried out using CDAP conjugation method of Lees et al (Vaccine 26: 190-198, 1996) with following modifications:
i) For preparing 19A conjugate, using a polysaccharide to CDAP ratio of 1:1 at 22°C with a period of activation of 4 min and using a polysaccharide to protein ratio of 1:1
ii)For preparing 19F conjugate, using a polysaccharide to CDAP ratio of 1:0.8 at 22°C with a period of activation of 9 to 10 min and a polysaccharide to protein ratio of 1:1 for 19F.
Table 3:
Comparison of conjugate results for 19A and 19F with "traditional CDAP conjugation method"(Lot 1) and "improved conjugation method" (Lot 2)

I)Conjugation Reaction details
Conjugates 19A conjugate 19F conjugate

Batch Number
Lot 1 Lot 2 Lot 1 Lot 2
PnPs Conc. (mg/ml) 9.5 9.5 9.5 9.5
PnPs
dissolution 2M NaCl 2M NaCl 2M NaCl 2M NaCl
Activation time (min) 4 4 4 9 to 10
Ratio PnPs/CDAP 1.0:1.5 1.0:1.0 1.0:1.5 1.0:0.8
CRM Cone. (mg/ml) 20 20 20 20
Initial Ratio Ps/CRM197 1.0:1.5 1.0:1.0 1.0:1.5 1.0:1.0
pHa:pHc:pHq 9.0:9.0:9.0 9.0:9.0:9.0 9.5:9.5:9.5 9.5:9.5:9.5

II) Final Conjugate Characteristics
Conjugates 19A con jugate 19F conjugate
Batch Number Lot 1 Lot 2 Lot 1 Lot 2
Final Ratio PnPs/CRM!97 0.53 0.86 0.52 0.96
CRMi97/PnPs 1.88 1.16 1.92 1.04
Free PnPs (%) 1.9 1.5 1.7 1.07
Free CRM197 (%) ND ND ND 2.2
Avg. Mol.
size (SEC-HP-
RI) (kDa) 1081 853 994 847
Avg.Mol. size
(UV/RI/MALS)
(kDa) 8212 6012 6393 4773
Results:
It was observed that modified Polysaccharide:CDAP ratio,CDAP activation time and initial polysaccharide:protein ratio, were found to minimize 4-dimethylamino-pyridine mediated degradation of sized polysaccharide during activation and also prevented subsequent polysaccharide-polysaccharide aggregation

thereby improving final conjugate characteristics with' respect to free polysaccharide content.
The improved conjugation method employed for preparing 19 A & 19F conjugates resulted in conjugates that did not show any phosphomonoester signal in respective conjugate profiles (31P Proton NMR)which indicated that modified conjugation method was found to be effective in preventing hydrolysis of polysaccharides across conjugation reactions.
Example 8
Preparation of remaining conjugates of PnPs
l,4,5,6A,6B,9V,14,and 23F
Conjugation of polysaccharide to carrier protein was carried out using CDAP conjugation method of Lees et al (Vaccine 26: 190-198, 1996).Mechanically size reduced polysaccharides (except for 6A which was used in native form or sized depending on size of 6A) were dissolved in NaCl 2M. CDAP(in acetonitrile)from a lOOmg/ml stock solution was added to the polysaccharide solution as per polysaccharide:CDAP ratio . Approximate 1 minute later , 2M NaOH was added to obtain the specific activation pH .The activation of the polysaccharide was performed at this pH during 4-10 minutes at 22°C.CRM 197 (the quantity depend on initial Ps/Protein ratio) was added to the activated polysaccharide and the coupling reaction was performed at the specific. pH for 3-8 hr depending on serotype. The reaction was then quenched with glycine for 1 hr at 22°C, and overnight at 12°C. The conjugates were then purified by 300kDa diafiltration followed by lOOkDa diafiltration. Further the polysaccharide and protein content of the purified 0.22um filtered conjugates were determined.
Example 9: Formulation of a 10 valent Pneumococcal Conjugate Vaccine
The 10-valent candidate vaccine includes the capsular polysaccharides from serotypes 1,5,6A,6B,7F,9V,14,19A, 19F and 23F. The conjugates were prepared as per disclosures of

Examples 1 to 8. Particularly 19A & 19F conjugates were prepared as per the improved conjugation method disclosed in Table 3.All the conjugates were diafiltered against 0.9% NaCl with 20mM L-Histidine pH 7.2 & concentration was made Img/ml with 0.9% NaCl.Further said conjugates were stored at 2 - 8 °C. The formulations were prepared in formulation buffer with aluminium phosphate gel (125 ug of aluminium content/dose).
Example 10: 19A & 19F conjugates-Immunogenic!ty study in rabbits
Two different formulations were prepared for this immunization programme. In one formulation, serotype 19A and 19F used were having PS:CDAP:CRM ratio (1:1.5:1.5) for conjugation and in second formulation, serotype 19A and 19F used were having PS:CDAP:CRM ratio (1:1:1 & 1:0.8:1) for conjugation. Both the formulations were prepared in formulation buffer with aluminium phosphate gel (125 ug of aluminium content/dose).
In the formulation having serotypes 19A and 19F [PS:CDAP:CRM ratio (1:1.5:1.5)], 6 rabbits were immunized (3 males and 3 females).The immunization schedule for the same formulation was kept on day 0 and day 14th (intramuscular injection)and bleeding was on 0 day and 28th day.Intramuscular injection were administered with 0 day and 28th day bleeding. 0.5ml of SHD (single human dose) was injected as per immunization schedule. Serum was separated from respective blood samples and sera samples were analyzed for total IgG estimation by multiplex bead based assay and OPA (opsonophagocytic assay) for functional antibody estimation.
In the formulation having serotypes 19A and 19F [PS:CDAP:CRM ratio 1:1:1 for 19A & 1:0.8:1 for 19F], 8 rabbits were immunized (4 males and 4 females).The immunization schedule for the same formulation was kept on day 0, day 14th and day 28th (intramuscular injection) and bleeding was on 0 day, 28th day and 42nd day. Intramuscular

injection were administered with 0 day, 28th day and 42nd day bleeding. 0.5ml of SHD (single human dose) was injected as per immunization schedule. Serum was separated from respective blood samples and sera samples were analyzed for total IgG estimation by multiplex bead based assay and OPA (opsonophagocytic assay) for functional antibody estimation.
Prevnarl3® vaccine was immunized in 2 rabbits (1 male and 1 female). The immunization schedule for the same formulation was 0 day and 14th day (intramuscular injection) and bleeding was on 0 day and 28th day. 0.5ml of SHD (single human dose) as mentioned in product insert was injected as per immunization schedule. Serum was separated from respective blood samples and sera samples were given for total IgG estimation by multiplex bead based assay and OPA (opsonophagocytic assay) for functional antibody estimation.Similar schedule was followed for Synflorix®.
Table 4:
Rabbit immunogenicity results of conjugates for 19A and 19F prepared by
I)"General CDAP conjugation method"
II)" Modified/Improved CDAP conjugation method"
III) Prevnar 13® and IV) Synflorix® -2 doses injection schedule

Pre- Pre-clinical
clinical Immunogenicity data
Serotypes Immunogenicity data OPA*- 28 days
IgG (GMT)- 28 days

19A (PnPs : CDAP : CRM197
4525 46
ratio 1:1.5:1.5)
General CDAP conjugation
method
19A (PnPs :CDAP:CRM197 4470 181
ratio 1:1:1)
Modified/Improved CDAP
conjugation method
19 F(PnPs :CDAP:CRM197 2770 44
ratio 1:1.5:1.5)
General CDAP conjugation
method
19F (PnPs: CDAP :CRM197 12721 226
ratio 1:0.8:1)
Modified/Improved CDAP
conjugation method
Prevnarl3® 19A 6477 298
Prevnarl3® 19F 7186 135
Synflorix® 19A(as cross 3062 14
protection from 19F)
Synflorix® 19F** 1 7 325 320
* OpsonoPhagocytic Assay ** 3yig/0.5ml SHD or 6)jg/lml
Table 5:
Rabbit immunogenicity results of conjugates for 19A and 19F
prepared by
I)"General CDAP conjugation method"
II)" Modified/Improved CDAP conjugation method"
III) Prevnar 13® and IV) Synflorix®: 3 doses injection
schedule

Pre- Pre- Pre- Pre-clinical
clinical clinical clinical Immunogenicity
Serotypes Immunogenicit y data Immunogenic ity data Immunogenic ity data data
OPA*- 42 days
IgG (GMT)- 28 IgG (GMT)- OPA*-
days 42 days 28 days

19A

(PnPs:CDAP:CRM
197 ratio
1:1.5:1.5) 4525 6041 46 109
General CDAP
conjugation
method
19A
(PnPs:CDAP:
CRM197 ratio
1:1:1) Modified / 5080 8063 294 345
Improved CDAP
conjugation
method

19 F(PnPs
:CDAP:CRMi97
ratio
1:1.5:1.5) 2770 6588 44 111
General CDAP
conjugation
method
19F
(PnPs:CDAP:
CKM197 ratio
1:0.8:1) Modified / 13958 46951 260 431
Improved CDAP
conjugation
method
Prevnarl3® 19A 9755 11593 341 241
Prevnarl3® 19F 9847 10500 178 86
*OpsonoPhagocytic Assay
Results:
Comparison of immunogenicity of 19A & 19F conjugates prepared by improved conjugation method of instant

invention with immunogenicity of 19A & 19F conjugates of Prevnarl3®:
IgG and OPA titers for serotypes 19A and 19F in formulation having PS:CDAP:CRM ratio (1:1:1 & 1:0.8:1) were found to be i)better titres than titres obtained for Prevnarl3© for 19F and ii) comparable titres to that of titres obtained by Prevnarl3 for 19A.
Whereas in case of formulation having serotypes 19A and 19F
with PS:CDAP:CRM ratio (1:1.5:1.5), IgG and OPA titres were
found to be on a lower side for both 19A & 19F as compared
to Prevnarl3®.
Comparison of immunogenicity of 19A & 19F conjugates prepared by improved conjugation method of instant invention with immunogenicity of 19A & 19F conjugates of Synflorix®:
IgG and OPA titers for serotype 19F in formulation having [PS:CDAP:CRM ratio (1:0.8:1)] was found to be comparable to that of titers obtained by Synflorix® .
Further IgG and OPA titers for serotype 19A in formulation having [PS:CDAP:CRM ratio (1:1:1)] was found to be more than that of titers obtained by Synflorix® for 19A through cross-protection from 19F.
Whereas in case of formulation having [PS:CDAP:CRM ratio (1:1.5:1.5)], IgG and OPA titers for serotype 19F were found to be on a lower-side as compared to Synflorix®.
Conclusions:
The molecular weight of sized 19A polysaccharide used for CDAP activation can have an effect on the amount of % free 19A PnPs in final conjugate as well as on storage. Thus the free polysaccharide content was found to be less than 1.7% when 19A polysaccharide having molecular weight between 150-170 Kda was used for activation.

The stability of polysaccharide (with respect to integrity of phosphodiester linkages) was not affected by sizing step or variation in pH conditions.
Only 19A polysaccharide was found to undergo degradation in presence of CDAP by-product (DMAP) whereas other phosphodiester containing PnPs (19F, 6A & 6B) were unaffected.
For 19A & 19F "Polysaccharide-polysaccharide cross linking /aggregation" was found to be dependent on "Duration of CDAP activation" .Whereas for other phosphodiester containing PnPs (6A & 6B) such cross-linking does not occur.
The modified conjugation method with following modifications:i)Using polysaccharide to CDAP ratio of 1:1 for 19A & 1:0.8 for 19F at 22°C with a period of activation of 4 min for 19A and 10 min for 19F and ii) using a polysaccharide to protein ratio of 1:1 for both 19A and 19F was found to minimize 4-dimethylamino-pyridin mediated degradation of sized polysaccharide during activation and also prevented subsequent polysaccharide-polysaccharide aggregation.
The modified ratios of polysaccharide to CDAP(1:1 for 19A and 1:0.8 for 19F)were found to prevent degradation and aggregation for both 19A and 19F PnPs as well as make process devoid of any diafiltration step thereby resulting in minimum loss of conjugate.
The 19A conjugates prepared by improved conjugation method were found to provide immunogenicity i)comparable to Prevnar 13® & ii) more than the cross-protection provided for 19A by Synflorix®.
The 19F conjugates prepared by improved conjugation method were found to provide immunogenicity i)better than Prevnar 13® & ii) comparable to Synflorix®.

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 method for making a conjugate with improved
immunogenicity and stability comprising a Streptococcus
pneumoniae polysaccharide containing a phosphodiester
linkage between repeat units covalently linked to a carrier
protein using cyanylation chemistry, the method comprising:
reacting polysaccharide having average size between 130 KDa and 190 KDa with a cyanylation agent in a ratio between 1:0.8 and 1:1 at about 22°C to about 25°C for a period of about 4 min to about 10 min, resulting in a cyanate-activated polysaccharide;and
contacting cyanate-activated polysaccharide with protein in a ratio of 1:1 at a pH of about 9 to about 9.5 for a period of 3hr to 5 hr followed by quenching with glycine,
wherein said conjugation process minimizes cyanylation agent by-product mediated degradation of sized polysaccharide and prevents subsequent polysaccharide-polysaccharide aggregation resulting in a conjugate having minimum free polysaccharide content.
2. The method of claim 1, wherein said conjugate has free
polysaccharide content less than 1.6 %.
3. The method of claim 1, wherein said polysaccharide
comprising a phosphodiester linkage between repeat units is
Streptococcus pneumoniae polysaccharide serotype 19A, 19F,
6A, or 6B.
4. The method of claim 1, wherein the protein carrier is selected from the group consisting of: CRM197, TT, DT, fragment C of TT, protein D of H. influenzae, pneumococcal PhtD, and pneumococcal Pneumolysin.
5. The method of claim 1, wherein the cyanylation agent comprisesl-cyano-4-dimethylaminopyridinium
tetrafluoroborate (CDAP).

6. The method of claim 1, wherein the cyanylation agent by-
product comprises 4-dimethylamino-pyridine.
7. The method of claim 1 , wherein the said conjugation
method does not require any diafiltration step for removal
of reaction by-products.
8. A method for making a conjugate with improved
immunogenicity and stability comprising a Streptococcus
pneumoniae serotype 19A polysaccharide covalently linked to
a carrier protein using cyanylation chemistry, the method
comprising:
reacting polysaccharide having average size between 130 KDa and 190 KDa with 1- cyano-4- dimethylammoniumpyridinium tetraf luoroborate in a ratio of 1:1 at 22°C for a period of 4 min, resulting in a cyanate-activated polysaccharide;and
contacting cyanate-activated polysaccharide with protein in a ratio of 1:1 at a pH of about 9 to about 9.5 for a period of 3hr to 5 hr followed by quenching with glycine,
wherein said conjugation process minimizes 4-dimethylamino-pyridine mediated degradation of sized polysaccharide and prevents subsequent polysaccharide-polysaccharide aggregation resulting in a conjugate having minimum free polysaccharide content.
9. The method of claim 8, wherein said conjugate has free ■ polysaccharide content less than 1.6 %.
10. The method of claim 8 , wherein the carrier protein is CRM 197.
11. The method of claim 8, wherein the said conjugation method does not require any diafiltration step for removal of reaction by-products.

12. A method for making a conjugate with improved
immunogenicity and stability comprising a Streptococcus
pneumoniae serotype 19F polysaccharide covalently linked to
a carrier protein using cyanylation chemistry, the method
comprising:
reacting polysaccharide having average size between 130 KDa and 180 KDa with 1- cyano-4- dimethylammoniumpyridinium tetraf luoroborate in a ratio of 1: 0.8 at 22°C for a period of 9 to 10 min, resulting in a cyanate-activated polysaccharide; and
contacting cyanate-activated polysaccharide with protein in a ratio of 1:1 at a pH of about 9 to about 9.5 for a period of 3hr to 5 hr followed by quenching with glycine,
wherein said conjugation process minimizes 4-dimethylamino-pyridine mediated degradation of sized polysaccharide and prevents subsequent polysaccharide-polysaccharide aggregation resulting in a conjugate having minimum free polysaccharide content .
13. The method of claim 12, wherein said conjugate has
free polysaccharide content less than 1.6 %.
14. The method of claim 12 , wherein the carrier protein is CRM i97.
15. The method of claim 1 , wherein the said conjugation method does not require any diafiltration step for removal of reaction by-products.
16. An immunogenic composition comprising at least 2
different S.pneumoniae polysaccharide-protein conjugates,
wherein i) atleast one S.pneumoniae polysaccharide-protein
conjugate is prepared by conjugation methods of claim 1 8
or 12 comprising of polysaccharides selected from 19A, 19F
, 6A and 6B, and ii) atleast one different S.pneumoniae

polysaccharide-protein conjugate - comprising of polysaccharides selected from 1, 2, 3, 4,5,7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 22F, 23F, 33F .
17. An immunogenic composition according to claim 16 comprising at least 2 different S.pneumoniae polysaccharide-protein conjugates, wherein i) atleast one S.pneumoniae polysaccharide-protein conjugate is prepared by conjugation methods of claim 1 8 or 12 comprising of polysaccharides selected from 19A, 19F, 6A and 6B , and ii) atleast one different S.pneumoniae polysaccharide-protein conjugate comprising of polysaccharides selected from 1,4,5,9V,14,and 23F.
18. An immunogenic composition according to claim 17 comprising 10 different S.pneumoniae polysaccharide-protein conjugates, wherein i) S.pneumoniae polysaccharide-protein conjugates comprising of polysaccharides 19A and 19F prepared by conjugation methods of claim 1, 8 or 12 , and ii) S.pneumoniae polysaccharide-protein conjugate comprising of polysaccharides from 1,4,5,6A,6B,9V,14,and 23F.
19. An immunogenic composition comprising one or more immunogenic conjugates of any one of claims 16 to 18 and a pharmaceutically acceptable carrier.
20. An immunogenic composition of claim 19, further comprising an adjuvant.
21. An immunogenic composition according to claim
20,comprising a step of adding aluminium salt adjuvant at
an amount of 100-125ug of Al+++ per 0.5 ml.
22.The immunogenic composition of claims 1-21,wherein the immunogenicity in terms of values of GMT antibody titre and opsonophagocytic assay for 19F is i) better than that

induced by Prevnar 13® vaccine ii) not significantly inferior to that induced by Synflorix® vaccine.
23. The immunogenic composition of claims 1-21,wherein the immunogenicity in terms of values of GMT antibody titre and opsonophagocytic assay for 19A is i) more than cross-protection value induced by Synflorix® vaccine ii) not significantly inferior to that induced by Prevnar 13® vaccine.
24. An immunogenic composition according to any preceding claim wherein the polysaccharide conjugates are in a buffered liquid form.
25. An immunogenic composition according to any preceding claim wherein the polysaccharide conjugates are in lyophilized form.