FORM-2
THE PATENTS ACT, 1970
(39 OF 1970)
AND
THE PATENTS RULES, 2003
(As Amended)
COMPLETE SPECIFICATION
(See section 10; rule 13)
"A Novel Quantification Method of Vaccines"
Serum Institute of India Ltd., an Institute organized 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 nature of this invention and the manner in which it is to be performed:

BACKGROUND OF THE INVENTION
Isolated bacterial polysaccharides from Haemophilus influenzae
serotype b, Streptococcus pneumoniae, Neisseria meningitidis have been successfully used as vaccines in adults.However, vaccines based on isolated polysaccharides are ineffective in young infants due to the T-helper cell independent (TI antigens) character of the Polysaccharide(PS) antigens.
Polysaccharides are T-independent antigens, and can not be processed or presented on MHC molecules to interact with T-cells and hence are poorly immunogenic. After they are coupled to proteins, however, the resulting construct becomes immunogenic. The conjugation of proteins to polysaccharides converts the polysaccharide from a weakly immunogenic T-cell independent antigen to a T-cell dependent antigen that recruits T-cell help, . and thus stimulates heightened immune responses. Note the discussion by J. M. Cruse, et al. (Editors), Conjugate Vaccines, Karger, Basel, (1989); and R. W. Ellis, et al. (Editors), Development and Clinical Uses of Haemophilus B Conjugate Vaccines, Marcel Dekker, New York (1994).
To produce a conjugate vaccine, the purified PS must first be chemically modified to generate reactive groups that can link to the protein.The PS should be of known molecular size before and after activation.Further there is a requirement to estimate the quantity of free polysaccharide which might be available in the monovalent bulk conjugate(s) or generated during the

vaccine formulation storage as a product of conjugate degradation over a period of time.Two critical measures after conjugation and purification are the PS to protein ratio and the percent non-conjugated saccharide (free saccharide). Typically, considerably less than 20% of the activated PS becomes conjugated. Thus two major measures used to follow vaccine stability are changes in molecular size and percent free (unbound) PS.
Current FDA specifications do not require that the free protein be removed from conjugate vaccines, but do set the allowable amount of free polysaccharide. Free polysaccharide, especially when greater than 10% of the total, has been shown to inhibit the humoral immune response to the conjugated polysaccharide. Peeters. C. et al. Vaccine, 10:833, 1992.
Also the purified protein-polysaccharide bulks are characterized for their molecular properties by routine QC methods. These routine QC tests include structural analyses such as: molecular size distribution, total protein content, total polysaccharide content, and use of said contents for determination of PS/carrier ratio, free PS content and free carrier content.
Guo, Yu-Yi et al (1998) discusses a simple and rapid method for quantitation of free capsular polysaccharide of Haemophilus influenza type b (polyribosyl ribitol phosphate, PRP) in

PRP-tetanus toxoid conjugate vaccine based on acid precipitation of tetanus toxoid (TT). Acid hydrolysis of PRP during the assay was not detected. The conditions used in the assay did not precipitate unconjugated PRP or adipic acid dihydrazide derivatized PRP.
Lei, Q. P et al(2000) discusses quantitation of low level unconjugated polysaccharide in tetanus toxoid-PRP conjugate vaccine by HPAEC/PAD(High performance anion exchange chromatography with pulsed amperometric detection) following rapid separation by deoxycholate-HCl.The unconjugated PRP was found to stay in the supernatant without' precipitation, while conjugated PRP-TT was fully precipitated. HPAEC-PAD has been applied for analysis of the PRP content in the supernatant after the separation. The said method has also been utilized for estimation of free and bound polysaccharide in conjugate vaccines prepared from Neisseria meningitidis serotypes A, C, W135 and Y.
The acid precipitation methods disclosed previously did not produce reproducible results due to incomplete precipitation of PRP-TT, especially when the vaccine is formulated in a salt-buffered solution. Also these methods are not time efficient for measuring free polysaccharide content in multivalent conjugate vaccines.

Yoo, Tae Hyeon et al (2003) discusses measurement of free polysaccharide in tetanus toxoid-conjugate vaccine using antibody/ammonium sulfate precipitation. The method involved adding anti-TT antibody and ammonium sulfate to precipitate PRP-TT conjugate and measuring free PRP in the supernatant. The said method has not been utilized for multivalent conjugate vaccine.Further the ammonium salt retained after protein precipitation can interfere in the assay. Salt could be removed by diafiltration ,thus requiring and additional step with definite losses.
Wu, Kai et al (2009) discloses a method for determination of free polysaccharide content in Streptococcus pneumoniae type 6B, 18C, 19F , 23F & Neisseria meningitidis group A , C . The samples of conjugate were pre-treated under various conditions to separate conjugated and free polysaccharides, then determined for total and free polysaccharide contents by an improved anthrone colorimetry, based on which the free polysaccharide content was calculated, and the developed method was verified. Compared with those pretreated under other conditions, both the separation rates of conjugated and free polysaccharides in the conjugate vaccine pre-treated with 5 mol/L sodium chloride and 85% ethanol and stored in frozen at -20°C for 72 h were high. The variation coeff. (CV) of separation rate of conjugated and free polysaccharides determined by the developed method was 1.7%, and that of

effectiveness was 5.6%.The CV value of free polysaccharide content in one batch of polysaccharide conjugate vaccine determined by the developed method was 4.2%.However serotype specific estimation of free polysaccharide in a multivalent conjugate vaccine cannot be measured by this method.
The methods cited above utilize acid or salt based precipitation and have been effective for monovalent conjugate bulks.
Previously rate nephelometry has been reported for measuring free polysaccharide content in multivalent conjugate.Rate nephelometry is an immunoassay based on rate of formation of immuno-precipitates.The said method however has a lower sensitivity which may not be suitable for determination of lowest amounts of free polysaccharides expected in a vaccine formulation especially pneumococcal conjugate vaccines having vary low quantity of each conjugate. Further the antibody volume required by the assay is significant and the assay has to be performed for each serotype separately.
Hence, a more efficient, alternative method is required to quantify free polysaccharide as well as total polysaccharide content in monovalent and multivalent conjugate vaccine.
Surprisingly we have found that when "anti-carrier protein antibody coupled beads" are added to a mixture containing

multivalent . protein-polysaccharide conjugates, free polysaccharide and free carrier protein, the antibody bound conjugates and free carrier protein get separated from free polysaccharides, wherein the free polysaccharide content in supernatant can be measured by a multiplex assay.
We have developed a novel system based on sepharose beads, which can be coupled with a anti-carrier protein antibody. The said antibody coupled beads can be mixed with sample containing multivalent protein polysaccharide conjugates to enable the separation of conjugated and unconjugated polysaccharide , thereby allowing estimation of free polysaccharide content from supernatant by multiplex competitive inhibition assay.Further total polysaccharide content and percent adsorption of polysaccharides can also be measured by instant method.
SUMMARY OF THE INVENTION
The invention relates to a simple,specific, reproducible, cost
effective and less time consuming method for quantifying free polysaccharide content in multivalent conjugate vaccines,wherein anti-carrier protein antibody coupled beads are mixed with sample containing multivalent protein polysaccharide conjugates to enable the separation of conjugated and unconjugated polysaccharide followed by estimation of free polysaccharide content from supernatant by multiplex competitive inhibition assay.The prior art methods

based on deoxycholate & HPAEC/PAD are only applicable to monovalent conjugates, whereas instant invention is particularly applicable for estimation of free polysaccharide from multivalent conjugate bulks.Additionally total polysaccharide content and percent adsorption of polysaccharides can also be measured by instant method.
DESCRIPTION OF THE DRAWINGS
Fig 1:Standard curve for estimation of type 19F serotype polysaccharide.
Fig 2:Standard curve for carrier protein CRM197.
Fig 3:Standard curve for estimation of type 5 serotype polysaccharide.
Fig 4:Standard curve for estimation of type 14 serotype polysaccharide.
DETAILED DESCRIPTION
The method of the instant invention for quantification of unconjugated polysaccharides in multivalent polysaccharide-protein conjugate vaccine compositions ,comprises following steps:

a)Covalently coupling microspheres to anti-carrier protein
antibody;
b)Dissolution of vaccine formulation followed by measuring of
the total serotype specific polysaccharide content by
multiplexed bead based competitive inhibition assay;
c)Adding the anti-carrier protein coupled microspheres to the
vaccine formulation containing multivalent
polysaccharide-protein conjugates, free polysaccharide and
free carrier protein;
d) Agitating the mixture of step (c) for a duration between ihr
and 4hr at 37°C;
e)Subjecting the mixture of step (d)to centrifugation at 3000
to 6000 rpm for a duration between 5 and 20 minutes;
f) Collecting the supernatant and measuring free polysaccharide
content by multiplexed bead based competitive inhibition
assay;and
g)Collecting the supernatant containing unadsorbed
polysaccharide-protein conjugates ,carrier protein and free
polysaccharide for measurement of percent adsorption by
multiplexed bead based competitive inhibition assay.
One preferred embodiment of the instant invention is that the
instant method is applicable for estimation of
i)free polysaccharide from multivalent polysaccharide-protein
conjugate vaccine formulation.
ii)total polysaccharide content and

iii)percent adsorption of polysaccharides.
One embodiment of the instant method is that said assay can
utilized for estimating free polysaccharide selected from gram
positive or gram-negative bacteria, wherein the said
polysaccharide can be with or without phosphodiester
bond.Polysaccharide can be selected from the group consisting
of Helicobacter pylori, Chlamydia pneumoniae. Chlamydia
trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae,
Staphylococcus spp., Staphylococcus aureus, Streptococcus spp.,
Streptococcus pyogenes, Streptococcus pneumoniae,
Streptococcus viridans, Enterococcusfaecalis, Neisseria
meningitidis, Neisseria gonorrhoeae, Bacillus anthracis,
Salmonella spp., Salmonella typhi, Vibrio cholerae,
Pasteurella pestis, Pseudomonas aeruginosa, Campylobacter spp .,
Campylobacter jejuni, Clostridium spp., Clostridium difficile,
Mycobacterium spp., Mycobacterium tuberculosis, Treponema spp.,
Borrelia spp., Borrelia burgdorferi, Leptospira spp.,
Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella
pertussis, Bordetella parapertussis, Bordetella
bronchiseptica, Hemophilus influenzae, Escherichia coli, Shigella spp., Erlichia spp., and Rickettsia spp.
Specifically, the instant method can be used for estimating free polysaccharide content in multivalent conjugates containing polysaccharides of Streptococcus pneumoniae type 1, 2, 3, 4,5,6A , 6B, IF, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C,

19F, 19A, 20, 22F, 23F, and 33F. N. meningitidis types A, B, C, D, W135 , X, Y , Z , 29E and ff. influenzae type b.
Microspheres, microparticles, microcapsules and beads, referred to herein collectively as "microspheres", are solid or semi-solid particles having a diameter of less than one millimeter, more preferably less than 100 microns, which can be formed of a variety of materials, including synthetic polymers, proteins, and polysaccharides, and can optionally have various surface chemistries selected from a group of free carboxylic acid, amine, or hydrazide groups, among many others.
Examples of suitable polymers for the formation of microspheres include polystyrenes, polyesters, polyethers, polyolefins, polyalkylene oxides, polyamides, polyacrylates, polymethacrylates, polyurethanes, celluloses, polyisoprenes, silica, and polysaccharides, particularly cross-linked polysaccharides, such agarose, which is available as Sepharose, dextran, available as Sephadex and Sephacryl, cellulose, starch, and the like. Preferably EAH-Sepharose and polystyrene based beads can be utilized.
According to the instant invention the anti-carrier protein antibody can be coupled to microsphere by carbodiimide conjugation chemistry based on EDC and S-NHS.

Another aspect of the instant invention is that total PS content and percent adsorption of the protein polysaccharide conjugates on the adjuvant can also be measured by carrying out multiplex analysis of the gel dissolved vaccine formulation or formulation supernatant, respectively.
Yet another aspect of the instant invention is that the said anti-protein antibody can be against a carrier protein selected from but not limited to DT,CRM197,TT,PD,FragmentC,Ply,PhtA, PhtB,PhtD,PhtE,OmpC and PorB.
In each of the embodiments of the invention, fluorescence may be measured through the use of a flow cytometer or a bead array reader. For example, a BioPlex-100, a BioPlex-200, a Luminex-100,a Luminex-200 or MagPix bead array reader may be used.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of

equivalency of the claims are/therefore intended to be embraced therein.
Examples:
Example 1:
Coupling of Anti-CRM197 antibody to polystyrene beads [Amine coupling kit PROCEDURE ,Bio-Rad]
The coupling procedure involves a two-step carbodiimide
reaction. The carboxyl groups on the surface of the polystyrene
beads must first be activated with a carbodiimide derivative
prior to coupling the anti-CRM197 antibody. EDC
(l-ethyl-3-[3-dimethylaminopropyl] carbodiimide
hydrochloride) reacts with carboxyl groups on the surface of the polystyrene beads to form an active O-acylisourea intermediate. This unstable intermediate is stabilized in aqueous solutions using S-NHS (A7-hydroxysulfosuccinimide) . EDC couples S-NHS to the carboxyl group resulting in an S-NHS activated site. Both the carbodiimide's, O-acylisourea intermediate and the S-NHS-ester formed are amine-reactive; however, S-NHS-ester has much greater stability at the physiological pH. These intermediates then reacts with primary amines of anti-CRM197 antibody to form amide bonds. The intermediate is hydrolyzed if it fails to react with an amine and the carboxyl is regenerated, releasing an N-unsubstituted urea.

The kit contents were brought to room temperature (RT) prior to their use. Beads were protected from light by covering the tubes with aluminium foil during the procedure. EDC and S-NHS were removed from -20°C deep freezer and refrigerator, respectively and kept at RT for approximately 10 min prior to their use.
a) The COOH beads were vortexed at high speed for approximately 1-2 min. 100 pi of properly vortexed beads was pipetted out from the vial in a 1.5 ml coupling reaction tube (approximately 1.25 x 106 beads) provided with the amine coupling kit and sonicated for 30 sec.
The mixture was centrifuged at 16500 x g for 5 min and supernatant was discarded .100 pi of bead wash buffer from the kit was added to bead pellet and vortex for 10 sec followed by sonication. Further the beads were centrifuged at 16500 x g for 5 min and , wherein the bead pellet was resuspended in 80 ul of bead activation buffer from the kit. The beads were vortexed for 30 second also sonicated for 30 sec.
100 ul each of 50 mg/ml of EDC and S-NHS were prepared by adding accurately weighed 5 mg each of EDC and S-NHS in 100 ul of bead activation buffer in two different tubes, immediately prior to their use.
10 ul of freshly made 50 mg/ml EDC was added in the bead suspension closely followed by addition of 10 pi of freshly made 50 mg/ml of. S-NHS. The beads were vortexed at high speed for

30 sec.The coupling reaction tube was covered with aluminium foil and kept on rotospin for 20 min at RT with 30 rpm for IX and 2X reactions. The tube was kept for 30 min on rotation while performing coupling of more than 2X scale reaction.
150 ul of PBS was added, pH 7.4 from the kit and the activated beads were vortexed at high speed for 10 sec followed by centrifugation at 16500 x g for 5 min and subsequently the supernatant was discarded. ,This step was repeated once.The activated beads were resuspended in 100 pi PBS and vortexed at medium speed for 30' sec followed by sonication for 15 sec.
a) The anti CRMl97-antibody sample (approximately 5 to 15 ug based on optimization experiments) was added to the tube of activated beads and the final volume was adjusted to 500 ul with PBS, pH 7.4. The covered tubes were were agitated on rotospin at RT for 2 hr with 30 rpm for IX and 2X reactions. The tube was kept for 21/2 hr while performing coupling on more than 2X scale reaction .
The coupled beads were centrifuged at 16500 x g for 5 min,wherein the supernatant was discarded.The beads were washed with 500 ul of PBS, pH 7.4 followed by centrifugation at 16500 x g for 5 min and subsequently discarding the supernatant.
The coupled beads were resuspended in 250 ul of blocking buffer from the kit and vortexed at medium speed for 15 sec. The covered tube was agitated on rotospin at RT for 30 min. with 30 rpm and

kept for 40-45 min while performing coupling on more than 2X scale reaction.
The beads were centrifuged at 16500 x g for 5 min. and supernatant was discarded.The coupled beads were washed with 500 pi of storage buffer from the kit and centrifuged at 19300 x g for 7 min.followed by discarding supernatant.
The coupled beads were resuspended in 150 ul of storage buffer by vortexing approximately for 1 min.
Example 2:
Coupling of Anti-CRM197 antibody to EAH SEPHAROSE 4B Beads
1 ml of EAH sepharose beads were washed with Milli Q water pH
4.5 and centrifuged at 5000 rpm for 10 minutes.The beads were further washed with 80 ml 0.5M NaCl/ml.The buffer of CRM MAb was exchanged with Milli Q water (pH 4.5).l ml CRM Mab (4mg/ml)was added to 1ml of EAH sepharose beads.1 ml of 0.3M EDC was added and pH was adjusted to 4.5 with 0.1M NaOH after 1 hour of reaction.The mixture was kept on tube rotor overnight at 4 Deg C.Further the beads were washed with 1M Acetic acid at 5000 rpm for 10 minutes.The beads were blocked with 1M Acetic acid for 2 hrs at room temperature on tube rotor. The beads were washed 3 times with 0 . 1M Acetate buffer having 0 . 5M NaCl (pH4 . 5) followed by washing with 0.1M Tris CI buffer having 0.5M NaCl (pH 8.0) The anti-CRMl97 antibody coupled beads were stored in antibody

storage buffer (20mM Phosphate buffer with 20% ethanol) at 4 Deg C for further use.
Example 3:
Reaction conditions for incubation of anti-CRMl97 coupled beads
with CRM197, PnPs-CRM197
i) lmg/ml CRM, formulation supernatant which is expected to contain free CRM and PnPs-CRM were incubated with anti-CRM197 coupled beads at 37 Deg C for 1 hr on tube rotator. The tube was centrifuged at 5000 rpm for 10 minutes. Followed by analysis of samples for unconjugated polysaccharide estimation by Bead based competitive inhibition assay (BBCIA),
Example 4:
Method for calculation of free PnPs
Concentration of Pneumococcal polysaccharide(PnPs) obtained after treatment with Anti-CRM197 antibody coupled beads/Concentration of Total PnPs*100
Example 5:
Estimation of Free OR unconjugated pneumococcal polysaccharide
in a multivalent vaccine formulation supernatant
Procedure:
ii) The EAH Sepharose beads were coupled with anti CRM197 antibody. Further 500 µl anti CRM197 antibody coupled beads were incubated with assay buffer and vaccine formulation supernatant

for 1 hr. at 37oC. with appropriate control (vaccine incubated with PBS).Also the supernatant was analyzed for CRM 197/PnPs content by multiplexed Bead based competitive inhibition assay (BBCIA). For analysis of CRM197, monoplex was performed taking CRM197 coupled polystyrene beads and anti CRM197 antibody.
Table 1:
CRM content in the sample with or without adsorption of the dissolved vaccine with anti-CRM MAb coupled beads

Samples CRM197 (µg/ml)
Multivalent Pneumococcal conjugate Vaccine 12.5
Multivalent Vaccine Formulation supernatant treated with anti CRM coupled beads N.D.
Table 2:

Determination of free polysaccharide in the supernatant of vaccine formulation

Total
Polysaccharide from Multivalent Free
Polysaccharide
from
Multivalent
Vaccine
Serotype Pneumococcal conjugate Vaccine (ng/ml) Formulation supernatant treated with anti CRM197 coupled beads(ng/ml) % Free PnPs
Type 1 402 N.D. -
Type 5 644 131 3.275
Type 6A 964 N.D. -
Type 6B 1016 N.D. -
Type 7F 398 105 2.625
Type 9V 369 N.D. -
Type 14 118 39 0.975
Type 19A 515 N.D. -
Type 19F 432 53 1.325
Type 23F 908 23 0.575
N.D: Not detectable
Result:

a) CRM197 content was found to reduce by close to 100% when multivalent pneumococcal conjugate vaccine formulation supernatant was incubated with anti CRM197 coupled EAH sepharose beads. Type 1, Type 6A, Type 6B, Type 9V Type 19A PnPs were not detected after above treatment. This indicates that free CRM and CRM-PnPs conjugate should have bound with anti CRM coupled EAH sepharose beads.
To check for the percent free polysaccharide in the supernatant of the adsorbed vaccine formulation, the formulation was centrifuged at 10000 rpm for 10 minutes and put for adsorption with anti-CRM antibody coupled polystyrene or EAH-Sepharose beads for 1 hour and the supernatant tested for the presence of remaining CRM and the free Polysaccharide.
Example 6:
Estimation of Free OR unconjugated pneumococcal polysaccharide
in a multivalent vaccine formulation (With adjuvant)
The formulated multivalent CRM-PnPs (with Adjuvant) were subjected to dissolution for free PnPs estimation. lml sample was centrifuged at l0,000 rpm for 10 minutes and supernatant was collected for further procedure.
Standards from SI to S8 were prepared in the first column of

96-well titer plate using assay buffer by serial 2 fold dilution as per Table 3. 125µl was discarded from the last well after mixing. Final total volume of each well was 125µl.
Table 3
Preparation of standards by dilution with assay buffer

Standard Working standard (µL) Reagent G (µL) Concentration (µg/mL)

Each
serotypes except 6B serotype 6B
SI 250 uL working stock solution — 4.4 8.8
S2 125 µL of SI solution 125 2.2 4.4
S3 125 µL of S2 solution 125 1.1 2.2
S4 125 µL of S3 solution 125 0.55 1.1
S5 125 uL of S4 solution 125 0.275 0.55
S6 125 µL of S5 solution 125 0.1375 0.275

S7
125 pL of S6 solution 125 0.06875 0.1375
S8 125 pL of S7 solution 125 0.03438 0.06875
Appropriately test sample{dissolved formulation for total polysaccharide, supernatant of formulation for percent adsorption, and the formulation supernatant after treatment with anti-CRM MAb coupled beads for free polysaccharide estimation) in column 3 , 5 etc was diluted with assay buffer. The sample dilutions of 1: 2, 1:4, 1: 8 and 1:16 were found to fit well in the standard curve. 100 pi of the standards and test sample were transferred to the respective adjacent columns in the same plate from the above series of dilution.
The sera/MAb was diluted with assay buffer as per optimized dilutions. 100 pi of suitably diluted antibody solution was added in to each respective well of standards and sample. Each antisera/monoclonal antibody used in this assay were used at their optimum dilution.
The plate was incubated at 37°C for 1 hour with shaking at 150 rpm for pre-adsorption.The required wells of multiscreen filter plate were prewetted with 50 µl of assay buffer for 2-3 minutes followed by aspiration of the plate using vacuum manifold.
Mixture of required volume of beads of each serotype in assay

buffer was- prepared to obtain ~4000 beads of each serotype per 50ul of final suspension. 4000 beads were added per well per 50ul in each required well of filter plate. The filter plate was aspirated using vacuum manifold.
50ul of incubated standards and sample were added in duplicates on filter plate containing beads with positive and negative control as per assay plate template planned and plate incubated at 37°C for 1 hour with shaking at 150 rpm in dark.The plate was washed with lOOµl of assay buffer followed by aspirating it on vacuum manifold. This step was performed 3 times.
Mixture of required volume of Anti Rabbit-PE and Anti-Mouse-PE with a final dilution of 1:250 and add 50 ul was added in each well containing standards and samples. Incubate the plate at 37°C for 30 minutes with shaking at 150 rpm in dark. Further the plate was washed 3 times.
lOOul of assay buffer was added and the plate was read on Bio-Plex 200 system.
Table 4:
Analysis of total PnPs estimation with adsorption study

Description FI FI Std %CV Obs Dilu Avg %
Bkgd Dev Cone -tio n Cone ng/m
1 Adsorp t-ion

Blank
3 3 1 47 ng/ml 1
Std 1 (4400ng/ml) 252 249 2 1 4396 1
Std 2 (2200ng/ml) 418 415 12 3 2261 1
Std 3 (llOOng/ml) 681 678 13 2 1066 1
Std 4 (550ng/ml) 999 996 22 2 529 1
Std 5 {275ng/ml) 1376 1373 15 1 264 1
Std 6 (138ng/ml) 1679 1676 107 6 160 1
Std 7 (69ng/ml) 2253 2250 134 6 66 1
Std 8 {34ng/ml) 2721 2718 43 2 33 1
Positive Control 6140 6137 783 13 *0.00 1
Formulation 1 509 506 10 2 3383 2 3850
Formulation 1 804 801 16 2 3194 4
Formulation 1 969 966 105 11 4495 8
Formulation 1 1363 1360 39 3 4327 16
Formulation 2 516 513 13 3 3321 2 3391
Formulation 2 768 765 28 4 3464 4
Formulation 2 1075 1072 55 5 3646 8
Formulation 2 1555 1552 64 4 3132 16
Formulation 3 484 481 11 2 3656 2 3884
Formulation 3 731 728 73 10 3779 4
Formulation 3 973 970 139 14 4457 8
Formulation 3 1464 1461 25 2 3643 16
Formulation 4 505 502 47 9 3423 2 3419
Formulation 4 789 786 31 4 3300 4
Formulation 4 1057 1054 98 9 3775 8
Formulation 4 1546 1543 70 5 3178 16
Formulation 5 544 541 1 0 3058 2 3211
Formulation 5 795 792 116 15 3256 4
Formulation 5 1111 1108 165 15 3404 8

Formulation 5 1556 1553 110 7 3125 16
Formulation 6 447 444 6 1 4108 2 4053
Formulation 6 747 744 18 2 3637 4
Formulation 6 1003 1000 23 2 4199 8
Formulation 6 1370 1367 83 6 4270 16
Formulation 1 (% Adsorption) 1247 1244 18 1 331 1 352 91
Formulation 1 (% Adsorption) 1586 1583 130 8 372 2
Formulation 2 (% Adsorption) 1213 1210 30 2 352 1 373 89
Formulation 2 (% Adsorption) 1551 1548 28 2 394 2
Formulation
3(%
Adsorption) 1073 1070 33 3 458 1 466 88
Formulation
3(%
Adsorption) 1440 1437 66 5 474 2
Formulation 4
(% Adsorption) 1049 1046 36 3 479 1 418 88
Formulation 4
(%
Adsorption) 1612 1609 76 5 356 2
Formulation 5
(% Adsorption) 828 825 93 11 757 1 849 74
Formulation 5 Adsorption) 1058 1055 76 7 942 2
Formulation 6 (% Adsorption) 792 789 48 6 819 1 835 79
Formulation 6 1111 1108 199 18 851 2

(%
Adsorption)
Table 5:
Analysis of different vaccine batches for % recovery in total
PnPs as well as % Adsorption for all PnPs

Serotype Vaccine Batch 1

Total PnPs Ag content recovery (%) % Adsorption

Formulation
1 Formulation 2 Formulation
1 Formulation
2
Type 1 94 129 84 85
Type 5 91 120 89 90
Type 6A 90 123 84 79
Type 6B 91 114 80 75
Type 9V 101 120 87 82
Type 14 86 101 81 75
Type 19A 99 113 95 95
Type 23F 93 109 78 69
Example 7:
Comparative estimation of free PnPs estimation in multivalent conjugate bulk by i) incubating multivalent conjugate bulk with anti-CRM coupled EAH sepharose beads & ii)incubating multivalent conjugate bulk with deoxycholate detergent.
EAH sepharose beads were coupled with anti-CRM197 Mab .500 ul anti CRM197 Mab coupled beads were incubated with Type 14 and Type 19F conjugate bulk for 1 hr. at 37°C. with appropriate control (Type 14 and Type 19F conjugate bulk were incubated with PBS) .The supernatant was analyzed for CRM/PnPs content by

multiplexed Bead based competitive inhibition assay (BBCIA) . For analysis of CRM, monoplex was performed taking CRM197 coupled polystyrene beads and anti CRM monoclonal antibody.
CRM197 content in the below mentioned conjugate bulks were determined before and after treatment with anti-CRM coupled beads
Table 6:
Estimation of CRM197 in monovalent bulk conjugate with and
without adsorption with Anti-CRM 197 MAb coupled beads

Samples CRM197 µg/ml
Type 14 Con: ugate bulk B#4 4982
Type 19F Conjugate bulk B#4 4915
Type 14 B#4treated coupled EAH Conjugate bulk with anti-CRM sepharose beads ND
Type 19F B#4treated coupled EAH Conjugate bulk with anti-CRM sepharose beads ND
Table 7:
Estimation of percent free polysaccharide in the monovalent

conjugate with or without adsorption with Anti-CRM Mab coupled beads

% Free PS % Free PS
determined determined
after anti after
Samples CRM197 deoxycholate
coupled treatment by
bead anthrone
treatment assay
Type 14 Conjugate bulk B#4treated ND (<15
with anti-CRM197 coupled EAH ng/ml i.e. 0.31
sepharose beads <0.75 %)
Type 19F Conjugate bulk B#4treated ND (<15
with anti-CRM197 coupled EAH ng/ml i.e. ND
sepharose beads <0.75 %)
CRM197 content in conjugate bulk for Type 14 and Type 19F B#4 was found to reduce by close to 100% when treated with anti-CRM coupled EAH sepharose beads. When anti-CRM197 bead treated conjugate bulks were analyzed for free polysaccharide content by BBCIA, none of them were found to be detected i.e. the value was below 15ng/ml i.e. <0.75 % in a conjugate containing 2000 ng/ml. Type 14 and Type 19F B#4 bulks were treated with deoxycholate detergent and analyzed by anthrone assay, Type 19F

was not found to be detected where as Type 14 was found to contain 0.31.% free PS.
Both the conjugate bulks showed similar kind of results which indicates that the claimed method used for quantification of free polysaccharide in a multivalent conjugate vaccine was equivalent to the previously existing deoxycholate detergent based methods.
We claim
1. A method for estimation of unconjugated polysaccharides from
a multiple antigenic polysaccharide protein conjugate molecule ,
comprising the steps of:
a)Covalently coupling microspheres to anti-carrier protein
antibody;
b)Dissolution of vaccine formulation followed by measuring of
the total serotype specific polysaccharide content by
multiplexed bead based competitive inhibition assay;
c)Adding the anti-carrier protein coupled microspheres to the
vaccine formulation containing multivalent
polysaccharide-protein conjugates, free polysaccharide and
free carrier protein;
d) Agitating the mixture of step (c) for a duration between lhr and 4hr at 37°C;
e) Subjecting the mixture of step (d)to centrifugation at 3000 to 6000 rpm for a duration between 5 and 2 0 minutes;and

f) Collecting the supernatant and measuring free polysaccharide content by multiplexed bead based competitive inhibition assay.
2. The method of claim 1, wherein coupling of anti-carrier protein antibody to microsphere is carried out using carbodiimide conjugation chemistry.
3. The method of claim 1 ,wherein the beads are selected from the group consisting of sepharose beads, agarose beads, polystyrene beads and polymeric microspheres.
4. The method of claim 1 ,wherein said anti-carrier protein antibody is against a carrier protein selected from group consisting of DT, CRM197, TT,PD, FragmentC, Ply,PhtA, PhtB, PhtD,PhtE,OmpC and PorB.
5. The method of claim 1, wherein said polysaccharide is a bacterial polysaccharide selected from the group consisting of Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus spp., Staphylococcus aureus, Streptococcus spp,, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Enterococcusfaecalis, Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis, Salmonella spp., Salmonella typhi, vibrio cholera, Pasteurella pestis, Pseudomonas aeruginosa, Campylobacter spp.,

Campylobacter jejuni, Clostridium spp., Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Escherichia coli, Shigella spp., Erlichia spp., and Rickettsia spp.
6.The method of claim 5, wherein said bacterial polysaccharide is from a Streptococcus pneumoniae 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 method of claim 5, wherein said N.meningitidis
polysaccharide is of a serotype selected from the group
consisting of A, B, C,D, W135 ,X, Y ,Z and 29E.
8. A method for estimation of unconjugated polysaccharides from
a multivalent polysaccharide protein conjugate composition ,
comprising the steps of:
a)Covalently coupling EAH Sepharose to anti-CRMl97 antibody; b)Dissolution of vaccine formulation followed by measuring of the total serotype specific polysaccharide content by multiplexed bead based competitive inhibition assay; c)Adding the anti-CRM197 coupled microspheres to the vaccine formulation containing multivalent polysaccharide-protein

conjugates, free polysaccharide and free carrier protein;
d)Agitating the mixture of step (c) for lhr at 37°C;
e)Subjecting the mixture of step (d)to centrifugation at 5000
rpm for 10 minutes;and
f) Collecting the supernatant and measuring free polysaccharide
content by multiplexed bead based competitive inhibition assay.
9. A method according to claim 1 or 8, further comprising:
a)Collecting the supernatant containing unadsorbed polysaccharide-protein conjugates ,carrier protein and free polysaccharide;and
b)Measurement of percent adsorption by multiplexed bead based competitive inhibition assay.
10. The method according to any of the previous claims, wherein
the multiplex system contains a bead array reader selected from
group consisting of BioPlex-100, a BioPlex-200, a Luminex-100, a
Luminex-200 or MagPix.