FORM-2
THE PATENT ACT,1970
(39 OF 1970)
AND
THE PATENT RULES, 2003
(As Amended)
COMPLETE SPECIFICATION (See section 10;rule 13)
"A METHOD FOR ASSAYING UNCONJUGATED POLYSACCHARIDE IN A POLYSACCHARIDE-PROTEIN CONJUGATE
PREPARATION"
SERUM INSTITUTE OF INDIA PVT LTD., a corporation organized and existing under the laws of India, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028, Maharashtra, India.

FIELD
The present disclosure relates to a method for assaying unconjugated polysaccharide (Ps) in a polysaccharide-protein conjugate vaccine.
BACKGROUND OF THE DISCLOSURE
Isolated bacterial capsular polysaccharides from Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis have been successfully used as vaccines in adults. Vaccines based on capsular polysaccharides stimulate humoral immunity but fail to induce immunologic memory, and thus are ineffective in young infants.
For a vaccine to be effective and induce long lasting immunity, induction of T cell memory is very important. Increased immunogenic response of an antigen can be achieved by converting the T-independent antigen to T-dependent antigen. The conjugation of polysaccharide to carrier protein renders the antigenic molecule to undergo the process of antigen presentation, and enhance the polysaccharide immunogenicity by eliciting the T-cell dependent response. 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). The proteins used for conjugation to polysaccharides include CRM197, tetanus toxoid, diphtheria toxoid, Neisseria meningitidis outer membrane complex, Haemophilus influenzae protein D, Pneumolysin, etc.
Neisseria meningitidis is a Gram-negative diplococcal bacterium that causes meningitis, septicemia and, rarely, pneumonia, carditis and septic arthritis. The 13 serogroups of N. meningitidis are classified according to the antigenic structure of the polysaccharide capsule. Six serogroups, A, B, C, Y, W135 and X, are responsible for virtually all cases of human disease. The polysaccharides from serotype A, C, W, Y & X have been conjugated to various carrier proteins to prepare a conjugated vaccine, effective against infection by these serotypes.
The polysaccharide component of polysaccharide-protein conjugate vaccines undergoes gradual depolymerization at a rate that depends on the type of conjugate, formulation components and storage conditions. This results in an increase in unconjugated polysaccharide content in the final vaccine formulation. Hence tests should be conducted to ensure stability of product. Polysaccharide-carrier protein conjugates are known to release unconjugated polysaccharide after conjugation

while it undergoes further processing, lyophilization or storage in liquid as well as solid formulations. Although only the meningococcal polysaccharide that is covalently bound to the carrier protein (i.e. Conjugated polysaccharide) is immunologically important for clinical protection, excessive levels of unbound (unconjugated) polysaccharide could potentially result in immunological hyporesponsiveness to group C polysaccharide (Gold, Lepow, Goldschneider, Draper, & Gotschlich, 1975; Granoff, Gupta, Belshe, & Anderson, 1998; Leach et al., 1997; MacDonald et al., 1998).
An acceptable value of unconjugated saccharide consistent with adequate immunogenicity, as shown in clinical trials, should be established for the particular product and each final lot must be shown to comply with this limit (Refer WHO TRS No.897, pg 17, 2000, A.3.3.5 section; WHO/TRS/924 Page No. 14, A.3.3.5; Page 145 A.3.6.4 WHO/TRS/962). Accordingly, a reliable and accurate determination of unconjugated polysaccharide in polysaccharide-protein conjugate vaccines is one of the important quality control parameters in development and production of these conjugate vaccines. Thus, it is a statutory requirement to quantify the concentration of unconjugated or unconjugated polysaccharide available in final vaccine formulation.

Previously rate nephelometry has been reported for measuring unconjugated 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 unconjugated polysaccharides expected in a vaccine formulation especially pneumococcal conjugate vaccines having very 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.
Hou, Ya-li et al. (2015) discloses use of differential rocket immunoelectrophoresis (DRIEP) for quantification of unconjugated polysaccharide content in tetravalent meningococcal conjugate vaccine (ACWY-TT). The said method however gives inconsistent unconjugated polysaccharide results in comparison to the DOC based method.
An acid precipitation method using deoxycholate (DOC)/HCl has been reported previously to quantify unconjugated polysaccharide from polysaccharide protein conjugate. The separation is based on the differences between acid precipitation properties of polysaccharide protein conjugate and polysaccharide alone. The above method has been used previously to determine

unconjugated polysaccharide in PRP-TT conjugate vaccines [Refer Guo, Y. Yet al 1998. Biologicals 26: 33-38; Lei, Q. P et al 2000.J. Pharmaceut. Biomed. 21: 1087-1091.] along with meningococcal polysaccharide-diphtheria toxoid conjugate vaccine ACWY-DT (Menactra) [Refer Lei, Q. P et al 2000. Dev. Biol. 103: 259-264.]. However, when the DOC/HCl method was applied to measure unconjugated polysaccharide of conjugate vaccine, unconjugated polysaccharide contents measured were inconsistent depending on formulation conditions. Especially, higher unconjugated Ps content was obtained in the formulation vaccine in a salt-buffered solution than before formulation, when the DOC/HCl method was used, and this in fact could be an obstacle in developing the vaccine in a liquid form or combining it with other vaccines including salt components such as a DTaP-HepB vaccine. [Refer Bae C. S et al; J. Microbiol. Biotechnol. (2002), 12: 787-792].
The method involving use of anti-TT antibody and ammonium sulfate to precipitate PRP-TT conjugate and measuring unconjugated PRP in the supernatant has been previously reported. However, the said method has not been utilized for formulated aluminum gel adsorbed multivalent conjugate vaccine. Further the ammonium salt retained after protein precipitation can interfere

in the assay. Refer: [Yoo et al; J. Microbiol. Biotechnol. (2003), 13(3), 469–472.]
Current typical quantification analysis of polysaccharides in vaccine formulations involves two major steps:(i) a hydrolysis step to release monosaccharides followed by (ii) resolving and quantifying monosaccharides with a high-performance anion-exchange chromatographic (HPAEC) method, using pulsed amperometric detection (PAD). Micoli et al; (2011) discloses HPAEC-PAD method for free Salmonella typhi polysaccharide (Vi) analysis in a Vi-CRM197 conjugate.
MenAfriVac™(Men A-TT), Menjugate™ , Meningitec™ and NeisVac-C™ Menactra™(Men ACWY-DT) , Menveo™(Men ACWY-CRM197), Nimenrix™(Men ACWY-TT)are the meningococcal monovalent and multivalent conjugate vaccines that have been approved for human use. Accordingly, free polysaccharide estimation for all of these vaccine(s) by HPAEC-PAD has been previously reported, however said vaccine(s) typically comprise of a single type of carrier protein and do not have Neisseria meningitidis serogroup X polysaccharide component. Additionally, HPAEC PAD method requires prior conversion of polysaccharide to Monomeric units by acid digestion and

the presence of high concentration of saccharide excipient such as sucrose (Glucose + fructose) may interfere with the determination of polysaccharide content of different meningococcal serogroup, which necessitates a development of a method to remove excipient prior to sample hydrolysis.
The existing meningococcal conjugate vaccines are based on A C Y W135 polysaccharides. However, the increase in incidence of MenX disease in African Meningitis Belt in the last 5 years has warranted development and introduction of a MenX polysaccharide conjugate vaccine in selected areas of the region to prevent and control future epidemics. The capsular polysaccharides of serogroup B, C, Y, and W135 meningococci are composed of sialic acid derivatives. Serogroup B and C meningococci express (α 2-8)- and(α 2-9 239)-linked polysialic acid, respectively, while alternating sequences of D-glucose or D-galactose and sialic acid are expressed by serogroup Y and W135 N. meningitidis. In contrast, the capsule of serogroup A meningococci is composed of(α 1-6 )-linked N-acetylmannosamine 6-phosphate , while N. meningitidis serogroup X synthesizes capsular polymers of( α 1-4)-linked N-acetylglucosamine 1-phosphate. Refer: [Yih-Ling Tzeng et al; Genetic Basis for Biosynthesis of the (134)-

Linked N-Acetyl-D-Glucosamine 1-Phosphate Capsule of Neisseria meningitidis Serogroup X; Infection And Immunity, Dec. 2003, p. 6712–6720; Vol. 71, No. 12.]
Also to increase the coverage of these vaccines across serotypes for a bacterium, conjugated polysaccharide from various serotypes is added to vaccine composition, inadvertently increasing the concentration of carrier protein. It is observed that co-administration of conjugate vaccines bearing the same carrier protein, reduces the immune response (due to immune interference) against polysaccharides conjugated with that particular protein [Ref: “Comparative effects of carrier proteins on vaccine-induced immune response” by Knuf M. et al; Vaccine. 2011 Jul 12;29(31):4881-90]. Also, it has been observed that the combination of polysaccharide from a particular serotype with a specific carrier protein shows better efficacy. [Ref: “Comparison of CRM197, diphtheria toxoid and tetanus toxoid as protein carriers for meningococcal glycoconjugate vaccines.” by Tontini M. et al; Vaccine. 2013 Oct 1; 31(42):4827-33].To circumvent the phenomenon of negative interference in multivalent vaccines composed of polysaccharide-protein conjugates and to provide better immune response, several vaccine manufacturers are developing vaccines comprising

multiple carrier proteins conjugated to different polysaccharides (Ps) so that the maximum load of each of the carrier proteins is not reached.
Further, a pentavalent Meningococcal conjugate vaccine comprising of Ps conjugated to two different carrier proteins is being developed, wherein polysaccharide from serotype A and X are individually conjugated to tetanus toxoid and polysaccharide from serotype C,Y,W are individually conjugated to recombinant CRM197 (Refer Moran et al ; Immunogenicity of the Meningitis Vaccine Project’s pentavalent MenACWYX polysaccharide conjugate vaccine MCV-5 20th International Pathogenic Neisseria Conference, 7th September 2016. Manchester, United Kingdom; Abstract ID: O35).
Previously reported DOC-HCl precipitation with a high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD)approach have been utilized for unconjugated polysaccharide (ACWY) estimation in monovalent/multivalent meningococcal conjugate vaccine(s) having a single type of carrier protein and have inherent limitations.
However, aforementioned methods do not discuss determination of unconjugated Ps content in a multivalent meningococcal conjugate vaccine having two

different types of carrier proteins. Also previously reported HPAEC-PAD chromatographic approach already utilized for unconjugated polysaccharide (ACWY) estimation in monovalent/multivalent meningococcal conjugate vaccine(s) have inherent limitations and is not applicable to unconjugated polysaccharide estimation in multivalent meningococcal conjugate vaccine(s) comprising of a structurally distinct phosphorylated sugar such as meningococcal X. Hence, there is a need for an alternative, highly reproducible method for accurate estimation of unconjugated Neisseria meningitidis serogroup X polysaccharide in pentavalent (ACWYX) polysaccharide-protein conjugate comprising of atleast two different types of carrier proteins and further overcoming the limitations of the previously reported methods.
Hence, a more efficient, alternative method is required to quantify unconjugated polysaccharide as well as total polysaccharide content in monovalent and multivalent conjugate vaccine.
OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows:

An object of the present disclosure is to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
Another object of the present disclosure is to provide a method to detect and quantitate individual concentration of unconjugated polysaccharide.
Still another object of the present disclosure is to provide a simple, rapid and cost-effective method for detecting individual concentration of unconjugated polysaccharide.
Yet another object of the present disclosure is to provide a method for detecting individual concentration of unconjugated polysaccharide, which generates reproducible results.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
Summary
The present disclosure relates to a robust method for assaying unconjugated polysaccharide (Ps) in a polysaccharide-protein conjugate vaccine compositions, preferably meningococcal multivalent (ACWYX) conjugate comprising of Neisseria meningitidis serogroup X

polysaccharide, atleast two different types of carrier proteins preferably Tetanus Toxoid (TT) and recombinant CRM197, a dissacharide excipient and salts wherein the method comprises of following steps-i) Optimal removal of interfering disaccharide excipients and salts by utilizing a centrifugal filtration prior to DOC–HCl precipitation and sample hydrolysis. ii) Subjecting the sample comprising of monovalent/multivalent polysaccharide protein conjugate to DOC-HCl precipitation wherein the conjugated polysaccharide - carrier proteins and two different types of unconjugated carrier proteins (TT and CRM197) are effectively precipitated out while the supernatant consist of unconjugated polysaccharide (Men ACYWX) only. iii) The above supernatant was further subjected to optimal acid or base hydrolysis for Neisseria meningitidis serogroup (ACYWX) polysaccharide preferably serogroup X to obtain a hydrolysate comprising the monomeric units only. The hydrolyzing agent concentration, temperature and time have been optimized to provide the best compromise between complete hydrolysis of the serogroup polysaccharides and the subsequent degradation of the resulting monomeric units only.

iv) Detection and quantitation of monomeric unit preferably glucosamine 1-phosphate in hydrolysate using High performance anion exchange chromatography column in conjunction with trap column and pulsed amperometric detection (HPAEC-PAD).
The said method can be utilized for determining
unconjugated saccharide, either residual saccharide
(generated across conjugation and conjugate
purification processes) and degraded saccharide
(generated on storage of conjugate).
Detailed Description
In the first embodiment of the present disclosure, the instant method was designed to estimate the individual concentration of unconjugated polysaccharide content in the monovalent or multivalent polysaccharide protein conjugate vaccine composition, wherein two or more carrier proteins are conjugated to one or more polysaccharides in a vaccine sample.
In one of the aspects of the said embodiment, the monovalent or multivalent polysaccharide protein conjugate bulk/vaccine composition comprises of one or more polysaccharide selected from groups consisting of 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., Ehrlichia spp., and Rickettsia spp. Polysaccharides of Streptococcus pneumoniae type 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, Polysaccharides of Meningococcal serogroup A, B, C, D, W135, X, Y, Z, 29E, H. influenzae type b etc. conjugated to two or more carrier protein selected from groups comprising of CRM197, diphtheria toxoid, Neisseria meningitidis outer membrane complex, 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 surface adhesin A (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).
In one of the preferred aspects of the said embodiment, the instant method was designed to estimate the individual concentration of unconjugated polysaccharide content in the following Mening multivalent conjugate vaccine composition comprising of:
Table 1- Contents of a single 5-Dose Vaccine vial of Pentavalent Conjugate Vaccine (ACWYX-TT/CRM197)(freeze-dried)

Component Quantity
Men A PS* 30 μg

Men C PS# 30 |μg
Men Y PS# 30 μg
Men W PS# 30 μg
Men X PS* 30 μg
Sucrose 15 mg
Sodium citrate (dihydrate) 2.5 mg
Tris (Trometamol) 0.61 mg
* - Conjugated to Tetanus Toxoid (TT); # - Conjugated to CRM197
Table 2- Contents of a single 5-Dose Diluent ampoule of Pentavalent Conjugate Vaccine (ACWYX-TT/CRM197)

Component Quantity
Sodium Chloride 0.9% w/v
Aluminium (Al+++) 125 μg/ Dose (0.5

Adjuvant ml)
Volume Between 3.00 to 3.20 ml
Table 3- Upon reconstitution final vaccine ready-for-injection to contain

Component Quantity
Men A PS* 5 μg
Men C PS# 5 μg
Men Y PS# 5 μg
Men W PS# 5 μg
Men X PS* 5 μg
Sucrose 2.42 mg
Sodium citrate (dihydrate) 0.4 mg
Tris (Trometamol) 0.098 mg
Aluminium (Al+++) Adjuvant 125 µg

Sodium Chloride q.s.
Table 4- Contents of a single 1-Dose Vaccine vial of Pentavalent Conjugate Vaccine (ACWYX-TT/CRM197) (freeze-dried)

Component Quantity
Men A PS* 6 μg
Men C PS# 6 μg
Men Y PS# 6 μg
Men W PS# 6 μg
Men X PS* 6 μg
Sucrose 15 mg
Sodium citrate (dihydrate) 2.5 mg
Tris (Trometamol) 1.21 mg
* - Conjugated to Tetanus Toxoid (TT); # - Conjugated to CRM197
Table 5- Contents of a single 1-Dose Diluent ampoule of Pentavalent Conjugate Vaccine (ACWYX-TT/CRM197)

Component Quantity
Sodium Chloride 0.9% w/v
Aluminium (Al+++) Adjuvant 125 µg/ Dose (0.5 ml)
Volume Between 0.60 to 0.66 ml
In the second embodiment of the disclosure, interfering disaccharide excipients and salts were filtered out from test sample by utilizing an advantageous centrifugal filtration/dialysis step.
The disaccharide excipients and salts were filtered out using the following protocol:
1. Vaccine vial was resuspended in 2 mL of solvent and vortexed gently, while for standard and bulk sample appropriate dilutions were prepared.
2. The resuspended content of vial was loaded onto the prewashed centrifugal filters (one vial on one filter). Centrifuge the filters at 4400 X g for 15

minutes. Carefully remove the filters and discard the filtrate.
3. The Centrifugal filters containing the carrier protein – polysaccharide retentate were washed with 2mL of solvent. The above washing step was repeated from seven to fifteen times.
4. Finally to concentrate the retentate, the centrifugal filters were centrifuged at 4400 X g for 15 minutes. The retentate from each filter were collected in microcentrifuge tube separately.
5. Finally each collected retentate was made up to 1 mL by adding solvent to it wherein, it can be further subjected to protein precipitation.
In one of the aspect of the second embodiment, the centrifugal filtration step was carried using a centrifugal filter membrane wherein, centrifugal filter membrane is selected from the group consisting of one or more of cellulose, cellulose ester or polyethersulphone membranes, having pore size selected from the range of 0.1µm to 1 µm. Preferably the

centrifugal filter membrane selected is made up of cellulose with the pore size of 0.1 to 0.2 µm.
In second aspect of the second embodiment, disaccharide excipients to be filtered out from the test sample consist of one or more excipients selected from the group of maltose, lactose, sucrose and trehalose.Preferably, excipient to be filtered out from the test sample is sucrose which is known to interfere with the estimation of Men Y serogroup polysaccharide and reduces load on detector which adds value in the analysis by maintaining the efficiency of the detector.
In third aspect of the second embodiment, salts to be filtered out from the test sample consist of one or more salts selected from the group of NaCl, KCl, Na2SO4, (NH4)2SO4, sodium phosphate and sodium citrate.
In fourth aspect of the second embodiment, conjugated polysaccharide - carrier proteins, unconjugated polysaccharide and unconjugated carrier proteins are effectively retained onto centrifuge filter membrane whereas the disaccharide excipients and salts are filtered out.

In another aspect of the second embodiment, the
filtered test sample retentate was washed several times
with solvent to rule out any presence of the
disaccharide excipients and salts in the retentate
(filtered test sample).
Yet the preferred solvent is selected from the group consisting of water for injection (WFI) and Milli-Q water.
In third embodiment of the disclosure, the resuspended retentate from the centrifugal filters were subjected to protein precipitation.
In a first aspect of the third embodiment, protein precipitation was carried out using one or more agent selected from a group consisting of trichloroacetic acid, DOC-HCl, ethanol, acetone, ammonium sulfate, PEG8000, and diethyl ether.
Preferably DOC having concentration in the range of 0.25% to 0.30% (w/v) (and all sub ranges there between) and titrated by conc. HCl to a pH between 6.2 and 6.9 was used for effective precipitation of two or more different types of carrier proteins.

In yet another preferred aspect, DOC- HCl 0.28% (w/v) having pH between 6.4 and 6.8 pH 6.6(±0.2) was used for effective precipitation of two or more different types of carrier proteins (TT conjugated to Men A & X and CRM197 conjugated to Men C, Y, W).
Table 6- Optimal DOC-HCl precipitation
conditions (% DOC and pH) for effective precipitation
of Polysaccharide-Protein Conjugate and Free carrier
protein.

Sample details Sample Volume (µL) DOC
addition
(0.28%
pH 6.6
(µL) Sample precipitation (1M HCL) (µL)
Carrier Protein : Tetanus toxoid
Men A 400 70 25
Men X 100 30 40
Carrier Protein : CRM197
Men C 250 250 50
Men Y 250 250 50
Men W 250 250 50

In another aspect of the third embodiment, the supernatant obtained after protein precipitation preferable consist of unconjugated polysaccharide only.
In fourth embodiment of the disclosure, the supernatant consisting of unconjugated polysaccharides were subjected to acid or base hydrolysis wherein the polysaccharides are converted to monomeric units only.
In a first aspect of the fourth embodiment, hydrolyzing agent concentration, temperature and time of exposure have been optimized to provide the best compromise between complete hydrolysis of the serogroup polysaccharides and the subsequent degradation of the resulting monomeric unit products.
The time period for optimal hydrolysis of
polysaccharides may last between 60 and 180 minutes
(and all sub ranges there between) and preferably 120
minutes and above.
The temperature for optimal hydrolysis of polysaccharides may be between 70°C and 110°C (and all
sub ranges there between) and preferably between 80°C
and 100°C.

In a second aspect of the fourth embodiment, hydrolyzing agent used was selected from a group consisting of Hydrochloric Acid (HCl), Trifluoroacetic acid (TFA), Sodium Hydroxide (NaOH), and Hydrogen peroxide (H2O2).
In another aspect of the fourth embodiment, TFA & HCl were used for optimal hydrolysis of polysaccharides.
In a yet another aspect of the fourth embodiment, TFA was used for optimal hydrolysis of Men (A, Y, W) polysaccharide & HCl was used for optimal hydrolysis of Men (C, X) polysaccharide.
Preferably the TFA & HCl hydrolyzing agent concentration, temperature and time of exposure have been optimized to provide the best compromise between complete hydrolysis of the Men (ACYW&X) serogroup polysaccharides and the subsequent degradation of the resulting monomeric units as shown below:
Table 7- Optimal acid or base hydrolysis conditions for Neisseria meningitidis serogroup A, C, Y, W and X

polysaccharide mainly hydrolyzing agent concentration, temperature and time

Sample details Acid used
for digestion Digestion
temperature
(°C) Digestion
time (minutes) Free
polysaccharide
Separation
Men AYW (8M) TFA 100 120 Centrifuge at 6000 X g
Men C (5M) HCl 80 120

for

5 minutes
Men X (5M) HCl 100 150 at 22°Cambient temperature
In fifth embodiment of the disclosure, the hydrolyzed polysaccharide sample obtained after acid digestion were analyzed using High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD)for a quantitative determination of individual concentration of unconjugated polysaccharide (Ps).
In one aspect of the fifth embodiment, High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was applied for a quantitative determination of individual concentration of unconjugated Ps content in the processed sample

comprising of Men-A, Men-C, Men-Y, Men-W and Men-X; wherein the preferred HPAEC columns may selected from the range of CarboPac columns such as PA1, PA100, PA20, PA10, PA200 or MA1, preferred temperature of auto
sampler may be between 22°C and 28°C (and all sub ranges
there between), preferred temperature of column may be
between 20°C and 35°C (and all sub ranges there
between), Flow rate of the column may be between 0.5 ml and 2ml (and all sub ranges there between), injection volume may be between 10µl and 60µl (and all sub ranges there between), the electrode used may be either silver or gold electrode.
Further, the operational conditions of HPAEC-PAD optimized for a quantitative determination of individual concentration of Unconjugated Ps content in the processed sample comprising of Men-A, Men-C, Men-Y, Men-W and Men-X were as mentioned below:
• Auto sampler Temperature : RT or 25°C
• Flow rate: 1 mL/min.
• Injection Volume: 25µL (Men A, C, Y, W), 50 µL (Men X)
• Run Time: 60 Minutes (Men A, Y, W) , 25 Minutes (Men C), 45 Minutes (Men X)

• Column: CarboPac PA1 with guard column Aminotrap
• Column temperature: 30°C
• Detection: Pulse Amperometric detection
• Reference Electrode: AgCl
• Waveform: Carbohydrates (standard quad. Potential)
Before loading samples and standards onto the HPAEC-PAD system, internal control were added in all the tubes of samples and standards
Table 8- Addition of Internal Control

Serogroup Standard
/ Sample
(µL) Internal control Internal
control
(µL) Total
volume
(µL)
Men A 460 Fucose 20 500
Men Y
Glucosamine-1-phosphate 20

Men W

Men C 480 Glucuronic Acid 20 500
Men X 480 Fucose 20 500
The instant assay can also be employed for other
multivalent conjugate vaccines that contain atleast
two different types of carrier proteins viz multivalent
pneumococcal polysaccharide-protein conjugate

vaccine(s) like i) Synflorix, a licensed 10 valent Pneumococcal polysaccharide conjugate vaccine comprises Ps conjugated to three different carrier proteins(DT,TT and protein D). Polysaccharide from serotype 1, 4, 5, 6B, 7F, 9V, 14 and 23F are conjugated to protein D (derived from non-typeable Haemophilus influenzae) carrier protein; Polysaccharide from serotype 18C is conjugated to tetanus toxoid carrier protein; and polysaccharide from serotype 19F is conjugated to diphtheria toxoid carrier protein ii)A 11 valent Pneumococcal conjugate vaccine comprising Ps conjugated to atleast two different carrier proteins(DT and TT) has also been reported (Ref: Rose-Marie Olandera et al 2002; Booster response to the tetanus and diphtheria toxoid carriers of 11-valent pneumococcal conjugate vaccine in adults and toddlers; Vaccine 20;336-341) wherein polysaccharide is selected from l,3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F and carrier proteins are Diphtheria toxoid and tetanus toxoid or iii) a 16 valent Pneumococcal conjugate vaccine comprising polysaccharide selected from serotypes 1, 2, 3,4,5, 6A, 6B,7F, 8,9V,9F,9N, 12F, 14, 15B, 17F,18C, 19A ,19F,20,22F,23F,33F and 45 and atleast two different carrier proteins selected from TT, CRM197, DT and pneumococcal surface adhesin A (PsaA).

In one of the embodiments, the percentage of unconjugated or unconjugated polysaccharide Ps was calculated by dividing the amount of unconjugated polysaccharide by the total amount of polysaccharide quantified in the sample.
In another aspect of the above embodiment, the total amount of polysaccharide was quantified by the instant method described in the disclosure without undergoing protein precipitation step.
The instant method described above was optimized for detection and quantitation of unconjugated polysaccharide in a monovalent/multivalent conjugate vaccine composition wherein, Lower limit of Detection (LLOD) Set: LLOD of Free polysaccharide: 0.17 ug/ml
In view of the many possible embodiments to which the principles of the disclosed disclosure may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the disclosure and should not be taken as limiting the scope of the disclosure.
Examples: Example 1:

The instant method was designed to estimate the individual concentration of unconjugated polysaccharide in the following Polysaccharide-Protein conjugate vaccine composition
Table 1- Contents of a single 5-Dose Vaccine vial of Pentavalent Conjugate Vaccine (ACWYX-TT/CRM197) (freeze-dried)

Component Quantity
Men A PS* 30 μg
Men C PS# 30 μg
Men Y PS# 30 μg
Men W PS# 30 μg
Men X PS* 30 μg
Sucrose 15 mg
Sodium citrate (dihydrate) 2.5 mg
Tris (Trometamol) 0.61 mg
* - Conjugated to Tetanus Toxoid (TT); # - Conjugated to CRM197

Table 2- Contents of a single 5-Dose Diluent ampoule of Pentavalent Conjugate Vaccine (ACWYX-TT/CRM197)

Component Quantity
Sodium Chloride 0.9% w/v
Aluminium (Al+++) Adjuvant 125 µg/ Dose (0.5 ml)
Volume Between 3.00 to 3.20 ml
Table 3- Upon reconstitution final vaccine ready-for-injection to contain

Component Quantity
Men A PS* 5 jug
Men C PS# 5 μg
Men Y PS# 5 μg
Men W PS# 5 μg
Men X PS* 5 μg
Sucrose 2.42 mg
Sodium citrate 0.4 mg

(dihydrate)
Tris (Trometamol) 0.098 mg
Aluminium (Al+++) Adjuvant 125 μg
Sodium Chloride q.s.
Table 4- Contents of a single 1-Dose Vaccine vial of Pentavalent Conjugate Vaccine (ACWYX-TT/CRM197) (freeze-dried)

Component Quantity
Men A PS* 6 μg
Men C PS# 6 μg
Men Y PS# 6 μg
Men W PS# 6 μg
Men X PS* 6 μg
Sucrose 15 mg
Sodium citrate (dihydrate) 2.5 mg

Tris (Trometamol) 1.21 mg
* - Conjugated to Tetanus Toxoid (TT); # - Conjugated to CRM197
Table 5- Contents of a single 1-Dose Diluent ampoule of Pentavalent Conjugate Vaccine (ACWYX-TT/CRM197)

Component Quantity
Sodium Chloride 0.9% w/v
Aluminium (Al+++) Adjuvant 125 µg/ Dose (0.5 ml)
Volume Between 0.60 to 0.66 ml
Example 2:
Sample Washing: (Excipient and Salt Removal Step)

The disaccharide excipients and salts were filtered out using the following protocol:
1. Vaccine vial was resuspended in 2 mL of WFI/MQW and vortexed gently, while for standard and bulk sample appropriate dilutions were prepared.
2. The resuspended content of vial was loaded onto the prewashed 0.1 to 0.2 µm polyethersulfone membrane based centrifugal filters (Pall) (one vial on one filter). Centrifuge the filters at 4400 X g for 15 minutes. Carefully remove the filters and discard the filtrate.
3. The Centrifugal filters containing the carrier protein – polysaccharide retentate were washed with 2mL of WFI/MQW. The above washing step was repeated seven more times (Total 08 washes).
4. Finally to concentrate the retentate, the centrifugal filters were centrifuged at 4400 X g for 15 minutes. The retentate from each filter were collected in microcentrifuge tube separately.
5. Finally each collected retentate was made up to 1 mL by adding WFI/MQW to it wherein, it can be further subjected to protein precipitation.
Example 3:

Table 6- Optimal DOC-HCl precipitation
conditions (% DOC and pH) for effective precipitation
of Polysaccharide-Protein Conjugate and Free carrier
protein.

Sample details Sample Volume (µL) DOC
addition
(0.28%
pH 6.6
(µL) Sample precipitation (1M HCL) (µL)
Carrier Protein : Tetanus toxoid
Men A 400 70 25
Men X 100 30 40
Carrier Protein : CRM197
Men C 250 250 50
Men Y 250 250 50
Men W 250 250 50

Table 7- Optimal acid or base hydrolysis conditions for Neisseria meningitidis serogroup A, C, Y, W and X polysaccharide mainly hydrolyzing agent concentration, temperature and time

Sample details Acid used
for digestion Digestion
temperature
(°C) Digestion
time (minutes) Free
polysaccharide
Separation
Men AYW (8M) TFA 100 120 Centrifuge at 6000 X g
Men C (5M) HCl 80 120

for

5 minutes
Men X (5M) HCl 100 150 at
22°C ambient
temperature

Further, the operational conditions of HPAEC-PAD optimized for a quantitative determination of individual concentration of Unconjugated Ps content in the processed sample comprising of Men-A, Men-C, Men-Y, Men-W and Men-X were as mentioned below:
• Chromatography system: High Performance Anion exchange chromatography system (Thermo Scientific -Model No. ICS5000 and ICS 5000+)
• Auto sampler Temperature : RT or 25°C
• Gradient elution: NaOH 400mM and Sodium Acetate
(CH3COONa) 320 mM
• Flow rate: 1 mL/min.
• Injection Volume: 25µL (Men A, C, Y, W), 50 µL (Men X)
• Run Time: 60 Minutes (Men A, Y, W) , 25 Minutes (Men C), 45 Minutes (Men X)
• Column: CarboPac PA1 guard 2x250mm with Aminotrap Trap column 2x50mm.
• Column temperature: 30°C
• Detection: Pulse Amperometric detection
• Reference Electrode: Silver chloride (AgCl)

• Waveform: Carbohydrates (standard quad. Potential)
• Lower limit of Detection (LLOD) Set: LLOD of Unconjugated polysaccharide: 0.17 ug/ml
Before loading samples and standards onto the HPAEC-PAD system, internal control were added in all the tubes of samples and standards
Table 8- Addition of Internal Control

Serogroup Standard
/ Sample
(µL) Internal control Internal
control
(µL) Total
volume
(µL)
Men A 460 Fucose 20 500
Men Y
Glucosamine-1-phosphate 20

Men W

Men C 480 Glucuronic Acid 20 500
Men X 480 Fucose 20 500

Table 9 - Running conditions (Buffers proportion): Men AYW

S.No. Retention (Min) % B (MQW) % C (320 mM Sodium Acetate) % D
1 0.000 95.5 0.0 0.0
2 0.500 95.5 0.0 0.0
3 16.000 95.5 0.0 0.0
4 18.100 43.7 31.3 0.0
5 42.000 5.0 70.0 0.0
6 42.100 0.0 0.0 0.0
7 52.000 0.0 0.0 0.0
8 52.100 95.5 0.0 0.0
9 65.000 95.5 0.0 0.0
Table 10 - Running conditions (Buffers proportion): Men C

S.No. Retention (Min) % B % C % D
1 0.000 34.4 15.6 0.0
2 0.500 34.4 15.6 0.0
3 25.000 34.4 15.6 0.0

Table 11 - Running conditions (Buffers proportion): Men X

S.No. Retention (Min) % B % C % D
1 0.000 87.5 0.0 0.0
2 0.500 87.5 0.0 0.0
3 13.000 87.5 0.0 0.0
4 13.100 56.2 31.3 0.0
5 25.000 17.5 70.0 0.0
6 25.100 0.0 0.0 0.0
7 35.000 0.0 0.0 0.0
8 35.100 87.5 0.0 0.0
9 45.000 87.5 0.0 0.0
Note: Remaining volume is % A is 400 mM NAOH

Claims:
1. A method for assaying unconjugated carbohydrate in a
carbohydrate-protein conjugate vaccine sample,
comprising the steps of:
i) Optimal removal of excipients and salts by utilizing a centrifugal filtration step to obtain a retentate
ii) Subjecting the retentate to protein precipitation to separate the conjugated and unconjugated carbohydrates.
iii) Subjecting the sample comprising carbohydrate to hydrolysis to obtain a hydrolysate
iv) Detection and quantitation of monomeric unit of carbohydrate preferably glucosamine 1-phosphate in hydrolysate using High performance anion exchange chromatography column in conjunction with trap column and pulsed amperometric detection (HPAEC-PAD).
2. The method according to claim 1, wherein the
carbohydrate is a natural or synthetic carbohydrate,
polysaccharide (Ps), oligosaccharide (Os), or
combination thereof.
3. The method according to claim 1, wherein the sample
to be assayed comprises of excipient preferably a

disaccharide selected from the group of maltose, lactose, sucrose and trehalose.
4. The method according to claim 1, wherein the sample to be assayed comprises of salt selected from the group of NaCl, KCl, Na2SO4, (NH4)2SO4, sodium phosphate and sodium citrate.
5. The method according to claim 1, wherein centrifugal filtration (step i) utilizes filter membrane selected from the group of one or more of cellulose, cellulose ester or polyethersulphone membranes, having pore size selected from the range of 0.1 µm to 1 µm.

6. The method according to claim 1 and 5, wherein centrifugal filtration (step i) utilizes polyethersulphone centrifugal filter membrane having pore size in the range of 0.1 µm to 0.5 µm.
7. The method as claimed in any one of the preceding claims, wherein the (step i) retentate comprises unconjugated carbohydrate and/or conjugated carbohydrate whereas the excipients and salts are filtered out.
8. The method as claimed in any one of the preceding
claims, wherein the sample to be assayed comprises
unconjugated carbohydrate and/or conjugated
carbohydrate selected from the group of Helicobacter

pylori, Chlamydia pneumoniae, Chlamydia trachomatis,
Ureaplasma urealyticum, Mycoplasma pneumoniae,
Staphylococcus spp., Staphylococcus aureus,
Streptococcus spp., Streptococcus pyogenes,
Streptococcus pneumoniae, Streptococcus viridans,
Enterococcus faecalis, 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., Ehrlichia spp., and Rickettsia spp. Polysaccharides of Streptococcus pneumoniae type 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9A, 9F, 9N, 9V, 10A, 11A, 12F, 14, 15B, 15C, 17F, 18C, 19F, 19A, 20, 22F, 23B, 23F, 24F, 33F, 35B, 38 and 45. Polysaccharides of Meningococcal serogroup A, B, C, D, W135, X, Y, Z, 29E, H. influenzae type b.
9. The method according to claim 1, wherein the (step ii) involves subjecting the retentate to protein precipitation to obtain a supernatant comprising the

unconjugated carbohydrate only to be further used as a sample.
10. The method according to claim 9, wherein the (step ii) involves protein precipitation using one or more agent selected from the group of trichloroacetic acid, Dimethoxy-4-chloroamphetamine - HCl (DOC-HCl), ethanol, acetone, ammonium sulfate, PEG8000, and diethyl ether.
11. The method according to claim 9 and 10, wherein the (step ii) involves protein precipitation using DOC having concentration in the range of 0.25% to 0.30% (w/v) and titrated by conc. HCl to a pH between 6.2 and 6.9.
12. The method as claimed in any one of the preceding
claim, wherein (step iii) involves subjecting the
carbohydrate to acid or base hydrolysis to obtain a
hydrolysate comprising monomeric unit of carbohydrate.
12. The method according to claim 11, wherein the carbohydrate is subjected to acid or base hydrolysis using one or more agent selected from the group of Hydrochloric Acid (HCl), Trifluoroacetic acid (TFA), Sodium Hydroxide (NaOH), and Hydrogen peroxide (H2O2).
13. The method according to claim 12, wherein the carbohydrate is subjected to acid hydrolysis using one

or more agent selected from the group of Hydrochloric Acid (HCl) and Trifluoroacetic acid (TFA).
14. The method as claimed in any one of the preceding claim, wherein the (step iv) involves chromatography columns comprising polystyrene substrate cross-linked with divinylbenzene.
15. The method as claimed in any one of the preceding claim, wherein the (step iv) involves trap columns comprising ethylvinylbenzene substrate 55% crosslinked with divinylbenzene.
16. The method as claimed in any one of the preceding claim, wherein (step iv) involves chromatography column having 10 µm diameter polystyrene substrate 2% crosslinked with divinylbenzene and agglomerated with 500 nm MicroBead quaternary ammonium functionalized latex.
17. A method for assaying total carbohydrate in a
carbohydrate-protein conjugate vaccine, wherein the
total carbohydrate content is measured by the method of
any preceding claim except (step ii) of claim 1, claim
9, claim 10 and claim 11; the unconjugated carbohydrate
content is measured as described in any one of
preceding claims, and thus the ratio of unconjugated to
total carbohydrate and ratio of conjugated to total
carbohydrate can be calculated.

18. The method as claimed in any one of the preceding claim, determine unconjugated carbohydrate in a meningococcal pentavalent (ACWYX) carbohydrate-protein conjugate vaccine composition comprising atleast two different types of carrier proteins, a dissacharide excipient and salts.
19. The method as claimed in any one of the preceding claim, determine unconjugated Men–X serotype carbohydrate in a meningococcal pentavalent (ACWYX) carbohydrate-protein conjugate vaccine composition comprising atleast two different types of carrier proteins, a dissacharide excipient and salts.
20. The method according to any one of the preceding claims, wherein the said unconjugated saccharide determined can be selected from i) residual saccharide generated across conjugation or conjugate purification processes and ii)degraded saccharide generated on storage of conjugate.