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 PROTEIN IN A CARBOHYDRATE-PROTEIN CONJUGATE COMPOSITION"
SERUM INSTITUTE OF INDIA PRIVATE LIMITED, a corporation 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 invention and the manner in which it is to be performed:

TECHNICAL FIELD:
The present disclosure relates to the field of biotechnology, more particularly to a method for analysis and quality control of polysaccharide-protein conjugate vaccine compositions.
BACKGROUND:
For a vaccine to be effective and induce long lasting immunity, induction of T cell memory is very important. Polysaccharide – protein conjugate vaccines play a pivotal role is inducing such T Cell memory, and hence they are preferred as vaccine antigens over polysaccharide vaccines. The conjugation of bacterial polysaccharide to carrier protein renders the antigenic molecule to undergo the process of antigen presentation, and enhances the polysaccharide immunogenicity by eliciting the T-cell dependent response against that particular bacterium. The carrier proteins used for conjugation with bacterial polysaccharides include CRM197, tetanus toxoid (TT), diphtheria toxoid (DT), Neisseria meningitidis outer membrane complex, Haemophilus influenzae protein D, and Pneumolysin.
The meningococcal meningitis is caused by Neisseria meningitidis, which is an aerobic Gram-negative encapsulated bacterium. To date, more than 10 serotypes of meningococcal have been characterized by differences in the polysaccharide capsule. The polysaccharides from Neisseria meningitidis serotype A, C, Y, W, and X have been conjugated to various carrier proteins to prepare a conjugated vaccine (Menactra®, Menveo®), effective against infection by these serotypes. Similarly for Streptococcus pneumoniae, more than 90 distinct serotypes have been identified throughout the world (WHO); of

which a small number of serotypes accounts for most diseases in infants. Pneumococcal conjugate vaccines containing polysaccharides from 7+ serotypes i.e. Prevnar® & Synflorix® PNEUMOSIL® are already into market. The polysaccharides from Pneumococcal serotype have been conjugated to various carrier proteins i.e. CRM197, Protein D, TT, DT, etc. to prepare a conjugated Polysaccharide-Protein (Ps-Pr) conjugate vaccine against these serotypes.
Polysaccharide-protein conjugate composition are known to be associated with contaminants such as un-reacted polysaccharide (free polysaccharide), un-reacted carrier protein (free protein), low molecular weight conjugates, and other chemicals used for affecting conjugation such as linkers, coupling agents etc. Such contaminants are highly undesirable in a product which is intended for use as a vaccine.
The amount of free polysaccharide and free carrier protein in the vaccine are key quality control specifications [Refer Peeters et al. 1992]. According to the WHO’s recommendations for the production & control of pneumococcal conjugate vaccines, the total protein content of the conjugate should be determined by means of an appropriately validated assay. [Ref: 54th meeting of the WHO Expert Committee on Biological Standardization, 17-21 November 2003]. Similarly, WHO’s Recommendations to assure the quality, safety and efficacy of group A meningococcal conjugate vaccines require each batch of bulk conjugate to be tested for conjugated and unconjugated protein by the methods agreed with the national regulatory authority.
High levels of free protein can indicate poor conjugation efficiency, product degradation caused by manufacturing inconsistencies, formulation, or storage conditions. Thus, particularly it is a statutory requirement to quantify the

concentration of free or unconjugated carrier protein available in final vaccine formulation because it is a key marker of process consistency an indirect measure of covalency, and could interfere with the immune response against the glycoconjugate. [Ref: Brian K. Nunnally, Vincent E. Turula, Robert D. Sitrin; Vaccine Analysis: Strategies, Principles, and Control; Springer-Berlin (2014)].
Various techniques for estimation of protein concentration in a sample are known in the art. The physico-chemical techniques like Lowry method, Bradford method, Biuret method, BCA etc. are the most widely used assays for the estimation of “total protein” concentration. These methods are non-specific and usually estimate total protein content in the final vaccine composition or conjugate bulks irrespective of the type of carrier protein.
Use of micellar electrokinetic chromatography (MEKC) as a routine method for quantitation of free protein for pneumococcal and meningococcal serotypes using DT or TT as carrier protein has been previously reported. However, limitations of the capillary electrophoresis technology still exist, which include poor reproducibility and low throughput. CE technology can be susceptible to instrumentation robustness issues (e.g. capillary performance, instrumentation errors, matrix effects, etc.) and suffers from low sample throughput.
Use of Hydrophobic Interaction Chromatography (HIC) by using Thermo HIC-10 columns for separating and quantifying free carrier protein in Staphylococcus aureus glycoconjugate has been disclosed previously. Same mentions QL for free carrier protein was demonstrated to be 0.056 mg/mL for CP5-CRM197 and 0.028 mg/mL for CP8-CRM197. Range is the region that the analytical procedure provides an acceptable degree of linearity, accuracy and precision when applied to samples

containing amounts of analyte within or at the extremes of the specified range of the analytical procedure. The range for free carrier protein was demonstrated from 0.056 to 0.199 mg/mL (5.7 – 19.8%) for CP5-CRM197 and 0.028 to 0.206 mg/mL (2.8 – 20.9%) for CP8-CRM197. % Recovery (90-94%).
Tetanus toxoid (TT) is a labile protein and when used in conjugate vaccines is polydispersed. TT has inherent tendency to form aggregate over the period of time. Frasch et al. 2015, presence of free PS or TT (100%–400%) had little effect on the potency of the conjugate and also had little effect on the SBA titer of the induced response.
Monitoring free carrier protein poses significant analytical challenges related to low throughput, multiple methods and technologies, sub-optimal resolution, and project-specific methods. Analytical methods used in conjugate vaccine development and production vary among manufacturers due to different components and conjugation chemistries. There is no individual method that can identify all proteins, and of course, a major limitation is the factor sensitivity.
Also, Unconjugated or free protein evaluation in composition comprising Neisseria Meningitidis Serogroup X polysaccharide conjugated to Tetanus toxoid is not known in the prior art.
Hence, there remains a significant need of a simple, accurate, repeatable, fast, convenient and robust assay for estimation of the concentration of unconjugated/free carrier protein in a monovalent or multivalent polysaccharide- protein conjugate composition.

OBJECT OF INVENTION:
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows:
It is an object of the present disclosure to provide a method for determining unconjugated or free carrier protein content in a carbohydrate-protein conjugate composition.
It is an object of the present disclosure to provide a method for determining unconjugated/free Tetanus toxoid (carrier protein) content in a conjugate vaccine composition.
It is an object of the present disclosure to provide a method for determining unconjugated/free Tetanus toxoid (carrier protein) content in vaccine comprising of N. meningitidis serogroup A- TT (Men A – TT) conjugate and N. meningitidis serogroup X- TT (Men X - TT)conjugate vaccine or composition.
It is an object of the present disclosure to provide a method for assessing the quality of the carbohydrate-protein conjugate composition.
It is an object of the present disclosure to provide a simple, accurate, repeatable, fast, convenient, highly sensitive, robust and non-destructive method devoid of any sample processing steps prior to analysis.
Other objects of the present disclosure is to ameliorate one or more problems of the prior art or to at least provide a useful alternative. Other objects and advantages of the present disclosure will be more apparent from the following description, and is not intended to limit the scope of the present disclosure.

The instant method is found to be suitable from 6.25µg/ ml to
100µg/ml i.e. 0.625 % to 10 % for the estimation of free
protein in Men A - TT and Men X - TT Conjugates. The lowest
standard concentration assessed during the development is 6.25
µg/mL i.e. 0.625 %. Our percent recovery is > 80 upto 120%.
SUMMARY OF INVENTION:
The present disclosure provides a method for evaluating unconjugated or free protein in a polysaccharide-protein conjugate composition (test sample) wherein the unconjugated or free protein is Tetanus toxoid (TT). The said method comprises of i) estimation of total protein concentration in a test sample by Folin - Lowry assay; ii) separation of unconjugated or free protein from a test sample by high performance size exclusion chromatography (HPLC-SEC); and iii) evaluation of unconjugated or free protein concentration by Photometric analysis. The method is applicable in evaluation of a unconjugated/free Tetanus toxoid in a monovalent/multivalent conjugate composition(s). The method is simple, accurate, repeatable, fast, robust and convenient and devoid of any sample processing steps prior to analysis.
The said method is found to be suitable from 6.25µg/ ml to 100µg/ml i.e. 0.625 % to 10 % for the estimation of free protein in Men A TT and Men X TT Conjugates. The lowest standard concentration assessed during the development is 6.25 µg/mL i.e. 0.625 %. Our percent recovery is > 80 upto 120%.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING:
The present disclosure will now be described with the help of the accompanying drawing, in which

Figure 1: Specificity of the method for estimation of unconjugated/free protein A: Blank ; B: Test sample
[Conjugate Bulk peak at Retention Time ~ 20 minutes; Free protein peak at Retention Time ~ 30 minutes; Matrix (3% sucrose + 0.5% Sodium Citrate + 10 mM Tris)]
Figure 2: Linearity Assay
Figure 3: Method assurance
Figure 4: Evaluation of total protein concentration in a Carbohydrate-Protein conjugate vaccine composition (test sample).
Figure 5: Evaluation of unconjugated/free protein concentration in a test sample.
DETAILED DESCRIPTION:
Source of Biological Material used in the present disclosure is as follows:

Organism Source
Neisseria meningitidis SynCoBiopartners (Netherlands)
Serogroup A M1027
Neisseria meningitidis CBER/FDA, USA
Serogroup X M8210
Clostridium Tetani (Harvard No Central research Institute
49205) for Tetanus toxoid (CRI), National Control
Authority, Kasauli, Himachal
Pradesh, India. Central
research Institute (CRI)
procured this strain from NVI,
Netherland

The present disclosure relates to a novel and efficient method for evaluation of free carrier protein content in a monovalent or multivalent conjugate composition. As envisaged in the present disclosure, the first step of the method involves evaluating the concentration of total carrier protein in the monovalent or multivalent conjugate preparation by Folin-Lowry protein assay, further followed by estimating the concentration of free carrier protein in the monovalent or multivalent conjugate composition by HPLC-SEC. Wherein the percentage of unconjugated free carrier protein is calculated by dividing the amount of free protein detected by HPLC-SEC by the total amount of protein quantified in the sample by Lowry protein assay. The method of the present disclosure overcomes the limitations of physico-chemical techniques such as Bradford method, Biuret method, DOC HCl precipitation, and other electrophoretic techniques such as SDS-PAGE, MEKC, CZE, for protein quantification and helps to determine the concentration of the “free carrier protein” in a monovalent or multivalent conjugate composition.
In an embodiment of present disclosure, the method is used to evaluate the percentage of free or unconjugated carrier protein in a monovalent or multivalent polysaccharide protein conjugate composition, wherein one or more polysaccharides are conjugated to same type of carrier protein.
In one of the aspect of this embodiment, the monovalent or multivalent polysaccharide protein conjugate composition comprises of one or more polysaccharide selected from group comprising of gram positive and gram-negative bacteria. In particular, the polysaccharide may be derived from the group of bacteria comprising of Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum,

Mycoplasma pneumoniae, Staphylococcus spp., Staphylococcus aureus, Streptococcus spp., Group A Streptococcus, Group B Streptococcus (group Ia, Ib, II, III, IV, V, VI, VII, VII, VIII, and IX) Streptococcus pyogenes, Streptococcus agalactiae , Streptococcus pneumoniae, Streptococcus viridans, Enterococcus faecalis, Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis, Salmonella spp., Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella typhimurium, 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, Haemophilus influenzae type B, Escherichia coli, Shigella spp., Ehrlichia spp., and Rickettsia spp. Polysaccharides of Streptococcus pneumoniae serotype type 1, 2, 3, 4,5,6, 6A, 6B, 6C, 6D,6E, 6G, 6H, 7A, 7B, 7C,7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13,14, 15A,15B, 15C, 15F, 16A, 16F, 17A,17F, 18,18C, 18F, 18A, 18B, 19F, 19A, 19B, 19C, 20, 20A, 20B, 21, 22A, 22F, 23A, 23F, 23B, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32A, 32F, 33A, 33B,33C, 33D, 33E, 33F, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, and 48; Neisseria meningitidis serotype A, B, C, D, H, I, K, L, M, W135, X, Y, Z, 29E; etc. In a preferred aspect of this embodiment, the polysaccharide is derived from Neisseria meningitidis serotype A, or Neisseria meningitidis serotype X. In other preferred aspect of this embodiment, the polysaccharide is derived from Streptococcus Pneumoniae.
It is understood by a person skilled in the art, the test sample may comprise of monovalent or multivalent conjugate

composition, wherein multivalent polysaccharide-protein conjugate may comprise of polysaccharide from one or more bacterial species.
In one of the aspect of this embodiment, the monovalent or multivalent polysaccharide protein conjugate composition comprises of one or more carrier proteins selected from group comprising of CRM197, diphtheria toxoid, Neisseria meningitidis outer membrane complex, fragment C of tetanus toxoid, tetanus toxoid, pertussis toxoid, flagellin (FliC), cholera toxin B subunit (CTB), 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), synthetic peptides, heat shock proteins (HSPs), pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen-derived antigens such as N 19, iron-uptake proteins, fHbp, Porins (Por A) toxin A or B from C. difficile and S. agalactiae. In a preferred aspect of this embodiment, the carrier protein is Tetanus Toxoid.
In an embodiment of present disclosure, the total protein concentration in a monovalent or multivalent conjugate composition is evaluated by Folin-Lowry assay. The evaluation process of the present embodiment can be modified as per requirement by the person skilled in the art. The other protein assays known in art to measure the total protein

concentration test sample may also be used by the person skilled in the art.
In an aspect of this embodiment, the folin-lowry assay is optimized to determine the total carrier protein concentration in a monovalent or multivalent conjugate composition wherein the composition comprised of one or more different types of bacterial capsular polysaccharides conjugated to a carrier protein. In a preferred aspect of this embodiment, the folin-lowry assay is optimized to determine the total Tetanus toxoid concentration in a monovalent or multivalent conjugate composition.
In other aspect of this embodiment, the folin-lowry assay is optimized to determine the total Tetanus toxoid concentration in a monovalent conjugate composition (test sample) comprising of Neisseria meningitidis serotype A polysaccharide conjugated to Tetanus toxoid.
In other aspect of this embodiment, the folin-lowry assay is optimized to determine the total Tetanus toxoid concentration in a monovalent conjugate composition (test sample) comprising of Neisseria meningitidis serotype X polysaccharide conjugated to Tetanus toxoid.
In other aspect of this embodiment, the folin-lowry assay is optimized to determine the total Tetanus toxoid concentration in a multivalent conjugate composition (test sample) comprising of Neisseria meningitidis serotype A polysaccharide conjugated to Tetanus toxoid; and Neisseria meningitidis serotype X polysaccharide conjugated to Tetanus toxoid;
In another aspect of this embodiment, the monovalent or
multivalent conjugate composition (test sample) further
comprises of excipient. In preferred aspect of this
embodiment, the excipient in a test sample comprises of one or

more agents selected from sucrose, Sodium citrate and Tris. In more preferred aspect of this embodiment, excipient comprises of 3% sucrose, 0.5% Sodium citrate and 10 mM Tris. In an aspect of this embodiment, the monovalent or multivalent conjugate composition (test sample) is devoid of excipient.
In an embodiment of present disclosure, the unconjugated or free carrier protein in a monovalent or multivalent polysaccharide - protein conjugate preparation is separated using High Performance Liquid Chromatography – Size exclusion chromatography (HPLC SEC). Size exclusion chromatography (SEC), otherwise known as gel filtration or gel permeation chromatography, relies on the penetration of macromolecules in a mobile phase into the pores of stationary phase particles. Differential penetration of the macromolecules is a function of the hydrodynamic volume of the particles. Size exclusion media exclude larger molecules from the interior of the particles while the smaller molecules are accessible to this volume. The order of elution can be predicted by the size of the protein as a linear relationship exists between elution volume and the log of the molecular weight of the protein being eluted.
In an aspect of the present embodiment, the size exclusion chromatographic resin used may be described in terms of pore size, wherein the pore size of the resin selected is between the range of 1 nm and 150 nm. In the preferred aspect, pore size of the size exclusion chromatographic resin selected is between 10 and 20 nm.
In an aspect of the present embodiment, the size exclusion chromatographic resin used in high performance size exclusion chromatography is based on agarose or polyacrylamide beads, and polymeric resins for medium-pressure systems. In a

preferred aspect of this embodiment, size exclusion chromatography resin used in high performance size exclusion chromatography is polyhydroxymethacrylate based resin.
In an aspect of the present embodiment, the size exclusion chromatographic column used in high performance size exclusion chromatography includes but is not limited to Phenomenex Yarra 3µm SEC-2000, Phenomenex Yarra 3µm SEC-3000, Phenomenex Yarra 3µm SEC-4000, Phenomenex Bio-Sep-SEC-S2000, Phenomenex Bio-Sep-SEC-S3000, Phenomenex Bio-Sep-SEC-S4000, Shodex™, HiPrep SEPHACRYL S100 HR, HiPrep SEPHACRYL S-200 HR, HiPrep SEPHACRYL S-300 HR, TSKgel SuperSW2000, TSKgel SuperSW3000, TSKgel UP-SW3000, TSKgel G5000PWXL, TSKgel G3000SWXL, TSKgel Ultra SW Aggregate.
In a preferred aspect of this embodiment, the size exclusion chromatographic column used in high performance size exclusion chromatography is a Shodex OHpak SB-800 HQ series column selected from the group comprising SB-802 HQ, SB-802.5 HQ, SB-803 HQ, SB-804 HQ, SB-805 HQ, SB-806 HQ, SB-806M HQ. The packing material for Shodex OHpak SB-800 HQ series resin is Polyhydroxymethacrylate based. In a preferred aspect the Shodex OHpak SB-800 HQ series resin is Shodex OHpak SB-804 HQ.
In an aspect of this embodiment, the mobile phase buffer used in the HPLC-SEC process is selected from phosphate buffer saline, Tris, MES, HEPES, citrate or combination thereof having a pH in the range 5.0 to 7.5. In preferred aspect, the mobile phase buffer used in the process is a phosphate buffer saline having a pH in the range 5.0 to 7.5. Yet another preferred aspect, wherein the mobile phase buffer is preferably a phosphate buffer saline having a pH 7.4.
In one of the aspect of present embodiment, the marker mix is selected from Salmon DNA, Thyroglobulin, BSA, Carbonic Anhydrase and Tyrosine having concentration in range of 0.2 –

1 mg/ml. Yet the preferred marker mix is selected from Salmon DNA, Thyroglobulin, BSA, Carbonic Anhydrase and Tyrosine having concentration of 0.5 mg/ml.
In one of the aspect of present embodiment, the loading of samples can be performed at a flow rate in the range 0.2 – 1 ml/minute. In yet another aspect, the loading of samples can be performed at flow rate in the range 0.2 – 0.8 ml/minute, 0.2 – 0.7 ml/minute, 0.2 – 0.6 ml/minute, 0.2 – 0.5 ml/minute, or 0.2 – 0.4 ml/minute. In one of the preferred aspect, the loading of samples is performed at a flow rate in the range 0.2 – 0.4 ml/minute.
In one of the aspect of present embodiment, preferred temperature of auto sampler may be between 22°C and 32°C. Yet more preferred auto sampler temperature is 25°C.
In one of the aspect of present embodiment, the injection volume of the sample can be in the range 100µl - 200µl. Yet the preferred injection volume of the sample is 100µl.
In one of the aspect of present embodiment, the method is a high performance size exclusion chromatography method (HPLC-SEC) in which the sample is delivered through a column resin and wherein the column is run at a temperature in between 25°C - 32°C. Yet the preferred column temperature is 30°C.
In one of the embodiments of the present disclosure, the eluate obtained by subjecting the test sample to high performance size exclusion chromatography is evaluated using photometric analysis comprising of refractive index detectors, wavelength absorbance detectors (spectroscopy), diode array detectors, chiral detectors, chemiluminescence detectors, circular dichroism detectors, light scattering detectors and fluorescence emission detectors. In a preferred aspect of this embodiment, wavelength absorbance detectors (spectroscopy) is

used to evaluate the elute obtained by HPLC-SEC. In one of the most preferred aspect of this embodiment, the detection is carried out at a wavelength in the range of 190 to 800 nm, more particularly 280 nm.
In one of the embodiments of the present disclosure, the photometric analysis is used to evaluate the concentration of unconjugated/free protein in a test sample. In one of the aspect of present embodiment, the percentage of unconjugated or free protein in a test sample is calculated using total amount of protein quantified in the test sample as reference. The total carrier protein concentration is used to determine the final percentage of unconjugated or free carrier protein in test sample. In an aspect of this embodiment, the concentration of unconjugated protein is in range between 6.25 µg/mL to 100 µg/mL.
According to one of the preferred embodiment, instant method is found to be suitable from 6.25µg/ ml to 100µg/ml i.e. 0.625 % to 10 % for the estimation of free protein in Men A TT and Men X TT Conjugates. The lowest standard concentration assessed during the development is 6.25 µg/mL i.e. 0.625 %. Our percent recovery is > 80 upto 120%.
TECHNICAL ADVANTAGES:
The present disclosure described herein above has several technical advantages including, but not limited to, the realization of an assay that estimates the free carrier protein content in a monovalent conjugate bulk. The method of the present disclosure provides the following advantages:
• The method is simple; and has reduced turnaround time per test sample.

• No sample pre-treatment is required prior to analysis (no sample filtration, no sample digestion, no heating, no voltage based separation, no specific pH requirement)
• The method demands simple buffer i.e. PBS pH 7.4;
• Low sample volume required for analysis;
• Keeps the same work flow as ELISA;
• Since the method doesn’t require any kind of sample pre-treatment prior to analysis and its stability is not affected at any stage of method the percentage recovery of the protein at the end of the method is quite high and hence the method provides accurate results consistently.
• The method has an advantage wherein the molecular weight of the free protein and the bacterial capsular polysaccharide conjugate has a large difference, hence the separation of free protein and bacterial polysaccharide protein conjugate by using size exclusion high performance liquid chromatography method, provided well separated peaks. This is essential for reliable quantitation. The carrier protein with the known concentration is used as a standard, so that the free protein content in the bacterial polysaccharide conjugate can be accurately determined.
• The presence of high concentration of excipient doesn’t affect with the determination of free or unconjugated protein content in a conjugate bulk, hence doesn’t affect the final outcome of the results.
• The Lowry protein assay is simplified method which provides reliable and stable results of total protein concentration.
• The Shodex OHpak SB-800HQ series packed with polyhydroxymethacrylate gels are designed for use with high-resolution, high-speed aqueous size exclusion chromatography which provide high capacity even for

larger proteins due to polymer matrix with large pores, sharper peaks, less sample loss, no unwanted silanol interactions, high thermal and chemical stability, high recovery rates, and easy scaleup.
• The method is applicable for quantifying labile carrier proteins like tetanus toxoid
• Instant method is found to be suitable from 6.25µg/ ml to 100µg/ml i.e. 0.625 % to 10 % for the estimation of free protein in Men A TT and Men X TT Conjugates. The lowest standard concentration assessed during the development is 6.25 µg/mL i.e. 0.625 %. Our percent recovery is > 80 upto 120%

EXAMPLES:
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skilled in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1: The instant method was designed to evaluate the concentration of unconjugated protein in the following Carbohydrate-Protein conjugate vaccine composition:

Table 1:
Pentavalent Meningococcal Conjugate Vaccine (0.5 ml dose)
Component Quantity
Men A Polysaccharide* 6 μg
Men C Polysaccharide # 6 μg
Men Y Polysaccharide # 6 μg
Men W Polysaccharide # 6 μg
Men X Polysaccharide * 6 μg
Sucrose 3 %
Sodium citrate (dihydrate) 0.5%
Tris (Trometamol) 10 mM
* - Conjugated to Tetanus Toxoid (TT);
# - Conjugated to CRM197

Table 2:
Monovalent Meningococcal A Conjugate Vaccine (Test sample
2)
Component Quantity
Men-A Polysaccharide conjugtated to Tetanus toxoid (TT) TT concentration to be determined
Sucrose 3 %
Sodium citrate (dihydrate) 0.5%
Tris (Trometamol) 10 mM

Table 3:
Monovalent Meningococcal X Conjugate Vaccine (Test
sample 3)
Component Quantity
Men-X Polysaccharide conjugtated to Tetanus toxoid(TT) TT concentration to be determined
Sucrose 3 %
Sodium citrate (dihydrate) 0.5%
Tris (Trometamol) 10 mM
The above mentioned compositions were evaluated for unconjugated/free protein concentration.
Example 2: Evaluation of total protein concentration in a Carbohydrate-Protein conjugate vaccine composition (test sample)
The total protein concentration of the compositions (test samples) was evaluated using Folin-lowry assay. The assay details are as follows:

1. Prepare standards (6.25–50.0 µg) in duplicate in Microcentrifuge test tube as shown below table by using 1 mg/mL Protein standard (Micro Standard-Liquid) (Bovine serum albumin) (Use fresh ampoule each time).

Table 4:
S.No. 1 mg/mL Protein standard
(Micro Standard-Liquid)
(BSA) ( µL) MQW (µL) Total
Volume
(µL)
1 0 400 400
2 12.5 387.5 400
3 25 375 400
4 50 350 400
5 75 325 400
6 100 300 400
2. From the above mentioned standard preparation Step 1,
transfer 200 µL standards (in duplicate) in
Microcentrifuge tube, to make working standards
concentration as mentioned in below table.

Table 5:
S.No. Standard Concentration
(µg) Working standard (µl)
1 Blank 0 200
2 S1 6.25 200
3 S2 12.5 200
4 S3 25 200
5 S4 37.5 200
6 S5 50 200
3. Take 200 µL of the prepared sample for analysis.
4. Take 100 µL of QC standard in duplicate and add 100 µL of MQW to it in microcentrifuge test tube.
5. Add 1 mL of reagent D (cupric-tartaric solution) to all the tubes i.e. standards, QC standard and sample. Vortex the tubes gently and briefly.

6. Incubate the standards, QC standard and sample for 10 minutes at room temperature.
7. Add 100 µL of reagent E (Folin- Ciocalteu’s phenol reagent) to all the tubes. Vortex the tubes gently and briefly.
8. Incubate the standards, QC standard and sample for 30 minutes at room temperature.
9. The samples were read on a 96 well plate by measuring the optical density of each sample within 30 minutes of completion of the assay.
Results & Interpretation:
Sample Preparation: MenX-TT conjugate bulk with sucrose, sodium citrate and Tris. Take 100 µL of MenX sample and diluted upto 1000 µL with MQW (Dilution factor is 10).

Standard/Sample ID Concentration (µg) Absorbance at 750nm
Blank 0.000 0.000
Blank-2 0.000 0.000
Blank-Average 0.000 0.000
Std 1 6.250 0.099
Std 1-2 6.250 0.103
Std 1-Average 6.250 0.101
Std 2 12.500 0.186
Std 2-2 12.500 0.188
Std 2-Average 12.500 0.187
Std 3 25.000 0.333
Std 3-2 25.000 0.342
Std 3-Average 25.000 0.337
Std 4 37.500 0.469
Std 4-2 37.500 0.486
Std 4-Average 37.500 0.478
Std 5 50.000 0.601
Std 5-2 50.000 0.604
Std 5-Average 50.000 0.602
Test Sample 0.301
Test Sample-2 0.305
Test Sample -Average 0.303

Note:
Correlation coefficient (r2) = 0.99484
Dilution factor: 10
Aliquot: 100 µL ( sample volume taken for analysis)
Protein content (mg/mL) = Concentration * dilution/ Aliquot
Total Protein content (mg/mL) = 2.19 mg/mL
The test complies with the assay validity criteria and the protein values obtained for the samples are well within the predefined limit. The test confirms the accurate estimation of the protein content in test samples (Refer Figure 4).
Example 3: Evaluation of unconjugated/free protein concentration in a Carbohydrate-Protein conjugate vaccine composition (test sample).
The unconjugated/free protein concentration of the compositions (test samples) was evaluated using HPLC-SEC followed by photometric analysis. HPLC-SEC resulted in the separation of the conjugated and free protein. The free protein was then estimated using photometric analysis.
Test Sample: MenX-TT conjugate bulk with sucrose, sodium citrate and Tris. The concentration of the sample for the analysis is 1 mg/mL based on protein content.
The details are as follows:
1. Working standards were prepared from 1 mg/mL from concentrated Purified Tetanus toxoid. PBS buffer was used to prepare these standards. The concentrations prepared were as follows:

Table 6:

Standard (µg/ml)
10 % 100
7.5 % 75
5 % 50
2.5 % 25
1.25 % 12.5
0.63 %(LOQ) 6.3
2. The working standards, test sample, & marker mix were subjected to HPLC-SEC using Column “Shodex OH Pak 804 HQ”.
3. Process parameters were set as follows:

■ Chromatography system: High Performance Size Exclusion Chromatography System (Waters/Thermo scientific)
■ Auto sampler Temperature: Upto 25°C
■ Flow rate: 0.3 mL/min
■ Injection Volume: 100 µL
■ Run Time: Upto 60 Minutes
■ Column: Shodex OH Pak SB-804 HQ
■ Column temperature: upto 30°C
■ Detection: PDA/UV.
■ Wavelength: 280 nm
■ Store the column by passing through 0.05% sodium azide.
■ Test Sample Preparation: Dilute sample in suitable volume of mobile phase to get approximately 1.0 mg/mL.
■ Working Standard: 1 mg/mL In-house produced Concentrated Purified Tetanus toxoid was used as a standard.
■ Internal control source: During the method development study the markers procured from Sigma i.e. Thyroglobulin, BSA, Carbonic Anhydrase and tyrosine was analyzed for resolution factor.
4. The readings were noted and the unconjugated/free protein
concentration of the compositions (test samples) was
determined based on the area of the standards obtained.

{The eluate obtained from the HPLC-SEC was subjected to photometric analysis to estimate the free protein in the test sample.}
Results and Interpretation:
Table 7:

Standard Concentration (µg/mL) Area @ 280nm
Blank 0.000 0.000
Std 1 6.25 145005
Std 2 12.5 290545
Std 3 25 554669
Std 4 50 978108
Std 5 75 1658414
Std 6 100 2201373
Test Sample 69333
Note: Correla tion coefficient (r2) = 0.995
The obtained chromatograms for standards were integrated to get the final area of an intended peak i.e. area of different concentration of TT standards which was used to establish linearity plot. The sample i.e. free protein area obtained is plotted against the standard curve and derives the concentration.
The unconjugated or free protein concentration was found to be 0.014 mg/mL
The test complies with the assay validity criteria and the protein values obtained for the samples are well within the proposed limit. The test confirms the accurate estimation of the free protein content in Test sample i.e. Men X-TT conjugate composition. Refer figure 5.

Example 4: Evaluation of percent unconjugated/free protein concentration in a Carbohydrate-Protein conjugate vaccine composition (test sample).
The percent unconjugated/free protein concentration in a Carbohydrate-Protein conjugate vaccine composition (test sample) was determined using following formula:

Example 5: Method assurance data wherein conjugated protein and unconjugated/free protein is differentiated
The method was explored to separate out unconjugated/free protein from test samples i.e. monovalent MenA-TT and MenX-TT. The polysaccharide (100-150 KDa) is conjugated to Tetanus Toxoid (150 KDa) to generate intended polysaccharide protein conjugate molecules which is bigger in size as compare to its starting material i.e. TT. In this method the molecules in solution were separated by their size. The conjugates molecules i.e. Ps + Pr were expected to elute out first followed by small molecules i.e. free Tetanus Toxoid (TT).
The big conjugate bulk molecules i.e. conjugated protein gets eluted at ~ 20 minutes as compare to that of the unconjugated protein molecule (Free protein) which gets eluted at ~ 30 minutes. This separation indicates the conjugated protein and

unconjugated protein is well differentiated from each other. Additionally, the sample was spiked with known amount of unconjugated protein and analyzed which also clearly indicate the differentiation and separation of conjugated protein and unconjugated protein with the good free TT recoveries. The graphical presentation provided below further proves the same.
Example 6: The method was validated for Specificity, Linearity and range, Limit of detection, Limit of Quantitation/Sensitivity, Precision and Accuracy.
■ Linearity:
The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.
Multiple assays were run over the period during the product development to establish the linearity and range. The details of the observed linearity and range are mentioned in the below table. (Area of individual standard along with correlation coefficient is presented below)

Table 8:
Std. conc. (µg/mL) Assay 1 Assay 2 Assay 3

Area RT Area RT Area RT
6.25 75660 29.527 145005 29.597 70353 29.563
12.5 199709 29.530 290545 29.570 163736 29.498
25 483629 29.566 554669 29.536 389302 29.516

50 1114473 29.536 978108 29.511 1034973 29.528
75 1712085 29.572 1658414 29.533 1482506 29.530
100 2196501 29.565 2201373 29.546 2129979 29.540
R2 0.998 - 0.995 - 0.996 -
The obtained R2 (correlation coefficient) values confirms the method is linear from standard 6.25 µg/mL to 100 µg/mL for the estimation of unconjugated/free protein in MenA-TT and MenX-TT Conjugates. Method was found to be suitable from 6.25µg/ ml to 100µg/ml i.e. 0.625 % to 10 %. Refer Figure 2.
This figure represents the standard linearity curve which is plotted using two variables i.e. area of standard on Y axis and known standard concentration on X-axis. The plot shows the correlation between the concentrations. By obtaining the correlation coefficient of more than or equal to 0.990 indicate the method is linear using the mentioned standard concentration.
■ Specificity:
Specificity is the ability to assess unequivocally the analyte in the presence of components, which may be expected to be present. Typically these might include impurities, degradants, matrix, etc. The method under discussion was demonstrated for the estimation of unconjugated/free protein in MenA-TT and MenX-TT conjugates as test sample. The test sample contains sucrose, Tris and sodium citrate. In this study during the method development, the sample matrix components i.e. sucrose, Tris and sodium citrate assessed at the optimized set method conditions. Concentrated Tetanus Toxoid (CTT) was used as positive control. The retention time of positive control, i.e. CTT is obtained at ~ 30 minutes whereas the conjugate bulk is eluted way before it i.e. at 20 minutes. In addition, No peak

observed of Sucrose, Tris and sodium citrate (Matrix) at the retention time of positive control i.e. CTT. This confirms the method is specific to estimate unconjugated/free protein. In addition, the test sample in this method analyzed are present in their final matrix and accuracy studies are performed by spiking the sample with known amount of analyte; recovery of the known amount of analyte further confirms the specific quantitation of unconjugated/free protein. Determination of unconjugated/free protein is done using UV/PDA detector. For the product related impurity like free polysaccharide, the choice of detector is RI and not UV/PDA. Hence, it is not expected to show any response in UV/PDA detector. Further, the absence of peak at the retention time of TT in RI confirms it.
This confirms the specificity of the method for the estimation of unconjugated/free protein in MenA-TT and MenX-TT Conjugates. Please see Figure 1
Figure 1 shows the buffer blank injection and the separation of unconjugated or free TT from the components present in the test samples i.e. conjugate bulk and excipient. This confirms the sample separation and specificity of the method.
■ Limit of Detection (LOD) And Limit Of Quantitation(LOQ) / Sensitivity
The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products.

The lowest standard concentration assessed during the development is 6.25 µg/mL i.e. 0.625 %, at this concentration the linearity of the standard based on area obtained was significant and maintains the linearity of the curve wrt to R2. This indicates that 6.25 µg/mL (lower standard) can be used and further opens the scope to go below the 6.25 µg/mL to estimate limit of quantitation. The method was able to quantitate the sample at 0.63% also, which prove the sensitivity of the method. The lowest standard i.e. 6.25 µg/mL was considered.
The data is presented below -

Table 9:
Details Standard Standard (µg/ml) Response Area
10 % 100 2129979
7.5 % 75 1482506
5 % 50 1034973
2.5 % 25 389302
1.25 % 12.5 163736
0.63 %(LOQ) 6.3 70353
Precision:
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility.
Intermediate precision:

Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc. These experiments are compiled to check the variation obtained by different analyst, different days, and different batches. (I.e. intermediate precision and reproducibility).MenA-TT and MenX-TT monovalent conjugate bulks were analyzed for unconjugated/free protein estimation. Each batch of both the serogroups (Men A and Men X) analyzed for free protein at least three times during the method development studies.
Note: No detectable free portion was observed in analyzed Men A-TT and X-TT. Therefore, the lowest standard was considered for the calculation and the result presented as per the table

Table 10
Details Assay 1 Assay 2 Assay 3
MenA-TT < 0.63 % < 0.63 % < 0.63 %
MenX-TT < 0.63 % < 0.63 % < 0.63 %
In addition, during the development all the samples were spiked with 1.5 % Free Tetanus toxoid spike and analyzed for recoveries. This indicated and supported the precision of the method.
ACCURACY:
The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. This is sometimes termed trueness. In accuracy studies, for both the serogroups, the monovalent conjugate bulk was spiked with 1.5% (i.e. 15µg in 1000 µg/mL of sample), 5% (i.e. 50µg in 1000 µg/mL of sample) and 9% (i.e. 90µg in 1000 µg/mL of sample) of CTT and analyzed for

unconjugated/free protein estimation. The recovery obtained for the serotypes was in the range of 80 to 120%.
The data is presented below:

Table 11
Spike % Test Sample % Recovery
1.5 MenA-TT Sample 1 97
5
103
9
99
1.5 MenA-TT Sample 2 80
5
108
9
106
1.5 MenX-TT Sample 1 100
5
104
9
102
1.5 MenX-TT Sample 2 106
5
108
9
106

We Claim:
l.A method for evaluating a carbohydrate-protein conjugate composition (test sample), comprising:
a. Subjecting the said test sample to protein assay to
determine the total protein concentration;
b. Subjecting the said test sample to dilution using a
buffer to 50-100 μl/ml of total protein concentration as
determined in step (a) ;
c. Subjecting the sample obtained in step (b) to a high
performance size exclusion chromatography (HPLC-SEC) to
obtain an eluate consisting of unconjugated protein;
d. Subjecting the said eluate consisting of unconjugated
protein to Photometric analysis, and evaluating the
eluate;
wherein, the method evaluates the unconjugated protein concentration in a test sample.
2. The method of claim 1, wherein the eluate is evaluated using
one or more of the following:
a. evaluating the unconjugated protein concentration by
determining the area of the unconjugated protein
containing peak or peaks;
b. evaluating the quality of the carbohydrate-protein
conjugate by assessing the symmetry of the carbohydrate-
protein conjugate containing peak or peaks;
c. evaluating the purity of the carbohydrate-protein
conjugate by dividing the area of the carbohydrate-protein
conjugate containing peaks by the area of all peaks of the
graphical representation.
3. The method of claim 1, wherein the carbohydrate is a natural or synthetic carbohydrate, polysaccharide (Ps), oligosaccharide (Os), or combination thereof.
4. The method of claim 1, wherein the carbohydrate is a bacterial capsular polysaccharide, and is obtained from

group comprising of Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus spp., Staphylococcus aureus, Streptococcus spp., Group A Streptococcus, Group B Streptococcus, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Enterococcus faecalis, Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis, Salmonella spp., Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella typhimurium, 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.. 5. The method of claim 4, wherein carbohydrate is obtained from one or more of the following:
a. Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6, 6A,
6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F,
9N, 9V, 10F, 10B, 10C, 10A, 11 A, 11F, 11B, 11C, 11D, 11E,
12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F,16A, 16F, 17A,
17F, 18, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A,
20B, 21, 22A, 22F, 23A,23B, 23F, 24A, 24B, 24F , 25F, 25A,
27, 28F, 28A, 29, 31, 32F, 32A, 33A, 33C, 33D, 33E, 33F,
33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38,
39,40,41F,41A,42,43,44,45, 46, 47F, 47A, 48.
b. Neisseria meningitidis serotypes A, B, C, D, W135, X, Y,
Z, and 2 9E.
c. Haemophilus influenzae type b.

d. Salmonella spp. including salmonella typhi, salmonella
paratyphi A, salmonella paratyphi B, salmonella paratyphi
C, salmonella typhimurium, and salmonella enteritidis.
e. Streptococcus spp. including Group A Streptococcus and
Group B Streptococcus, Streptococcus group (Ia, Ib, II,
III, IV, V, VI, VII, VIII, and IX.).
6. The method of claim 1, wherein the carbohydrate-protein conjugate sample to be evaluated comprises unconjugated protein and/or conjugated protein selected from the group of CRM197, diphtheria toxin/toxoid, Neisseria meningitidis outer membrane complex, fragment C of tetanus toxoid, tetanus toxin/toxoid, pertussis toxin/toxoid, flagellin (FliC), cholera toxin B subunit (CTB), protein D of H. influenzae, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins( Por A), 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), NTHi high molecular weight protein, fHbp and purified protein derivative of tuberculin (PPD).
7. The method of claim 1, wherein the method evaluates unconjugated tetanus toxoid concentration in a test sample.
8. The method of claim 1, wherein the HPLC-SEC is performed using columns selected from the group conmprising of Phenomenex Yarra 3µm SEC-2000, Phenomenex Yarra 3µm SEC-3000, Phenomenex Yarra 3µm SEC-4000, Phenomenex Bio-Sep-SEC-S2000, Phenomenex Bio-Sep-SEC-S3000, Phenomenex Bio-Sep-SEC-S4000, Shodex™, HiPrep SEPHACRYL S100 HR, HiPrep SEPHACRYL S-200 HR, HiPrep SEPHACRYL S-300 HR, TSKgel SuperSW2000,

TSKgel SuperSW3000, TSKgel UP-SW3000, TSKgel G5000PWXL, TSKgel G3000SWXL, and TSKgel Ultra SW Aggregate.
9. The method of claim 1, wherein the HPLC-SEC is performed
using polyhydroxymethacrylate based chromatography column
having pore size between 5 and 150 nm.
10. The method of claim 1, wherein the HPLC-SEC is performed using columns is a Shodex OHpak SB-800 HQ series column selected from the group comprising SB-802 HQ, SB-802.5 HQ, SB-803 HQ, SB-804 HQ, SB-805 HQ, SB-806 HQ, SB-806M HQ; preferably Shodex OHpak SB-804 HQ .
11. The method of claim 1, wherein the Photometric analysis is a absorption spectroscopy using wavelength between 190 nm and 800 nm.
12. The method of claim 1, wherein the Photometric analysis is refractive index measurement.
13. The method of claim 1, wherein the Photometric analysis is diode array detection.
14. The method of claim 1, wherein the Photometric analysis is fluorescence emission detection.
15. The method of claim 1, wherein the method evaluates the concentration of unconjugated tetanus toxoid;
Wherein the concentration of unconjugated tetanus toxoid is in range between 6.25 µg/mL to 100 µg/mL.
16. The method of claim 1, wherein the buffer used in the HPLC-SEC process is selected from phosphate buffer saline, Tris, MES, HEPES, citrate or combination thereof having a pH in the range 5.0 to 7.5.
17. The method of claim 1, wherein the buffer is Phosphate buffer saline.