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 LOW AMOUNT OF UNCONJUGATED PROTEIN IN A SAMPLE COMPRISING
POLYSACCHARIDE-PROTEIN CONJUGATE"
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 samples, particularly intermediate bulk & vaccine compositions.
BACKGROUND OF THE INVENTION:
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 account 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.
Further, for Haemophilus influenzae conjugate vaccine, polyribosylribitol phosphate (PRP) isolated from the Hib capsule is conjugated to a carrier protein. Four different types of carrier protein have been used – diphtheria toxoid (PRP-DT), tetanus toxoid (PRP-TT), CRM197 (a non-toxic variant of diphtheria toxin HbOC), and the outer membrane protein complex of

serogroup B Neisseria meningitidis (PRP-OMP). Thiomersal and adjuvants have been used in some preparations. Vaccines in which poly(ribose-ribitol-phosphate) from H. influenzae (HIB) is conjugated to a suitable carrier have been licensed for use in infants in many countries (eg. ProHIBIT, HibTITER, Hiberix, OmniHIB, ActHIB, VaximHib, PedvaxHIB, Sii HibPro, BioHib, PedaHib), and have dramatically reduced the incidence of HIB disease worldwide. Considering the success of HIB vaccines, it is not surprising that vaccine manufacturers are actively pursuing development and licensure of conjugate vaccines against other bacterial pathogens. Linking polysaccharides to protein carriers produces vaccines with heterogeneous populations of large molecules. However, sources of heterogeneity include variability in the length and number of polysaccharide chains attached to individual carrier protein molecules and often, variability in the number of attachment sites for each polysaccharide chain.
Polysaccharide-protein conjugate(s) 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. High levels of unconjugated carrier protein hinder the selective immune response including shortened antibody persistence leading to reduced vaccine potency [Lee LH, Blake MS. Clin Vaccine Immunol 2012;19:551–6]. The intrinsic heterogeneity of toxoids and conjugate vaccines provides challenges for biochemical characterization by traditional methods. Hence, analysis of stability indicating parameters like % free protein has become a critical quality attribute and needs to be quantified and monitored in the purified conjugate (drug substance) and formulated drug product.
The amount of free polysaccharide and free carrier protein in the vaccine are key quality control specifications. 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].
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 which include ion exchange chromatography, capillary electrophoresis and indicate

the amounts of group A meningococcal polysaccharide and carrier protein contained in each single human dose labelled on the carton or the leaflet accompanying the container. [Ref: WHO Expert Committee on Biological Standardization Fifty-seventh report; WHO Technical Report Series No. 962, 2011].
As per WHO (Refer WHO TRS 897) and Indian Pharmacopoeia (Refer Indian Pharmacopoeia (IP 2022)) for licensed conjugate vaccine, there is a strong/must have requirement for the estimation of free(unconjugated/unbound) protein less than or equal to 1%, to which most of the other vaccine manufacturers are proving based on the dilution theories or by showing and monitoring the conversion of individual carrier protein and polysaccharide into the formation of conjugate (conjugation reaction) due to lack of the methodology to estimate such a low amount of free protein. WHO (Refer WHO TRS 897) specifications also suggest measuring unconjugated protein in Hib conjugate bulks
Quantification of unconjugated polysaccharide (free polysaccharide) and protein (free protein) in the purified conjugates and formulated final product are important quality control parameters. In addition, the bulk conjugates are monitored for free protein as stability indicating parameter. Since the purified conjugates contain low amounts of free protein, highly sensitive methods are needed.
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 an intermediate conjugate bulk as well as 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)].
The regulatory requirement/specification itself is very stringent i.e. Free protein shall be less than 1%. This itself invites for an analytical method which provides an ultrasensitive estimation. The method has to be quantitative i.e. to be able to provide absolute concentration of free protein in sample. The method has to be sensitive, specific and able to distinguish between total or conjugated protein from free protein. Excipient interference should not be a concern and affect overall quantitation of free/unconjugated protein. To maintain the sample in its native form is very important so that the free protein being estimated is a true estimation

i.e. not derived as a result of sample processing by any means physical/chemical. No sample processing is preferred or encouraged because sample processing can have an effect on conjugated sample also which in turns resulting into generation of free protein from the conjugated part and will be difficult to differentiate between presence of actual free protein and generated free protein during analysis leading to false results.
Conjugation chemistry which generates large and labile conjugates makes it difficult to separate free protein from the conjugated protein. Free protein is considered as stability indicating parameter therefore the method should have an ability to support the stability studies. The conjugates formed/manufactured using conjugation chemistries like cyanogen bromide activation of PRP, carbodiimide mediated coupling, reductive amination one step method of n-hydroxyl succinimide activation and by formation of thio ether bond yield the conjugates which are anticipated bigger in size and less stable as compared to conjugates formed by cyanylation (CDAP/CPIP) chemistries making it difficult to separate out free protein from the conjugated molecule at such a low level.
A protein can occur in different “size forms” (monomeric, aggregated, degraded, complexed) and those different forms exhibit different functions. As many protein “size forms” are held together by non-covalent means, they are often weak and could be easily broken if using non-native (i.e., denaturing) conditions.
Tetanus toxoid protein is known to be a globular protein with a tendency to form aggregates. Polymeric Tetanus Toxoid (TT) is plain TT which is polymeric in nature and contains high molecular weight aggregates, dimers, monomers and low molecular weight fragments. The existence of dimers can affect the efficiency of the conjugation process – presumably through steric hindrance at the protein surface – leading to a loss of activity. Due to aggregation and low purity, bulk tetanus toxoid is usually purified prior to conjugation, either by size exclusion chromatography or tangential flow filtration, to prepare a mostly conjugation suitable grade fraction. Tetanus toxoid used for conjugation with polysaccharide is monomeric in nature. It is difficult to separate and quantitate or estimate free monomeric TT in very low quantity present in conjugate vaccine at bulk stage or release or during stability studies. Similarly, other proteins like Diphtheria toxoid, CRM197 which are polymeric in nature are difficult to separate and quantitate.
Various techniques for estimation of protein concentration in a sample are known in the art. The physicochemical techniques like Kjeldahl method, Lowry method, Bradford method,

Biuret method, BCA, Ultraviolet (UV) Absorbance at 280 nm. 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. MEKC assay involves use of detergent above its critical micellar concentration (CMC) for hydrophobic separation. The said method suffers from shifting peak migration times and is not suitable for polysaccharide-protein conjugates containing inherent traces of detergent (in Hib case, CTAB traces arising due to downstream processing) which interfere with quantification of free protein.
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.
Different resin types are commercially available today, such as agarose, silica, and methacrylate. Silica-based and agarose-based resins are the two most used types of SEC resins for analytical purposes. Particle size is important for the resolution in SEC; a rule of thumb is that smaller particle and narrow size distribution usually give the highest resolution. For achieving high resolution, buffers and sample composition are important parameters to be considered. Other parameters that affect resolution is flow rate (especially for large proteins). Further, resins must be stable in wide range of buffers thereby providing means for effective resolution and separation of sample/analytes. Interactions between resins and buffers might

affect the retention time, peak area, resolution and thereby complete recovery of the sample to be analyzed/quantitated. Further, the resin columns for analyzing biopolymers by GFC/SEC, resin/columns must feature attributes like high pore volume per unit column volume, low sample adsorption and excellent column efficiency, all contributing to unsurpassed sample resolution.
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.
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.
The method reported for estimating low amount of free Tetanus Toxoid particularly in Hib conjugate bulk using SEC-HPLC is that of Burki Rajendar et al, 2021 using Shodex Protein KW-802.5 SEC column, however same estimates free TT "above 3.7%" but fails to disclose estimation of <1% free TT , fails to disclose Column - TSK Gel G4000SWXL, fails to acknowledge Low Molecular Weight Hib Conjugate interference while estimating free TT, fails to acknowledge interference of CTAB traces emerging from polysaccharide purification processes.
Use of deoxycholate (DOC) or HCI-induced precipitation for evaluating the protein content of the Hib conjugate bulk and the Hib conjugate product has been previously reported. [Refer Hyun Sung Kim et al 2006]. Use of DOC affects the protein structure and hence may interfere with the analysis of the free protein in a conjugate vaccine.
Kay Lockyer et al (2018) discloses quantification of low amount of Free TT i.e. <0.5% in Men C-TT Conjugate using SEC HPLC columns (U-3000 Dionex system + Tosoh Bioscience TSK PWXL guard + TSK gel G4000PWXL). Also, Bastos et al (2017) (Bio-Manguinhos) discloses TSK-G® 4000 PWxL for “Polysaccharide-Protein conjugate” analysis. TSKgel G4000 PWxl column is polymethacrylate resin based column. However, both the above fail to disclose 1) estimation of free TT in HibTT bulk conjugates 2) estimation of free TT in presence of impurities like HMW, aggregate, trimer, dimer, LMW Hib conjugate & detergent traces (CTAB).

EP1015027 (Andrew Lees/1997) has disclosed estimation of free protein using size-exclusion HPLC on a Beckman SEC G2000 column / S400HR column. However same fails to disclose 1) estimation of free TT in HibTT bulk conjugates 2) estimation of free TT in presence of impurities like HMW, aggregate, trimer, dimer, LMW Hib conjugate & detergent traces (CTAB).
IN201721018117 / IN202023023720 disclose estimating free carrier protein (0.625% to 10%) by using SEC-HPLC (Shodex OH Pak 804 HQ & Phenomenex SEC Yarra 2000) in Meningococcal Bulk Conjugates (CWY- CRM, AX-TT). However same fails to disclose estimation of free TT in Hib conjugate bulk in presence of impurities like HMW, aggregate, trimer, dimer, LMW Hib conjugate & detergent traces (CTAB) & use of Column - TSK Gel G4000SWXL.
CN101000351 (2006) discloses free protein estimation (CRM) in Pneumococcal conjugates by using SEC-HPLC (columns- TSKgel G5000PW, TSKgelG5000PWxl, TSKgel G4000PWxl, TSKgel G4000SWxl). This method fails to disclose 1) estimation of free TT in HibTT bulk conjugates 2) estimation of free TT in presence of impurities like HMW, aggregate, trimer, dimer, LMW Hib conjugate & detergent traces (CTAB).
CN110018253 (2019) discloses free protein estimation (CRM) by using SEC-HPLC (columns- TSKgel G3000SW or TSKgel G5000PWXL). However same fails to disclose 1) estimation of free TT in HibTT bulk conjugates 2) estimation of free TT in presence of impurities like HMW, aggregate, trimer, dimer, LMW Hib conjugate & detergent traces (CTAB).
Ian Hartzel et al 2019 discloses quantification of free carrier protein in conjugate vaccines (Staph aureus conjugate) using Tosoh G3000 SWxl. However same fails to disclose 1) estimation of free TT in HibTT bulk conjugates 2) estimation of free TT in presence of impurities like HMW, aggregate, trimer, dimer, LMW Hib conjugate & detergent traces (CTAB).
Thus, in past, estimation of free carrier protein has been disclosed, however none of the prior art disclose estimation of free Tetanus Toxoid in presence of impurities like HMW, aggregate, trimer, dimer, LMW Hib conjugate & detergent traces (CTAB).
Estimating Free Tetanus Toxoid (<1%) in Hib-TT conjugate bulk is challenging & peculiar from the perspective of obtaining improved resolution in presence of other impurities like

HMW, aggregate, trimer, dimer, LMW Hib conjugate & detergent traces (carried over from Downstream process e.g. CTAB); Conjugates if subjected to harsh conditions may give erroneous results (due to undesirable protein degradation). Hence methods reported previously for estimating free protein from N. meningitidis conjugates cannot be simply extrapolated to arrive at a method that successfully estimates significantly low amount of free TT i.e. < 1% free or unconjugated TT (monomeric TT) from Hib-TT bulk conjugates of molecular size 800-900 KDa.
Also, unconjugated or free protein evaluation in bulk or composition comprising polysaccharide conjugated to a monomeric carrier protein 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 low amount of unconjugated/free carrier protein in a polysaccharide- protein conjugate bulk or final vaccine 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 low amount or level of unconjugated or free carrier monomeric protein content in a polysaccharide-protein conjugate sample (bulk or final vaccine composition).
• It is an object of the present disclosure to provide a method for determining low amount or level of unconjugated/free monomeric Tetanus toxoid (carrier protein) content in a conjugate sample (bulk or final vaccine composition).
• It is an object of the present disclosure to provide a method for determining low amount or level of unconjugated/free monomeric Tetanus toxoid (carrier protein) content in vaccine comprising of Haemophilus influenzae type b PRP - TT (Hib–TT) conjugate sample (bulk or final vaccine composition).
• It is an object of the present disclosure to provide a method for assessing the quality of the polysaccharide-protein conjugate bulk or final vaccine or 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 are 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.
SUMMARY OF INVENTION:
The present disclosure provides a method for evaluating low amount of unconjugated or free protein in a polysaccharide-protein conjugate sample (bulk or final vaccine composition) 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 for the estimation of free protein in Haemophilus influenzae type b PRP - TT (Hib–TT) Conjugates; wherein the free protein is monomeric in nature and concentration is less than 1%; wherein free protein is estimated in presence of other impurities like HMW, aggregate, trimer, dimer, LMW Hib conjugate, detergent traces (e.g. CTAB from DSP), and wherein spiking ensures LMW Hib conjugate & free TT are differentiated, bulk sample is not subjected to any harsh condition thereby avoiding protein degradation & subsequently correct quantification. The method is particularly useful for quantifying low amounts of free carrier protein present in the polysaccharide-protein conjugate vaccines.
Proposed method measures < 1% free protein (TT) from Hib-TT bulk conjugates (800-900 KDa) using SEC-HPLC (TSK Gel G4000SWXL) wherein TSK Gel G4000SWXL provides better resolution of monomeric TT particularly in Hib bulk conjugates (as compared to previously reported SEC HPLC Columns i.e. Shodex, Phenomenex columns) that too in presence of other impurities like HMW, aggregate, trimer, dimer, LMW Hib conjugate, detergent traces (e.g. CTAB from DSP).

Shodex columns are not suitable for the resolution of monomeric TT whereas Phenomenex Yarra 3μm SEC-2000 was tried for TT analysis but noted that it has a resin consistency issue and found not rugged which gives variable results from column lot to lot while the Tosoh columns were a preferred choice and under consideration. Whereas, TSK gel 4000 SWXL (7.8 ID x 30 cm, 8μM) was found to provide improved separation/resolution between conjugate and TT as compared to Acclaim SEC-1000 (7 μm 1000 A°, 7.8 X 300 mm), TSK gel SP- 5 PW (7.5 ID X 7.5 cm 10μM), Anion exchange resin DEAE Toypearl & Shodex 806 SB OH PAK.
BREIF DESCRIPTION OF DRAWING:
The present disclosure will now be described with the help of the accompanying drawing, in which:
Figure 1: Representative chromatogram for free protein determination in Hib conjugate bulk
by SEC-HPLC
Figure 2: Representative chromatogram for free protein determination in Hib conjugate bulk
by SEC-HPLC where Conjugate bulk is at around 12 minutes and free protein peak is at
around 22 minutes
Figure 3: Representative chromatogram for free protein determination in Hib conjugate bulk
by SEC-HPLC where Conjugate bulk is at around 12 minutes and free protein peak is at
around 22 minutes)
Figure 4: Representative chromatogram of TT by SEC-HPLC using Acclaim SEC-1000
column
Figure 5: Representative chromatogram of Conjugate Bulk (Hib-TT) by SEC-HPLC using
Acclaim SEC-1000 column
Figure 6: Representative chromatogram of TT and Conjugate Bulk (Hib-TT) by SEC-HPLC
using Acclaim SEC-1000 column
Figure 7: Representative chromatogram of TT by SEC-HPLC using TSK gel SP- 5 PW
column
Figure 8: Representative chromatogram of Conjugate bulk (Hib-TT) by SEC-HPLC using
TSK gel SP- 5 PW column
Figure 9: Representative chromatogram of TT by SEC-HPLC using Shodex 806 SB OH
PAK column

Figure 10: Representative chromatogram of Conjugate bulk (Hib-TT) by SEC-HPLC using Shodex 806 SB OH PAK column
Figure 11: Representative chromatogram of TT and Conjugate bulk (Hib-TT) by SEC-HPLC using Shodex 806 SB OH PAK column
Figure 12: Representative chromatogram for system suitability Figure 13: Calibration curve for Linearity
Figure 14: Representative chromatogram of Blank by SEC-HPLC
Figure 15: Representative chromatogram of Hib Conjugate Bulk + 0.25% spike of TT by SEC-HPLC
Figure 16: Representative chromatogram of Hib Conjugate Bulk + 0.5 % spike of TT by SEC-HPLC
Figure 17: Representative chromatogram of Hib Conjugate Bulk + 1 % spike of TT by SEC-HPLC
Figure 18: Representative chromatogram of Hib Conjugate Bulk + 1.5 % spike of TT by SEC-HPLC
Figure 19: Representative chromatogram of Hib Conjugate Bulk + 2 % spike of TT by SEC-HPLC
DETAILED DESCRIPTION:
Although the present disclosure may be susceptible to different embodiments, certain embodiments are shown in the drawing and following detailed discussion, with the understanding that the present disclosure can be considered an exemplification of the principles of the disclosure and is not intended to limit the scope of disclosure to that which is illustrated and disclosed in this description.
Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and processes, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, well-known composition, well-known processes, and well-known techniques are not described in detail.

The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms "a,” "an," and "the" may be intended to include the plural forms as well, unless the context clearly suggests otherwise.
The terms "comprises," "comprising," “including,” and “having,” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the process of the present disclosure is not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.
The terms first, second, third, etc., should not be construed to limit the scope of the present disclosure as the aforementioned terms may be only used to distinguish one element, component, region, layer or section from another component, region, layer or section. Terms such as first, second, third etc., when used herein do not imply a specific sequence or order unless clearly suggested by the present disclosure. The present disclosure provides an immunogenic composition and a process for preparing the same.
It is understood that each feature or embodiment, or combination, described herein is a nonlimiting, illustrative example of any of the aspects of the invention and, as such, is meant to be combinable with any other feature or embodiment, or combination, described herein. For example, where features are described with language such as “one embodiment”, “some embodiments”, “certain embodiments”, “further embodiment”, “specific exemplary embodiments”, and/or “another embodiment”, each of these types of embodiments is a non-limiting example of a feature that is intended to be combined with any other feature, or combination of features, described herein without having to list every possible combination. Such features or combinations of features apply to any of the aspects of the invention.
As used herein, ‘free monomeric protein’ refers to an unbound or unconjugated monomeric protein present in the polysaccharide-carrier protein conjugate bulk which is to be quantified by HPLC-SEC method and can be interchangeably used with the term ‘unconjugated

monomeric protein’. The ‘free monomeric protein’ or ‘unconjugated monomeric protein’ may also refers to the ‘free or unconjugated carrier protein’.
Source of Biological Material (Refer Table 1) used in the present disclosure is as follows:
Table1: Source of Biological Material

Organism Source
Haemophilus influenzae type b (Harward Netherlands Vaccines Institute (NVI, The
No. 760705) Netherlands)
Clostridium Tetani (Harvard No 49205) Central research Institute (CRI), National
for Tetanus toxoid 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 or quantification of free or unconjugated monomeric carrier protein content in a monovalent or multivalent bulk conjugate may comprise of polysaccharide -protein conjugate bulk, protein-protein conjugate bulk or antibody-protein conjugate bulk 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 bulk preparation by Folin - Lowry protein assay, further followed by estimating the concentration of free or unconjugated monomeric carrier protein in the monovalent or multivalent conjugate bulk composition by HPLC-SEC. Wherein the percentage of unconjugated or 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 physicochemical 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 bulk composition.
In an embodiment of present disclosure, the total protein concentration in a monovalent or multivalent conjugate bulk composition may be 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. Other protein assays known in art to measure the total protein concentration of test sample may also be used by the person skilled in the art.
In an aspect of this embodiment, the Folin - Lowry assay may be optimized to determine the total carrier protein concentration in a monovalent or multivalent conjugate bulk 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 may be optimized to determine the total carrier protein concentration in a monovalent or multivalent conjugate bulk composition.
In other aspect of this embodiment, the Folin - Lowry assay may be optimized to determine the total Tetanus toxoid concentration in a monovalent conjugate bulk composition (test sample) comprising of Haemophilus influenzae type B (PRP) polysaccharide conjugated to Tetanus toxoid.
In other aspect of this embodiment, the Folin - Lowry assay may be optimized to determine the total Tetanus toxoid concentration in a multivalent conjugate bulk composition (test sample) comprising of Haemophilus influenzae type B (PRP) polysaccharide conjugated to Tetanus toxoid.
In an embodiment of the present disclosure, Test sample may comprise of polysaccharide -protein conjugate bulk, protein-protein conjugate bulk or antibody-protein conjugate bulk composition.
In an embodiment of the present disclosure, Test sample may comprise of free or unconjugated monomeric carrier protein. The said free or unconjugated carrier protein could be monomeric in nature. The test sample typically contains high molecular weight aggregates, dimer, monomer and low molecular weight fragments.
In another aspect of this embodiment, the monovalent or multivalent conjugate bulk composition (test sample) may further comprises of excipients and stabilizers.
In an aspect of this embodiment, the monovalent or multivalent conjugate composition bulk (test sample) may be devoid of excipients and stabilizers.
In an embodiment of present disclosure, the unconjugated or free carrier protein in a monovalent or multivalent polysaccharide - protein conjugate preparation may be 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 embodiment of present disclosure, the method may be used to evaluate or quantify the low level (amount or percentage) of free or unconjugated monomeric carrier protein in a monovalent or multivalent polysaccharide protein conjugate bulk composition, wherein one or more polysaccharides are conjugated to same type of carrier protein.
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 could be between the range of 1 nm and 150 nm. In the preferred aspect, pore size of the size exclusion chromatographic resin selected may be between 10 and 50 nm.
In an aspect of the present embodiment, the size exclusion chromatographic resin used in high performance size exclusion chromatography is silica based, agarose or polyacrylamide beads, methacrylate based 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 could be silica based resin, polyhydroxymethacrylate based resin, acrylamide based resin and methacrylate based resin. In a preferred aspect of this embodiment, size exclusion chromatography resin used in high performance size exclusion chromatography (HPLC-SEC) is silica based resin preferably columns (with resins) based on Silica gel bonded with hydrophilic groups.
In an aspect of the present embodiment, the size exclusion chromatographic column used in high performance size exclusion chromatography (HPLC-SEC) includes but is not limited to Phenomenex Yarra 3μm SEC-2000, Phenomenex Yarra 3 μm SEC-3000, Phenomenex Yarra 3μm SEC-4000, Phenomenex Bio-Sep-SEC-S2000, Phenomenex Bio-Sep-SEC13 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 G4000SWXL, Shodex Protein KW-802.5,

Shodex OH Pak SB-800 HQ, Phenomenex Yarra 3μm SEC-2000, TSK Gel G3000SWXL, TSKgel G5000PWXL, TSKgel G5000SWXL, TSKgel Ultra SW Aggregate Acclaim SEC-1000; TSK gel SP- 5 PW; Anion exchange resin DEAE Toypearl; Shodex 806 SB OH PAK.
In another aspect, the column may be selected such that the column has large pore size resolving power, preferably pore size of column is between 400A° to 500A° and particle size is between 7 to 9µm. In another aspect, the column may be selected such that the column has large pore size resolving power, preferably pore size of column is 450A° and particle size is 8µm.
In a preferred aspect of this embodiment, the size exclusion chromatographic column used in high performance size exclusion chromatography (HPLC-SEC) could be TSK Gel G4000SWXL column. The other columns that may be used includes but not limited to TSK-GEL SW, Super SW and TSK-GEL SWXL series (Tosoh).
In one of the embodiments of the present disclosure, the eluate obtained by subjecting the test sample to high performance size exclusion chromatography (HPLC-SEC) may be 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) could be used to evaluate the elute obtained by HPLC-SEC. In a preferred aspect of this embodiment, the photometric analysis may be carried out using UV/PDA detector. 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, preferably 200 to 400 nm, more preferably 214 nm and 280 nm.
In an aspect of this embodiment, the mobile phase buffer used in the HPLC-SEC process may be 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 aspects of present embodiment, a marker mix may be used in the HPLC-SEC process 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 concentration is 0.5 mg/ml.
In one of the aspects of present embodiment, the loading of samples can be performed at a flow rate in the range 0.2 - 2 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, 0.5 - 1.0 ml/minute, or 0.2 - 0.4 ml/minute. In one of the preferred aspects, the loading of samples is performed at a flow rate in the range 0.5 - 1.0 ml/minute.
In one of the aspects 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 aspects of present embodiment, the injection volume of the sample can be in the range 10μl - 200μl. Yet the preferred injection volume of the sample is between 50 μl and 100 μl.
In one of the aspects 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 aspects of present embodiment, the method is a high performance size exclusion chromatography method (HPLC-SEC) in which the injection run time may be up to 60 minutes.
In one of the embodiments of the present disclosure, the photometric analysis may be used to evaluate the concentration of unconjugated/free protein in a test sample. In one of the aspects 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 or free monomeric protein may be less than 3% or less than 2% or less than 1%. In an aspect of this embodiment, the concentration of unconjugated or free monomeric protein may be less than 1%. The limit of quantitation of free protein may be 2 μg/ml. The limit of detection of free protein may be 1 μg/ml.
In one of the embodiments of the present disclosure, the present method may be suitable for the estimation of free protein in Haemophilus influenzae type b PRP - TT (Hib-TT)

Conjugates; wherein the free protein is monomeric in nature and concentration is less than 1%; wherein free protein is estimated in presence of other impurities like HMW, aggregate, trimer, dimer, LMW Hib conjugate, detergent traces (e.g. CTAB from DSP), and wherein spiking ensures LMW Hib conjugate & free TT are differentiated, bulk sample is not subjected to any harsh condition thereby avoiding protein degradation & subsequently correct quantification. The method may be particularly useful for quantifying low amounts of free carrier protein present in the polysaccharide-protein conjugate vaccines. The method is simple, accurate, repeatable, fast, robust and convenient and devoid of any sample processing steps prior to analysis.
In one of the embodiments of the present disclosure, detergent traces present in the conjugate bulk may be selected from the group comprising of cetyltrimethylammonium bromide (CTAB), tetrabutylammonium salt, myristyltrimethylammonium salt, hexadimethrine bromide, alkyl sulfates, sodium dodecyl sulfate, sodium deoxycholate, sodium dodecyl sulfonate, sodium s-alkyl sulfates, sodium fatty alcohol polyoxyethylene ether sulfates, sodium oleyl sulfate, N-oleoyl poly (amino acid) sodium, sodium alkylbenzene sulfonates, sodium α olefin sulfonates, sodium alkyl sulfonates, α-sulfo monocarboxylic acid esters, fatty acid sulfoalkyl esters, succinate sulfonate, alkyl naphthalene sulfonates, sodium alkane sulfonates, sodium ligninsulfonate, and sodium alkyl glyceryl ether sulfonates.
In one of the embodiments of the present disclosure, the molecular weight of the conjugate bulk may be selected from the group ranging from 100 to 2000 kDa, 200 to 1500 kDa, 200 to 1000 kDa, 300 to 1500 kDa, 400 to 1500 kDa or 500 to 1000 kDa. In an aspect of this embodiment, the molecular weight of the conjugate bulk is preferably ranging from 800 to 900 kDa.
According to one of the preferred embodiments, instant method may be found to be suitable for the estimation of free or unconjugated monomeric protein in Hib-TT Conjugates; wherein the free or unconjugated monomeric protein concentration may be less than 1%. The percent recovery is >70% up to 130%.
In a preferred embodiment of the present disclosure, test sample or conjugate bulk samples may comprise of a Haemophilus influenzae conjugate manufactured using cyanogen bromide activation of Haemophilus influenzae type B PRP, carbodiimide mediated coupling with Tetanus toxoid.

In a yet another preferred embodiment of the present disclosure, the test sample may be combination vaccine comprising one or more antigens selected from Tetanus Toxoid, Diphtheria Toxoid, Whole Cell Pertussis, HBsAg, Hib Conjugate and/or inactivated poliovirus.
In one of the aspects of this embodiment, the instant method may found to be suitable for the estimation of free or unconjugated monomeric protein other than Tetanus Toxoid in monovalent or multivalent polysaccharide protein conjugate bulk or final vaccine composition selected from the group comprising of CRM197, diphtheria toxin/toxoid (DT), Neisseria meningitidis outer membrane complex, fragment C of tetanus toxoid, recombinant full length tetanus toxin with mutations (8MTT), 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, Por B), transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA),PhtA, PhtB, PhtE, 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), C5a peptidase group A or group B Streptococcus, human serum albumin, bovine serum albumin (BSA), NTHi high molecular weight protein, fhbp 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, toxin A or B from C. difficile and S. agalactiae.
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 aspects of this embodiment, the instant method may be found to be suitable for the estimation of free or unconjugated monomeric protein other than Tetanus Toxoid in monovalent or multivalent polysaccharide protein conjugate bulk or final vaccine composition wherein the polysaccharide could be 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, Ureaplasmaurealyticum, 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, Klebsiella pneumoniae, 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. such as Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Shigella boydii; Ehrlichia spp., and Rickettsia spp. Polysaccharides of Streptococcus pneumoniae serotype type 1, 2, 3, 4,5,6, 6A, 6B, 6C, 6D,6E, 6F, 6G, 6H, 7A, 7B, 7C, 7D, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10D, 10A, 11, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 13F, 14, 15A,15B, 15C, 15F, 16, 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, 35A, 35B, 35C, 35D, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, and 48; Neisseria meningitidis serotype A, B, B16, B6, C, D, E29, H, I, K, K454, L, M, W135, X, Y, Z.
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 low amount of free carrier protein content in a polysaccharide-protein conjugate bulk or final vaccine composition. The method of the present disclosure provides the following advantages:
• Proposed method measures low amount of free protein (TT) i.e. <1% from Hib-TT bulk conjugates (800-900 KDa) using SEC-HPLC (TSK Gel G4000SWXL) wherein TSK Gel G4000SWXL provides better resolution of monomeric TT particularly in Hib bulk conjugates (as compared to previously reported SEC HPLC Columns i.e. Shodex, Phenomenex columns) that too in presence of other impurities like HMW, aggregate, trimer, dimer, LMW Hib conjugate, detergent traces (e.g. CTAB from

DSP), wherein spiking ensures LMW Hib conjugate & free TT are differentiated, bulk sample is not subjected to any harsh condition thereby avoiding protein degradation & subsequently correct quantification. The method is particularly useful for quantifying low amounts of free carrier protein present in the polysaccharide-protein conjugate vaccines.
• 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 7.2 ± 0.2;
• 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 HPLC-SEC 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 TSK Gel G4000SWXL column is designed for use with high-resolution, highspeed aqueous size exclusion chromatography which provides 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 polymeric carrier proteins like tetanus toxoid.
• Instant method is found to be suitable for estimation of free protein concentration of less than 1%.
EXAMPLES
The foregoing description of the embodiments has been provided for purposes of illustration and not intended to limit the scope of the present disclosure. Individual components of a particular embodiment are generally not limited to that particular embodiment, but, are interchangeable. Such variations are not to be regarded as a departure from the present disclosure, and all such modifications are considered to be within the scope of the present disclosure.
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 compositions and 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. The present disclosure is further described in light of the following examples which are set forth for illustration purpose only and not to be construed for limiting the scope of the disclosure.
However, those of skill 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: Polysaccharide-Protein Conjugate bulk
Conjugate bulk samples comprise of a Haemophilus influenzae conjugate manufactured using cyanogen bromide activation of Haemophilus influenzae type B PRP, carbodiimide mediated coupling with Tetanus toxoid.
The conjugate bulk is present in 20 mM Tris. The process related residuals are separately estimated on every batch and confirmed its absence in the purified sample.

EXAMPLE 2: Evaluation of total protein concentration in a Polysaccharide-Protein conjugate bulk composition (test sample) using Folin-Lowry assay
The total protein concentration in a Polysaccharide-Protein conjugate bulk composition (test sample) was evaluated using Folin-Lowry assay. The assay details are as follows: Step 1: Introduction:
First the proteins are treated with copper ion in alkaline solution and then aromatic amino acid in the treated sample is reduced by phosphomolybdate and phosphotungstic acid which is present in the Folin- Ciocalteu reagent. The end product of the reaction has a blue to violet color. The amount of the protein in the sample is estimated by reading absorbance at 750nm by UV-Vis Spectrophotometer.
Step 2: Working Standard and Sample Preparation:
Bovine serum albumin (BSA) (Working standard): The BSA standard is prepared from 2 mg/ml NIST BSA standard readily available from Thermo fisher Scientific. Store at 2°C to 8°C. (Valid upto: 5 years). Sample to be appropriately diluted and aliquot is used for analysis.
Prepare standards (6.25–50.0 µg) in duplicate in Microcentrifuge test tube as shown below in Table 2 by using above mentioned 2 mg/mL Protein standard 2 (Bovine serum albumin) (Use fresh ampoule each time).
Table 2: Standard solutions for Folin - Lowry method using 2mg/ml NIST BSA

Standard 2 mg/ml NIST BSA from ampoule (µl) MQW (µl) Total Volume (µl)
Blank 0 800 800
STD 1 25 1575 1600
STD 2 50 1550 1600
STD 3 100 1500 1600
STD 4 75 725 800
STD 5 100 700 800
Vortex all the standards in the micro centrifuge tubes. From the above-mentioned standard preparation, transfer 200µl of standard in duplicate in micro centrifuge tube. It gives working standards concentration as mentioned below in Table 3;
Table 3: Working Standard solutions for Folin - Lowry method after centrifugation

Standard Working Standard (µl) MQW (µl) Content (µg)
Blank 0 200 0
STD 1 200 0 6.25
STD 2 200 0 12.5
STD 3 200 0 25.0
STD 4 200 0 37.5
STD 5 200 0 50.0
Step 3: Protocol
a) Take the 2 mg/ml NIST Bovine serum albumin 1ml ampule in duplicate in test tube or micro centrifuge tube and prepared standard curve from 6.25 µg to 50 µg.
b) Vortex all the standards in the micro centrifuge tubes (Working standard).
c) Prepare a standard curve each time the assay is performed.
d) Standard, control and a sample dilution are prepared in duplicate.
e) Place samples (pre-diluted) and QC control (200 µl) in duplicate.
f) Add 1 ml of Cupric-tartaric solution and vortex gently & incubate for 10 minutes at room temperature.
g) Add 100 µl of pre-diluted Folin-Ciocalteu reagent (1ml of Folin-Ciocalteu reagent + 1ml of MQW) and vortex gently and incubate for 30 minutes at room temperature and read the absorbance at 750 nm (total sample volume = 1.3 ml).
h) Open the method in the Gen 5 secure software. (e.g.: method name: Lowry(750nm) /
Protein by Lowry(750nm)). i) Prepare the plate layout and enter the sample and control details. j) After completion of the incubation place the 96 well plate in proper orientation as per
the layout prepared. k) Now through the Gen-5 secure software initiates the reading. l) Read the plate within 30 minutes of completion of the assay. m) The reader would measure the optical density of the assay/wells in plate. n) After completion of the reading save the result remove the plate and print the report. o) The remaining sample(s), intermediate dilution(s) and any additional sample shall be
disposed in copious amount of water. p) Verify the raw data printed and then log off the system.
Step 4: Calculation

Data analysis should be done manually. Check absorbance of standards, control and samples in raw data from spectrophotometer. If the absorbance of any sample falls outside the values of the standard curve, the assay should be repeated using a more appropriate pre-dilution, or aliquot, for that sample.
EXAMPLE 3: Evaluation of unconjugated/free protein concentration in a Polysaccharide-Protein conjugate bulk 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.
Reagents and their source:
1. WFI/Milli Q Water (WFI/MQW)
2. Potassium dihydrogen orthophosphate (e.g. Fisher Scientific 7778-77-0)
3. Di-Sodium hydrogen orthophosphate anhydrous (e.g. Fisher Scientific 7558-79-4)
4. Sodium Chloride (e.g. Fischer Scientific7647-14-5).
5. Potassium chloride (e.g. Fischer Scientific 7447-40-7)
6. Sodium azide (e.g.Sigma S8032).
7. Thyroglobulin (e.g. Sigma T101)
8. Bovine serum albumin (e.g. Sigma A3069)
9. P-amino benzoic acid (e.g Sigma A9878)
Step 1: Introduction:
The estimation of free TT conjugate bulk does not require any sample processing (i.e. no sample filtration, no sample digestion, no heating, no voltage-based separation, no specific pH requirement the method demands only simple buffer i.e. PBS pH 7.2 ± 0.2, no detergents required) Ultra-sensitive estimation of free protein.
Step 2: Working Standard and Sample Preparation:
The standard used is Ultra filtered Tetanus Toxoid produced at SIIPL. No sample preparation (The sample used is either neat or diluted to 1 mg/mL based on the final protein concentration obtained in conjugate bulk).

Prepare standards i.e. working Standard from 1 mg/mL from In-house produced Purified Tetanus toxoid. The concentrations prepared are 0.25% to 2% in PBS buffer.
Table 4: Working Standard solutions for HPLC-SEC method

Standard (µg/ml)
0.25% 2.5
0.50% 5
1.0% 10
1.5% 15
2.0% 20
Step 3: Protocol
Refer Table 5 for process parameters.
Table 5: Process parameters and Instrumental method details

Sr No Parameters Details
1 Sample volume Sample volume required 1 mL sample to prepare the dilution (50 to 100 µL injection).
2 Injection volume 50 to 100µL
3 Flow rate 0.5 to 1.0 mL/min
4 Column TSK Gel 4000 SWxL (Column- ID: 7.8mm and Length: 30 cm, 8µM, Part No. – 0008023)
5 Auto sampler Temperature Up to 30°C
6 Column temperature Up to 30°C
7 Retention time Depends on column state/lot
8 Detector UV/PDA detector (214 nm and 280 nm)
9 Time upto 60 minutes/injection
10 Buffer Phosphate buffer saline (pH 7.4)
11 pH 7.4 (7.2 ± 0.2)
12 Column storage 0.05% Sodium azide for column storage
13 HPLC-SEC Waters/Thermo
a) Switch on the main power of the computer and the instrument.
b) Prepare the mobile phases and fill in the bottles meant for them. Fill up one eluent bottle with Milli Q Water and insert lines not being used for run, into it, fill up second eluent bottle with PBS. Similarly fill up third eluent bottle with 0.05% Sodium Azide and insert respective line C or A into it (as required) and dip the other line into Milli Q Water.
c) Prime the HPLC line with the mobile phase PBS.
d) Set the flow rate and connect to the column inlet, ensuring that flow is in the direction indicated by the arrow on the column.

e) Tighten the end fitting going into the guard followed by main column.
f) Connect column outlet to flow cell.
g) Increase the flow rate slowly.
h) Give the set up as per defined instrument program.
i) Equilibrate column for at least two column volumes at 0.50 mL/min while observing
the back pressure. j) After equilibration, prepare and add the blank/s, system suitability and sample/s in
labeled HPLC vials and cap it properly. k) The same instrument method set conditions are applicable for goodnight i.e. column
storage method with the change in solvent to 0.05% sodium azide. l) On completion of the run: Perform the integration for PBS Blank, Standards and
Sample/s. m) Click on report format and print the report by selecting the required chromatographic
scale and in the table select columns as per requirement n) Store the column by passing through 0.05% sodium azide if column is not to be used
for a long time. (Conditioning time: at least one column volume) or otherwise
equilibrate the column with PBS and use. o) After completion of testing, switch off the HPLC system after disconnecting the
column from the system. Close column`s two ends by end frits. Keep the column back
in place. Remove the vials from the auto sampler. The remaining samples, dilutions
and additional sample shall be disposed in copious amount of water. p) Finally calculate the concentration of free protein in mg/mL based on the area of the
standards obtained. The obtained concentration of free protein is divided by the total
protein and finally the value is presented in percentage.
Step 4 Calculation of percent unconjugated/free protein concentration in a Polysaccharide-Protein conjugate bulk composition (test sample):
Calculation of unconjugated/free monomeric protein concentration in a Polysaccharide-Protein conjugate bulk composition (test sample) is performed as follows:
Approach 1 (Limit test – Semi quantitative): Inject 1% Protein standard and 1 mg/mL conjugate bulk sample. Calculate the area of free carrier protein peak in sample conjugate and 1 % tetanus toxoid peak, which is run as a control/standard. If the free protein area in sample is obtained less than the free protein area in standard, the result shall be presented “Free

protein <1%” i.e. if the peak area of sample observed is less than the peak are of control, the sample is reported to contain free protein less than 1%. In case the free protein area in sample obtained more than they are in standard solution of 1% the result shall be presented as “Free protein > 1%” (This is based on the observation of area value).
Approach 2 (Quantitative): Prepare the linearity using Ultra filtered Tetanus Toxoid (UFTT) i.e. plot the standard curve from 2.5 µg/mL to 20 µg/mL (0.25 % to 2%) and sample 1 mg/mL and perform the SEC-HPLC analysis. Estimate the presence of free protein in sample based on area obtained in the form of µg/ml. Based on the concentration obtained the calculation shall be performed as follows
Total protein concentration estimated by Lowry is: 2000 µg/mL
Free protein concentration estimated by SEHPLC is: 5 µg/mL
Dilution factor: 2 (2000 µg /mL sample is diluted to 1000 µg/mL for analysis)
% Free Protein = Free protein concentration estimated by SEC-HPLC X 100
Total protein concentration estimated by Lowry
= 2 X 5 X 100 2000
= 10 X 100 2000
= 0.5% (Free protein present in sample)
Step 5: Observations and Interpretations:
Figure 1 shows a typical profile of conjugate bulk wherein conjugate bulk peak is observed at
12 minutes and presence of free protein is expected at 22 minutes but since the sample does
not contain any residual free protein no peak is observed at 22 minutes. This interprets the
sample does not contain any free protein and result presented as “not detectable”.
Figure 2 shows a typical profile of Hib conjugate bulk wherein conjugate bulk peak is
observed at 12 minutes and presence of free protein is noted at 22 minutes. The area obtained
for the free protein peak is noted and compare with the peak obtained in 1% protein standards
(e.g. if peak area obtained in sample is 6000 AU and the peak area obtained for 1% standard

is 12000, this indicates the sample contains less than 1% free protein (<1%) and if the area obtained is more than 12000 the sample contains free protein more than 1% (>1%) Figure 3 explains same observations considering the chromatogram
EXAMPLE 4: Comparison of Methods and Columns
A. Comparison of Methods:
The method of invention disclosing estimation or quantification of low level of monomeric carrier protein in polysaccharide-carrier protein conjugate bulk using SEC-HPLC comprising TSK gel G4000 SWXL was compared with different methods used for estimation of protein. Different methods are mentioned below:
a. SEC-HPLC method using TSK gel G4000 SWXL vs MEKC: MEKC method uses
harsh condition (i.e. borate buffer with high pH) and therefore not suitable or preferred for free protein estimation
b. SEC-HPLC method using TSK gel G4000 SWXL vs SDS-PAGE: SDS-PAGE
approach is not used for free protein quantitation.
c. SEC-HPLC method using TSK gel G4000 SWXL vs CZE: CZE method uses harsh
condition (i.e. borate buffer with high pH) and therefore not suitable or preferred for free protein estimation
d. SEC-HPLC method using TSK gel G4000 SWXL vs Bradford method: Bradford
method is use for total protein estimation
e. SEC-HPLC method using TSK gel G4000 SWXL vs Biuret method: Biuret method is
use for total protein estimation.
f. SEC-HPLC method using TSK gel G4000 SWXL vs DOC HCl precipitation: This
DOC-HCl method is use for free polysaccharide estimation
B. Comparison of Columns:
In general, although the below mentioned/ studied columns are used for the separation of biomolecules i.e. for size exclusion chromatography are considered inappropriate in our case. The vendors cited i.e. Shodex/Tosoh/Phenomenex have a vast variety of series of columns and selection is completely based on the application, principle and the conjugation chemistry of the product intended to work on. Though these columns are SEC columns, the selection is not generalized and demands approach based column selection.

Unsuitability of the below mentioned columns is supported and considered based on the knowledge about the separation techniques, knowing the nature of TT i.e. monomeric, size of conjugates, the applicable conjugation chemistries and the analytical experience gained during the analysis of glycoconjugate vaccine produced with various chemistries which utilizes TT as carrier protein.
In addition, during the development, the following columns were explored considering the monomeric nature of TT and the conjugation chemistries before finalizing the credible approach which can work for the estimation of free monomeric protein in this case. Refer Table 6 for comparison of the columns.
a. TSK gel 4000 SWXL (Tosoh biosciences) vs Acclaim SEC-1000, 7 µm 1000 A°, 7.8 X 300 mm (Thermo Fisher scientific): The chromatograms in the Figure 4, 5 and 6 indicates, no separation between conjugate and free protein was observed, hence this column is not suitable for the application.
b. TSK gel 4000 SWXL (Tosoh biosciences) vs TSK gel SP- 5 PW, 7.5 ID X 7.5 cm
10µM (Tosoh corporation (ion exchange column)): The chromatograms in the Figure
7 and 8 indicates, no required separation was observed, hence this column is not
suitable for the application.
c. TSK gel 4000 SWXL (Tosoh biosciences) vs Shodex 806 SB OH PAK (Shodex):
The chromatograms in the Figure 9, 10 and 11 indicates no required separation was observed; hence, this column is not suitable for the application.
d. TSK gel 4000 SWXL (Tosoh biosciences) vs Anion exchange resin DEAE Toypearl
(Tosoh biosciences (ion exchange column)): Our method required SEC column. This
is an ion exchange column and therefore not explored hence becomes not applicable in
this case. There was no proper chromatogram observed using Anion exchange resin
DEAE Toypearl column hence not displayed.
Concluding, the TSK Gel G4000SWXL indicated and supported the given application principally.
Other columns:

a. TSK gel 4000 SWXL (Tosoh biosciences) vs Shodex OH PAK 804 HQ (Shodex):
Shodex column are explored but found not suitable with respect to desired separation.
b. TSK gel 4000 SWXL (Tosoh biosciences) vs Phenomenex Yarra 3µm SEC-2000:
This column is suitable for the molecules that are comparatively in monomeric form i.e.
molecules like CRM.
c. TSK gel 4000 SWXL (Tosoh biosciences) vs U-3000 Dionex + Tosoh Bioscience
TSK PWXL guard + TSK gel 4000 PWXL (polymethacrylate): This column can be
used for free protein estimation and data related to carrier protein i.e. GFC TT
separation.
Table 6: Comparison of columns (Acclaim SEC-1000; TSK gel SP- 5 PW; Anion exchange resin DEAE Toypearl; TSK gel 4000 SWXL; Shodex 806 SB OH PAK)

Columns and Source Details Observations
Acclaim SEC-1000, 7 Resin material: Silica Only TT peak showed resolution w.r.t
µm 1000 A°, 7.8 X based three peaks comprising HMW or dimer,
300 mm Particle size: 7µm monomer and fragments/LMW but
(Thermo Fisher Pore size: 1000A or 100 when the same is spiked in the
Scientific) nm conjugate bulk resolution observed compromised affecting separation and ultimately quantitation.
TSK gel SP- 5 PW, Resin material: Due to conjugation chemistry;
7.5 ID X 7.5 cm Methacrylate based No binding of Conjugate as well as free
10µM Particle size: 20-30µm protein observed.
(Tosoh Bioscience Pore size: 1000A or 100 Early elution observed.
(cation exchange nm No required resolution noted.
resin))
Anion exchange resin Resin material: Due to conjugation chemistry;
DEAE Toypearl Methacrylate based No binding of Conjugate as well as free
(Tosoh Bioscience) Particle size: 100 µm protein observed
Pore size: 1000A or 100 No separation between conjugate bulk
nm and TT noted.
TSK gel 4000 SWXL Resin material: Silica Observed desired separation.
(Tosoh Bioscience) based Specifically used for separation and
Particle size: 8 µm Pore size: 450A or 45 nm quantification low level of proteins
Shodex 806 SB OH Resin material: Though the separation in TT peaks
PAK (Shodex) Methacrylate based observed but cannot be quantitated due
Particle size: 13 µm merged peak appeared between
Pore size: 15000A or Conjugate bulk and free TT peaks.
1500nm Incomplete elution was observed.

Based on past experience the Shodex columns are not suitable for the resolution of monomeric TT whereas Phenomenex Yarra 3µm SEC-2000 was tried for TT analysis but noted that it has a resin consistency issue and found not rugged which gives variable results from column lot to lot while the Tosoh columns were a preferred choice and under consideration.
These columns may be able to separate the molecules having reasonable amount of monomeric/ highly purified TT in absence of other impurities like HMW, aggregate, trimer, dimer and LMW components i.e. monomeric TT and supporting conjugation chemistries.
While in case of selecting suitable Tosoh columns from the vast available series, we were very particular about selection wherein the particle size of resin/bead and bore size was taken into consideration. The column which has a large resin particle size with bigger bore size was selected i.e. TSK Gel G4000SWXL with an intension to achieve desired separation.
EXAMPLE 5: Method validation
The method and procedure followed for System suitability, Linearity and Range, LOD/ LOQ and recovery is same as mentioned in Example 3. The only difference is with reference to preparation of the solutions to use during the respective experiment.
a. System Suitability:
System suitability solution: Dilute 50 µL of 5.0 mg/mL thyroglobulin, 200 µL of 5.0
mg/mL BSA and 100 µL of 0.1g/L PABA in 650 µL of PBS. Prepare freshly. Perform
the analysis as per the procedure mentioned in HPLC analysis section.
1% tetanus toxoid control: Dilute In-coming tetanus toxoid coming before conjugation
based upon protein content by Lowry method to 20 µg/mL in PBS. Perform the analysis
as per the procedure mentioned in HPLC analysis section.
Figure 12 depicts system suitability of the method.
b. Linearity and Range of the method, LOD and LOQ:
Dilute the protein standard based upon protein content by Lowry method to 0.25% to 2% in PBS. i) Linearity and range of the method: Refer Table 7 and Figure 13.
Table 7: Experimental data on Linearity and Range of the method

Standard Curve
Conc. In percentage (%) Concentration in µg/mL
0.25 2.5
0.50 5
1.00 10
1.50 15
2.00 20
ii) LOD and LOQ of the method:
LOD and LOQ calculation at 280 nm and 214 nm produced approximately similar
results.
Limit of detection is 0.10% i.e. 1 µg/mL
Limit of quantitation is 0.20% i.e. 2 µg/ml
Table 8: Experimental data on LOD and LOQ of the method at 214 nm and 280 nm

LOD/LOQ calculation at 214 nm and 280 nm
Conc Area Area Avg
0.10 0.000 0.000 0.000
0.20 8160 8587 8373.5
0.40 17203 16578 16890.5
0.60 44393 43997 44195
0.80 58490 61359 59924.5
1.00 79731 76459 78095
1.20 94999 88869 91934
1.40 111831 110455 111143
1.60 127506 125554 126530
1.80 144955 143375 144165
2.0 173113 160739 166926
c. Recovery of the method:
For recovery analysis three samples are prepared and injected which are as follows -i) conjugate bulk (1 mg/mL) ii) spike alone (10 µL of 1mg/mL TT i.e. 10 µg TT)

iii) sample (1mg/mL conjugate bulk) + spike (10 µg TT).
The above three injections are performed and analyzed by HPLC. Integrate the obtained chromatogram i.e. the peak observed at 22 minutes. If the sample i.e. conjugate bulk contains peak area of 1000 AU, 6000 AU for spike alone and 6500 AU for sample + spike the recovery is calculated as per the following formula
% Recovery = (Area for Sample + spike)
(Area of sample + Area of Spike alone)
= (6500 AU) X 100
(1000 AU + 6000 AU)
= (6500 AU) X 100 (7000 AU)
= 0.928 X 100
= 92.9
= 93% (Recovery is 93%)
Table 9: Experiment 1 of Recovery of the method

Sr No Conc % Observed area ultra TT Expected area ultra TT % Spike recovery
1 0.25 65219 71688 91
2 0.5 150681 161387 93
3 1 352216 340786 103
4 1.5 552085 520185 106
5 2 673430 699584 96
Table 10: Experiment 2 of Recovery of the method

Sr No Conc % Observed area ultra TT Expected area ultra TT % Spike recovery
1 0.25 94503 65340 145
2 0.5 213785 243829 88
3 1 605098 600805 99
4 1.5 937259 957782 101
5 2 1331870 1314759 101
Table 11: Experiment data for Spike study with Ultra Purified TT in Hib conjugate
Bulk with 2 mg/mL

Sr No Conc % Observed area ultra TT Observed area of Spike TT % Spike recovery
1 0.25 261325 331051 79
2 0.5 508885 499836 102
3 1 1003897 764231 131
4 1.5 1592441 1221179 130
5 2 2264063 1598357 142
EXAMPLE 6: Estimation of less than 1% of monomeric free protein
Inject 1% Protein standard and 1 mg/mL conjugate bulk sample. If the free protein area in sample is obtained less than the free protein area in standard, the result shall be presented “Free protein <1%”. In case the free protein area in sample obtained more than they are in standard solution of 1% the result shall be presented as “Free protein > 1%” (This is based on the observation of area value)
Following data in Table 12 and Figures 14 to 19 presents and support that the method represents invention and its capability to estimate free protein up to 0.25% indicating comfortable estimation way below 1%.
Table 12: Experimental data for Estimation of less than 1% of monomeric free protein

TT Spike Area
0.25% 9032
0.50% 19712

1.0% 41140
1.5% 60522
2.0% 92663
The instant method of quantifying low amount of carrier protein (less than 1%) in the presence of aggregates and other impurities that has been utilized for free TT quantification in Hib-TT conjugate vaccine can also be employed for quantifying other types of free carrier proteins (for instance quantifying free DT in a polysaccharide protein conjugate vaccine).

We Claim:
1. A method for quantifying low level of unconjugated or free monomeric protein in a
test sample; wherein
a) the test sample comprises of polysaccharide-carrier protein conjugate bulk or protein-protein conjugate bulk or antibody-protein conjugate bulk; and
b) the method comprising the following steps:
i) subjecting the test sample to a protein assay to quantify the total protein
concentration; ii) subjecting the test sample to a high-performance size exclusion
chromatography (HPLC-SEC) to obtain an eluate comprising unconjugated
monomeric protein; iii) passing the eluate through a detector; and iv) evaluating the eluate by Photometric analysis for quantifying the unconjugated
or free monomeric protein in a test sample;
2. The method as claimed in claim 1, wherein the protein assay is Folin-Lowry assay.
3. The method as claimed in claim 1, wherein the HPLC-SEC is performed using size exclusion chromatographic columns selected from the group comprising of methacrylate based chromatography columns, acrylamide based chromatography columns and silica based chromatography columns, preferably columns based on Silica gel bonded with hydrophilic groups.
4. The method as claimed in claim 1, wherein the HPLC-SEC is performed using size exclusion chromatographic columns selected from the group comprising 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, TSKgel SuperSW2000, TSKgel SuperSW3000, TSKgel UP-SW3000, TSKgel G4000SWXL, Shodex Protein KW-802.5, TSK Gel G3000SWXL, TSKgel Ultra SW Aggregate, Acclaim SEC-1000, TSKgel G5000PWXL, Shodex OH Pak SB-800 HQ series comprising SB-802 HQ, SB-802.5 HQ, SB-803 HQ, SB-804 HQ, SB-805 HQ, SB-806 HQ and SB-806M HQ, HiPrep SEPHACRYL S100 HR, HiPrep SEPHACRYL S-200 HR, HiPrep SEPHACRYL S-300 HR, TSK gel SP- 5 PW, Anion exchange resin DEAE Toypearl.
5. The method as claimed in claim 4, wherein the HPLC-SEC is performed using size exclusion chromatographic column comprising is TSKgel G4000SWXL having pore

size of column between 400A° to 500A°, particle size between 7 to 9µm and the column is run at a temperature in between 25°C - 32°C
6. The method as claimed in claim 1, wherein the HPLC-SEC is performed using a buffer selected from the group comprising of phosphate buffer saline, Tris, MES, HEPES, citrate and combination thereof, having a pH in the range of 5.0 to 7.5; more preferably the buffer is phosphate buffer saline having pH ranging from 7.2 to 7.5, preferably pH 7.4; a flow rate of the buffer ranging from 0.2 to 2 ml per minute, preferably 0.5 to 0.1 ml per minute and the HPLC-SEC injection run time of up to 60 minutes.
7. The method as claimed in claim 1, wherein the eluate is evaluated by Photometric analysis selected from the group 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; more preferably by wavelength absorption detectors (spectroscopy) comprising of UV/PDA detector using wavelength between 190 nm to 800 nm, preferably 200 to 400 nm.
8. The method as claimed in claim 1, comprises of quantifying unconjugated or free monomeric protein in a test sample, wherein the low level of monomeric unconjugated protein is less than 3% or wherein the low level of monomeric unconjugated protein is less than 2% or wherein the low level of monomeric unconjugated protein is less than 1%.
9. The method as claimed in claim 1, wherein the test sample comprises of polysaccharide-protein conjugate bulk, including high molecular weight molecules, aggregate, monomer, dimer, trimer, low molecular weight molecules and detergent traces.
10. The method as claimed in claim 9, wherein the detergent traces comprises of cetyltrimethylammonium bromide (CTAB), tetrabutylammonium salt, myristyltrimethylammonium salt, hexadimethrine bromide, alkyl sulfates, sodium dodecyl sulfate, sodium deoxycholate, sodium dodecyl sulfonate, sodium s-alkyl sulfates, sodium fatty alcohol polyoxyethylene ether sulfates, sodium oleyl sulfate, N-oleoyl poly (amino acid) sodium, sodium alkylbenzene sulfonates, sodium α olefin sulfonates, sodium alkyl sulfonates, α-sulfo monocarboxylic acid esters, fatty acid sulfoalkyl esters, succinate sulfonate, alkyl naphthalene sulfonates, sodium alkane sulfonates, sodium ligninsulfonate, and/or sodium alkyl glyceryl ether sulfonates.

11. The method as claimed in claim 9, wherein the polysaccharide is a bacterial capsular polysaccharide, and is obtained from group comprising of Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasmaurealyticum, 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, Klebsiella pneumoniae, 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. such as Shigella sonnei, Shigella flexneri, Shigella dysenteriae; Shigella boydii; Ehrlichia spp., and Rickettsia spp..
12. The method as claimed in claim11, wherein polysaccharide is obtained from one or more of the following:
a. Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6, 6A, 6B, 6C, 6D, 6E, 6F, 6G,
6H, 7A, 7B, 7C, 7D, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10D, 10A, 11,
11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 13F, 14, 15A, 15C, 15B, 15F,
16, 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, 35A, 35B, 35C, 35D, 35F, 36,
37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, 48;
b. Neisseria meningitidis serotypes A, B, B16, B6, C, D, E29, H, I, K, K454, L, M,
W135, X, Y, Z;
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).

13. The method as claimed in claim 9, wherein the carrier protein is selected from the group comprising of CRM197, diphtheria toxin/toxoid, Neisseria meningitidis outer membrane complex, fragment C of tetanus toxoid, recombinant full length tetanus toxin with mutations (8MTT), 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, Por B), transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), PhtA, PhtB, PhtE, pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, M. catarrhalis uspA, protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), C5a peptidase group A or group B Streptococcus, human serum albumin, bovine serum albumin (BSA), NTHi high molecular weight protein, fHbp, 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, toxin A or B from C. difficile and S. agalactiae.
14. The method as claimed in any one of the preceding claims, wherein the test sample comprises of Haemophilus influenzae type b (Hib) - tetanus toxoid (TT) conjugate bulk and wherein in the molecular weight of Haemophilus influenzae type b (Hib) -tetanus toxoid (TT) conjugate bulk is the range from 100 kDa to 2000 kDa, preferably 800 kDa to 900 kDa.
15. The method as claimed in claim 14, wherein the Haemophilus influenzae type b (Hib) - tetanus toxoid (TT) conjugate bulk manufactured using cyanogen bromide activation of Haemophilus influenzae type b PRP, carbodiimide mediated coupling with Tetanus toxoid.