Novel reference standard and improved method for quantification of saccharide in vaccine

Abstract

A method for quantification of O-specific polysaccharide (OSP) content in a sample, the method comprises: (a) adding a chromogen to the sample to obtain a mixture, where the sample is optionally dilut

Description

The present disclosure relates to analytical methods. Particularly, the present disclosure relates to a method for quantification of saccharide or polysaccharide content in polysaccharide protein conjugates or vaccine composition. BACKGROUND OF THE DISCLOSURE All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art. The development of vaccines against polysaccharide-encapsulated pathogens is challenging due to an insufficient immune response to the polysaccharides. Capsular polysaccharide (CPS) is a component related to immune response on bacterial cells, capsular polysaccharides are composed of monosaccharides bonded together by glycosidic bonds, they have excellent biocompatibility and unique biological activity and have been proven to be the excellent targets for bacterial vaccine development. Bactericidal and / or opsonic antibodies directed against capsular polysaccharide (PS) glycotopes can prevent invasive diseases caused by capsular bacteria. Bacterial polysaccharides are composed of monosaccharide units as their building blocks. Different saccharides / sugars / sugar derivatives have been identified as building blocks, such as glucose, mannose, galactose, xylose, arabinose, rhamnose, fucose, fructose, ribose (Rib), glyceraldehyde, paratose, glucuronic acid, galacturonic acid, N-acetyl-glucosamine, and N-acetyl-galactosamine. Polysaccharides differ from each other based on monosaccharide compositions, length of the chain, and the extent of their branching system. Conjugate vaccines have been developed to induce a robust immune response against bacterial capsular polysaccharides (CPSs). CPSs are long polymers composed of many repeating units of simple sugars and serve as a protective external layer for many bacteria. Depending on the chemical composition of the repeating unit (usually composed of one to seven monosaccharides), bacteria can synthesize hundreds of chemically and immunologically different polysaccharides. Other than capsular polysaccharide (CPS), lipopolysaccharide (LPS) and exopolysaccharide (EPS) are also considered as virulence factors. The LPS observed in the structure of enteric bacteria consists of three domains; i) an inner fatty acid rich domain (Lipid A), ii) an intermediate core-domain and iii) an outer domain (O-antigen). The O-antigen consists of repeating units of sugar oligomers, are highly variable. O-antigen specific polysaccharides or O-specific polysaccharides (OSP) are critical for bacterial evasion from some effectors of the host immune response, particularly from the alternative complement cascade. Also, sensitivity to bactericidal proteins and peptides and complement in non-immune serum is affected by the integrity or the length of O-polysaccharides. The immunogenicity of the O-specific polysaccharide (O-SP) moiety of LPS when coupled to a carrier protein may protect against bacterial pathogens. In principle, protein that carries a human T cell epitope and can transform a T cellindependent polysaccharide-specific immune response into a T helper cell response can be used as a carrier for polysaccharide conjugate vaccine. In conjugate vaccines, polysaccharides are covalently conjugated to carrier proteins. Chemical conjugation with carrier protein can effectively enhance the immunogenicity of poorly immunogenic antigens. This method has been successfully used to produce polysaccharide conjugate vaccines against infectious diseases. The methods used to prepare the conjugated vaccine mainly include reductive amination, amidation and etherification reactions, and the conjugated vaccine obtained by the method shows a high degree of stability. Polysaccharide protein conjugates involve several important aspects, such as cellular metabolism, structure, assembly and identification, and are information carriers through biological interaction. In the study of the structure of these compounds, it is important to analyze the composition of the carbohydrate moiety and ensure its physical and chemical properties. WHO provides excellent guidance for key quality control tests at each stage of conjugate vaccine. Vaccines require a comprehensive analytical characterization with a panel of quality control tests to guarantee consistent manufacturing and immunogenicity. Therefore, after the successful coupling of polysaccharides and proteins, various tests are required to ensure its stability, safety and effectiveness. Different tests are performed to evaluate or quantify or estimate the factors like polysaccharide content, polysaccharide size, protein content, identity verification, O-acetyl content, purification assays and immunogenicity. The polysaccharide component of polysaccharide-protein conjugate vaccines undergoes gradual depolymerization at a rate that depends on the type of conjugate, formulation components and storage conditions. This increases unconjugated polysaccharide (Free polysaccharide) content in the final vaccine formulation. Hence tests should be conducted to ensure stability of product. Polysaccharide-carrier protein conjugates are known to release unconjugated polysaccharide after conjugation while it undergoes further processing, lyophilization or storage in liquid as well as solid formulations. Polysaccharide content is considered as important control to guarantee the vaccine immunogenicity. The polysaccharide that is covalently bound to the carrier protein (i.e. conjugated polysaccharide) is immunologically important for clinical protection. Also, the content of free polysaccharide (level of unconjugated or “free” polysaccharide), has a critical impact on the quality of the vaccine in particular on vaccine efficacy, since it has a negative effect on the final immunogenicity, as the immune responses can be directed to these free polysaccharides rather than to conjugated forms. Thus, it is difficult to achieve desired / optimum immunogenicity when the amount of free polysaccharide exceeds that of the conjugated polysaccharide. Free polysaccharide is used to assess the vaccine stability and integrity, determining, among other parameters, the product shelflife. Accurate quantification of the free (unconjugated) polysaccharide is one of the most critical quality attributes to be monitored during vaccine manufacture and storage. Accordingly, a reliable and accurate determination of total and free polysaccharide in polysaccharide-protein conjugate vaccines is one of the important quality control parameters in development and production of these conjugate vaccines for product release and stability monitoring to ensure appropriate immune response. An acceptable value of unconjugated / free saccharide consistent with adequate immunogenicity, as shown in clinical trials, should be established for the particular product and each final lot must be shown to comply with the prescribed limit (Refer WHO TRS No.897, pg 17, 2000, A. 3.3.5 section; WHO / TRS / 924 Page No. 14, A.3.3.5; Page 145 A.3.6.4 WHO / TRS / 962). Accordingly, a reliable and accurate determination of unconjugated 3 polysaccharide in polysaccharide-protein conjugate vaccines is one of the important quality control parameters in development and production of these conjugate vaccines. Thus, it is a statutory requirement to quantify the concentration of unconjugated polysaccharide available in final vaccine formulation. The assays in prior art for quantification of polysaccharides are diverse and typically involve complex procedures. Many different classes of assays have been utilized to quantify saccharide / polysaccharide content, including ELISA, liquid and gas chromatography, fluorescence, infrared spectroscopy, capillary electrophoresis, mass spectrometry and colorimetry. Refractive index has been used in conjunction with HPLC for many years to estimate sugar content. However, without the added purification and normalization provided by time-consuming chromatography, this approach has proven exceedingly sensitive to chemical, thermal, and hydraulic interference. Due to the ubiquity of spectrophotometers and relative simplicity of the measurement, colorimetric assays have remained popular and include assays based on phenol sulfuric acid for hexoses / pentoses, anthrone for hexoses / pentoses, resorcinol for sialic acid, ascorbic acid for phosphates, and Purpald for glycols. Sources of variability within the various colorimetric assays for saccharide concentration determination include elements related to sample preparation and measurement by a human operator, such as sample handling, reagent dilutions, instability of reagents, variation of heating and cooling times. Other methods involving phenol, 1-napthosulfonate, and aniline phthalate / Trichloroacetic acid (TCA) have been proposed but suffer from toxicity, interference, and limited reactivity with ketoses. All of the aforementioned methods suffer from a combination of large sample requirements, complex sample preparations and derivatizations, highly toxic chemicals, low throughput, insufficiently broad reactivity and substantial interference. Several inventors have recognized the analytical bottleneck posed by sugar quantitation and devised high throughput methods. Procedures based on anthrone, phenol sulfuric acid, and purpald have been scaled-down to microplates. However, these assays possess many of the same weaknesses as the original progenitor assays such as reagent instability, poor reactivity and undesirable handling of highly toxic reagents. Anthrone tends to increase background noise, thus decreased sensitivity and requires to be used in cold conditions. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) has also been employed as a quantification method with high sensitivity; however, samples require hydrolysis and carrier protein release prior to analysis, and the equipment is still relatively expensive. Alternatively, the immunoassay-based methods mentioned previously may be used with suitable standards for quantification but comes with the aforementioned drawbacks in addition to reagent development burden and its associated increased animal use. While multiplexed immunoassay-based methods such as those involving bead-based technology have been reported for direct antigen detection in urine for diagnostic purposes and can be developed for vaccine antigen characterization, the lack of availability of off-the-shelf, validated kits places much of the burden of assay development and optimization on individual manufacturers. Among many colorimetric methods for carbohydrate determination, the phenol sulfuric acid method is the easiest and most reliable method for measuring neutral sugars in oligosaccharides, proteoglycans, glycoproteins, and glycolipids. The phenol sulfuric acid method is used widely because of its sensitivity and simplicity. Other methods using anthrone, orcinol, or resorcinol may be sensitive but are not convenient. Xionggang Xi et al. 2010 (Shanghai Normal University, PR China) discloses Phenol-sulfuric acid method for determination of tea polysaccharides in Camellia sinensis; monosaccharide mixture solution (MMS) was used as a standard solution for the calibration curves. K. Stojilkovski et al. 2019 (University of Ljubljana, Slovenia) discloses phenol-sulfuric acid method for the determination of polysaccharides in herbal syrup. In the industrial production process of polysaccharide protein conjugate vaccines, it is often necessary to measure and monitor the content of polysaccharides. Due to the wide variety of polysaccharides, the content and existing forms of polysaccharides are also variable. Therefore, choosing an appropriate detection method is crucial. The total sugar represents the sum of reducing sugars (glucose, fructose, lactose, etc.) and oligosaccharides (sucrose, etc.) that can be hydrolyzed into reducing sugars under the measurement conditions. The determination methods of total sugar content mainly include phenol-sulfuric acid method, anthrone-sulfuric acid method, full-wavelength enzyme labelling method, and direct titration method. Phenol-sulfuric acid method can also measure methylated sugars, pentoses and polysaccharides, and is not affected by proteins. The determination principle of the phenol-sulfuric acid method is that concentrated sulfuric acid dehydrates polysaccharides to form uronic acid and hydroxyurea formaldehyde, and then condenses with phenol to form orange-red compounds. Within a certain concentration range, the color depth is proportional to the sugar content, which can be determined at specific wavelength. However, experimental conditions, such as color development wavelength, color development time, color development temperature, and detection range play an important role in the accuracy of the determination of total sugar content. In addition, the calculation of the specific total sugar content needs to be obtained according to the standard curve drawn by standard monosaccharides. At present, glucose is often used as a standard in experiments, which leads to large errors in the determination of sugar content, especially for heteropolysaccharides. WO2022143716 discloses that selection of reference substance is particularly important when measuring the polysaccharide content by colorimetry. The commonly used detection methods for polysaccharide content mainly use a monosaccharide, such as glucose, as the standard, but the detection results are often inconsistent with the actual situation. Apart from glucose, the hydrolysis of heteropolysaccharides may also generate other monosaccharides, and different monosaccharides have significant differences in structure and physicochemical properties. For example, in terms of functional groups, glucose is aldohexose and fructose is ketohexose. Also, ribose, xylose, and arabinose are pentose. And glucose, mannose, galactose, and fructose are hexose. In addition, in the process of phenol-sulfuric acid method, pentoses are more likely to react than hexose, the reaction rate of uronic acid is relatively slow, and uronic acid cannot be completely converted. Therefore, it is speculated that there may be significant differences in color development ability of different monosaccharides after condensation with phenol. Although a great deal of research has been done to improve the phenol-sulfuric acid method, little attention has been paid to the errors caused by monosaccharides standard used in the process. Phenol-sulfuric acid assay or method are colorimetric assays which work best if the standards and sample are of the same nature, i.e., behaviour of sample must be similar to that of the standard. The uniqueness of a molecule is exploited for designing the colorimetric assay mainly the Orcinol assay reacts with the ribose unit in the Hib polysaccharide estimation method. In case of PS estimation in Meningococcal vaccines, Phosphorous backbone of the polysaccharide is used as a unique character and polysaccharide content is estimated by determining the Phosphorous assay. In some methods it is noted that, excipients, such as mannitol, 2-phenoxyethanol, tris, polysaccharide like Vi polysaccharide, carrier proteins like diphtheria toxoid and tetanus toxoid interfere with “phenol sulfuric assay”. Rehab H. Bahy et al. 2016 (Egypt Cairo University, Egypt) discloses; Conjugation of pneumo polysaccharide (CPS was prepared from S. pneumoniae strain (6A / B or 19F)) and their evaluation; Estimation of polysaccharide content (Phenol-sulfuric acid method) using serial dilution of glucose standard solution. Canaan-Haden et al. 2006 (Center for Genetic Engineering and Biotechnology, Cuba) discloses method using high-performance reverse phase (RP) chromatography with fluorescence detection to determine the composition and identity of Streptococcus pneumoniae capsular polysaccharide. For polysaccharide quantification, a monosaccharide reference mixture (Gal, Glc, Rha,ManNAc, GlcNAc, and GalNAc) was used as standard for routine analysis. M.C. Cook et al. 2013 (Centre for Vaccine Evaluation, Biologies and Genetic Therapies Directorate Health Canada, Canada) discloses using HPAEC-PAD method along with mixed standard for quantification of meningococcal polysaccharides; Mixed standard solutions were made using monosaccharides (N-acetyl neuraminic acid (A0812), glucose (G8270) and galactose (G0750), respectively, for serogroups C, Y and W135). Polysaccharides typically contain repeating units of different types of sugars. For polysaccharides like O-specific polysaccharide (OSP), the analysis of polysaccharides is done using Dubois method which uses glucose as a standard (Phenol sulfuric acid assay). Phenol Sulfuric acid assay is a commonly used assay for determination of polysaccharide content containing hexoses sugars. Such polysaccharides have repeating units with multiple sugars. These sugars react in the process of Dubois method with different degree of reaction for each sugar. This is because there is no uniqueness which can be exploited and all the present hexoses are general sugars with varied reactivity hence accurate quantification of polysaccharide content is not possible. Polysaccharides with no uniqueness regarding the sugars present in their structure, when analysed using Dubois method (Phenol sulfuric acid assay) with glucose as a standard tends to undergo a reaction with recoveries on lower side (-60%). If O-specific polysaccharides (OSP) e.g. OSP of Salmonella Paratyphi A is to be estimated there are no international or reference standards available for quantification of the same. N. Ravenscroft et al. 2015 (University of Cape Town, South Africa; University of Trieste, Italy; Novartis Vaccines Institute for Global Health, Italy) discloses O-acetylated O-polysaccharide isolated from Salmonella paratyphi A and it use in vaccine preparation; the ratios of 1.00:1.00:1.00:0.76:0.17 ofRha, Man, Gal, Glc, Par present in the O-polysaccharide structure of S. paratyphi A', In the absence of a commercially available monomer standard, Par was quantified by 1H NMR analysis which yielded the expected molar ratio of 1:1 with respect to Rha from the ratio of the corresponding H-6 resonances. Incomplete glucosylation agrees with HPAEC-PAD data (74%) previously reported. GLC analysis of the chiral glycosides of the OPS showed that the hexoses were in the D absolute configuration and Rha in the L absolute configuration. As noted above, the absence of a commercial standard meant that analysis for Par could not be performed, and its D configuration was established by NMR glycosylation shifts. In OSP of Salmonella Paratyphi A, structure is determined by NMR method. NMR has several disadvantages, such as it is expensive, requires dedicated space having large area, expert evaluations, it is not user friendly and requires more turnaround time. Further, Dubois method (Phenol sulfuric acid assay) gives inaccurate results of polysaccharide content in the OSP. Ali A et al 2014 discloses Salmonella enterica serovar Paratyphi A O-specific polysaccharide conjugated to diphtheria toxoid, wherein Sephadex G-75 profiles of S. Paratyphi A OSP are mentioned. Elution volume was plotted against refractive index (RI) detector readings and polysaccharides (PS) concentrations as measured by Anthrone assay. Francesca Micoli et al 2012 discloses that Phenol sulfuric assay was used for total sugar content, using Glc as standard for O:2-CRM197 Conjugates against Salmonella Paratyphi A. Laura B. Martin et al 2023 highlights the gaps for Paratyphi vaccine development and likewise highlights urgent need for availability of reference standard to be used in vaccine characterization and standardized immunological assays. Therefore, there is unmet need for a simple, fast, safe, accurate, high throughput, nearuniversal method, more efficient, convenient, cost effective, less turnaround time, highly sensitive, robust, non-destructive stable and reliable alternative method for estimation of both total polysaccharides (from Polysaccharide, in-process, bulk conjugates and Final Lot) and free polysaccharides (from Bulk conjugate and final lot) from monovalent / multivalent vaccine(s), particularly by using 1) a suitable mixed monosaccharide reference standard that mimics the respective saccharide’s original structural composition that overcomes the overestimation or underestimation problem reported previously; 2) a sample pre-treatment for removal of excipient that may interfere with subsequent phenol sulfuric acid assay; 3) optimized concentrations of Phenol and Sulfuric acid; and 4) utilizing a sequence of addition of phenol followed by sulfuric acid (addition in the centre of the test tube). OBJECTS OF THE DISCLOSURE: Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows: An object of the present disclosure is to ameliorate one or more problems of the prior art or to at least provide a useful alternative. Another object of the present disclosure is to provide a method for quantifying saccharide or polysaccharide content in a composition. Another object of the present disclosure is to provide a method for quantification of total polysaccharide as well as free polysaccharide content in vaccine composition. Yet another object of the present disclosure is to provide a method for quantification of total polysaccharide as well as free polysaccharide content in monovalent or multivalent conjugate bulk. Yet another object of the present disclosure is to provide a method for quantification of total polysaccharide as well as free polysaccharide content by colorimetric assay in monovalent or multivalent conjugate bulk. Yet another object of the present disclosure is to provide a method for quantification or estimation of both total polysaccharides (from different stages, such as Polysaccharide, in-process, bulk conjugates and Final Lot) and free polysaccharides (from different stages, such as Bulk conjugate and final lot) from monovalent / multivalent vaccine(s). Yet another object of the present disclosure is to provide a method involving use of novel standard comprising more than one sugar for quantification of total polysaccharide as well as free polysaccharide content by colorimetric assay in the monovalent or multivalent conjugate bulk or vaccine composition. Yet another object of the present disclosure is to provide a method involving use of more than one sugar i.e. mixture or combination of sugars as standard for quantification of total polysaccharide as well as free polysaccharide content by colorimetric assay in the monovalent or multivalent conjugate bulk or vaccine composition. Yet another object of the present disclosure is to provide a method involving use of more than one sugar i.e. mixture or combination of sugars as standard for quantification of total polysaccharide as well as free polysaccharide content by colorimetric assay at different stages such as purified polysaccharides, purified polysaccharide protein conjugate bulk and final vaccine formulation in the monovalent or multivalent conjugate bulk or vaccine composition. Still another object of the present disclosure is to provide a method for assessing the quality and stability of the polysaccharide-protein conjugate vaccine composition. Still another object of the present disclosure is to provide a simple, fast, safe, accurate, high throughput, near-universal method, more efficient, convenient, cost effective, less turnaround time, highly sensitive, robust, non-destructive stable and reliable alternative method for quantification or estimation of total polysaccharide as well as free polysaccharide content. Still another object of the present disclosure is to provide a suitable mixed monosaccharide reference standard that mimics the respective saccharide’s original structural composition and overcomes the overestimation or underestimation problem reported previously. Still another object of the present disclosure is to provide optimized concentrations of Phenol and Sulfuric acid for quantification of total polysaccharide as well as free polysaccharide content. Still another object of the present disclosure is to provide a method utilizing a sequence of addition of phenol followed by sulfuric acid (addition in the centre of the test tube) for quantification of total polysaccharide as well as free polysaccharide content. 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 with technical advancement. 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 THE DISCLOSURE The present invention provides stable and efficient method for quantification of saccharide or polysaccharide content in polysaccharide protein conjugates or vaccine composition. Accordingly, in one aspect, the present invention is directed to a method for quantifying saccharide content in a composition, the method comprising: adding a chromogen to a sample to obtain a mixture, wherein the sample is optionally diluted with a buffer prior to the addition of the chromogen; mixing an acid with the mixture to obtain a solution; incubating the solution; subjecting the incubated solution to a spectrophotometric assay or colorimetry method; and quantifying the saccharide content in the sample using mixed saccharide standard solution. The mixed saccharide standard solution comprises at least two saccharides. Accordingly, in another aspect, the present invention is directed to a method for quantification of O-specific polysaccharide content in a sample. The method for quantification of O-specific polysaccharide content in a sample, the method comprising: diluting the sample with the buffer to obtain the diluted sample comprising O-specific polysaccharide in the range from 0 pg / ml to 50.0 pg / ml; adding the phenol in the range from 70.0% to 90.0% to the diluted sample to obtain the mixture; mixing the sulphuric acid in range from 15.0 M to 20.0 M to mixture to obtain the solution; incubating the solution at temperature in range from 20.0 °C to 30.0 °C for duration in range of 20.0 minutes to 50.0 minutes; subjecting the incubated solution to the spectrophotometric assay at 480 nm; quantifying the O-specific polysaccharide content in the sample using mixed saccharide standard solution. The mixed saccharide standard solution is a combination of rhamnose, mannose, galactose, glucose and paratose in a ratio of 1: 1: 1: 0.76:1. The sample is purified O-specific polysaccharides, purified O-specific polysaccharide protein conjugate bulk or a final vaccine formulation. The purified O-specific polysaccharide protein conjugate bulk and final vaccine formulation is additionally subjected to pre-treatment by passing through desalting column and membrane filter and treating with DOC-HC1, prior to the dilution of the sample. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS The present invention will now be described with the help of the accompanying drawing, in which: Figure 1 illustrates PD-10 desalting column preparation. Figure 2 illustrates PD-10 desalting column equilibration. Figure 3 illustrates aample application to PD-10 desalting column. Figure 4 illustrates membrane filter setup (3KDa). Figure 5 illustrates sample elution through membrane filter setup (3 KDa) Figure 6 illustrates Linearity of phenol sulphuric acid method Figure 7 illustrates NMR Data of samples comprising OSP Purified Polysaccharide (simple molecules) Figure 8 illustrates NMR Data of samples comprising OSP-DT Conjugate Bulk (complex molecules) DETAILED DESCRIPTION OF THE DISCLOSURE 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, unless stated otherwise. 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. 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 nonlimiting 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. More particularly, as regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification clearly and unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise. Furthermore, the ranges defined throughout the specification include the end values as well, i.e. a range of 1 to 10, between 1 to 10 imply that both 1 and 10 are included in the range. For the avoidance of doubt, the applicant shall be entitled to any equivalents according to applicable law. As used herein, the term “about” when qualifying a value of a stated item, number, percentage, or term refers to a range of plus or minus 10 percent, 9 percent, 8 percent, 7 percent, 6 percent, 5 percent, 4 percent, 3 percent, 2 percent or 1 percent of the value of the stated item, number, percentage, or term. Preferred is a range of plus or minus 10 percent. In case numerical ranges are used herein such as “in a concentration between 1 and 5 micromolar”, the range includes not only 1 and 5 micromolar, but also any numerical value in between 1 and 5 micromolar, for example, 2, 3 and 4 micromolar. The term “in vitro” as used herein denotes outside, or external to, the animal or human body. The term “in vitro” as used herein should be understood to include “ex vivo”. The term “ex vivo” typically refers to tissues or cells removed from an animal or human body and maintained or propagated outside the body, e.g., in a culture vessel. The term “in vivo” as used herein denotes inside, or internal to, the animal or human body. The subject matter of and information disclosed within the publications and patents or patent applications mentioned in this specification are incorporated by reference herein. Definitions: In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth through the specification. As used herein ‘total polysaccharide’ refers to a polysaccharide which is not conjugated or unbound to the carrier protein and conjugated or bound to carrier protein. In some embodiments, as used herein the terms ‘total polysaccharide’ and ‘total saccharide’ refers to a total O-specific polysaccharide (OSP or O-SP). In some embodiments, the term ‘total polysaccharide’ or ‘total OSP polysaccharide’ also refers to the total amount of polysaccharide or OSP-polysaccharide present in an OSP-conjugate bulk or vaccine formulation. As used herein the term ‘polysaccharide’ can be interchangeably used with the term ‘saccharide’. As used herein ‘free polysaccharide’ refers to a polysaccharide which is not conjugated or bound to the carrier protein and can be present in the polysaccharide protein conjugate bulk or polysaccharide protein conjugate vaccine. In some embodiments, as used herein the terms ‘free polysaccharide’ and ‘free saccharide’ refers to a free O-specific polysaccharide (OSP or O-SP). The term ‘free polysaccharide’ also refers to ‘unconjugated polysaccharide’. As used herein ‘standard solution’ refers to ‘saccharide standard solution’ or ‘mixed saccharide standard solution’. As used herein the term ‘colorimetry’ can be interchangeably used with the term ‘ spectrophotometry’. As used herein the term ‘mixture of saccharides’ refers to ‘combination of sugars’. As used herein the term ‘sample’ refers to ‘test sample’. The term "vaccine" refers to "immunogenic composition" and vice versa. As used herein, the term “quantification” is used interchangeably with “estimation” or “quantitative estimation” and refers to the act of measuring the amount or concentration of a substance. In the context of the present invention, it refers to the measurement of the amount or concentration of polysaccharide or O-specific polysaccharide in a sample. The terms “immunogen” and “immunogenic” refer to substances capable of producing or generating an immune response in an organism directed specifically against the polysaccharide. The terms “antigenic” and “antigenicity” refer to the capability of a polysaccharide / saccharide to be specifically bound by an antibody to the polysaccharide. The term “immunospecific” means that the antibodies corresponding to the polysaccharide / saccharide antigens exhibit a substantially greater affinity for the PSs attached to solid supports and biomolecules compared to the affinity for other antigens. Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, 15 or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps and can mean "includes”, "including”, and the like; “consisting essentially of’ or “consists essentially” likewise is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. The use of the expression “one or more” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results. While certain embodiments of the inventions have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Variations or modifications to the composition of this invention, within the scope of the invention, may occur to those skilled in the art upon reviewing the disclosure herein. Such variations or modifications are well within the spirit of this disclosure. In an aspect, the present invention is directed to a method for determining saccharide content in a composition. In an embodiment, the present invention is directed to a method for quantifying saccharide content in a composition, the method comprising: a) providing a sample; b) diluting the sample with the buffer to obtain a diluted sample; c) adding a chromogen to the diluted sample to obtain a mixture; d) mixing an acid with the mixture to obtain a solution; e) incubating the solution; and f) subjecting the incubated solution to a spectrophotometric assay or colorimetry assay; and g) quantifying the saccharide content in the sample using mixed saccharide standard solution wherein the mixed saccharide standard solution comprises at least two saccharides. In an embodiment, the present invention is directed to a method for quantifying saccharide content in a composition, the method comprising: a) adding a chromogen to a sample to obtain a mixture, wherein the sample is optionally diluted with a buffer prior to the addition of the chromogen; b) mixing an acid with the mixture to obtain a solution; c) incubating the solution; and d) subjecting the incubated solution to a spectrophotometric assay or colorimetry assay; e) quantifying the saccharide content in the sample using mixed saccharide standard solution, wherein the mixed saccharide standard solution comprises at least two saccharides. The saccharide content includes but not limited to monosaccharide content, disaccharide content, oligosaccharide content, total polysaccharide content, free polysaccharide or combinations thereof. The sample incudes but not limited to a purified polysaccharide, a purified polysaccharide protein conjugate bulk, a final vaccine formulation, an immunogenic composition, a vaccine or combinations thereof. The vaccine composition includes but not limited to a monovalent vaccine, a multivalent conjugate bulk, a mixture of different vaccines against different diseases. The present disclosure provides the method for quantification or estimation of total polysaccharide as well as free polysaccharide content in the conjugate bulk or vaccine composition. In an embodiment, the method provides quantification or estimation of total polysaccharide as well as free polysaccharide content in the monovalent or multivalent conjugate bulk. In an embodiment, the method provides quantification or estimation of total polysaccharide as well as free polysaccharide content by spectrophotometry in the monovalent or multivalent conjugate bulk or vaccine composition. In another embodiment, the method provides quantification or estimation of total polysaccharide as well as free polysaccharide content by colorimetry in the monovalent or multivalent conjugate bulk or vaccine composition. In another embodiment, the method uses alternative standard to glucose as standard for quantification of total polysaccharide as well as free polysaccharide content by colorimetry in the monovalent or multivalent conjugate bulk or vaccine composition. In another embodiment, the method provides quantification or estimation of total polysaccharide as well as free polysaccharide content by colorimetry at different stages such as purified polysaccharides, purified polysaccharide protein conjugate bulk and final vaccine formulation in the monovalent or multivalent conjugate bulk or vaccine composition. In another embodiment, the method provides quantification or estimation of total polysaccharide as well as free polysaccharide content by spectrophotometry or colorimetry in the monovalent or multivalent conjugate bulk or vaccine composition comprising following non-limiting steps: a) providing the sample; b) diluting the sample; c) adding the chromogen to the sample to obtain the mixture; d) mixing the acid with the mixture to obtain the solution; e) incubating the solution; f) subjecting the incubated solution to the spectrophotometric assay or colorimetric assay; and g) quantifying the saccharide content in the sample using mixed saccharide standard solution. In another embodiment, the method for quantification or estimation of total polysaccharide as well as free polysaccharide content by spectrophotometry or colorimetry in the monovalent or multivalent conjugate bulk or vaccine composition comprises: a) adding the chromogen to the sample to obtain the mixture, wherein the sample is optionally diluted with a buffer prior to the addition of the chromogen; b) mixing the acid with the mixture to obtain the solution; c) incubating the solution; d) subjecting the incubated solution to the spectrophotometric assay or colorimetric assay; and e) quantifying the saccharide content in the sample using mixed saccharide standard solution. In another embodiment, the present invention is directed to the method for quantifying saccharide content in the composition, the method comprising: a) providing the sample; b) diluting the sample; c) adding the chromogen to the sample to obtain the mixture; d) mixing the acid with the mixture to obtain the solution; e) incubating the solution; f) subjecting the incubated solution to a spectrophotometric assay or colorimetry assay; and g) quantifying the saccharide content in the sample using mixed saccharide standard solution as standard solution. In another embodiment, the method for quantifying saccharide content in the composition, comprises: a) adding the chromogen to the sample to obtain the mixture, wherein the sample is optionally diluted with a buffer prior to the addition of chromogen; b) mixing the acid with the mixture to obtain the solution; c) incubating the solution; d) subjecting the incubated solution to a spectrophotometric assay or colorimetry assay to obtain an absorbance value; and e) quantifying the saccharide content in the sample by comparing the absorbance value with a reference standard of a mixed saccharide standard solution. In another embodiment, the present invention is directed to the method for quantifying or estimating total polysaccharide content in the composition, the method comprising: a) providing the sample; b) diluting the sample; c) adding the chromogen to the sample to obtain the mixture; d) mixing the acid with the mixture to obtain the solution; e) incubating the solution; f) subjecting the incubated solution to the spectrophotometric or colorimetric assay, alongside the standard solution; and g) quantifying the total polysaccharide content in the sample using mixed saccharide standard solution. In another embodiment, the method for quantifying or estimating total polysaccharide content in the composition comprises: a) adding the chromogen to the sample to obtain the mixture, wherein the sample is optionally diluted with a buffer prior to the addition of chromogen; b) mixing the acid with the mixture to obtain the solution; c) incubating the solution; d) subjecting the incubated solution to a spectrophotometric assay or colorimetry assay to obtain an absorbance value; and e) quantifying the saccharide content in the sample by comparing the absorbance value with a reference standard of a mixed saccharide standard solution. In another embodiment, the present invention is directed to the method for quantifying free polysaccharide content in the composition, the method comprising: a) providing the sample; b) diluting the sample; c) adding the chromogen to the sample to obtain the mixture; d) mixing the acid with the mixture to obtain the solution; e) incubating the solution; f) subjecting the incubated solution to a spectrophotometric or colorimetric assay, alongside a mixed saccharide standard solution; and g) quantifying the free polysaccharide content in the sample using the mixed saccharide standard solution. In another embodiment, the method for quantifying free polysaccharide content in the composition comprises: a) adding the chromogen to the sample to obtain the mixture, wherein the sample is optionally diluted with a buffer prior to the addition of chromogen; b) mixing the acid with the mixture to obtain the solution; c) incubating the solution; d) subjecting the incubated solution to a spectrophotometric assay or colorimetry assay alongside a mixed saccharide standard solution to obtain an absorbance value; and e) quantifying the saccharide content in the sample by comparing the absorbance value with a reference standard of the mixed saccharide standard solution. In an embodiment, the present invention is directed to the method for quantification of saccharide content in a sample, the method comprising: a) diluting the sample with a buffer to obtain a diluted sample; b) adding a chromogen to the diluted sample to obtain a mixture; c) mixing an acid to mixture to obtain a solution; d) incubating the solution; e) subjecting the incubated solution to a spectrophotometric assay or colorimetry assay; and f) quantifying the saccharide content in the sample using a mixed saccharide standard solution, wherein the mixed saccharide standard solution comprises at least two saccharides. In an embodiment, the method as disclosed herein is directed to estimate the individual concentration of unconjugated polysaccharide as well as total polysaccharide content in monovalent or multivalent polysaccharide protein conjugate bulk / vaccine composition, wherein the composition comprise of one or more polysaccharides. In an embodiment, the method as disclosed herein is directed to estimate the individual concentration of unconjugated polysaccharide as well as total polysaccharide content in monovalent or multivalent O-specific polysaccharide protein conjugate bulk / vaccine composition, wherein the composition comprises of one or more O-specific polysaccharides. In another embodiment, polysaccharide as a component of polysaccharide-protein conjugate vaccine component is selected from Streptococcus spp. such as Group A Streptococcus, Group B Streptococcus (group la, lb, II, III, IV, V, VI, VII, VII, VIII, and IX); Streptococcus pneumoniae (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, IOC, 10A, HA, HF, HB, HC, HD, HE, 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, 32A, 32F, 3 3A, 33C, 3 3D, 33E, 33F, 33B, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 4IF, 41 A, 42, 43, 44, 45, 46, 47F, 47A, and 48) , Streptococcus pyogenes. Streptococcus agalactiae; Streptococcus viridans; Salmonella spp. such as, Salmonella typhi; Salmonella paratyphi'. Salmonella enteritidis; Salmonella typhimuriunr, Shigella spp. such as Shigella sonnei, Shigella flexneri, Shigella dysenteriae; Shigella boydii; E.coli; Neisseria meningitidis (serotypes such as A, B, B16, B6, C, D, E29, H, I, K, K454 L, M, W135, X, Y, and Z etc); Neisseria gonorrhoeae; Haemophilus influenzae', Haemophilus pneumonia, Helicobacter pylori', Chlamydia pneumoniae', Chlamydia trachomatis', Ureaplasma urealyticunr, Mycoplasma pneumoniae', Staphylococcus spp. such as Staphylococcus aureus, Staphylococcus aureus type 5, Staphylococcus aureus type 8', Enterococcus faecalis; Enterococcus faeciunr, Bacillus anthracis; Vibrio cholerae; Pasteurella pestis; Pseudomonas aeruginosa, Campylobacter spp. such as jejuni', Clostridium spp. such as Clostridium difficile', Mycobacterium spp. such as Mycobacterium tuberculosis', Moraxella catarrhalis; Klebsiella pneumoniae', Treponema spp.; Borrelia spp.; Borrelia burgdorferi', Leptospira spp.; Hemophilus ducreyi', Corynebacterium diphtheria, Bordetella pertussis', Bordetella parapertussis', Bordetella bronchiseptica, Ehrlichia spp.; and Rickettsia spp. In another embodiment, O-specific polysaccharide as a component of O-specific polysaccharide-protein conjugate vaccine component is selected from group of Gramnegative bacteria comprising Salmonella spp. such as, Salmonella typhi, Salmonella paratyphi A, Salmonella enteritidis, Salmonella typhimuriunr, Shigella spp. such as Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Citrobacter spp. such as, C. freundii, C. werkmanii; Cronobacter species (former Enterobacter sakazakii); Shigella boydii', Escherichia coli; Klebsiella pneumoniae; Vibrio cholerae; Pseudomonas aeruginosa; Plesiomonas shigelloides; Campylobacter jejuni. In another embodiment, the carrier protein of polysaccharide-protein conjugate vaccine isselected from group comprising but not limited to CRM197, diphtheria toxoid, tetanus toxoid, Neisseria meningitidis outer membrane complex, fragment C of tetanus toxoid, recombinant full-length tetanus toxin with eight individual amino acid mutations (8MTT), pertussis toxoid, protein D of H. influenzae, E. coli LT, E. coli ST, exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), Outer Membrane Protein T2554 from Salmonella spp, Pilus proteins from Streptococcus spp, porins, transferrin binding proteins (Tbp-B, Tbp-A etc), 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), purified protein derivative of tuberculin (PPD), Cholera toxin subunit B, fHbp, Por A and Por B. In another embodiment, the carrier protein of polysaccharide-protein conjugate vaccine is selected from CRM197, diphtheria toxoid, tetanus toxoid. In another embodiment, the carrier protein of polysaccharide-protein conjugate vaccine is diphtheria toxoid (DT). In another embodiment, the sample used in the method for quantification or estimation of total polysaccharide as well as free polysaccharide content by colorimetry is purified polysaccharides, purified polysaccharide protein conjugate bulk or final vaccine formulation. In another embodiment, the purified polysaccharides is O-specific polysaccharides, the purified polysaccharide protein conjugate bulk is O-specific polysaccharide - diphtheria toxoid conjugate bulk, final vaccine formulation is monovalent or multivalent vaccine formulation. The multivalent vaccine formulation comprises Vi polysaccharide - tetanus toxoid conjugate, O-specific polysaccharide - diphtheria toxoid conjugate and excipients. In another embodiment, the excipients in multivalent vaccine formulation are selected from a group comprising maltose, lactose, sucrose, mannitol, trehalose or combinations thereof. In another embodiment, the dilutions of samples are performed to obtain the concentrations ranging from about 0 to 50 pg / ml, including all the values in the range, for instance, 0.1 pg / ml, 0.2 pg / ml, 0.3 pg / ml, 0.4 pg / ml and so on and so forth, up until 50 pg / ml and including subranges of the range 0 pg / ml to 50 pg / ml. In another embodiment, the sample comprising purified polysaccharides are not to be subjected to pre-treatment. In another embodiment, the sample comprising purified polysaccharide protein conjugate bulk and vaccine formulation including polysaccharide protein conjugate is subjected to pretreatment before dilution. In another embodiment, the pre-treatment process includes passing the test sample through columns, filters or combination thereof. In another embodiment, the pre-treatment process includes passing the test sample through columns or the pre-treatment process includes passing the test sample through filters or the pre-treatment process includes passing the test sample through columns followed by filters to obtain a pre-treated sample. In another embodiment, the pre-treatment process is performed to remove the impurities such as excipients or salts, present in the sample. The excipients or salts present in the sample can interfere or cause error in the quantification of saccharide content. In another embodiment, the pre-treatment process is performed to remove or filter out excipients selected from a group comprising maltose, lactose, sucrose, mannitol and trehalose, preferably sucrose or mannitol. Excipient are known to interfere with the estimation of polysaccharide and reduces load on detector; therefore, removal of excipients helps in maintaining the efficiency of the detector. In an embodiment, mannitol interferes with the estimation or quantification of polysaccharide content. In another aspect of the embodiment, the salts to be filtered out from the test sample consist of one or more salts selected from the group of NaCl, KC1, Na2SO4, (NH4)2SO4, sodium phosphate and sodium citrate. In an embodiment, the columns in pre-treatment process include use of desalting columns and membrane filters. The pre-treatment of the sample is performed by desalting followed by filtration. Desalting is a simple and fast method that rapidly remove unwanted salts, low molecular weight contaminants and other small molecules from a sample due to their size. Use of desalting column provides a more convenient and quicker alternative to dialysis. In another embodiment, desalting columns are disposable or reusable plastic or glass columns prepacked with beads of a suitable media, typically polysaccharide (Sephadex), or porous polyacrylamide that are more resistant to enzymatic degradation. The beads have defined molecular weight cut-off (MWCO) exclusion points that trap, retain, and slow down the elution of small molecules below the MWCO, but exclude and therefore do not impede the more rapid elution of larger macromolecules such as proteins. In another embodiment, desalting columns are selected from a group comprising but not limited to cross linked dextran based, polysaccharide based, porous polyacrylamide based and polypropylene based. In another embodiment, desalting columns are selected from but not limited to PD-10 desalting column, Thermo Fisher Zeba Spin, Biorad Desalting column, Avantor -Gtrap and Pierce desalting columns. In yet another embodiment, desalting columns selected is PD-10 desalting column. In another embodiment, column equilibration of desalting columns is performed using equilibration buffer i.e. milliQ water. The equilibration buffer is allowed to enter the packed bed of column completely and the flow-through is discarded. In another embodiment, sample application is performed by adding the sample in the equilibrated column. The sample is allowed to enter the packed bed of column completely and the flow-through is discarded. In another embodiment, the filters in pre-treatment process include membrane filters. Membrane filters are a good tool for fast and efficient filtration of liquid samples. In another embodiment, membrane filters are centrifugal membrane filters selected from but not limited to Sartorius-Vivaspin turbo 15 / 4 / 2, Amicon Ultra centrifugal filters 15 / 4 / 2, Pall centrifugal devices and Thermo Centrifugal devices, preferably Sartorius-Vivaspin turbo 15 / 4 / 2. In another embodiment, membrane filters are made from cellulose acetate, cellulose nitrate (collodion), polycarbonate, polypropylene, and polyamide (nylon). The filter used for membrane filtration has a molecular weight cut-off (MWCO) ranging from about 1 to 10 kDa. In an embodiment, the filter has pore size of about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6, kDa, about 7 kDa, about 8 kDa, about 9 kDa or about 10 kDa. In another embodiment, the filters used in pre-treatment process include centrifugal filter membrane wherein, centrifugal filter membrane is selected from the group consisting of one or more of cellulose, regenerated cellulose, cellulose ester, polyethersulphone, modified polyethersulphone or water-wettable Polytetrafluoroethylene., having pore size selected from the range of about 0.1pm to 1 pm or about IkDa to 5kDa filter. Preferably the centrifugal filter membrane is made up of cellulose or polyethersulphone with the pore size of about 0.1 to 0.2 pm or about 3kDa filter. In an embodiment, centrifugal membrane filter is assembled below desalting column for sample collection i.e. eluate collection. The filtrate is collected when a sample is passed to desalting column followed by centrifugal membrane filter to obtain the retentate of sample. The pre-treatment step is repeated to obtain the desired volume of retentate. In yet another embodiment, the filtered test sample retentate is washed several times with solvent preferably water for injection (WFI), Milli-Q water or buffer such as tris to rule out any presence of the disaccharide excipients and salts in the retentate (filtered test sample). The buffer such as Tris is added to the retentate for the final volume makeup and the pretreated samples are obtained. In another embodiment, the pre-treated samples comprising purified polysaccharide protein conjugate bulk and final vaccine formulation are diluted using buffer and the diluted samples are ready for analysis to quantify or estimate total polysaccharide content or free polysaccharide content. In another embodiment, buffer used for dilution of pre-treated sample is selected from tris, histidine, pyridine, citrate, phosphate, acetate, Disodium phosphate, monopotassium phosphate, sodium chloride, sodium borate, Succinic acid, nitrate, sodium hydroxide, HEPES (4-(2-hydroxy ethyl)-1 -piperazineethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate), MOPS (3-(N-morpholino)propanesulfonic acid), Bicine, Bis-Tris, Carbonate, Tricine, ACES (N-(2-acetamido)-2-aminoethanesulfonic acid) or combinations thereof. In another embodiment, the samples comprising purified polysaccharide protein conjugate bulk and vaccine formulation are further treated with protein precipitating agent along with addition of acid. In another embodiment, the samples comprising purified polysaccharide protein conjugate bulk and vaccine formulation are treated with protein precipitating agent along with addition of acid to separate the free or unconjugated polysaccharide and conjugated polysaccharide. Protein precipitating agent used precipitate the free protein molecules along with conjugated protein molecules (polysaccharide protein conjugate) which makes easy to quantify free polysaccharide content or total polysaccharide content. In another embodiment, protein precipitation is carried out using one or more protein precipitating agent selected from a group consisting of trichloroacetic acid, deoxycholatehydrochloric acid (D0C-HC1), ethanol, acetone, ammonium sulfate, PEG8000, and diethyl ether. In another embodiment, D0C-HC1 is preferred protein precipitating agent comprising DOC addition first followed by HC1 addition. D0C-HC1 comprising DOC having concentration in the range of 0.2% to 2%, 0.25% to 1.5% (w / v) or 0.5% to 1.5%. In another embodiment, for samples comprising polysaccharide protein conjugate bulk, DOC in the DOC-HC1 is 1% and for samples comprising final vaccine formulation, DOC in the DOC-HC1 is 1.2%. In another embodiment, after addition of DOC to the sample in the test tubes or Eppendorf tubes, the tubes are incubated at 2 to 8°C for 20 to 40 minutes, preferably 30 minutes. Further, HC1 is added (titrated) in the tubes and centrifuged at 5000 to 15000 rpm, preferably 10000, for 10 to 15 minutes, preferably 15 minutes, at 2 to 8°C, preferably 5°C. In another embodiment, protein precipitation is obtained by addition of DOC and titrated by cone. HC1 having concentration in the range of IM to 5M, preferably 3M, which is used for effective precipitation of different types of carrier proteins. The pH during precipitation step is in the range of about 6.0 to 8.0, about 6.5 to 7.5 or about 7.0 to 7.5, preferably about 7.2. In an embodiment, the pH during precipitation step is 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0. In an embodiment, for polysaccharide protein conjugate bulk samples, 1.0% DOC and 3M HC1 is used and for Bivalent Vaccine, 1.2% DOC and 3M HC1 is used for protein precipitation which cause separation of Free PS or Free OPS. In an embodiment, after addition of DOC-HC1 in the tubes containing sample, the supernatant is taken out without disturbing the precipitated pellet and further diluted. In a deoxycholate (DOC) precipitation method, free saccharide is recovered in the supernatant of DOC-precipitated protein samples, which has also been shown to measure reproducible % free saccharide levels. In an embodiment, the blank sample comprising Na DOC is passed through the quantification process and then it is subtracted from the total estimated final value. In another embodiment, the untreated sample comprising purified polysaccharide and pretreated sample is used for analysis to quantify or estimate free polysaccharide content by colorimetric assay or method. In another embodiment, the untreated sample comprising purified polysaccharide and pretreated sample is further diluted to obtain serial dilutions ranging from 0 to 50 pg / ml to obtain a calibration curve. In an embodiment, the untreated sample comprising purified polysaccharide and pre-treated sample is diluted to obtain serial dilutions ranging from about 0.1 pg / ml to 50 pg / ml, including all the values in the range, for instance, 0.2 pg / ml, 0.3 pg / ml, 0.4 pg / ml, 0.5 pg / ml and so on and so forth, up until 50 pg / ml, and including subranges of the range 0.1 pg / ml to 50 pg / ml. In another embodiment, a method for quantification or estimation of total polysaccharide as well as free polysaccharide content by colorimetry require the standard or standard solution. In another embodiment, the present invention is directed to a method for quantification or estimation of total polysaccharide as well as free polysaccharide content by colorimetry at different stages such as purified polysaccharides, purified polysaccharide protein conjugate bulk and final vaccine formulation in the monovalent or multivalent conjugate bulk or vaccine composition comprising following non-limiting steps; a) providing the sample; b) diluting the sample; c) adding the chromogen to the sample to obtain a mixture; d) mixing the acid with the mixture to obtain the solution; e) incubating the solution; f) subjecting the incubated solution to the spectrophotometric assay or colorimetry assay ; and g) quantifying the saccharide content in the sample using mixed saccharide standard solution as standard solution. In an embodiment, the present invention is directed to the method for quantification of O-specific polysaccharide content in a sample, the method comprising: a) diluting the sample with the buffer to obtain the diluted sample; b) adding the chromogen to the diluted sample to obtain the mixture; c) mixing the acid to the mixture to obtain the solution; d) incubating the solution; e) subjecting the incubated solution to the spectrophotometric assay; f) quantifying the O-specific polysaccharide content in the sample using a mixed saccharide standard solution, wherein the mixed standard solution comprises at least two saccharides. In an embodiment, the method for quantification of O-specific polysaccharide content in a sample comprises: a) adding the chromogen to the sample to obtain the mixture, wherein the sample is optionally diluted with a buffer prior to addition of the chromogen; b) mixing the acid to the mixture to obtain the solution; c) incubating the solution; d) subjecting the incubated solution to the spectrophotometric assay to obtain an absorbance value; e) quantifying the O-specific polysaccharide content in the sample by comparing the absorbance value with a reference standard obtained by using a mixed saccharide standard solution, wherein the mixed standard solution comprises at least two saccharides. In another embodiment, the sample is selected from purified O-specific polysaccharides, purified O-specific polysaccharide protein conjugate bulk and vaccine formulation including O-specific polysaccharide protein conjugate. In another embodiment, O-specific polysaccharide is selected from of Gram-negative bacteria comprising Salmonella spp. such as, Salmonella typhi, Salmonella paratyphi. Salmonella enteritidis, Salmonella typhimurium', Shigella spp. such as Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Citrobacter spp. such as, C. freundii, C. werkmanii; Cronobacter species (former Enterobacter sakazakii); Shigella boydir, Escherichia coli; Klebsiella pneumoniae; Vibrio cholerae; Pseudomonas aeruginosa; Plesiomonas shigelloides; Campylobacter jejuni. In another embodiment, O-specific polysaccharide as a component of O-specific polysaccharide-protein conjugate vaccine component is selected from group of Gramnegative bacteria comprising Salmonella spp. such as, Salmonella typhi. Salmonella paratyphi A, Salmonella enteritidis, and Salmonella typhimurium. In another embodiment, O-specific polysaccharide as a component of O-specific polysaccharide-protein conjugate vaccine component is O-specific polysaccharide of Salmonella paratyphi A. As of date, no international or any reference standard is available for O-specific polysaccharides. Further, there are no international or any reference standard available for O-specific polysaccharide of Salmonella paratyphi A. In another embodiment, the standard or standard solution comprising the mixture of saccharides or polysaccharides (combination of sugars or mono sugars) is provided in the exact molar ratios comparable to actual O-specific polysaccharide of Salmonella paratyphi A. In another embodiment, the present invention is directed to a method for quantification of saccharide content in the composition as disclosed herein, wherein the standard or standard solution comprise of saccharide or sugar molecules to obtain mixed saccharide standard solution. In another embodiment, the present invention is directed to a method for quantification of saccharide content in the composition as disclosed herein, wherein a novel reference standard is a mixed saccharide standard solution. In another embodiment, the standard or standard solution comprises saccharide or sugar molecules. In another embodiment, the standard or standard solution comprises more than one or at least two saccharide molecules or combination of saccharide molecules. In another embodiment, the standard is a mixed saccharide standard solution, wherein the mixed saccharide standard solution includes at least two saccharides. In another embodiment, the saccharide used for mixed saccharide standard solution is selected from but not limited to monosaccharides, di saccharides, oligosaccharides, and polysaccharides. The mentioned saccharides are further classified depending upon the 30 number of carbon atoms in each molecule. Monosaccharides comprise of glyceraldehyde (triose), ribose (pentose) glucose (hexose), fructose (hexose), galactose (hexose), tagatose, mannose, arabinose, xylose, erythrose (tetrose), sedoheptulose (heptose), rhamnose, paratose and others. Disaccharides comprise of sucrose, isomaltulose, lactose, maltose, trehalose, and others. Oligosaccharides comprise of maltose gluco-oligosaccharides, raffinose, stachyose, fructo-oligosaccharides (FOS), arabino-oligosaccharides (AXOS), amylose, amylopectin, modified starches and others. Polysaccharides comprise of pectin, cellulose, hemicellulose and hydrocolloids (arabic gum, guar gum, others). In another embodiment, the saccharide used for preparation of mixed saccharide standard solution is selected depending on the structure or the backbone of polysaccharide to be quantified. In another embodiment, the mixed saccharide standard solution is prepared by mixing one or more saccharides. In an embodiment, the present invention is directed to a method for determining saccharide content in the composition as disclosed herein, wherein a novel reference standard is a mixed saccharide standard solution comprising rhamnose, mannose, galactose, paratose and glucose. In another embodiment, the mixed saccharide standard solution is a combination of rhamnose, mannose, galactose, paratose and glucose. In another embodiment, the mixed saccharide standard solution is prepared by mixing of rhamnose, mannose, galactose, paratose and glucose in milliQ water. In another embodiment, the mixed saccharide standard solution is prepared by mixing of rhamnose, mannose, galactose, glucose and paratose in the ratio of about 1: 1: 1: 0.76:1. Dehydration of polysaccharides takes place when it reacts with concentrated sulphuric acid (H2SO4) and it produces furfural derivatives. These furfural derivatives further react with phenol and produce orange coloured complex. The intensity of the colour is directly proportional to the concentration of polysaccharides present in the sample. In another embodiment, dehydration of O-Specific polysaccharides takes place when it reacts with concentrated sulphuric acid (H2SO4) and it produces furfural derivatives. These furfural derivatives further react with phenol and produce orange colored complex. The intensity of the color is directly proportional to the concentration of O-specific polysaccharides present in the sample. In another embodiment, the dilution of sample and mixed saccharide standard solution is carried out with the buffer selected from a group comprising tris, histidine, pyridine, citrate, phosphate, acetate, Disodium phosphate, monopotassium phosphate, sodium chloride, sodium borate, Succinic acid, nitrate, sodium hydroxide, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate), MOPS (3-(N-morpholino) propanesulfonic acid), Bicine, Bis-Tris, Carbonate, Tricine, ACES (N-(2-acetamido)-2-aminoethanesulfonic acid) and combinations thereof. The dilution of sample and mixed saccharide standard solution is carried out with Tris buffer. In another embodiment, the mixed saccharide standard solution is further diluted to obtain serial dilutions ranging from 0 to 50 pg / ml to obtain a calibration curve and to prepare a novel reference standard, especially for O-specific polysaccharides. In an embodiment, the mixed saccharide standard solution is diluted to obtain serial dilutions ranging from 0.1 pg / ml to 50 pg / ml, including all the values in the range, for instance, 0.2 pg / ml, 0.3 pg / ml, 0.4 pg / ml, 0.5 pg / ml and so on and so forth, up until 50 pg / ml, and including subranges of the range 0.1 pg / ml to 50 pg / ml. In another embodiment, the concentration of sample containing polysaccharide is in the range of 1 pg / ml to 50 pg / ml, preferably about 2.5 pg / ml to 35 pg / ml. In an embodiment, the concentration of sample containing polysaccharide is range of about 1 pg / ml to 50 pg / ml, including all the values in the range, for instance, 1.1 pg / ml, 1.2 pg / ml, 1.3 pg / ml, 1.4 pg / ml and so on and so forth, up until 50 pg / ml, and including subranges of the range 1 pg / ml to 50 pg / ml. In another embodiment, the chromogen is added to the dilutions of sample to obtain a mixture. Chromogens are used in colorimetric detection for direct visualization of results without the need for a film or imaging instrument. They are simple and easy to use. Since each chromogen has their unique optical and chemical properties, they have totally different colorimetric sensing mechanisms. In an embodiment, the chromogen is added to the dilutions of mixed saccharide standard solution to obtain a mixture. In an embodiment, the chromogen is selected from phenol, resorcinol, 2,6-dimethylphenol, Orcinol, DNSA (3,5-dinitrosalicylaldehyde) and TNBS (2,4,6-Trinitrobenzene sulfonic acid), preferably phenol. In an embodiment, the chromogen is phenol and the concentration of phenol is in the range of 70% to 90%, preferably 80%; and the volume of phenol is in the range of 5pl to lOOpl. In an embodiment, the concentration of chromogen is about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89% or about 90%. In another embodiment, the chromogen is added to the dilutions of sample and mixed saccharide standard solution followed by addition of acid for sample digestion. In another embodiment, the acid is mineral acid. In another embodiment, the mineral acid is selected from hydrochloric acid, sulphuric acid, perchloric acid, carbonic acid hydrofluoric acid, phosphoric acid, trifluoroacetic acid, acetic acid and nitric acid, preferably sulphuric acid. In another embodiment, the concentration of sulphuric acid is in the range of about 15 M to 20 M, preferably 18 M; and the volume of sulphuric acid is in the range of about 1ml to 10 ml, preferably 2.5 ml. In some embodiments, the concentration of mineral acid is about 15 M, about 16 M, about 17 M, about 18 M, about 19 M or about 20 M. In an embodiment of the present disclosure, the ratio of sulphuric acid to phenol is in the range of about 10: 1 to 200: 1, about preferably 100: 1. In an embodiment, the ratio of sulphuric acid to phenol is ranging from about 10:1 to 200: 1, including all the values in the range, for instance, 11: 1, 12: 1, 13: 1, 14: 1 and so on and so forth, up until 200: 1 and including subranges of the range 10:1 to 200: 1. In another embodiment, the acid addition is a critical step, the addition is done in one stroke using a pipette and in the centre of the reaction mixture for the optimal reaction process. In another embodiment, the solution comprising test sample, chromogen and mineral acid is incubated at about 20 to 30°C for about 20 to 50 mins to obtain incubated sample solution. In an embodiment, the solution comprising test sample, chromogen and mineral acid is incubated at a temperature of about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C or about 30 °C. In an embodiment, the solution comprising test sample, chromogen and mineral acid is incubated for a duration of about 20 minutes to 50 minutes, including all the values in the range, for instance, 21 minutes, 22 minutes, 23 minutes, 24 minutes and so on and so forth, up until 50 minutes and including subranges of the range 20 minutes to 50 minutes. In an embodiment, the solution comprising test sample, chromogen and mineral acid is incubated for 30 minutes. In another embodiment, the incubated sample solution is added or transferred to the plates of 96-well plate. The plates are read at a range of about 450 to 550 nm in a spectrophotometer, including all the values in the range, for instance, 451 nm, 452 nm, 453 nm, 454 nm, and so on and so forth, up until 550 nm. The plates are read at a 480 nm i.e. absorbance is obtained at 480 nm. The remaining sample, intermediate dilutions and any additional sample is disposed or discarded. In another embodiment, the total polysaccharide concentration or the content in the test sample is calculated using below equation; Polysaccharide = Calculated content (pg / ml) (From Raw Data) x Dilution concentration 1000 (mg / mL) Consider the dilution factor 1 In another embodiment, the method involves use of more than one saccharide / sugar i.e. mixture or combination of saccharides / sugars as standard for quantification of total polysaccharide as well as free polysaccharide content by colorimetric assay at different stages such as purified polysaccharides, purified polysaccharide protein conjugate bulk and vaccine formulation in the monovalent or multivalent conjugate bulk or vaccine composition is qualified and validated considering parameters such as specificity, linearity, range, precision, accuracy, robustness, LOD (limit of detection) and LOQ (limit of quantification). In another embodiment, the phenol sulphuric acid method is validated. The method is linear in the range from 2.5 pg / mL to 35 pg / mL with the RSQ value of >0.99. No interference indicates that the method is specific for estimation of OSP-polysaccharide in all samples. Precision shows that % CV of the total PS values is <10 %. The method is accurate for polysaccharide content estimation in purified OSP polysaccharide, OSP-DT purified conjugate bulk and bivalent typhoid vaccine samples as the % recovery is within 80 - 120 %. LOQ is 2.5 pg / mL. The method is robust and the % CV obtained is 4 % indicating the repeatable nature of the assay or method. In another embodiment, the method of the present invention for the quantification of saccharide content or O-specific polysaccharide content in a sample is an in-vitro method. In another embodiment, the method of the present invention for the quantification of saccharide content in a sample is a non-diagnostic and non-therapeutic method. In another embodiment, the method of the present invention for the quantification of O-specific polysaccharide content in a sample is a non-diagnostic and non-therapeutic method. In another embodiment, total polysaccharide and free polysaccharide content quantification by the method of the present invention is for both gram-positive and gram-negative pathogenic bacteria. In another embodiment, the polysaccharide has a molecular weight ranging from about 20 kDa to 100 kDa, preferably ranging from about 30 kDa to 50 kDa and contain mannose, glucose, and galactose as repeating units. In an embodiment, the polysaccharide has a molecular weight ranging from about 20 kDa to 100 kDa, including all the values in the range, for instance, 21 kDa, 22 kDa, 23 kDa, 24 kDa and so on and so forth, up until 100 kDa, and including subranges of the range 20 kDa to 100 kDa. In another embodiment, the polysaccharide further comprise at least one saccharide / sugar selected from fucose, rhamnose, paratose, arabinose, and glucosamine. The polysaccharide further preferably comprises rhamnose and paratose. According to an embodiment of the present invention, the polysaccharide comprises repeating units of rhamnose, mannose, galactose, glucose and paratose. In an embodiment, the present invention is directed to a method for quantification of saccharide content in the composition as disclosed herein, wherein the mixed saccharide standard solution includes rhamnose, mannose, galactose, glucose and paratose in ratio of about 1:1:1:0.76:1. In another embodiment, polysaccharide or O-specific polysaccharide content of Salmonella paratyphi A is estimated by phenol sulphuric acid method and using mixed monosaccharide solution as standard i.e., combination of rhamnose, mannose, galactose, glucose and paratose in the ratio of 1: 1: 1: 0.76:1 that provides the polysaccharide content (concentration) in-line i.e. comparable with that of the theoretical / expected concentration. In an embodiment, the present invention is directed to a method for quantification of O-specific polysaccharide content in a sample, said method comprising: a) diluting the sample with the buffer to obtain the diluted sample comprising O-specific polysaccharide in the range from about 1 pg / ml to 50.0 pg / ml; b) adding the phenol in the range from about 70.0% to 90.0% to the diluted sample to obtain the mixture; c) mixing the sulphuric acid in range from about 15.0 M to 20.0 M to mixture to obtain the solution; d) incubating the solution at temperature in range from about 20.0 °C to 30.0 °C for duration in range of about 20.0 minutes to 50.0 minutes; e) subjecting the incubated solution to the spectrophotometric assay at 480 nm; f) quantifying the O-specific polysaccharide content in the sample using mixed saccharide standard solution; wherein the mixed saccharide standard solution comprises at least two saccharides; and wherein the sample is a purified O-specific polysaccharide, a purified O-specific polysaccharide protein conjugate bulk or a vaccine formulation; and wherein the purified O-specific polysaccharide protein conjugate bulk and final vaccine formulation is additionally subjected to pre-treatment by passing through desalting column and membrane filter and treating with DOC-HC1, prior to the dilution of the sample. In an embodiment, the method for quantification of O-specific polysaccharide content in a sample comprises: a) adding the phenol in the range from about 70.0% to 90.0% to a sample to obtain the mixture, wherein the sample is diluted with Tris buffer to obtain the diluted sample comprising O-specific polysaccharide in the range from about 1 pg / ml to 50.0 pg / ml; b) mixing the sulphuric acid in range from 15.0 M to 20.0 M to mixture to obtain the solution; c) incubating the solution at temperature in range from 20.0 °C to 30.0 °C for duration in range of 20.0 minutes to 50.0 minutes; d) subjecting the incubated solution to the spectrophotometric assay at 480 nm to obtain an absorbance value; e) quantifying the O-specific polysaccharide content in the sample by comparing the absorbance value with a reference standard of a mixed saccharide standard solution; wherein the mixed saccharide standard solution comprises rhamnose, mannose, galactose, glucose and paratose; and wherein the sample is a purified O-specific polysaccharide, a purified O-specific polysaccharide protein conjugate bulk or a vaccine formulation; and wherein the purified O-specific polysaccharide protein conjugate bulk and final vaccine formulation is additionally subjected to pre-treatment by passing through desalting column and membrane filter and treating with 1% or 1.2% DOC-HC1, prior to the dilution of the sample. In an embodiment, the method for quantification of O-specific polysaccharide content in a sample comprises: a) adding the 80.0% phenol to a sample to obtain the mixture, wherein the sample is diluted with lOmM Tris buffer to obtain the diluted sample comprising O-specific polysaccharide in the range from about 1 pg / ml to 50.0 pg / ml; b) mixing the 18 M sulphuric acid to the mixture to obtain the solution; c) incubating the solution at temperature of about 25 °C for duration of about 30 minutes; d) subjecting the incubated solution to the spectrophotometric assay at 480 nm to obtain an absorbance value (OD); e) quantifying the O-specific polysaccharide content in the sample by comparing the absorbance value (OD) with a reference standard (OD) of a mixed saccharide standard solution; wherein the mixed saccharide standard solution comprises rhamnose, mannose, galactose, glucose and paratose in a ratio of about 1: 1: 1: 0.76:1; and wherein the sample is a purified O-specific polysaccharide, a purified O-specific polysaccharide protein conjugate bulk or a vaccine formulation; and wherein the purified O-specific polysaccharide protein conjugate bulk and final vaccine formulation is additionally subjected to pre-treatment by passing through desalting column and membrane filter and treating with 1% or 1.2% D0C-HC1, prior to the dilution of the sample. In an embodiment, the present invention is directed to a method for quantification of 0-specific polysaccharide content of Salmonella Paratyphi A in a sample, said method comprising: a) diluting the sample with the buffer to obtain the diluted sample comprising O-specific polysaccharide in the range from about 1 pg / ml to 50.0 pg / ml; b) adding the phenol in the range from about 70.0% to 90.0% to the diluted sample to obtain the mixture; c) mixing the sulphuric acid in range from about 15.0 M to 20.0 M to mixture to obtain the solution; d) incubating the solution at temperature in range from about 20.0 °C to 30.0 °C for duration in range of 20.0 minutes to 50.0 minutes; e) subjecting the incubated solution to the spectrophotometric assay at 480 nm; f) quantifying the O-specific polysaccharide content in the sample using mixed saccharide standard solution; wherein the mixed saccharide standard solution comprises at least two saccharides; wherein the sample is purified O-specific polysaccharides, purified O-specific polysaccharide protein conjugate bulk or a final vaccine formulation; and wherein the purified O-specific polysaccharide protein conjugate bulk and vaccine formulation is additionally subjected to pre-treatment by passing through desalting column and membrane filter and treating with DOC-HC1, prior to the dilution of the sample. In an embodiment, the method for quantification of O-specific polysaccharide content of Salmonella paratyphi A in a sample comprises: a) adding the phenol in the range from about 70.0% to 90.0% to a sample to obtain the mixture, wherein the sample is diluted with Tris buffer to obtain the diluted sample comprising O-specific polysaccharide in the range from about 1 pg / ml to 50.0 pg / ml; b) mixing the sulphuric acid in range from 15.0 M to 20.0 M to mixture to obtain the solution; c) incubating the solution at temperature in range from 20.0 °C to 30.0 °C for duration in range of 20.0 minutes to 50.0 minutes; d) subjecting the incubated solution to the spectrophotometric assay at 480 nm to obtain an absorbance value; e) quantifying the O-specific polysaccharide content in the sample by comparing the absorbance value with a reference standard of a mixed saccharide standard solution; wherein the mixed saccharide standard solution comprises rhamnose, mannose, galactose, glucose and paratose; and wherein the sample is a purified O-specific polysaccharide, a purified O-specific polysaccharide protein conjugate bulk or a vaccine formulation; and wherein the purified O-specific polysaccharide protein conjugate bulk and final vaccine formulation is additionally subjected to pre-treatment by passing through desalting column and membrane filter and treating with 1% or 1.2% DOC-HC1, prior to the dilution of the sample. In an embodiment, the method for quantification of O-specific polysaccharide content of Salmonella paratyphi A in a sample comprises: a) adding the 80.0% phenol to a sample to obtain the mixture, wherein the sample is diluted with lOmM Tris buffer to obtain the diluted sample comprising O-specific polysaccharide in the range from about 1 pg / ml to 50.0 pg / ml; b) mixing the 18 M sulphuric acid to the mixture to obtain the solution; c) incubating the solution at temperature of about 25°C for duration of about 30 minutes; d) subjecting the incubated solution to the spectrophotometric assay at 480 nm to obtain an absorbance value (OD); e) quantifying the O-specific polysaccharide content in the sample by comparing the absorbance value (OD) with a reference standard (OD) of a mixed saccharide standard solution; wherein the mixed saccharide standard solution comprises rhamnose, mannose, galactose, glucose and paratose in a ratio of about 1: 1: 1: 0.76:1; and wherein the sample is a purified O-specific polysaccharide, a purified O-specific polysaccharide protein conjugate bulk or a vaccine formulation; and wherein the purified O-specific polysaccharide protein conjugate bulk and final vaccine formulation is additionally subjected to pre-treatment by passing through desalting column and membrane filter and treating with 1% or 1.2% D0C-HC1, prior to the dilution of the sample. In another embodiment, the present invention is directed to the method for determining the saccharide content in the composition as presently disclosed, wherein the saccharide content includes a total polysaccharide or a free polysaccharide / unconjugated polysaccharide, or conjugated polysaccharide, or combination thereof. In another embodiment, the present invention is directed to the method for determining the saccharide content in the composition as presently disclosed, wherein the saccharide content includes a total polysaccharide or total O-specific polysaccharide. In another embodiment, the present invention is directed to the method for determining the saccharide content in the composition as presently disclosed, wherein the saccharide content includes a free polysaccharide / unconjugated polysaccharide or free O-specific polysaccharide / unconjugated O-specific polysaccharide. In another embodiment, the present invention is directed to the method for determining the saccharide content in the composition as presently disclosed, wherein the saccharide content includes conjugated polysaccharide or conjugated O-specific polysaccharide. In another embodiment, the present invention is directed to the method for determining the saccharide content in the composition as presently disclosed, wherein the method further includes determining the ratio of a. the free polysaccharide content to the total polysaccharide content or b. the conjugated polysaccharide content to the total polysaccharide content c. the free polysaccharide content and the conjugated polysaccharide content to the total polysaccharide content in the composition. In another embodiment, the phenol sulphuric acid method for polysaccharide or OSP polysaccharide quantification using mixed saccharide standard solution as standard is applicable for determining the potency and shelf life of the vaccine with low variations in the output or results. In another embodiment, the phenol sulphuric acid method for polysaccharide or OSP polysaccharide quantification using mixed saccharide standard solution as standard is not applicable for diagnostic and therapeutic methods. The method does not use reagents (e.g. anthrone reagent) which are unstable and give rise to give higher background (less sensitivity). The method does not use volatile solvents (e.g. ethanol), does not use chromatography steps. The method is easy, cost effective and can be performed within a small wet lab. The present invention is also directed towards the use of the mixed saccharide standard solution comprising at least two saccharides for quantification of O-specific polysaccharide content in a sample. In some embodiments, the present invention relates to a mixed saccharide standard solution comprising at least two saccharides for use in the quantification of O-specific polysaccharide content in a sample, wherein the sample is selected from a group comprising a purified O-specific polysaccharide, a purified O-specific polysaccharide protein conjugate bulk, or a vaccine formulation including O-specific polysaccharide protein conjugate. In another embodiment, the mixed saccharide standard solution for use in the quantification of O-specific polysaccharide content in a sample, comprises rhamnose, mannose, galactose, glucose and paratose. In another embodiment, the mixed saccharide standard solution for use in the quantification of O-specific polysaccharide content in a sample, comprises rhamnose, mannose, galactose, glucose and paratose present in a ratio of about 1: 1: 1: 0.76:1. In some embodiments, the present invention is directed towards the use of the mixed saccharide standard solution comprising at least two saccharides for quantification of O-specific polysaccharide content of Salmonella paratyphi A in a sample. The present invention is also directed towards a kit for quantification of O-specific polysaccharide content in a sample. More particularly, the present invention provides a kit for quantification of O-specific polysaccharide content in a sample, comprising: a chromogen; an acid; a reference standard of a mixed saccharide standard solution comprising at least two saccharides; and optionally, i) a means for obtaining an absorbance value, ii) a package insert or label providing instructions to perform the quantification of O-specific polysaccharide content in the sample, or both i) and ii). In an embodiment, the kit comprises: a chromogen selected from a group comprising phenol, resorcinol, 2,6-dimethylphenol, orcinol, DNSA (3,5-dinitrosalicylaldehyde), TNBS (2,4,6-Trinitrobenzene sulfonic acid), or any combination thereof; an acid selected from a group comprising hydrochloric acid, sulphuric acid, perchloric acid, carbonic acid hydrofluoric acid, phosphoric acid, trifluoroacetic acid, acetic acid, nitric acid, or any combination thereof; a reference standard of a mixed saccharide standard solution comprising at least two saccharides selected from a group comprising monosaccharide, disaccharide, oligosaccharide, polysaccharide, or a combination thereof; optionally, i) a means for obtaining an absorbance value, ii) a package insert or label providing instructions to perform the quantification of O-specific polysaccharide content in the sample, or both i) and ii); and optionally wherein O-specific polysaccharide is of Salmonella paratyphi A. Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps and can mean "includes”, "including”, and the like; “consisting essentially of’ or “consists essentially” likewise is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. The numerical values given for various physical parameters, dimensions and quantities are only approximate values and it is envisaged that the values higher than the numerical value assigned to the physical parameters, dimensions and quantities fall within the scope of the invention unless there is a statement in the specification to the contrary. Similarly, the components used in purification, e.g., filters, columns, are not intended to be in any way limiting or exclusionary, and can be substituted for other components to achieve the same purpose at the discretion of the practitioner. While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustration of the disclosure and not as a limitation. EXAMPLES: A. Source of Biological Material used in the present disclosure is as follows: 1. Salmonella enterica subsp. enterica serovar Typhi str. Ty2 Salmonella enterica serovar typhi deposited atNCMR-NCCS; Pune, an International Depositary Authority having assigned Accession No. MCC 0193; Strain designation- PDL-1, S. typhi: ATCC 19430; C6524 (NICED, Kolkata, India): Identified by Geneombio Technologies Private Limited, Pune and isolated from stool sample of typhoid confirmed patient at Villoo Poonawalla Memorial Hospital, Pune. 2. Salmonella enterica subsp. enterica serovar Paratyphi A: ATCC 9150 procured from Chromachemie Laboratory Private Limited, Bangalore 3. Tetanus toxoid (TT) Strain Clostridium tetani Harvard Strain No.49205: TT was procured from Clostridium tetani (Harvard No 49205) obtained from Central research Institute (CRI), National Control Authority, Kasauli, Himachal Pradesh, India. Central research Institute (CRI) procured this strain from NVI, Netherland. 4. Diphtheria Toxoid (DT) Strain Corynebacterium diphtheriae PW8 CN2000 Al: The strain was obtained from the Wellcome Research Laboratory, London, United Kingdom by the National Control Authority Central Research Institute (C.R.I.) Kasauli, Himachal Pradesh, India. B. Vaccine Formulation: 1. Vi-TT (Vi polysaccharide - Tetanus toxoid): 50 pg / mL 2. OSP-DT (O-specific polysaccharide - diphtheria toxoid): 50 pg / mL 3. Tris Buffer: 0.60 mg / mL 4. Mannitol: 50 mg / mL 5. 2-Phenoxy ethanol: 5 mg / mL (only in multidose presentation) C. Materials and Equipment: • D (+) Glucose (e.g. Sigma cat.no. G5767) • Mannose (e.g. Arcos cat.no. 150601000) • Rhamnose (e.g. Arcos cat.no. 174081000) • Galactose (e.g. Sigma cat.no. G5388) • Sulfuric acid (e.g. Sigma cat.no. 258105) • Milli Q Water (MQW) • Laboratory vortex mixer (e.g. Borosil) • Microplate Spectrophotometer (e.g. Molecular devices) • Weighing balance (e.g. Sartorius) • Test tube stand / rack (e.g. Tarsons) • 96 wells plate (e.g. Nunc., Thermo) • Microcentrifuge Tubes (e.g. Eppendorf) • Variable volume micropipette and tips (e.g. Eppendorf) • Variable volume beakers (e.g. Borosil) • Variable volume measuring cylinders (e.g. Borosil) • Variable volume bottles (e.g. Borosil) • Phenol (e.g. Sigma Cat No.33517) • Tris base / Trizma base (Sigma Cat No. 102459657) D. Solution / Reagent preparation: a) Mixed monosaccharide solution / OSP Sugar Standard Solution (0.1 mg / mL): 49.46 mg rhamnose, 44.31 mg mannose, 44.92 mg galactose, 36.10 mg of Paratose, and 33.68 mg of glucose was dissolved in 1000 ml of Milli q water. The final volume was made to 2000 mL with Milli Q water and stored at -20° C. The prepared standard solution is stable and fit to be used for at least about 5 years. b) 80 % Phenol Reagent: The phenol was heated in a water bath at 40°C and 80 g of phenol was weighed in the beaker. The final volume was made to 100 mL with Milli Q water and stored it in an amber-coloured bottle. The prepared phenol reagent is stable and fit to be used for at least about 3 months. c) 18M Sulphuric acid: Commercially available concentrated sulphuric acid with 18M concentration. d) lOmM Tris: 2.42 gm of Tris base was dissolved in 1800 mL of Milli Q water. pH 7.0 was adjusted with concentrated HC1. The final volume was made to 2000 mL. The prepared Tris is stable and fit to be use for at least about 3 months. EXAMPLE 1: OPTIMIZATION OF DEOXYCHOLATE (DOC) AND HYDROCHLORIC ACID (HCL) CONCENTRATIONS Spike recovery or % Recovery were estimated for optimization of DOC and HC1 concentrations required for separation of free polysaccharide (Free PS) or free O-specific polysaccharide (Free OPS) from conjugate bulk (OSP-DT) and final Lot of Bivalent vaccine (BTCV - Bivalent typhoid conjugate vaccine). From Table 1, it was found that for conjugate bulk 1.0% DOC and 3M HC1 and for Bivalent vaccine 1.2% DOC and 3M HC1 are the optimized concentrations required for separation of Free PS or Free OPS. Table 1: Spike recovery or % Recovery estimation for Optimization of DOC and HC1 concentrations Sr. No. Sample Parameter Sample % Recovery Remark 1 Conjugate Bulk Bulk with 1.0% DOC and 3M HC1 DT Tris +OSP PS 3 pg / mL 3MHC1 124 1.0% DOC and 3M HO selected for Conjugate bulk 2 Conjugate Bulk Bulk with 1.0 % DOC and 3M HC1 DT Tris +OSP PS 30 pg / mL 3 M HC1 91 3 Conjugate Bulk Bulk with 1.0 % DOC and IM HC1 DT Tris +OSP PS 3 pg / mL 1MHC1 195 4 Bivalent Vaccine Bivalent Vaccine with 1.0 % DOC and 3MHC1 DT TT Vi Tris +OSP PS 3 gg / mL 3 M HC1 153 5 Bivalent Vaccine Bivalent Vaccine with 1.0 % DOC and 3MHC1 DT TT Vi Tris +OSP PS 30 gg / mL 3 M HC1 103 6 Conjugate Bulk Bulk with 2.0% DOC and 3M HC1 DT Tris +OSP PS 3 gg / mL 3MHC1 90 7 Conjugate Bulk Bulk with 2.0% DOC and 3M HC1 DT Tris +OSP PS 30 gg / mL 3 M HC1 62 8 Bivalent Vaccine Bivalent Vaccine with 2.0 % DOC and 3MHC1 DT TT Vi Tris +OSP PS 3 gg / mL 3 M HC1 122 9 Bivalent Vaccine Bivalent Vaccine with 2.0 % DOC and 3MHC1 DT TT Vi Tris +OSP PS 30 gg / mL 3 M HC1 79 10 Bivalent Vaccine Bivalent Vaccine with 1.5 % DOC and 3MHC1 DT +Vi-TT CB+OSP PS (3 gg / mL) 112 11 Bivalent Vaccine Bivalent Vaccine with 1.5 % DOC and 3MHC1 DT +Vi-TT CB+OSP PS (20 gg / mL) 80 12 Bivalent Vaccine Bivalent Vaccine with 1.5 % DOC and 3MHC1 DT +Vi-TT CB+OSP PS (30 gg / mL) 82 13 Bivalent Vaccine Bivalent Vaccine with 1.2 % DOC and 3MHC1 DT +Vi-TT CB+OSP PS (3 gg / mL) 1.2% DOC 121 1.2% DOC and 3M HCL selected for Final Lot of Bivalent Vaccine 14 Bivalent Vaccine Bivalent Vaccine with 1.2 % DOC and 3MHC1 DT +Vi-TT CB+OSP PS (20 gg / mL) 1.2% DOC 91 15 Bivalent Vaccine Bivalent Vaccine with 1.2 % DOC and 3MHC1 DT +Vi-TT CB+OSP PS (30 gg / mL) 1.2% DOC 90 16 Bivalent Vaccine Bivalent Vaccine with 1.4 % DOC and 3MHC1 DT +Vi-TT CB+OSP PS (3 gg / mL) 1.4% DOC 111 17 Bivalent Vaccine Bivalent Vaccine with 1.4 % DOC and 3MHC1 DT +Vi-TT CB+OSP PS (20 gg / mL) 1.4% DOC 82 18 Bivalent Vaccine Bivalent Vaccine with 1.4 % DOC and 3MHC1 DT +Vi-TT CB+OSP PS (30 gg / mL) 1.4% DOC 83 19 Bivalent Vaccine Bivalent Vaccine with 1.2 % DOC and 3MHC1 BV + SPK (20 gg / mL) 1.2 %DOC 99 20 Bivalent Vaccine Bivalent Vaccine with 1.3 % DOC and 3MHC1 BV + SPK (20 pg / mL) 1.3 %DOC 96 21 Bivalent Vaccine Bivalent Vaccine with 1.4 % DOC and 3MHC1 BV + SPK (20 pg / mL) 1.4 %DOC 85 EXAMPLE 2: SELECTION OF MEMBRANE FILTERS FOR SAMPLE PRETREATMENT Selection of membrane filters were carried out using two types of membrane filters. Filtration in pre-treatment was carried out for the purified conjugate bulk (OSP-DT) and bivalent typhoid conjugate vaccine (BTCV) (Final lot) samples. Two types of membrane filters were used (Refer Table 2); i. Sartorius Vivaspin 3Kda, MOC: PES membrane. ii. Merck Amicon Ultra 3kDa MOC: Regenerated cellulose When BTCV and OSP-DT sample were filtered through two different make filters, % recovery obtained was 100% and 94%, respectively which indicated that both the make membrane filters can be used for sample pre-treatment. Table 2: Comparison of two membrane filters Sr. No. Manufacturer of Filter Polysaccharide cone. (pg / mL) % Recovery BTCV Merck Amicon Ultra 3kDa Filter 112 100 % Sartorius Vivaspin 3Kda Filter 112 OSP-DT Merck Amicon Ultra 3kDa Filter 428 94% Sartorius Vivaspin 3Kda Filter 454 EXAMPLE 3: PHENOL SULPHURIC ACID METHOD FOR O-SPECIFIC POLYSACCAHRIDE QUANTIFICATION A. Sample preparation / Sample Pre-treatment: a) Sample: Purified Polysaccharide (For Typhoid Conjugate Vaccine - Salmonella paratyphi - O-specific Polysaccharide (TCV-SPT O-specific Polysaccharide)): The sample(s) was diluted 200 times. 100 pL of liquid sample(s) was taken and 900 pL of 10 mM Tris was added........(1) 100 pL of (1) was added in 3900 pL of 10 mM Tris...................... (2) b) Sample: OSP-DT conjugate (For TCV-SPT O-specific Purified Bulk conjugate / Final Lot): The Conjugate samples have salts and mannitol as part of the matrix. To clarify the same, the sample clarification steps were conducted as follows: 1. PD-10 treatment: i. PD-10 desalting columns were used for sample clarification. ii. Different sample matrix components e.g. mannitol may interfere in the phenol sulfuric acid assay, which were removed by PD-10 treatment. Below procedure was followed to clarify the OSP-DT conjugate samples using PD-10 desalting columns. i. PD-10 Desalting column preparation (Refer Figure Ik 2 columns were prepared for under analysis sample (5mL per sample = 2.5 mL x 2 PD-10 columns) The top cap was removed and the column storage solution was poured off. The sealed end of the column was cut at a notch using scissors. ii. Column equilibration (Refer Figure 2): The column was filled with 5 mL of equilibration buffer i.e. MilliQ water (MQW) and the equilibration buffer was allowed to enter the packed bed completely. The flow-through was discarded. The above process was repeated for another 5 times. iii. Sample application (Refer Figure 3)^ 2.5 ml of sample was added per column. The sample was allowed to enter the packed bed completely. The flow-through was discarded. 2. Membrane filter setup (3KDa): The membrane filter cup was removed from the membrane holder tube by unscrewing the cap. To this membrane filter cup 2 mL MQW was added and a short wash of 1950 x g was given for 5 mins. If any retentate was observed, it was discarded. This is referred as filter-wetting step. (Refer figure 4). i. Sample Elution: 3 KDa membrane filter cup was placed for sample collection under the 1st PD-10 column. The 1st PD-10 column was filled with 3.5 mL of MQW and the eluate was collected completely (Refer to Figure 5) in the filter. - Using the same 3KDa filter, the above step was repeated for the 2nd PD-10 column (duplicate) and the filtrate was collected. A total of - 7.0 mL of sample was collected in a single membrane filter cup. - Milli Q water was added up to 15 mL mark. The 3 KDa unit was reassembled (Membrane filter cup + membrane holder tube + cap) The sample was centrifuged at 1950 X g for 60 min (Set centrifuge at 22°C). The retentate was collected from the membrane cup in a 5mL volumetric flask by carefully removing the entire retentate from the membrane cup. 500 pL or less 10 mM Tris was added in the membrane cup and vortexed for 10 seconds such that all the contents of the filter were removed. The above step was repeated for one more time and the contents were collected in the volumetric flask. The final volume was made up to 5mL with 10 mM Tris. The above solution was used for further analysis. a. Dilution of eluted sample for quantification of Total Polysaccharide content in conjugate bulk: The sample was diluted 20 times. 200 pL of liquid samples were taken and 1800 pL of 10 mM Tris was added to it.... (1) 1500 pL of (1) was added in 1500 pL of 10 mM Tris...................... (2) b. Dilution of eluted sample and separation of Free PS for Free Polysaccharide Content quantification in conjugate bulk: i. The sample was diluted to 50 pg / mL based on total protein concentration as per the following table, e.g.: Total Protein Cone. 200 pg / mL (Refer Table 3) Table 3: Parameters for separation of free polysaccharide Tube No Total Protein conc.(pg / mL) Sample volume (pL) lOmM Tris (pL) 1% DOC (pL) Incubation 3M HC1 Total Volume (pL) Total protein Cone, obtained (pg / mL) 1 200 500 1200 200 30 mins at 2 - 8°C 100 2000 50 2 200 500 1200 200 100 2000 ii. After the acid addition step (3M HC1), all the tubes were vortexed briefly. iii. The tubes were centrifuged at 10000 rpm at 5 °C (2- 8°C) for 15 mins. iv. The tubes were removed carefully without disturbing the formed pellet settled at the bottom of the tube. Approx. 1500 pL was taken (pipette out) from one tube such that the pellet was not disturbed. v. The same activity was performed for the second tube (of the same batch) and 1000 pL (from each tube) was used for analysis. c. Dilution of eluted sample for Total Polysaccharide content quantification in bivalent typhoid vaccine / final lot: The sample was diluted 2.5 times. 800 pL of liquid samples was added in 1200 pL of 10 mM Tris.............(1) d. Dilution of eluted sample and separation of Free PS for Free Polysaccharide Content quantification in bivalent typhoid vaccine / final lot: i. 1700 pL of sample was taken into 2 Eppendorf tubes and 200 pL of 1.2 % DOC was added into it. ii. The tubes were vortexed gently and kept them at 2-8° C for 30 minutes. iii. 100 pL of 3M HC1 was added into it and the tubes were vortexed gently. iv. The tubes were centrifuged at 10000 rpm for 15 minutes at 5 °C in a precooled centrifuge. v. 1000 pL of supernatant was pooled up from 2 tubes without disturbing the pellet. B. Quantification of Total and Free Polysaccharide content by Phenol Sulfuric acid method: i. Standards (2.5 - 35 pg / mL) were prepared in the borosilicate test tube as shown below in Table 4 by using 0.1 mg / mL OSP sugar standards or mixed monosaccharide standard solution of the present invention. Table 4: Preparation of standards using 0.1 mg / mL OSP sugar standards Sr. No. Standard 0.1 mg / mL OSP sugar Standard (pL) 10 mM Tris Total Volume (pL) Cone. (pg / mL) 1 BLANK 0 1000 1000 0 2 SI 25 975 1000 2.5 3 S2 50 950 1000 5 4 S3 100 900 1000 10 5 S4 150 850 1000 15 6 S5 200 800 1000 20 7 S6 250 750 1000 25 8 S7 300 700 1000 30 9 S8 350 650 1000 35 ii. Similarly, diluted 1000 pL of the PS sample(s) were taken in duplicate. iii. 25 pL of 80% Phenol was added in each tube and vortexed for few seconds. iv. 2.5 mL Sulfuric acid was added in each tube with a stepper pipette and care was taken that the acid addition was done at the centre of the tube with one stroke addition for optimal reaction. This was an exothermic reaction, and the test tubes were heated. v. This addition was performed in a fume hood for safe handling and vortexed gently. vi. The tubes were incubated at room temperature for 30 mins. vii. Tubes were vortexed gently for approx. 10 seconds / tube. viii. 250 pL of reaction volume from each tube was transferred to a 96-well plate. This plate was ready for reading. ix. The plate was read at 480 nm in a microplate spectrophotometer. x. The remaining sample(s), intermediate dilution(s), and any additional sample was disposed of in copious amounts of water. Calculations: The polysaccharide concentration was calculated in the given sample by using the formula given below (Refer Table 5); Polysaccharide Concentration (mg / mL) Calculated Content (pg / mL) (From Raw Data) X dilution 1000 Consider the dilution factor 1. if it is updated in the protocol Table 5: Calculation of polysaccharide concentration Sr. No. Mixed monosaccharide standard solution (pg / mL) Mean OD Cone. (pg / mL) 1 Blank-(0.0) 0.0 2 Std 1-(2.5) 0.016 3 Std 2-(5.0) 0.073 4 Std 3 -(10.0) 0.143 5 Std 4-(15.0) 0.220 6 Std 5 - (20.0) 0.296 7 Std 6-(25.0) 0.370 8 Std 7 - (30.0) 0.437 9 Std 7-(35.0) 0.519 Sample Sample (OSP-PS) 0.301 20.534 Polysaccharide Concentration (mg / mL) Calculated Content (pg / mL) (From Raw Data) X dilution 1000 = 20.534 *250 Polysaccharide Concentration _____________________________________________________ (mg / mL) 1000 Polysaccharide Concentration (mg / mL) = 5.134 = 5.1 Expected concentration (Dry weight) = 5 mg / mL Obtained concentration = 5.1 mg / mL % Recovery = 102 % EXAMPLE 4: POLYSACCHARIDE OR O-SPECIFIC POLYSACCHARIDE ESTIMATION BY PHENOL SULPHURIC ACID METHOD USING GLUCOSE AND MIXED MONOSACCAHRIDES SOLUTION AS STANDARD a. Phenol Sulfuric method and Anthrone method - Glucose as standard Polysaccharide or O-specific polysaccharide content was estimated by phenol sulphuric acid method and anthrone method. Standard used was 0.1 mg / mL Glucose standard Table 6: Phenol Sulfuric method and Anthrone method - Glucose as standard Sample Details Expected cone. As per dry weight (mg / mL) Anthrone method (mg / mL) Phenol-Sulfuric method (mg / mL) Sample 1 (Batch 1) 5 3.1 6.5 Sample 2 (Batch 1) 5 3.3 - Sample 3 (Batch 1) Liquid sample 4.3 8.9 Actual concentration of the sample (dry-weight basis) was 5 mg / mL. The polysaccharide was assumed to be pure and the concentration obtained by biochemical method i.e., phenol sulphuric acid method and anthrone method should be ~ 5 mg / mL. From Table 6, the polysaccharide content observed by both anthrone and phenol sulfuric acid method (Dubois method) showed variable values. The difference in expected concentration and the concentration obtained was indicative that there is over estimation by phenol sulfuric acid assay. This missing link of the weight / concentration could be attributed to having a non-similar standard (Glucose). The anthrone method showed under estimation, while the Dubois method showed over estimation of polysaccharide content. The results indicated that the standard used for the analysis (glucose) reacts differently in both the assays in comparison to the sample i.e. since the monomeric unit of OSP has 5 sugars it was supposed that all sugars must be behaving differently and not in line with the glucose hence their difference in expected concentration and the concentration obtained. The similar over estimation of polysaccharide content was observed when mannose was used as standard. The standard needs to be harmonized in relation to the reaction efficiency of sugars. To accurately quantitate the O antigen specific PS the standard should be of the same composition. b. Phenol sulfuric method - Mixed monosaccharide solution (i.e. 5 Mono sugars / monosaccharides in combination) as standard There are no international or reference standards available for O-specific polysaccharide of Salmonella paratyphi A, hence finding the right standard for O-specific polysaccharide estimation or quantification was a challenge. Polysaccharide or O-specific polysaccharide content was estimated by phenol sulphuric acid method. Standard used was mixed monosaccharide solution i.e., combination of rhamnose, mannose, galactose, glucose and paratose present in the ratio of 1: 1: 1: 0.76:1. Mixed monosaccharide solution as standard was prepared. Table 7: Phenol Sulfuric method - Mixed monosaccharide solution as standard Sample Details Expected cone. As per dry weight (mg / ml) Phenol-Sulfuric method (mg / mL) Sample 1 (Batch 1) 5 4.6 Sample 2 (Batch 1) 5 4.9 Sample 3 (Batch 1) 5 4.7 Sample 4 (Batch 1) 5 4.7 Sample 5 (Batch 1) 5 4.7 Sample 6 (Batch 1) 5 5.1 From Table 7, it was observed that the polysaccharide content (concentration) obtained was in-line i.e., comparable with that of the theoretical / expected concentration i.e. 5 mg / mL. EXAMPLE 5: VALIDATION OF METHOD Validation of phenol sulphuric acid method using mixed saccharide / monosaccharide solution as standard for quantification of OSP polysaccharide content in conjugate bulk (OSP-DT) and vaccine samples of the bivalent typhoid conjugate vaccine (BTCV) was carried out as per the following parameters: a. Range and Linearity: Range and Linearity were performed for validation of phenol sulphuric acid method using mixed saccharide / monosaccharide solution as standard for quantification of OSP polysaccharide content in the bivalent typhoid conjugate vaccine (BTCV) sample. (Refer Table 8) Table 8: Range and Linearity data Parameter Details RSQ (r Squared value) Acceptance criteria Precision (Repeatability) Run 1 1.000 RSQ Should be >0.99 Run 2 1.000 Run 3 0.999 Run 4 0.999 Run 5 0.999 Run 6 0.999 Precision (Intermediate precision) Run 2 1.000 Run 3 1.000 Run 4 1.000 Run 5 1.000 Run 6 0.999 Accuracy Run 1 1.000 Run 2 0.999 Run 3 0.999 Analytical method validation was performed for O-Specific polysaccharide estimation by phenol sulfuric acid method in BTCV samples. A linear regression is confirmed if the data points display an RSQ value >than 0.99. A total of 14 Assays were performed and in all the assays the RSQ obtained was well beyond 0.99. From Table 8 and Figure 6, it was observed that the method was linear in the range from 2.5 pg / mL to 35 pg / mL with the RSQ value of >0.99. b. Specificity: Specificity was performed for validation of phenol sulphuric acid method using mixed saccharide / monosaccharide solution as standard for quantification of OSP polysaccharide content in the purified OSP polysaccharide (OSP-PS), purified conjugate bulk (OSP-DT) and bivalent typhoid conjugate vaccine (Final lot) sample. (Refer Table 9) Table 9: Specificity Data Sr. No Sample OD Average OD Average Blank OD Acceptance criteria (OD-Optical density) 1 OSP (Positive Control) 0.332 0.338 0.335 0.000 The O.D values for positive controls should be more than the O.D. of the first standard. 2 OSP-PS -0.004 -0.005 0.000 The O.D. values of the Sample matrix (WFI) -0.005 sample matrix should be close to blank (below 1st standard). 3 10 mM Tris 0.003 0.000 0.000 The O.D. values of the sample matrix should be close to blank (below 1st standard). -0.003 4 OSP-DT (Positive control) 0.205 0.200 0.00 The O.D values for positive controls should be more than the O.D. of the first standard. 0.195 5 OSP-DT Sample matrix 0.005 0.004 0.00 The O.D. values of the sample matrix should be close to blank (below 1st standard). 0.003 6 GFC-DT * (Negative control) 0.006 0.005 0.00 The O.D. values of the negative control should be close to blank (below 1st standard). 0.003 7 BTCV (positive control) 0.303 0.307 0.00 The O.D values for positive controls should be more than the O.D. of the first standard. 0.310 8 BTCV sample matrix 0.012 0.012 0.00 The O.D. values of the sample matrix should be close to blank (below 1st standard). 0.012 9 GFC-TT ** (negative control) 0.004 0.004 0.00 The O.D. values of the negative control should be close to blank (below 1st standard). 0.003 10 Vi-TT (negative control) 0.018 0.016 0.00 The O.D. values of the negative control should be close to blank (below 1st standard). 0.014 11 Std_2.5*** 0.027 0.028 0.000 The O.D. values of the negative control should be close to blank (below 1st standard). 0.029 *GFC-DT is Gel filtration chromatography-Diphtheria toxoid i.e. purified DT. **GFC-TT is Gel filtration chromatography-Tetanus Toxoid i.e. purified TT. *** Std 2.5 is the lowest concentration of standard at which the color change in the solution is initiated. Specificity was performed on purified polysaccharide, purified conjugate bulk and bivalent typhoid vaccine, along with samples negative control and sample matrix also taken during experiment. From table 8, the results indicated that the method was specific for estimation or quantification of OSP polysaccharide content in the purified OSP polysaccharide samples, purified bulk conjugate and bivalent typhoid conjugate vaccine. No interference was observed for sample matrix, negative control. Hence method was specific for estimation of OSP-polysaccharide in all three stages / samples. c. Precision: Precision was performed for validation of phenol sulphuric acid method using mixed saccharide / monosaccharide solution as standard for quantification of OSP polysaccharide content in the purified OSP polysaccharide, purified conjugate bulk and bivalent typhoid vaccine (Final lot) sample. (Refer Table 10). % CV (Coefficient of variation) was estimated. A total of 6 replicates were analysed. Table 10: Precision Data Sample type Assay 1 Assay 2 Assay 3 Assay 4 Assay 5 Assay 6 % CV OSP-PS 5.4 5.5 5.4 5.4 5.6 5.4 1 % OSP-DT 0.472 0.442 0.435 0.479 0.446 0.447 4% Bivalent Vaccine 113 115 115 109 110 108 3 % Acceptance criteria The % CV of the total PS values of the six determinations should be <10 %. The degree of precision is estimated by the statistical value of % CV i.e. % coefficient of variation. A total of 6 assays were performed for each of the samples mentioned above. In all the samples a max % CV of 4% was observed. The acceptance criteria set is <10% and all samples meet the criteria. This indicates that the method is precise in determining the sample consistently. d. Accuracy: Accuracy was performed for validation of phenol sulphuric acid method using mixed saccharide / monosaccharide solution as standard for quantification of OSP polysaccharide content in the purified OSP polysaccharide, purified conjugate bulk and bivalent typhoid vaccine (Final lot) sample. (Refer Table 11). Table 11: Accuracy Data Sample type Assay No Sample % Recovery OSP 1 Low Spike Cone.(5 pg / mL) 95 Middle Spike Cone.(20 pg / mL) 101 High Spike Cone. (30 pg / mL) 101 2 Low Spike Cone.(5 pg / mL) 101 Middle Spike Cone.(20 pg / mL) 97 High Spike Cone. (30 pg / mL) 101 3 Low Spike Cone.(5 pg / mL) 94 Middle Spike Cone.(20 pg / mL) 98 High Spike Cone. (30 pg / mL) 102 OSP-DT 1 Low Spike Cone.(5 pg / mL) 102 Middle Spike Cone.(20 pg / mL) 97 High Spike Cone. (30 pg / mL) 97 2 Low Spike Cone.(5 pg / mL) 116 Middle Spike Cone.(20 pg / mL) 95 High Spike Cone. (30 pg / mL) 98 3 Low Spike Cone.(5 pg / mL) 102 Middle Spike Cone.(20 pg / mL) 99 High Spike Cone. (30 pg / mL) 96 BTCV 1 Low Spike Cone.(5 pg / mL) 106 Middle Spike Cone.(20 pg / mL) 101 High Spike Cone. (30 pg / mL) 100 2 Low Spike Cone.(5 pg / mL) 109 Middle Spike Cone.(20 pg / mL) 103 High Spike Cone. (30 pg / mL) 103 3 Low Spike Cone.(5 pg / mL) 105 Middle Spike Cone.(20 pg / mL) 109 High Spike Cone. (30 pg / mL) 103 Accuracy parameter was performed on purified polysaccharide, purified bulk conjugate and bivalent typhoid conjugate vaccine and found that the method is accurate for polysaccharide content estimation in purified OSP polysaccharide, OSP-DT purified conjugate bulk and bivalent typhoid vaccine samples as the % recovery was within 80 -120 %. e. Limit of Quantification (LOQ): LOQ was performed for validation of phenol sulphuric acid method using mixed saccharide / monosaccharide solution as standard for quantification of OSP polysaccharide content. Table 12: LOQ data Standard (pg / ml) Minimum % Recovery Maximum % Recovery %CV Std 1 (2.5) 91 101 3.9 Total six assays performed for LOQ (2.5 pg / mL) with standard curve 2.5 pg / mL to 35.0 pg / mL. The lowest standard minimum and maximum % Recovery is well within 80-120%. (Refer Table 12) f. Robustness: Robustness was performed for validation of phenol sulphuric acid method using mixed saccharide / monosaccharide solution as standard for quantification of OSP polysaccharide content considering following parameters (Refer Tables 13 to 19); Case 1: Holding sample in Tris for 2.5 hrs and 5 Hrs Table 13: Holding sample in Tris for 2.5 hrs and 5 Hrs Case Parameters % Recovery Acceptance criteria Remark Case - 1 Sample Hold in 10 mM Tris 2.5 Hours 97.96 % Recovery should be between 80 -120 % w.r.tthe Control condition Complies 5 Hours 99.46 Complies Case 2: Delaying in reading time (30 and 60 mins read time) Table 14: Delaying in reading time (30 and 60 mins read time) Case Parameters % Recovery Acceptance criteria Remark Case - 2 Plate Read Time 30 mins 99.56 % Recovery should be between 80 -120 % w.r.tthe control condition Complies 60 mins 99.43 Case 3: Holding conjugate post buffer exchange for 24 hours Table 15: Holding conjugate post buffer exchange for 24 hours Case Parameter % Recovery Acceptance criteria Remark Case- 3 Holding sample post buffer exchange in 10 mM Tris At 2-8°C for 24 Hours 104 % Recovery should be between 80 - 120 % w.r.t the control condition Complies Case 4: Using Different make of 3KD Filter (Merck instead of Sartorius) Table 16: Using Different make of 3KD Filter (Merck instead of sartorius) Case Parameter % Recovery Acceptance criteria Remark Case-4 By using Another Filter Manufacturer (Merck) 94 (Conjugate) % Recovery should be between 80 - 120 % w.r.t the control condition Complies 100 (Bivalent) Case 5: Reading the sample plate using adhesive film Table 17: Reading the sample plate using adhesive film Case Parameter % Recovery Acceptance criteria Remark Case- 5 Read the microplate with Adhesive Film 96 % Recovery should be between 80 - 120 % w.r.t the control condition Complies Case 6: For Free PS: Holding the supernatant up to 4 days and then testing Table 18: For Free PS: Holding the supernatant up to 4 days and then testing Case- 6 Post Collection of the supernatant holding the sample at 2-80C Day % Recovery Acceptance criteria Remark 1 102 % Recovery should be between 80 -120 % w.r.t the control condition Complies 2 102 3 96 4 100 Case 7: For Free PS: Removal of supernatant immediately after 0.5 and 1 hours. Table 19: For Free PS: Removal of supernatant immediately after 0.5 and 1 hours Case-7 Post DOC precipitation removal of the supernatant % Recovery Acceptance criteria Remark Immediately 105 % Recovery should be between 80 -120 % w.r.t the control condition Complies After 0.5 Hour 108 After 1 Hour 112 g. Repeatability: Repeatability was performed for validation of phenol sulphuric acid method using mixed saccharide / monosaccharide solution as standard for quantification of OSP polysaccharide content using conjugate bulk OSP-DT samples. The standard used is mixed saccharide / monosaccharide (5 mono sugar combination). The % CV obtained was 4 % indicating the repeatable nature of the assay or method (Refer Table 20). Table 20: Repeatability data Repeatability OSP-DT (pg / mL) 1 472.097 2 441.745 3 434.996 4 479.28 5 445.978 6 447.019 Average 0.454 STD 0.018 % CV 4 EXAMPLE 6: Comparison of Phenol sulphuric acid method with NMR for quantification of O-specific polysaccharide Refer to Table 21 below for comparison of Phenol sulphuric acid method with NMR for quantification of O-specific polysaccharide Table 21: Comparison of Phenol sulphuric acid method with NMR Parameter NMR Phenol sulphuric acid method Advantage method (Day to day usage) Cost Effectiveness No Yes Phenol sulphuric acid method Expertise / Experts required for NMR output readouts. Yes No Phenol sulphuric acid method Hardware Dedicated Space and large area required Can be done within a small wet lab Phenol sulphuric acid method Readouts Up to molecular level finger printing. Only Content can be derived NMR Routine Commercial level impact for large sample analysis Not viable, as turnaround time is large. Viable and practical option Phenol sulphuric acid method Variations in output Large dependency on experts evaluations Low Phenol sulphuric acid method Characterization Yes Partial (Only concentration NMR estimations) Simple molecular analysis Provides High amount of resolution and separation of Polysaccharides into individual sugars which helps in exact quantification of PS. (Refer Figure 7) No interference of secondary molecules (Buffer components). Method specific for polysaccharide / sugars. Hence accurate and robust quantification of PS possible NMR and Phenol sulphuric acid method Complex Molecules Complex molecules such as Conjugates and Multivalent vaccines are difficult to evaluate because of resolution and interference issues. More complex the molecule more difficult it is to evaluate in NMR. Hence PS quantification becomes a challenge in such complex molecules. (Refer Figure 8) This is a simpler method. Method is specific for polysaccharides / Sugars. Hence in case of conjugates the Protein interference is not observed and only the Polysaccharide component of the sample will respond. Phenol Sulfuric acid method TECHNICAL ADVANTAGES Applicant’s alternative method (efficient, robust, minimum turnaround time, cost effective overcoming limitations of previously known polysaccharide quantification methods) for quantification of free polysaccharide and total polysaccharide content from monovalent conjugate bulk and multivalent conjugate bulk or monovalent conjugate vaccine and multivalent conjugate vaccine (in particular polysaccharide-protein conjugate vaccine) provides following advantages over previously reported methods; 1. Mixture of sugars or saccharides present in the standard solution, when used as reference standard provides accurate quantification of polysaccharide content. There are no international or reference standards available for O-specific polysaccharide (OSP) content quantification. 2. The O-specific polysaccharide (OSP) has a repeat unit with 05 sugars namely Rhamnose, Mannose, Galactose, Glucose and Paratose present in a ratio of 1:1:1:0.76:1. In the present method of quantification, the mixed saccharide / monosaccharide standard 62 solution mimics the same and comprises of a combination of sugars - Rhamnose: Mannose: Galactose: Glucose: Paratose in a ratio of 1: 1: 1:0.76:1. 3. Instant method enables quantification of total polysaccharide as well as free polysaccharide. 4. Use of mannose or glucose as monosaccharide standards as previously used either overestimates OR underestimates polysaccharide content. 5. The method for quantification of O-specific polysaccharide content in a sample as disclosed is used for estimating both total polysaccharides (during namely purified polysaccharides (in process), polysaccharide protein conjugate bulk and Final Lot i.e. final vaccine formulation) and free polysaccharides (polysaccharide protein conjugate bulk and final lot i.e. final vaccine formulation) 6. Standard prepared is associated with precision in optical analysis and less deviation / error. 7. Applicable for estimation of O-specific polysaccharide (for e.g. OSP of Salmonella enterica Paratyphi A). 8. Pre-treatment step using desalting columns and membrane filters (PD-10 desalting columns along with 3KDa membrane filter) for sample clarification for removal of mannitol / other excipients avoids subsequent interference with phenol sulfuric acid assay. 9. No interference of excipients / impurities or other antigens like polysaccharides or carrier proteins. 10. The method is validated considering parameters such as specificity, linearity, range, precision, accuracy, robustness, LOD (limit of detection) and LOQ (limit of quantification). 11. The method is applicable for determining the potency and shelflife of the vaccine. 12. Low variations were observed in the output / results. 13. Do not use reagents (e.g. anthrone reagent) which are unstable and give rise to give higher background (less sensitivity) which requires to be used in cold conditions. 14. The present method of quantification involves sequential addition of phenol followed by sulfuric acid (addition in the centre of the test tube), incubation: about 30 min at RT and absorbance at 480nm. 15. Does not use volatile solvents (e.g. ethanol) 16. Does not use / require chromatography. 17. Easy, cost effective method that can be performed in a laboratory. The present invention is further explained by the following numbered embodiments. These embodiments represent important aspects of the present disclosure, but should not be construed to limit its scope: I.A method for quantification or estimation of total polysaccharide content in a sample, said method comprising: i. adding a chromogen to the sample to obtain a mixture; ii. mixing an acid with the mixture to obtain a solution; iii. incubating the solution; and iv. subjecting the incubated solution to a spectrophotometric or colorimetric assay to quantify or estimate the total polysaccharide content in the sample. II. A method for quantification or estimation of free polysaccharide content in a sample, said method comprising: i. adding a chromogen to the sample to obtain a mixture; ii. mixing an acid with the mixture to obtain a solution; iii. incubating the solution; and iv. subjecting the incubated solution to a spectrophotometric or colorimetric assay to quantify or estimate the free polysaccharide content in the sample. III.The method as defined in embodiments I or II, wherein the method comprises diluting the sample prior to adding the chromogen to the sample. IV.The method as defined in embodiment III, wherein the dilution of sample is carried out to obtain concentration ranging from about 0 pg / ml to 50 pg / ml. V.The method as defined in embodiments I or II, wherein the method comprises employing a standard solution for quantifying or estimating the total polysaccharide content or free polysaccharide content, wherein the standard solution comprises more than one saccharide molecule or combination of saccharide molecules. VI.The method as defined in embodiment V, wherein the standard solution comprises more than one saccharide selected from a group comprising monosaccharide, disaccharide, oligosaccharide, polysaccharide and combinations thereof. VII.The method as defined in embodiment VI, wherein the monosaccharide is selected from a group comprising glyceraldehyde (triose), ribose (pentose) glucose (hexose), fructose (hexose), galactose (hexose), tagatose, mannose, arabinose, xylose, erythrose (tetrose), sedoheptulose (heptose), rhamnose, paratose and combinations thereof; the disaccharide is selected from a group comprising sucrose, isomaltulose, lactose, maltose, trehalose and combinations thereof; the oligosaccharide is selected from a group comprising maltose gluco-oligosaccharides, raffinose, stachyose, fructo-oligosaccharides (FOS), arabino-oligosaccharides (AXOS), amylose, amylopectin, modified starches and combinations thereof; and the polysaccharide is selected from a group comprising pectin, cellulose, hemicellulose, hydrocolloids (arabic gum and guar gum) and combinations thereof. VIII.The method as defined in embodiment VI, the standard solution is prepared by mixing rhamnose, mannose, galactose, glucose and paratose, wherein the rhamnose, mannose, galactose, glucose and paratose is in a ratio of about 1: 1: 1: 0.76:1. IX.The method as defined in embodiments I or II, wherein the chromogen is at a concentration ranging from about 70% to 90%, wherein the chromogen is selected from a group comprising phenol, resorcinol, 2,6-dimethylphenol, Orcinol, DNSA (3,5-dinitrosalicylaldehyde), TNBS (2,4,6-Trinitrobenzene sulfonic acid) and combinations thereof. X.The method as defined in embodiments I or II, wherein the acid is at a concentration ranging from about 15 M to 20 M; wherein the acid is selected from a group comprising hydrochloric acid, sulphuric acid, perchloric acid, carbonic acid hydrofluoric acid, phosphoric acid, trifluoroacetic acid, acetic acid, nitric acid and combinations thereof. XI.The method as defined in embodiments I or II, wherein ratio of the chromogen to the acid is ranging from about 10:1 to 200: 1. XII.The method as defined in embodiments I or II, wherein the solution is incubated at a temperature ranging from about 20 °C to 30 °C, for a duration ranging from about 20 minutes to 50 minutes. XIII.The method as defined in embodiments I or II, wherein the sample is selected from a group comprising purified polysaccharides, purified polysaccharide protein conjugate bulk, final vaccine formulation and combinations thereof. XIV.The method as defined in embodiment XIII, wherein the sample comprising the purified polysaccharide protein conjugate is subjected to pre-treatment prior to subjecting to the quantification or estimation of total polysaccharide content or free polysaccharide content, wherein the pre-treatment comprising- passing the purified polysaccharide protein conjugate to desalting column, membrane filter or a combination thereof. XV.The method as defined in embodiment XIV, wherein the desalting column is PD-10 desalting column selected from a group comprising Thermo Fisher Zeba Spin, Biorad Desalting column and Avantor -Gtrap, Pierce desalting column; and the membrane filter is selected from a group comprising Sartorius-Vivaspin turbo 15 / 4 / 2, Amicon Ultra centrifugal filters 15 / 4 / 2, Pall centrifugal devices and Thermo Centrifugal devices. XVI.The method as defined in embodiment XIV, wherein the pre-treatment removes excipients present in the sample, wherein the excipient is selected from a group comprising maltose, lactose, sucrose, mannitol and trehalose. XVII.The method as defined in embodiment XIV, wherein the pre-treated sample is diluted by employing buffer prior to quantification or estimation of total polysaccharide content or free polysaccharide content; wherein the buffer is selected from a group comprising tris, histidine, pyridine, citrate, phosphate, acetate, Disodium phosphate, monopotassium phosphate, sodium chloride, sodium borate, Succinic acid, nitrate, sodium hydroxide, HEPES (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate), MOPS (3-(N- morpholino)propanesulfonic acid), Bicine, Bis-Tris, Carbonate, Tricine, ACES (N-(2-acetamido)-2-aminoethanesulfonic acid) and combinations thereof. XVIII.The method as defined in embodiment XIII, wherein the sample comprising purified polysaccharide protein conjugate bulk and final vaccine formulation is treated with protein precipitating agent and acid, for separating free or unconjugated polysaccharide and conjugated polysaccharide; wherein the protein precipitating agent is selected from a group comprising trichloroacetic acid, D0C-HC1, ethanol, acetone, ammonium sulfate, PEG8000, diethyl ether and combinations thereof. XIX.The method as defined in embodiment I, wherein the method for quantification or estimation of total polysaccharide content in the sample comprises: i. diluting the sample to obtain a concentration ranging from about 0 pg / ml to 50 pg / ml; ii. adding chromogen at a concentration ranging from about 70% to 90% to the diluted sample to obtain a mixture; iii. adding acid at concentration ranging from about 15 M to 20 M to the mixture to obtain a solution; iv. incubating the solution at a temperature ranging from about 20 °C to 30 °C for a duration ranging from about 20 minutes to 50 minutes; and v. subjecting the incubated solution to a spectrophotometric or colorimetric assay alongside a standard solution comprising more than one saccharide or combination of saccharides to quantify or estimate the total polysaccharide content. XX.The method as defined in embodiment I, wherein the method for quantification or estimation of free polysaccharide content in the sample comprises: i. diluting the sample to obtain a concentration ranging from about 0 pg / ml to 50 pg / ml; ii. adding chromogen at a concentration ranging from about 70% to 90% to the diluted sample to obtain a mixture; iii. adding acid at concentration ranging from about 15 M to 20 M to the mixture to obtain a solution; iv. incubating the solution at a temperature ranging from about 20 °C to 30 °C for a duration ranging from about 20 minutes to 50 minutes; and v. subjecting the incubated solution to a spectrophotometric or colorimetric assay alongside a standard solution comprising more than one saccharide or combination of saccharides to quantify or estimate the free polysaccharide content. XXI.The method as defined in embodiment I, wherein the method for quantification or estimation of free polysaccharide content or total polysaccharide content in the sample comprising purified polysaccharide protein conjugate bulk and final vaccine formulation, said method comprises: i. pre-treating the sample with desalting column, membrane filter or combination thereof, followed by optionally treating with protein precipitating agent and acid to obtain pre-treated sample. ii. diluting the pre-treated sample to obtain a concentration ranging from about 0 pg / ml to 50 pg / ml; iii. adding chromogen at a concentration ranging from about 70% to 90% to the diluted sample to obtain a mixture; iv. adding acid at concentration ranging from about 15 M to 20 M to the mixture to obtain a solution; v. incubating the solution at a temperature ranging from about 20 °C to 30 °C for a duration ranging from about 20 minutes to 50 minutes; and vi. subjecting the incubated solution to a spectrophotometric or colorimetric assay alongside a standard solution comprising more than one saccharide or combination of saccharides to quantify or estimate the free polysaccharide content or total polysaccharide content in the sample. XXII.A method for quantification of O-specific polysaccharide content in a sample, the method comprising: a) diluting the sample with a buffer to obtain a diluted sample; b) adding a chromogen to the diluted sample to obtain a mixture; c) mixing an acid to the mixture to obtain a solution; d) incubating the solution; e) subjecting the incubated solution to a spectrophotometric assay; f) quantifying the O-specific polysaccharide content in the sample using a mixed saccharide standard solution, wherein the mixed saccharide standard solution comprises at least two saccharides. XXIII.A method for quantification of O-specific polysaccharide content in a sample, said method comprising: a) diluting the sample with the buffer to obtain the diluted sample in the range from 1 pg / ml to 50.0 pg / ml; b) adding the phenol in the range from 70.0% to 90.0% to the diluted sample to obtain the mixture; c) mixing the sulphuric acid in range from 15.0 M to 20.0 M to mixture to obtain the solution; d) incubating the solution at temperature in range from 20.0 °C to 30.0 °C for duration in range of 20.0 minutes to 50.0 minutes; e) subjecting the incubated solution to the spectrophotometric assay at 480 nm; f) quantifying the O-specific polysaccharide content in the sample using mixed saccharide standard solution; wherein the mixed saccharide standard solution comprises at least two saccharides; and wherein the sample is a purified O-specific polysaccharide, a purified O-specific polysaccharide protein conjugate bulk or a vaccine formulation; and wherein the purified O-specific polysaccharide protein conjugate bulk and final vaccine formulation is additionally subjected to pre-treatment by passing through desalting column and membrane filter and treating with DOC-HC1, prior to the dilution of the sample.

Claims

1. A method for quantification of O-specific polysaccharide content in a sample, the method comprising:a) adding a chromogen to the sample to obtain a mixture, wherein the sample is optionally diluted with a buffer prior to the addition of the chromogen;b) mixing an acid to the mixture to obtain a solution;c) incubating the solution;d) subjecting the incubated solution to a spectrophotometric assay or a colorimetric assay; ande) quantifying the O-specific polysaccharide content in the sample using a mixed saccharide standard solution,wherein the mixed saccharide standard solution comprises at least two saccharides.

2. The method as claimed in claim 1, wherein the buffer is selected from a group comprising tris, histidine, pyridine, citrate, phosphate, acetate, disodium phosphate, monopotassium phosphate, sodium chloride, sodium borate, succinic acid, nitrate, sodium hydroxide, HEPES (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate), MOPS (3-(N-morpholino)propanesulfonic acid), Bicine, bis-Tris, carbonate, Tricine, ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), or any combination thereof.

3. The method as claimed in any one of claims 1 or 2, wherein the chromogen is selected from a group comprising phenol, resorcinol, 2,6-dimethylphenol, orcinol, DNSA (3,5-dinitrosalicylaldehyde), TNBS (2,4,6-Trinitrobenzene sulfonic acid), or any combination thereof, at a concentration ranging from about 70% to 90%.

4. The method as claimed in any one of claims 1 to 3, wherein the acid is selected from a group comprising hydrochloric acid, sulphuric acid, perchloric acid, carbonic acid hydrofluoric acid, phosphoric acid, trifluoroacetic acid, acetic acid, nitric acid, or any combination thereof, at a concentration ranging from about 15 M to 20 M.

5. The method as claimed in any one of claims 1 to 4, wherein the solution is incubated in step c) at a temperature ranging from about 20 °C to 30 °C, for a duration ranging from about 20 minutes to 50 minutes.

6. The method as claimed in any one of claims 1 to 5, wherein the mixed saccharide standard solution comprises at least two saccharides selected from a group comprising monosaccharide, disaccharide, oligosaccharide, polysaccharide, or any combination thereof;optionally, wherein the mixed saccharide standard solution comprises at least two monosaccharides selected from a group comprising glyceraldehyde (triose), ribose (pentose) glucose (hexose), fructose (hexose), galactose (hexose), tagatose, mannose, arabinose, xylose, erythrose (tetrose), sedoheptulose (heptose), rhamnose, paratose, or any combination thereof.

7. The method as claimed in claim 6, the mixed saccharide standard solution comprises rhamnose, mannose, galactose, glucose and paratose in a ratio of about 1: 1: 1: 0.76:1.

8. The method as claimed in claim 1, wherein the sample is selected from a group comprising a purified O-specific polysaccharide, a purified O-specific polysaccharide protein conjugate bulk, or a vaccine formulation including O-specific polysaccharide protein conjugate.

9. The method as claimed in claim 8, wherein the sample comprising O-specific polysaccharide protein conjugate is subjected to pre-treatment prior to the dilution of the sample, for removal of excipients selected from a group comprising maltose, lactose, sucrose, mannitol and trehalose;optionally, wherein the pre-treatment comprises passing the sample to desalting column, membrane filter, or any combination thereof.

10. The method as claimed in claim 9, wherein the desalting column is selected from a group comprising cross linked dextran based, polysaccharide based, porous polyacrylamide based and polypropylene based.

11. The method as claimed in claim 9, wherein the membrane filter is selected from a group comprising polyethersulfone based, modified polyethersulfone based, regenerated cellulose based, and water-wettable polytetrafluoroethylene based; wherein the membrane has a molecular weight cut-off (MWCO) from about 1 to 10 kDa.

12. The method as claimed in any one of claims 8 to 11, wherein the sample comprising purified O-specific polysaccharide protein conjugate bulk and vaccine formulation is treated with a protein precipitating agent, to separate free or unconjugated O-specific polysaccharide and conjugated O-specific polysaccharide;wherein the protein precipitating agent is selected from a group comprising trichloroacetic acid, deoxycholate-hydrochloric acid (DOC-HC1), ethanol, acetone, ammonium sulfate, PEG8000, diethyl ether, or any combinations thereof.

13. The method as claimed in any one of claims 1 to 12, wherein the O-specific polysaccharide content in the sample is free O-specific polysaccharide content and total O-specific polysaccharide content.

14. The method as claimed in any one of claims 1 to 13, wherein the O-specific polysaccharide is selected from a group of Gram-negative bacteria comprising: Salmonella spp., Salmonella typhi, Salmonella paratyphi. Salmonella enter itidis, Salmonella typhimurium; Shigella spp., Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Citrobacter spp., C. freundii, C. werkmanii; Cronobacter species (former Enterobacter sakazakii); Shigella boydir, Escherichia coli; Klebsiella pneumoniae; Vibrio cholerae; Pseudomonas aeruginosa; Plesiomonas shigelloides; or Campylobacter jejuni.

15. A method for quantification of O-specific polysaccharide content in a sample, said method comprising:i. diluting the sample with a buffer to obtain the diluted sample comprising the O-specific polysaccharide in the range from about 1 pg / ml to 50.0 pg / ml;ii. adding phenol in the range from about 70.0% to 90.0% to the diluted sample to obtain a mixture;iii. mixing sulphuric acid in range from about 15.0 M to 20.0 M to the mixture to obtain a solution;iv. incubating the solution at temperature in range from about 20.0 °C to 30.0 °C for duration in range of about 20.0 minutes to 50.0 minutes;v. subjecting the incubated solution to a spectrophotometric assay at 480 nm;vi. quantifying the O-specific polysaccharide content in the sample using a mixed saccharide standard solution;wherein the mixed saccharide standard solution comprises at least two saccharides; andwherein the sample is a purified O-specific polysaccharide, a purified O-specific polysaccharide protein conjugate bulk, or a vaccine formulation; andwherein the purified O-specific polysaccharide protein conjugate bulk and final vaccine formulation is additionally subjected to pre-treatment by passing through a desalting column and a membrane filter and treating with DOC-HC1, prior to the dilution of the sample.

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