DESC:TECHNICAL FIELD
The present invention relates to the field of saccharide-protein conjugate vaccine(s) and particularly to saccharide coupled microspheres used to evaluate the saccharide-protein conjugates in vaccines/ immunogenic compositions.

BACKGROUND OF THE DISCLOSURE
The background information herein below relates to the present invention but is not necessarily prior art.
Saccharides (hereinafter interchangeably referred to as PSs, saccharides, polysaccharides, capsular saccharides, oligosaccharides) are carbohydrates and are abundantly found in variety of organisms. Saccharides are seen on capsule, cell walls and other cell surfaces of the organisms, microorganisms, bacteria, yeast, fungi, and viruses. Capsular saccharides have epitope motifs usually not found in mammals and are used to mediate immunogenicity. Such saccharides are therefore useful for the preparation of vaccines against bacterial diseases such as meningitis, pneumonia, and typhoid fever.

Combination and polyvalent vaccines not only provide protection against several different pathogens at the same time but can also increase vaccine protection against pathogens that have closely related pathogenic strains or serotypes. In particular, the use of polyvalent conjugate vaccines for Streptococcus pneumoniae is important due to the organism’s many serotypes, each with a distinct polysaccharide capsule.

However, testing the vaccine immune response to each serotype can be extremely time-consuming and laborious if each component must be assessed in an individual serological immunoassay. Therefore, multiplexed serological testing methods have been developed for determining the efficacy of combination and polyvalent vaccines.

Multiplexed immunoassays reduce the number of assays needed to confirm immune responses and cross-reactivity, use less serum, and can be performed faster. Testing in multiplex reduces the variables that must be controlled when performing individual tests and can thus be more reliable, and ultimately are more cost-effective than single-plex methods.
Immunoassays as well, rely on detection of antibodies corresponding to saccharide antigens to diagnose infectious diseases and to assess safety and efficacy of saccharide-based vaccines. Diagnostic assays/ immunoassays that detect antibodies corresponding to saccharide antigens (such as antigen content determination assays, identity assays, potency assays, immunofluorescent assays, Western blotting, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassay (RIAs), and the like), use solid phase matrix to which saccharides are bound/ immobilised or employ a liquid phase with suspended polysaccharides. For better results and detection purposes, the saccharides are coupled with microspheres.

Bead-based suspension array technologies are often used for development of multiplexed serological assays to simultaneously assess immune responses to multiple antigens and have been used in vaccine trials, testing in clinical laboratories, epidemiological studies, and in basic immunological research.

In particular, the xMAP® microsphere technology from Luminex has been used extensively for multiplexed serological assays to detect immune responses to a variety of antigens, including pathogens, autoimmune markers, as well as human leukocyte antigen (HLA) and alloantigens, which is important for donor and recipient testing in transplantation (Das and Dunbar, 2020).

The immobilization of biomolecules or any other such entities can be achieved by coupling by (a) ionic interactions; (b) adsorption; (c) complexation (such as “metal-coordination” mediated coupling); and (d) covalent bond formation between active/stable reactive groups on the surface and specific functional groups on the entity to be immobilized. For example, particles such as micro- and nano-spheres; nanotubes; metal particles including one or more metals with any size, shape, or composition; semiconductor particles; molecularly imprinted polymers (MIPS); magnetic particles; other dyed materials and the like and microtiter plates are common solid matrices in many immobilization systems.

Preparing and maintaining the active, functionalized surface of the solids are important to assure immobilization of biological material for development of a sufficiently sensitive assay.
Current procedures for immobilization of biomolecules on solid surfaces generally involve reactions of activated carboxyl, amino-, hydroxyl- or thiol-groups on the solid surfaces with the biomolecules. After activation of, or introduction of a functionalized spacer to, these groups, the activated groups provide sites on the solid surface for direct attachment of the biomolecules.

While immobilisation (on solid supports) or for formation of the saccharide coupled microspheres, the saccharides are bound by either non-covalent bonds or by covalent bonds. The selection of immobilization by either non-covalent bond or covalent bond depends on several factors like nature and function of biomolecule, compatibility as well as operating and storage conditions. Non-covalent chemical bonding/ chemistry includes attachment by van der Waals forces, hydrophobic interdigitation, physical adsorption, ionic bonding, affinity binding and the like. Covalent binding includes binding through sharing of valence electrons between an atom on the solid surface and an atom on the saccharides by forming strong chemical bonds.

Non-covalent immobilization of saccharides onto solid surfaces (coating) is generally time, reagent, and labour consuming because the optimal coating conditions vary among saccharides from different bacterial strains as well as between serotypes of the same bacteria. It also results in low loading capacity, high leaching and is sensitive to environmental changes.

Variability in conditions for non-covalent methods impacts accuracy and reproducibility of quantitative determinations as well as makes immobilization difficult for two or more different saccharides on the same surface. Saccharides also tend to aggregate and render stability of the bonded surface or couples unpredictable and for a shorter shelf life. Aggregation is overcome by adding detergents, however such addition cause variation in further assays and determinations.

Since non-covalent methods of coupling generally relate to physical adsorption of saccharides to bead surfaces, the bonding is fragile in nature. Additionally, saccharides with neutral charge are not able to couple with beads using physical adsorption methods.

Covalent couplings overcome some of the problems associated with classical non-covalent coupling methods. However, covalent chemistries include oxidization and other chemical modifications of saccharides for coupling polysaccharides.

Modification in saccharide structure possibly shields the epitopes and can lead to loss of sensitivity or challenges in assessing the true antigenicity/immunogenicity. Modifications in antigen structures via covalent reactions can cause limitations and challenges in functions of analytics. Covalent modification of saccharide also introduces new structures which can lead to additional challenges of cross reactivity. Modification of the structure of the saccharide leads to challenges in multiplex assays having higher valencies.

Currently used functional groups for providing direct attachment sites, have a number of disadvantages. For example, most of these functional groups (such as N-hydroxysuccinimide (NHS) esters, isothiocyanates, and the like) are prone to hydrolysis in an aqueous environment and become non-reactive (i.e., chemically inactive) in a matter of less than an hour. Therefore, the use of such functional groups for attaching the biomolecules to the surface of solids may lead to undesirable issues such as time-dependent variations in the quantity, repeatability, and uniformity of the attachment process.

Fray et al., Bioconjugate Chem., 1999, 10, 562-571 have reported a strategy in which particles are pre-activated with hydrolysis-resistant aldehyde functional groups, but low reaction yields of less than 8% have been observed with these microspheres. U.S. Pat. No. 6,146,833 to Milton describes a reaction between an acyl fluoride activated polymer-surface and an amino derivatized biomolecule at room temperature. The use of fluorophenyl resins in the solid phase synthesis of amides, peptides, hydroxamic acids, amines, urethanes, carbonates, sulfonamides, and alpha-substituted carbonyl compounds has been described in International Publication No. WO 99/67228 to Clerc et al.

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/ EDC/ EDAC/ EDCI/ water soluble carbodiimide mediated coupling is currently the major mode of covalent immobilization of biomolecules to solid surfaces as described by Hermanson, G. T., in Bioconjugate Techniques, Academic Press, NY, 1996; Frey, A. et al., Bioconjugate Chem., 1999, 10, 562-571; Gilles, M. A. et al., Anal. Biochem., 1990, 184, 244-248; Chan V. W. F. et al., Biochem. Biophys. Res. Communications, 1988, 151(2), 709-716; and Valuev, I. L. et al., Biomaterials, 1998, 19, 41-43. The most frequently used method to immobilize biomolecules (such as oligonucleotides, proteins, and carbohydrates) onto fluorescent microspheres is by activating carboxy groups present on the surface of the microspheres. The activation requires excess N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) and a coupling pH of 4 to 6. The reaction between the carbodiimide and carboxyl functional groups forms an activated O-acylurea derivative reaction intermediate. A subsequent nucleophilic attack of the reaction intermediate by the primary nitrogen of the amino-groups of the biomolecule being attached to the microspheres releases the substituted urea and produces an amide linkage between the reaction intermediate and the biomolecule.

There are, however, a number of disadvantages to such activation of the carboxy groups. For example, the reaction intermediate has an extremely short half-life and rapidly undergoes hydrolysis or rearranges to produce the N-acylurea adduct. In addition, the optimum pH for the formation of O-acylurea is about 4 to 5. However, the primary amino group of the nucleophile is predominantly protonated at a pH of about 4 to 5 and is thus mostly unreactive. These limitations of the reaction intermediate can severely restrict coupling yields of biomolecules to microspheres.

Covalent coupling reagents such as N-hydroxysulfosuccinimide (NHS)/ sulfo-NHS or carbodiimide based reactions oxidize the polysaccharides/protein structure at specific locations which alters the native state of polysaccharides. Coupling reagents are observed to be toxic and require lengthy and difficult laboratory practices, especially to comply with good manufacturing practices. “9.2 Premises and equipment” in Annex 2, TRS No 999 of “WHO good manufacturing practices for biological products” requires documented quality risk management of additional product in manufacturing facility, including potency and toxicological evaluation on cross-contamination risks. In some instances, standard EDC/sulfo-NHS coupling procedures may be somewhat problematic. For example, EDC and sulfo-NHS are hygroscopic solids that react with moisture in the air, and special precautions must be used to keep the surface modifier in the bottle fresh. Working solutions of the surface modifiers must be made immediately before use. The urea side products from EDC activation are sometimes hard to remove from the bead suspension and can interfere with subsequent coupling reactions or assays.

Some covalent coupling methods include manual amine coupling method that use 2 step carbodiimide reaction that chemically modifies the polysaccharide. Such methods require usage of cross-linking agents like cyanuric chloride and requires freshly prepared reagents. Moreover, the reagents and chemicals used are considered toxic/ hygroscopic.

Further, microsphere/ bead coupling involving use of toxic chemistries also limits the use of technologies for automating the process of coupling (for example use of robotic liquid handling systems) for increasing throughput of laboratories.
Covalent reaction methods are reported to be variable and lab to lab reproducibility of results is difficult.

IN276304 (Serum Institute of India Private Limited) discloses a method for simultaneously detecting the presence of multiple anti-polysaccharide antibodies in a single test sample. The invention discloses use of four different chemistries (Poly-L-Lysine, EDC-ADH, DMTMM-NH2 and DMTMM-COOH) for coupling of each of pneumococcal polysaccharides to the carboxylated microspheres. Thus, it discloses use of conventional chemical coupling methods for Streptococcus polysaccharides.

IN491469 (Serum Institute of India Private Limited) discloses modified Sandwich ELISA for determining the antigen content and percent adsorption. The modified sandwich ELISA uses optimized parameters to quantify conjugated polysaccharide in the presence of 9 other conjugated antigens in a 10 valent vaccine.

Modified Amine Coupling of Pneumococcal polysaccharides to Beads (using EDC/NHS) (particularly for 6B, 9V and 19A polysaccharide) for preparing pneumococcal polysaccharide coupled beads is known.

Pickering et al., 2002; Lal et al., 2005; Whitelegg et al., 2012 describe a PLL (poly-L-lysine) conjugation reaction. Biagini et al., 2003 desribe conventional chemistry method based on sodium periodate oxidation/ADH. Schlottmann et al., 2006 provide for DMTMM (4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) based method. Covalent coupling reactions are indicated to be associated with limitations. The reactions oxidize the saccharide structure which alters the native state of polysaccharide. The coupling of DMTMM to microspheres performs well, but the microspheres coupled to DMTMM are more hydrophobic than microspheres activated with the commonly used surface modifier, sulfo-N-hydroxysuccinimide (sulfo-NHS). In other words, the DMTMM modified microspheres exhibited a propensity to stick to each other rather than dispersing in an aqueous solution. In contrast, microspheres with sulfo-NHS groups attached thereto retain a water-loving (i.e., hydrophilic) group (the sulfo) on the surface thereof when the sulfo-NHS is reacted with the original carboxyl group on the microspheres. The microspheres, therefore, stay well dispersed in water and aqueous solutions and solvents. In contrast, DMTMM is soluble in water because of the quaternary ammonium salt moiety that it contains. After reaction with a carboxyl group on the surface of a microsphere, this positive charge is lost due to its solubility in water. In this manner, hydrophilic carboxyl groups on the surface of the microsphere are replaced with hydrophobic aromatic rings thereby reducing the hydrophilicity of the microspheres.

WHO mentions (for e.g. WHO TRS 977 in case of Pneumococcal Vaccines) that for polysaccharide protein conjugate vaccines, antigen content determination is crucial as a good manufacturing practice.

Thus, it would be advantageous to develop a method for altering the surface characteristics of a microsphere without one or more of the disadvantages described above. There is an unmet need for a method/ process to couple microsphere to saccharides that is rapid, simple, repeatable, cost effective, scalable, nontoxic and provides stable saccharide coupled microspheres with structure of saccharides remaining intact/unaffected.

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 provide a rapid, simple, repeatable, cost effective, scalable, non-toxic method/ process to couple microsphere to saccharides to obtain stable saccharide couple microsphere that overcomes the drawbacks associated with previously reported conventional/chemical coupling methods.

Another object of the present disclosure is to provide an improved method of coupling that does not modify the structure of saccharides and is applicable for wide range of bacterial polysaccharides.

Yet another object of the present disclosure is to provide a method of non-covalent (electrostatic) coupling of polysaccharides to microspheres to form couples where structure of saccharides remains unaffected/intact and retains epitope confirmation.

Still another object of the present disclosure is to provide a method of coupling microsphere with streptococcal saccharides.

Yet another object of the present disclosure is to provide a method of coupling microsphere with meningococcal saccharides.

Another object of the present disclosure is to provide a method of coupling microsphere with Haemophilus saccharides.

Yet another object of the present disclosure is to provide a method of coupling microsphere with Salmonella saccharides.

Still another object of the present disclosure is to provide assays based on the saccharide coupled microspheres to determine potency, identity, immune response associated with the saccharides.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY OF THE DISCLOSURE
The present invention provides a rapid, simple, cost effective, scalable method of coupling microspheres with saccharides to obtain the saccharide coupled microspheres which are used to determine antigen content, identity, free polysaccharide estimation, and antibody concentration.

The present invention is directed to a method of non-covalent (electrostatic) coupling polysaccharides to microspheres to form couples under specific reaction conditions (specific size and concentration of polysaccharides, specific chemical composition) where the structure of polysaccharides remains unaffected. Beads surfaces are activated using bead reagent, wherein the bead reagent includes metal ions. The activated beads are used for the coupling of saccharides using coupling buffer for specific incubation temperature and time. The prepared bead mixture is utilized in pneumococcal, Hib vaccine development assays such as antigen content determination, identity assay, free Ps estimation in drug product, estimation of antibody concentration (IgG) in clinical (human) sera samples, estimation of IgG titer in animal sera sample and the like.

Accordingly, in one aspect, the present invention is directed to a method of coupling a saccharide to a microsphere, the method comprising:
a. providing the microsphere;
b. providing the saccharide;
c. diluting the saccharide with a buffer at pH in range of 3.0 to 9.0;
d. mixing the microsphere with the saccharide to form the saccharide coupled microsphere;
wherein the mixing further includes incubation at temperature in range of 20? to 40? for incubation time in range of 30 mins to 180 mins.

In accordance with the embodiments of the present invention,
the mixing is performed with the coupling ratio of the microsphere to the saccharide in range of 50 to 12500, and/ or
optionally, the microsphere is activated with a bead reagent before mixing with the saccharide, wherein the bead reagent includes metal ions,
optionally, the saccharide coupled microsphere is formed by non-covalent electrostatic coupling, and/ or
optionally, the saccharide remains intact, retains epitope confirmation and the saccharide coupled microsphere is stable.

In an embodiment of the present invention, the buffer includes phosphate buffered saline with tween (PBST) buffer, (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES) buffer, tris-aminomethane (Tris) buffer, 2-(N-morpholino)ethane sulfonic acid (MES) buffer, 3-(N-morpholino) propane sulfonic acid (MOPS) buffer.

In an embodiment of the present invention, the saccharide is bacterial saccharide including Group A Streptococcus, Group B Streptococcus, Streptococcus pneumoniae/ pneumococcus, Haemophilus bacteria, Haemophilus influenzae bacteria, Haemophilus influenzae type b bacteria (Hib), Salmonella, Typhoidal salmonella, Non-typhoidal salmonella, Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi, Streptococcus pyogenes, Streptococcus agalactiae, Shigella, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Escherichia coli, or Neisseria meningitidis/ meningococcus saccharide.

In an embodiment of the present invention, the saccharide is Streptococcus pneumoniae saccharide.

In an embodiment of the present invention, the Streptococcus pneumoniae saccharide has a molecular size in range of 50 kDa to 3000 kDa, is diluted with PBST buffer at a pH in range of 3.0 to 9.0 and has concentration of 0.05 mg/mL to 50.0 mg/mL.

In an embodiment of the present invention, the Streptococcus pneumoniae saccharide is mixed with the microsphere and incubated at temperature of 23? to 39?, for incubation time of 60 mins to 120 mins.

In an embodiment of the present invention, the Streptococcus pneumoniae saccharide is selected from serotypes 1, 2, 3, 4, 5, 6, 6A, 6B, 6C, 6D, 7, 7B, 7C, 7F, 8, 9, 9A, 9L, 9N, 9V, 10, 10A, 10B, 10C, 10F, 10X, 11, 11A, 11B, 11C, 11F, 12, 12A, 12B, 12F, 13, 14, 15, 15A, 15B, 15BC, 15C, 15F, 16, 16F, 17, 17A, 17F, 18, 18A, 18B, 18C, 18F, 19, 19A, 19B, 19F, 20, 20A, 20B, 20F, 21, 22, 22A, 22F, 23, 23A, 23B, 23F, 24, 24F, 25, 25F, 26, 27, 28, 28A, 28F, 29, 30, 31, 32, 33, 33A, 33B, 33C, 33D, 33F, 34, 35, 35A, 35B, 35C, 35D, 35F, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, and/ or 48.

In an embodiment of the present invention, the Streptococcus pneumoniae saccharide is selected from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 24F and/ or 33F.

In an embodiment of the present invention, the saccharide is Haemophilus influenzae type b bacteria (Hib) saccharide.

In an embodiment of the present invention, the Haemophilus influenzae type b bacteria (Hib) saccharide has a molecular size in range of 0.05 kDa to 5 kDa, is diluted with PBST buffer at a pH in range of 4 to 6 and has concentration of 1.0 µg/mL to 50.0 µg/mL.

In an embodiment of the present invention, the Haemophilus influenzae type b bacteria (Hib) saccharide is mixed with the microsphere and incubated at temperature of 20? to 30?, for incubation time of 60 mins to 120 mins.

In an embodiment of the present invention, the saccharide is Neisseria meningitidis saccharide.

In an embodiment of the present invention, the saccharide is Neisseria meningitidis saccharide serotypes selected from meningococcal serotypes A (type I and III), B (type II), B6, B16, C (type II-alpha), D (type IV), Z’/ E, E29, H, I, K, K454, L, M, W135, X, Y, Z.

In an embodiment of the present invention, the Neisseria meningitidis polysaccharide has a molecular size in range of 75 kDa to 3000 kDa, is diluted with PBST buffer at a pH in range of 4 to 7 and has concentration of 0.01 mg/mL to 10.0 mg/mL.

In an embodiment of the present invention, the Neisseria meningitidis polysaccharide is mixed with the microsphere and incubated at temperature of 20 to 40?, for incubation time of 60 mins to 120 mins.

Accordingly, in one aspect, the present invention is directed to a method of coupling a saccharide to a microsphere, the method comprising:
a. providing the microsphere;
b. providing the saccharide;
c. diluting the saccharide with the buffer at pH in range of 3.0 to 9.0; wherein and the saccharide is in range of 0.1 mg/ml to 50.0 mg/ml;
d. mixing the microsphere with the saccharide to form the saccharide coupled microsphere;
wherein the saccharide has a size in the range of 50.0 kDa to 3000 kDa; and
wherein the mixing includes incubation at temperature in range of 20? to 40? for incubation time in range of 30 mins to 180 mins.

In another aspect, the present invention is directed to the saccharide coupled microsphere obtained by the method as disclosed herein.

In an embodiment of the present invention, the saccharide coupled microsphere has Mean Florescence intensity/ MFI value in the range of 200 to 20000.

In an embodiment of the present invention, the saccharide coupled microsphere is a Streptococcus pneumoniae saccharide coupled microsphere.

In an embodiment of the present invention, the saccharide coupled microsphere is a Haemophilus influenzae type b bacteria (Hib) saccharide coupled microsphere.

In an embodiment of the present invention, the saccharide coupled microsphere is a Neisseria meningitidis saccharide coupled microsphere.

In another aspect, the present invention is directed to a method of evaluating immunogenicity of immunogenic composition, the method comprising:
a. providing a test sample corresponding to the saccharide in the immunogenic composition;
b. providing the saccharide coupled microsphere corresponding to the saccharide in the immunogenic composition; and
c. evaluating the immunogenicity of the immunogenic composition by antigen content determination, or estimating antibody concentration (IgG), or identity assay, free polysaccharide estimation using the test sample and the saccharide coupled microsphere,
wherein the saccharide coupled microsphere is obtained by the method of the present disclosure.

In another aspect, the present invention is directed to an antigen content determination method using the saccharide coupled microsphere obtained by the method as disclosed herein.

In another aspect, the present invention is directed to an identity assay method using the saccharide coupled microsphere obtained by the method as disclosed herein.

In another aspect, the present invention is directed to a free saccharide estimation method using the saccharide coupled microsphere obtained by the method as disclosed herein.

In another aspect, the present invention is directed to a method of estimating antibody concentration (IgG) in sera sample using the saccharide coupled microsphere obtained by the method as disclosed herein (human/ animal).

In another aspect, the present invention is directed to the saccharide coupled microsphere to determine antibody titre of an immunogenic composition/ a vaccine.

In another aspect, the present invention is directed to apparatus comprising the saccharide coupled microsphere.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING
The present invention will now be described with the help of the accompanying drawing, in which:
FIG. la and 1b illustrate an embodiment with Streptococcus pneumoniae polysaccharide coupled microsphere with details of MFI value against Type 1 serotype samples;
FIG. 2a and 2b illustrate an embodiment with Streptococcus pneumoniae coupled microsphere with details of MFI value against Type 2 serotype samples;
FIG. 3a and 3b illustrate an embodiment with Streptococcus pneumoniae coupled microsphere with details of MFI value against Type 4 serotype samples;
FIG. 4a and 4b illustrate an embodiment with Streptococcus pneumoniae coupled microsphere with details of MFI value against Type 7F serotype samples;
FIG. 5a and 5b illustrate an embodiment with Streptococcus pneumoniae polysaccharide coupled microsphere with details of MFI value against Type 8 serotype samples;
FIG. 6a and 6b illustrate an embodiment with Streptococcus pneumoniae polysaccharide coupled microsphere with details of MFI value against Type 10A serotype samples;
FIG. 7a and 7b illustrate an embodiment with Streptococcus pneumoniae polysaccharide coupled microsphere with details of MFI value against Type 24F serotype samples;
FIG. 8a to 8q illustrate embodiments with details of MFI value against saccharide coupled microspheres with saccharides of pneumococcal serotypes 3, 5, 6A, 6B, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 24F, 33F, respectively;
FIG. 9 illustrates an embodiment with details of MFI value against saccharide coupled microspheres with saccharides of Haemophilus influenzae type b; and
FIG. 10a to 10e illustrate embodiments with details of MFI value against saccharide coupled microspheres with saccharides of Neisseria meningitis serotypes (A, C, W, X, Y, respectively).

DETAILED DESCRIPTION OF THE DISCLOSURE
Before the present compositions and formulations of the invention are described, it is to be understood that this invention is not limited to particular compositions and formulations described, since such compositions and formulation may, of course, vary. It is also to be understood that the terminology used herein is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open -ended and do not exclude additional, non-recited members, elements or method steps. It will be appreciated that the terms "comprising", "comprises" and "comprised of" as used herein comprise the terms "consisting of", "consists" and "consists of". More specifically, the term “comprise” as used herein means that the claim encompasses all the listed elements or method steps, but may also include additional, unnamed elements or method steps. For example, a method comprising steps a), b) and c) encompasses, in its narrowest sense, a method which consists of steps a), b) and c). The phrase "consisting of" means that the composition (or device, or method) has the recited elements (or steps) and no more. In contrast, the term “comprises” can encompass also a method including further steps, e.g., steps d) and e), in addition to steps a), b) and c).

Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. In case the terms "first", "second", "third" or “(A)”, “(B)” and “(C)” or "(a)", "(b)", "(c)", "(d)", "i", "ii" etc. relate to steps of a method or use or assay there is no time or time interval coherence between the steps, that is, the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below.
In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may do. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some, but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art.

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 term "vaccine" is optionally substitutable with the term "immunogenic composition" and vice versa.
The term “microsphere” is optionally substitutable with the term "bead" and vice versa.

The terms microsphere-polysaccharide conjugation and microsphere-polysaccharide coupling are interchangeably used throughout the specification.
DEFINITIONS
The term "saccharide" throughout this specification may indicate polysaccharide, saccharide or oligosaccharide, or combinations thereof. The capsular saccharide antigen may be a full-length polysaccharide or it may be extended to bacterial 'sized-saccharides' and 'oligosaccharides' (which naturally have a low number of repeat units, or which are polysaccharides reduced in size for manageability, but are still capable of inducing a protective immune response in a host).

The term “MFI” is associated with Mean Fluorescence Intensity.

Other “biomolecules” which can be conjugated to polysaccharide (interchangeably referred to as PSs) include enzymes, enzyme substrates, enzyme inhibitors, hormones, antibiotics, antibodies, antigens, peptides, polypeptides, proteins, other polysaccharides, nucleic acids, nucleosides, nucleotides, polynucleotides, and the like.

The terms “covalent” and “valence” refer to a chemical bond between two atoms in a molecule created by the sharing of electrons, usually in pairs, by the bonded atoms and may involve single bonds or multiple bonds. The term “covalent” does not include hydrophobic/hydrophilic interactions, hydrogen-bonding, and van der Waals interactions.

The term “non-covalent” refers to interactions between two or more molecules and/or by two or more parts of the same molecule which are not “covalent” in nature. Such “non-covalent” interactions include electrostatic interactions such as, hydrogen bonds, hydrophobic/hydrophilic interactions, salt bridges, and van der Waals interactions.

The term “coating” as used herein refers to non-covalent immobilization of polysaccharides on solid surfaces, e.g., through adsorption. The nature of passive adsorption predominantly involves multiple hydrophobic interactions between solid phase and the polysaccharide.

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. It is also generally desirable that the affinity of antibodies corresponding to the polysaccharide / saccharide antigens toward PSs attached to solid supports and biomolecules is similar to that toward the corresponding unattached PSs.
DISCLOSURE

METHOD OF COUPLING
In one aspect, the present invention is directed to a method of coupling a saccharide to a microsphere to obtain a saccharide coupled microsphere, the method comprising:
a. providing the microsphere;
b. providing the saccharide;
c. diluting the saccharide with a buffer at pH in range of 3.0 to 9.0;
d. mixing the microsphere with the saccharide to form the saccharide coupled microsphere;
wherein the mixing further includes incubation at temperature in range of 20? to 40? for incubation time in range of 30 mins to 180 mins.

In one aspect, the present invention is directed to a method of coupling a saccharide to a microsphere, the method comprising:
a. providing the microsphere;
b. providing the saccharide;
c. diluting the saccharide with the buffer at pH in range of 3.0 to 9.0; wherein and the saccharide is in range of 0.1 mg/ml to 50.0 mg/ml;
d. mixing the microsphere with the saccharide to form the saccharide coupled microsphere;
wherein the saccharide has a size in the range of 50.0 kDa to 3000 kDa; and
wherein the mixing further includes incubation at temperature in range of 20? to 40? for incubation time in range of 30 mins to 180 mins.

The present invention pertains to the method for coupling saccharides to microsphere such that the non-conventional saccharide to bead coupling uses metal activated beads and the coupling is non-covalent electrostatic coupling.

The present invention pertains to the method for coupling saccharides to microsphere using non-covalent coupling of saccharides to microspheres to form the saccharide coupled microsphere under specific reaction conditions wherein the structure of polysaccharides remains intact/unaffected and retains epitope confirmation.

The present invention pertains to the method for coupling saccharides which is based on principles of metal coordination chemistry and electrostatic mode of interaction-based coupling of antigens wherein bead surface is first activated using metal solutions of Nickel to introduce suitable metal ions on the bead surface.

The saccharide coupled microsphere obtained by the method of present invention finds application developing immunochemical assays for
a) determining Antigen content,
b) identity assays,
c) determining antibody/ IgG or serology applications,
d) estimating stability of saccharides or estimating free saccharide content,
e) other serological analysis.
The method has optimized a specific combination of concentration of polysaccharide, size of polysaccharide, reaction time, temperature, pH of buffer /PBST to allow development of electron donor sites which allow electrostatic attachment of Ps on to activated beads structure that too with polysaccharide structure remaining intact.
In an embodiment, the present invention pertains to the method of coupling saccharides to microspheres, the method comprising:
a. providing at least one microsphere;
b. providing at least one saccharide; and
c. mixing the at least one microsphere with the at least one saccharide to form a saccharide coupled microsphere;
wherein the saccharide has a size in the range of 0.01 kDa to 3000 kDa,
optionally wherein the at least one microsphere includes at least one activated microsphere and
optionally wherein the coupling of saccharides to microsphere is non-covalent electrostatic coupling.

In an embodiment, the method includes providing at least one or more microsphere.

In an embodiment, the method includes providing at least one or more saccharide.

In an embodiment, the method includes mixing the at least one or more microspheres with the at least one or more saccharides to form a saccharide coupled microsphere.
In an embodiment of the present invention, the method comprises
mixing is performed with the coupling ratio of the microsphere to the saccharide is in range of 50 to 12500, and/or
optionally, the microsphere is activated with a bead reagent before mixing with the saccharide, wherein the bead reagent includes metal ions, and
optionally, the saccharide coupled microsphere is formed by non-covalent electrostatic coupling, .and/or
optionally, the saccharide remains intact, retains epitope confirmation and the saccharide coupled microsphere is stable.

PROVIDING MICROSPHERE

In an embodiment, the method of coupling saccharides to microspheres includes step of providing at least one microsphere.
MICROSPHERES
Microspheres, microparticles, microcapsules, polymer particles and beads, referred to herein collectively as “microspheres”, are solid or semi-solid particles. In an embodiment, the microsphere has a diameter of less than one millimetre, more preferably less than 100 microns, which can be formed of a variety of materials, including synthetic polymers, proteins, and polysaccharides. Microspheres are used for separations, diagnostics, and drug delivery. Microspheres for separations techniques are formed of polymers of synthetic or protein origin, such as polyacrylamide, hydroxyapatite or agarose. Polymeric microspheres are used to separate molecules including proteins based on molecular weight and/or ionic charge or by interaction with molecules chemically coupled to the microparticles. In the diagnostic area, microspheres are frequently used to immobilize an enzyme, substrate for an enzyme, or labelled antibody, which is then interacted with a molecule to be detected, either directly or indirectly. In the controlled drug delivery area, molecules are encapsulated within microparticles or incorporated into a monolithic matrix for subsequent release.

Microspheres have been commercially available as a tool for biochemists for many years. For example, antibodies conjugated to beads create relatively large particles specific for particular ligands. The large antibody-coated particles are routinely used to crosslink receptors on the surface of a cell for cellular activation, are bound to a solid phase for immunoaffinity purification, and may be used to deliver a therapeutic agent that is slowly released over time, using tissue or tumour-specific antibodies conjugated to the particles to target the agent to the desired site.

A number of different techniques are routinely used to make these microspheres from synthetic polymers, natural polymers, proteins and polysaccharides, including phase separation, solvent evaporation, emulsification, and spray drying. Examples of suitable polymers for the formation of microspheres include polystyrenes, polyesters, polyethers, polyolefins, polyalkylene oxides, polyamides, polyacrylates, polymethacrylates, polyurethanes, celluloses, polyisoprenes, silica, and polysaccharides, particularly cross-linked polysaccharides, such agarose, which is available as Sepharose, dextran, available as Sephadex and Sephacryl, cellulose, starch, and the like. Exemplary polymers used are addition polymers, such as polystyrene, polyvinyl alcohol, homopolymers and copolymers of derivatives of acrylate and methacrylate, particularly esters and amides having free hydroxyl functionalities. However, the availability and cost of these other polymeric particles make the use of polystyrene particles preferred. Other considerations which favour polystyrene are uniformity in the size and shape of the particles which are to be conjugated. The size of the polymer particles ranges from about 0.1 to about 100.0 µm. The preferred particle size is in the range of about 0.5 to 20.0 µm.

Other polymers used for the formation of microspheres include (a) homopolymers and copolymers of lactic acid and glycolic acid (PLGA) as described in U.S. Pat. No. 5,213,812 to Ruiz; U.S. Pat. No. 5, 417,986 to Reid et al.; U.S. Pat. No. 4,530,840 to Tice et al.; U.S. Pat. No. 4,897,268 to Tice et al.; U.S. Pat. No. 5,075,109 to Tice et al.; U.S. Pat. No. 5,102,872 to Singh et al.; U.S. Pat. No. 5,384,133 to Boyes et al.; U.S. Pat. No. 5,360,610 to Tice et al.; and European Patent Application Publication Number 248,531 to Southern Research Institute; (b) block copolymers such as tetronic 908 and poloxamer 407 as described in U.S. Pat. No. 4,904,479 to Illum; and (c) polyphosphazenes as described in U.S. Pat. No. 5,149,543 to Cohen et al.

Microspheres may be of a latex type. The term “latex,” as used herein, pertains to a stable colloidal dispersion of a polymeric substance in an aqueous medium. “Latex” is intended to mean an emulsion consisting substantially of latex mixed with water as a medium, but may also include additional ingredients such as bulking agents, fixing agents, adhesives, dyes and plasticizers, such latex compounds requiring heating to remove moisture and ensure effective adhesion. Also considered within the scope of the present invention are embodiments wherein the dispersion medium comprises an organic solvent. The dispersed particles preferably have an average particle size of about 0.1-100 µm, more preferably about 0.5-20 µm. The particle size distribution of the dispersed particles is not particularly limited, and the particles may have either wide particle size distribution or monodispersed particle size distribution. The polymer latex used in the present invention may be latex of the so-called core/shell type other than ordinary polymer latex having a uniform structure. In this case, use of different glass transition temperatures of core and shell may be preferred.

The naturally occurring or synthetic latex polymers are preferably derived from one or more unsaturated monomers which are capable of polymerizing in an aqueous environment. Particularly preferred are the use of any of the following monomers: (meth)acrylic based acids and esters, acrylonitrile, styrene, divinylbenzene, vinyl esters including but not limited to vinyl acetate, acrylamide, methacrylamide, vinylidene chloride, butadiene and vinyl chloride. The polymers that are produced may take the form of homopolymers (i.e., only one type of monomer selected) or copolymers (i.e., mixtures of two or more types of monomer are selected; this specifically includes terpolymers and polymers derived from four or more monomers). In one form, the copolymer could be a random, a block, or an alternating copolymer. Crosslinking is useful in many polymers for imparting structural integrity and rigidity to the microparticle.
Latex microspheres can be based on a range of synthetic polymers, such as polystyrene, polyvinyltoluene, polystyrene-acrylic acid, polyacrolein, and poly(meth)acrylate esters and their copolymers. The monomers used are normally water-insoluble, and are emulsified in aqueous surfactant so that monomer droplets and/or micelles are formed, which are then induced to polymerize by the addition of initiator to the emulsion. Substantially spherical monodisperse polymer particles are produced. By controlling the conditions, a variety of size ranges can be provided.

Microspheres for use in conjugates and methods of coupling are commercially available. Microspheres include xMAP™ from Luminex Corporation (Austin, Tex.). xMAP™ microspheres are 5.6 µm in diameter and composed of polystyrene, divinylbenzene and methacrylic acid, which provides surface carboxylate functionality for covalent attachment of Saccharides and biomolecules. The microspheres include microsphere dyed with red- and/or infrared-emitting fluorochromes. By proportioning the concentrations of each fluorochrome, spectrally addressable microsphere sets are obtained. The microsphere sets are mixable, and are analysed using the Luminex100™ instrument (Luminex). Each set are identified and classified by a distinct fluorescence signature pattern.

When particles are used as the solid phase, one means of separating bound from unbound species is to use particles that are made of or that include a magnetically responsive material. Such a material is one that responds to a magnetic field. Magnetically responsive materials include paramagnetic materials, ferromagnetic materials, ferrimagnetic materials, and metamagnetic materials. In an embodiment, the metals include Nickel, Iron, Copper, Magnesium, Chromium, Zinc, cobalt, as well as metal oxides such as Fe3O4, BaFe12O19, CoO, NiO, Mn2O3, Cr2O3, CoFe2O4, and CoMnP. The magnetically responsive material may constitute the entire particle. In an embodiment, the magnetically responsive material is one component of the particle, the remainder being a polymeric material to which the magnetically responsive material is affixed.

In an embodiment, the microsphere surface is activated using metal solutions of Nickel, wherein the suitable metal ions are introduced on the bead surface.

In an embodiment, the metals are used in the metal induced reaction.

The magnetically responsive materials are used and quantity of material is not critical and can vary over a wide range. The quantity can affect the density of the particle, however, and both the quantity and the particle size can affect the ease of maintaining the particle in suspension.

In an alternate embodiment, the concentration of magnetically responsive material is low enough to facilitate estimations/ testing/ assays. The magnetically responsive material in a particle is in range from 0.1% to 75.0% by weight of the particle as a whole, or in range of 2.0% to 50.0%, or in range of 3.0% to 25.0%, or in range of 5.0% to 15.0%. In another embodiment, the magnetically responsive material can be dispersed throughout the polymer, applied as a coating on the polymer surface or as one of two or more coatings on the surface, or incorporated or affixed in any other manner that secures the material in the polymer matrix.

In an embodiment, the microsphere for conjugation to a saccharide is a polymer selected from the group consisting of a polystyrene, a polyester, a polyether, a polyolefin, a polyalkylene oxide, a polyamide, a polyacrylate, a polymethacrylate and a polyurethane, or a mixture thereof. In a preferred embodiment, the microsphere is a polystyrene.

In an alternate embodiment, the microsphere contains carboxyl groups and includes both magnetic microspheres and non-magnetic microspheres.

In yet another alternate embodiment, the microsphere is coupled to a linker molecule prior to reacting the microsphere with the activated polysaccharide. In another embodiment, the linker compound is selected from the group consisting of a,?-diaminoalkane, adipic acid dihydrazide and a,?-alkanedihydrazide.

In an embodiment the microsphere includes specific concentration of internal dyes that correlate to a specific bead region. Internal dyes differ between regions, the outer coating of carboxyl groups is same across all bead regions.

In a preferred embodiment, the bead includes optically detectable beads, fluorescence beads. xMAP beads. The beads include magnetic beads and non-magnetic beads.
In a preferred embodiment, the microsphere is a magnetic microsphere.

The magnetic microspheres have mean microsphere diameter in range of 1 µm to 100 µm, or in range of 1 µm to 80 µm, or in range of 1 µm to 60 µm or in range of 1 µm to 40 µm or in range of 1 µm to 20 µm. In a more preferred embodiment the microsphere has mean microsphere diameter in range of 2 µm to 15 µm.

In another more preferred embodiment, the microsphere has mean microsphere diameter in range of 2 µm to 10 µm. In a more preferred embodiment, the microsphere is a magnetic bead microsphere that are fluorescently dyed magnetic microspheres. The magnetic bead microsphere act as both identifier and solid surface to build assay.

In another preferred embodiment, the microsphere is a non-magnetic microsphere. In a more preferred embodiment, the microsphere is a polystyrene microsphere.

The non-magnetic microspheres have a mean microsphere diameter are in range of 1 µm to 100 µm, or in range of 1 µm to 80 µm, or in range of 1 µm to 60 µm or in range of 1 µm to 40 µm or in range of 1 µm to 20 µm. In a more preferred embodiment the microsphere has mean microsphere diameter in range of 2 µm to 15 µm.

In another more preferred embodiment, the microsphere has mean microsphere diameter in range of 2 µm to 10 µm. In a more preferred embodiment, the microsphere is a non-magnetic microsphere that is internally labelled with fluorescent dyes and contains surface carboxyl groups.

In an embodiment, the microspheres are carboxylated microparticles (“beads”) that are color-coded into 100 spectrally distinct sets, or “regions.” Each of these bead regions are distinguishable by an xMAP® instrument that performs interrogation of up to 100 different analytes simultaneously from a single sample.

In a preferred embodiment, the at least one microsphere provided is an activated microsphere.
In an embodiment, the blank beads/ microspheres are provided. The bead/ microsphere surface is activated using metals.

In a more preferred embodiment, the at least one microsphere includes an activated magnetic microsphere.

In another embodiment, the bead/ microsphere surface is activated using buffers.

BEAD REAGENT

In an embodiment, the microspheres/ beads are activated using bead reagent (hereinafter interchangeably referred to as the activation buffer).

In an embodiment, the method of coupling the microspheres with the saccharide includes providing at least one microsphere, wherein the at least one microsphere is activated.

The bead reagent activates bead surfaces forming a co-ordination bond with metal ions. The bead reagent facilitates metal ions to bind the microsphere. The role of bead reagent is to activate the blank beads under an incubation period. The activated beads are used for the coupling of saccharides.

In an embodiment the bead reagent includes solutions with at least one divalent cation, trivalent cation, or tetravalent cations including metals or their metal oxides. In another embodiment, the bead reagents include at least one divalent cation. In another embodiment, the bead reagents include gold, silver, nickel, iron, copper, magnesium, chromium, zinc, cobalt, Cd2+, Co2+, Ni2+, Pb2+, Zn2+, Ca2+, Cr2+, as well as metal oxides such as Fe3O4, BaFe12O19, CoO, NiO, Mn2O3, Cr2O3, CoFe2O4, and CoMnP.

In a preferred embodiment, the metal ions include nickel ions, chromium ions, and gold ions.

In a more preferred embodiment, the metal ions in the bead reagent are chromium ions.

In another more preferred embodiment, the at least one microsphere is activated using the buffer reagent/ the activation reagent. The buffer reagent includes Chromium perchlorate, 2-(N-morpholino) ethnesulphonic acid, sodium chloride, polypropylene/ polyethlylene glycol copolymer, isothiazolinones, and water.

In an embodiment, the bead reagent includes Anteotech regent A-CMPARA1. The microsphere is activated with the bead reagent.

In an embodiment, the at least one microsphere is activated using the bead reagent or the activation buffer for time in range of 30 mins to 90 mins.

In a preferred embodiment, the at least one microsphere is activated using the bead reagent for time in range of 30 mins to 80 mins, or in range of 30 mins to 70 mins.

In another preferred embodiment, the at least one microsphere is activated using the bead reagent for time in range of 40 mins to 70 mins, in range of 50 mins to 70 mins.

In a more preferred embodiment, the at least one microsphere is activated using the bead reagent for 60 mins.

In a preferred embodiment, the at least one microsphere provided is an activated microsphere activated using the bead reagent or the activation reagent.

In a more preferred embodiment, the at least one microsphere includes an activated magnetic microsphere activated using the bead reagent or the activation reagent.

PROVIDING SACCHARIDES

In an embodiment, the method includes providing at least one saccharide.

Saccharide herein after is interchangeably referred to as oligosaccharide, polysaccharides, short chained saccharides, capsular saccharides.
In an embodiment, the present invention is directed to the method of coupling saccharides to microspheres, wherein the method includes step of providing at least one saccharide.

In another embodiment, this invention provides a method for coupling a saccharide to a microsphere or a biomolecule.

The saccharide includes immunogenic and/or antigenic polysaccharides. The method for coupling a saccharide to a microsphere or a biomolecule does not change the immunogenicity and/or the antigenicity of the polysaccharide.
In another embodiment, the saccharide includes capsular polysaccharides, polysaccharides/ saccharides derived from lipopolysaccharides (LPS) and lipooligosaccharides (LOS) of Gram-negative bacteria cell-wall, such as the O-specific side chain, and also fungal cell-wall polysaccharides.

Polysaccharides are composed of repeat units. For use in conjugates of the invention, in certain embodiments a saccharide comprises at least about 4 repeat units preferably up to about 3,000. Thus, the number-average degree of polymerization (the average number of glycose rings contained in one molecule) of the saccharide is at least about 4, with no particular upper limit, though it is preferably at most about 3,000. Especially for use as in an immunoassay, the number-average degree of polymerization of a saccharide is between 4 to 1,000, and particularly between 4 to 700, and more particularly between 50 to 200.

In an embodiment, the saccharide has no repeat units.

In another embodiment the saccharide has repeat units. A repeat unit is characteristic of a given saccharide and thus the composition and molecular weight of the repeat unit greatly vary from one polysaccharide/ saccharide to another. For example, while the repeat unit of most capsular polysaccharides/ saccharides contains hydroxyl, carboxyl, and/or phosphoryl groups, some polysaccharides/ saccharides also contain amino groups (e.g. Streptococcus pneumoniae serotype 1), whereas others do not (e.g. Streptococcus pneumoniae serotype 14) and some contain N-acetyls (e.g. Streptococcus pneumoniae serotype 14), whereas others do not (e.g. Streptococcus pneumoniae serotype 6B). Also, as a matter of example, the molecular weight of capsular polysaccharides of Streptococcus pneumoniae serotypes 3 and 4 is 360 and 847, respectively. Thus, there is no general correspondence between the number of repeat units and the molecular weight of the polysaccharide that may be globally applied, irrespective of the polysaccharide composition. In an embodiment, the molecular weight saccharide is in the average range of 1,000 to 5,500,000 Daltons. The molecular weight of a saccharide is expressed as a mean value, since a polysaccharide/ saccharide is constituted by a population of molecules of heterogeneous size.

Polysaccharides may be either chemically synthesized, purified from a natural source according to conventional methods, or natural PSs can be further chemically modified. For example, in the case of bacterial or fungal polysaccharides, these latter may be extracted from the microorganisms and treated to remove the toxic moieties, if necessary. A particularly useful method is described by Gotschlich et al., J. Exp. Med., 129: 1349 (1969).

Polysaccharides may be used as synthesized or purified. They may be also depolymerized prior to use. Indeed, native capsular polysaccharides usually have a molecular weight greater than 10,000 Daltons. When it is preferred to use capsular polysaccharides of lower molecular weight, e.g. 10,000 to 20,000 Daltons on average, polysaccharides as purified may be submitted to fragmentation. To this end, conventional methods are available. For example, WO 93/07178 describes a fragmentation method using an oxidation-reduction depolymerization reaction.

The term “polysaccharide” as used herein is meant to include compounds made up of many hundreds or even thousands of monosaccharide units per molecule. These units are held together by glycosidic linkages. Their molecular weights are normally greater than about 5,000 and can range up to millions of Daltons. They are normally naturally-occurring, such as, for example, starch, glycogen, cellulose, gum arabic, agar, and chitin. The polysaccharide should have one or more reactive functional groups, such as hydroxyl, carboxyl, amino, phosphoryl, etc. The polysaccharide may be straight or branched chain.

The hydroxyl, carboxyl, phosphoryl, or amino groups of the polysaccharide that are involved in the linkage may be native functional groups. Alternatively, they may have been introduced artificially by chemical modification. Amino groups may be generated by controlled acidic or basic hydrolysis of native N-acyl groups such as N-acetyl groups. Hydrazide groups may be introduced by coupling the polymer with a linker, such as, e.g., adipic acid dihydrazide using conventional EDC-mediated coupling chemistry or other suitable means.
Polysaccharides that can be covalently linked according to methods described herein include starch-like and cellulosic material, but the present method is especially suitable for conjugating microbial polysaccharides that are haptens or immunogens. It is noted that the term “polysaccharides” as used herein comprises sugar-containing polymers and oligomers, whether they only contain glycosidic linkages or also phosphodiester or other linkages. They may also contain non-sugar moieties such as acid groups, phosphoryl groups, amino groups, hydroxyls and amino acids, and are optionally depolymerized.

Bacterial polysaccharides are described in details in Lennart Kenne and Bengt Lindberg, “Bacterial polysaccharides” in The polysaccharides, Vol. 2, Ed. G. O. Aspinall, 1983, Academic Press, pp. 287-363.

In another embodiment, the saccharide is a bacterial polysaccharide. In another embodiment, the bacterial polysaccharide is isolated from bacteria selected from the group consisting of Streptococcus spp., Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae (pneumococcus, (1, 2, 3, 4, 5, 6, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 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, 33A, 33C, 33D, 33E, 33F, 33B, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, and 48), Streptococcus viridans, Salmonella spp., Salmonella typhi, Salmonella Paratyphi, Salmonella enteritidis, Salmonella Typhimurium, Neisseria meningitidis/ meningococcus (serotypes such as A, B, B16, B6, C, D, E29, H, I, K, K454 L, M, W135, X, Y, and Z etc), Neisseria gonorrhoeae, Shigella spp., Haemophilus bacteria, Haemophilus influenzae bacteria (type a, b, c, or d), Haemophilus influenzae type b bacteria (Hib), Helicobacter pylori, Nontyphoidal salmonella, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus spp., Staphylococcus aureus, Enterococcus faecalis, Bacillus anthracis, Vibrio cholera, Shigella spp (Shigella sonnei, Shigella flexneri, Shigella dysenteriae; Shigella boydii) Klebsiella pneumoniae, Pasteurella pestis, Pseudomonas aeruginosa, Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Hemophilus influenzae, Escherichia coli, Erlichia spp., and Rickettsia spp and from fungi such as Candida albicans, Candida kefyr, Cryptococcus neoformans, Hansenula anomala, and Hansenula arabitolgens.
In an embodiment, the saccharide is bacterial saccharide including Group A Streptococcus, Group B Streptococcus, Streptococcus pneumoniae/ pneumococcus, Haemophilus bacteria, Haemophilus influenzae bacteria, Haemophilus influenzae type b bacteria (Hib), Salmonella, Typhoidal salmonella, Non-typhoidal salmonella, Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi, Streptococcus pyogenes, Streptococcus agalactiae, Shigella, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Escherichia coli, or Neisseria meningitidis/ meningococcus saccharide.

The polysaccharide/ saccharide is associated with bacteria, Streptococcus, Streptococcus pneumoniae/ pneumococcus, Haemophilus bacteria, Haemophilus influenzae bacteria, Haemophilus influenzae type b bacteria (Hib), and Neisseria meningitidis/ meningococcus.

In an embodiment, the saccharide is native or is modified. The modified saccharide includes size reduced saccharides. In an embodiment, the saccharide from stock is diluted with the buffer at a particular pH range corresponding to the saccharide and condition required for conjugation to the microsphere to obtain optimal MFI’s to positive samples.

In an embodiment, the saccharide is a native saccharide.

In another embodiment, the saccharide is a size reduced saccharide.

In another embodiment, the saccharide has a size in the range of 0.01 kDa to 3000 kDa.

In a preferred embodiment, the saccharide has a size in the range of 0.05 kDa to 2800 kDa, or 0.10 kDa to 2800 kDa, or 0.5 kDa to 2800 kDa, or 1.0 kDa to 2800 kDa, or 2.0 kDa to 2800 kDa, or 5.0 kDa to 2800 kDa, or 10.0 kDa to 2800 kDa, or 15.0 kDa to 2800 kDa, or 20.0 kDa to 2800 kDa, or 25.0 kDa to 2800 kDa, or 30.0 kDa to 2800 kDa, or 35.0 kDa to 2800 kDa, or 40.0 kDa to 2800 kDa, or 45.0 kDa to 2800 kDa, or 50.0 kDa to 2800 kDa.

In another preferred embodiment, the saccharide has a size in the range of 50.0 kDa to 2800 kDa, or 50.0 kDa to 2700 kDa, or 50.0 kDa to 2600 kDa, or 50.0 kDa to 2500 kDa, or 50.0 kDa to 2400 kDa, or 50.0 kDa to 2300 kDa, or 50.0 kDa to 2200 kDa, or 50.0 kDa to 2100 kDa, or 50.0 kDa to 2000 kDa, or 50.0 kDa to 1900 kDa, or 50.0 kDa to 1800 kDa, or 50.0 kDa to 1900 kDa, or 50.0 kDa to 1800 kDa, or 50.0 kDa to 1700 kDa, or 50.0 kDa to 1600 kDa, or 50.0 kDa to 1500 kDa, or 50.0 kDa to 1400 kDa, or 50.0 kDa to 1400 kDa, or 50.0 kDa to 1300 kDa, or 50.0 kDa to 1250 kDa.

In a more preferred embodiment, the saccharide has a size in the range of 50.0 kDa to 1200 kDa.

In a further more preferred embodiment, the saccharide has a size in the range of 50.0 kDa to 1150 kDa.
In a further more preferred alternate embodiment, the saccharide has a size in the range of 50.0 kDa to 1100 kDa.

In a further more preferred alternate embodiment, the saccharide has a size in the range of 50.0 kDa to 1000 kDa.
In a further more preferred alternate embodiment, the saccharide has a size in the range of 50.0 kDa to 800 kDa.

In a further more preferred alternate embodiment, the saccharide has a size in the range of 50.0 kDa to 600 kDa.

DILUTION OF SACCHARIDE

In an embodiment, the stock of native saccharide is diluted with a buffer prior to coupling.

BUFFER
In an embodiment, the saccharide is diluted with the buffer. The buffer acts as microsphere - polysaccharide coupling component at a pH and ion strength to enable efficient immobilization of polysaccharide to activated magnetic beads/ microspheres.

In another embodiment, the present invention is directed to the method of coupling saccharides to microspheres, wherein the buffer includes phosphate-buffered saline with Tween 20 (PBST) buffer, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, Tris-aminomethane (Tris) Buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, and 3-(N-morpholino)propanesulfonic acid (MOPS) buffer.

In another embodiment, the buffer used is phosphate buffer.

In a preferred embodiment, the buffer used is PBST buffer.

PH

In an embodiment, the present invention is directed to the method of coupling saccharides to microspheres, where the saccharide is diluted with a buffer at pH in range of 3.0 to 9.0.
In a preferred embodiment, the mixing is performed at pH in the range of 3.0 to 8.0, or in range of 3.0 to 7.5.

CONCENTRATION OF SACCHARIDE
In another embodiment, the present invention is directed to the method of coupling saccharides to microspheres, wherein concentration of saccharide is in range of 0.1 mg/mL to 50.0 mg/mL.

In another embodiment, the present invention is directed to the method of coupling saccharides to microspheres, wherein concentration of saccharide is in range of 0.1 mg/mL to 40.0 mg/mL, or in range of 0.1 mg/mL to 30.0 mg/mL, or in range of 0.1 mg/mL to 20.0 mg/mL, or in range of 0.1 mg/mL to 10.0 mg/mL.

In an embodiment, the native saccharide (herein after referred interchangeably as PnPS) is provided in stock concentration range of 5 to 10 mg/ml. Size reduced saccharide is provided in stock concentration range of 11.0 to 15.0 mg/ml.

MIXING

In an embodiment, the method includes mixing the at least one microsphere with the at least one saccharide to form a saccharide coupled microsphere.
In an embodiment, the method of coupling utilises metal induced reaction sites on surface of the bead/ microsphere. The reaction sites create multivalent binding with target molecule/ polysaccharide/ ligand through chelation to the electron donating groups of the ligand/ target molecule/ saccharide to be coupled.

In another embodiment, the method includes mixing the at least one microsphere with the at least one saccharide at a concentration and size.
In an embodiment, the method includes mixing the least one microsphere with the at least one saccharide, wherein the concentration of the polysaccharide is in range of 0.1 to 10.0 mg/mL, or 0.1 to 8.0 mg/mL or 0.1 to 6.0 mg/mL or 0.1 to 4.0 mg/mL.
In an embodiment, the method includes mixing the least one microsphere with the at least one saccharide, wherein the polysaccharide has a size in range of 0.01 kDa to 3000 kDa.
In a preferred embodiment, the method includes mixing the least one microsphere with the at least one saccharide, the polysaccharide has a size in the range of 0.05 kDa to 2800 kDa, or 0.10 kDa to 2800 kDa, or 0.5 kDa to 2800 kDa, or 1.0 kDa to 2800 kDa, or 2.0 kDa to 2800 kDa, or 5.0 kDa to 2800 kDa, or 10.0 kDa to 2800 kDa, or 15.0 kDa to 2800 kDa, or 20.0 kDa to 2800 kDa, or 25.0 kDa to 2800 kDa, or 30.0 kDa to 2800 kDa, or 35.0 kDa to 2800 kDa, or 40.0 kDa to 2800 kDa, or 45.0 kDa to 2800 kDa, or 50.0 kDa to 2800 kDa.

In another embodiment, the method includes mixing the at least one microsphere with the at least one saccharide in a buffer.

In another embodiment, the method includes mixing the at least one microsphere with the at least one saccharide at a pH in range of 3 to 9.

pH is one of the conjugation condition and pH of 4.5 can work for specific conditions related to saccharide-microsphere couples of Streptococcus pneumoniae Serotype Polysaccharides 2, 6A, 8, 23F, and the like.

In another embodiment, the method includes mixing the at least one microsphere and the at least one saccharide with a chemical composition.

In another embodiment, the step of mixing further includes incubation.

In a more preferred embodiment, the at least one microsphere is activated using the bead reagent for 60 mins.

MIXING: COUPLING RATIO
In an embodiment, the microspheres and the saccharides are mixed with each other in a specific coupling ratio.
The specific ratios and assay conditions associated with the saccharide coupled microspheres is provided below.
The coupling ratio is dependent on the requirement of saccharide concentration and blank microsphere/ beads per 100µL. The ratio is calculated as follows:
Coupling ratio = No. of microspheres (Beads) (00 µl) / Concentration of Saccharides (PS) required for coupling (100µl).
The significance of maximum ratio indicates requirement of less concentration of saccharide for coupling. The calculated broadest coupling ratio for bacterial saccharides is in range of 50 to 12500.
In an embodiment, the mixing is performed with the coupling ratio of the microsphere to the saccharide is in range of 50 to 12500.

In an embodiment, the mixing is performed with the coupling ratio of the microsphere to the pneumococcal saccharides is in range of 50 to 500. In a preferred embodiment, the mixing is performed with the coupling ratio of the microsphere to the pneumococcal saccharides is in range of 50 to 250.

In an embodiment, the mixing is performed with the coupling ratio of the microsphere to the Hib saccharide is in range of 5000 to 12500. In a preferred embodiment, the mixing is performed with the coupling ratio of the microsphere to the Hib saccharide is in range of 8000 to 12500

In an embodiment, the mixing is performed with the coupling ratio of the microsphere to the Neisseria meningitis saccharide is in range of 50 to 12500.

MIXING: INCUBATION:
In an embodiment, the mixing further includes incubation.

In a preferred embodiment, the step of mixing further includes incubation at a temperature of 20? to 40?.

In a preferred embodiment, the step of mixing further includes incubation at a temperature of 23? to 39?.
In another embodiment, the present invention is directed to the method of coupling saccharides to microspheres, wherein the mixing is done for incubation temperature in range of 23? to 39?.
In another embodiment, the present invention is directed to the method of coupling saccharides to microspheres, wherein the mixing is done for incubation time of 30 mins to 180 mins.
In an embodiment, the step of mixing further includes incubation time in range of 30 mins to 180 mins. In a preferred embodiment, the incubation time is in range of 30 mins to 150 mins, or in range of 30 mins to 120 mins, or in range of 30 mins to 90 mins. In another preferred embodiment, the incubation time is in 30 mins to 70 mins.

In an embodiment, the method includes mixing the at least one microsphere with the at least one saccharide with the buffer and at the pH in range of 3.0 to 9.0. In an embodiment, the pH of mixing the saccharide with the microsphere is same as the dilution pH of the saccharide.

In an embodiment, the method includes mixing the at least one microsphere with the at least one saccharide with the buffer, at the pH in range of 3 to 9, and incubation.

In an embodiment, the method includes mixing the at least one microsphere with the at least one saccharide with the buffer, at the pH in range of 3 to 9, and incubation at a temperature of 23? to 39?.

In an embodiment, the method includes mixing the at least one microsphere with the at least one saccharide with the buffer, at the pH in range of 3 to 9, and incubation at a temperature of 23? to 39? for 30 mins to 180 mins.

In a preferred embodiment, method includes mixing the saccharide and microsphere to form saccharide coupled microsphere wherein the saccharide is provided at concentration of 0.1 mg/mL to 10 mg/mL, at a size in range of 0.01 kDa to 3000 kDa, at a pH in range of 3.0 to 9.0, in the buffer for incubation time of 30 mins to 180 mins and at incubation temperature in range of 23oC to 39oC to allow development of sites which allow electrostatic attachment of saccharide on to activated beads/ microsphere.

CHEMICAL COMPOSITION

In another embodiment, the method includes mixing the at least one microsphere and the at least one saccharide having a chemical composition.

The chemical composition includes at least one component from nitrogen, phosphorus, uronic acids, o-acetyl, methyl-pentoses, hexosamines, or a combination thereof.
In a preferred embodiment, the method includes mixing the at least one microsphere and the at least one saccharide having the chemical composition, wherein the chemical composition includes nitrogen and phosphorus.

In another preferred embodiment, the method includes mixing the at least one microsphere and the at least one saccharide having the chemical composition, wherein the chemical composition includes nitrogen content in range of 0.01 mg/ ml to 10 mg/ ml. The nitrogen content is present as 0-6 (%).

In another preferred embodiment, the method includes mixing the at least one microsphere and the at least one saccharide having the chemical composition, wherein the chemical composition includes phosphorus in range of 0.01 mg/ ml to 10 mg/ ml. The phosphorus is present in form of 0-7 (%).

In another preferred embodiment, the method includes mixing the at least one microsphere and the at least one saccharide having the chemical composition, wherein the chemical composition includes nitrogen and phosphorus in the ratio of 1: 100 to 100: 1.

In another preferred embodiment, the method includes mixing the at least one microsphere and the at least one saccharide having the chemical composition, wherein the chemical composition includes nitrogen, phosphorus and at least one component selected from methyl pentose, hexosamines, uronic acids, O-acetyl.

In another preferred embodiment, the method includes mixing the at least one microsphere and the at least one saccharide having the chemical composition, wherein the chemical composition includes nitrogen, phosphorus and methyl pentose.

In another preferred embodiment, the method includes mixing the at least one microsphere and the at least one saccharide having the chemical composition, wherein the chemical composition includes nitrogen, phosphorus and hexosamines.

In another preferred embodiment, the method includes mixing the at least one microsphere and the at least one saccharide having the chemical composition, wherein the chemical composition includes nitrogen, phosphorus and O-acetyl.
In another preferred embodiment, the method includes mixing the at least one microsphere and the at least one saccharide having the chemical composition, wherein the chemical composition includes nitrogen, phosphorus and uronic acid.

In an embodiment, the method of coupling saccharides to microspheres, the method comprising:
a. providing the microsphere;
b. providing the saccharide;
c. diluting the saccharide with the buffer at pH in range of 3.0 to 9.0; wherein and the saccharide is in range of 0.1 mg/ml to 50.0 mg/ml
d. mixing the microsphere with the saccharide to form the saccharide coupled microsphere;
wherein the saccharide has a size in the range of 50.0 kDa to 3000 kDa; and
wherein the mixing further includes incubation at incubation temperature in range of 20? to 40? for incubation time in range of 30 mins to 180 mins.

In another embodiment, the method of coupling saccharides to microspheres, the method comprising:
a. providing the microsphere;
b. providing the saccharide;
c. diluting the saccharide with the PBST buffer at pH in range of 3.0 to 6.0; wherein and the saccharide is in range of 0.1 mg/ml to 10.0 mg/ml
d. mixing the microsphere with the saccharide to form the saccharide coupled microsphere;
wherein the saccharide has a size in the range of 50.0 kDa to 3000 kDa; and
wherein the mixing further includes incubation at incubation temperature in range of 23? to 39? for incubation time in range of 60 mins to 120 mins.

In another embodiment, the method of coupling saccharides to microspheres, the method comprising:
a. providing the microsphere;
b. providing the saccharide;
c. diluting the saccharide with the PBST buffer at pH in range of 3.0 to 6.0; wherein and the saccharide is in range of 0.1 mg/ml to 10.0 mg/ml
d. mixing the microsphere with the saccharide to form the saccharide coupled microsphere;
wherein the saccharide is a size reduced saccharide and has a size in the range of 50.0 kDa to 3000 kDa; and
wherein the mixing further includes incubation at incubation temperature in range of 23? to 39? for incubation time in range of 60 mins to 120 mins.

In an embodiment, the method of coupling the saccharide to the microsphere, the method comprising:
a. providing the microsphere;
b. providing the saccharide;
c. diluting the saccharide with the buffer at pH in range of 3.0 to 9.0;
d. mixing the microsphere with the saccharide to form the saccharide coupled microsphere;
wherein the mixing further includes incubation at temperature in range of 20? to 40? for incubation time in range of 30 mins to 180 mins.
wherein Streptococcus pneumoniae polysaccharide is selected from serotypes 1, 2, 3, 4, 5, 6, 6A, 6B, 6C, 6D, 7, 7B, 7C, 7F, 8, 9, 9A, 9L, 9N, 9V, 10, 10A, 10B, 10C, 10F, 10X, 11, 11A, 11B, 11C, 11F, 12, 12A, 12B, 12F, 13, 14, 15, 15A, 15B, 15BC, 15C, 15F, 16, 16F, 17, 17A, 17F, 18, 18A, 18B, 18C, 18F, 19, 19A, 19B, 19F, 20, 20A, 20B, 20F, 21, 22, 22A, 22F, 23, 23A, 23B, 23F, 24, 24F, 25, 25F, 26, 27, 28, 28A, 28F, 29, 30, 31, 32, 33, 33A, 33B, 33C, 33D, 33F, 34, 35, 35A, 35B, 35C, 35D, 35F, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, and/ or 48, and
wherein the Streptococcus pneumoniae polysaccharide has a molecular size in range of 50 kDa to 3000 kDa, is diluted with PBST buffer at a pH in range of 3.0 to 9.0 and has concentration of 0.05 mg/mL to 50.0 mg/mL,
wherein the Streptococcus pneumoniae polysaccharide is mixed with the microsphere and incubated at temperature of 23? to 39?, for incubation time of 60 mins to 120 mins,
wherein
the mixing is performed with the coupling ratio of the microsphere to the saccharide is in range of 50 to 500, and/ or
optionally, the microsphere is activated with a bead reagent before mixing with the saccharide, wherein the bead reagent includes metal ions,
optionally, the saccharide coupled microsphere is formed by non-covalent electrostatic coupling, and/ or
optionally, the saccharide remains intact, retains epitope confirmation and the saccharide coupled microsphere is stable.
In another embodiment, the method of coupling the saccharide to the microsphere, the method comprising:
a. providing the microsphere;
b. providing the saccharide;
c. diluting the saccharide with the buffer at pH in range of 3.0 to 9.0;
d. mixing the microsphere with the saccharide to form the saccharide coupled microsphere;
wherein the mixing further includes incubation at temperature in range of 20? to 40? for incubation time in range of 30 mins to 180 mins,
wherein the saccharide is Haemophilus influenzae type b bacteria (Hib) polysaccharide,
wherein the Haemophilus influenzae type b bacteria (Hib) polysaccharide has a molecular size in range of 0.1 kDa to 5 kDa, is diluted with PBST buffer at a pH in range of 4 to 6 and has concentration of 1.0 µg/mL to 50.0 µg/mL,
wherein the Haemophilus influenzae type b bacteria (Hib) polysaccharide is mixed with the microsphere and incubated at temperature of 20? to 30?, for incubation time of 60 mins to 120 mins,
wherein
the mixing is performed with the coupling ratio of the microsphere to the saccharide is in range of 5000 to 12500, and/ or
optionally, the microsphere is activated with a bead reagent before mixing with the saccharide, wherein the bead reagent includes metal ions,
optionally, the saccharide coupled microsphere is formed by non-covalent electrostatic coupling, and/ or
optionally, the saccharide remains intact, retains epitope confirmation and the saccharide coupled microsphere is stable.

In another embodiment, the method of coupling the saccharide to the microsphere, the method comprising:
a. providing the microsphere;
b. providing the saccharide;
c. diluting the saccharide with the buffer at pH in range of 3.0 to 9.0;
d. mixing the microsphere with the saccharide to form the saccharide coupled microsphere;
wherein the mixing further includes incubation at temperature in range of 20? to 40? for incubation time in range of 30 mins to 180 mins,
wherein the polysaccharide is Neisseria meningitidis polysaccharide serotypes selected from meningococcal serotypes A (type I and III), B (type II), B6, B16, C (type II-alpha), D (type IV), Z’/ E, E29, H, I, K, K454, L, M, W135, X, Y, Z, and
wherein the Neisseria meningitidis polysaccharide has a molecular size in range of 75 kDa to 3000 kDa, is diluted with PBST buffer at a pH in range of 4 to 7 and has concentration of 0.01 mg/mL to 10.0 mg/mL
wherein the mixing of the Neisseria meningitidis polysaccharide with the microsphere is done for incubation temperature of 20 to 39?, for incubation time of 60 mins to 120 mins,
wherein
the mixing is performed with the coupling ratio of the microsphere to the saccharide is in range of 50 to 12500, and/ or
optionally, the microsphere is activated with a bead reagent before mixing with the saccharide, wherein the bead reagent includes metal ions,
optionally, the saccharide coupled microsphere is formed by non-covalent electrostatic coupling, and/ or
optionally, the saccharide remains intact, retains epitope confirmation and the saccharide coupled microsphere is stable.

SACCHARIDE COUPLED MICROSPHERE

In another aspect, the present invention is directed to the saccharide coupled microsphere obtained by the method as disclosed in herein.

In an embodiment, the saccharide coupled microsphere is obtained by the method as disclosed herein, wherein the saccharide coupled microsphere is associated with optimal/ percentage range of antibody activity/ value corresponding to optimal Mean Fluorescence Intensity/ MFI values. MFI corresponds to average fluorescence intensity of microspheres after the microsphere have been stained with a fluorescently labelled antibody. Fluorescently labelled microspheres are coupled with the saccharides that act as antigens. When an antibody binds to a microsphere, such binding increases fluorescence intensity of the saccharide coupled microsphere. MFI values is thus obtained for the binding between the microspheres/ beads (fluorescent microsphere/ beads coupled with the saccharides) and the antibodies as well as the MFI value obtained determines strength of coupling of the saccharide and the microsphere.
In an embodiment of the present invention, the saccharide coupled microsphere has Mean Florescence intensity/ MFI value in the range of 200 to 20000.

Optimal MFI values are calculated with blank MF less than 100. Optimal MFI value is associated with MFI of 1st standard being 2 to 5 times more than blank, preferably 3 times more than blank. Bead control MFI value is more than at least 5 to 20% of last standard, preferably at least by 10% of last standard. In antigen content assay, for first standard optimal MFI range 200-500 and eight standard optimal MFI range is from 4000 to 20,000.

Optimum antibody dilution range is from 1:5000 to 1:50,000 and optimal antibody concentration ranges from 20 ng to 200 ng required for 4000 beads per well per 50µL solution.

For 4000 beads per 50µL of solution, 0.5% to 5% of antibody activity is required for optimal MFI value.

In antigen content assay, for first standard optimal MFI value ranges from 200 to 500 and eight standard optimal MFI ranges from 4000 to 20,000.

The saccharide coupled microsphere obtained by disclosed method is associated with preservation of epitopes even after coupling. Preservation of the epitopes and other parameters of the saccharide coupled microsphere are determined by back fit reference to standards, duplicate % CV, assay blank, and percentage gradations between the MFIs.

The saccharide coupled microsphere obtained by the method as disclosed herein is associated with improved MFI value. The saccharide coupled microsphere in comparison to beads obtained by conventional methods (for e.g. amine coupling) is associated with better/ improved MFI’s data. The improved MFI data indicates that the saccharide coupled microsphere have higher sensitivities and higher dynamic range.
In an embodiment of the present invention, the saccharide coupled microsphere is a Streptococcus pneumoniae saccharide coupled microsphere.

In an embodiment of the present invention, the saccharide coupled microsphere is a Haemophilus influenzae type b bacteria (Hib) saccharide coupled microsphere.

In an embodiment of the present invention, the saccharide coupled microsphere is a Neisseria meningitidis saccharide coupled microsphere.
The saccharide coupled microsphere obtained by the method as disclosed herein is associated with improved stability of beads at different temperature conditions, improved specificity of assay using inhibition experiments, consistent linearity, accuracy, precision, LOQ parameters.

During qualification stage the system suitability parameters are monitored such as Back fit of the reference standards, duplicate % CV, assay blank and % gradation between the MFI’s. When all the parameters are complying to the corresponding specification indicate that the epitopes are intact, as the assessment method is based on the ‘antigen-antibody reaction’. If epitopes are damaged antibody binding to antigen will be inappropriate and impact on the standard curve formation and duplicate %CV of the assay.
STREPTOCOCCAL POLYSACCHARIDES & COUPLING

In another embodiment, the present invention is directed to the method of coupling saccharides to microspheres, wherein the saccharide is Streptococcus polysaccharide.

In another embodiment, the present invention is directed to the method of coupling saccharides to microspheres, wherein the saccharide from Streptococcus pneumoniae including Group A Streptococcus, Group B Streptococcus.

In another embodiment, the present invention is directed to the method of coupling saccharides to microspheres, wherein the saccharide is Streptococcus pneumoniae polysaccharide.

In another embodiment, the present invention is directed to the method of coupling polysaccharides to microspheres, wherein the polysaccharide is Streptococcus pneumoniae polysaccharide selected from serotypes 1, 2, 3, 4, 5, 6, 6A, 6B, 6C, 6D, 7, 7B, 7C, 7F, 8, 9, 9A, 9L, 9N, 9V, 10, 10A, 10B, 10C, 10F, 10X, 11, 11A, 11B, 11C, 11F, 12, 12A, 12B, 12F, 13, 14, 15, 15A, 15B, 15BC, 15C, 15F, 16, 16F, 17, 17A, 17F, 18, 18A, 18B, 18C, 18F, 19, 19A, 19B, 19F, 20, 20A, 20B, 20F, 21, 22, 22A, 22F, 23, 23A, 23B, 23F, 24, 25, 25F, 26, 27, 28, 28A, 28F, 29, 30, 31, 32, 33, 33A, 33B, 33C, 33D, 33F, 34, 35, 35A, 35B, 35C, 35D, 35F, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, and/ or 48.

In another embodiment, the present invention is directed to the method of coupling polysaccharides to microspheres, wherein the polysaccharide is Streptococcus pneumoniae polysaccharide selected from serotypes 1, 2, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 24F and/ or 33F.

In another embodiment, the present invention is directed to the method of coupling polysaccharides/saccharides to microspheres, wherein the Streptococcus pneumoniae polysaccharide has molecular size in the range of 50 kDa to 3000 kDa, preferably in the range of 50 kDa to 1500 kDa.
In an embodiment, the polysaccharide/ saccharide used as is native polysaccharide/ saccharide.

In a preferred embodiment, the saccharide of serotype 2, 6A, 8 are of native size.

In an alternate embodiment, the saccharide is reduced in size.

In a preferred embodiment, the saccharide of serotype 2, 6A, 8 or all are reduced in size.

In a more preferred embodiment, the saccharide is a modified saccharide. The Streptococcus pneumoniae serotype 2 modified saccharide has size 80kDa and after coupling was observed to be associated with the lesser MFI value compared with the native saccharide (Size 1011 kDa) which gives a good increase in the MFI value.

In an embodiment, the present invention is directed to the method of coupling saccharides to microspheres, wherein concentration of saccharide is in range of 0.05 mg/mL to 50.0 mg/mL.

In an embodiment, the present invention is directed to the method of coupling saccharides to microspheres, wherein concentration of saccharide is in range of 0.05 mg/mL to 10.0 mg/mL.

In a preferred embodiment, the present invention is directed to the method of coupling saccharides to microspheres, wherein concentration of saccharide is in range of 0.05 mg/mL to 8.0 mg/mL, or in range of 0.05 mg/mL to 6.0 mg/mL, or in range of 0.05 mg/mL to 5.0 mg/mL, or in range of 0.05 mg/mL to 4.0 mg/mL.

In another preferred embodiment, the present invention is directed to the method of coupling saccharides to microspheres, wherein concentration of saccharide is in range of 0.1 mg/mL to 4.0 mg/mL, or in range of 0.1 mg/ ml to 3.0 mg/ml, or in range of 0.1 mg/ ml to 2.5 mg/ml.

In an embodiment, the method includes diluting the at least one microsphere with the at least one Streptococcus pneumoniae polysaccharide with the buffer and at the pH in range of 3.0 to 9.0.

In an embodiment, the method includes diluting the at least one microsphere with the at least one Streptococcus pneumoniae polysaccharide with the buffer and at the pH in range of 4.0 to 7.0.

In an embodiment, the method includes diluting the at least one microsphere with the at least one Streptococcus pneumoniae polysaccharide with the buffer, at the pH in range of 3.0 to 9.0, and then mixing.

In an embodiment, the method includes mixing the at least one microsphere with the at least one Streptococcus pneumoniae polysaccharide with incubation at a temperature of 23? to 39?.

In an embodiment, the method includes mixing the at least one microsphere with the at least one Streptococcus pneumoniae polysaccharide with incubation at a temperature of 23? to 39? for incubation time in range of 30 mins to 180 mins.

In an embodiment, the method includes mixing the at least one microsphere with the at least one Streptococcus pneumoniae polysaccharide with incubation at a temperature of 23? to 39? for incubation time in range of 60 mins to 180 mins.

In an embodiment, the method includes mixing the at least one microsphere with the at least one Streptococcus pneumoniae polysaccharide with incubation at a temperature of 25? to 37? for incubation time in range of 60 mins to 180 mins.

In an embodiment, the method includes mixing the at least one microsphere with the at least one Streptococcus pneumoniae polysaccharide with incubation at a temperature of 25? to 37? for incubation time in range of 60 mins to 120 mins.

In a preferred embodiment, the stock of native Streptococcus pneumoniae polysaccharide saccharide is diluted in a buffer prior to coupling.
In another embodiment, the saccharide is Streptococcus pneumoniae polysaccharide/ saccharide selected from a group of polysaccharides associated with Streptococcus pneumoniae/ pneumococcal strains associated with global Streptococcus pneumoniae/ pneumococcal sequence cluster (GPSC) selected from GPSC 1 to 131.
In another embodiment, a composition comprising Streptococcus pneumoniae polysaccharide/ saccharide coupled microsphere is obtained by the method as disclosed herein.

The Streptococcus pneumoniae polysaccharide/ saccharide coupled microsphere obtained by the method as disclosed herein is associated with improved MFI value.

The Streptococcus pneumoniae polysaccharide/ saccharide coupled microsphere in comparison to beads obtained by conventional methods (for e.g. amine coupling) is associated with better/ improved MFI’s data. The improved MFI data indicates that the Streptococcus pneumoniae polysaccharide saccharide coupled microsphere has higher sensitivity and higher dynamic range.

In an embodiment, the Streptococcus pneumoniae polysaccharide/ saccharide coupled microsphere for serotypes 7F, 8, 10A and 24F are associated with improved higher MFI’s than the beads obtained by the conventional methods indicating higher sensitivity.

HEAMOPHILUS SACCHARIDE & COUPLING
In another embodiment, in the method as disclosed herein, the saccharide is associated Haemophilus bacteria, Haemophilus influenzae bacteria, or Haemophilus influenzae type b bacteria (Hib).

In another embodiment, in the method as disclosed herein, the saccharide is Haemophilus influenzae type a, type b, type c, type d, type e, or type f bacteria polysaccharide.

In another embodiment, in the method as disclosed herein, the saccharide is Haemophilus influenzae type b bacteria (Hib) polysaccharide.

In another embodiment, in the method as disclosed herein, the saccharide is Haemophilus influenzae type b bacteria (Hib) saccharide selected from a group of saccharides associated with type I and type II Hib strains.

In another embodiment, in the method as disclosed herein, the Haemophilus influenzae type b bacteria (Hib) saccharide is associated with a molecular size of 0.05 kDa to 3000 kDa.

In another embodiment, in the method as disclosed herein, the Haemophilus influenzae type b bacteria (Hib) saccharide is associated with a molecular size of 0.05 kDa to 5 kDa.

In another embodiment, in the method as disclosed herein, the Haemophilus influenzae type b bacteria (Hib) saccharide is associated with a molecular size of 0.1 kDa to 1 kDa.

In another embodiment, in the method as disclosed herein, the Haemophilus influenzae type b bacteria (Hib) saccharide is associated with a molecular size of 0.3 kDa for more than 50 % of the saccharides.

In an embodiment, the method includes diluting the at least one microsphere with the at least one Hib polysaccharide with the PBST buffer at the pH in range of 3.0 to 9.0.

In an embodiment, the method includes diluting the at least one microsphere with the at least one Hib polysaccharide with the PBST buffer at the pH in range of 4.0 to 6.0.

In another embodiment, in the method as disclosed herein, concentration of Haemophilus influenzae type b bacteria (Hib) saccharide is in range of 1.0 µg/mL to 50.0 µg/mL.
In another embodiment, in the method as disclosed herein, concentration of Haemophilus influenzae type b bacteria (Hib) saccharide is in range of 5 µg/mL to 15 µg/mL.

In another embodiment, in the method as disclosed herein, the saccharide is Haemophilus influenzae type b bacteria (Hib) saccharide selected from a group of saccharide associated with Haemophilus influenzae type b bacteria (Hib) strains associated variation of hcsA gene.

In an embodiment, the method includes mixing the at least one microsphere at incubation temperature of 20? to 30?.

In an embodiment, the method includes mixing the at least one microsphere at incubation temperature of 20? to 30? for 30 mins to 180 mins.
In an embodiment, the method includes mixing the at least one microsphere at incubation temperature of 25? for 60 mins to 120 mins.

In another embodiment, the composition comprising Haemophilus influenzae type b bacteria (Hib) saccharide coupled microsphere is obtained by the method as disclosed herein.

MENINGOCOCCAL SACCHARIDES & COUPLING

In another embodiment, in the method as disclosed herein, the saccharide is associated with Neisseria meningitidis/ meningococcus.

In another embodiment, in the method as disclosed herein, the saccharide is Neisseria meningitidis polysaccharide.

In another embodiment, in the method as disclosed herein, the saccharide is Neisseria meningitidis saccharide selected from meningococcal serotypes A (type I and III), B (type II), B6, B16, C (type II-alpha), D (type IV), Z’/ E, E29, H, I, K, K454, L, M, W135, X, Y, Z.

In another embodiment, in the method as disclosed herein, the saccharide is Neisseria meningitidis saccharide selected from meningococcal serotypes A, B, C, W, X, and Y.

In another embodiment, in the method as disclosed herein, the Neisseria meningitidis saccharide is associated with a size of 75 kDa to 3000 kDa.

In another embodiment, in the method as disclosed herein, concentration of Neisseria meningitidis saccharide is in range of 0.01 mg/mL to 10.0 mg/mL.

In an embodiment, the method includes mixing the at least one microsphere with the at least one Neisseria meningitidis polysaccharide with the buffer and at the pH in range of 3.0 to 9.0.

In an embodiment, the method includes mixing the at least one microsphere with the at least one Neisseria meningitidis polysaccharide with the buffer, at the pH in range of 3.0 to 9.0, and incubation.

In an embodiment, the method includes mixing the at least one microsphere with the at least one Neisseria meningitidis polysaccharide with the buffer, at the pH in range of 3.0 to 9.0, and incubation at a temperature in range of 20 oC to 40 oC, preferably in range of 23 oC to 39oC.
In an embodiment, the method includes mixing the at least one microsphere with the at least one Neisseria meningitidis polysaccharide with the buffer, at the pH in range of 3.0 to 9.0, and incubation at a temperature of 23 oC to 39 oC for 30 mins to 180 mins.

In another embodiment, in the method as disclosed herein, the saccharide is Neisseria meningitidis saccharide selected from a meningococcal strain associated with variation in lpxL1, fHbp, and tps genes.

In an embodiment, the composition comprising Neisseria meningitidis saccharide coupled microsphere is obtained by the method as disclosed herein.

ANOTHER ASPECT: METHOD OF EVALUATING IMMUNOGENICITY

In another aspect, the present invention is directed to the saccharide coupled microspheres as and when used to determine antibody titre of an immunogenic composition/ a vaccine.

In an embodiment, the present invention is directed to a method of evaluating immunogenicity of immunogenic composition, the method comprising;
a. providing a test sample corresponding to the saccharide in the immunogenic composition;
b. providing the saccharide coupled microsphere corresponding to the saccharide in the immunogenic composition; and
c. evaluating the immunogenicity of the immunogenic composition by antigen content determination, or estimating antibody concentration (IgG), or identity assay, free polysaccharide estimation using the test sample and the saccharide coupled microsphere.

ANTIGEN CONTENT DETERMINATION

In another aspect, the present invention is directed to an antigen content determination method using the saccharide coupled microsphere obtained by the method as disclosed herein.

In an embodiment, the antigen content determination method using the saccharide coupled microsphere comprises:
i. desorbing the test sample and the standard,
ii. preparing the percent adsorption sample,
iii. preparing mixture of antisera/ Monoclonal antibody (mAb) for at least one serotype specific saccharide,
iv. diluting standard, test sample and percent adsorption sample,
v. transferring the standard, the sample, and the bead control into respective wells containing mixture of sera/ Mab, incubate the dilution plate,
vi. providing the saccharide coupled microsphere/ bead for at least one serotype specific polysaccharide,
vii. preparing assay plate by adding the coupled beads in each well of the plate, followed by adding the standard and the sample from dilution plate to wells in the assay plate having beads as well as bead blanks, incubating and washing,
Washing and dilution is performed using reagent D. (i.e. Luminex Assay Buffer, LAB),
viii. preparing anti rabbit phycoerythrin and anti-mice phycoerythrin and adding in each well,
ix. adding reagent D to the plate, and
x. reading the plate on protein suspension array system.

In an embodiment, the desorption is performed with a desorption buffer. The desorption buffer includes sodium citrate and /or 1M Sodium Hydroxide (~140µL for 2mL).

In an embodiment, reagent D is interchangeably referred to as Luminex Assay Buffer. Reagent D includes bovine serum albumin (BSA), Tween 20 PBS, reagent C, reagent B. solution is maintained at pH 7 +/- 0.2 with 1 N NaOH or 1 M Citric acid and filtered through 0.22 µ filter.

In a preferred embodiment, the antigen content determination method using the saccharide coupled microsphere comprises:
i. providing a standard solution of polysaccharides,
ii. providing a test sample,
iii. desorbing the test sample and the standard,
iv. preparing the percent adsorption sample,
v. preparing mixture of antisera/ Monoclonal antibody (Mab) for at least one serotype specific saccharide,
vi. dilute test sample and percent adsorption sample,
vii. transfer the standard, the sample, and the bead control into respective wells containing mixture of sera/ mAb, incubate the dilution plate,
viii. providing the saccharide coupled microsphere/ bead for at least one serotype specific polysaccharide,
ix. preparing assay plate by adding the beads in each well of the plate, followed by adding the standard and the sample from dilution plate to wells in the assay plate having beads as well as bead blanks, incubating and washing,
x. preparing anti rabbit phycoerythrin and anti-mice phycoerythrin and adding in each well,
xi. incubating the plate,
xii. washing the plate,
Washing and dilution is performed using reagent D,
xiii. adding reagents D to the plate, and
xiv. reading the plate on protein suspension array system.

In an embodiment, reagent D is interchangeably referred to has Luminex Assay Buffer. Reagent D includes bovine serum albumin (BSA), Tween 20 PBS, reagent C, reagent B. solution is maintained at pH 7 +/- 0.2 with 1 N NaOH or 1 M Citric acid. Filtered through 0.22 µ filter.

See more detailed method of antigen content determination assay in the “METHODS” herein.

IDENTITY ASSAY
In another aspect, the present invention is directed to an identity assay method using the saccharide coupled microsphere obtained by the method as disclosed herein.

In an embodiment, the identity assay method using the saccharide coupled microsphere comprises:
i. desorbing the test sample and the standard,
ii. preparing mixture of antisera/ Monoclonal antibody (Mab) for at least one serotype specific saccharide,
iii. dilute test sample (with Luminex Assay Buffer),
iv. transfer the standard, the sample, and the bead control into respective wells containing mixture of sera/ Mab, incubate the dilution plate,
v. providing the saccharide coupled microsphere/ bead for at least one serotype specific polysaccharide,
preparing assay plate by adding the beads in each well of the plate, followed by adding the standard and the sample from dilution plate to wells in the assay plate having beads as well as bead blanks, incubating and washing
Washing and dilution is performed using reagent D.
vi. preparing anti rabbit phycoerythrin and anti-mice phycoerythrin and adding in each well,
vii. adding reagents D to the plate, and
viii. reading the plate on protein suspension array system.

In an embodiment, the desorption is performed with a desorption buffer. The desorption buffer includes sodium citrate.

In an embodiment, reagent D is interchangeably referred to has Luminex Assay Buffer. Reagent D includes bovine serum albumin (BSA), Tween 20 PBS, reagent C, reagent B. solution is maintained at pH 7 +/- 0.2 with 1 N NaOH or 1 M Citric acid and filtered through 0.22 µ filter)
In a preferred embodiment, the identity assay method using the saccharide coupled microsphere comprises:
i. providing a standard solution of polysaccharides,
ii. providing a test sample,
iii. desorbing the test sample and the standard,
iv. preparing mixture of antisera/ Monoclonal antibody (Mab) for at least one serotype specific saccharide,
v. dilute test sample (with Luminex Assay Buffer),
vi. transfer the standard, the sample, and the bead control into respective wells containing mixture of sera/ mAb, incubate the dilution plate,
vii. providing the saccharide coupled microsphere/ bead for at least one serotype specific polysaccharide,
viii. preparing assay plate by adding the beads in each well of the plate, followed by adding the standard and the sample from dilution plate to wells in the assay plate having beads as well as bead blanks, incubating and washing,
ix. preparing anti rabbit phycoerythrin and anti-mice phycoerythrin and adding in each well,
x. incubating the plate,
xi. washing the plate,
xii. adding reagents D to the plate, and
xiii. reading the plate on protein suspension array system.

See more detailed method of identity assay in the “METHODS” herein.

FREE SACCHARIDE ESTIMATION

In another aspect, the present invention is directed to a free saccharide estimation method using the saccharide coupled microsphere obtained by the method as disclosed herein.

In an embodiment, the free saccharide estimation method using the saccharide coupled microsphere comprises:
i. preparing free saccharides samples,
ii. preparing mixture of antisera/ Monoclonal antibody (Mab) for at least one serotype specific saccharide,
iii. dilute test sample (with Luminex Assay Buffer),
iv. transfer the standard, the sample, and the bead control into respective wells containing mixture of sera/ mAb, incubate the dilution plate,
v. providing the saccharide coupled microsphere/ bead for at least one serotype specific polysaccharide,
vi. preparing assay plate by adding the beads in each well of the plate, followed by adding the standard and the sample from dilution plate to wells in the assay plate having beads as well as bead blanks, incubating and washing,
vii. preparing anti rabbit phycoerythrin and anti-mice phycoerythrin and adding in each well,
viii. adding reagents D to the plate and
ix. reading the plate on protein suspension array system.

In a preferred embodiment, the free saccharide estimation method using the saccharide coupled microsphere comprises:
i. providing a standard solution of polysaccharides,
ii. providing a test sample,
iii. preparing free Saccharides samples,
iv. preparing mixture of antisera/ Monoclonal antibody (Mab) for at least one serotype specific saccharide,
v. dilute test sample (with Luminex Assay Buffer),
vi. transfer the standard, the sample, and the bead control into respective wells containing mixture of sera/ mAb, incubate the dilution plate,
vii. providing the saccharide coupled microsphere/ bead for at least one serotype specific polysaccharide,
viii. preparing assay plate by adding the beads in each well of the plate, followed by adding the standard and the sample from dilution plate to wells in the assay plate having beads as well as bead blanks, incubating and washing,
ix. preparing anti rabbit phycoerythrin and anti-mice phycoerythrin and adding in each well,
x. incubating the plate,
xi. washing the plate,
xii. adding reagent D to the plate and
xiii. reading the plate on protein suspension array system.
See more detailed method of the free saccharide estimation assay method in the “METHODS” herein.

ESTIMATING ANTIBODY CONCENTRATION (IGG) IN SERA SAMPLE

In another aspect, the present invention is directed to a method of estimating antibody concentration (IgG) in sera sample, the method using the saccharide coupled microsphere obtained by the method as disclosed herein.

In an embodiment, the method of estimating antibody concentration (IgG) in sera sample using the saccharide coupled microsphere comprises:
i. providing the saccharide coupled microsphere/ bead for at least one serotype specific polysaccharide,
ii. preparing assay plate by adding the beads in each well of the plate, followed by adding the standard and the sample from dilution plate to wells in the assay plate having beads as well as bead blanks, incubating and washing,
iii. preparing anti rabbit phycoerythrin and anti-mice phycoerythrin and adding in each well,
iv. adding reagents to the plate and
v. reading the plate on protein suspension array system.

In a preferred embodiment, the method of estimating antibody concentration (IgG) in sera sample using the saccharide coupled microsphere comprises:
i. providing a standard solution of polysaccharides,
ii. providing a test sample,
iii. providing the saccharide coupled microsphere/ bead for at least one serotype specific polysaccharide,
iv. preparing assay plate by adding the beads in each well of the plate, followed by adding the standard and the sample from dilution plate to wells in the assay plate having beads as well as bead blanks, incubating and washing,
v. preparing anti rabbit phycoerythrin and anti-mice phycoerythrin and adding in each well,
vi. incubating the plate,
vii. washing the plate,
viii. adding reagents to the plate and
ix. reading the plate on protein suspension array system.

See more detailed method of estimating antibody concentration (IgG) in sera sample using the saccharide coupled microsphere in the “METHODS” herein.

In an embodiment, for the method of estimating antibody concentration (IgG), the sera sample includes a human sera sample.

In a preferred embodiment, for the method of estimating antibody concentration (IgG), the sera sample includes an animal sera sample.

In another preferred embodiment, for the method of estimating antibody concentration (IgG), the sera sample includes a human sera sample and an animal sera sample.

ANOTHER ASPECT: APPARATUS
In another aspect, the present invention is directed to an apparatus including the saccharide coupled microsphere.

ANOTHER ASPECT: APPLICATION
In another aspect, the present invention is directed to the saccharide coupled microspheres as and when used to determine antibody titre of an immunogenic composition/ a vaccine.

METHODS
METHOD OF EVALUATING IMMUNOGENICITY
In an embodiment, the present invention is directed to a method of evaluating immunogenicity of immunogenic composition, the method comprising;
a. providing a test sample corresponding to the saccharide in the immunogenic composition;
b. providing the saccharide coupled microsphere corresponding to the saccharide in the immunogenic composition; and
c. evaluating the immunogenicity of the immunogenic composition by antigen content determination, or estimating antibody concentration (IgG), or identity assay, free polysaccharide estimation using the test sample and the saccharide coupled microsphere.

ANTIGEN CONTENT DETERMINATION

The antigen content determination method using the saccharide coupled microsphere comprises:

Preparation of standard:
Prepare 21 Valent Reference standard: Mix all the serotypes (except 6B) at 4.4µg/ml and serotype 6B at 8.8µg/ml. Prepare the series of standard from S1 to S8 in the first column of 96-well titer plate using reagent D by serial 2-fold dilution as per the following table. Discard 125 µl from the last well after mixing. Final total volume of each well should be 125 µl.

Preparation of sample:
Take 2ml of test sample.

Desorption of sample and standard:
Desorption is done with desorption buffer. Desorption buffer includes sodium citrate.
Add 200mg sodium citrate per 2ml of sample and standard, incubate for 2Hrs at 37°C on roto-spin for complete desorption.

Preparation of % adsorption sample:
Take 1.0 mL of sample in tube and centrifuged it at 10000 g for 10 minutes and collect supernatant.

Preparation mixture of sera/mAb:
Prepare the 21 Valent mixture of Antisera/ Mab using the optimized dilution for each serotype Procedure:

Preparation dilution plate:
Prepare the series of standard from S1 to S8 by serial 2-fold dilution. Appropriately dilute the test sample & percent adsorption sample with LAB to fit in the standard curve.

Transfer 100 µl of the standards, sample and bead control to the respective wells, containing sera/mAb in the same plate.

Incubate the plate in shaking incubator at 37°C for 1 hour with shaking at 150 rpm.

Preparation mixture of Beads:
Prepared the 21 valent mixture of beads in Luminex Assay Buffer.

Preparation of Assay Plate:
Add 4000 beads per well per 50µl in each required wells of filter plate.
Add 50µl of incubated standards and samples from dilution plate in duplicates on filter plate containing beads with bead control and blank
Incubate the plate in shaking incubator at 37oC for 1 hour with shaking at 150 rpm.
Wash the plate with 100ul of LAB. This step should be performed 3 times.

Preparation of Phycoerythrin (PE):
Prepare the mixture of required volume of anti-Rabbit-phycoerythrin and anti-mice-phycoerythrin.
Add 50 µl in each well. Incubate the plate in shaking incubator at 37oC for 30 minutes with shaking at 150 rpm.
Wash the plate with 100 µl of reagent D, 3 times.
Add 100ul of reagent D and read the plate on Protein Suspension Array System (Bio-Plex 200).
Adding reagents D (Luminex Assay Buffer: Dissolve 2.0 gm bovine serum albumin (BSA), 2.5 mL reagent C and 1 mL Tween 20 in 800 mL of 1X PBS. Make up the volume to 1000 mL with reagent B. Adjust the pH of the solution to 7.2 ± 0.1 with 1 N NaOH or 1 M Citric acid. Filter through 0.22 µ filter.

IDENTITY ASSAY

Preparation of standard:
Prepare 21 Valent Reference standard: Mix all the serotypes at 4.4µg/ml except 6B and serotype 6B at 8.8µg/ml

Preparation of sample:
Take 2ml of test sample.

Desorption of sample and standard:
Desorption is done with desorption buffer. Desorption buffer includes sodium citrate and or 1M sodium hydroxide.
Add 200mg sodium citrate per 2ml of sample and standard, incubate for 2Hrs at 37°C on roto-spin for complete desorption.

Preparation mixture of sera/mAb:
Prepare the 21 Valent mixture of Antisera/ Mab using the optimized dilution for each serotype.

Procedure:
Preparation dilution plate:
Prepare the series of standard from S1 to S8 by serial 2-fold dilution. Appropriately dilute the test sample to fit in the standard curve.
Transfer 100 µl of the standards, sample and bead control to the respective wells, containing sera/mAb in the same plate.
Incubate the plate in shaking incubator at 37°C for 1 hour with shaking at 150 rpm.

Preparation mixture of Beads:
Prepared the 21 valent mixture of beads in Luminex Assay Buffer

Preparation of Assay Plate:
Add 4000 beads per well per 50µl in each required well of filter plate.
Add 50µl of incubated standards and samples from dilution plate in duplicates on filter plate containing beads with bead control and blank
Incubate the plate in shaking incubator at 37oC for 1 hour with shaking at 150 rpm.
Wash the plate with 100ul of LAB. This step should be performed 3 times.

Preparation of phycoerythrin (PE):
Prepare the mixture of required volume of anti-Rabbit-phycoerythrin and anti-mice-phycoerythrin
Add 50 µl in each well. Incubate the plate in shaking incubator at 37oC for 30 minutes with shaking at 150 rpm.

Wash the plate with 100 µl of reagent D, 3 times.

Add 100ul of reagent D and read the plate on Protein Suspension Array System (Bio-Plex 200).
Adding reagents D (Luminex Assay Buffer: Dissolve 2.0 gm bovine serum albumin (BSA), 2.5 mL reagent C and 1 mL Tween 20 in 800 mL of 1X PBS. Make up the volume to 1000 mL with reagent B. Adjust the pH of the solution to 7.2 ± 0.1 with 1 N NaOH or 1 M Citric acid. Filter through 0.22 µ filter).

FREE SACCHARIDE ESTIMATION IN DRUG PRODUCT

Preparation of standard:
Prepared 21 Valent Reference standard: Mix all the serotypes at 4.4µg/ml except 6B and serotype 6B at 8.8µg/ml
Preparation of sample:
Take 2ml supernatant of test sample by centrifuged at 5000g for 5min.

Preparation of Free PS sample:
A) Method-A: Preparation for free saccharide content using deoxycholate and hydrochloric acid. From the final sample after DOC precipitation, take the appropriate aliquot so as to fit in the standard curve range.

B) Method-B: Preparation of free saccharide content using the three different mAb coupled with Beads are incubated with the test solution for 1Hr at 37°C at 150rpm. Further centrifuge the sample at 5000g for 5min and collect the supernatant.

Preparation mixture of sera/mAb:
Prepare the 21 Valent mixture of Antisera/ Mab using the optimized dilution for each serotype.

Procedure:
Preparation dilution plate:
Prepare the series of standard from S1 to S8 by serial 2-fold dilution. Appropriately dilute the test sample.
Transfer 100 µl of the standards, sample and bead control to the respective wells, containing sera/mAb in the same plate.
Incubate the plate in shaking incubator at 37°C for 1 hour with shaking at 150 rpm.

Preparation mixture of Beads:
Prepared the 21 valent mixture of beads in Luminex Assay Buffer

Preparation of Assay Plate:
Add 4000 beads per well per 50µl in each required well of filter plate.
Add 50µl of incubated standards and samples from dilution plate in duplicates on filter plate containing beads with bead control and blank
Incubate the plate in shaking incubator at 37oC for 1 hour with shaking at 150 rpm.
Wash the plate with 100ul of LAB. This step should be performed 3 times.
Preparation of PE:
Prepare the mixture of required volume of anti-Rabbit-phycoerythrin and anti-mice-phycoerythrin
Add 50 µl in each well. Incubate the plate in shaking incubator at 37oC for 30 minutes with shaking at 150 rpm.
Wash the plate with 100 µl of reagent D, 3 times.
Add 100ul of reagent D and read the plate on Protein Suspension Array System (Bio-Plex 200).
Adding reagents D (Luminex Assay Buffer: Dissolve 2.0 gm bovine serum albumin (BSA), 2.5 mL reagent C and 1 mL Tween 20 in 800 mL of 1X PBS. Make up the volume to 1000 mL with reagent B. Adjust the pH of the solution to 7.2 ± 0.1 with 1 N NaOH or 1 M Citric acid. Filter through 0.22 µ filter).

ESTIMATION OF ANTIBODY CONCENTRATION (IGG) IN CLINICAL (HUMAN) SERA SAMPLES.

Preparation of standard:
Prepare the working reference standard by diluting the 007SP reference sera from 1:500 to 1:64000

Preparation of sample:
Generally, test sample dilutions scheme as 1:200, 1:400, 1:800, 1:1600 fits well in the standard curve.

Procedure:
Preparation mixture of Beads:
Prepared the 21 valent mixture of beads in Luminex Assay Buffer

Preparation of Assay Plate:
Prewet the required wells of multiscreen filter plate with 100 µl of reagent D and aspirate the plate using vacuum manifold
Add 4000 beads per well per 50µl in each required well of filter plate. Aspirate the filter plate using vacuum manifold
Add 50µl of incubated standards and samples from dilution plate in duplicates on filter plate containing beads with bead control and blank
Incubate the plate in shaking incubator at 37oC for 1 hour with shaking at 150 rpm.
Wash the plate with 100ul of LAB. This step should be performed 3 times.

Preparation of PE:
Prepare the mixture of required volume of anti-Rabbit-phycoerythrin and anti-mice-phycoerythrin
Add 50 µl in each well. Incubate the plate in shaking incubator at 37oC for 30 minutes with shaking at 150 rpm.

Wash the plate with 100 µl of reagent D, 3 times.

Add 100ul of reagent D and read the plate on Protein Suspension Array System (Bio-Plex 200).
Adding reagents D (Luminex Assay Buffer: Dissolve 2.0 gm bovine serum albumin (BSA), 2.5 mL reagent C and 1 mL Tween 20 in 800 mL of 1X PBS. Make up the volume to 1000 mL with reagent B. Adjust the pH of the solution to 7.2 ± 0.1 with 1 N NaOH or 1 M Citric acid. Filter through 0.22 µ filter).

ESTIMATION OF IGG TITER IN ANIMAL SERA SAMPLE

Preparation of standard:
Prepare the working reference standard by diluting the 007SP reference sera from 1:500 to 1:64000

Preparation of sample:
Generally, test sample dilutions scheme as 1:200, 1:400, 1:800, 1:1600 fits well in the standard curve.

Procedure:
Preparation mixture of Beads:
Prepare the 21 valent mixture of beads in Luminex Assay Buffer
Preparation of Assay Plate:
Prewet the required wells of multiscreen filter plate with 100 µl of reagent D and aspirate the plate using vacuum manifold.
Add 4000 beads per well per 50µl in each required well of filter plate. Aspirate the filter plate using vacuum manifold.
Add 50µl of incubated standards and samples from dilution plate in duplicates on filter plate containing beads with bead control and blank.

Incubate the plate in shaking incubator at 37oC for 1 hour with shaking at 150 rpm.
Wash the plate with 100ul of LAB. This step should be performed 3 times.

Preparation of PE:
Prepare the mixture of required volume of anti-Rabbit-phycoerythrin and anti-mice-phycoerythrin
Add 50 µl in each well. Incubate the plate in shaking incubator at 37oC for 30 minutes with shaking at 150 rpm.

Wash the plate with 100 µl of reagent D, 3 times.
Add 100ul of reagent D and read the plate on Protein Suspension Array System (Bio-Plex 200). Adding reagents D (Luminex Assay Buffer: Dissolve 2.0 gm bovine serum albumin (BSA), 2.5 mL reagent C and 1 mL Tween 20 in 800 mL of 1X PBS. Make up the volume to 1000 mL with reagent B. Adjust the pH of the solution to 7.2 ± 0.1 with 1 N NaOH or 1 M Citric acid. Filter through 0.22 µ filter).

The present invention is an alternative method for coupling of saccharides like antigens to microsphere. The proposed method is based on principles of metal coordination chemistry and electrostatic mode of interaction-based coupling of purified bacterial saccharides to microsphere, such coupled microsphere is subsequently used for saccharide-protein related conjugate vaccine. Bio- assays are selected from but not limited to a) antigen content and b) IgG determination-serology-based applications, c) identity assays and d) stability assays (Free saccharide estimation).
The improved method of coupling the microsphere with the saccharides utilizes specific advantageous parameters related to buffer type, pH, incubation time, chemicals used, size of the polysaccharides, serotype of bacterial polysaccharides, as compared to previous conventional covalent coupling assays.

EMBODIMENTS
The present invention is illustrated in more detail by the following embodiments and combinations of embodiments which result from the corresponding dependency references and links:
I. A method of coupling a saccharide to a microsphere to obtain a saccharide coupled microsphere, the method comprising:
a. providing the microsphere;
b. providing the saccharide;
c. diluting the saccharide with a buffer at pH in range of 3.0 to 9.0; and
d. mixing the microsphere with the saccharide to form the saccharide coupled microsphere;
wherein the mixing further includes incubation at temperature in range of 20? to 40? for incubation time in range of 30 mins to 180 mins.
II. The method of coupling a saccharide to a microsphere as disclosed in embodiment I, wherein the method comprises:
a. providing the microsphere;
b. providing the saccharide;
c. diluting the saccharide with the buffer at pH in range of 3.0 to 9.0; optionally wherein and the saccharide concentration is in range of 1.0 µg/ml to 50.0 mg/ml, preferably in the range of 1.0 µg/ml to 10 mg/ml:
d. mixing the microsphere with the saccharide to form the saccharide coupled microsphere;
optionally, wherein the saccharide has a size in the range of 0.05 kDa to 3000 kDa, preferable in the range of 0.05 kDa to 1500 kDa; and
optionally, wherein the mixing includes incubation at temperature in range of 20? to 40? for incubation time in range of 30 mins to 180 mins.
III. The method as disclosed in embodiment II, wherein
optionally, the mixing is performed with the coupling ratio of the microsphere to the saccharide is in range of 50 to 12500, preferably in range of 50 to 250, and/ or
optionally, the metal ions are adsorbed on surface of the microsphere during activation of the microsphere before mixing with the saccharide,
optionally, the saccharide coupled microsphere is formed by non-covalent electrostatic coupling, and/ or
optionally, the saccharide remains intact, retains epitope confirmation and the saccharide coupled microsphere is stable.
IV. The method as disclosed in any one of embodiments I to III, wherein the buffer includes phosphate buffered saline with tween (PBST) buffer, (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES) buffer, tris-aminomethane (Tris) buffer, 2-(N-morpholino)ethane sulfonic acid (MES) buffer, 3-(N-morpholino) propane sulfonic acid (MOPS) buffer.
V. The saccharide coupled microsphere obtained by the method as disclosed in any one of embodiments I to III.
VI. The saccharide coupled microsphere as disclosed in embodiment IV, wherein the saccharide coupled microsphere has Mean Fluorescence Intensity/ MFI value in range of 200 to 20,000.
VII. The method as disclosed in any one of embodiments I to IV, wherein the saccharide is bacterial saccharide including Group A Streptococcus, Group B Streptococcus, Streptococcus pneumoniae/ pneumococcus, Haemophilus bacteria, Haemophilus influenzae bacteria, Haemophilus influenzae type b bacteria (Hib), Salmonella, Typhoidal salmonella, Non-typhoidal salmonella, Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi, Streptococcus pyogenes, Streptococcus agalactiae, Shigella, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Escherichia coli, or Neisseria meningitidis/ meningococcus saccharide.
VIII. The method as disclosed in embodiment VII, wherein the saccharide is Streptococcus pneumoniae polysaccharide.
IX. The method as disclosed in embodiment VIII, wherein Streptococcus pneumoniae polysaccharide is selected from serotypes 1, 2, 3, 4, 5, 6, 6A, 6B, 6C, 6D, 7, 7B, 7C, 7F, 8, 9, 9A, 9L, 9N, 9V, 10, 10A, 10B, 10C, 10F, 10X, 11, 11A, 11B, 11C, 11F, 12, 12A, 12B, 12F, 13, 14, 15, 15A, 15B, 15BC, 15C, 15F, 16, 16F, 17, 17A, 17F, 18, 18A, 18B, 18C, 18F, 19, 19A, 19B, 19F, 20, 20A, 20B, 20F, 21, 22, 22A, 22F, 23, 23A, 23B, 23F, 24, 24F, 25, 25F, 26, 27, 28, 28A, 28F, 29, 30, 31, 32, 33, 33A, 33B, 33C, 33D, 33F, 34, 35, 35A, 35B, 35C, 35D, 35F, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, and/ or 48.
X. The method as disclosed in embodiment VIII, wherein the Streptococcus pneumoniae polysaccharide is selected from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 24F and/ or 33F.
XI. The method as disclosed in any one of embodiments VIII to X, wherein the Streptococcus pneumoniae polysaccharide has a molecular size in range of 50 kDa to 3000 kDa, preferably in range of 50 kDa to 1500 kDa, is diluted with PBST buffer at a pH in range of 3.0 to 9.0, preferably at pH in range of 4.0 to 7.0, and has concentration in range of 0.05 mg/mL to 50.0 mg/mL, preferably in range of 0.05 mg/ mL to 50 mg/ mL, more preferably in range of 0.05 mg/ mL to 10 mg/mL.
XII. The method as disclosed in any one of embodiments VIII to XI, wherein the Streptococcus pneumoniae polysaccharide is mixed with the microsphere and incubated at temperature in range of 20 to 40?, preferably in range of 23? to 39?, for incubation time of 60 mins to 120 mins.
XIII. A Streptococcus pneumoniae saccharide coupled microsphere obtained by the method as disclosed in any one of embodiments VIII to XII.
XIV. The method as disclosed in any one of embodiments I to IV, wherein the saccharide is Haemophilus influenzae type b bacteria (Hib) polysaccharide.
XV. The method as disclosed in embodiment XIV, wherein the Haemophilus influenzae type b bacteria (Hib) polysaccharide has a molecular size in range of 0.05 kDa to 5 kDa, is diluted with PBST buffer at a pH in range of 4 to 6 and has concentration of 1.0 µg/ml to 50.0 µg/ml.
XVI. The method as disclosed in any one of embodiments XIV or XV, wherein the Haemophilus influenzae type b bacteria (Hib) polysaccharide is mixed with the microsphere and incubated at temperature of 20? to 30?, for incubation time of 60 mins to 120 mins.
XVII. A Haemophilus influenzae type b bacteria (Hib) saccharide coupled microsphere obtained by the method as disclosed in any one of embodiments XIV to XVI.
XVIII. The method as disclosed in any one of embodiments I to IV, wherein the saccharide is Neisseria meningitidis saccharide.
XIX. The method as disclosed in embodiment XVIII, wherein the polysaccharide is Neisseria meningitidis polysaccharide serotypes selected from meningococcal serotypes A (type I and III), B (type II), B6, B16, C (type II-alpha), D (type IV), Z’/ E, E29, H, I, K, K454, L, M, W135, X, Y, Z.
XX. The method as disclosed in any one of embodiments XVIII to XIX, wherein the Neisseria meningitidis polysaccharide has a molecular size in range of 75 kDa to 3000 kDa, is diluted with PBST buffer at a pH in range of 4.0 to 7.0 and has concentration of 0.01 mg/mL to 10.0 mg/mL.
XXI. The method as disclosed in any one of embodiments XVIII or XX, wherein the mixing of the Neisseria meningitidis polysaccharide with the microsphere is done for incubation temperature of 20? to 40? for incubation time of 60 mins to 120 mins.
XXII. A Neisseria meningitidis polysaccharide saccharide coupled microsphere obtained by the method as disclosed in any one of embodiments XVIII to XXI.
XXIII. A method of evaluating immunogenicity of immunogenic composition, the method comprising;
a. providing a test sample corresponding to the saccharide in the immunogenic composition;
b. providing the saccharide coupled microsphere corresponding to the saccharide in the immunogenic composition;
c. evaluating the immunogenicity of the immunogenic composition by antigen content determination, or estimating antibody concentration (IgG), or identity assay, free polysaccharide estimation using the test sample and the saccharide coupled microsphere,
wherein the saccharide coupled microsphere is obtained by the method as disclosed in any one of embodiments I to IV.
XXIV. An antigen content determination method using the saccharide coupled microsphere obtained by the method as disclosed in any one of embodiments I to IV.
XXV. An identity assay method using the saccharide coupled microsphere obtained by the method as disclosed in any one of embodiments I to IV.
XXVI. A free polysaccharide estimation method using the saccharide coupled microsphere obtained by the method as disclosed in any one of embodiments I to IV.
XXVII. A method of estimating antibody concentration (IgG) in sera sample, the method using the saccharide coupled microsphere obtained by the method as disclosed in any one of embodiments I to IV.
XXVIII. The method of estimating antibody concentration (IgG) in sera sample as disclosed in embodiment XXVII, wherein the sera sample includes a human sera sample and an animal sera sample.
XXIX. An apparatus comprising the saccharide coupled microsphere obtained by the method as disclosed in any one of embodiments I to IV.
XXX. The saccharide coupled microsphere as and when used in assay or evaluation of a vaccine or an immunogenic composition, wherein the saccharide coupled microsphere is obtained by the method as disclosed in any one of embodiments I to IV.
XXXI. A method of coupling a saccharide to a microsphere, the method comprising:
a. providing at least one microsphere;
b. providing at least one saccharide; and
c. mixing the at least one microsphere with the at least one saccharide to form a saccharide coupled microsphere;
wherein the saccharide has a size in the range of 0.01 kDa to 3000 kDa.
XXXII. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein the microsphere is either magnetic or non-magnetic.
XXXIII. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein the microsphere is activated before mixing with the saccharide.
XXXIV. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein the microsphere is activated using a bead reagent having metal ions.
XXXV. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein the microsphere is activated using a nickel based bead reagent.
XXXVI. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein the saccharide is native saccharide or size reduced saccharide.
XXXVII. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein the saccharide is size reduced saccharide.
XXXVIII. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein the saccharide is diluted with PBST buffer before mixing.
XXXIX. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein the mixing is aqueous based reaction.
XL. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein the method allows development of electron donor sites forming electrostatic attachment of the saccharide on to the microsphere, wherein the saccharide is intact.
XLI. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein the saccharide concentration, the size of polysaccharide, mixing incubation temperature, mixing incubation time, pH of buffer is optimised to allow development of electron donor sites forming electrostatic attachment of the saccharide on to the microsphere, wherein the saccharide is intact.
XLII. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein
optionally, the mixing is performed with the coupling ratio of the microsphere to the saccharide is in range of 50 to 12500, and/ or
optionally, the metal ions are adsorbed on surface of the microsphere during activation of the microsphere before mixing with the saccharide, and/ or
optionally, the saccharide coupled microsphere is formed by non-covalent electrostatic coupling, and/ or
optionally, the saccharide remains intact, retains epitope confirmation and the saccharide coupled microsphere is stable.
XLIII. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein
the mixing is performed with the coupling ratio of the microsphere to the saccharide is in range of 50 to 12500, and
the saccharide coupled microsphere is formed by non-covalent electrostatic coupling.
XLIV. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein
the mixing is performed with the coupling ratio of the microsphere to the saccharide is in range of 50 to 12500,
the metal ions are adsorbed on surface of the microsphere during activation of the microsphere before mixing with the saccharide,
the saccharide coupled microsphere is formed by non-covalent electrostatic coupling,
the saccharide remains intact, retains epitope confirmation and the saccharide coupled microsphere is stable.
XLV. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein the saccharide is pneumococcal polysaccharide saccharide selected from a group of polysaccharides associated with Streptococcus pneumoniae/ pneumococcal strains associated with global Streptococcus pneumoniae/ pneumococcal sequence cluster (GPSC) selected from GPSC 1 to 131.
XLVI. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein the saccharide is associated Haemophilus bacteria type a, type b, type c, type d, type e, or type f.
XLVII. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein the saccharide is Haemophilus influenzae type b bacteria (Hib) saccharide selected from a group of saccharides associated with type I and type II Hib strain.
XLVIII. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein the polysaccharide is Haemophilus influenzae type b bacteria (Hib) polysaccharide selected from a group of polysaccharides associated with variation of hcsA gene.
XLIX. The method of coupling a saccharide to a microsphere as disclosed in any one of previous embodiments, wherein the polysaccharide is meningococcal polysaccharide selected from a group of polysaccharides associated with variation in lpxL1, fHbp, and tps genes.
L. An apparatus comprising the saccharide coupled microsphere obtained by the method as disclosed in any one of previous embodiments.

EXAMPLES
The present invention is further illustrated in combination with the following examples. These examples are provided to exemplify the present invention but are not intended to restrict the scope of the presently claimed invention in any way. The terms and abbreviations in the examples have their common meanings. For example, “%”, “Eq. wt.”, “Eq.”, "° C", “wt. %”, "% w/w", “% w/v” and "gm" represent “percentage”, “Equivalent Weight”, “Equivalents”, "degree Celsius", “percent by weight”, "percent weight by weight", “percent weight by volume” and "gram" respectively.

REAGENTS/ SOLUTIONS/ INSTRUMENTS/ EQUIPMENT:
A. Coupling reagents/ Chemicals:
• Activation reagent: Anteotech
• Storage buffer: PBST with 1% BSA (SIIPL – Inhouse). BSA from Sigma Aldrich, India
• Beads: Magnetic beads (Luminex Corporation), MicroPlex® Microspheres, MagPlex® Microspheres
• Trypan Blue (Sigma)
• PBST (Phosphate-buffered saline with Tween 20) buffer: Sodium Chloride (Qualigens), Potassium Chloride (Qualigens), Di-sodium hydrogen phosphate (Merck), Potassium dihydrogen phosphate (Sigma-Aldrich), Tween 20 (SDFCL), Sodium hydroxide (Qualigens), Hydrochloric acid (Sigma-Aldrich).
• Blocking Buffer: BSA/ Bodvine Serum Albumin (Sigma Aldrich, India)
• PBS Buffer: Sodium Chloride (Qualigens), Potassium Chloride (Qualigens), Di-sodium hydrogen phosphate (Merck), Potassium dihydrogen phosphate (Sigma-Aldrich), Sodium hydroxide (Qualigens), Hydrochloric acid (Sigma-Aldrich).

B. Testing reagents/ Chemicals:
• 10X PBS buffer
• 20# W/V Sodium azide (NaN3): Sodium Azide (Sigma-Aldrich),
• Luminex Assay Buffer: 1X PBS, BSA (Sigma Aldrich), Tween 20 (SDFCL), Sodium hydroxide/ NaOH (Qualigens), Citric acid (Qualigens).
• Formulation Buffer (Aluminium Phosphate buffer): 20mM Histidine, Succinate buffer, Thiomerseal, Tween 20, Adjuvant.
• Phycoerythrin: Human Phycoerythrin, Rat Phycoerythrin, Rabbit Phycoerythrin, Mice Phycoerythrin from Jackson Immunoresearch, USA.
• Reference standard: Respective Streptococcus Pneumoniae (Pneumococcal) monovalent conjugate bulks of serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 24F. (Serotype 24F is from Kempegowda Institute of Meidcal Sciences, KIMS, Bangalore, India. Rest all Pneumococcal serotypes are from Center for Disease Control and Prevention (CDC), Atlanta, USA)
• Reference standard: Haemophilus influenzae type b saccharides. The Hib strain 760705 was obtained from Netherlands Vaccines Institute (NVI, The Netherlands)
• Reference standard: Neisseria meningitis (A, C, W, Y, X) saccharides. Neisseria meningitidis A (strain M1027 from SynCo Biopartners, Netherlands), Neisseria meningitidis C (strain C11(60E) from CBER/FDA, USA), Neisseria meningitidis W (strain S877 from CBER/FDA, USA), Neisseria meningitidis Y (strain M10659 from CDC, USA), Neisseria meningitidis (strain X M8210 from CBER/FDA, USA)
• Monoclonal Antibody: Antibody against Pneumococcal monovalent conjugate bulks of serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 24F. (Staten Serum Institute)
• Carboxylated coupled beads: Carboxylated beads coupled with serotypes Pneumococcal monovalent conjugate bulks of serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 24F. (SIIPL-In-house).
• For animal testing license: 41/PO/RcBi/S/99/CPCSEA dated:08/03/2022\
• Ethical committee approval: 17/30-09/2024-MT.
• No any external sample were used for human, the international human reference std i.e. NIBSC (WHO) international reference standard i.e. 007Sp were used
• Rat Tox sera samples proposed from Advenous laboratory which were vaccinated with PCV 21 valent at Advenous Lab.
C. Instruments:
• Automated Cell counter: Bio-Rad
• Inverted microscope: Nikon
• Analytical weighing balance: Sartorius
• pH meter: Thermo Orian
• Centrifuge: Thermo scientific
• Shaker Incubator: Titramax
• Protein Suspension array system (Bioplex-200): Bio-Rad
• Vaccum manifold: Millipore
• Sonicator: Amkette industries
• Micropipette, Multi-channel (30-300µL and 10-100µL), Single-channel (0.5-10µL, 10-100µL, 20-200 L, 100-1000µL and 500-5000µL): All from Eppendorf
• Magnetic separator: Bio-Rad

EXAMPLE 1: DILUTION OF NATIVE POLYSACCHARIDES (STREPTOCOCCAL PNEUMONIAE POLYSACCHARIDES)
Native polysaccharide available stock concentration range for all the serotypes was between 5 to 10 mg/ml and for size reduced polysaccharide between 11 to 15mg/ml. As per Table 1 below, each serotype required the optimized concentration range, hence stock of polysaccharide was diluted in the mentioned respective buffer condition prior to coupling.

TABLE 1: DILUTION OF NATIVE POLYSACCHARIDES
Sr. No. Sero-type Chemical composition Conc. of PS require for coupling PnPS Reconstitution Solution/Buffer
1 1 Nitrogen, Phosphorus, Uronic acids, O-acetyl 0.5 to 1.0 mg/mL PBST (pH 5.5 ± 0.5), 5-6
2 2 Nitrogen, Phosphorus, Uronic acids, Methyl-pentoses 1.5 to 2.0 mg/mL PBST (pH 4.5 ± 0.5), 4-5
3 4 Nitrogen, Phosphorus, O-acetyl, Hexosamines 0.5 to 1.0 mg/mL PBST (pH 6.5 ± 0.5), 6-7
4 5 Nitrogen, Phosphorus, Uronic acids, Hexosamines 2.0 to 2.5 mg/mL PBST (pH 6.5 ± 0.5), 6-7
5 6A Nitrogen, Phosphorus, Methyl pentose 1.5 to 2.0 mg/mL PBST (pH 4.5 ± 0.5), 4-5
6 6B Nitrogen, Phosphorus, Methyl pentose 0.5 to 1.0 mg/mL PBST (pH 5.5 ± 0.5), 5-6
7 7F Nitrogen, Phosphorus, Hexosamines, Methyl pentose, O-acetyl 0.5 to 1.0 mg/mL PBST (pH 6.5 ± 0.5), 6-7
8 8 Nitrogen, Phosphorus, Uronic acid 1.5 to 2.0 mg/mL PBST (pH 4.5 ± 0.5), 4-5
9 9V Nitrogen, Phosphorus, Uronic acid, Hexosamines, O-acetyl 0.5 to 1.0 mg/mL PBST (pH 5.5 ± 0.5), 5-6
10 10A Nitrogen, Phosphorus, Hexosamines 0.5 to 1.0 mg/mL PBST (pH 6.5 ± 0.5), 6-7
11 11A Nitrogen, Phosphorus, O-acetyl 2.0 to 2.5 mg/mL PBST (pH 5.5 ± 0.5), 5-6
12 12F Nitrogen, Phosphorus, Uronic acid, Hexosamines, Methyl pentose, 0.5 to 1.0 mg/mL PBST (pH 6.5 ± 0.5), 6-7
13 14 Nitrogen, Phosphorus, Hexosamines 2.0 to 2.5 mg/mL PBST (pH 6.5 ± 0.5), 6-7
14 15B Nitrogen, Phosphorus, Hexosamines, O-acetyl 2.0 to 2.5 mg/mL PBST (pH 5.5 ± 0.5), 5-6
15 18C Nitrogen, Phosphorus, Methyl pentose, O-acetyl 2.0 to 2.5 mg/mL PBST (pH 6.5 ± 0.5), 6-7
16 19A Nitrogen, Phosphorus, Hexosamines, Methyl pentose 0.5 to 1.0 mg/mL PBST (pH 5.5 ± 0.5), 5-6
17 19F Nitrogen, Phosphorus, Hexosamines, Methyl pentose 0.5 to 1.0 mg/mL PBST (pH 5.5 ± 0.5), 5-6
18 22F Nitrogen, Phosphorus, Uronic acid, Methyl pentose, O-acetyl 2.0 to 2.5 mg/mL PBST (pH 5.5 ± 0.5), 5-6
19 23F Nitrogen, Phosphorus, Methyl pentose, O-acetyl 2.0 to 2.5 mg/mL PBST (pH 4.5 ± 0.5), 4-5
20 24F Nitrogen, Phosphorus, Hexosamines, Methyl pentose 0.5 to 1.0 mg/mL PBST (pH 6.5 ± 0.5), 6-7
21 33F Nitrogen, Phosphorus, O-acetyl 2.0 to 2.5 mg/mL PBST (pH 6.5 ± 0.5), 6-7

EXAMPLE 2: COMPOSITION ASSOCIATED WITH STREPTOCOCCAL POLYSACCHARIDE COUPLED MICROSPHERES
Each Streptococcal polysaccharide saccharide contain different functional groups, sizes and structure. The specific conditions applied to different Saccharides during dilution of the saccharide stock and further conjugation to the beads gave the optimal MFI’s to positive samples. The following table 2 represents these conditions.

TABLE 2: SEROTYPES AND COMPOSITIONS

SR Chemical composition Steps applied during dilution of PnPS stock Steps applied during coupling for incubation

Conc. Of PS require for coupling Size of Ps (kDa) Dilution of PnPS stocks Time Temp
1 Nitrogen, Phosphorus, Uronic acids, O-acetyl 0.5 to 1.0 mg/mL 80 PBST (pH 5.5 ± 0.5), 5-6 1Hr 25°C
2 Nitrogen, Phosphorus, Uronic acids, Methyl-pentoses 1.5 to 2.0 mg/mL 1011 PBST (pH 4.5 ± 0.5), 4-5 2Hr 37°C
4 Nitrogen, Phosphorus, O-acetyl, Hexosamines 0.5 to 1.0 mg/mL 87 PBST (pH 6.5 ± 0.5), 6-7 1.5Hr 25°C
5 Nitrogen, Phosphorus, Uronic acids, Hexosamines 2.0 to 2.5 mg/mL 75 PBST (pH 6.5 ± 0.5), 6-7 1.5Hr 25°C
6A Nitrogen, Phosphorus, Methyl pentose 1.5 to 2.0 mg/mL 551 PBST (pH 4.5 ± 0.5), 4-5 2Hr 37°C
6B Nitrogen, Phosphorus, Methyl pentose 0.5 to 1.0 mg/mL 107 PBST (pH 5.5 ± 0.5), 5-6 1Hr 25°C
7F Nitrogen, Phosphorus, Hexosamines, Methyl pentose, O-acetyl 0.5 to 1.0 mg/mL 135 PBST (pH 6.5 ± 0.5), 6-7 1.5Hr 25°C
8 Nitrogen, Phosphorus, Uronic acid 1.5 to 2.0 mg/mL 565 PBST (pH 4.5 ± 0.5), 4-5 2Hr 37°C
9V Nitrogen, Phosphorus, Uronic acid, Hexosamines, O-acetyl 0.5 to 1.0 mg/mL 108 PBST (pH 5.5 ± 0.5), 5-6 1.5Hr 25°C
10A Nitrogen, Phosphorus, Hexosamines 0.5 to 1.0 mg/mL 119 PBST (pH 6.5 ± 0.5), 6-7 1Hr 25°C
11A Nitrogen, Phosphorus, O-acetyl 2.0 to 2.5 mg/mL 100 PBST (pH 5.5 ± 0.5), 5-6 1.5Hr 25°C
12F Nitrogen, Phosphorus, Uronic acid, Hexosamines, Methyl pentose, 0.5 to 1.0 mg/mL 99 PBST (pH 6.5 ± 0.5), 6-7 1.5Hr 25°C
14 Nitrogen, Phosphorus, Hexosamines 2.0 to 2.5 mg/mL 143 PBST (pH 6.5 ± 0.5), 6-7 1.5Hr 25°C
15B Nitrogen, Phosphorus, Hexosamines, O-acetyl 2.0 to 2.5 mg/mL 136 PBST (pH 5.5 ± 0.5), 5-6 1.5Hr 25°C
18C Nitrogen, Phosphorus, Methyl pentose, O-acetyl 2.0 to 2.5 mg/mL 79 PBST (pH 6.5 ± 0.5), 6-7 1.5Hr 25°C
19A Nitrogen, Phosphorus, Hexosamines, Methyl pentose 0.5 to 1.0 mg/mL 91 PBST (pH 5.5 ± 0.5), 5-6 1.5Hr 25°C
19F Nitrogen, Phosphorus, Hexosamines, Methyl pentose 0.5 to 1.0 mg/mL 85 PBST (pH 4.5 ± 0.5), 4-5 1.5Hr 25°C
22F Nitrogen, Phosphorus, Uronic acid, Methyl pentose, O-acetyl 2.0 to 2.5 mg/mL 106 PBST (pH 5.5 ± 0.5), 5-6 1Hr 25°C
23F Nitrogen, Phosphorus, Methyl pentose, O-acetyl 2.0 to 2.5 mg/mL 94 PBST (pH 4.5 ± 0.5), 4-5 1Hr 25°C
24F Nitrogen, Phosphorus, Hexosamines, Methyl pentose 0.5 to 1.0 mg/mL 81 PBST (pH 6.5 ± 0.5), 6-7 1Hr 25°C
33F Nitrogen, Phosphorus, O-acetyl 2.0 to 2.5 mg/mL 191 PBST (pH 6.5 ± 0.5), 6-7 1Hr 25°C

EXAMPLE 3: STREPTOCOCCUS PNEUMONIAE/ PNEUMOCOCCAL SEROTYPE - TYPE 1
Efficient coupling was outcome of correct saccharide size, concentration and pH
Efficiently coupled beads have following characteristics:
a) Higher sensitivity
b) Less noise/background
c) Good precision (CV among duplicates) and accuracy (back fitted recoveries)

The specific conditions applied to Pneumococcus type 1 serotype Saccharides during dilution of the saccharide stock and further conjugation to the beads/ microspheres gave the optimal MFI’s to positive samples.

TABLE 3a: Serotype 1 conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.
Type 1 pH (PBST) Concentration of Ps Molecular size (kDa) Incubation time, temperature
A (pH 4.5 ± 0.5), 4-5 0.1 mg/mL 80 1 Hr/25deg C
B (pH 4.5 ± 0.5), 4-5 0.3 mg/mL 80 1 Hr/25deg C
C (pH 5.5 ± 0.5), 5-6 0.5 mg/mL 80 1 Hr/25deg C
D (pH 5.5 ± 0.5), 5-6 1.0 mg/mL 80 1 Hr/25deg C

TABLE 3b: Serotype 1 conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.
Condition pH (PBST) Concentration of PS Molecular size
(kDa) Incubation time,
Temperature
A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 mg/mL 540 1 Hr/25deg C
B (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 mg/mL 540 1 Hr/25deg C
C (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 mg/mL 80 1 Hr/25deg C
D (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 mg/mL 80 1 Hr/25deg C

See FIG. 1a and 1 b & TABLE 3a and 3b for details of MFI value against Type 1 serotype in conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.

Inference: Four different conditions (A, B, C, D) were studied to obtain the maximum signal (MFI) of type 1 Saccharide coupled microspheres (PS coupled beads). During coupling, the pH range, Saccharide (Ps) Concentration, Molecular size and Incubation time was critically studied. From the above table the maximum MFI signal were observed for condition-D compared with other 3 conditions. If Higher the MFI values is directly proportional to the higher sensitivity of the assay, hence parameters mentioned in the condition-D are optimal for coupling serotype serotype-1 antigen to microsphere beads.

EXAMPLE 4: STREPTOCOCCUS PNEUMONIAE/ PNEUMOCOCCAL TYPE 2 SEROTYPE

The specific conditions applied to Pneumococcus type 2 serotype Saccharides during dilution of the saccharide stock and further conjugation to the beads/ microspheres gave the optimal MFI’s to positive samples.

TABLE 4a: STREPTOCOCCUS PNEUMONIAE/ PNEUMOCOCCAL TYPE 2 Serotype conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.
Type 2 Buffer pH Concentration of Ps Molecular size (kDa) Incubation time, temperature
A PBST PBST (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 mg/mL 80 1 Hr/25deg C
B PBST PBST (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 mg/mL 80 1 Hr/37deg C
C PBST PBST (pH 4.5 ± 0.5), 4-5 1.5 to 2.0 mg/mL 80 2 Hr/37deg C
D PBST PBST (pH 4.5 ± 0.5), 4-5 1.5 to 2.0 mg/mL 1011 2 Hr/37deg C

TABLE 4b: STREPTOCOCCUS PNEUMONIAE/ PNEUMOCOCCAL TYPE 2 Serotype conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.
Condition pH Concentration of PS Molecular size
(kDa) Incubation time, temperature
A PBST (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 mg/mL 95 1 Hr/25deg C
B PBST (pH 4.5 ± 0.5), 4-5 1.5 to 2.0 mg/mL 95 1 Hr/37deg C
C PBST (pH 4.5 ± 0.5), 4-5 1.5 to 2.0 mg/mL 1011 1 Hr/37deg C
D PBST (pH 4.5 ± 0.5), 4-5 1.5 to 2.0 mg/mL 1011 2 Hr/37deg C

See FIG. 2a and 2b & TABLE 4a and 4b for details of MFI value against Type 2 serotype in conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.

The pneumococcal serotype 2 modified saccharide has size 95kDa and after coupling was observed to be associated with the lesser MFI value compared with the Native saccharide (Size 1011kDa) which gives a good increase in the MFI value.

Inference: Four different conditions (A, B, C, D) were studied to obtain the maximum signal (MFI) of serotype-2 Saccharide coupled microspheres (PS coupled beads). During coupling, the pH range, Saccharide (Ps) Concentration, Molecular size and Incubation time was critically studied. From the above table the maximum MFI signal were observed for condition-D compared with other 3 conditions. Higher the MFI values is directly proportional to the higher sensitivity of the assay, hence parameters mentioned in the condition-D are optimal for coupling serotype-2 antigen to microsphere beads.

EXAMPLE 5: STREPTOCOCCUS PNEUMONIAE/ PNEUMOCOCCAL TYPE 4 SEROTYPE.
The specific conditions applied to Pneumococcus type 4 serotype Saccharides during dilution of the saccharide stock and further conjugation to the beads/ microspheres gave the optimal MFI’s to positive samples.

TABLE 5a: STREPTOCOCCUS PNEUMONIAE/ PNEUMOCOCCAL TYPE 4 SEROTYPE conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.
Type 4 Buffer pH Concentration of Ps Molecular size
(kDa) Incubation time, temperature
A PBST PBST (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 mg/mL 87 1 Hr/25deg C
B PBST PBST (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 mg/mL 87 1 Hr/25deg C
C PBST PBST (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 mg/mL 87 1.5 Hr/25deg C
D PBST PBST (pH 6.5 ± 0.5), 6-7 0.5 to 1.0 mg/mL 87 1.5 Hr/25deg C

TABLE 5b: STREPTOCOCCUS PNEUMONIAE/ PNEUMOCOCCAL TYPE 4 SEROTYPE conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.
Condition pH Concentration of PS Molecular size
(kDa) Incubation time,
temperature
A PBST (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 mg/mL 318 1 Hr/25deg C
B PBST (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 mg/mL 318 1 Hr/25deg C
C PBST (pH 6.5 ± 0.5), 6-7 0.5 to 1.0 mg/mL 87 1.5 Hr/37deg C
D PBST (pH 6.5 ± 0.5), 6-7 0.5 to 1.0 mg/mL 87 1.5 Hr/25deg C

See FIG. 3a and 3b and TABLE 5a and 5b for details of MFI value against Type 4 serotype in conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.

Inference: Four different conditions (A, B, C, D) were studied to obtain the maximum signal (MFI) of serotype-4 Saccharide coupled microspheres (PS coupled beads). During coupling, the pH range, Saccharide (Ps) Concentration, Molecular size and Incubation time was critically studied. From the above table the maximum MFI signal were observed for condition-D compared with other 3 conditions. Higher the MFI values is directly proportional to the higher sensitivity of the assay, hence parameters mentioned in the condition-D are optimal for coupling serotype-4 antigen to microsphere beads.
The saccharide coupled microsphere obtained herein above was further qualified using the serological assay (Multiplex-Immunoassay). During qualification stage the system suitability parameters were monitored such as Back fit of the reference standards, duplicate % CV, assay blank and % gradation between the MFI’s. To achieve these parameters within the specifications the coupling optimization was performed and no any interference was observed. Accordingly, no interference was observed in the method.

EXAMPLE 6: COMPARISON OF BEADS OF THE PRESENT INVENTIONAND CONVENTIONAL BEADS
Saccharide coupled microspheres were prepared for PCV-21 serotypes. The coupled beads were evaluated for Human sera i.e. 007SP (007SP is for use in the enzyme-linked immunosorbent assay protocol for quantification of human IgG antibodies specific for Streptococcus pneumoniae capsular polysaccharides (Pn PS ELISA). 007SP is a pooled serum from 278 healthy volunteers following vaccination with 23 valent pneumococcal polysaccharide vaccine to evaluate the coupling procedure. The beads coupled as per present invention was compared with a conventional method (i.e. modified amine coupling) method. It was observed that the obtained MFI’s data by metal activated beads were found to give higher sensitivities and higher dynamic range for serotypes 7F, 8, 10A and 24F which was observed higher MFI’s than the conventional method. Accordingly, the sensitivity of the assay for the serotypes has increased which has improved the efficiency of the assay.

See FIG. 4a and 4b (for serotype 7), FIG 5a and 5b (for serotype 8), FIG 6 (for serotype 10A), and FIG 7 (for serotype 24F) for details of MFI value against Type 7F, 8, 10A and 24F serotypes respectively in conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.

TABLE 6: STREPTOCOCCUS PNEUMONIAE/ PNEUMOCOCCAL TYPE 7F SEROTYPE conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.
Condition pH Concentration of PS Molecular size (kDa) Incubation time, temperature
A PBST (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 mg/mL 511 1 Hr/25deg C
B PBST (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 mg/mL 511 1.5 Hr/25deg C
C PBST (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 mg/mL 135 1.5 Hr/25deg C
D PBST (pH 6.5 ± 0.5), 6-7 0.5 to 1.0 mg/mL 135 1.5 Hr/25deg C

Inference: Four different conditions (A, B, C, D) were studied to obtain the maximum signal (MFI) of serotype-7F Saccharide coupled microspheres (PS coupled beads). During coupling, the pH range, Saccharide (Ps) Concentration, Molecular size and Incubation time was critically studied. From the above table the maximum MFI signal were observed for condition-D compared with other 3 conditions. Higher the MFI values is directly proportional directly proportional to the higher sensitivity of the assay, hence parameters mentioned in the condition-D are optimal for coupling serotype-7F antigen to microsphere beads.

TABLE 7: STREPTOCOCCUS PNEUMONIAE/ PNEUMOCOCCAL TYPE 8 SEROTYPE conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.
Condition pH Concentration of PS Molecular size
(kDa) Incubation time, temperature
A PBST (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 mg/mL 80 1 Hr/25deg C
B PBST (pH 5.5 ± 0.5), 4-5 0.5 to 1.0 mg/mL 80 1 Hr/25deg C
C PBST (pH 4.5 ± 0.5), 4-5 1.5 to 2.0 mg/mL 565 1 Hr/37deg C
D PBST (pH 4.5 ± 0.5), 4-5 1.5 to 2.0 mg/mL 565 2 Hr/37deg C

Inference: Four different conditions (A, B, C, D) were studied to obtain the maximum signal (MFI) of serotype-10A Saccharide coupled microspheres (PS coupled beads). During coupling, the pH range, Saccharide (Ps) Concentration, Molecular size and Incubation time was critically studied. From the above table the maximum MFI signal were observed for condition-D compared with other 3 conditions. Higher the MFI values is directly proportional to the higher sensitivity of the assay, hence parameters mentioned in the condition-D are optimal for coupling serotype-10A antigen to microsphere beads.

TABLE 8: STREPTOCOCCUS PNEUMONIAE/ PNEUMOCOCCAL TYPE 10A SEROTYPE conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.
Condition pH Concentration of PS Molecular size
(kDa) Incubation time, temperature
A PBST (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 mg/mL 990 1 Hr/25deg C
B PBST (pH 6.5 ± 0.5), 6-7 0.5 to 1.0 mg/mL 990 1 Hr/25deg C
C PBST (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 mg/mL 119 1 Hr/25deg C
D PBST (pH 6.5 ± 0.5), 6-7 0.5 to 1.0 mg/mL 119 1 Hr/25deg C

Inference: Four different conditions (A, B, C, D) were studied to obtain the maximum signal (MFI) of serotype-10A Saccharide coupled microspheres (PS coupled beads). During coupling, the pH range, Saccharide (Ps) Concentration, Molecular size and Incubation time was critically studied. From the above table the maximum MFI signal were observed for condition-D compared with other 3 conditions. Higher the MFI values is directly proportional to the higher sensitivity of the assay, hence parameters mentioned in the condition-D are optimal for coupling serotype-10A antigen to microsphere beads.

TABLE 9: STREPTOCOCCUS PNEUMONIAE/ PNEUMOCOCCAL TYPE 24F SEROTYPE conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.
Condition pH Concentration of PS Molecular size
(kDa) Incubation time, temperature
A PBST (pH 5.5 ± 0.5), 4-5 0.5 to 1.0 mg/mL 519 1 Hr/25deg C
B PBST (pH 6.5 ± 0.5), 6-7 0.5 to 1.0 mg/mL 519 1 Hr/37deg C
C PBST (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 mg/mL 81 1 Hr/25deg C
D PBST (pH 6.5 ± 0.5), 6-7 0.5 to 1.0 mg/mL 81 1 Hr/25deg C

Inference: Four different conditions (A, B, C, D) were studied to obtain the maximum signal (MFI) of serotype-24F Saccharide coupled microsphere beads. During coupling, the pH range, Saccharide (Ps) Concentration, Molecular size and Incubation time was critically studied. From the above table the maximum MFI signal were observed for condition-D compared with other 3 conditions. Higher the MFI values is directly proportional to the higher sensitivity of the assay, hence parameters mentioned in the condition-D are optimal for coupling serotype-24F antigen to microsphere beads.
EXAMPLE 7: STREPTOCOCCUS PNEUMONIAE/ PNEUMOCOCCAL SEROTYPES 3, 5, 6A, 6B, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 24F, 33F

The specific conditions applied to each pneumococcal serotype 3, 5, 6A, 6B, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 24F, 33F saccharides during dilution of the saccharide stock and further conjugation to the beads/ microspheres gave the optimal MFI’s to positive samples.
TABLE 10: STREPTOCOCCUS PNEUMONIAE/ PNEUMOCOCCAL SEROTYPES 3, 5, 6A, 6B, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 24F, 33F provides for conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.
Serotype Condition pH (PBST) PS Conc.
mg/mL Mol size (kDa) Incubation time
3 A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 911 1 Hr/ 25?
3 B (pH 5.5 ± 0.5), 5-6 1.0 to 1.5 911 1 Hr/ 25?
3 C (pH 5.5 ± 0.5), 5-6 1.5 to 2.0 191 1 Hr/ 25?
3 D (pH 6.5 ± 0.5), 6-7 2.0 to 2.5 191 1 Hr/ 25?
5 A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 75 1 Hr/ 25?
5 B (pH 5.5 ± 0.5), 5-6 1.0 to 1.5 75 2 Hr/ 25?
5 C (pH 6.5 ± 0.5), 6-7 1.5 to 2.0 75 2 Hr/ 25?
5 D (pH 6.5 ± 0.5), 6-7 2.0 to 2.5 75 2 Hr/ 37?
6A A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 551 1 Hr/ 25?
6A B (pH 4.5 ± 0.5), 4-5 1.0 to 1.5 551 1.5 Hr/ 25?
6A C (pH 4.5 ± 0.5), 4-5 1.0 to 1.5 551 2 Hr/ 25?
6A D (pH 4.5 ± 0.5), 4-5 1.0 to 1.5 511 2 Hr/ 37?
6B A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 706 1 Hr/ 25?
6B B (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 706 1.5 Hr/ 25?
6B C (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 107 1 Hr/ 25?
6B D (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 107 1.5 Hr/ 25?
9V A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 108 1 Hr/ 25?
9V B (pH 5.5 ± 0.5), 4-5 1.0 to 1.5 108 2 Hr/ 25?
9V C (pH 4.5 ± 0.5), 4-5 1.0 to 1.5 108 1 Hr/ 25?
9V D (pH 4.5 ± 0.5), 4-5 1.0 to 1.5 108 1.5 Hr/ 25?
10A A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 119 1 Hr/ 25?
10A B (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 119 1.5 Hr/ 25?
10A C (pH 6.5 ± 0.5), 6-7 0.5 to 1.0 119 1 Hr/ 25?
10A D (pH 6.5 ± 0.5), 6-7 0.5 to 1.0 119 1 Hr/ 25?
11A A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 100 1 Hr/ 25?
11A B (pH 5.5 ± 0.5), 5-6 1.0 to 1.5 100 1.5 Hr/ 25?
11A C (pH 5.5 ± 0.5), 5-6 1.5 to 2.0 100 1.5 Hr/ 25?
11A D (pH 5.5 ± 0.5), 5-6 2.0 to 2.5 100 1.5 Hr/ 25?
12F A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 99 1 Hr/ 25?
12F B (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 99 1.5 Hr/ 25?
12F C (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 99 1.5 Hr/ 25?
12F D (pH 6.5 ± 0.5), 6-7 0.5 to 1.0 99 1.5 Hr/ 25?
14 A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 143 1 Hr/ 25?
14 B (pH 5.5 ± 0.5), 5-6 1.0 to 1.5 143 1.5 Hr/ 25?
14 C (pH 5.5 ± 0.5), 5-6 1.5 to 2.0 143 1.5 Hr/ 25?
14 D (pH 6.5 ± 0.5), 6-7 2.0 to 2.5 143 1.5 Hr/ 25?
15B A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 136 1 Hr/ 25?
15B B (pH 5.5 ± 0.5), 5-6 1.0 to 1.5 136 1.5 Hr/ 25?
15B C (pH 5.5 ± 0.5), 5-6 1.5 to 2.0 136 1.5 Hr/ 25?
15B D (pH 5.5 ± 0.5), 5-6 2.0 to 2.5 136 1.5 Hr/ 25?
18C A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 79 1 Hr/ 25?
18C B (pH 5.5 ± 0.5), 5-6 1.0 to 1.5 79 1.5 Hr/ 25?
18C C (pH 5.5 ± 0.5), 5-6 1.5 to 2.0 79 1.5 Hr/ 25?
18C D (pH 6.5 ± 0.5), 6-7 2.0 to 2.5 79 1.5 Hr/ 25?
19A A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 91 1 Hr/ 25?
19A B (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 91 1 Hr/ 37?
19A C (pH 4.5 ± 0.5), 4-5 1.0 to 1.5 91 1.5 Hr/ 25?
19A D (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 91 1.5 Hr/ 25?
19F A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 583 1 Hr/ 25?
19F B (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 583 1.5 Hr/ 25?
19F C (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 85 1 Hr/ 25?
19F D (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 85 1.5 Hr/ 25?
22F A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 106 1 Hr/ 25?
22F B (pH 5.5 ± 0.5), 5-6 1.0 to 1.5 106 1 Hr/ 25?
22F C (pH 5.5 ± 0.5), 5-6 1.5 to 2.0 106 1 Hr/ 25?
22F D (pH 5.5 ± 0.5), 5-6 2.0 to 2.5 106 1 Hr/ 25?
23F A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 94 1 Hr/ 25?
23F B (pH 4.5 ± 0.5), 4-5 1.0 to 1.5 94 1 Hr/ 25?
23F C (pH 4.5 ± 0.5), 4-5 1.5 to 2.0 94 1 Hr/ 25?
23F D (pH 4.5 ± 0.5), 4-5 2.0 to 2.5 94 1 Hr/ 25?
24F A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 519 1 Hr/ 25?
24F B (pH 5.5 ± 0.5), 5-6 1.0 to 1.5 519 1 Hr/ 25?
24F C (pH 6.5 ± 0.5), 6-7 1.5 to 2.0 81 1 Hr/ 25?
24F D (pH 6.5 ± 0.5), 6-7 2.0 to 2.5 81 1 Hr/ 25?
33F A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 191 1 Hr/ 25?
33F B (pH 5.5 ± 0.5), 5-6 1.0 to 1.5 191 1 Hr/ 25?
33F C (pH 5.5 ± 0.5), 5-6 1.5 to 2.0 191 1 Hr/ 25?
33F D (pH 6.5 ± 0.5), 6-7 2.0 to 2.5 191 1 Hr/ 25?

See FIG. 8a to 8q for details of MFI value against pneumococcal serotypes 3, 5, 6A, 6B, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 24F, 33F respectively in conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.
Inference: Four different conditions (A, B, C, D) were studied to obtain the maximum signal (MFI) of type 3 Saccharide coupled microspheres (PS coupled beads). During coupling the pH range, Saccharide (Ps) Concentration, Molecular size and Incubation time was critically studied. From the above table the maximum MFI signal were observed for condition-D compared with other 3 conditions. If higher the MFI values proportional to the higher sensitivity of the assay, hence parameters mentioned in condition-D is mandatory during coupling of Pneumococcal serotype 3 antigen to microsphere beads.

EXAMPLE 8: HAEMOPHILUS INFLUENZAE SACCHARIDES

The specific conditions applied to Haemophilus influenzae saccharides during dilution of the saccharide stock and further conjugation to the beads/ microspheres gave the optimal MFI’s to positive samples.

TABLE 11: HAEMOPHILUS INFLUENZAE SACCHARIDES provides for conditions of pH and Concentration of Polysaccharides, molecular size (0.3KD for more than 50% of PRP), incubation temperature and time.

Condition pH of PBST Ps Conc. (µg/mL) Incubation time, temperature
A (pH 4.5 ± 0.5), 4-5 5 to 10 1 Hr/ 25?
B (pH 4.5 ± 0.5), 4-5 10 to 15 1.5 Hr/ 25?
C (pH 4.5 ± 0.5), 4-5 10 to 15 2 Hr/ 25?
D (pH 5.5 ± 0.5), 5-6 10 to 15 1 Hr/ 25?

See FIG. 9 for details of MFI value against Haemophilus influenzae saccharides in conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.

EXAMPLE 9: STABILITY DATA FOR SACCHARIDE COUPLED MICROSPHERE

The stability of coupled beads was monitored at two different temperature conditions i.e. 2-8°C (Real time) and 25°C (Accelerated). The CV of 30% reduction in the MFI was considered as sign off instability. The data clearly suggests that there is no change in the MFI values throughout the stability the overall % CV was observed <25 indicating the stability of saccharide coupled microspheres using the said process. The stability data for Streptococcus serotypes and Haemophilus type b are presented below.

TABLE 12: MFI's (Real time stability at 2°C to 8°C.) & MFI's (Accelerated at 25° C.) for Pneumococcal Saccharide coupled microspheres
SR= Serotype, ST= Standard, D0/ D5/ D10/ D15 = 0/ 5/ 10/ 15 Days duration of the stability test, MN = Mean, SD = Standard deviation, % CV = % of coefficient of variation, 1M/ 3M/ 6M = 1/ 3/ 6 Months duration of stability test, BC = Bead Control, S1 to S8 = Standards

The assay format was a multiplex in a nature. For antigen content assay format included reference standard, assay blank, Bead control and test sample. Generally, the standard concentration range required for all serotypes except 6B, 4400ng/ml to 34.38ng/ml, for 6B 8800ng/ml to 68.75ng/ml with serial two-fold dilutions as described below:
Prepared the series of standard from S1 to S8 with serial two fold dilutions as per the following table. Discarded 125 µl from the last well after mixing. Final total volume of each well was 125 µl.
TABLE 12A: STANDARD DILUTIONS
Standard Working standard (µL) Reagent G (µL) Concentration (ng/mL)
Each serotype except 6B serotype 6B
S1 250 µL working stock solution
- 4400 8800
S2 125 µL of S1 solution
125 2200 4400
S3 125 µL of S2 solution
125 1100 2200
S4 125 µL of S3 solution
125 550 1100
S5 125 µL of S4 solution
125 275 550
S6 125 µL of S5 solution 125 137.5 275
S7 125 µL of S6 solution 125 68.75 137.5
S8 125 µL of S7 solution 125 34.38 68.75

Bead Control: Bead Control is mixture of all 21 valent coupled beads with their respective antisera. As there is direct binding between antigen and respective antibodies which gives maximum MFI signal compared with S1 to S8. As this is a competitive inhibition assay the antibody mixture first incubated with standard and sample. After incubation, unbound antibody is further incubated with coupled beads. Hence, the signal from S1 to S8 appears increase in order and comparatively less with bead control. Hence, during antibody dilution optimization the dilutions were selected which showed at least minimum of 10% gradation between S8 and bead control to avoid saturation in standard curve formation. Hence, in every assay along with standard bead control value also monitored.

TABLE 12B: Stability data for Pneumococcal Saccharide coupled microspheres for Serotypes 1, 2 and 3.

The stability study for serotype 1 and 2 coupled beads were performed at real time (2°C to 8°C) condition and for serotype 1, 2 and 3 at Accelerated (25°C) condition. The MFI of reference standard from S1 to S8 were monitored and compiled. The difference in the MFI throughout the stability were calculated in the form of % coefficient of variance (CV) at each standard level. It was observed that serotype 1 and 2 at real time (2°C to 8°C) condition and for serotype 1, 2 and 3 at Accelerated (25°C) condition the %CV were less than 25%. Based on stability study it was concluded that the coupled beads were stable for 6M at real time condition and 15 days at accelerated condition.

SR ST 1M 3M 6M MN %CV D0 D5 D10 D15 MN %CV
Real time stability at 2°C to 8°C Accelerated at 25° C
1 S1 271 193 201 221 19 216 255 204 211 221 10
1 S2 556 390 405 450 20 409 487 373 391 415 12
1 S3 1029 660 735 808 24 732 720 725 701 720 2
1 S4 1503 1118 1252 1291 15 1228 1021 1146 1165 1140 8
1 S5 2287 1803 2037 2042 12 1716 1394 1744 1679 1633 10
1 S6 2950 2568 2723 2747 7 2408 1926 2251 2335 2230 10
1 S7 3840 3315 3509 3554 7 2950 2211 2634 3109 2726 15
1 S8 4765 4232 4047 4348 9 3578 2356 3118 3635 3172 19
1 BC - - - - - 5411 3209 4238 4602 4365 21
2 S1 465 439 465 456 3 495 608 550 482 534 11
2 S2 783 740 787 770 3 814 1073 866 788 885 15
2 S3 1319 1216 1290 1275 4 1361 1668 1475 1359 1466 10
2 S4 2018 1822 2186 2009 9 2227 2490 2292 2128 2284 7
2 S5 3382 3126 3739 3416 9 3298 4208 3561 3074 3535 14
2 S6 5141 5188 6480 5603 14 5490 7349 5441 4934 5804 18
2 S7 9450 8761 10331 9514 8 8590 10223 7638 8187 8659 13
2 S8 13863 13631 12786 13426 4 11233 12867 10978 11360 11609 7
2 BC - - - - - 13545 14293 13779 13146 13691 3
3 S1 - - - - - 186 203 191 183 191 4
3 S2 - - - - - 252 302.3 284 257 264 6
3 S3 - - - - - 359 376 426 369 383 8
3 S4 - - - - - 502 533 567 566 542 6
3 S5 - - - - - 629 672 761 681 686 8
3 S6 - - - - - 747 810 865 872 823 7
3 S7 - - - - - 871 829 991 966 914 8
3 S8 - - - - - 987 - 1158 1100 1082 8
3 BC - - - - - 1262 1104 1345 1237 1237 8

TABLE 12C: Stability data for Pneumococcal Saccharide coupled microspheres for Serotypes 4, 5, 6A, 6B, and 7F.
The stability study for serotype 4, 5, 6A, 6B and 7F coupled beads were performed at 2 different conditions i.e. real time (2°C to 8°C) and Accelerated (25°C). The MFI of reference standard from S1 to S8 were monitored and compiled. The difference in the MFI throughout the stability were calculated in the form of % coefficient of variance (CV) at each standard level. It was observed that for serotype 4, 5, 6A, 6B and 7F the obtained %CV were less than 25%. Based on stability study it was concluded that the coupled beads were stable for 6M at real time condition and 15 days at accelerated condition.
SR ST 1M 3M 6M MN %CV D0 D5 D10 D15 MN %CV
Real time stability at 2°C to 8°C Accelerated at 25° C
4 S1 134 129 144 136 6 162 175 122 135 148 16
4 S2 273 248 277 266 6 273 325 202 221 255 22
4 S3 536 444 550 510 11 471 511 447 445 468 7
4 S4 891 846 1016 917 10 866 748 752 704 767 9
4 S5 1515 1490 1802 1602 11 1270 1133 1198 1137 1185 5
4 S6 2239 2216 2479 2311 6 1965 1728 1795 1776 1816 6
4 S7 3092 3057 3233 3127 3 2461 2279 2240 2467 2362 5
4 S8 4103 3959 3879 3980 3 3316 2897 2974 3197 3096 6
4 BC - - - - - 9661 9140 8525 8364 8923 7
5 S1 171 142 141 151 11 158 206 138 151 163 18
5 S2 378 298 302 326 14 306 405 268 281 315 20
5 S3 766 553 603 641 17 604 654 563 580 600 7
5 S4 1241 993 1133 1122 11 1125 1028 1021 986 1040 6
5 S5 2065 1768 1927 1920 8 1694 144 1547 1586 1243 59
5 S6 2781 2630 2788 2733 3 2516 2020 2281 2404 2305 9
5 S7 3710 3411 3642 3588 4 3149 2375 2727 3094 2836 13
5 S8 4741 4486 4396 4541 4 3882 2540 3343 3918 3421 19
5 BC - - - - - 5886 3762 4729 5141 4880 18
6A S1 85 75 86 82 7 87 107 90 88 93 10
6A S2 173 138 156 155 11 148 193 153 143 159 14
6A S3 313 248 278 280 12 284 296 293 277 287 3
6A S4 522 415 525 487 13 499 466 535 452 488 8
6A S5 901 780 918 866 9 755 710 861 753 770 8
6A S6 1332 1187 1332 1284 7 1161 1077 1367 1118 1181 11
6A S7 1968 1754 2070 1931 8 1627 1459 1969 1665 1680 13
6A S8 3789 2995 3687 3490 12 2626 2637 3311 2633 2802 12
6A BC - - - - - 8610 8045 7473 8364 8123 6
6B S1 446 361 359 389 13 397 406 389 397 2
6B S2 839 643 642 708 16 662 811 667 642 695 11
6B S3 1318 1008 1045 1124 15 1065 1123 1085 1098 1093 2
6B S4 1867 1488 1611 1655 12 1604 1444 1598 1607 1563 5
6B S5 2613 2242 2334 2396 8 2132 1817 2110 2093 2038 7
6B S6 3214 2899 2916 3010 6 2708 2328 2787 2649 2618 8
6B S7 3975 3631 3550 3718 6 3187 2650 2942 3186 2991 9
6B S8 4470 4414 3948 4277 7 3631 3461 3753 3615 4
6B BC - - - - - 4973 3318 4174 4283 4187 16
7F S1 203 170 155 176 14 150 202 142 149 161 17
7F S2 432 345 317 365 16 291 388 258 267 301 20
7F S3 817 640 597 685 17 534 644 528 507 553 11
7F S4 1326 1077 1074 1159 12 944 934 906 874 914 3
7F S5 2181 1899 1869 1983 9 1449 1455 1451 1334 1422 4
7F S6 2863 2739 2653 2752 4 2241 1979 2068 2032 2080 5
7F S7 3836 3600 3515 3650 5 2811 2537 2618 2759 2681 5
7F S8 4990 4576 4017 4527 11 3342 2924 3203 3570 3260 8
7F BC - - - - - 6376 4928 5186 5430 5480 12

TABLE 12D: Stability data for Pneumococcal Saccharide coupled microspheres for Serotypes 8, 9V, and 10A.
The stability study for serotype 8, 9V, and 10A coupled beads were performed at 2 different conditions i.e. real time (2°C to 8°C) and Accelerated (25°C). The MFI of reference standard from S1 to S8 were monitored and compiled. The difference in the MFI throughout the stability were calculated in the form of % coefficient of variance (CV) at each standard level. It was observed that for serotype 8, 9V, and 10A the obtained %CV were less than 25%. Based on stability study it was concluded that the coupled beads were stable for 6M at real time condition and 15 days at accelerated condition.
SR ST 1M 3M 6M MN %CV D0 D5 D10 D15 MN %CV
Real time stability at 2°C to 8°C Accelerated at 25° C
8 S1 582 687 678 649 9 811 824 626 688 737 13
8 S2 1139 1181 1202 1174 3 1301 1502 1070 1203 1269 14
8 S3 1988 1986 2060 2011 2 2120 2337 1981 2077 2129 7
8 S4 3080 2873 3684 3212 13 3285 3440 2916 3350 3248 7
8 S5 4796 4719 5401 4972 8 4548 4988 4200 4859 4649 8
8 S6 6365 6617 7666 6883 10 6285 7049 5447 6451 6308 10
8 S7 8384 8404 9207 8665 5 7457 7217 6067 8349 7272 13
8 S8 9282 10120 10103 9835 5 8425 6508 9211 8048 17
8 BC - - - - - 11410 9537 8703 10954 10151 12
9V S1 752 625 774 717 11 781 1005 801 857 861 12
9V S2 875 721 917 838 12 929 1206 875 962 993 15
9V S3 1072 862 1044 993 11 1159 1325 1117 1193 1198 8
9V S4 1249 995 1325 1189 15 1485 1473 1343 1399 1425 5
9V S5 1577 1370 1657 1535 10 1629 1657 1616 1638 1635 1
9V S6 1836 1636 1995 1822 10 1999 1944 1989 1952 1971 1
9V S7 2246 2042 2385 2224 8 2205 2053 2160 2377 2199 6
9V S8 2578 2572 2732 2627 3 2490 2180 2487 2641 2450 8
9V BC - - - - - 3214 2672 2865 3051 2951 8
10A S1 700 679 735 705 4 706 801 710 739 7
10A S2 1201 1150 1329 1226 8 1118 1432 1169 1197 1229 11
10A S3 1831 1788 2064 1894 8 1811 1966 1857 1938 1893 4
10A S4 2649 2538 3120 2769 11 2775 2468 2732 2693 2667 5
10A S5 3556 3683 4269 3836 10 3448 3105 3401 3612 3392 6
10A S6 4346 4703 5309 4786 10 4404 3908 4286 4477 4269 6
10A S7 5214 5639 5951 5601 7 4711 3792 4439 5362 4576 14
10A S8 5691 6493 6272 6152 7 5486 4139 4951 5741 5079 14
10A BC - - - - - 6532 4844 5688 6006 5768 12

TABLE 12E: Stability data for Pneumococcal Saccharide coupled microspheres for Serotypes 11A, 12F, 14, 15B and 18C.
The stability study for serotype 11A, 12F, 14, 15B and 18C coupled beads were performed at 2 different conditions i.e. real time (2°C to 8°C) and Accelerated (25°C). The MFI of reference standard from S1 to S8 were monitored and compiled. The difference in the MFI throughout the stability were calculated in the form of % coefficient of variance (CV) at each standard level. It was observed that for serotype 11A, 12F, 14, 15B and 18C the obtained %CV were less than 25%. Based on stability study it was concluded that the coupled beads were stable for 6M at real time condition and 15 days at accelerated condition.
SR ST 1M 3M 6M MN %CV D0 D5 D10 D15 MN %CV
Real time stability at 2°C to 8°C Accelerated at 25° C
11A S1 409 385 406 400 3 598 717 586 563 616 11
11A S2 759 699 733 730 4 981 1283 965 941 1042 15
11A S3 1313 1130 1244 1229 8 1605 1853 1643 1615 1679 7
11A S4 1870 1745 2103 1906 10 2506 2398 2461 2411 2444 2
11A S5 2663 2683 3065 2804 8 3389 3145 3378 3312 3306 3
11A S6 3522 3601 4000 3707 7 4589 3882 4397 4454 4331 7
11A S7 4396 4714 5257 4789 9 4937 4383 5136 5433 4972 9
11A S8 5808 5929 6127 5955 3 6148 4926 5663 6499 5809 12
11A BC - - - - - 8188 5859 7025 7178 7062 14
12F S1 218 205 225 216 5 249 331 244 247 268 16
12F S2 412 395 469 425 9 475 635 468 469 512 16
12F S3 807 711 792 770 7 898 927 854 906 896 3
12F S4 1285 1164 1423 1291 10 1495 1406 1470 1499 1467 3
12F S5 2038 1935 2395 2123 11 2135 1922 2172 2077 2077 5
12F S6 2588 2895 3121 2868 9 2988 2671 3063 3046 2942 6
12F S7 3650 3694 4207 3850 8 3825 2817 3628 3887 3539 14
12F S8 4387 4785 4829 4667 5 4560 3191 4064 4717 4133 17
12F BC - - - - - 6328 4116 5388 5387 5305 17
14 S1 189 168 178 178 6 176 235 176 196 17
14 S2 378 323 327 343 9 309 394 255 282 310 19
14 S3 608 538 567 571 6 527 573 465 475 510 10
14 S4 953 851 983 929 7 858 793 721 784 789 7
14 S5 1491 1401 1534 1475 5 1181 1134 1028 1069 1103 6
14 S6 1900 1899 1986 1928 3 1661 1510 1435 1630 1559 7
14 S7 2464 2513 2620 2533 3 2010 1649 1539 1935 1783 13
14 S8 3039 2978 2780 2932 5 2318 1726 1975 2294 2078 14
14 BC - - - - - 4178 2918 3086 3259 3360 17
15B S1 224 215 249 229 8 236 294 234 237 250 12
15B S2 450 425 464 446 4 422 537 391 414 441 15
15B S3 773 674 828 758 10 742 735 740 733 737 1
15B S4 1137 1126 1347 1204 10 1127 1052 1102 1089 1092 3
15B S5 1667 1743 1983 1798 9 1546 1296 1497 1529 1467 8
15B S6 2153 2263 2483 2300 7 2115 1719 1904 2015 1938 9
15B S7 2618 2657 2851 2709 5 2474 1902 2153 2509 2259 13
15B S8 2954 3094 3113 3054 3 2707 1976 2477 2868 2507 16
15B BC - - - - - 3538 2485 2927 3231 3045 15
18C S1 92 74 70 78 15 75 91 74 78 79 10
18C S2 175 128 124 142 20 110 150 109 120 122 16
18C S3 234 183 181 199 15 176 187 192 183 185 4
18C S4 431 284 328 348 22 248 253 283 266 263 6
18C S5 620 388 455 488 25 347 351 404 347 362 8
18C S6 729 593 572 631 14 486 412 206 457 390 32
18C S7 950 677 669 765 21 575 454 566 587 545 11
18C S8 1063 861 717 881 20 612 497 615 695 605 13
18C BC - - - - - 1040 804 1043 908 949 12

TABLE 12F: Stability data for Pneumococcal Saccharide coupled microspheres for Serotypes 19A, 19F, and 22F.
The stability study for serotype 19A, 19F, and 22F coupled beads were performed at 2 different conditions i.e. real time (2°C to 8°C) and Accelerated (25°C). The MFI of reference standard from S1 to S8 were monitored and compiled. The difference in the MFI throughout the stability were calculated in the form of % coefficient of variance (CV) at each standard level. It was observed that for serotype 19A, 19F, and 22F the obtained %CV were less than 25%. Based on stability study it was concluded that the coupled beads were stable for 6M at real time condition and 15 days at accelerated condition
SR ST 1M 3M 6M MN %CV D0 D5 D10 D15 MN %CV
Real time stability at 2°C to 8°C Accelerated at 25° C
19A S1 267 231 245 248 7 242 310 235 233 255 14
19A S2 520 452 453 475 8 426 539 421 431 454 12
19A S3 922 741 779 814 12 743 778 764 770 764 2
19A S4 1253 1149 1234 1212 5 1159 1063 1112 1156 1123 4
19A S5 1883 1725 1846 1818 5 1617 1410 1532 1566 1531 6
19A S6 2251 2197 2265 2238 2 2111 1757 2039 1952 1965 8
19A S7 2927 2731 2761 2806 4 2402 1878 2227 2690 2299 15
19A S8 3359 3295 3081 3245 4 2742 2062 2609 2898 2578 14
19A BC - - - - - 4039 2559 3301 3409 3327 18
19F S1 337 283 305 308 9 317 402 313 305 334 14
19F S2 628 539 583 583 8 566 709 543 568 596 13
19F S3 1138 896 1020 1018 12 966 1025 1003 990 996 2
19F S4 1573 1362 1620 1518 9 1534 1382 1560 1498 1493 5
19F S5 2276 2011 2419 2235 9 2055 1813 2113 2161 2035 8
19F S6 2830 2808 3000 2879 4 2829 2372 2674 2684 2640 7
19F S7 3644 3438 2860 3314 12 3226 2679 3166 3537 3152 11
19F S8 4544 4389 4359 4431 2 3795 2793 3747 3982 3579 15
19F BC - - - - - 7523 4839 5191 6084 5909 20
22F S1 134 134 158 142 9 222 274 184 203 221 18
22F S2 282 251 297 276 8 366 505 359 358 397 18
22F S3 479 461 585 508 13 690 751 679 646 692 6
22F S4 796 774 1037 869 17 1209 1111 1103 1071 1124 5
22F S5 1371 1323 1655 1450 12 1666 1517 1598 1664 1611 4
22F S6 1827 1967 2351 2048 13 2376 2159 2544 2245 2331 7
22F S7 2578 2680 3264 2840 13 3073 2616 2737 3030 2864 8
22F S8 3706 3651 4485 3948 12 3628 3042 3585 3851 3527 10
22F BC - - - - - 9291 6159 8809 7595 7963 18

TABLE 12G: Stability data for Pneumococcal Saccharide coupled microspheres for Serotypes 23F, 24F, and 33F.
The stability study for serotype 23F, 24F, and 33F coupled beads were performed at 2 different conditions i.e. real time (2°C to 8°C) and Accelerated (25°C). The MFI of reference standard from S1 to S8 were monitored and compiled. The difference in the MFI throughout the stability were calculated in the form of % coefficient of variance (CV) at each standard level. It was observed that for serotype 23F, 24F, and 33F the obtained %CV were less than 25%. Based on stability study it was concluded that the coupled beads were stable for 6M at real time condition and 15 days at accelerated condition
SR ST 1M 3M 6M MN %CV D0 D5 D10 D15 MN %CV
Real time stability at 2°C to 8°C Accelerated at 25° C
23F S1 161 133 130 141 12 135 169 118 130 138 16
23F S2 328 258 259 282 14 237 326 226 234 256 18
23F S3 658 486 503 549 17 470 495 448 463 469 4
23F S4 1013 807 917 912 11 848 746 797 757 787 6
23F S5 1605 1468 1529 1534 4 1222 997 1158 1166 1136 9
23F S6 2105 2007 2085 2066 3 1822 1331 1564 1780 1624 14
23F S7 2661 2679 2517 2619 3 2179 1643 1909 2240 1993 14
23F S8 3534 3349 3056 3313 7 2654 1748 2431 2800 2408 19
23F BC - - - - - 4023 2530 3164 3443 3290 19
24F S1 249 225 290 255 13 374 467 356 365 390 13
24F S2 475 483 515 491 4 674 851 609 642 694 16
24F S3 951 774 951 892 11 1229 1281 1202 1160 1218 4
24F S4 1342 1208 1696 1415 18 1938 1764 1841 1833 1844 4
24F S5 2048 2113 2567 2243 13 2564 2412 2681 2514 2543 4
24F S6 2729 2943 3647 3106 15 3726 3301 3604 3611 3560 5
24F S7 3746 3909 4672 4109 12 4236 3566 4156 4696 4164 11
24F S8 4631 5195 5459 5095 8 5268 3807 5127 5460 4915 15
24F BC - - - - - 7672 5423 6736 6782 6653 14
33F S1 517 517 555 530 4 752 936 753 740 795 12
33F S2 849 889 953 897 6 1173 1479 1133 1131 1229 14
33F S3 1339 1296 1384 1339 3 1768 1992 1832 1819 1853 5
33F S4 1795 1775 2176 1915 12 2467 2468 2473 2541 2487 1
33F S5 2565 2524 3018 2702 10 3073 3137 3102 3326 3159 4
33F S6 3078 3453 3952 3494 13 4178 3875 4093 4062 4052 3
33F S7 4088 4228 5034 4450 11 4767 4242 4595 5150 4689 8
33F S8 4987 5551 5610 5383 6 5341 4761 5303 5873 5319 9
33F BC - - - - - 7201 5464 6455 6582 6425 11

TABLE 12H: Stability data for Haemophilus influenzae.
The stability study for Hib PRP coupled beads were performed at 2 different conditions i.e. real time (2°C to 8°C) and Accelerated (25°C). The MFI of reference standard from S1 to S8 were monitored and compiled. The difference in the MFI throughout the stability were calculated in the form of % coefficient of variance (CV) at each standard level. It was observed that for Hib PRP the obtained %CV were less than 25%. Based on stability study it was concluded that the coupled beads were stable for 8M at real time condition and 3M at accelerated condition
STD 1M 3M 8M Mean %CV Initial 1M 3M Mean %CV
Real time stability at 2°C to 8°C Accelerated at 25° C
S1 4627 4458 4366 4554 4 4609 4891 4458 4653 5
S2 2502 2456 2365 2376 6 2645 3047 2456 2716 11
S3 1255 1254 1090 1140 12 1418 1525 1254 1399 10
S4 673 672 514 574 21 698 737 672 702 5
S5 315 334 245 284 17 344 403 334 360 10
S6 152 172 126 141 18 169 199 172 180 9
S7 62 82 57 65 17 77 102 82 87 15
S8 31 37 31 30 22 36 69 37 47 40

EXAMPLE 10: ANTIGENIC INTEGRITY OF SACCHARIDE COUPLED MICROSPHERE

The antigenic integrity of the saccharide coupled microspheres/ antigens were assessed using serological reactivity with specific antibodies generated in the different species such as mice, rat, rabbit and human. The antigenic integrity was confirmed by using following approaches:

a) No cross reactions on beads: The negative sera value was near to baseline.
b) Dilutional linearity with positive sera: The dilution linearity was determined by monitoring the % gradation between each dilution. The acceptable percentage gradation during each dilution should be more than 10%.

Below table indicate that the saccharide coupled microspheres showed no cross reactions as all the MFI’s with negative sera MFI value was <100. Also, in the case of positive sera the beads showed good dilution linearity from 1:100 to 1:102400 dilution supporting that the epitopes of each antigen were unaffected during coupling process.

TABLE 13 (TABLE 13A to 13E): ANTIGENIC INTEGRITY OF SACCHARIDE COUPLED MICROSPHERES OF PNEUMOCOCCOL POLYSACCHARIDES OF SEROTYPES 1, 2, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 24F, 33F.

Table 13 (A to E) provides for the method evaluating immunogenicity of immunogenic composition where the saccharide coupled microsphere corresponding to the saccharide in the immunogenic composition are used.

TABLE 13A: For Pneumococcal Serotypes 1, 2 and 4
SR = Serotype, NS = Negative Sera
Parameter MFI Gradation (%) between each dilution
SR Species sera Mice Rabbit Rat Human Mice Rabbit Rat Human
1 NS 16 16 17 4 --- --- --- ---
1 1:100 9559 10999 13358 15400 --- --- --- ---
1 1:200 6699 8261 10069 6298 30 25 25 59
1 1:400 4869 6101 7587 4307 27 26 25 32
1 1:800 2693 5178 5269 1770 45 15 31 59
1 1:1600 1410 3692 3920 1261 48 29 26 29
1 1:3200 824 2707 2949 527 42 27 25 58
1 1:6400 388 1857 1984 297 53 31 33 44
1 1:12800 217 1262 1648 156 44 32 17 47
1 1:25600 126 816 1122 98 42 35 32 37
1 1:51200 69 406 537 67 46 50 52 32
1 1:102400 43 185 230 24 37 54 57 64
2 NS 8 13 14 4 --- --- --- ---
2 100 2049 22383 21062 27326 --- --- --- ---
2 200 1334 17894 16273 24308 35 20 23 11
2 400 973 13339 11940 20756 27 25 27 15
2 800 610 10087 7594 9674 37 24 36 53
2 1600 360 7127 5764 6372 41 29 24 34
2 3200 232 5298 4353 2975 36 26 24 53
2 6400 108 3855 3263 1553 54 27 25 48
2 12800 60 2768 2879 945 44 28 12 39
2 25600 35 1965 2110 536 42 29 27 43
2 51200 21 1122 1223 338 40 43 42 37
2 102400 12 575 544 155 43 49 56 54
4 NS 16 13 13 9 --- --- --- ---
4 100 6837 15247 12260 12997 --- --- --- ---
4 200 5144 10205 7186 10034 25 33 41 23
4 400 3863 6439 4056 5830 25 37 44 42
4 800 2345 4157 2162 2798 39 35 47 52
4 1600 1291 2179 1149 1779 45 48 47 36
4 3200 784 1259 557 958 39 42 52 46
4 6400 357 604 287 486 55 52 49 49
4 12800 184 288 183 266 48 52 36 45
4 25600 104 140 108 149 44 52 41 44
4 51200 54 62 48 66 48 56 56 56
4 102400 33 35 28 30 39 44 42 55

TABLE 13B: For Pneumococcal Serotypes 5, 6A, 6B and 7F
SR Species sera Mice Rabbit Rat Human Mice Rabbit Rat Human
5 NS 14 10 11 9 --- --- --- ---
5 100 7572 17177 19337 21039 --- --- --- ---
5 200 5207 12610 14506 10728 31 27 25 49
5 400 3625 9453 10663 6758 30 25 26 37
5 800 1940 7222 7054 3099 46 24 34 54
5 1600 851 5259 5263 1972 56 27 25 36
5 3200 557 3744 3556 822 35 29 32 58
5 6400 212 22911 2250 457 62 -512 37 44
5 12800 124 1461 1813 262 42 94 19 43
5 25600 71 817 1112 140 43 44 39 47
5 51200 38 331 479 87 46 59 57 38
5 102400 26 145 166 39 33 56 65 55
6A NS 10 18 18 10 --- --- --- ---
6A 100 2806 21802 11160 9670 --- --- --- ---
6A 200 1783 18371 8727 7650 36 16 22 21
6A 400 1206 16173 6085 4581 32 12 30 40
6A 800 611 14049 3899 2179 49 13 36 52
6A 1600 282 9915 2628 1359 54 29 33 38
6A 3200 164 6735 1622 704 42 32 38 48
6A 6400 68 4117 971 322 59 39 40 54
6A 12800 40 2643 757 179 41 36 22 44
6A 25600 25 1659 431 94 38 37 43 47
6A 51200 16 920 222 44 36 45 48 53
6A 102400 13 502 93 21 19 45 58 52
6B NS 11 47 47 11 --- --- --- ---
6B 100 5074 13258 13559 18222 --- --- --- ---
6B 200 3223 9423 10185 8454 36 29 25 54
6B 400 2263 7070 8070 7197 30 25 21 15
6B 800 1263 5900 6030 3050 44 17 25 58
6B 1600 573 4397 4716 2014 55 25 22 34
6B 3200 412 3505 3494 832 28 20 26 59
6B 6400 171 2655 2706 360 58 24 23 57
6B 12800 95 1931 2229 262 44 27 18 27
6B 25600 55 1309 1619 127 42 32 27 52
6B 51200 32 816 986 62 42 38 39 51
6B 102400 23 460 460 34 28 44 53 45
7F NS 15 15 15 17 --- --- --- ---
7F 100 9813 17952 20970 13051 --- --- --- ---
7F 200 8882 14193 17660 9777 9 21 16 25
7F 400 7636 10034 13987 5364 14 29 21 45
7F 800 5304 7120 9688 2443 31 29 31 54
7F 1600 3634 4539 6488 1528 31 36 33 37
7F 3200 2629 2821 3960 807 28 38 39 47
7F 6400 1510 1503 2094 417 43 47 47 48
7F 12800 905 769 1486 224 40 49 29 46
7F 25600 481 399 828 112 47 48 44 50
7F 51200 257 142 368 49 47 64 56 56
7F 102400 126 71 152 21 51 50 59 57

TABLE 13C: For Pneumococcal Serotypes 8, 9V, 10A, 11A
SR Species sera Mice Rabbit Rat Human Mice Rabbit Rat Human
8 NS 9 20 20 17 --- --- --- ---
8 100 1350 23847 23946 14182 --- --- --- ---
8 200 999 22621 22291 11238 26 5 7 21
8 400 553 17901 16970 6607 45 21 24 41
8 800 254 13322 11816 3161 54 26 30 52
8 1600 119 9761 8145 1987 53 27 31 37
8 3200 78 6621 5614 1071 35 32 31 46
8 6400 34 4685 3841 550 56 29 32 49
8 12800 23 2899 3045 312 32 38 21 43
8 25600 15 2118 2018 166 35 27 34 47
8 51200 12 1019 1143 78 20 52 43 53
8 102400 12 578 509 35 0 43 55 55
9V NS 14 19 19 6 --- --- --- ---
9V 100 5335 15518 14491 26041 --- --- --- ---
9V 200 4348 12626 10955 23384 19 19 24 10
9V 400 3503 10827 8405 15373 19 14 23 34
9V 800 2306 8237 5675 6962 34 24 32 55
9V 1600 1442 5803 4439 4462 37 30 22 36
9V 3200 934 3957 3035 2036 35 32 32 54
9V 6400 408 2742 2245 1148 56 31 26 44
9V 12800 242 2001 1965 642 41 27 12 44
9V 25600 130 1388 1429 358 46 31 27 44
9V 51200 60 775 810 236 54 44 43 34
9V 102400 34 427 383 108 43 45 53 54
10A NS 9 18 19 18 --- --- --- ---
10A 100 6294 22246 21230 13271 --- --- --- ---
10A 200 4233 16623 16084 10479 33 25 24 21
10A 400 3141 12411 10682 6016 26 25 34 43
10A 800 1745 9075 6590 2986 44 27 38 50
10A 1600 871 5349 4212 1858 50 41 36 38
10A 3200 537 3402 2263 999 38 36 46 46
10A 6400 210 1846 1159 486 61 46 49 51
10A 12800 114 976 829 265 46 47 28 45
10A 25600 59 527 416 145 48 46 50 45
10A 51200 35 222 181 68 41 58 56 53
10A 102400 18 118 80 32 49 47 56 53
11A NS 15 18 18 17 --- --- --- ---
11A 100 11656 23861 23923 15049 --- --- --- ---
11A 200 10694 20808 23523 8637 8 13 2 43
11A 400 8985 17201 19529 4839 16 17 17 44
11A 800 6334 14756 14438 2978 30 14 26 38
11A 1600 4211 10865 12536 1620 34 26 13 46
11A 3200 3321 8914 8982 883 21 18 28 45
11A 6400 1713 6648 6766 527 48 25 25 40
11A 12800 1123 5094 6338 258 34 23 6 51
11A 25600 580 3817 4471 91 48 25 29 65
11A 51200 307 2114 2729 62 47 45 39 32
11A 102400 157 1276 1317 31 49 40 52 50

TABLE 13D: For Pneumococcal Serotypes 12F, 14, 15B, 18C,
SR Species sera Mice Rabbit Rat Human Mice Rabbit Rat Human
12F NS 4 15 14 2 --- --- --- ---
12F 100 4387 15695 17720 13709 --- --- --- ---
12F 200 2391 13958 12997 8509 45 11 27 38
12F 400 1726 10942 9886 4812 28 22 24 43
12F 800 753 9136 7213 2862 56 17 27 41
12F 1600 352 6842 5795 1543 53 25 20 46
12F 3200 216 4898 4508 786 39 28 22 49
12F 6400 75 3550 3191 500 65 28 29 36
12F 12800 53 2232 2581 225 30 37 19 55
12F 25600 25 1392 1700 80 52 38 34 64
12F 51200 17 648 878 57 32 53 48 29
12F 102400 9 303 349 28 47 53 60 51
14 NS 15 16 17 14 --- --- --- ---
14 100 22490 23201 24261 24924 --- --- --- ---
14 200 19266 18259 21247 21508 14 21 12 14
14 400 21518 13229 15104 14928 -12 28 29 31
14 800 19386 9119 7829 6695 10 31 48 55
14 1600 15626 5708 4651 4257 19 37 41 36
14 3200 12974 3041 1986 2018 17 47 57 53
14 6400 7865 1764 985 1113 39 42 50 45
14 12800 6079 949 677 626 23 46 31 44
14 25600 4050 504 329 348 33 47 51 44
14 51200 2247 221 146 214 45 56 56 39
14 102400 1304 110 63 99 42 50 57 54
15B NS 9 18 19 3 --- --- --- ---
15B 100 3256 12046 11743 18097 --- --- --- ---
15B 200 2203 9147 8356 11524 32 24 29 36
15B 400 1590 6288 5491 7094 28 31 34 38
15B 800 948 4697 3061 4109 40 25 44 42
15B 1600 515 2755 1712 2279 46 41 44 45
15B 3200 357 1464 947 1127 31 47 45 51
15B 6400 166 859 476 683 54 41 50 39
15B 12800 98 454 325 362 41 47 32 47
15B 25600 56 245 195 135 43 46 40 63
15B 51200 31 115 95 86 45 53 52 36
15B 102400 19 63 52 42 39 45 45 51
18C NS 10 19 19 7 --- --- --- ---
18C 100 3268 3287 3458 25369 --- --- --- ---
18C 200 2235 2193 2119 23669 32 33 39 7
18C 400 1740 1580 1233 15624 22 28 42 34
18C 800 1301 1114 720 9211 25 29 42 41
18C 1600 826 509 471 4920 36 54 35 47
18C 3200 634 323 271 2492 23 36 42 49
18C 6400 354 194 164 1458 44 40 39 41
18C 12800 221 112 137 702 38 42 16 52
18C 25600 124 76 91 303 44 32 34 57
18C 51200 72 43 52 214 42 43 43 29
18C 102400 42 30 29 126 42 30 44 41

TABLE 13E: For Pneumococcal Serotypes 19A, 19F, 22F, 23F, 24F, 33F
SR Species sera Mice Rabbit Rat Human Mice Rabbit Rat Human
19A NS 14 14 15 15 --- --- --- ---
19A 100 9262 16223 15051 25021 --- --- --- ---
19A 200 7402 12199 11870 22824 20 25 21 9
19A 400 6617 10296 8851 16862 11 16 25 26
19A 800 4932 8488 5883 8017 25 18 34 52
19A 1600 3212 5552 4126 5346 35 35 30 33
19A 3200 2380 3550 2638 2393 26 36 36 55
19A 6400 1176 2384 1635 1309 51 33 38 45
19A 12800 728 1496 1221 776 38 37 25 41
19A 25600 380 915 853 449 48 39 30 42
19A 51200 189 441 408 292 50 52 52 35
19A 102400 86 199 178 127 54 55 56 57
19F NS 9 17 19 22 --- --- --- ---
19F 100 8436 17089 18148 16622 --- --- --- ---
19F 200 5605 13338 13470 6150 34 22 26 63
19F 400 4072 8954 9763 4410 27 33 28 28
19F 800 2157 7582 6068 1913 47 15 38 57
19F 1600 1137 4781 4330 1212 47 37 29 37
19F 3200 710 3485 3270 566 38 27 24 53
19F 6400 293 2382 225 309 59 32 93 45
19F 12800 178 1554 1838 144 39 35 -717 53
19F 25600 83 929 1170 86 53 40 36 40
19F 51200 49 434 597 47 42 53 49 45
19F 102400 24 211 228 21 51 51 62 55
22F NS 16 15 17 7 --- --- --- ---
22F 100 13123 22741 23765 23136 --- --- --- ---
22F 200 11800 20543 21698 13704 10 10 9 41
22F 400 11177 16673 18897 8473 5 19 13 38
22F 800 10090 12869 15837 5090 10 23 16 40
22F 1600 8626 9881 13785 2644 15 23 13 48
22F 3200 7449 7124 11245 1308 14 28 18 51
22F 6400 5466 4845 7399 816 27 32 34 38
22F 12800 3694 2875 6312 420 32 41 15 49
22F 25600 2657 2183 4163 136 28 24 34 68
22F 51200 1566 978 2033 90 41 55 51 34
22F 102400 998 535 978 47 36 45 52 48
23F NS 14 16 15 14 --- --- --- ---
23F 100 3221 14898 16421 15723 --- --- --- ---
23F 200 2000 10997 13505 7198 38 26 18 54
23F 400 1203 8337 9590 4339 40 24 29 40
23F 800 520 6058 6288 2123 57 27 34 51
23F 1600 238 3803 3972 1297 54 37 37 39
23F 3200 154 2366 2501 674 35 38 37 48
23F 6400 67 1364 1414 309 56 42 43 54
23F 12800 47 807 986 176 31 41 30 43
23F 25600 32 460 574 92 31 43 42 48
23F 51200 23 158 250 54 28 66 57 41
23F 102400 17 79 95 22 26 50 62 59
24F NS 10 21 19 16 --- --- --- ---
24F 100 5029 23586 23536 9782 --- --- --- ---
24F 200 3051 22799 19700 7529 39 3 16 23
24F 400 2064 18496 15186 4269 32 19 23 43
24F 800 950 14444 11404 2063 54 22 25 52
24F 1600 491 11401 9372 1308 48 21 18 37
24F 3200 284 9081 7561 694 42 20 19 47
24F 6400 112 6672 5833 340 61 27 23 51
24F 12800 77 4876 5063 182 31 27 13 46
24F 25600 40 3426 3859 95 48 30 24 48
24F 51200 27 2094 2292 45 33 39 41 53
24F 102400 17 1216 1252 20 37 42 45 56
33F NS 9 20 21 48 --- --- --- ---
33F 100 3760 21128 22505 22398 --- --- --- ---
33F 200 2171 17035 16919 15045 42 19 25 33
33F 400 1281 13558 13855 9537 41 20 18 37
33F 800 537 10987 10337 6267 58 19 25 34
33F 1600 249 9244 8356 3327 54 16 19 47
33F 3200 170 7151 6977 1820 32 23 17 45
33F 6400 78 5365 5181 1139 54 25 26 37
33F 12800 43 3921 4265 537 45 27 18 53
33F 25600 31 2550 2913 212 28 35 32 61
33F 51200 20 1353 1647 119 35 47 43 44
33F 102400 14 674 690 59 30 50 58 50

EXAMPLE 11: COMPARISON OF CONVENTIONAL METHOD OF COUPLING AND METHOD OF PRESENT INVENTION

Below is comparison of the present method of coupling compared with the conventional method of coupling, i.e. Two step carbodiimide reaction.

TABLE 14: CONVENTIONAL AND PRESENT METHOD OF COUPLING MICROSPHERE WITH SACCHARIDE

Parameters Conventional coupling Chemistries Method of Present invention
Concentration of Saccharides 2mg Variable based on Bacterial serotypes, for e.g. 0.5-1 mg/ml, 1.5-2.0mg/ml and 2.0-2.5mg/ml serotype-wise.
Size of the Saccharides Native Saccharides Requires specific Saccharides sizes to allow electro-static interactions
Principle of the method Two step carbodiimide reaction which involves chemical modification PnPS were conjugated to poly-l-lysine or oxidation chemistry Utilises metal polymer complexes binding of the target molecule through chelating to the electron donating groups of the ligand to be coupled. No modification of Saccharides
Conditions require for Bead Coupling The antigens (protein) to be coupled were prepared using conjugation buffer.
Protein molecule: 50 to 100 µg/ml), conjugation buffer pH 5.2 Antigen (polysaccharide) were diluted using different PBST buffers with different pH as listed in tables listed herein for different Bacterial saccharides and different serotypes of same bacteria
Incubation time and temperature Antigen (protein) were mixed with the activated beads and incubated at room temperature for 60 min Antigens (polysaccharide) were mixed with the activated beads and incubated at different temperature as listed in tables listed herein for different Bacterial saccharides and different serotypes of same bacteria
Blocking Agent 2.0% BSA 1.0% BSA
Cross linking agent (Cyanuric chloride) Required Not required
Toxic/ hygroscopic chemicals Yes No toxic materials used
Freshly prepared reagents Requires fresh reagents Not required
Bead Scale-up Up to 4 mL Up to 100 mL
Time duration for coupling 15-20 days 1-2 days
Time duration for Qualification 7-10 days 2-4 days
Microsphere Non-Magnetic/magnetic Non-Magnetic/Magnetic

EXAMPLE 12: PERFROMANCE OF SACCHARIDE COUPLED MICROSPHERES
The performance of the saccharide coupled microspheres of present invention was compared with saccharide coupled microspheres formed using known reagents and the result is summarized in Table 15. The performance was compared by calculating the % difference values between MFI’s. The saccharide coupled microspheres using the method of present invention showed higher MFI’s (more than 50%) for all the serotypes at each dilution level. The MFI difference with the method of present invention lead to higher sensitivity in the assay.
The comparison of the data indicates the unaffected epitopes during coupling by the two processes.
Inference: The performance is compared by calculating the % difference values between MFI’s. The microsphere/ bead coupled with the saccharides using the method as disclosed herein showed higher MFI’s (more than 50%) for all the microsphere coupled with saccharides of serotypes at each dilution level. This MFI difference with the said process will lead to higher sensitivity in the assay.
TABLE 15: SACCHARIDE COUPLED MICROSPHERE PERFORMANCE
SR= Serotypes, B = Blank, R = Dilution reference, P = Prior art, I = Applicants invention saccharide coupled microsphere, D = Percentage (%) Difference
SR R B 100 200 400 800 1600 3200 6400 12800 25600 51200 102400
1 P 4 15400 6298 4307 1770 1261 527 297 156 98 67 24
1 I 19 3430 2567 1455 598 441 190 87 52 45 29 22
1 D 78 59 66 66 65 64 71 67 54 57 8
2 P 4 27326 24308 20756 9674 6372 2975 1553 945 536 338 155
2 I 8 13506 10792 6133 2736 1742 942 480 279 156 72 34
2 D 51 56 70 72 73 68 69 70 71 79 78
4 P 9 12997 10034 5830 2798 1779 958 486 266 149 66 30
4 I 20 7740 4892 3221 2030 1340 595 584 287 73 59 35
4 D 40 51 45 27 25 38 -20 -8 51 11 -17
5 P 9 21039 10728 6758 3099 1972 822 457 262 140 87 39
5 I 19 10164 7942 4710 2221 1404 731 355 192 103 46 23
5 D 52 26 30 28 29 11 22 27 26 47 41
6A P 10 9670 7650 4581 2179 1359 704 322 179 94 44 21
6A I 20 6111 2717 1758 794 545 188 96 60 24 14 4
6A D 37 64 62 64 60 73 70 66 74 68 81
6B P 11 18222 8454 7197 3050 2014 832 360 262 127 62 34
6B I 10 8935 6908 3951 1946 1239 666 345 188 100 46 21
6B D 51 18 45 36 38 20 4 28 21 26 38
7F P 17 13051 9777 5364 2443 1528 807 417 224 112 49 21
7F I 14 4797 2886 1686 835 490 262 107 63 47 28 14
7F D 63 70 69 66 68 68 74 72 58 43 33
8 P 17 14182 11238 6607 3161 1987 1071 550 312 166 78 35
8 I 9 4433 2399 2871 1737 675 422 260 102 49 34 20
8 D 69 79 57 45 66 61 53 67 70 56 43
9V P 6 26041 23384 15373 6962 4462 2036 1148 642 358 236 108
9V I 9 16514 13147 7709 3768 2370 1293 661 367 200 96 42
9V D 37 44 50 46 47 36 42 43 44 59 61
10A P 18 13271 10479 6016 2986 1858 999 486 265 145 68 32
10A I 7 7436 5413 3254 2149 936 669 494 181 67 42 24
10A D 44 48 46 28 50 33 -2 32 54 38 25
11A P 17 15049 8637 4839 2978 1620 883 527 258 91 62 31
11A I 13 13438 10518 6121 2995 1867 1010 512 287 159 78 35
11A D 11 -22 -26 -1 -15 -14 3 -11 -75 -26 -13
12F P 2 13709 8509 4812 2862 1543 786 500 225 80 57 28
12F I 3 7941 5922 3372 1561 983 508 237 129 71 32 15
12F D 42 30 30 45 36 35 53 43 11 44 46
14 P 14 24924 21508 14928 6695 4257 2018 1113 626 348 214 99
14 I 9 24373 23767 13975 6370 3921 2077 1059 609 342 165 75
14 D 2 -11 6 5 8 -3 5 3 2 23 24
15B P 3 18097 11524 7094 4109 2279 1127 683 362 135 86 42
15B I 8 8262 6484 3812 1824 1173 602 301 162 86 40 18
15B D 54 44 46 56 49 47 56 55 36 53 57
18C P 7 25369 23669 15624 9211 4920 2492 1458 702 303 214 126
18C I 17 12371 9859 5979 3017 1889 1042 529 307 166 79 39
18C D 51 58 62 67 62 58 64 56 45 63 69
19A P 15 25021 22824 16862 8017 5346 2393 1309 776 449 292 127
19A I 9 10537 9121 6608 3886 2654 1507 845 497 293 146 69
19A D 58 60 61 52 50 37 35 36 35 50 46
19F P 22 16622 6150 4410 1913 1212 566 309 144 86 47 21
19F I 11 5904 4695 2826 1428 908 502 254 134 69 32 15
19F D 64 24 36 25 25 11 18 7 20 32 29
22F P 7 23136 13704 8473 5090 2644 1308 816 420 136 90 47
22F I 14 13862 10878 6051 2909 1851 958 488 264 145 67 31
22F D 40 21 29 43 30 27 40 37 -7 26 34
23F P 14 15723 7198 4339 2123 1297 674 309 176 92 54 22
23F I 11 10202 7846 4361 2072 1286 662 310 171 91 43 19
23F D 35 -9 -1 2 1 2 0 3 1 20 14
24F P 16 9782 7529 4269 2063 1308 694 340 182 95 45 20
24F I 34 4974 3049 2286 1457 795 441 274 157 56 32 29
24F D 49 60 46 29 39 36 19 14 41 29 -45
33F P 48 22398 15045 9537 6267 3327 1820 1139 537 212 119 59
33F I 11 17645 13648 7891 3783 2372 1293 659 363 199 90 41
33F D 21 9 17 40 29 29 42 32 6 24 31

EXAMPLE 13: COUPLING RATIO OF THE POLYSACCHARIDE TO BEAD IN FORMATION OF SACCHARIDE COUPLED MICROSPHERES

The specific ratios and assay conditions associated with the saccharide coupled microspheres is provided below.
The coupling ratio is dependent on the requirement of saccharide concentration and blank microsphere/ beads per 100µL. The ratio is calculated as follows:
Coupling ratio = No. of microspheres (Beads) (00 µl) / Concentration of Saccharides (PS) required for coupling (100µl).
The significance of maximum ratio indicates requirement of less concentration of saccharide for coupling.

TABLE 16: COUPLING RATIOS OF SACCHARIDES AND MICROSPHERES
Conc. = Conc. Of PS require for coupling for 100µl, BN = No. of Beads in millions, BL = No. of Beads per 100 µl.
The calculation uses Bead Stock solution of 100 µl beads for the Desired Bead conditions.
Number of beads is 1.25 million.
Type Desired polysaccharide conditions Desired bead conditions PS to bead ratio Bead recovery after coupling
Conc. (µg) Size of Ps (kDa) (PBST) Dilution of PnPS stocks Bead stock solution BN BL No. of Beads per µl Recovery (%)
1 50 to 100 80 (pH 5.5 ± 0.5), 5-6 100 µl beads 1.25 12500 125-250 10000 80
2 150 to 200 1011 (pH 4.5 ± 0.5), 4-5 100 µl beads 1.25 12500 62.5- 83.3 10200 82
4 50 to 100 87 (pH 6.5 ± 0.5), 6-7 100 µl beads 1.25 12500 125-250 9940 80
5 200 to 250 75 (pH 6.5 ± 0.5), 6-7 100 µl beads 1.25 12500 50-62.5 10500 84
6A 150 to 200 551 (pH 4.5 ± 0.5), 4-5 100 µl beads 1.25 12500 62.5- 83.3 9940 80
6B 50 to 100 107 (pH 5.5 ± 0.5), 5-6 100 µl beads 1.25 12500 125-250 10500 84
7F 50 to 100 135 (pH 6.5 ± 0.5), 6-7 100 µl beads 1.25 12500 125-250 11200 90
8 150 to 200 565 (pH 4.5 ± 0.5), 4-5 100 µl beads 1.25 12500 62.5- 83.3 10400 83
9V 50 to 100 108 (pH 5.5 ± 0.5), 5-6 100 µl beads 1.25 12500 125-250 10100 81
10A 50 to 100 119 (pH 6.5 ± 0.5), 6-7 100 µl beads 1.25 12500 125-250 11300 90
11A 200 to 250 100 (pH 5.5 ± 0.5), 5-6 100 µl beads 1.25 12500 50-62.5 9960 80
12F 50 to 100 99 (pH 6.5 ± 0.5), 6-7 100 µl beads 1.25 12500 125-250 10500 84
14 200 to 250 143 (pH 6.5 ± 0.5), 6-7 100 µl beads 1.25 12500 50-62.5 10800 86
15B 200 to 250 136 (pH 5.5 ± 0.5), 5-6 100 µl beads 1.25 12500 50-62.5 10400 83
18C 200 to 250 79 (pH 6.5 ± 0.5), 6-7 100 µl beads 1.25 12500 50-62.5 10700 86
19A 50 to 100 91 (pH 5.5 ± 0.5), 5-6 100 µl beads 1.25 12500 125-250 10000 80
19F 50 to 100 85 (pH 4.5 ± 0.5), 4-5 100 µl beads 1.25 12500 125-250 11400 91
22F 200 to 250 106 (pH 5.5 ± 0.5), 5-6 100 µl beads 1.25 12500 50-62.5 10300 82
23F 200 to 250 94 (pH 4.5 ± 0.5), 4-5 100 µl beads 1.25 12500 50-62.5 11500 92
24F 50 to 100 81 (pH 6.5 ± 0.5), 6-7 100 µl beads 1.25 12500 125-250 10100 81
33F 200 to 250 191 (pH 6.5 ± 0.5), 6-7 100 µl beads 1.25 12500 50-62.5 10700 86
3 200 to 250 191 (pH 6.5 ± 0.5), 6-7 100 µl beads 1.25 12500 50-62.5 11200 90
Hib 1 to 1.5 0.3 KD (pH 5.5 ± 0.5), 5-6 100 µl beads 1.25 12500 8333-12500 10500 84

EXAMPLE 14: NEISSERIA MENINGITIS SACCHARIDES

The specific conditions applied to Neisseria meningitis saccharides during dilution of the saccharide stock and further conjugation to the beads/ microspheres gave the optimal MFI’s to positive samples.

TABLE 17: NEISSERIA MENINGITIS SACCHARIDES provides for conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.
Serotype Condition pH (PBST) Saccharide Conc. (mg/mL) Mol size (kDa) Time, Temperature
Men A A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 136 1 Hr/ 25?
Men A B (pH 5.5 ± 0.5), 5-6 1.0 to 1.5 136 1 Hr/ 25?
Men A C (pH 5.5 ± 0.5), 5-6 1.5 to 2.0 136 1 Hr/ 25?
Men A D (pH 6.5 ± 0.5), 6-7 0.5 to 1.0 136 1.5 Hr/25?
Men C A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 116 1 Hr/ 25?
Men C B (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 116 1 Hr/37?
Men C C (pH 4.5 ± 0.5), 4-5 1.0 to 1.5 116 1.5 Hr/25?
Men C D (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 116 1.5 Hr/25?
Men W A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 123 1 Hr/37?
Men W B (pH 5.5 ± 0.5), 5-6 1.0 to 1.5 123 2 Hr/25?
Men W C (pH 6.5 ± 0.5), 6-7 1.5 to 2.0 123 2 Hr/25?
Men W D (pH 6.5 ± 0.5), 6-7 0.5 to 1.0 123 1.5 Hr/25?
Men X A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 116 1 Hr/ 25?
Men X B (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 116 1 Hr/25?
Men X C (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 116 1 Hr/ 25?
Men X D (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 116 1.5Hr/37?
Men Y A (pH 4.5 ± 0.5), 4-5 0.5 to 1.0 106 1 Hr/ 25?
Men Y B (pH 5.5 ± 0.5), 5-6 0.5 to 1.0 106 1 Hr/ 25?
Men Y C (pH 6.5 ± 0.5), 6-7 0.5 to 1.0 106 1 Hr/ 25?
Men Y D (pH 6.5 ± 0.5), 6-7 0.5 to 1.0 106 1.5 Hr/37?

See FIG. 10 (10a to 10e) for details of MFI value against Neisseria meningitis saccharides of serotypes (A, C, W, X, Y respectively) in conditions of pH and Concentration of Polysaccharides, molecular size, incubation temperature and time.

KEY FEATURES
• The method provides non-covalent coupling of polysaccharides to microspheres to form couples under specific reaction conditions where the structure of polysaccharides remains unaffected.
• The method includes specific reaction condition (Ps antigen specific) which allows efficient coupling of Ps on to the polystyrene beads.
• The method includes specific combination of concentration of PnPS, size of Ps, reaction time, pH of buffer to allow development of electron donor sites which allow electrostatic attachment of Ps on to activated beads.
• Method is associated with efficient coupling as an outcome of correct saccharide size, concentration and pH.
• Efficiently coupled beads have following characteristics:
a) Higher sensitivity
b) Less noise/background
c) Good precision (CV among duplicates) and accuracy (back fitted recoveries)
d) Stable saccharide microsphere coupling
• Method is aqueous based and highly stable and does not require fresh use of toxic chemicals
• Method is fast and scalable to higher volumes
• Method allows stable saccharide coupled microsphere/ coupled beads that are robust and carries native antigens with no loss of activity
• Saccharide coupled microsphere formed allows higher sensitivity of detection leading to development of assays with higher sensitivity.
• Saccharide coupled microspheres showed good dilution linearity and no cross reactions
• The sensitivity of the assay for the serotypes is increased with the saccharide coupled microsphere that subsequently lead to improved efficiency of the assays for vaccine potency determination.
• Obtained saccharide coupled beads are stable at different temperatures.
• The saccharide coupled microspheres is prepared using an activation buffer to activate the metal
• The method provides following advantages
o Allows formation of stable saccharide coupled beads which are robust and carries saccharide with no loss of activity
o Highly stable aqueous based reaction.

ADVANTAGES:
• Method is simple and easy to perform is replicable with different Saccharides and buffers.
• Method is cost effective and do not need toxic compounds.

APPLICATIONS
• Method of forming the saccharide coupled microsphere is applicable for both magnetic and non-magnetic microspheres.
• Method of forming the saccharide coupled microsphere is applicable for Saccharides of different microorganisms, bacteria, especially for streptococcal, meningococcal, Haemophilus, Salmonella saccharides.
• Method of forming the saccharide coupled microsphere facilitates forming couples that are usable in serological testing of human and animals’ sera samples, for testing of vaccines, for developing serological testing of vaccines in human sera samples.
• The method is applicable to develop immunoassays for Pneumococcal Purified Ps (PCV10, PCV21, PCV22), as well as other conjugate vaccines (Hib, Meningococcal, Typhi/Paratyphi and the like)
• Saccharide coupled microspheres can be used for
o Antigen content estimation of PCV 10 (Pneumosil), PCV 21, PCV22.
o Serological testing of PCV 10, PCV 21, and PCV22 for use with animal sera and human sera samples
o Identity testing of PCV 10, PCV-21 PCV22, Hib, samples. ,CLAIMS:WE CLAIM:
1. A method of coupling a saccharide to a microsphere to obtain a saccharide coupled microsphere, the method comprising:
a. providing the microsphere;
b. providing the saccharide;
c. diluting the saccharide with a buffer at pH in range of 3.0 to 9.0; and
d. mixing the microsphere with the saccharide to form the saccharide coupled microsphere;
wherein the mixing further includes incubation at temperature in range of 20? to 40? for incubation time in range of 30 mins to 180 mins.

2. The method as claimed in claim 1, wherein
the mixing is performed with the coupling ratio of the microsphere to the saccharide is in range of 50 to 12500, and/ or
optionally, the microsphere is activated with a bead reagent before mixing with the saccharide, wherein the bead reagent includes metal ions,
optionally, the saccharide coupled microsphere is formed by non-covalent electrostatic coupling, and/ or
optionally, the saccharide remains intact, retains epitope confirmation and the saccharide coupled microsphere is stable.

3. The method as claimed in claim 1, wherein the buffer includes phosphate buffered saline with tween (PBST) buffer, (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES) buffer, tris-aminomethane (Tris) buffer, 2-(N-morpholino)ethane sulfonic acid (MES) buffer, and 3-(N-morpholino) propane sulfonic acid (MOPS) buffer.

4. The method as claimed in any one of claims 1 to 3, wherein the saccharide is bacterial saccharide including Group A Streptococcus, Group B Streptococcus, Streptococcus pneumoniae/ pneumococcus, Haemophilus bacteria, Haemophilus influenzae bacteria, Haemophilus influenzae type b bacteria (Hib), Salmonella, Typhoidal salmonella, Non-typhoidal salmonella, Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi, Streptococcus pyogenes, Streptococcus agalactiae, Shigella, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Escherichia coli, or Neisseria meningitidis/ meningococcus saccharide.

5. The method as claimed in claim 4, wherein the saccharide is Streptococcus pneumoniae saccharide.

6. The method as claimed in claim 5, wherein the Streptococcus pneumoniae saccharide has a molecular size in range of 50 kDa to 3000 kDa, is diluted with PBST buffer at a pH in range of 3.0 to 9.0 and has concentration of 0.05 mg/mL to 50.0 mg/mL.

7. The method as claimed in any one of claims 5 to 6, wherein the Streptococcus pneumoniae saccharide is mixed with the microsphere and incubated at temperature of 23? to 39?, for incubation time of 60 mins to 120 mins.

8. The method as claimed in any one of claims 5 to 7, wherein Streptococcus pneumoniae polysaccharide is selected from serotypes 1, 2, 3, 4, 5, 6, 6A, 6B, 6C, 6D, 7, 7B, 7C, 7F, 8, 9, 9A, 9L, 9N, 9V, 10, 10A, 10B, 10C, 10F, 10X, 11, 11A, 11B, 11C, 11F, 12, 12A, 12B, 12F, 13, 14, 15, 15A, 15B, 15BC, 15C, 15F, 16, 16F, 17, 17A, 17F, 18, 18A, 18B, 18C, 18F, 19, 19A, 19B, 19F, 20, 20A, 20B, 20F, 21, 22, 22A, 22F, 23, 23A, 23B, 23F, 24, 24F, 25, 25F, 26, 27, 28, 28A, 28F, 29, 30, 31, 32, 33, 33A, 33B, 33C, 33D, 33F, 34, 35, 35A, 35B, 35C, 35D, 35F, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, and/ or 48.

9. The method as claimed in claim 8, wherein the Streptococcus pneumoniae polysaccharide is selected from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 24F and/ or 33F.

10. The method as claimed in claim 4, wherein the saccharide is Haemophilus influenzae type b bacteria (Hib) saccharide.

11. The method as claimed in claim 10, wherein the Haemophilus influenzae type b bacteria (Hib) saccharide has a molecular size in range of 0.05 kDa to 5 kDa, is diluted with PBST buffer at a pH in range of 4 to 6 and has concentration of 1.0 µg/mL to 50.0 µg/mL.

12. The method as claimed in any one of claims 10 to 11, wherein the Haemophilus influenzae type b bacteria (Hib) saccharide is mixed with the microsphere and incubated at temperature of 20? to 30?, for incubation time of 60 mins to 120 mins.

13. The method as claimed in claim 4, wherein the saccharide is Neisseria meningitidis saccharide.

14. The method as claimed in claim 13, wherein the saccharide is Neisseria meningitidis saccharide serotypes selected from meningococcal serotypes A (type I and III), B (type II), B6, B16, C (type II-alpha), D (type IV), Z’/ E, E29, H, I, K, K454, L, M, W135, X, Y, Z.

15. The method as claimed in any one of claims 13 to 14, wherein the Neisseria meningitidis saccharide has a molecular size in range of 75 kDa to 3000 kDa, is diluted with PBST buffer at a pH in range of 4 to 7 and has concentration of 0.01 mg/mL to 10.0 mg/mL.

16. The method as claimed in any one of claims 13 to 15, wherein the Neisseria meningitidis saccharide is mixed with the microsphere and incubated at temperature of 20 to 40?, for incubation time of 60 mins to 120 mins.

17. A saccharide coupled microsphere obtained by the method as claimed in any one of the claims 1 to 16.

18. The saccharide coupled microsphere as claimed in claim 17, wherein the saccharide coupled microsphere has Mean Florescence intensity/ MFI value in the range of 200 to 20000.

19. The saccharide coupled microsphere as claimed in any one of claims 17 to 18, wherein the saccharide coupled microsphere is a Streptococcus pneumoniae saccharide coupled microsphere.

20. The saccharide coupled microsphere as claimed in any one of claims 17 to 18, wherein the saccharide coupled microsphere is a Haemophilus influenzae type b bacteria (Hib) saccharide coupled microsphere.
21. The saccharide coupled microsphere as claimed in any one of claims 17 to 18, wherein the saccharide coupled microsphere is a Neisseria meningitidis saccharide coupled microsphere.

22. A method of evaluating immunogenicity of immunogenic composition, the method comprising;
a. providing a test sample corresponding to the saccharide in the immunogenic composition;
b. providing the saccharide coupled microsphere corresponding to the saccharide in the immunogenic composition; and
c. evaluating the immunogenicity of the immunogenic composition by antigen content determination, or estimating antibody concentration (IgG), or identity assay, free polysaccharide estimation using the test sample and the saccharide coupled microsphere,
wherein the saccharide coupled microsphere is obtained by the method as claimed in any one of claims 1 to 3.

23. An antigen content determination method using the saccharide coupled microsphere obtained by the method as claimed in any one of claims 1 to 3.

24. An identity assay method using the saccharide coupled microsphere obtained by the method as claimed in any one of claims 1 to 3.

25. A free polysaccharide estimation method using the saccharide coupled microsphere obtained by the method as claimed in any one of claims 1 to 3.

26. A method of estimating antibody concentration (IgG) in sera sample, the method using the saccharide coupled microsphere obtained by the method as claimed in any one of claims 1 to 3.

27. The method of estimating antibody concentration (IgG) in sera sample as claimed in claim 26, wherein the sera sample includes a human sera sample and an animal sera sample.

28. An apparatus comprising the saccharide coupled microsphere obtained by the method as claimed in any one of claims 1 to 3.

29. The saccharide coupled microsphere as and when used in assay or evaluation of a vaccine or an immunogenic composition, wherein the saccharide coupled microsphere is obtained by the method as claimed in any one of claims 1 to 3.
Dated this 02nd Day of April 2025

_______________________________
MOHAN RAJKUMAR DEWAN, IN/PA – 25
OF R. K. DEWAN & CO.
AUTHORIZED AGENT OF APPLICANT