FORM-2
THE PATENT ACT,1970
(39 OF 1970)
AND
THE PATENT RULES, 2003
(As Amended)
COMPLETE SPECIFICATION (See section 10;rule 13)
"PROCESS FOR STABILIZATION OF BACTERIAL PROTEIN AND ITS USE IN VACCINE MANUFACTURING THEREOF"
SERUM INSTITUTE OF INDIA PVT LTD., a corporation organized and existing under the laws of India, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028, Maharashtra, India.
The following specification particularly describes the invention and the manner in which it is to be performed:

FIELD OF THE INVENTION
The present invention relates to field of Biotechnology and is applicable to vaccine manufacturing industry.
BACKGROUND OF THE INVENTION
The background information herein below relates to the present disclosure but is not necessarily prior art.
Formaldehyde treatment was introduced in 1920’s to detoxify diphtheria toxin (bacterial protein) for vaccine use. Since its introduction, the method has been extensively used for inactivation of other bacterial toxins and preparation of many bacterial and viral vaccines. Such vaccines have proved to be very efficacious, and their use has been instrumental in the eradication of several diseases.
Bacterial toxins such as Cholera toxin, Diphtheria toxin, CRM197, Pertussis toxin, E. coli heat-labile toxin LT, Shiga toxin, Pseudomonas Exotoxin A, Botulinum toxin, Tetanus toxin, Anthrax toxin LF, Bordetella pertussis AC toxin, Staphylococcus aureus Exfoliatin B, etc. are known to be successfully inactivated/detoxified using formaldehyde treatment in the past. Many of these toxoids are effective immunizing agents against respective pathogens that are part of the individual and combination/multivalent vaccines. Diphtheria and Tetanus Toxoids are not only effectively used as one of the component in new generation multivalent vaccines like Bivalent, Trivalent, Quadrivalent, Pentavalent, Hexavalent and/or Heptavalent vaccines, but are also used as a carrier protein to boost immunogenicity of bacterial polysaccharides in case of conjugate vaccines against Neisseria meningitidis and Streptococcus pneumoniae. Polysaccharide – carrier protein conjugate vaccines are preferred over conventional polysaccharide vaccines given the advantage of induction of T cell dependent long lasting immunological memory against encapsulated bacteria.
Toxoids used for vaccine preparation should be purified in order to eliminate constituents of growth medium and metabolites which tend to provoke undesirable reactions. Conventionally, commercial purification of toxoid for vaccine preparation is carried out by fractional precipitation using ammonium sulphate (WHO Manual for the production and control of Vaccines. Tetanus toxoid. BLG/UNDP/77.2 Rev1.). Tetanus toxoid purified by this

method very well achieves WHO requirement of purity which is 1000 Lf/mg of protein nitrogen. However, this method is not designed to remove aggregates and most importantly the criterion of purity determination doesn’t consider presence or absence of low molecular weight impurities (Doshi J.B. et. al. 2003).
Although, detoxification of toxins by treatment with Formaldehyde (WHO recommended method) is highly effective in production of detoxified toxin (toxoid), it also results in extensive modification and heterogeneity of the molecule leading to complexities in product characterization and acts as hindrance for accessibility to some amino acids during subsequent conjugation reaction. It has been reported that upon formaldehyde inactivation of toxin and purification of toxoid thereof, a residual amount of formalin remains bound to the toxoid in the form of an active electrofile (reactive imine groups). Such active imine groups are prone for further cross-linking with other amino acid of same or other protein molecules, leading to formation of multimeric/aggregated toxoid and loss of the conformational properties, further altering the antigenic and immunogenic properties.
Latham W. C. and his group (1965) first evaluated use of chromatographic technique for the purification of tetanus toxoid. The technique was further modified by the same team for the large scale purification of toxoid in 1967, wherein they used sephadex G-100 as a gel-filtration media, recovery was just 50% and toxoid had 50-55% monomeric content. Immuno-purification of tetanus toxoid using affinity chromatography was tried by M. Hughes et. al., in 1974. Sheppard and his group employed similar technique for the purification of tetanus toxin in 1987. In both the cases, purity achieved was very high as well as recovery was also good, but said methods could not get acceptance due to its limitations pertaining to use at large scale and high production cost. In 1997 Vancetto M.D.C. and his group (Vancetto M.D.C.; et. al., 1997) reported a method wherein, first toxin was ultrafiltered followed by formalin treatment, diafiltration and lastly gel filtration using Sephadex G-50. Though method achieved about 92% recovery, the final average purity was not substantially high as compared to that reported earlier by Latham W.C., 1965. In 1999 and 2001 Prado S. M. and his group reported sephacryl-HR-100 and HR-200 as a better media for the chromatographic purification of the toxoid (Prado S. M. et.al; 1999 and 2001). They employed this technique only at small scale along with ultrafiltration by tangential flow filtration method but reported that gel-filtration chromatography shows extremely poor separation Very recently, hydrophobic chromatography and immobilized metal affinity chromatography has been tried for the purification of tetanus toxoid. Results of hydrophobic chromatography were similar to

that of ammonium sulphate precipitation method while results of immobilized metal affinity chromatography were very poor. Massachusetts Health Research Institute has previously reported purified Tetanus Toxoid with 60% monomer by using Hydrophobic Interaction Chromatography (Phenyl sepharose) or Hydrophobic Interaction Chromatography followed by Ion exchange (DEAE Sepharose).
Attempts have been made to avoid or remove aggregates during the preparation of the toxoid by employing various purification schemes like centrifugation, chromatography columns, and other techniques, but during storage cross-linking/aggregation event still continues, due to residual reactive imine groups which reacts with second amino acid (Refer fig no.1, Rappuoli R., 1997; Schwendeman S.P., et al.,1995), rendering the molecule less favourable for further use for conjugation purpose. This phenomenon is a limiting factor for the use of highly purified tetanus toxoid (>80% monomer) for longer duration as within few months monomer content reduces below 80%. Though, known methods are capable of recovering/ regaining the conformational properties, a decrease in the antigenic and immunogenic properties is still observed.
Important pre-requisite criteria, shared with all carrier proteins, are cGMP manufacturability at large scale with regards to yield, production cost and quality of the product. It should be possible for large scale production of protein having high yield using a process that is viable in a cGMP environment. Further, the process should also be capable of producing the proteins having a desirable purity, such as >80% monomer content. Carrier protein should contain a sufficient number of exposed amino acids targeted for the selected conjugation process; should be stable and have good solubility in the buffers and concentrations at which polysaccharide-carrier protein conjugation reactions take place. Carrier proteins should be sufficiently surface exposed to allow efficient in vitro glyco-conjugation.
The licensed conjugate vaccines are composed of a carbohydrate moiety i.e. antigen and is covalently linked to a carrier protein. In polysaccharide-carrier protein conjugate vaccine, due consideration is given to overall molecular size of the conjugate molecule; exposure/availability of the antigenic sites; pre-exposure or co-exposure to a protein carrier; dual role of protein as carrier and protective antigen; and new ways of presenting polysaccharide antigens to immune system.
It has been reported that conjugation of dimerized, or aggregated carrier protein to a polysaccharide antigen may affect:

i. Efficiency of the conjugation process, presumably through steric hindrance at the
protein surface; ii. Molecular size of the conjugate molecule, resulting into uncertainty in predicting efficacy and quality control parameters of the polysaccharide- protein conjugate vaccine; iii. Loss of conformational properties, resulting into decrease in antigenic and immunogenic properties of the polysaccharide-protein conjugate vaccine.
Applicant has found that the aggregated and dimeric tetanus toxoid is responsible for inconsistency in size of conjugate, lower conjugation efficiency, poor stability and lower potency for polysaccharide-protein conjugate vaccine(s), wherein tetanus toxoid is used as a carrier protein. Further, it is desirable that a low amount of protein should be present in a conjugate and can elicit a high immune response.
It has also been reported that formation of aggregates with repetitive epitopes might induce anti-drug antibodies (ADAs) leading to tolerance breakdown; hence use of aggregated carrier protein in vaccines is undesirable.
There is a long felt need for an improved process of stabilization of monomer content of proteins (preferably, toxoids) during storage for preventing formation of multimeric / aggregated proteins, and process for preparation of polysaccharide–carrier protein conjugate vaccine elucidating better vaccine manufacturing consistency, stability and potency in vaccine.

Summary of Invention:
Present invention provides composition and process to address the issue of short shelf life of proteins that are to be utilized as either carrier protein for polysaccharide-protein conjugation or as an antigen of monovalent / multivalent / combination vaccines. Present invention particularly provides composition and process for stabilization of proteins during storage, wherein proteins with minimum aggregates/dimers are obtained, in order to manufacture efficacious, potent, stable vaccines with improved homogeneity.
The Present invention relates to a composition and process of stabilization of proteins, and its use in preparation of ‘polysaccharide-carrier protein’ conjugate vaccines. Additionally proteins of the Present invention can also be used as an antigen for monovalent and multivalent/combination vaccines.
The Present invention prevents formation of multimeric/aggregated proteins during storage and thus prevents loss of conformational properties which alters the antigenic and immunogenic properties of the vaccine antigen, thereby providing a stable, homogeneous vaccine. The vaccine prepared according to present invention results into increased yields of polysaccharide-protein conjugate due to improved conjugation efficiency. The invention enables better predictability, safety, consistency, stability and potency of vaccines and minimizes induction of anti-drug antibodies (ADAs) against the vaccine.

Brief Description of the Accompanying Drawing:
The present invention will now be described with the help of the accompanying drawing, in which:
Figure 1: SDS PAGE Analysis of purified monomeric toxoid;
Figure 2: Stability Analysis of purified monomeric Tetanus toxoid without stabilizers A) Graphical Analysis & B) HPLC Analysis;
Figure 3: Stability Analysis of purified monomeric Tetanus toxoid in presence of Glycine as stabilizers A) Graphical Analysis & B) HPLC Analysis;
Figure 4: Stability Analysis of purified monomeric Tetanus toxoid in presence of Lysine as stabilizers A) Graphical Analysis & B) HPLC Analysis;
Figure 5: Stability Analysis of purified monomeric Tetanus toxoid in presence of Tween-80 as stabilizers A) Graphical Analysis & B) HPLC Analysis;
Figure 6: Stability Analysis of purified monomeric Tetanus toxoid in presence of Histidine as stabilizers A) Graphical Analysis & B) HPLC Analysis;
Figure 7: Reducing SDS PAGE Analysis of Tetanus toxoid (stabilized) using 8% gel;
Figure 8: % Monomer Content of Tetanus Toxoid stored at 2-8 °C with varying concentration of Histidine as stabilizer; and
Figure 9: % Monomer Content of Tetanus Toxoid stored at 37 °C with varying concentration of Histidine as stabilizer.

Description of Invention:
Geographical origin of the biological material used in present inventions is as follows:

Serial # Name of Organism Source
1 Clostridium tetani (Harvard No 49205) The Rijks Institute Voor de Volksgezondheid (Netherlands)
2 Haemophilus influenzae type b (Harward No. 760705) Netherlands Vaccines Institute (Netherlands).
Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and methods, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.
The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms "a,” "an," and "the" may be intended to include the plural forms as well, unless the context clearly suggests otherwise. The terms "comprises," "comprising," “including,” and “having,” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the method and process of the present disclosure is not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.
The terms first, second, third, etc., should not be construed to limit the scope of the present disclosure as the aforementioned terms may be only used to distinguish one element, component, region, layer or section from another component, region, layer or section. Terms such as first, second, third etc., when used herein do not imply a specific sequence or order unless clearly suggested by the present disclosure.

The present invention is in the field of Biotechnology and is applicable in vaccine manufacturing industry. The invention relates to process of preparation/ isolation, stabilization of proteins and conjugation of proteins with bacterial polysaccharides, suitable for use as an antigen for monovalent, multivalent combination, or as polysaccharide-protein conjugate vaccine preparation.
In an embodiment of the present invention, protein is a bacterial protein. Proteins are organic molecules found in living organisms. Proteins are complex molecules that play many critical roles in the biological functions. They do most of the work in cells and are required for the structure, function, and regulation of the biological functions. In an aspect of this embodiment, microorganisms produce toxins to promote infection and disease by directly damaging host tissues and by disabling the immune system. These toxins can be modified to suppress the toxicity, but these modified toxins (i.e. toxoids) shows antigenicity and can be further used as vaccine antigens.
In an embodiment of present invention, toxoid is prepared. In an aspect of this embodiment, exotoxin such as Cholera toxin, Diphtheria toxin, CRM197, Pertussis toxin, E. coli heat-labile toxin LT, Shiga toxin, Pseudomonas Exotoxin A, Botulinum toxin, Tetanus toxin, Anthrax toxin LF, Bordetella pertussis AC toxin, Staphylococcus aureus Exfoliatin B, etc. may be obtained by culturing respective microbial cultures. In other aspect of this embodiment, toxoid can be obtained by subjecting exotoxin to inactivation using one or more suitable inactivation process selected from treatment with Heat, UV, Formalin / Formaldehyde, Acetylethyleneimine, etc. In a preferred aspect of this embodiment, exotoxin is inactivated using Formalin / Formaldehyde.
In one of the embodiment of the present invention, Diphtheria toxoid is prepared. Diphtheria is an infectious disease caused by the bacterium Corynebacterium diphtheria, which primarily infects the throat and upper airways, and produces a toxin affecting other organs. Diphtheria toxin is an exotoxin secreted by Corynebacterium diphtheria, possesses antigenic properties and is toxic in nature. To reduce toxicity, the toxin is converted to the toxoid by subjecting it to inactivation. In an aspect of this embodiment, diphtheria toxin (exotoxin) is obtained from Corynebacterium diphtheria and detoxified using a suitable inactivating agent. In preferred aspect of this embodiment, diphtheria toxin (exotoxin) is inactivated using Formalin / Formaldehyde.

In another embodiment of present invention, Tetanus toxoid is prepared. Tetanus is an acute infectious disease caused by toxigenic strains of the bacterium Clostridium tetani (C.tetani), a gram-positive, spore-forming, strictly anaerobic bacterium. Tetanus toxin is an exotoxin secreted by Clostridium tetani, possesses antigenic properties and is toxic in nature. To reduce toxicity, the toxin is converted to the inactive toxoid by subjecting it to inactivation. In an aspect of this embodiment, Tetanus toxin (exotoxin) is obtained from Clostridium tetani and detoxified using a suitable inactivating agent. In preferred aspect of this embodiment, tetanus toxin (exotoxin) is inactivated using Formalin / Formaldehyde.
In one of the embodiment of present invention, the crude toxoid obtained by inactivation and is subjected to purification. In one of the aspects of this embodiment, crude toxoid obtained by inactivation can be further subjected to purification by using WHO suggested method (WHO Manual for the production and control of Vaccines. Tetanus toxoid. BLG/UNDP/77.2 Rev1) i.e. conventional method.
The conventional method involves the following steps:
(i) Exotoxin obtained from fermentation is detoxified and subjected to 80-90 times concentration by ultrafiltration using tangential flow filtration system through 30 kD cutoff membrane;
(ii) Two step fractional ammonium sulphate precipitation;
(iii) Diafiltration for the removal of ammonium sulphate.
The toxoid obtained comprises 50-60% monomeric toxoid and rest in the form of dimers and multimers.
In other aspect of this embodiment, the toxoid obtained after ultrafiltration, fractional ammonium sulphate precipitation and diafiltration, is further subjected to purification based on separation of molecules on the basis of their molecular size.
In other aspect of this embodiment, different critical process parameters like column bed height, flow rate, sample volume and sample protein content may be optimized to achieve best separation of desired toxoid molecules. Preferably, Gel permeation/filtration chromatography may be used leading to elution of toxoids in the sequential manner starting with toxoid aggregates followed by dimers; monomeric toxoid and low molecular weight proteins. In preferred aspect of this embodiment, gel filtration media such as PLgel, Sephacryl S-100 HR, Sephacryl S-200HR, Sephacryl S-HR 300, BPG - 450 columns

(Sephacryl -HR gel-filtration media from GE healthcare) Sephadex, Superdex 200) Bio-Gel (cross linked polyacrylamide), agarose gel and/or Styragel may be used for the purpose of purification using Gel permeation/filtration chromatography. In more preferred aspect of this embodiment, gel filtration media Sephacryl S-HR 300 may be used for the purpose of purification using Gel permeation/filtration chromatography. The method may further comprise multi-column large scale setup packed with gel-filtration media having fractionation range of 1×104 to 1.5×106, for the purification of (multimeric) toxoid to obtain highly pure monomeric toxoid. In other aspect of this embodiment, crude Tetanus toxoid is subjected to one or more purification steps selected from ultrafiltration, fractional ammonium sulphate precipitation, and diafiltration. The purified Tetanus toxoid may further be subjected to purification using Gel permeation/filtration chromatography to obtain highly pure monomeric tetanus toxoid. The term “Monomeric protein / toxoid” refers to a purified protein / toxoid having monomer content more than 70 %, preferably having more than 80% monomer content. In another aspect of this embodiment, crude diphtheria toxoid is subjected to one or more purification steps selected from ultrafiltration, fractional ammonium sulphate precipitation, diafiltration. The purified diphtheria toxoid may further be subjected to purification using Gel permeation/filtration chromatography to obtain highly pure monomeric diphtheria toxoid. The term “Monomeric protein / toxoid” refers to a purified protein / toxoid having monomer content more than 70 %, preferably having more than 80% monomer content.
The GPC purified fraction can be further subjected to other chromatography steps selected from but not limited to hydrophobic interaction chromatography, size exclusion chromatography, ion exchange chromatography and combinations thereof.
In one of the embodiments of present invention, highly pure monomeric toxoid obtained may be subjected to quality control tests such as monomer content detection using HPLC, protein content using DC – protein assay, purity, Lf/ml, endotoxin content, pH and sterility.
In one of the embodiments of present invention, a composition is provided. The composition comprises highly pure monomeric toxoid solution and a stabilizing agent. Formaldehyde inactivated toxoids tend to aggregate upon storage, and hence the stabilizing agent may be used to stabilize the highly pure monomeric toxoid solution and prevent its aggregation. The stabilizing agent prevents formation of multimeric or aggregated toxoid during toxoid storage. The composition may be a liquid composition or a lyophilized composition.

The term “Stabilized protein” refers to a protein composition comprising of a stabilizing agent, wherein the stabilizing agent prevents formation of multimeric or aggregated protein during storage.
The present invention further provides a process for stabilization of monomeric protein during storage. The process comprises addition of a stabilizing agent to the purified monomeric protein. The addition of the stabilizing agent to purified monomeric protein prevents the formation of multimeric or aggregated protein during storage.
In an aspect of the present invention, the protein is a toxoid. In another aspect, the toxoid is derived from Cholera toxin, Diphtheria toxin, CRM197, Pertussis toxin, E. coli heat-labile toxin LT, Shiga toxin, Pseudomonas Exotoxin A, Botulinum toxin, Tetanus toxin, Anthrax toxin LF, Bordetella pertussis AC toxin, and Staphylococcus aureus Exfoliatin B. In a preferred aspect the protein is selected from Tetanus toxoid and/or Diphtheria toxoid.
In accordance with the present invention, the stabilizing agent is selected from an amino acid buffer solution or a polysorbate solution. In one of the aspects of this embodiment, one or more amino acids selected from the group comprising Glycine, Lysine, Histidine, and/or Arginine solution can be added to highly pure monomeric toxoid solution having monomer content 80-99% and stored till further use. In another aspect, one or more polysorbate selected from the group comprising polysorbate 20, polysorbate 40, polysorbate 60 and/or polysorbate 80 can be added to highly pure monomeric toxoid solution having monomer content 80-99% and stored till further use. In other aspect of this embodiment, the final concentration of stabilizing agent in highly pure monomeric toxoid solution may be selected from the range of 1 mM to 500 mM, from 1 mM to 400 mM, preferably from the range of 50 mM to 400 mM and still preferably from 100 mM to 300 mM. In a more preferred aspect, the final concentration of stabilizing agent in highly pure monomeric toxoid solution may be in the range of 100 mM to 200mM. In another aspect of this embodiment, the stabilizing agent added highly pure monomeric toxoid solution may be stored at temperature selected from -20 °C, 0°C, 2-8 °C, 25 °C, 37 °C, and 40 °C. In preferred aspect of this embodiment, Glycine solution is added to highly pure monomeric toxoid, at a final concentration selected from the range of 20 mM to 300 mM, and stored at temperature selected from -20 °C, 0°C, 2-8 °C, 25 °C, 37 °C, and 40 °C, till further use, wherein the monomer content across storage is maintained above 80%. In other preferred aspect of this embodiment, Lysine solution is added to highly pure monomeric toxoid, at a final concentration selected from the range of 20

mM to 300 mM, and stored at temperature selected from -20 °C, 0°C, 2-8 °C, 25 °C, 37 °C, and 40 °C, till further use. In a more preferred aspect of this embodiment, Histidine solution is added to highly pure monomeric toxoid, at a final concentration selected from the range of 1 mM to 400 mM, and stored at temperature selected from -20 °C, 0°C, 2-8 °C, 25 °C, 37 °C, and 40 °C, till further use. In another aspect Histidine solution is added to highly pure monomeric toxoid, at a final concentration selected from the range of 100 mM to 200 mM, and stored at temperature selected from -20 °C, 0°C, 2-8 °C, 25 °C, 37 °C, and 40 °C, till further use. In a preferred aspect of the present invention, the % monomer reduction of the protein stabilized using 200 mM Histidine stored at 37 °C for 6 months is less than 10% and the % monomer reduction of the protein stabilized using 50 mM Histidine stored at 2-8 °C for 6 months is less than 10%. In more preferred aspect of this embodiment, Arginine solution is added to highly pure monomeric toxoid at a final concentration selected from the range of 1 mM to 400 mM, and stored at temperature selected from -20 °C, 0°C, 2-8 °C, 25 °C, 37 °C, and 40 °C, till further use. In a preferred aspect of this embodiment, polysorbate 80 (Tween-80) solution is added to highly pure monomeric toxoid at a final concentration selected from the range of 0.01 to 20 %, and stored at temperature selected from -20 °C, 0°C, 2-8 °C, 25 °C, 37 °C, and 40 °C, till further use.
In one of the embodiments of the present invention, Tetanus toxin (exotoxin) is obtained from Clostridium tetani and detoxified using formaldehyde to obtain crude tetanus toxoid. Further, crude Tetanus toxoid is subjected to one or more purification steps selected from ultra-filtration, fractional ammonium sulphate precipitation, and dia-filtration to obtain a toxoid preparation having 50-60% monomeric content, followed by gel filtration chromatography to obtain a toxoid preparation having more than 80% monomeric content. In one embodiment, Histidine may be added to stabilize freshly purified toxoid preparation having more than 80% monomeric content and stored at temperature in range of -20 to +40 °C, till further use, wherein the monomer content across storage is maintained between 80 – 99 %, preferably above 80%. Quality control tests such as monomer content detection using HPLC, protein content using DC – protein assay, purity, Lf/ml, endotoxin content, pH and sterility may be carried out.
In one of the embodiments of present invention, Diphtheria toxin (exotoxin) is obtained from Corynebacterium diphtheria and detoxified using formaldehyde to obtain crude diphtheria toxoid. Further, crude diphtheria toxoid is subjected to one or more purification steps selected from ultra-filtration, fractional ammonium sulphate precipitation, and dia-filtration to obtain a

toxoid preparation having 50-60% monomeric content, followed by gel filtration chromatography to obtain a toxoid preparation having >80% monomeric content. In one embodiment, Histidine may be added to stabilize freshly purified toxoid preparation having >80% monomeric content and stored at temperature in range of -20 to +40 °C, till further use. Quality control tests such as monomer content detection using HPLC, protein content using DC – protein assay, purity, Lf/ml, endotoxin content, pH and sterility may be carried out.
In one of the embodiments of present invention, other toxins such as CRM197, Cholera toxin, Pertussis toxin, E. coli heat-labile toxin LT, Shiga toxin, Pseudomonas Exotoxin A, Botulinum toxin, Anthrax toxin LF, Bordetella pertussis AC toxin, Staphylococcus aureus Exfoliatin B, etc. may be obtained by microbial fermentation and detoxified using formaldehyde to obtain crude toxoid. Further, crude toxoid may be subjected to one or more purification steps selected from ultra-filtration, fractional ammonium sulphate precipitation, and dia-filtration to obtain a toxoid preparation having 50-60% monomeric content, followed by gel filtration chromatography to obtain a toxoid preparation having >80% monomeric content. Amino acids, such as Histidine, glycine, arginine and lysine or polysorbate, such as polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80 may be added to stabilize freshly purified toxoid preparation having >80% monomeric content and stored at temperature in range of -20 to +40 °C, till further use. Quality control tests such as monomer content detection using HPLC, protein content using DC – protein assay, purity, Lf/ml, endotoxin content, pH and sterility may be carried out.
In one of the embodiments of present invention, stored toxoid may optionally be subjected to buffer exchange (prior to conjugation) using any suitable method known in the art. In one of the aspect of this embodiment, buffers used in buffer exchange is preferably a conjugation compatible buffer and may include one or more buffers selected from the group comprising Phosphate buffer, MOPS, NaCl, TRIS, TRIS-HCl, TRIS-NaCl, MES, MES-NaCl, etc. In one of the aspect of this embodiment, Buffer exchange may be carried out using Tangential flow filtration, Ultrafiltration, Diafiltration, Centrifugation, Chromatographic techniques like Gel filtration chromatography, etc. In one of the aspect of this embodiment, buffer exchange may be carried out using ultrafiltration using 1–50 kDa molecular weight cut off membrane. In one of the aspect of this embodiment, buffer exchange step is carried out immediately before conjugation reaction.

In one of the embodiments of present invention, toxoid obtained as per above discussed embodiments may further be used as monovalent vaccine, or as a component of multivalent combination vaccines or as carrier protein for manufacturing polysaccharide-carrier protein conjugate vaccine.
In one of the embodiments of present invention, the polysaccharide antigen may be obtained by fermentation of microorganisms selected from group of Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus spp., Staphylococcus aureus, Streptococcus spp., Group A Streptococcus, Group B Streptococcus, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus viridans, Enterococcus faecalis, Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis, Salmonella spp., Salmonella typhi, Salmonella paratyphi, Salmonella typhimurium, Salmonella enteritidis, Vibrio cholerae, Pasteurella pestis, Pseudomonas aeruginosa, Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Escherichia coli, Shigella spp., Ehrlichia spp., and/or Rickettsia spp.
In one of the aspect of this embodiment, polysaccharide antigen may be obtained from Haemophilus influenzae type B (Hib-B).
In another aspect of this embodiment, the multivalent vaccine composition comprises polysaccharide antigen derived from Streptococcus pneumoniae serotypes selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9A, 9F, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F, 45, 38, 35B , 23B, 24F, 15A, and/or 15C and a carrier protein selected from tetanus toxoid, diphtheria toxoid and CRM197.
In another aspect of this embodiment, the multivalent vaccine composition comprises polysaccharide antigen derived from N. meningitidis serotypes A, B, C, D, W135, X, Y, Z and/or 29E and a carrier protein selected from tetanus toxoid and CRM197.
In another aspect of this embodiment, the multivalent vaccine composition comprises polysaccharide antigen derived from Salmonella serovar strains selected from group comprising of Salmonella Enterica serovar typhi TY2 strain with "tviB" gene specific for Vi

polysaccharide; S. typhi: ATCC 19430; C6524 (NICED, Kolkata, India); S. paratyphi A: ATCC 9150, CMCC50073, CMCC50973; S. enteritidis: ATCC 4931; ATCC 13076; S. enteritidis R11; S. enteritidis D24359; S. enteritidis 618; S. enteritidis 502; S. enteritidis IV3453219;S. typhimurium: S. typhimurium 2192; ATCC 14208; S. typhimurium 2189; S. typhimurium D23580; ATCC 19585; ATCC 700408; (LT2/SL134 (ST19)); S. typhimurium 177(ST19) CDC 6516-60; ATCC 700720.
In another aspect of this embodiment, the multivalent vaccine composition comprises polysaccharide antigen derived from Group A Streptococcus, or Group B Streptococcus (group Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX).
In another aspect of this embodiment, the multivalent vaccine composition comprises polysaccharide antigen derived from Shigella spp. selected from Shigella sonnei, Shigella dysenteriae, Shigella flexneri, and Shigella boydii.
In another aspect of this embodiment, the multivalent vaccine composition comprises polysaccharide antigen derived from Staphylococcus spp. selected from Staphylococcus aureus, Staphylococcus aureus type 5, and Staphylococcus aureus type 8.
In one of the embodiments of present invention, polysaccharide- protein conjugate is provided. The polysaccharide- protein conjugate may be obtained using following protocol:
1. Preparation of processed polysaccharide;
2. Preparation of protein and addition of the stabilizing agent selected from Histidine, lysine, glycine, arginine, or polysorbate solution to protein before storage.
3. Buffer exchange of stabilized protein using conjugation compatible buffer.
4. Conjugation of processed polysaccharide with protein.
5. Purification of crude conjugate by Ammonium sulphate precipitation
6. Gel permeation chromatographic purification
7. 0.22 micron filtration
In one of the embodiments of present invention, conjugation of a polysaccharide and toxoid/carrier protein may be carried out using cyanylation conjugation chemistry, Carbodiimide conjugation chemistry, reductive amination conjugation chemistry, or any other conjugation methods known in the art. In preferred aspect of this embodiment, conjugation reagent may be selected from Cyanogen Bromide (CNBr), 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP), p-nitrophenylcyanate and N-

cyanotriethylammonium tetrafluoroborate ('CTEA'), 1 – cyano – 4 – pyrrolidinopyridinium tetrafluorborate (CPPT), 1 - cyanoimidazole namely (1 - CI), 1 – cyanobenzotriazole (1 -CBT) or 2 – cyanopyridazine -3(2H) one (2 -CPO), or a functional derivative or modification thereof.
In one of the embodiments of present invention, said activated polysaccharide or carrier protein, particularly polysaccharide is reacted with hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide, a mixture thereof, preferably with adipic acid dihydrazide.
In one of the embodiments of present invention, conjugation reaction utilizes linkers selected from the group consisting of adipic acid dihydrazide, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine and p-nitrophenylethyl amine.
In one of the embodiments of present invention, polysaccharide antigen and toxoid are used for the conjugation process (using Cyanogen Bromide chemistry) as shown below:
i. The polysaccharide antigen may be required to be of optimum concentration for the conjugation process. The polysaccharide may be subjected to concentration adjustment using any of the methods known in the art, preferably ultrafiltration.
ii. The polysaccharide may be depolymerized under mild alkaline conditions, preferably using carbonate-bicarbonate buffer.
iii. After target polysaccharide size is reached, the depolymerized polysaccharide may be activated using Cyanogen Bromide.
iv. Freshly prepared adipic acid dihydrazide (ADH) solution may be added within 6-10 minutes to the reaction mixture obtained from above step.
v. The reaction may be carried out for NLT 16 hours at 2-10 °C. The role of the ADH linker is to provide amine groups in polysaccharide required for conjugation reaction.
vi. The reaction mixture obtained may be concentrated and diafiltered volume by volume with phosphate buffer saline (PBS) using 10 kD NMWCO UF membrane to remove free ADH. The removal of ADH is monitored on HPLC and diafiltration may be continued till free ADH level reaches below 5%. The resulting retentate may further be diafiltered with NLT 5X MES-NaCl buffer. This may further be concentrated to achieve a concentration of NLT 20 mg/mL. This concentrated processed polysaccharide may be kept at 2 - 8 °C till further use. vii. The retentate is passed through a 0.22 μm filter, which serves as a clarification step. This also ensures that bioburden levels are controlled during the process, which is

typically performed in grade C area. The filtered activated polysaccharide may be collected, sampled, aliquoted and stored at 2-8 °C till further processing. A sample may be drawn from the processed polysaccharide pool for analysis, which includes polysaccharide molecular size (kD), polysaccharide content, and polysaccharide degree of activation.
viii. The conjugation reaction requires two components viz. processed/activated polysaccharide and the carrier protein with more than 80% monomeric toxoid content. The toxoid/carrier protein stored in amino acid solution (Histidine, glycine, lysine, and arginine) or polysorbate solution as per embodiments of the present invention may be subjected to buffer exchange and concentration adjustment using any conjugation friendly buffer, preferably ultrafiltration using 10 kDa nominal molecular weight cut off ultrafiltration membrane. ix. The carrier protein may be concentrated and diafiltered with conjugation compatible buffer, preferably MES-NaCl buffer using 10 kD UF NMWCO membrane. This diafiltered carrier protein/toxoid may be further concentrated to NLT 20 mg/mL using the same membrane. x. The activated polysaccharide and diafiltered carrier protein (toxoid) may be mixed in appropriate quantities (for example, ratio of polysaccharide: Toxoid = 1:1), in presence of EDC under stirring at 3±1 °C. The conjugation reaction may be monitored on HPLC and continued till ≥ 85% conversion of protein (based on the free protein conversion to conjugate) is reached. xi. After the conjugation reaction has proceeded to its acceptance criteria for conversion, the reaction may be terminated by quenching, preferably the conjugation reaction may be quenched using phosphate EDTA buffer.
xii. The conjugate obtained from above step may be filtered through a 30 SP CUNO filter followed by 0.22 μm filtration, ensuring removal of any large aggregates.
xiii. The diafiltration may be performed to remove conjugation reagents and unreacted carrier protein. The conjugation reaction mixture obtained from above step may be diafiltered with 0.05% saline using 300 kD UF NMWCO membrane.
xiv. The retentate obtained may be further passed through 0.22μ filter ensuring bioburden levels are controlled during the process. The filtered crude conjugate may be collected, sampled and stored at 2-8 °C till further processing.
xv. The crude conjugate may be further diluted with W.F.I. to targeted concentration, if required.

xvi. The diluted conjugate reaction mixture may be further processed to remove free polysaccharide using ammonium sulphate (50% w/v stock solution). The precipitation step may be carried out at less than 15 °C under stirring. The precipitation step drives the conjugate in the precipitate, and leaves the free polysaccharides in the supernatant. After addition of ammonium sulphate the resulting suspension may be stored at less than 15 °C without stirring for 10 to 48 hours.
xvii. The suspension obtained from above step may be centrifuged at ~7000 g at 2-8 °C. The supernatant may be discarded by decantation and the pellet obtained may be dissolved in Tris-saline. xviii. The resulting solution from above step may be filtered through 30 SP depth filter and diafiltered with Tris -Saline using ultrafiltration membrane, as a clarification step.
xix. The resulting solution from above step may be loaded on GPC column containing size exclusion chromatography matrix. The use of GPC chromatography for processed conjugate (post-ammonium sulphate step) reduces the free polysaccharide levels in the resulting material. The column may be eluted with 20 mM Tris in 0.9% NaCl, and fractions are collected based on Absorption at 280 nm. Appropriate fractions based on acceptance criteria with respect to free polysaccharide, Ratio and molecular size are pooled. xx. The resulting pooled conjugate eluate from above step may be diafiltered with 20 mM Tris using 300 kD UF NMWCO membrane. This retentate volume can be targeted such that the polysaccharide content in it is approximately 1 mg/mL.
xxi. The bulk conjugate obtained from above step may be filtered through 0.22 μm filter under grade A environment to ensure sterility.
xxii. The filtered conjugate may be further stored at 2-8 °C.
In one of the aspect of this embodiment, sample from the filtered bulk conjugate may be subjected to quality control tests for complete analysis of free polysaccharide content, polysaccharide to protein ratio, average molecular weight of the conjugate, etc.
In one of the embodiments of present invention, polysaccharide antigen and toxoid is used for the conjugation process using CDAP chemistry. Polysaccharide may be conjugated to carrier protein using conjugation process comprising steps including depolymerisation of polysaccharide using alkaline buffer to achieve size reduced PRP; treatment with cyanylation agent like CDAP (1-cyano-4-dimethylamino pyridinium tetrafluoroborate) to form a cyanate ester; coupling of activated cyanylated polysaccharide to amino group of carrier protein; and

purification of final conjugate using ultrafiltration. The degree of conversion of polysaccharide – carrier protein conjugate may be confirmed by offline testing using HPLC. The conjugation reaction may be quenched after achieving desired level of conversion of conjugate with specification of not less than 60% conversion of conjugate, and then conjugate reaction may be neutralized by Glycine (2M) addition. The polysaccharide - carrier protein conjugate may be purified on ultra filtration membrane filters (300kDa and 100kDa) to remove non-reactive reagents and by-products. Final conjugate bulk may be further purified by using 0.22 μm filters and stored at 2-8 °C. In other aspects of this embodiment, polysaccharide may be conjugated to carrier protein wherein the saccharide: protein ratio (w/w) is between 0.4 and 1; and the free polysaccharide content in final polysaccharide – carrier protein conjugate bulk is not more than 5%, more preferably is less than 2%.
In one of the embodiments of present invention, polysaccharide - carrier protein conjugate may be Haemophilus influenza PRP – Tetanus toxoid conjugate. Haemophilus influenza PRP – Tetanus toxoid conjugate may be obtained as per the protocol given below:
i. Haemophilus influenza PRP is processed.
ii. Tetanus toxoid is obtained from toxin by inactivation using formalin/formaldehyde. Toxoid is purified using Gel filtration chromatography to obtain highly pure monomeric Tetanus toxoid (>80%). Highly pure monomeric Tetanus toxoid (>80%) is subjected to addition of an amino acid solution (Histidine, lysine, glycine, arginine) or polysorbate solution at a final concentration of 5 – 300 mM, and stored at temperature -20 to +40 °C till further use.
iii. The stabilized Tetanus toxoid is subjected to buffer exchange, to be further used in conjugation process. Buffer exchange may be carried out by ultrafiltration using 10 kDa molecular weight cut off ultrafiltration membrane.
iv. Conjugation of processed Haemophilus influenza PRP with Tetanus toxoid.
v. Purification of crude conjugate by Ammonium sulphate precipitation.
vi. Gel permeation chromatographic purification.
vii. 0.22 micron filtration to obtain final Haemophilus influenza PRP – Tetanus toxoid conjugate to be further used as vaccine.

In one of the embodiments of present invention, polysaccharide - carrier protein conjugate may be Haemophilus influenza PRP – Tetanus toxoid obtained as per any of the above discussed embodiments.
In one of the embodiments of present invention, polysaccharide - carrier protein conjugate may be Haemophilus influenza PRP – Diphtheria toxoid obtained as per any of the above discussed embodiments.
In one of the embodiments of present invention, polysaccharide - carrier protein conjugate may be Neisseria meningitidis polysaccharide – Tetanus toxoid obtained as per any of the above discussed embodiments. Neisseria meningitidis polysaccharide may be obtained from one or more serotypes selected from the group of A, B, C, D, W135, X, Y, Z and 29E.
In one of the embodiments of present invention, polysaccharide - carrier protein conjugate may be Neisseria meningitidis polysaccharide– Diphtheria toxoid obtained as per any of the above discussed embodiments. Neisseria meningitidis polysaccharide may be obtained from one or more serotypes selected from the group of A, B, C, D, W135, X, Y, Z and 29E.
In one of the embodiments of present invention, polysaccharide - carrier protein conjugate may be Streptococcus pneumoniae polysaccharide – Tetanus toxoid obtained as per any of the above discussed embodiments. Streptococcus pneumoniae polysaccharide may be obtained from one or more serotypes selected from the group of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9A, 9F, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F, 45, 38, 35B, 23B, 24F, 15A, 15C.
In one of the embodiments of present invention, polysaccharide - carrier protein conjugate may be Streptococcus pneumoniae polysaccharide – Diphtheria toxoid obtained as per any of the above discussed embodiments. Streptococcus pneumoniae polysaccharide may be obtained from one or more serotypes selected from the group of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9A, 9F, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F, 45, 38, 35B, 23B, 24F, 15A, 15C.
The vaccine prepared according to Present invention results into increased yields of polysaccharide-protein conjugate due to improved conjugation efficiency. In an embodiment the yield of Hib conjugate can be in the range of 10- 50%

In another embodiment, the free PRP value in the conjugate prepared in accordance with the Present invention can be in the range of 1 - 5% at release and during storage it can go up to about 20% until one year.
In one of the embodiments of present invention, toxoid obtained as per any of the embodiments of the present invention may be used as a monovalent vaccine. In one aspect of this embodiment, tetanus toxoid obtained as per any of the embodiments of the present invention may be used as a Tetanus vaccine. In one aspect of this embodiment, diphtheria toxoid obtained as per any of the embodiments of the present invention may be used as a diphtheria vaccine.
In one of the embodiments of present invention, toxoid obtained as per any of the embodiments of the present invention may be used as a component of multivalent combination vaccine. In one aspect of this embodiment, diphtheria toxoid or tetanus toxoid obtained as per any of the embodiments of the present invention may be used as an antigen in a multivalent combination vaccine. The combination vaccine may comprise one or more antigens selected from Diphtheria Toxoid (D), Tetanus toxoid (T), Inactivated B. pertussis antigen (wP), acellular pertussis antigen, HBs antigen, Hib PRP-protein conjugate antigen, Salmonella typhi ViPs- protein conjugate antigen, Streptococcus pneumoniae Polysaccharide-protein conjugate antigen, Neisseria meningitidis Polysaccharide-protein conjugate antigen, Inactivated polio virus (IPV), Inactivated Rotavirus (IRV) etc. In one of the preferred aspect, the toxoid obtained as per any of the embodiments of the present invention may be used as a component of combination vaccine comprising Diphtheria Toxoid (D), Tetanus toxoid (T), and Inactivated B. pertussis antigen (wP). In another aspect of this invention, the toxoid obtained as per any of the embodiments of the present invention may be used as a component of multivalent /combination vaccine composition comprising of two or more antigens in combination as DTP, DTP-Hib, DTP-Hib-HepB, DTP-Hib-HepB-IPV, or DTP-Hib-HepB-IPV-IRV. In another aspect of the embodiment, the toxoid has an immune boosting effect in a multivalent combination effect.
In one of the embodiments of present invention, the vaccine composition prepared using components disclosed in any of the embodiments of the present invention is a liquid composition.

In one of the embodiments of present invention, the vaccine composition prepared using components disclosed in any of the embodiments of the present invention is a lyophilized composition.
The composition and process for stabilization of bacterial protein and its use in vaccine manufacturing as described in the present invention, prevents formation of multimeric/aggregated toxoid, which in turn aids in attributing better predictability, safety, consistency, stability and potency in vaccine.
The present invention is further described in light of the following examples. In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only examples of the invention and should not be taken as limiting the scope of the invention.
Examples:
Example 1: Preparation of Tetanus toxoid by conventional Method
Tetanus toxoid was prepared from the toxin produced by the growth of a highly toxigenic strain of Clostridium tetani in a suitable medium (Source of Clostridium tetani (Harvard No 49205): The Rijks Institute Voor de Volksgezondheid (Netherlands)). The supernatant fluid, which contains the toxin, was separated from the organisms; the toxin so separated from the organisms was detoxified and purified (as per protocol given in WHO Manual for the production and control of Vaccines. Tetanus toxoid. BLG/UNDP/77.2 Rev1. / Conventional Method).
The purified Tetanus toxoid was tested for purity and monomer content. The purified Tetanus toxoid shows presence of monomers, dimmers and aggregates. The monomer content was found to be 50 - 55%. Refer Figure 1, wherein Lane 1: Molecular weight marker; Lane 2: Tetanus toxoid (Conventional Method).
Example 2: Preparation of highly pure monomeric Tetanus toxoid

Tetanus toxoid obtained by process as described in example 1 (conventional process) was further purified using Sephacryl High Resolution size exclusion chromatography resins (Sephacryl S-300 HR).
Monomer content for purified tetanus toxoid was determined using HPLC system (Class VP, UV-Vis Shimadzu system) equipped with protein Pak-300 column (Water’s). Sample elution was done using phosphate buffer (0.1 M, pH 7.2 + 0.1) at 1 ml/min flow rate. 280 nm wavelength was used to detect the protein peak. The purified toxoid was tested for monomeric content and was found to be more than 80%.
Further, to confirm the purity of monomeric toxoid, SDS page (reducing 8% gel) analysis was carried out for 3 different batches of purified monomeric toxoid along with TT (obtained by conventional method) as control.
Results and Inference:
In SDS PAGE Analysis, Control sample i.e. TT (conventional method) showed considerable amount of aggregates and dimmer. The monomeric content of purified Tetanus toxoid obtained was found to be more than 80% with average molecular size of 150 kDa. Please refer Figure 1, wherein Lane 1: Molecular weight marker; Lane 2: Tetanus toxoid (Conventional Method); and Lane 3, 4 and 5: Purified Monomeric toxoid (3 batches).
Example 3: Study to identify suitable stabilizer for stabilization of highly pure monomeric toxoid (obtained in example 2) at 37 °C.
Different protein stabilizing agents were evaluated for their potential to stabilize monomeric toxoid from aggregation at 37 °C.
Materials:
1. Tetanus toxoid (150 kD) (Protein concentration = 2 - 2.5 mg/ml, Monomer content : > 80 %)
2. Stabilizing agents tested: Histidine, Glycine, lysine (Concentration 200 mM each); and Tween-80 (0.05%)

3. Instrument used: HPLC system (Binary gradient, Protein Pac-300 column, UV-Vis detector)
Procedure:
1. Monomeric toxoid (having more than 80% monomeric content, obtained as per example 2), was subjected to addition of stabilizer as per Table 1.

Table 1
Sample # Test Sample Stabilizer Total Volume
Control Tetanus toxoid (2.5 mg/ml) - 50 ml
Control 1 Normal saline Histidine (200 mM) 50 ml
Control 2 Normal saline Glycine (200 mM) 50 ml
Control 3 Normal saline Lysine (200 mM) 50 ml
Control 4 Normal saline Tween 80 (0.05%) 50 ml
Test 1 Tetanus toxoid (2.5 mg/ml) Glycine (200 mM) 50 ml
Test 2 Tetanus toxoid (2.5 mg/ml) Lysine (200 mM) 50 ml
Test 3 Tetanus toxoid (2.5 mg/ml) Tween 80 (0.05%) 50 ml
Test 4 Tetanus toxoid (2.5 mg/ml) Histidine (200 mM) 50 ml
2. All solutions prepared as per Table no. 1 were sterile filtered using sterile 0.2 micron disc filter and stored in incubator at 37 °C (±1 °C).
3. 1 ml sample was removed aseptically and was tested by HPLC system for percent monomer content.
4. Sample analysis was performed for day zero followed by week 1, week 2, week 3, week 4 and week 8).
Results and Inference:
1. Control: Tetanus toxoid (without any stabilizer)
• Tetanus toxoid (without any stabilizer) at Day Zero showed 88.41% monomer content).
• Within a week monomer content reduced below 80%.
• After 8th week monomer content found to be drastically reduced to 60.83%. This high speed of aggregation is responsible for short shelf life of monomeric tetanus

toxoid. This suggested the need to investigate potential stabilizing agent to inhibit aggregation and enhance shelf life of monomeric tetanus toxoid.
• Please refer figure 2.
2. Control 1, Control 2 Control 3 & Control 4 showed no activity on HPLC Analysis.
3. Test 1: Tetanus toxoid (with 200 mM Glycine as stabilizer)

• Tetanus toxoid (with 200 mM Glycine as stabilizer) at Day Zero showed 88.30 % monomer content).
• As seen in Figure 3, Tetanus toxoid with Glycine as a stabilizer showed slower rate of aggregation as compared to toxoid without stabilizer.
• After 8th week monomer content found to be drastically reduced to 65.55%.
• Over all, there is difference of about 5% less aggregation at every testing stage which remains consistent till end. It indicates that though not substantial but there is reduction in aggregation speed due to use of glycine.
• Please refer figure 3.
4. Test 2: Tetanus toxoid (with 200 mM Lysine as stabilizer)
• Tetanus toxoid (200 mM Lysine as stabilizer) at Day Zero showed 88.53% monomer content).
• As seen in Figure 4, Tetanus toxoid with Lysine as a stabilizer showed slower rate of aggregation as compare to toxoid without stabilizer.
• After 8th week monomer content found to be reduced to 76.52%.
• There are other two aspects noted i.e. main peak broadening and appearance of additional big size low molecular weight peak probably of lysine (as seen in lysine control sample).
• Please refer figure 4.
5. Test 3: Tetanus toxoid (with 0.05% Tween-80 as stabilizer)
• Tetanus toxoid (0.05% Tween-80 as stabilizer) at Day Zero showed 86.13%
monomer content).

• No stabilization activity was observed for Tween-80. Aggregation profile (Change in monomer content) of control sample almost exactly overlaps with that of toxoid with Tween-80.
• Please refer Figure 5.
6. Test 4: Tetanus toxoid (with 200 mM Histidine as stabilizer)
• Tetanus toxoid (200 mM Histidine as stabilizer) at Day Zero showed 88.30% monomer content). Monomer content is well maintained above 80% till 8th week at 37 °C. After initial reduction of about 3 % monomer content remained stable at 85% in presence of Histidine.
• Experiments concluded that Histidine strongly/significantly inhibits aggregation process in monomeric tetanus toxoid during storage.
• Please refer figure 6.
Example 4: SDS PAGE Analysis Study to confirm the stabilization effect.
Reducing SDS-PAGE analysis using 8% gel was performed after eight weeks of incubation of toxoid samples at 37 °C (Ref table no.1). All samples were loaded (50 μl/well) with samples as mentioned below:
Results and Inference:
Please refer Figure 7, wherein Lane 1: Molecular weight marker; Lane 2: Tetanus toxoid without stabilizer; Lane 3: Tetanus toxoid with Histidine; Lane 4: Tetanus toxoid with Glycine and Lane 5: Tetanus toxoid with lysine.
It was observed that:
• Tetanus toxoid without stabilizer (Lane 2) showed high degree of dimmer and aggregate formation after incubation at 37 °C for eight weeks.
• Tetanus Toxoid stabilized with 200 mM Histidine (Lane 3) showed no presence of aggregates.
• Tetanus Toxoid stabilized with 200 mM Glycine (Lane 4) showed presence of aggregates.

• Tetanus Toxoid stabilized with 200 mM Lysine (Lane 5) showed inhibition of aggregation but smearing effect was observed on the gel.
Example 5: Study to determine optimum concentration of Histidine to be used as a stabilizer for stabilization of monomeric tetanus toxoid at 2 - 8 °C.
Various concentrations of Histidine were studied to determine the optimum concentration of Histidine to be used as a stabilizer for stabilization of monomeric tetanus toxoid at 2 - 8 °C. Below are the details of the studied parameter.
Tetanus toxoid obtained as per example 1 was used for further studies. All solutions prepared as per below table were sterile filtered using pre-sterile 0.2 micron disc filter.
• For each analysis 1 ml sample was removed aseptically and it was tested by HPLC system for percent monomer content on first day, after one week followed by after every month.
Results and Inference:

Table 3: % Monomer Content of Tetanus Toxoid stored at 2-8 °C
Sample 0 Day 1 Week 1 Month 2 Month 3 Month 4 Month 5 Month 6 Month
Test 1.1 92.95 93.19 93.53 93.61 93.85 94.08 94.14 94.23
Test 1.2 93.16 93.28 93.71 93.89 94.09 94.15 94.16 94.49
Test 1.3 93.85 93.99 94.36 94.71 94.9 95.07 95.14 95.52
Test 1.4 94.01 94.33 94.87 94.98 95.09 95.2 95.33 95.52
Test 1.5 94.35 94.63 95.25 95.46 95.63 95.94 96.23 96.85

Real time studies for stabilizer concentration optimization at 4 °C (±1 °C) showed that for Histidine containing toxoid samples, all concentrations of Histidine showed aggregation inhibition effect i.e. from 25 mM till 200 mM. All samples prepared showed stable monomer content at 4 °C (±1 °C). Please refer Figure 8.
Example 6: Study to determine optimum concentration of Histidine to be used as a stabilizer for stabilization of monomeric tetanus toxoid at 37 °C.
Various concentrations of Histidine were studied to determine the optimum concentration of Histidine to be used as a stabilizer for stabilization of monomeric tetanus toxoid at 37 °C. Below are the details of the studied parameter.
Tetanus toxoid obtained as per example 1 was used for further studies. All solutions prepared as per table 4 were sterile filtered using pre-sterile 0.2 micron disc filter.

Table 4
Sample # Test Sample Stabilizer Total Volume
Control Tetanus toxoid (2.3 mg/ml) - 50 ml
Test 1.6 Tetanus toxoid (2.3 mg/ml) Histidine (25mM) 50 ml
Test 1.7 Tetanus toxoid (2.3 mg/ml) Histidine (50 mM) 50 ml
Test 1.8 Tetanus toxoid (2.3 mg/ml) Histidine (100 mM) 50 ml
Test 1.9 Tetanus toxoid (2.3 mg/ml) Histidine (150 mM) 50 ml
Test 1.10 Tetanus toxoid (2.3 mg/ml) Histidine (200 mM) 50 ml
• For each analysis 1 ml sample was removed aseptically and it was tested by HPLC system for percent monomer content on first day, after one week followed by after every month.
Results and Inference:

Table 5: % Monomer Content of Tetanus Toxoid
Sample 0 Day 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week
Control 92.80 83.84 76.85 74.22 69.06 66.67 64.79
Test 1.6 92.95 88.10 80.90 79.13 78.04 72.75 71.74
Test 1.7 93.16 89.35 82.26 80.77 78.68 77.35 74.57
Test 1.8 93.85 90.41 84.77 82.53 80.69 77.82 75.45
Test 1.9 94.01 91.82 86.77 83.53 81.74 81.02 80.09
Test 1.10 94.35 93.94 92.79 91.73 91.40 91.09 90.90

Real time studies for stabilizer concentration optimization at 37 °C (±1 °C) showed that control toxoid sample without Histidine had considerable reduction in monomer within 6 months. While in case of Histidine containing toxoid samples aggregation inhibition effect kept on increasing from 25 mM till 200 mM. With 25 mM Histidine concentration, monomer content reduced < 80% after 4 weeks of incubation, at 50 mM concentration monomer content reduced < 80% after 5 weeks of incubation and for 100 mM it took 6 weeks incubation. For 150 mM and 200 mM Histidine concentration monomer content did not reduce below 80% even after 6 weeks incubation. Please refer Figure 9.
Example 7: Conjugation of stabilized monomeric toxoid to a capsular polysaccharide to obtain a polysaccharide-carrier protein conjugate.
Procedure:
a. Preparation of processed Carrier protein / toxoid
Highly pure monomeric Tetanus toxoid (>80%) obtained as per process of Example 1, was subjected to addition of a stabilizing agent i.e. Histidine at final concentration of 50 mM, and stored at temperature 2-8 °C till further use.
The toxoid/carrier protein stored in Histidine was subjected to buffer exchange and concentration adjustment using a conjugation compatible buffer immediately before the conjugation reaction, preferably ultrafiltration using 10 kD UF NMWCO membrane and MES-NaCl buffer to a concentration of NLT 20 mg/ml.
b. Preparation of processed polysaccharide
Purified Haemophilus influenza PRP polysaccharide was subjected to concentration adjustment using ultrafiltration (Source of Haemophilus influenzae type b (Harward No. 760705): Netherlands Vaccines Institute (Netherlands)). The polysaccharide was depolymerized under mild alkaline conditions using carbonate-bicarbonate buffer. After target polysaccharide size was reached, the depolymerized polysaccharide was activated using Cyanogen Bromide. Freshly prepared adipic acid dihydrazide (ADH) solution was added within 6-10 minutes to the reaction mixture obtained from above step. The reaction was carried out for NLT 16 hours at 2-10 °C. The reaction mixture

obtained was concentrated and diafiltered volume by volume with phosphate buffer saline (PBS) using 10 kD NMWCO UF membrane to remove free ADH. The removal of ADH is monitored on HPLC and diafiltration was continued till free ADH level reaches below 5%. The resulting retentate was further diafiltered with NLT 5X MES-NaCl buffer. This was further concentrated to achieve a concentration of NLT 20 mg/mL. This concentrated processed polysaccharide was kept at 2 - 8 °C till further use.
c. Conjugation Process:
The processed polysaccharide (step b) and processed carrier protein (toxoid) (Step a) were mixed in ratio of polysaccharide: Toxoid = 1:1, in presence of EDC under stirring at 3 (±1) °C. The conjugation reaction was monitored on HPLC and continued till ≥ 85% conversion of protein (based on the free protein conversion to conjugate) was reached. After the conjugation reaction proceeded to its acceptance criteria for conversion, the reaction was terminated by quenching using phosphate EDTA buffer. The conjugate thus obtained was filtered through a 30 SP CUNO filter followed by 0.22 µm filtration, ensuring removal of any large aggregates. The diafiltration was performed to remove conjugation reagents and unreacted carrier protein. The conjugation reaction mixture obtained was diafiltered with 0.05% saline using 300 kD UF NMWCO membrane. The retentate obtained was further passed through 0.22µ filter ensuring bioburden levels were controlled during the process. The conjugation reaction mixture obtained was further processed to remove free polysaccharide using ammonium sulphate (50% w/v stock solution) based precipitation and centrifugation, followed by dissolution of pellet in Tris-saline. The resulting solution was optionally subjected to depth filtration, diafiltration and gel permeation (size exclusion) column chromatography. The resulting conjugate was diafiltered with 20 mM Tris using 300 kD UF NMWCO membrane. This retentate volume was targeted such that the polysaccharide content in it is approximately 1 mg/mL. The filtered conjugate was further stored at 2-8 °C.

Example 8:
Stabilized monomeric (> 80%) tetanus toxoid of the present application was evaluated for suitability of conjugation with Haemophilus influenza PRP polysaccharide (3 batches were carried out).
Haemophilus influenza PRP polysaccharide was conjugated with
1. Tetanus toxoid was purified as per example 1. The Tetanus toxoid had monomeric content of 50-55% (Control Sample).
2. Test: Tetanus toxoid was purified as per example 2. The Tetanus toxoid had monomeric content of more than 80 %. (3 batches: batch 1, batch 2 and batch3)
The conjugates prepared were evaluated for below mentioned parameters. The results observed are given below for each the respective parameter tested.

Table 6
Test parameter Acceptance criteria Haemophilus influenza PRP polysaccharide Conjugates prepared using carrier protein

Control Batch 1 Batch 2 Batch 3
Conjugation efficiency - 92.02 92.22 92.19 93.35
PRP content (mg/ml) Not less than 0.25 mg/ml 1.01 1.063 1.14 0.97
Protein content (mg/ml) Not less than 0.46 mg/ml 2.93 2.91 3.29 2.83
PRP/Protein ratio 0.30 to 0.55 0.34 0.36 0.35 0.34
Free PRP (%) Not more than 20% 1 1 1 1
Molecular size by
HPLC MP/MW
(kDa) For information 817 / 768 797 / 731 798 / 718 783 / 688
Molecular size
distribution (%
conjugate eluted
<0.2kD) More than 60% 70.03 69.71 72.39 77.26

The conjugates prepared using stabilized monomeric (> 80%) tetanus toxoid of the present application as carrier protein for conjugation with Haemophilus influenza PRP polysaccharide were found to be within acceptance limits. Conjugates prepared using stabilized monomeric (> 80%) tetanus toxoid were comparable with that of control.
Example 9: Samples prepared as per example 7 were further evaluated for stability at 2-8 °C and 25 °C for a period of 3 months.
The results observed are given below for each of the respective parameter tested.

Table 7: % Free PRP Quantification
Test parameter Stability Time Point Haemophilus influenza PRP polysaccharide Conjugates prepared using carrier protein

Control Batch 1 Batch 2 Batch 3
2-8 °C 3 Months 1 1.03 1.26 1
25 °C 1 Month 4.64 6.21 6.76 5.37

2 Months 5.02 6.25 6.8 5.76

3 Months 5.09 6.47 8.33 5.12
Conjugates prepared using stabilized monomeric (> 80%) tetanus toxoid were within the desired limit.

Table 8: HPLC-SEC MOL. WT. (MP/MW) (kDa)
Test parameter Stability Time Point Haemophilus influenza PRP polysaccharide Conjugates prepared using carrier protein

Control Batch 1 Batch 2 Batch 3
2-8 °C 3 Months 760/675 741/631 740/637 742/634
25 °C 1 Month 698/590 693/567 686/577 683/571

2 Months 638/488 629/459 624/468 636/481

3 Months 606/433 594/406 593/422 614/444

Inference :
The stability data of conjugates confirmed that conjugates prepared using stabilized monomeric (> 80%) tetanus toxoid of the present application as carrier protein were found as stable conjugates at different temperature for up to 3 month and within acceptance limits.
Example 10: Samples prepared as per example 7 were further evaluated for humoral immune response in Wistar rats.
1:4 single human dose was injected on Day 0 and Day 28 in 10 Wistar rats and humoral
immune response was analysed on day 0 and day 42.

Table 9: Analysis of humoral immune response in Wistar rats
Test parameter Time Point Haemophilus influenza PRP polysaccharide Conjugates prepared using carrier protein

Control Batch 1 Batch 2 Batch 3
Total IgG Day 0 80 80 80 80

Day 42 640 1280 1280 1810
SBA Day 0 4 4 4 4

Day 42 14 32 39 192
From the above results, it is inferred that total IgG and Functional IgG titres (SBA) (for Batch 1, 2 & 3) were found to be greater than 2 fold as compared to control sample. This shows that conjugates prepared using present invention i.e. conjugates prepared using monomeric Tetanus toxoid having more than 80% monomeric content elicits better humoral immune response as compared to control sample i.e. conjugates prepared using Tetanus toxoid having 50-55% monomeric content.

TECHNICAL ADVANCEMENTS
The present disclosure described herein above has several technical advantages including, but not limited to, the realization of a process for stabilization of bacterial protein and its use in vaccine manufacturing wherein the process prevents formation of multimeric/aggregated toxoids, resulting in better predictability, safety, consistency, stability and potency in vaccine. Further, applicant has also found that a conjugate prepared as per present invention is capable of eliciting a better immune response.
The foregoing description of the specific embodiments fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein has been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired object or results.
Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.

The numerical values mentioned for the various physical parameters, dimensions or quantities are only approximations and it is envisaged that the values ten percent higher/lower than the numerical values assigned to the parameters, dimensions or quantities fall within the scope of the disclosure, unless there is a statement in the specification specific to the contrary.
While considerable emphasis has been placed herein on the components and component parts of the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other changes in the preferred embodiment as well as other embodiments of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

We Claim :
1. A process for stabilization of a protein during storage, comprising addition of a
stabilizing agent to a protein;
Wherein, the protein is a monomeric protein, and stabilization is obtained by preventing formation of multimeric or aggregated protein.
2. The process as claimed in claim 1, wherein the protein is a toxoid.
3. The process as claimed in claim 2, wherein the toxoid is derived from Cholera toxin, Diphtheria toxin, CRM197, Pertussis toxin, E. coli heat-labile toxin LT, Shiga toxin, Pseudomonas Exotoxin A, Botulinum toxin, Tetanus toxin, Anthrax toxin LF, Bordetella pertussis AC toxin, and Staphylococcus aureus Exfoliatin B.
4. The process as claimed in claims 1 or 2, wherein the protein is selected from Tetanus toxoid and/or Diphtheria toxoid.
5. The process as claimed in claim 1, wherein the stabilizing agent is an amino acid.
6. The process as claimed in claim 5, wherein the amino acid is selected from a group comprising of Histidine, Lysine, Glycine and combinations thereof.
7. The process as claimed in claims 5 and 6, wherein the stabilizing agent is Histidine.
8. The process as claimed in claim 1, wherein concentration of the stabilizing agent is between 1 - 400 mM.
9. The process as claimed in claim 1, wherein concentration of the stabilizing agent is between 100 - 300 mM.
10. The process as claimed in any of the preceding claims, wherein stabilizing agent is Histidine at a concentration of 100-200 mM.
11. The process as claimed in claims 1-10, wherein the % monomer content of said protein during storage is between 80 - 99 %.
12. The process as claimed in claims 1-10, wherein the % monomer reduction of said protein stabilized using 200 mM Histidine stored at 37 °C for 6 months is less than 10% and the % monomer reduction of said protein stabilized using 50 mM Histidine stored at 2-8 °C for 6 months is less than 10%.
13. The process as claimed in any of the preceding claims, wherein the protein is a vaccine composition comprising monomeric toxoid and stabilizing agent.
14. The process as claimed in claim 1, wherein prior to addition of a stabilizing agent, protein is purified using gel permeation chromatography to obtain a protein having 80-99 % monomeric content.

15. The process as claimed in claims 1 or 14, wherein the protein is subjected to chromatography selected from the group comprising of hydrophobic interaction chromatography, size exclusion chromatography, ion exchange chromatography and combinations thereof ; to obtain a protein having 80-99 % monomeric content.
16. A process for conjugation of a protein to a capsular polysaccharide to obtain a polysaccharide - protein conjugate, comprising of following steps:
a. Preparation of processed polysaccharide;
b. Subjecting protein to buffer exchange using conjugation compatible buffer
prior to use as carrier protein in a conjugation reaction;
c. Conjugating the processed polysaccharide obtained in step (a) with protein
obtained in step (b);
d. Subjecting the conjugate obtained in step (c) to purification, to obtain a
purified polysaccharide- protein conjugate;
Wherein the protein is stabilized protein having monomer content of more than 80% and is obtained by a process as claimed in claim 1.
17. The process as claimed in claim 16, wherein the stabilized protein is a monomeric toxoid.
18. The process as claimed in claim 17, wherein the monomeric toxoid is derived from Cholera toxin, Diphtheria toxin, CRM197, Pertussis toxin, E. coli heat-labile toxin LT, Shiga toxin, Pseudomonas Exotoxin A, Botulinum toxin, Tetanus toxin, Anthrax toxin LF, Bordetella pertussis AC toxin, and Staphylococcus aureus Exfoliatin B.
19. The process as claimed in claims 16 -18, wherein the protein is a monomeric toxoid selected from Tetanus toxoid and Diphtheria toxoid.
20. The process as claimed in claim 16, wherein the conjugation is carried out using conjugation chemistry selected from group comprising of cyanylation conjugation chemistry, Carbodiimide conjugation chemistry, and reductive amination conjugation chemistry.
21. The process as claimed in claim 16, wherein the processed polysaccharide is obtained by fermentation of at least one microorganism selected from group comprising Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus spp., Streptococcus spp., Group A Streptococcus, Group B Streptococcus, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus viridans,

Enterococcus faecalis, Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis, Salmonella spp., Salmonella typhi, Salmonella paratyphi, Salmonella typhimurium, Salmonella enteritidis, Staphylococcus aureus, Vibrio cholerae, Pasteurella pestis, Pseudomonas aeruginosa, Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Escherichia coli, Shigella spp., Ehrlichia spp., and Rickettsia spp.
22. The process as claimed in claim 16, wherein the processed polysaccharide is obtained by fermentation of Neisseria meningitidis serotype selected from the group comprising of N. meningitidis serotype A, B, C, D, W135, X, Y, Z and/or 29E.
23. The process as claimed in claim 16, wherein the processed polysaccharide is obtained by fermentation of Streptococcus pneumoniae serotype selected from the group comprising of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9A, 9F, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F, 45, 38, 35B, 23B, 24F, 15A, and/or 15C.
24. The process as claimed in claim 16, wherein the processed polysaccharide is obtained by fermentation of Haemophilus influenza.
25. The process as claimed in claim 16, wherein the processed polysaccharide is obtained by fermentation of Salmonella serovar strains selected from group comprising of Salmonella Enterica serovar typhi TY2 strain with "tviB" gene specific for Vi polysaccharide; S. typhi: ATCC 19430; C6524 ; S. paratyphi A: ATCC 9150, CMCC50073, CMCC50973; S. enteritidis: ATCC 4931; ATCC 13076; S. enteritidis R11; S. enteritidis D24359; S. enteritidis 618; S. enteritidis 502; S. enteritidis IV3453219; S. typhimurium: S. typhimurium 2192; ATCC 14208; S. typhimurium 2189; S. typhimurium D23580; ATCC 19585; ATCC 700408; (LT2/SL134 (ST19)); S. typhimurium 177 (ST19) CDC 6516-60; and ATCC 700720.
26. The process as claimed in claim 16, wherein the processed polysaccharide is obtained by fermentation of Group A Streptococcus, or Group B Streptococcus (group Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX).
27. The process as claimed in claim 16, wherein the processed polysaccharide is obtained by fermentation of Shigella spp. selected from Shigella sonnei, Shigella dysenteriae, Shigella flexneri, and Shigella boydii.

28. The process as claimed in claim 16, wherein the processed polysaccharide is obtained by fermentation of Staphylococcus spp. selected from Staphylococcus aureus, Staphylococcus aureus type 5, and Staphylococcus aureus type 8.
29. The process as claimed in claim 16, wherein the conjugation compatible buffer is selected from the group comprising Phosphate buffer, MOPS, NaCl, TRIS, TRIS-HCl, TRIS-NaCl, MES and MES-NaCl.
30. A composition of a protein, comprising;
a. a monomeric protein; and
b. a stabilizing agent;
Wherein, the stabilizing agent prevents formation of multimeric or aggregated protein during storage.
31. The composition as claimed in claim 30, wherein the protein is a monomeric toxoid.
32. The composition as claimed in claim 31, wherein the monomeric toxoid is derived from Cholera toxin, Diphtheria toxin, CRM197, Pertussis toxin, E. coli heat-labile toxin LT, Shiga toxin, Pseudomonas Exotoxin A, Botulinum toxin, Tetanus toxin, Anthrax toxin LF, Bordetella pertussis AC toxin, and Staphylococcus aureus Exfoliatin B.
33. The composition as claimed in claims 30-32, wherein the protein is a monomeric toxoid selected from Tetanus toxoid and Diphtheria toxoid.
34. The composition as claimed in claim 30, wherein the stabilizing agent is an amino acid.
35. The composition as claimed in claim 34, wherein the amino acid is selected from a group comprising of Histidine, Lysine, Glycine and combinations thereof.
36. The composition as claimed in claims 30 and 34-35, wherein the stabilizing agent is Histidine.
37. The composition as claimed in claim 30, wherein concentration of the stabilizing agent is between 1 - 400 mM.
38. The composition as claimed in claim 30, wherein concentration of the stabilizing agent is between 100 - 300 mM.
39. The composition as claimed in claims 30-38, wherein stabilizing agent is Histidine at a concentration of 100-200 mM.
40. The composition as claimed in claims 30-39, wherein the % monomer content of said protein during storage is between 80 - 99 %.

41. The composition as claimed in claims 30-39, wherein the % monomer content of said protein during storage at 37 °C is more than 80 %.
42. The composition as claimed in claims 30-41, wherein the composition is a vaccine composition comprising monomeric protein and stabilizing agent.
43. The composition as claimed in claim 42, wherein the composition is a vaccine composition comprising monomeric tetanus toxoid and stabilizing agent.
44. The composition as claimed in claim 42, wherein the stabilized toxoid is a vaccine composition comprising monomeric tetanus toxoid and Histidine.
45. The composition as claimed in claim 30, wherein the protein is a vaccine antigen.
46. The composition as claimed in claim 30, wherein the protein is a carrier protein in polysaccharide-protein conjugate vaccine.
47. The composition as claimed in claim 44, wherein the monomeric tetanus toxoid is an antigen in a monovalent vaccine composition.
48. The composition as claimed in claim 44, wherein the monomeric tetanus toxoid is an antigen in a multivalent combination vaccine composition.
49. The composition as claimed in claim 48, wherein multivalent vaccine composition comprises of two or more antigens selected from Tetanus (T), diphtheria (D), Pertussis (P), Haemophilus influenza PRP - protein conjugate (Hib), Hepatitis B (HepB), Inactivated polio virus (IPV), inactivated rotavirus (IRV).
50. The composition as claimed in claim 49, wherein multivalent vaccine composition comprises of two or more antigens in combination as DTP, DTP-Hib, DTP-Hib-HepB, DTP-Hib-HepB-IPV, or DTP-Hib-HepB-IPV-IRV.
51. The composition as claimed in claim 48, wherein the monomeric Tetanus toxoid has an immune boosting effect.
52. The composition as claimed in claims 42-51, wherein composition is a liquid composition.
53. The composition as claimed in claims 42-51, wherein composition is a lyophilized composition.
54. A polysaccharide – protein conjugate, comprising;
a. a polysaccharide;
b. a protein;
Wherein polysaccharide – protein conjugate is prepared by a process as claimed in claim 16.

55. The polysaccharide – protein conjugate as claimed in claim 54, wherein the polysaccharide is obtained by fermentation of at least one microorganism selected from group comprising Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus spp., Streptococcus spp., Group A Streptococcus, Group B Streptococcus, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus viridans, Enterococcus faecalis, Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis, Salmonella spp., Salmonella typhi, Salmonella paratyphi, Salmonella typhimurium, Salmonella enteritidis, Staphylococcus aureus, Vibrio cholerae, Pasteurella pestis, Pseudomonas aeruginosa, Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Escherichia coli, Shigella spp., Ehrlichia spp., and Rickettsia spp.
56. The polysaccharide – protein conjugate as claimed in claim 54, wherein the polysaccharide is obtained by fermentation of Neisseria meningitidis serotype selected from the group comprising of N. meningitidis serotype A, B, C, D, W135, X, Y, Z and/or 29E.
57. The polysaccharide – protein conjugate as claimed in claim 54, wherein the polysaccharide is obtained by fermentation of Streptococcus pneumoniae serotype selected from the group comprising of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9A, 9F, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F, 45, 38, 35B, 23B, 24F, 15A, and/or 15C.
58. The polysaccharide – protein conjugate as claimed in claim 54, wherein the polysaccharide is obtained by fermentation of Haemophilus influenza.
59. The polysaccharide – protein conjugate as claimed in claim 54, wherein the polysaccharide is obtained by fermentation of Salmonella Enterica serovar typhi TY2 strain with "tviB" gene specific for Vi polysaccharide; S. typhi: ATCC 19430; C6524; S. paratyphi A: ATCC 9150, CMCC50073, CMCC50973; S. enteritidis: ATCC 4931; ATCC 13076; S. enteritidis R11; S. enteritidis D24359; S. enteritidis 618; S. enteritidis 502; S. enteritidis IV3453219; S. typhimurium: S. typhimurium 2192; ATCC 14208; S. typhimurium 2189; S. typhimurium D23580; ATCC 19585; ATCC

700408; (LT2/SL134 (ST19)); S typhimurium 177(ST19) CDC 6516-60; ATCC 700720.
60. The polysaccharide - protein conjugate as claimed in claim 54, wherein the polysaccharide is obtained by fermentation of Group A Streptococcus, or Group B Streptococcus (group Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX).
61. The polysaccharide - protein conjugate as claimed in claim 54, wherein the polysaccharide is obtained by fermentation of Shigella spp. selected from Shigella sonnei, Shigella dysenteriae, Shigella flexneri, and Shigella boydii.
62. The polysaccharide - protein conjugate as claimed in claim 54, wherein the polysaccharide is obtained by fermentation of Staphylococcus spp. selected from Staphylococcus aureus, Staphylococcus aureus type 5, and Staphylococcus aureus type 8.
63. The polysaccharide - protein conjugate as claimed in claim 54, wherein the protein is a monomeric toxoid.
64. The polysaccharide - protein conjugate as claimed in claim 63, wherein the monomeric toxoid is derived from Cholera toxin, Diphtheria toxin, CRM197, Pertussis toxin, E. coli heat-labile toxin LT, Shiga toxin, Pseudomonas Exotoxin A, Botulinum toxin, Tetanus toxin, Anthrax toxin LF, Bordetella pertussis AC toxin, and Staphylococcus aureus Exfoliatin B.
65. The polysaccharide - protein conjugate as claimed in claims 54 and 63, wherein the protein is a monomeric toxoid selected from Tetanus toxoid and Diphtheria toxoid.
66. The polysaccharide - protein conjugate as claimed in claims 54-65, wherein the polysaccharide - protein conjugate is a vaccine composition comprising Haemophilus influenza PRP conjugated to Tetanus toxoid.
67. The polysaccharide - protein conjugate as claimed in claim 66, wherein the Haemophilus influenza PRP conjugated to Tetanus toxoid is a vaccine antigen in a monovalent vaccine composition.
68. The polysaccharide - protein conjugate as claimed in claim 66, wherein the Haemophilus influenza PRP conjugated to Tetanus toxoid is a vaccine antigen in a multivalent combination vaccine composition.
69. The vaccine composition as claimed in claim 66, wherein composition is a liquid composition.
70. The vaccine composition as claimed in claim 66, wherein vaccine composition is a lyophilized composition.