FORM 2 THE PATENTS ACT, 1970 (39 of 1970) AND THE PATENTS RULES, 2003 COMPLETE SPECIFICATION (See Section 10; Rule 13) MENINGOCOCCAL PROTEIN BASED VACCINE FORMULATIONS AND METHODS FOR MANUFACTURING THEREOF SERUM INSTITUTE OF INDIA PVT. LTD. an Indian Company of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune-411028, Maharashtra, India THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED. 2 FIELD The present invention relates to field of vaccine formulations and methods for manufacturing thereof. Particularly, the present invention relates to Upstream, Downstream and formulation development of Neisseria meningitidis (meningococcal) serogroup B based recombinant/ 5 chimeric protein antigens, methods of preparing such chimeric protein-based formulations and use of these formulations for prevention and/ or treatment of subjects with Neisseria meningitidis (meningococcal) serogroup B infections. BACKGROUND All publications herein are incorporated by reference to the same extent as if each individual 10 publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art. 15 Neisseria meningitidis is an important pathogen, particularly in children and young adults. Septicemia and meningitis are the most life-threatening forms of invasive meningococcal disease (IMD). Case fatality rates remain around 10% for disseminated disease, while a third of survivors of meningococcal disease suffer from significant debilitating, and long-term sequelae. This disease has become a worldwide health problem because of its high morbidity 20 and mortality. Toxoid-based vaccines have almost eliminated diphtheria and tetanus in wealthy countries, while capsule-based vaccines have substantially reduced disease caused by Haemophilus influenzae, Streptococcus pneumoniae, and some strains of Neisseria meningitidis. However, challenges remain in developing vaccines against pathogens for which toxoid and capsule25 based vaccines are not feasible. These pathogens include non-typeable strains of H. influenzae and S. pneumoniae, un-encapsulated pathogens such as Neisseria gonorrhoeae and Moraxella catarrhalis and encapsulated serogroup B N. meningitidis, for which a capsulebased vaccine is not feasible. Given the rise in the emergence of multi-drug resistant bacteria, new approaches for vaccine development are required. The development of a vaccine against 30 serogroup B meningococcus presents particular difficulties because the polysaccharide capsule is poorly immunogenic owing to its immunologic similarity to human neural cell 3 adhesion molecule. Also, strategies for generating successful vaccines are hampered by pathogen diversity and the difficulties associated with presenting epitopes from membraneembedded surface proteins to the immune system. Neisseria meningitidis (N. meningitidis) is a Gram-negative bacterium which colonizes the 5 human upper respiratory tract and is responsible for worldwide sporadic and cyclical epidemic outbreaks of, most notably, meningitis and sepsis. N. meningitidis typically possess a cytoplasmic membrane, a peptidoglycan layer, an outer membrane which together with the capsular polysaccharide constitute the bacterial wall, and pili, which project into the outside environment. Neisseria meningitidis (Nm) remains a leading cause of sepsis and bacterial 10 meningitis in children and young adults. There are approximately 500,000 cases of meningococcal disease each year with around 50,000 deaths. In developed countries, the bacterium is a leading cause of mortality among children, has important public health impacts during outbreaks in schools and universities, and can cause profound disability in survivors. Encapsulated strains of N. meningitidis are a major cause of bacterial meningitis and 15 septicemia in children and young adults. N meningitidis can be classified into at least 12 serogroups (including serogroups A, B, C, H, I, K, L, 29E, W135, X, Y and Z) based on chemically and antigenically distinctive polysaccharide capsules. The most common serogroups being A, B and C which are responsible for 90% of disease worldwide. Serogroup B is the most common cause of meningococcal disease in Europe, USA and several countries 20 in Latin America and causes epidemics in sub-Saharan Africa every 5-10 years. Meningococcal is a devastating disease that can kill children and young adults within hours despite the availability of antibiotics. Therefore, prophylactic immunisation is the best way to protect individuals from meningococcal infection. Vaccines are available based on the bacterial polysaccharide capsule, but the polysaccharide 25 capsule of N. meningitidis serogroup B is poorly immunogenic as it has structural identity with a human glycoprotein in neural tissue and could induce autoimmunity if used as a vaccine. Therefore, serogroup B ‘humanization’ put on-hold early efforts towards the development of a safe and immunogenic conjugate polysaccharide vaccine against B group since the polysaccharide does not elicit serum bactericidal antibodies and in vitro anti-capsule 30 B antibodies recognize neural cell adhesion molecules in fetal brain tissue. Two main approaches have been used to develop vaccines against serogroup B N. meningitidis; outer membrane vesicle vaccines (OMVV) and recombinant protein subunit 4 vaccines. OMVVs were first developed in the 1980s. The immunodominant antigen in meningococcal OMVVs is PorA, an abundant outer-membrane porin with eight surfaceexposed loops. Loops one and four are termed variable region 1 and 2 (VR1 and VR2), respectively, as they generate immune responses and are subject to antigenic variation. The 5 VR2 loop dominates PorA-specific immunity elicited by OMVVs, which offer limited or no cross-protection against strains expressing PorA with a different VR2. When longer follow up periods were assessed including an OMV vaccine studied over 20 months in Chile, the efficacy decreased to 50%, indicating poor longevity of response (A.L. Wilkins, M.D. Snape et al 2017). 10 To broaden coverage, OMVVs containing multiple PorAs have been developed and selected for the prevalence of PorA sequences in circulating strains. However, OMVVs present complex manufacturing and regulatory issues. Prior art vaccines have often made use of purification of so called "blebs" which represent vesicles shed from the cell surface of the particular organism of interest. However, such a crude product carries many problems. For 15 example, there is wide variation in the composition of these blebs. There is no reliable way of controlling which proteins are included or excluded from these blebs. These blebs may or may not include polysaccharide- coating elements of the organism of interest. The proportions of the various components of the blebs in relation to one another cannot be reliably determined. The composition of these blebs cannot be easily determined or 20 controlled. The OMV in Bexsero® (GSK Vaccines) is highly reactogenic, so the vaccine is routinely given with paracetamol due to which parents become concerned about adverse effects (Prymula, R. 2014). Bexsero® provides uncertain coverage as 1) antigens are derived from a single meningococcal strain (Tan, L., et al. 2010) 2) most studies of immunogenicity were 25 done with a 3+1 schedule and not the 2+1 schedule planned for the UK (doses at 2, 4 and 12 months) 3) indirect not direct correlates of protection have been mostly measured in 13 month olds, and not in infants who are at the greatest risk. N. meningitidis serogroup B vaccines based on Outer membrane vesicle (OMVs) have been used successfully to prevent epidemics (most recently in New Zealand) but only confer 30 protection against strains expressing the same variant of PorA (an outer membrane porin) as in the OMV. OMVs as immunogens are not favoured because consistency and toxicity can be 5 problematic during manufacture. For example, OMVs may contain toxic lipopolysaccharide (LPS). PorA is the most abundant meningococcal outer membrane protein (OMP), which also elicits SBA, and is the main target of immune responses elicited by OMV vaccines which have been 5 used successfully in outbreaks. PorA variants differ in their variable regions (VRs), surface exposed loops which are the target of immune responses. Of note, VR2 is responsible for most SBA elicited by OMV vaccines. However, PorA is an integral OMP with multiple hydrophobic domains. This makes PorA difficult to produce as a recombinant protein in its native conformation, limiting its use as an antigen in subunit vaccines. 10 Factor H binding protein (fHbp) is also an antigen that elicits serum bactericidal antibody responses in immunised individuals and is a key component of investigational vaccines for the prevention of meningococcal, in particular serogroup B, disease that are currently being evaluated in clinical trials. Factor H Binding Protein (fHbp, also referred to in the art as lipoprotein 2086 (Fletcher et al (2004) Infect Immun 72:2088-2100), Genome-derived 15 Neisserial antigen (GNA) 1870 (Masignani et al. (2003) J Exp Med 197:789-99) or "741") is a surface-exposed lipoprotein expressed in the N. meningitidis bacterium. Based on differences in the nucleotide and predicted amino acid sequences, fHbps from different Neisseria meningitidis strains have been categorised using several schemes. These include two subfamilies (A and B) (Murphy E, et al. (2009) The Journal of Infectious Diseases 200: 20 379- 389) or three variant groups (V1, V2, and V3) (Masignani V, et al. (2003) The Journal of Experimental Medicine 197: 789-799), with subfamily A corresponding to V2 and V3, and subfamily B corresponding to V1 (which is the most abundant). The recombinant subunit vaccines Bexsero® (GSK vaccines) and Trumenba® (Wyeth Pharmaceuticals) contain an important meningococcal antigen, factor H binding protein 25 (fHbp), which is a lipoprotein composed of two β-barrels that tightly bind domains 6 and 7 of human complement factor H (CFH). fHbp is antigenically variable; databases of genome sequences contain more than 900 different fHbp peptides, which fall into three variant groups or two subfamilies: V1 (subfamily B), V2 and V3 (both subfamily A). In general, immunisation with a particular fHbp induces cross-protection against strains that express 30 fHbp belonging to the same, but not a different, variant group, although there can be crossprotection between fHbp variant groups 2 and 3 (subfamily A). Bexsero® contains a single fHbp peptide (V1.1), with two other recombinant antigens as well as an OMV, while 6 Trumenba®is composed solely of two fHbp peptides (V1.55 and V3.45). To date, no vaccine studies have included V2 fHbp even though strains expressing this variant account for around 20-30% of all isolates (38% of UK cases). There is no vaccine with a v2 fHbp as this is an inherently unstable antigen. fHbp on Neisseria meningitidis is a 27 KDa lipoprotein that 5 consists of two beta barrels (an N terminal barrel and a C terminal barrel) joined by a short amino acid linker. The reason for the lack of V2 fHbp in current licensed vaccines is the instability of its N terminal β-barrel (Prymula, R. 2014; Johnson, S 2012). Further, protein stability is important during vaccine manufacturing as it affects yield and is a significant issue for quality control. Also, antigens in Bexsero® and Trumenba® have exact sequence matches 10 to 36 and 4.8%, respectively of serogroup B N. meningitidis disease isolates currently circulating in the UK, leading to concerns about their ability to provide broad coverage against an antigenically diverse pathogen. Further, modified V2 fHbp having increased stability over wild type V2 fHbp has been disclosed wherein modified V2 fHbp comprising atleast six mutations at Ser35, Aspl07, Vall l2, Leul l4, Serl37 and Glyl38. Since, a single 15 fHbp does not provide universal protection against meningococcal disease, therefore, immunisation with a vaccine comprising representatives from each of the three variants, VI, V2 and V3, is necessary for a broad-N meningitidis serogroup B protection. Both Bexsero® and the Trumenba® vaccine(s) were developed before it was appreciated that Complement factor H (CFH) binds the meningococcus via fHbp at high affinity, and that this 20 can impair immune responses (Schneider, M 2009). Although fHbp has been demonstrated to be an important protective antigen, the extent to which fHbp interacts with fH upon immunization, and whether any fHbp-fH interaction affects the overall immunogenicity of fHbp in humans is currently unknown. Studies in hfH transgenic mice and infant rhesus macaques, the latter having a polymorphism in the fH gene that allows for either high or low 25 binding to fHbp (Konar M 2015), have demonstrated that binding of fH to fHbp lowers the immunogenicity of fHbp (Beemink PT 2011) (Costa 2014) (Giuntini S 2015) (Granoff DM 2015) (Rossi R 2013). Further, Upstream, Downstream and formulation development can often be the rate-limiting step in early introduction of biopharmaceuticals into the market and in meeting the demands 30 of the population. Upstream process development includes scale-up of a fermentation process to ensure a similar product yield with quality at large scale as is produced at small scale. Various 7 cultivation parameters, such as media composition, pH, agitation, aeration, temperature, cell density, the concentration of inducers, induction time, and feeding strategies affect the protein expression level depending upon expression systems. Thus, it is essential to evaluate each of the cultivation conditions for the expression of every recombinant protein and the 5 development of effective bioprocesses. Escherichia coli is the most widely used bacterial host for the production of recombinant proteins due to: (1) its fast growth rate with a generation time spanning 20 min under optimized conditions (Clark and Maaløe, 1967), (2) well-developed tools of molecular manipulations along with in-depth knowledge of its biology, and (3) the ability to achieve 10 high cell density using inexpensive culture reagents. But in practice, a number of obstacles encountered along the pipeline must be overcome. These include poor growth of the host strain, protein instability or toxicity, aggregation and inclusion body formation, unsuitability of environmental conditions (temperature, pH, salt concentration, etc) and even no amplified expression at all. When the protein of interest cannot be detected or it is detected but at very 15 low levels (less than micrograms per liter of culture), this could be due to slower growth rate, low final cell density, and death. E. coli cannot perform post-translational modifications, limiting the product range that can be produced in a soluble and active form in this host organism. Furthermore, E. coli cannot secrete recombinant proteins. Consequently, recombinant E. coli cells need to be disrupted to access the intracellular product, which is 20 then usually purified by several steps of filtration. In addition, high level expression of recombinant proteins in E. coli results in aggregation of expressed proteins into inclusion bodies (IB's). This poses a serious challenge for producing soluble recombinant proteins with proper biological function at the industrial scales, as it requires extensive processing involving isolation from cell, solubilization, refolding and 25 purification to produce the bioactive proteins. Altering culture conditions usually present the simplest solution to reduce IBs formation in E. coli. However, culture conditions favourable for soluble protein production may vary depending on the involved proteins of interest and the used host strains of E. coli, and thus require experimental optimization. Factors such as expression strain, fermentation medium, and operating conditions, all play important roles in 30 the process upscaling to maintain or improve yield on a larger scale and eventually at an industrial scale to provide large quantities of the protein through a cost-effective, commercially viable manufacturing process. It is important, therefore, to identify an appropriate parameter or set of parameters that will be critical to the specific process. 8 High-level production of recombinant proteins will subsequently require an efficient purification process at the industrial scales since it contributes to the approval of therapeutic products for human use. Cell disruption is required for recovery of the desired proteins, expressed as intracellular IBs. Cell disruption can be very effective in small scale work; 5 however, upscaling is very poor. Sonication has high energy requirements, as well as high health and safety issues, due to noise. It is not continuous. Chemical Cell Lysis pose significant health and safety risks to the user and cost of using large volumes of reagents needed for large-scale production can be prohibitive. Further, the presence of salts and detergents may not be compatible with protein assays. It may also affect the results of 10 downstream applications (e.g., mass spectrometry). Hence, appropriate cell lysis method needs to be considered for optimum results during upscaling. It is well-established that an increased product concentration in the upstream process leads to a higher volume of chromatography resin and a higher buffer requirement. Host cell proteins (HCPs) and DNA are the main source of impurities, and the HCPs of each process vary 15 significantly from each other in their molecular mass, charge, hydrophobicity, and structure. Therefore, they present a challenge for chromatographic purification. Where a purified soluble active recombinant protein is needed, it is invaluable to have means to (i) detect it along the expression and purification scheme, (ii) attain maximal solubility, and (iii) easily purify it from the E. coli cellular milieu. The expression of a stretch of amino 20 acids (peptide tag) or a large polypeptide (fusion partner) in tandem with the desired protein to form a chimeric protein may allow these three goals to be straightforwardly reached. Being small, peptide tags are less likely to interfere when fused to the protein. However, in some cases they may provoke negative effects on the tertiary structure or biological activity of the fused chimeric protein. Hence, peptide tags should be removed too because it can affect 25 protein conformation, hamper the interaction with a partner molecule or decrease the biological activity. Indeed, when these tags are removed, the final solubility of the desired product is unpredictable. In the case of tag removal by enzyme digestion, expression vectors possess sequences that encode for protease cleavage sites downstream of the gene coding for the tag. Choosing among the different proteases is based on specificity, cost, number of 30 amino acids left in the protein after cleavage and ease of removal after digestion C-type cysteine protease from Tobacco Etch Virus (TEV) is among the most widely used. However, expression of TEV protease in Escherichia coli faced difficulties regarding protein yield 9 (product yield is reduced) or low solubility of the protein at the industrial scales which means that large volumes and often long incubation times are required for efficient cleavage. Vaccines based on recombinant protein antigens generally require an adjuvant to achieve protection from the associated disease. Aluminum salts are the most prevalent adjuvants in 5 vaccines approved for human use by the U.S. Food and Drug Administration. The point of zero charge (PZC) of the adjuvant is the point at which the net surface charge is zero; the PZC for aluminum oxyhydroxide is approximately 11, whereas the PZC for aluminum phosphate is approximately 4–5.5. Protein adsorption to the adjuvant surfaces is generally maximized when the sign of the net charge of the protein is opposite that of the adjuvant 10 surface, allowing for an electrostatic attraction. Therefore, the protein vaccine formulation is formulated with adsorption buffers to improve adsorption to the surface. However, protein conformation can change when proteins bind to liquid-solid interfaces. Furthermore, conformational changes induced by binding to adjuvant could alter protein stability during long-term storage. For example, if adsorption is essentially complete, aggregation via 15 pathways that occur in bulk solution are not likely to occur. Conversely, unfolding upon binding may expose normally buried residues to solvent, promoting degradation processes such as oxidation. (J Pharm Sci. 2009 September; 98(9): 2970–2993). Physical and covalent stabilization upon long-term storage of
WE CLAIM: 1. A modified factor H binding protein (fHbp) comprising wild type fHbp variant and at least one exogenous loop(s), wherein • the modified factor H binding protein (fHbp) is selected from amino acid 5 sequence with at least 75% identity with any one of sequences of SEQ ID Nos 6 to 10, • the at least one exogenous peptide loop(s) is immunogenic, • the at least one exogenous peptide loop(s) is derived from a bacterial membrane protein, 10 • the modified fHbp is a fusion protein, • the fHbp variant is selected from v1, v2 and v3, modified with at least one PorA loop comprising at least 10 amino acids inserted into a β-turn region in fHbp; and • the PorA loop is selected from VR1, and VR2. 15 2. The modified factor H binding protein as claimed in claim 1, wherein the modified fHbp is modified to reduce factor H binding activity. 20 3. A nucleic acid sequence encoding the modified fHbp, wherein the nucleic acid sequence is with at least 75% identity with any one of sequences of SEQ ID NO. 1 to 5. 4. An immunogenic composition comprising at least one modified fHbp as claimed in 25 any one of the claims 1 to 2 or the nucleic acid sequence encoding the modified fHbp as claimed in claim 3. 5. The immunogenic composition as claimed in claim 4, wherein the composition comprises two or more different modified fHbp. 30 6. The immunogenic composition as claimed in any one of the claims 4 to 5, wherein the composition comprises a pharmaceutically acceptable carrier. 136 7. The immunogenic composition as claimed in any one of the claims 4 to 6, wherein the composition further comprises an adjuvant. 8. The immunogenic composition as claimed in any one of the claims 4 to 7, wherein the 5 composition further comprises at least one other prophylactically or therapeutically active molecule comprising a monovalent carrier protein: capsule polysaccharide conjugate vaccine. 9. The immunogenic composition as claimed in any one of the claims 4-8, wherein the 10 fHbp scaffold bearing exogenous peptide loops is incorporated as the protein carrier molecule in the conjugate vaccine. 10. The immunogenic composition as claimed in any one of the claims 4 to 9 comprising a recombinant protein/ modified fHbp fusion protein in combination with at least one 15 additional antigen selected from: - a protein antigen from PorB, Fet A, OmpC, NHBA, NadA, meningococcal antigen 287, NspA, HmbR, NhhA, App, 936, - a saccharide or conjugate antigen from N. meningitidis serogroup A, C, W, Y and/or X, 20 - a saccharide or conjugate antigen from Streptococcus pneumoniae, - a diphtheria antigen, such as a diphtheria toxoid e.g. the CRM197 mutant, - a tetanus antigen, such as a tetanus toxoid, - an antigen from Bordetella pertussis, acellular or whole cell pertussis antigens, - a saccharide or conjugate antigen from Haemophilus influenzae B, 25 - polio antigen(s) such as IPV, - measles, mumps and/or rubella antigens, - influenza antigen(s), such as the haemagglutinin and/or neuraminidase surface proteins, 137 - antigen (protein or saccharide or conjugate) from Streptococcus agalactiae (group B streptococcus), - antigen (protein or saccharide or conjugate) from Streptococcus pyogenes (group A streptococcus), 5 - antigen (protein or saccharide or conjugate) from Staphylococcus aureus, - antigen (protein or saccharide or conjugate) from Salmonella Spp. 11. The immunogenic composition as claimed in any one of the claims 4 to 10, wherein the fHbp scaffold bearing exogenous peptide loops is incorporated as the protein 10 carrier molecule in the polysaccharide conjugate vaccine selected from Monovalent (A, X, C, W, Y), Bivalent (A-C, A-B, X-B, C-B), Trivalent (AC-B, AC-Hib), Quadrivalent (AC-Hib-B), Pentavalent (ACWYX), or Hexavalent (ACWYX-B). 12. The modified fHbp as claimed in any one of claims 1 to 2, a nucleic acid as claimed 15 in claim 3, or a composition as claimed in any one of claims 4 to 8, for use as a medicament, or in treatment or prevention of a pathogenic infection or colonization in a subject. 13. A combination of the modified fHbp as claimed in any one of claims 1 to 2, a nucleic 20 acid as claimed in claim 3, or a composition as claimed in any one of claims 4 to 8, and at least one other prophylactically or therapeutically active molecule. 14. The combination as claimed in claim 13 or composition as claimed in claim 8, wherein the at least one other prophylactically or therapeutically active molecule 25 comprises a conjugate vaccine, comprising any of serogroup capsular polysaccharides selected from A, C, Y, W, or X strains, or combinations thereof. 15. The combination as claimed in claim 14 or the composition as claimed in claim 8, wherein the protein: capsule polysaccharide vaccine comprises any of serogroup C or 30 A capsule with bacterial toxoids, bivalent vaccines (with serogroup C and A capsular polysaccharide conjugated to bacterial toxoids), quadrivalent (serogroups A, C, Y, W 138 polysaccharides conjugated to bacterial toxoids) or pentavalent (serogroups A, C, Y, W, X polysaccharides conjugated to bacterial toxoid) conjugate vaccines. 16. The factor H binding protein (fHbp) as claimed in claims 1 to 2 as and when used as 5 an epitope display scaffold. 17. A vaccine formulation comprising at least one recombinant protein/ modified fHbp fusion protein, an adjuvant and one or more pharmaceutically acceptable excipient, wherein in the recombinant protein/ modified fHbp fusion protein the one or more 10 exogenous loops is selected from but not limited to group consisting of Transferrin Binding Protein, Neisserial Heparin Binding Protein, Neisserial Surface Protein A, PorA, meningococcal enterobactin receptor FetA, Neisserial Adhesin A, the fHbpfHbp fusion protein as claimed in any one of the previous claims or a combination thereof. 15 18. The vaccine formulation as claimed in claim 17, wherein the recombinant protein/ modified fHbp fusion protein is fHbp as claimed in any one of claims 1-2, or the nucleic acid sequence encoding the modified fHbp as claimed in claim 3, and wherein the fHbp is derived from Neisseria meningitidis serogroup A, B, C, H, I, K, L, 29E, 20 W135, X, Y and Z. 19. The vaccine formulation as claimed in claims 17 or 18, wherein the recombinant protein/ modified fHbp fusion protein is derived from Neisseria meningitidis serogroup B. 25 20. The vaccine formulation as claimed in any one of the claims 17-19, wherein the recombinant protein/ modified fHbp fusion protein is fHbp has molecular weight in the range of 10 kDa to 200 kDa, preferably up to 50 kDa. 30 21. The vaccine formulation as claimed in any one of the claims 17-20, wherein the adjuvant is selected from the group consisting of aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and potassium aluminum sulfate, MF-59, a liposome, a lipopolysaccharide, a saponin, lipid A, lipid A derivatives, Monophosphoryl lipid A, GLA, 3–deacylated monophosphoryl lipid A, AS01, AS03, 139 AF3, IL-2, RANTES, GM- CSF, TNF-a, IFN-g, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL, an oligonucleotide, an oligonucleotide comprising at least one unmethylated CpG and/or a liposome, Freund’s adjuvant, Freund’s complete adjuvant, Freund’s incomplete adjuvant, polymers, co-polymers such as 5 polyoxyethylene-polyoxypropylene copolymers, including block co-polymers, polymer p 1005, CRL-8300 adjuvant, muramyl dipeptide, such as an agonist of TLR1/2 TLR2, TLR3, TLR-4 agonists, TLR5, TLR7, TLR7/8, TLR8, TLR9, ODN 2216 (type A), TLR11/12, TLR-4 agonists, flagellin, flagellins derived from gram negative bacteria, TLR-5 agonists, fragments of flagellins capable of binding to TLR10 5 receptors, Alpha-C-galactosylceramide, Chitosan, Interleukin-2, QS-21, squalene, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, mycobacterium cell wall preparations, mycolic acid derivatives, non-ionic block copolymer surfactants, OMV, fHbp, saponin combination with sterols and lipids, dmLT, 1,25- dihydroxyvitamin D3, CAF01, poly [di (carboxylatophenoxy)- phosphazene] (PCPP) 15 and Venezuelan equine encephalitis (VEE) replicon particles or a combination thereof. 22. The vaccine formulation as claimed in any one of the claims 17-21, wherein the adjuvant is aluminium hydroxide having particle size > 500 nm. 20 23. The vaccine formulation as claimed in any one of the claims 17-20, wherein the one or more pharmaceutically acceptable excipient is a. a buffering agent selected from carbonate, phosphate, acetate, HEPES, 25 Succinate, TRIS, borate, citrate, lactate, gluconate, tartrate, or a combination thereof; b. a sugar selected from trehalose, mannose, raffinose, lactobionic acid, glucose, maltulose, iso- maltulose, maltose, lactose, dextrose, fructose, or a combination thereof; 30 c. a sugar alcohol or polyol selected from mannitol, lactitol, sorbitol, glycerol, xylitol, maltitol, lactitol, erythritol, isomalt and hydrogenated starch hydrolysates or a combination thereof; d. a surfactant selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 85, nonylphenoxypolyethoxethanol, 140 octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin, a poloxamer, copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), 5 octoxynols, phospholipids, nonylphenol ethoxylates, polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols, sorbitan esters or a combination thereof; e. a polymer selected from dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin or a combination thereof; 10 f. a salt selected from NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaC12, MgC12, or a combination thereof; g. an amino acid selected from tricine, leucine, iso-leucine, glycine, glutamine, L-arginine, L-arginine hydrochloride, lysine, L-alanine, Tryptophan, Phenylalanine, Tyrosine, Valine, Cysteine, Glycine, Methionine, Proline, 15 Serine, Threonine, or a combination thereof; h. a hydrolysed protein selected from gelatin, lactalbumin hydrolysate, monosodium glutamate, collagen hydrolysate, keratin hydrolysate, peptides, Casein hydrolysate, whey protein hydrolysate, serum albumin or a combination thereof; 20 i. a preservative selected from phenoxyethanol, Benzethonium chloride (Phemerol), Phenol, m-cresol, Thiomersal, Formaldehyde, paraben esters, benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chlor-m-cresol, benzyl alcohol or a combination thereof; and j. a liquid carrier selected from water for injection (WFI) or saline. 25 24. The vaccine formulation as claimed in any one of the claims 17-23, wherein the vaccine formulation comprises: - at least one at least one recombinant protein/ at least one modified fHbp as claimed in any one of previous claims 30 - aluminium hydroxide; - mannitol; - phosphate; and - polysorbate. 141 25. The vaccine formulation as claimed in any one of the claims 17-24, wherein the vaccine formulation comprises: - at least one modified fHbp of the amino acid sequence with at least 75% identity 5 with from any one of amino acid sequences SEQ ID NO. 6 to 10; or - at least one modified fHbp encoded the nucleic acid sequence is with at least 75% identity with by any one of nucleic acid sequence SEQ ID NO. 1 to 5; and - aluminium hydroxide; - mannitol; 10 - phosphate; and - polysorbate. 26. The vaccine formulation as claimed in any one of the claims 17-25, comprising (i) at least one recombinant protein/ modified fHbp fusion protein; (ii) aluminium 15 hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; (iii) mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; (iv) phosphate buffer in an amount in the range of 1 mM to 10 mM; and (v) polysorbate 20 in an amount in the range of 0.01 mg/ml to 2 mg/ml; wherein each recombinant protein/ modified fHbp fusion protein is present in an amount in the range of 15 µg/ml to 150 µg/ml. 20 27. The vaccine formulation as claimed in any one of the claims 17-25, comprising (i) at least two recombinant proteins/ modified fHbp fusion proteins; (ii) aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; (iii) mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; (iv) phosphate buffer in an amount in 25 the range of 1 mM to 10 mM; and (v) polysorbate 20 in an amount in the range of 0.01 mg/ml to 2 mg/ml; wherein each recombinant protein/ modified fHbp fusion protein is present in an amount in the range of 15 µg/ml to 150 µg/ml. 28. The vaccine formulation as claimed in any one of the claims 17-25, comprising (i) at 30 least three recombinant proteins/ modified fHbp fusion proteins; (ii) aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; (iii) mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; (iv) phosphate buffer in an amount in the range of 1 mM to 10 mM; and (v) polysorbate 20 in an amount in the range of 142 0.01 mg/ml to 2 mg/ml; wherein each recombinant protein/ modified fHbp fusion protein is present in an amount in the range of 15 µg/ml to 150 µg/ml. 29. The vaccine formulation as claimed in any one of the claims 17-25, comprising (i) at 5 least four recombinant proteins/ modified fHbp fusion proteins; (ii) aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; (iii) mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; (iv) phosphate buffer in an amount in the range of 1 mM to 10 mM; and (v) polysorbate 20 in an amount in the range of 0.01 mg/ml to 2 mg/ml; wherein each recombinant protein/ modified fHbp fusion 10 protein is present in an amount in the range of 15 µg/ml to 150 µg/ml. 30. The vaccine formulation as claimed in any one of the claims 17-24, wherein the vaccine formulation comprises: 15 - at least one modified fHbp of the amino acid sequence with at least 75% identity with from any one of amino acid sequences SEQ ID NO. 6 to 10; or - at least one modified fHbp encoded the nucleic acid sequence is with at least 75% identity with by any one of nucleic acid sequence SEQ ID NO. 1 to 5; and 20 - aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; - mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; - phosphate buffer in an amount in the range of 1 mM to 10 mM; and - polysorbate 20 in an amount in the range of 0.01 mg/ml to 2 mg/ml. 25 31. The vaccine formulation as claimed in any one of the claims 17-30, comprising: a. fHbpV3.45 M5:PorA316-320/exP1.14 (of SEQ ID NO. 6 or encoded by SEQ ID NO. 1) in an amount in the range of 15 µg/ml to 150 µg/ml; or b. fHbpV2.19 M6:PorA316-320/exP1.4 (of SEQ ID NO. 7 or encoded by SEQ ID NO. 2) in an amount in the range of 15 µg/ml to 150 µg/ml; or 30 c. fHbpV1.14:PorA307-311/exP1.9 (of SEQ ID NO. 8 or encoded by SEQ ID NO. 3) in an amount in the range of 15 µg/ml to 150 µg/ml; or d. fHbpV1.1:PorA307-311/exP1.4 (of SEQ ID NO. 9 or encoded by SEQ ID NO. 4); in an amount in the range of 15 µg/ml to 150 µg/ml; and e. aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; 143 f. mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; g. phosphate buffer in an amount in the range of 1 mM to 10 mM; and h. polysorbate 20 in an amount in the range of 0.01 mg/ml to 2 mg/ml. 5 32. The vaccine formulation as claimed in any one of the claims 17-30, comprising: a. fHbpV3.45 M5:PorA316-320/exP1.14 (of SEQ ID NO. 6 or encoded by SEQ ID NO. 1) in an amount in the range of 15 µg/ml to 150 µg/ml; b. fHbpV2.19 M6:PorA316-320/exP1.4 (of SEQ ID NO. 7 or encoded by SEQ ID NO. 2) in an amount in the range of 15 µg/ml to 150 µg/ml; 10 c. fHbpV1.14:PorA307-311/exP1.9 (of SEQ ID NO. 8 or encoded by SEQ ID NO. 3) in an amount in the range of 15 µg/ml to 150 µg/ml; d. fHbpV1.1:PorA307-311/exP1.4 (SEQ ID NO. 9 or encoded by SEQ ID NO. 4) in an amount in the range of 15 µg/ml to 150 µg/ml; e. aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; 15 f. mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; g. phosphate buffer in an amount in the range of 1 mM to 10 mM; and h. polysorbate 20 in an amount in the range of 0.01 mg/ml to 2 mg/ml. 33. The vaccine formulation as claimed in any one of the claims 17-30, comprising: 20 a. fHbpV3.45 M5:PorA316-320/exP1.14 (of SEQ ID NO. 6 or encoded by SEQ ID NO. 1) in an amount in the range of 15 µg/mL to 150 µg/mL; or b. fHbpV2.19 M6:PorA316-320/exP1.4 (of SEQ ID NO. 7 or encoded by SEQ ID NO. 2) in an amount in the range of 15 µg/mL to 150 µg/mL; or c. fHbpV1.1:PorA307-311/exP1.9 (of SEQ ID NO. 10 or encoded by SEQ ID NO. 25 5) in an amount in the range of 15 µg/mL to 150 µg/mL; or d. fHbpV1.14:PorA307-311/exP1.9 (of SEQ ID NO. 8 or encoded by SEQ ID NO. 3) in an amount in the range of 15 µg/mL to 150 µg/mL; and e. aluminium hydroxide in an amount in the range of 0.5 mg/mL to 4.5 mg/mL; f. mannitol in an amount in the range of 5 mg/mL to 100 mg/mL; 30 g. phosphate buffer in an amount in the range of 1 mM to 10 mM; and h. polysorbate 20 in an amount in the range of 0.01 mg/mL to 2 mg/mL. 34. The vaccine formulation as claimed in any one of the claims 17-30, comprising: 144 a. fHbpV3.45 M5:PorA316-320/exP1.14 (of SEQ ID NO. 6 or encoded by SEQ ID NO. 1) in an amount in the range of 15 µg/mL to 150 µg/mL; b. fHbpV2.19 M6:PorA316-320/exP1.4 (of SEQ ID NO. 7 or encoded by SEQ ID NO. 2) in an amount in the range of 15 µg/mL to 150 µg/mL; 5 c. fHbpV1.1:PorA307-311/exP1.9 (of SEQ ID NO. 10 or encoded by SEQ ID NO. 5) in an amount in the range of 15 µg/mL to 150 µg/mL; d. fHbpV1.14:PorA307-311/exP1.9 (of SEQ ID NO. 8 or encoded by SEQ ID NO. 3)in an amount in the range of 15 µg/mL to 150 µg/mL; e. aluminium hydroxide in an amount in the range of 0.5 mg/mL to 4.5 mg/mL; 10 f. mannitol in an amount in the range of 5 mg/mL to 100 mg/mL; g. phosphate buffer in an amount in the range of 1 mM to 10 mM; and h. polysorbate 20 in an amount in the range of 0.01 mg/mL to 2 mg/mL. 35. The vaccine formulation as claimed in any one of the claims 17-34, wherein the 15 formulation comprises 2-phenoxyethanol in an amount in range of 1 mg/mL to 10 mg/ml. 36. The vaccine formulation as claimed in any one of the claims 17-35, wherein the vaccine composition is stable at 2-8°C, 25°C and 40°C for over a period of six 20 months. 37. The vaccine formulation as claimed in any one of the claims 17-35, having Zeta potential in the range of -16 mV to -30 mV; osmolality in the range of 200 mOsmol/kg to 500 mOsmol/kg. 25 38. The vaccine formulation as claimed in any one of the claims 17-35, further comprising one or more antigen selected from Diphtheria toxoid (D), Tetanus toxoid (T), Whole cell pertussis (wP), hepatitis B virus surface antigen (HbsAg), Haemophilus influenzae b PRP-Carrier protein conjugate (Ηib), Haemophilus 30 influenzae (a, c, d, e, f serotypes and the unencapsulated strains), Neisseria meningitidis A antigen(s), Neisseria meningitidis C antigen(s), Neisseria meningitidis W-135 antigen(s), Neisseria meningitidis Y antigen(s), Neisseria meningitidis X antigen(s), Streptococcus Pneumoniae antigen(s), Neisseria meningitidis B bleb or purified antigen(s), Staphylococcus aureus antigen(s), Anthrax, BCG, Hepatitis (A, C, 145 D, E, F and G strains) antigen(s), Human papilloma virus, HIV, Salmonella typhi antigen(s), acellular pertussis, modified adenylate cyclase, Malaria Antigen (RTS,S), Measles, Mumps, Rubella, Dengue, Zika, Ebola, Chikungunya, Japanese encephalitis, rotavirus, Diarrheal antigens, Flavivirus, smallpox, yellow fever, Shingles, Varicella 5 virus antigens, and combinations thereof. 39. The vaccine formulation as claimed in any one of the claims 17-35, 38 comprising (i) at least one fusion protein comprising stable non-functional/ non-lipidated fHbp and PorA VR2 loop, and (ii) at least one polysaccharide-protein conjugate. 10 40. The vaccine formulation as claimed in any one of the claims 17-39, wherein the recombinant protein/ modified fHbp fusion protein is co-administered with one or more vaccine selected from BEXSERO, MENVEO, MENACTRA, NIMENRIX, MenQuadFi, MENFIVE, MenAfriVac, Men AC, and Men ACHib. 15 41. The vaccine formulation as claimed in any one of the claims 17-40, wherein the recombinant protein/ modified fHbp fusion protein is co-administered with MENFIVE. 20 42. The vaccine formulation as claimed in any one of the claims 17-41 comprising i) at least one fusion protein comprising stable non-functional /non-lipidated fHbp and PorA VR2 loop and at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) capsular saccharide of serogroup C N. meningitidis and (ii) CRM197; 25 (c) a conjugate of (i) capsular saccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) capsular saccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) capsular saccharide of serogroup X N. meningitidis and (ii) tetanus toxoid. 30 43. The vaccine formulation as claimed in any one of the claims 17-41 comprising comprises i) at least two fusion proteins, each consisting of one fHbp variant type coupled to one PorA VR2 loop and at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) capsular saccharide of serogroup C N. 146 meningitidis and (ii) CRM197; (c) a conjugate of (i) capsular saccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) capsular saccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) capsular saccharide of serogroup X N. meningitidis and (ii) tetanus toxoid. 5 44. The vaccine formulation as claimed in any one of the claims 17-41 comprising i) at least three fusion proteins, each consisting of one fHbp variant type coupled to two PorA VR2 loop and at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a 10 conjugate of (i) capsular saccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) capsular saccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) capsular saccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) capsular saccharide of serogroup X N. meningitidis and (ii) tetanus toxoid. 15 45. The vaccine formulation as claimed in any one of the claims 17-41 comprising i) at least four fusion proteins, each consisting of one fHbp variant type coupled to three PorA VR2 loop and at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a 20 conjugate of (i) capsular saccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) capsular saccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) capsular saccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) capsular saccharide of serogroup X N. meningitidis and (ii) tetanus toxoid. 25 46. The vaccine formulation as claimed in any one of the claims 17-45 for use in the treatment or prevention of infection and/or disease caused by Neisseria meningitidis serogroup B. 30 47. The vaccine formulation as claimed in any one of the claims 17-45, wherein the vaccine formulation elucidates cross protection against Neisseria gonorrhea strains and Neisseria meningitidis serogroups ACWYX. 147 48. The vaccine formulation as claimed in any one of the claims 17-45, wherein the percent adsorption of the recombinant protein/ modified fHbp fusion protein on to an adjuvant is in the range of 70 % to 100 %. 5 49. The vaccine formulation as claimed in claim 48, wherein percent adsorption of fHbp V3.45 M5 PorA 316-320 exP1.14 on to an adjuvant is in the range of 80 % to 100 %. 50. The vaccine formulation as claimed in claim 48, wherein the percent adsorption of fHbp V1.14 PorA 307-311 exP1.9 on to an adjuvant is in the range of 80 % to 90 %. 10 51. The vaccine formulation as claimed in claim 48, wherein the percent adsorption of fHbp V2.19 PorA 316-320 exP1.4 on to an adjuvant is in the range of 80 % to 90 %. 52. The vaccine formulation as claimed in claim 48, wherein the percent adsorption of fHbp V1.1 PorA 307-311 exP1.4 15 on to an adjuvant is in the range of 80 % to 90 %. 53. The vaccine formulation as claimed in claim 48, wherein the percent adsorption of fHbp V1.1 PorA 307-311 exP1.9 on to an adjuvant is in the range of 70 % to 80 %. 20 54. A method for manufacturing a vaccine formulation as claimed in any one of the claims 17-53, the method comprising the following steps: 25 (a) growing host cells comprising the expression vector in nutrient medium; (b) inducing the host cells for expressing protein; (c) harvesting and separating the host cells; (d) lysing the harvested cells and separating host cell debris to obtain tagged proteins; 30 (e) purifying the tagged proteins; (f) removing tags from the tagged proteins to obtain recombinant proteins/ modified fHbp fusion proteins; (g) purifying the recombinant proteins/ modified fHbp fusion proteins; and (h) preparing the vaccine formulation comprising the purified recombinant 35 proteins/ modified fHbp fusion proteins. 148 55. The method as claimed in claim 54, wherein the host cell is a bacterial expression host system. 56. The method as claimed in claim 55, wherein the bacterial expression host system is an 5 Escherichia coli strain selected from the group consisting of BL21 (DE3), BL21 (DE3) pLysS*, BL21 (DE3) pLysE*, BL21 star (DE3), BL21-A1, BLR (DE3), HMS174 (DE3)**, Tuner (DE3), Origami2 (DE3)**, Rosetta2 (DE3)*, Rosettagami (DE3), Lemo21 (DE3)*, T7 Express, m15 pREP4*, C41(DE3), C43(DE3) or B834(DE3). 10 57. The method as claimed in claim 54, wherein the nutrient medium is selected from Undefined medium, Terrific Broth (TB) Medium, Lysogenia Broth, Luria Broth or Luria-Bertani medium, chemically defined medium, M9 Minimal Medium, Chemically Defined M9 Modified Salt Medium, 2xYT medium or Super Optimal 15 broth with Catabolite repression (SOC) Medium and combinations thereof. 58. The method as claimed in claim 54, wherein the concentration of L-methionine during growth of host cells in step (a) is maintained in the range of 1 mM to 10 mM, wherein 20 the fermentation is in fed batch mode. 59. The method as claimed in any claim 54, wherein the host cells are grown at a temperature in the range of 35℃ to 39℃; pH in the range of 5.0 to 9.0; dissolved oxygen in the range of 10 to 100%; agitation in the range of 100–1800 rpm; gas flow 25 rate in the range of 0-2 volume of gas per unit volume of liquid per minute (VVM). 60. The method as claimed in claim 54, wherein the host cells are induced using an inducer selected from lactose and its non-hydrolyzable analog isopropyl β-D-1- thiogalactopyranoside (IPTG). 30 61. The method as claimed in claim 60, wherein the concentration of lactose is in the range of 1 g/L to 50 g/L and the concentration of IPTG is in the range of 1 mM to 10 mM. 149 62. The method as claimed in claim 54, wherein the host cells are lysed using a method selected from chemical mode, biological mode, physical mode, mechanical mode, and a combination thereof. 5 63. The method as claimed in claim 62, wherein the host cells are lysed using a combination of chemical and mechanical mode. 64. The method as claimed in any one of the claims 62-63, wherein the host cells are lysed using a lysis buffer having a pH in the range of 7 to 9, followed by mechanical 10 lysis at a pressure in the range of 1000-1500 Bar for 3 to 8 cycles. 65. The method as claimed in claim 64, wherein the mechanical lysis is carried out using a homogenizer. 15 66. The method as claimed in claim 54, wherein the tagged protein in step (e) is purified using chromatography step, followed by concentration and diafiltration. 67. The method as claimed in claim 54, wherein the tags are removed using TEV protease 20 having a protein: TEV protease ratio in the range of 5:1 to 30:1. 68. The method as claimed in claim 54, wherein the recombinant protein/ modified fHbp fusion protein in step (g) is purified using chromatography step, followed by 25 concentration and diafiltration. 69. The method as claimed in claimed in claim 68, wherein the chromatography is selected from column chromatography, ion-exchange chromatography, anion exchange chromatography, cation exchange chromatography, column 30 chromatography, flash chromatography, gel filtration/ size-exclusion/ gel-permeation (molecular sieve) chromatography, affinity chromatography, paper chromatography, thin-layer chromatography, gas chromatography, dye-ligand chromatography, hydrophobic interaction chromatography, pseudoaffinity chromatography, liquid chromatography, high-pressure liquid chromatography (HPLC), immobilized metal 35 affinity chromatography, anion exchange chromatography, cation exchange 150 chromatography, multimodal chromatography, multimodal anion exchange chromatography, electrostatic interaction chromatography, hydrogen bonding chromatography, reverse phase chromatography, and combinations thereof 5 70. The method as claimed in any one of the claims 54-69, wherein the tagged proteins are expressed as inclusion bodies (IB) and are purified by urea unfolding of protein and on column refolding of the protein. 71. The method as claimed in any claim 54, wherein the vaccine formulation is prepared 10 by adsorbing individual recombinant protein/ modified fHbp fusion protein on to an adjuvant, followed by addition to an excipient mixture comprising a sugar alcohol, a buffering agent, a stabilizer, and a liquid carrier. 72. The method as claimed in claim 71, wherein the excipient mixture comprises a 15 preservative. 73. The method as claimed in claim 67, wherein TEV protease is produced by a method comprising the following steps: (a) growing host cells comprising the expression vector in nutrient medium; 20 (b) inducing the host cells for expressing TEV protease; (c) harvesting and separating the host cells; (d) lysing the harvested and separating host cells to obtain TEV protease; (e) purifying the TEV protease; and (f) concentrating, diafiltration and storing the purified TEV protease. 25 74. The method as claimed in claim 73, wherein the host cell for expressing TEV protease is an Escherichia coli strain selected from the group consisting of BL21 (DE3), BL21 (DE3) pLysS*, BL21 (DE3) pLysE*, BL21 star (DE3), BL21-A1, BLR (DE3), HMS174 (DE3)**, Tuner (DE3), Origami2 (DE3)**, Rosetta2 (DE3)*, Rosettagami 30 (DE3), Lemo21 (DE3)*, T7 Express, m15 pREP4*, C41(DE3), C43(DE3), Rosetta™(DE3)pLysS or B834(DE3). 151 75. The method as claimed in any one of the claims 54-74, wherein the glucose feed is stopped, and glycerol feed is started when OD at 590/ 600 nm is 20-100 and inducing the culture by adding and/ or maintaining lactose at 1-50 g/L in fed batch mode. 5 76. A method for inducing an immune response against Neisseria meningitidis serogroup B strain in an individual by administering to the individual a vaccine formulation as claimed in any one of the previous claims, wherein the step of administration induces an immune response against the Neisseria meningitidis serogroup B strain.