OA20569A - Improved methods for enterovirus inactivation, adjuvant adsorption and dose reduced vaccine compositions obtained thereof. - Google Patents

Abstract

The present invention is directed to improved methods of Enterovirus inactivation by formaldehyde in presence of tromethamine buffer resulting in maximum recovery of D-antigen. Subsequent adsorption of said sIPV on aluminium hydroxide provides significantly dose reduced sIPV compositions.

Description

IMPROVED METHODS FOR ENTEROV1RUS INACTIVATION, ADJUVANT ABSORPTION AND DOSE REDUCED VACCINE COMPOSITIONS OBTAINED

THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No. 15/517,225, which is the U.S. National Phase of International Application No. PCT/IN2015/000376, filed Oct. 6, 2015 and published in English, which claims priority to Indian Provisional Application No. 31.80/MUM/2014, filed Oct. 7, 2014. The disclosurcs of the abovc-idcntificd applications arc incorporated by reference in their entireties.

BACKGROUND

The prevalence of polio virus has largely been decreased by the use of Oral Polio Vaccine (OPV), based on live-attenuated Sabîn polio strains. However, OPV has limitations for the post-eradication era. Therefore, development of Sabin-IPV plays an important rôle in the WHO polio éradication strategy. The use of attenuated Sabin instead of wild-type Salk polio 15 strains will provide additional safety during vaccine production. Moreover, to prevent the emergence of circulating vaccine-derived polioviruses (cVDPVs), the use of OPV should be discontinued following polio éradication, and replaced by IPV. These cVDPVs are transmissible and can becomc ncurovirulent (similar to wild polioviruses) resultîng in vaccine associated paraiytic poliomyelîtis. Such strains can potentially re-seed the world with 20 polioviruses and negate the éradication accomplïshrnents.

IPV is delivercd by întramuscular (IM) or deep subcutaneous (SC) injection. IPV is currendy available either as a non-adjuvanted stand-alone formulation, or in varions combinations, including DT-IPV (with diphtheria and tetanus toxoids) and hexavalentDTPHepB- Hib-IPV vaccincsfadditionally with pertussis, hepatitis B, and Haemophilus influenzae b.Thc currently 25 acceptable standard dose of polio vaccines contains D antigens as 40 Units of înactivated poliovirus type 1 (Mahoney), 8 units of înactivated poliovirus type 2 (MEF-I) and 32 nuits of înactivated poliovirus type 3 (Saukctt) (e.g. Infanrix-IPV™).Existing préparations of standalone IPV do not contain adjuvant.

Most experts agréé that worldwide use of IPV is préférable because of its proven protective 30 track-rccord and safety. However, when compared to OPV, the cost-prizc for IPV is significantly higher. This is mainly due to requirements for: (i) more virus per dose; (ii) additional downstream processing (î.e. concentration, purification and inactivation), and the related QC-testing (iii) loss of antigen or poor recovery in downstream and iv) contaminent, Unrîl now, the fînancial challenge has been a major drawback for IPV innovation and implémentation in low and middle-income coLintries. The production costs of sIPV are 5 currently estimated équivalent to that for IPV, which is about 20-foid more expensive than

OPV. The future global demand for IPV following éradication of polioviruses couid increase from the current level of 80 million doses to 450 million doses per year. Consequently, approaches to “stretch” supplies of IPV are likely to be required.

Reduccd-dosc cffïcacious vaccine formulations which provide protection against infection 10 using a lower dose of IPV antigen are désirable in situations where the supply of conventional vaccine is insufficient to meet global needs or where the cost of manufacture of the conventional vaccine prevents the vaccine being sold at a price which is affordable for developing countries. Also the exposure to lower dose of IPV; compared to the existing marketed formulations couid be more safer. Thus, varions strategies to make IPV available at 15 more affordable prices need to be evaîuated.

In case of pandémie influenza vaccines the use of adjuvants has permitted dose réduction, increased die availability and reduced cost of the vaccine. Therefore, it has been spcculated that an adjuvanted vaccine formulation of sIPV would reducc cost and also increase the number of available sIPV doses worldwide.

Globally different research groups hâve been évaluai!ng dose sparing for vaccines (Influenza vaccines in particularjby employing severai adjuvants namdy Alum, Emulsion, TLRagonists (MPL, CpG, poly-IC, imiquimod) ,dmLT,l,25- diliydroxyvitamin D3,CAF()1, poly [di (carboxylatophenoxy)- phosphazene] (PCPP) and Venezuelan equine encephalitis (VEE) replicon particles. Most of the adjuvant types being studied hâve encountered following hurdles i) Unknown safety or classifîed as toxic by regulatory agencies ii) having limitations regards to route of administration iii) lacking manufacturing reproducibitity iv) stability of adjuvant.

Emulsion adjuvants (MF-59,AS03,AF3) hâve been previously reported to provide a strong dose-reduction effect (> 30foîd) for Influenza and Hepatitis B vaccines. These adjuvants 30 work by formîng a depot at tbe site of injection, enabling the meted release of antigenic material and the stimulation of antîbody producing plasma cells. However, these adjuvants hâve been dccmcd too toxic for widespread human prophylactic vaccine use and are usually reserved for those severe and/or terminal conditions such as cancer where there is a higher tolérance of side-effccts.

Further, aluminium salts hâve been considered safe, are already being used in combination vaccines containing sIPV, hâve the lowest development hurdles and are inexpensive to 5 manufacture. However aluminium adjuvants are not known for permitting significant dosereduction.

One of the most critical steps in the production of vaccines against pathogens, in particular viral vaccines, is viral inactivation. In the case of virus inactivation, forma lin is lhe most frequently used inactivating agent in the manufacture of vaccines. Formaldéhyde inactivâtes a 10 virus by irrcversibly cross-linking primary amine groups in surface proteins with other nearby nitrogen atoms in protein or DNA through a —CH2-linkagc.A potential probiem with using formalin for viral inactivation is that this involves a sériés of Chemical reactions that produce réactivé products that can induce cross-linking of viral proteins and aggregation of virus particles. This could hamper the inactivating effîciency of the formalin and could also resuit 15 in the partial destruction of the immunogcnicity of the antigen in vaccine. Accordingly, it has been reported previously that formalin inactivation of polioviruses could affect the viral immunogcnicity as well as antigenicity. Refer Morag Ferguson et al Journal of General Virology (1993), 74, 685-690. Most importantly, previously disclosed formaldéhyde inactivation methods were particularly carried oui in presence of phosphate buffer wherein 20 significant D-antigen losses were observed along with epitope modification for Sabin Type

I/II/III (D-antigen recovery post inactivation :22% for sabin type I, 15% for sabin type fl, 25% for sabin type 111), thereby failing to préservé the epitopic conformation. It is therefore possible that antibodies produced by récipients of formai in-inactivated polioviruses (in presence of phosphate buffer) may not contribute to the protcctive immune response.

By combining formalin and UV-inactivation, scientists tried to overcome the limitations of isolated UV-inactivation or formai in-inactivation, respectively, when inactivating the particularly résilient poliovirus. See, e.g., McLean, et al., “Expériences in the Production of Poliovirus Vaccines,” Prog. Med. Virol., vol 1, pp. 122-164 (1958.) Taylor et al. (J. Immunol. (1957) 79:265-75) describe the inactivation of poliomyelitis virus with a formalin and 30 ultraviolet combination. Molner et al. (Am. J. Pub. Health (1958) 48:590-8) describe the formation of a mcasurable Ievel of circulating antibodies in the blood of subjects vaccînated with ultraviolet-formalin inactivated poliomyelitis vaccine. Truffelli et al. (Appl. Microbiol.

(1967) 15:516-27) report on the inactivation of Adenovirus and Simian Virus 40 Tumorigenicity in hamsters by a three stage inactivation process consisting of fonnalin, UV light and β-propiolactone (BPL). Miyamac (Microbiol. Immunol. (1986) 30:213-23) describes the préparation of immun ogens of Sendai virus by a treatment with UV ray s and 5 formalin. However previously discussed promising alternatives for formaldéhyde like βpropiolactone (BPL) hâve been reported to produce an immune complex-reaction when combined with other components of the rabies vaccine, Addîtionally, it has been shown to produce squamous cell carcmomas, lymphomas and hepatomas in mice.

It is therefore particularly désirable to cmploy favorable formaldéhyde inactivation conditions 10 that maintain the structural întegrîty of antigenic structures of Sabin strains as well as utilize safe and eost-effective adjuvants that can resuit in significantly dose reduced (i.e. 8 to 10 fold) sIPV (Sabin IPV) vaccine compositions thereby reducing cost of manufacture, increasing vaccine supplies and making vaccines affordable for developing countrics.

The présent inventors hâve surprisingly found that D-antigen losses post-formaldehyde 15 inactivation could be due to présence of phosphate buffer that unexpcctedly causes undesirable aggregatîon of polio viruses. The instant invention provides an improved process of formaldéhyde inactivation in presence of TRIS buffer thereby ensuring minimal cpitopic modifications and subsequently minimizing D-antigen losses. Subsequently significantly dose reduced Sabin IPV vaccine compositions with at least 8 fold dose réduction for Sabin 20 Type I and 3 fold dose réduction for Sabin Type III can be obtained.

SUMMARY OF THE INVENTION

One aspect of the application is directed to a dose reduced Inactivated Polio vaccine (IPV) composition. The composition incîudes an inactivated polio virus antigen selected from the group consisting of IPV Type i at a dose less than 15 D-antigen units (DU), IPV Type 2 at a 25 dose less than 18 D-antigen unit (DU), and IPV Type 3 at a dose less than 17 D-anrigen unit (DU), wherein the inactivated polio virus antigen is per 0.5 ml. The composition also incîudes one or more antigens selected from the group consisting of Diphtheria toxoid (D), Tetanus toxoid (T), Whole cell pertussis (wP), hepatitis B virus surface antigen (HBsAg), Haemophilus influenzae b PRP-Carrier protein conjugale (Hib), Haemophilus influenzae (a, 30 c, d, e, f serotypes and the unencapsulated strains), Neisseria meningitidis A antigen(s),

Neisseria meningitidis C antigen(s), Neisseria meningitidis W-135 anfigenjs), Neisseria meningitidis Y antigen(s), Neisseria meningitidis X antigen(s), Streptococcus Pneumoniae antigen(s), Neisseria meningitidis B hleb or purified antigen(s), Staphylococcus aureus antigen(s), Anthrax, BCG, Hepatitis (A, C, 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, 5 Chikungunya, Japanese encephalitis, rotavirus, Diarrheal antigens, Flavivirus, smallpox, yellow fever, Shingles, and Varicella virus antigens.

Another aspect of the application is directed to a dose reduced Inactivated Polio vaccine (IPV) composition. The composition includes an inactivated polio virus antigen selected from the group consisting of IPV Type 1 at a dose less than 15 D-antîgcn units (DU), IPV Type 2 10 at a dose less than 18 D-antigen unit (DU), and IPV Type 3 at a dose less than 17 D-antigen unit (DU), wherein the inactivatedpolio virus is per 0.5 ml. The composition also includes a diphtheria toxoid, (D) antigen in an amount of 1 to 50 Lf per 0.5 ml, a tetanus toxoid, (T) antigen in an amount of 1 to 30 Lf per 0.5 ml, a whole cell pertussis, (wP) antigen in an amount of 1 to 50 IOU per 0.5 ml or acellular pertussis, (aP), a hepatitis B virus surface 15 antigen, (HBsAg) in an amount of 1 to 20 gg per 0.5 ml, and a Haemophilus influenzae type b antigen, (Hib) in an amount of 1 to 20 μ g per 0.5 ml.

Yet another aspect of the application is directed to a process of manufacturing a dose reduced Inactivated Polio vaccine (IPV) composition. The composition includes an inactivated polio virus antigen selected from the group consisting of IPV Type 1 at a dose less than 15 D20 antigen units (DU), IPV Type 2 at a dose less than 18 D-antigen unit (DU), and IPV Type 3 at a dose less rhan 17 D-antigen unit (DU), per 0.5 ml. The composition also includes a diphtheria toxoid, (D) antigen in an amount of 1 to 50 Lf per 0.5 ml, a tetanus toxoid, (T) antigen in an amount of ] to 30 Lf per 0.5 ml, a whole cell pertussis, (wP) antigen in an amount of i to 50 IOU per 0.5 ml or acellular pertussis, (aP) comprising one or more of 25 modified adenylate cyclase, Pertussis toxoid (PT) l-50gg, Fi lamentons hemagglutinin (FHA) l-50gg, Pertactin (P69 or PRN) l-2Ûgg or Fimbrial proteins (FIM 1 , 2 and 3) 2-25gg; per 0,5ml, a hepatitis B virus surface antigen, (HBsAg) in an amount of 1 to 20 gg per 0.5 ml, and a Haemophilus influenzae type b antigen, (Hib) in an amount of 1 to 20 gg per 0.5 ml. The process comprises the steps of: a) adsorbing the IPV (Sabin/Salk strain) bulk mdividually 30 on aluminium hydroxide, followed by pH adjustment to 6.2 - 6.6, more preferably 6.5; b) adsorbing the D on aluminium phosphate, followed by pH adjustment to 5.5 - 6.5; c) adsorbing the T on aluminium phosphate, followed by pH adjustment to 5.5 - 6.5; d) adsorbing the HBsAg on aluminium phosphate, followed by pH adjustment to 6.0 - 6.5;

e) blending a mixture obtained from steps b), c), and d) by agitation at room température for 18-24 hours; f) blending a content of step (a) with the mixture obtained in steps b), c) and d), followed by pli adjustment to 6,4 - 6.6 and agitation at room température for 60 minutes; g) adding an inactivated wP antigen/acellular pertussis antîgen and a stabilizer (Histidîne 5 amino acid buffer solution 100-300mM) to the mixture in step Ij, followed by agitation for minutes and left in static condition for ovemight at 2 - 8 °C; h) adding tire Hib antigen to the mixture obtained in step g) at 2 - 8 °C, followed by pli adjustment to 6.4 - 6.6; and i) adjustîng the pH to 6.0 to 7.0 with sodium hydroxide / sodium carbonate and adding normal saline (0.9% NaCI) or WFI (q,s.) to make up a volume of the mixture obtained in step h), 10 followed by agitation for 2 hours.

Yel even another aspect of the application is directed to a method for producing a composition comprising Entcroviral poliovims particles. The method includes the steps of: a)purifying the Enteroviral poliovirus particles in a medium comprising a phosphate buffer;

b) exchanging the phosphate buffer of the purified Enteroviral poliovirus particles of step a) 15 for a non-phosphate buffer selected from the group consisting of TRIS, TES, MOPS, HEPES or bicarbonate buffer and a combination thereof; c) stabilizing the purified and bufferexchanged Enteroviral poliovirus particles of step b); d) inactivating the Enteroviral poliovirus particles of step c) by; (i) adding forma lin to the purified Enteroviral poliovirus particles and stirrîng for 7 days; (ii) filtering the product of step (i); and (iii) adding formalin 20 to the product of step (ii) and stirrîng for 6 days; and (iv) filtering the product of step (iii).

The non-phosphate buffer is présent at a concentration of 30-70 mM. Aggregation of the Enteroviral poliovims particles is prevented or reduced. The D-antigen iosses are reduced post inactivation by 8 to 10 fold as compared to inactivation in phosphate buffer.

These aspects will bc described in further detail below.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1: Alum phosphate gel prepared in 0.9% NaCI (pH Vs Zêta potential at different concentrations of Alum phosphate gel).

Fig. 2: Alum phosphate gel prepared în WWfpH Vs Zêta potential at different concentrations of Alum phosphate gel).

Fig. 3: Alum Hydroxide gel prepared in 0.9% NaCI (pH Vs Zêta potential at different concentrations of Aluni hydroxide gel).

Fig. 4: Akim Hydroxide gel prepared in WF1 (pH Vs Zêta potential at different concentrations of Aluni hydroxide gel).

Fig. 5: Diphtheria Toxoîd Purification - Chromatogram of Gel Filtration Chromatography Scphacryl S-300 HR, Column XK 26/70. X axis represents volume (ml) and Y axis 5 represents UV at 280 nm (mALJ).

DETAILED DESCRIPTION

Although the present disclosure may be susceptible to different embodiments, certain embodiments are shown in the following detailed discussion, with the understanding that the présent disclosure can be considered an exemplification of the principles of the disclosure 10 and is not intended to limit the scope of disclosure to that which is illustrated and disclosed in dus description. Embodiments are provided so as to llioroughly and fully convey the scope of the présent disclosure to the person skilled in the art. Numerous details are set forth, rclating to spécifie components, and methods, to provide a complété understanding of embodiments of the présent disclosure. IL will be apparent to the person skilled in the art that the details 15 provided in the embodiments should not be constmed to limit the scope of the present disclosure. In some embodiments, well-known composition, well-known processes, and wellknown techniques are not described in detail.

The tcnninology 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 20 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 first, second, third, etc., should not be construcd to limit the scope of the present disclosure as the aforementîoned terms may be only used to distinguish one element, componcnt, région, layer or section from another componcnt, région, layer or section. Terms 25 such as first, second, third etc., when used herein do not imply a spécifie sequence or order unless clearly suggesled by the present disclosure. The present disclosure provides an immunogenic composition and a process for preparing the same.

The term vaccine is optionally substitutable with the term immunogenic composition and vice versa.

D-antigen units (also referred to as international units or HJ): The D aniigenic form ofthe poliovirus induces protective neutralising antîbodies. D antîgen units referred to herein (for instance in the vaccines of the invention) arc the measured total D antigen units of each unadsorbed bulk IPV antigen type prior to formulation of the final vaccine which are added 5 in each human dose of formaiated vaccine (typically 0,5mL final volume). Reliable methods of measuring D-antigen units are well known in the art and are published, for instance, by the European Pharmacopoeia. For instance, D-antigcn units may be measured using Clic EL1SA test (D-antîgen quantification by ELISA) below^r. European Pharmacopoeia provides a test sample (European Pharmacopoeia Biological Référencé Préparation - available from Ph, Eur. 10 Secrétariat, e.g. Code P 216 0000) for standardisation of such methods between manufacturera (Pharmeuropa Spécial Issue, Βίο 96-2). Thus the D-antigen unit value is well understood in the art.

The term dose herein is typically one administration of the vaccine of the invention, which is typically one injection. A typical human dose is 0.5mL. Of course valions doses may be 15 admînistered in a vaccine administration schedule.

The term ”IPV or an immunogenic composition comprising these components herein is intended to mean inactivatcd polio virus type I (e.g. Mahoncy, as preferably used), type 2 (e.g. MEF-1), or type 3 (e.g. Saukett), or a Sabin Sérotype 1, 2, 3 combination of either two or ail three of these types. An example of a full (or standard) dose (40-8-32 D antigen units of 20 Salk based IPV types l, 2 and 3 respectively) IPV immunogenic composition for the purposes of this invention could be Poliovac® (Sérum Institute of India Pvt. Ltd.). Thus, where it is stated herein that one, two, three fold dose réduction (reduced) as compared to standard dose of Salk based IPV is présent in an immunogenic composition of the invention it is meant D-antigen units cquating to X% of réduction of dose of 40, 8, and/or 32 D-antigen 25 units of IPV types 1,2 and/or 3 respectively (as measured in each bulk IPV antigen type) are formulated within each dose of said vaccine.

The term saccharide throughout this spécification may indicate polysaccharide or oligosaccharide and includes both. The capsular saccharide antigen may be a full-lcngth polysaccharide or it may be extended to bacterial ‘sized-saccharides’ and 'oligosaccharides' 30 (which naturally hâve a low number of repeat units, or which are polysaccharides reduced in size for manageability, but are still capable of inducing a protective immune response in a host.

An important aspect of the instant invention is that said improved process of formalin inactivation and adsorption on aluni sait comprises of following steps:

a) Adding Sabin IPV purified bulk to TRIS buffer (30 to 50mM) having pH between 6.8 to 7.2,

b) Adding M-199 medium containing glycine (5gm/'l) to mixture of (a),

c) Adding 0.025% formaldéhyde while mixing,

d) Incubating mixture obtained in Step (c) at 37°C from 5 to 13 days on magnetic stirrer,

e) Subjecting post-incubation mixture to intermediate 0.22μ filtration on day 7 and final filtration on day 13,

f) Storing bulk obtained after step (e) at 2-8°C,

g) Performing D-Ag ELISA for D-Antigen unit détermination,

h)Taking the desired volume of autoclaved Al(0H)i to gct the final concentration of Alum(Al^l’⁺⁺) between 0.8 to 1.2 mg/dose in a 50 ml Container,

i) Adding IPV bulk with adjusted D-Ag unit and making up the volume with diluent (ΙΟχ ΜΙ 99+0.5 Glycine %),

j) Adjusting the final formulation pH and obtaining final formulation with pi-1 between 6 and 6.5,

k) Subjecting the formulation bulk to magnetic stirring overnight at 2-8°C and wherein formalin inactivation of step (a) does not occur in presence of phosphate buffer.

A first embodiment of instant invention is that said non-phosphate buffer to be used during formaldéhyde inactivation can be selected from the group consisting of TRIS, TBS, MOPS, HEP ES, and bicarbonate buffers.

A preferred aspect of first embodiment is that said formaldéhyde inactivation can occur in presence of TRIS Buffer or TBS(TRIS Buffered saline) having concentration selected from 30mM,40mM and 50mM, preferably 40mM and at a pH selected from 6.8,6.9,7,7.1 and 7.2 ,preferably between 6.8 and 7.2 wherein said inactivation does not utilize any phosphate buffer.

A second embodiment of the instant invention is that adsorption of formalin inactivated s IPV can be done on aluminium hydroxide having concentration selected from 1.5mg/dose, 1.8mg/dose,2.2 mg/dose, preferably between 2mg/dose to 2.4 mg/dose and at a pH selected from 6.2,6.3,6.4 and 6.5, preferably 6.5.

A third embodiment of instant invention is that said improved process of formalin inactivation and aluminium hydroxide adsorption can resuit in D- Antigen recovery postinactivation between 50% and 80% and percent adsorption of aluminium hydroxide can be between 85 and 99% ,

One aspect of third embodiment is that présent invention provides an improved process of 10 formalin inactivation and aluminium hydroxide adsorption resulting in dose réduction of at least 8 fold for Sabin Type 1, at least 3 fold for Sabin Type 3 as compared to standard dose of 40 DU-8DU-32DU.

Second aspect of third embodiment is that instant invention provides improved formaldéhyde inactivation and aluminium hydroxide adsorption methods that resuit in vaccine compositions 15 comprising of i) inactivated poliovirtis type l at a dose of at least 5D-antigen units, ii) inactivated poliovirus type 2 at a dose of at least 8D-antigcn units and iii) inactivated poliovirtis type 3 at a dose of at Least lOD-antigen units.

A fourth embodiment of instant invention is that said aluminium sait adjuvant is an aluminium hydroxide having concentration between 1.5mg/0.5 ml dose and 2.5 mg/0.5 ml 20 dose, preferably between 2.100 mg/0.5ml dose and 2.4mg/0.5 ml dose at a pH of about 6.5.

One aspect of fourth embodiment is that total aluminium content in the tri va lent vaccine (Type 1, 2 and 3) can be between 800-1000pg, preferably 800pg ΑΡ⁺ per 0.5mL dose ,characterized in that at least 400 pg AP’ for Type l,atleast 200 pg Al³⁺ for Type 2,atleast 200 pg Al³⁺for Type 3.

Another aspect of fourth embodiment is that said dose reduced polio virus vaccine composition can consist of Type 1 and Type 3 and is devoid of Type 2 wherein the dose volume can be between 0.1 and 0.4 ml.

A fifth embodiment of instant invention is that the dose reduced vaccine compositions prepared by instant methods can be “Standalonc sIPV” wherein the antigens may comprise of sIPV type 1 or sIPV type 2 or sIPV type 3, or sIPV types 1 and 2, or sIPV types 1 and 3, or sIPV types 2 and 3, or sIPV types 1, 2 and 3.

In first aspect of the fifth embodiment, Inactivated polio virus is selected from tire group of Salk and Sabin strain; and the concentration of individual Type 1, Type 2, and Type 3 of Salk or Sabin strain based IPV is not more than 20 D antigen units.

In second aspect of the fifth embodiment, inactivated polio virus is a Salk strain and the concentration of individual Type 1, Type 2, or Type 3 of Salk strain based IPV is selected from the group of dose composition comprising of 7.5-16-10, 8-2-5, 10-2-5, 10-2-10, 10-212, 10-2-16, 7.5-16-10, 5-2-5 D antigen units; more particularly concentration of individual Type 1, Type 2, or Type 3 of Salk strain based IPV is selected from group of 8-2-5 and 10-2 - 10 D antigen units.

In third aspect of the fifth embodiment, inactivated polio viras is a Sabin strain and the concentration of individual Type 1, Type 2, and Type 3 of Sabin strain based IPV is selected from the group of dose composition comprising of 5-16-10, 2.5-8-5, 5-8-10 D antigen units; more particularly concentration of individual Type 1, Type 2, and Type 3 of Sabin strain based IPV is 5-16-10 D antigen units.

A sixth embodiment of instant invention is that the dose reduced vaccine compositions prepared by instant methods can be “Combination Vaccines containing dose reduced IPV” wherein said non-IPV antigens of combination vaccines can be selected from but not limited to Diphtheria loxoid (D), Tetanus toxoid (T), Whole cell pertussis (wP), hepatitîs B virus surface antigen (HBsAg), Haemophilus influenzae b PRP-Carrier protein conjugale (Hib), Haemophilus 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 auretts antigen(s), Anthrax, BCG, Hepatitîs (A, C, 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 virus antigens.

A preferred aspect of the sixth embodiment, wherein the Combination Vaccines comprising dose reduced IPV may comprise of antigens selected from Diphtheria toxoid (D), Tetanus toxoid (T). Whole cell pertussis (wP), acellular pertussis, hepatitis B virus surface antigen (HBsAg), Haemophilus influenzae b PRP-Carrier protein conjugate (Hib) other than dose 5 reduced IPV as disclosed in earlier embodiment.

A preferred aspect of the sixth embodiment is that the Combination Vaccines comprising dose reduced IPV may comprise of IPV type 1 or IPV type 2 or IPV type 3, or IPV type 1 and 2, or !PV type 1 and 3, or IPV type 2 and 3, or IPV type I, 2 and 3.

In preferred aspect of the sixth embodiment, inactivated polio virus is a Salk strain and the 10 concentration of individual Type 1, Type 2, or Type 3 of Salk strain based IPV is selected from the group of dose composition comprising of 7.5-16-10, 8-2-5, 10-2-5, 10-2-10, 10-212, 10-2-16, 7.5-16-10, 5-2-5 D antigen units; more particularly concentration of individual Type I, Type 2, or Type 3 of Salk strain based IPV is selected from group of 8-2-5 and 10-2 - 10 D antigen units.

In preferred aspect of the sixth embodiment, inactivated polio virus is a Sabin strain and tlic concentration of individual Type I, Type 2, and Type 3 of Sabin strain based IPV is selected from the group of dose composition comprising of 5-16-10, 2.5-8-5, 5-8-10 D antigen units; more particularly concentration of individual Type 1, Type 2, and Type 3 of Sabin strain based IPV is 5-16-10 D antigen units.

Components of the Combination Vaccine composition:

• Inactivated Polio Virus (IPV)

Polio (Poliomyelitis) is a highly infections virus. The poliovirus invades the nervous system and can cause irréversible paralysis in a matter of hours. Three types of poliovirus exist globally i.e. Type 1, Type 2, & Type 3.

Sevenlh embodiment of the présent invention comprises of împroved methods of Polio virus (salk or sabin strains) inactivation by formaldéhyde in présence of Non-Phosphate Buffer (TRIS, TBS, MOPS, HEPES, and bicarbonate) resulting in maximum recoveiy of D-antigen. Subséquent adsorption of said IPV on aluminium hydroxide provides significantly dose reduced IPV compositions. The instant invention provides an improved process of formalin inactivation and aluminium hydroxide adsorption, wherein the D -Antigen recovery post inactivation is in the range of 50-80% and percent adsorption of aluminium hydroxide is in the range of 70-99 %.

!n one ofthe aspects ofthe seventh embodiment, CCL81-VERO (Monkey kidney) cell were used as host cells for the growîng of polio viras i.e. Sabin and Salk strains. After infection of host cells with dcsircd strain of polio virus and incubation of 72 hours, the medium containing the virus and cell débris was pooled and collected in a single container.

In second aspect of the seventh embodiment, the harvcst may be subjected to sériés of filtration (6 μ and 0.45 μ assembly); and filtrate was collected in a glass container. The filtrate was subjected to tangential flow filtration with lOOkDa cassette; diafrltered using phosphate buffer and purified using an ion exchange chromatography.

In third aspect of the seventh embodiment, the purified virus pool may be further subjected to buffer cxchangc from Phosphate buffer to Non-Phosphate Buffer (TRIS, TBS, MOPS, HEPES, and bicarbonate) (30 to 70 mM, pl-I: 7 - 7.5) with TFF system (100 kDa, 0.1 m²) followed by addition of M-199 and 0.1 - 1.0 % glycine.

In fourth aspect of the seventh embodiment purified virus pool in Non-Phosphate Buffer (TRIS, TBS, MOPS, HEPES, and bicarbonate) (30 to 70 mM, pH: 7 - 7.5) was subjected to inactivation by 0.01 — 1.0% formaim with continuons stirring of virus bulk and incubated at ambient température for 13 days, with intermediate filtration on day 7. Further, the inactivated bulk was subjected to final filtration and later stored at 2-8 ^ÜC.

In fifth aspect of the seventh embodiment, final purified bulk may be subjected to adsorption on AI(OH)3 , wherein the final alum (Al³¹) concentration is between 0.1 - I.5 mg/dose, M199+0.5% glycine was used to make up the volume and pH was adjusted to 6 - 7 using IN HCl/NaOH, further subjected to overnight stirring at 2 - 8 °C.

The présent inventors hâve surprisingly observed that D-amigen loss, post-formaldehyde inactivation was due to presence of phosphate buffer that unexpectedly causes undesirable aggregation of polio viruses. The instant invention provides an improved process of formaldéhyde inactivation in presence of Non-Phosphate Buffer (TRIS, TBS, MOPS, HEPES, and bicarbonate) (30 to 70 mM, pH: 7 - 7.5) thereby ensuring minimal epîtopic modifications and subsequently minimizing D-anligen loss.

• Haemophilus influenzae b (Hib) PRP - Protein Conjugate:

Haemophilus influenzae type b is a Gram-negative bactenum that causes meningitis and acute respiratory infections, mainly in children. The outermost structure of H. influenzae type b is composcd of polyribosyl-ribitol-phosphatc (PRP), a polysaccharide that is rcsponsiblc for virulence and immunity. PRP is a hapten which is considered as poor inununogenic in nature hence PRP is linked covalently with a carrier protein to make highly inununogenic Hib antigen. This process changes the polysaccharide from a T-independent to a T-depcndcnt antigen and greatly improves immunogenicity, particularly in young children.

Eighth embodiment of the présent invention comprises of préparation of Hib PRP-protein conjugate. It may be noted that the carrier proteins used for the conjugation of the Hib antigen may bc selected from group comprising of Tetanus toxoid, CRM en (Cross Reactive Material 197, a genetieally detoxified form of diphtheria toxoid), Diphtheria toxoid, Neisseria meningitidis outer membrane complex, fragment C of tetanus toxoid, pertussis toxoid, protein D of H. influenzae, E. coli LT, E, coli ST, and exotoxin A from Pseudomonas acruginosa, outer membrane complex e (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumocoecal surface adhesin A (PsaA), pncumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11 , protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and léthal factor (LF) of Bacillus anthracis, ovalbumin, keyliole limpet hemoeyanin (KLH), human sérum albumin. bovine sérum albumin (BSA) and purified protein dérivative of tubercuïin (PPD), synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins comprising multiple human CD4+ T cell epitopes front various pathogenderived antigens such as N 19, iron-uptake proteins, toxin A or B from C. difficile and S.agalactiae proteins or any équivalents thereof. Preferably the carrier protein in conjugale is selected from TT or CRM 197.

In first aspects of the eighth embodiment, Hib antigen may be derivcd from the capsulaipolysaccharide of Haemophilus influenzae type b strain. To produce the PRP polysaccharide, H. Influenzae type-b bacteria was grown in semi synthetic media under certain conditions of température, agitation and optical density etc. PRP is an outer membrane bound polysaccharide, gets released into the medium during the fermentation under agitation condition. Fermented biomass separated broth contains crude PRP, which was again purified by précipitation using a detergentN, N, N-tiimethyl-l-hexadecanaminium bromide, followed by éthanol gradient précipitation and filtration. Final purified PRP polysaccharide was tested for meeting the spécifications lîke endotoxin, nucleic acid and protein as per the WHO, BP, EP, IP etc.

In second aspect of the eighth embodiment, polysaccharide - protein conjugate may be prepared by coupling of polysaccharide (PRP) with a carrier protein. Hib PRP was 5 conjugated to carrier protein using conjugation process comprising of steps including depolynierization of PRP using alkaline buffer to achieve size reduced PRP; treatment with a cyanylation agent like CDAP (l-cyano-4-dimethylamino pyridinium tetrafluoroborate) to form a cyanale ester; coupling of activated cyanylated polysaccharide to amino group of carrier protein; purification of final conjugate using ultrafiltration.

More preferably, the optimal input ratio of reactants i.e. PRP, CDAP and CRM 197 may be selected at 1:1.5:1 ratio for conjugation reaction. During conjugation, purified PRP polysaccharide was depolymerized using an alkaline buffer (0.4M Carb-Bicarbonate buffer, pH 10.5 ±0.1) to achieve size reduced PRP. Size reduced PRP was treated for cyanylation using CDAP (l-cyano-4-dimethylamino pyridinium tetrafluoroborate) chemistry to form a cyanatc ester. The activated cyanylated polysaccharide was couplcd directly with amino group on the carrier protein CRM197. The degree of conversion of Hib conjugale was confirmed by the offline testing using HPLC. The conjugation réaction was quenched by achieving the desired level of conversion of conjugate with the spécification of not less than 65% conversion of Hib conjugate, and then conjugale reaction was neLitralized by Glycine (2M) addition. The Hib PRP-CRM197 Conjugate was further purified 011 ultra-filtration membrane filters (300kDa and lOOkDa) to remove nonreactive reagents and byproducts. Final conjugate bulk is 0.22 pm filtered and stored at 2-8%?.

More preferably, Hib PRP may be conjugated to carrier protein wherein the saccharide: protein ratio (w/w) may be between 0.4 and 1; and the free PRP content in final Hib PRP 25 protein conjugate bulk may be not more than 5%, more preferably is less than 2%.

In third aspect of the eighth embodiment, the Hib PRP may be conjugated to tetanus toxoïd (TT) by CNBr chemisüy, Rcductive amination chemistry , Cyanylation chemistry or any other chemistry already discloses in Kniskem et al., “Conjugation: design, chemistry, and analysis” in Ellis et al.. Development and cl i ni cal uses of Haemophilus influenzae type B 30 conjugate vaccines. New York: Marcel Dekkcr, 1994: 37-69

In forth aspect of the eighth embodiment. the carrier protein may be présent in both free and conjugated form in a composition of the présent disclosure, the unconjugated form is preferably no more than 20% of the total amount of the carrier protein in the composition as a whole, and more preferably présent at less than 5% by weîght, more preferably is less than 5 2%.

In fifth aspect of the eighth embodiment, the Hib antigen is not subs tan liai ly adsorbed on to any adjuvant.

In sixth aspect of the eighth embodiment, the Hib antigen may not be subjectcd to deliberate or intentional adsorption on any adjuvant.

In seventh aspect of the eighth embodiment, the percentage of adsorption of Hib antigen on to any adjuvant may be less than 20%.

In eighth aspect of the eighth embodiment, the Hib antigen used in the combination vaccine of (lie présent disclosure is derived from the capsular polysaccharide of Haemophihis influenzae type b (Hib) strain 760705.

· Diphtheria toxoid

Diphtheria is an infectious disease caused by the bacterium Corynebacterium diphtheria, which primarily infects the tliroat and upper airways, and produces a toxin affecting other organs. Diphtheria toxin is an exotoxin secreted by Corynebacterium diphtheria, possesses antigcnic propertïes and is toxic in nature. To reduce toxicity, the toxin is converted to the 20 inactive toxoid by subjecting it to inactivation. The inactivation process may be selected from one or more of treatment with Heat, UV, Formalin /Formaldéhyde, Acetylethyleneimine, etc. To increase immunogenicity, the toxoid is adsorbed to an adjuvant. The toxoid thus formed is able to induce anti toxin antibodies against C diphtheria. Existence of dimers can lead to adverse reactions.

In ninth embodiment of the présent invention, Diphtheria toxoid was prepared.

In first aspect of the ninth embodiment, diphtheria toxin (exotoxin) may be obtained from Corynebacterium diphtheria and detoxificd using a suitable inactivating agent. The example of suitable inactivating agent includes Formaldéhyde.

In second aspect of the ninth embodiment, diphtheria toxoid obtained may be purified using Gel filtration chromatography with Sephacryl S-300 HR. as resin with linear flow rate of 2 - 5 ml/min and stabilizcd by addition of an amino acid buffer solution (Histidinc, lysine, glycine, arginine) or polysorbate solution at a final concentration of 5 - 300 mM. and stored at 5 température -20 to +40 °C till further use. The purified D thus obtained comprises of homogcnous fraction devoid of undesirable aggregatcs (Rcfcr Figure 1) with at least 80 - 90 % monomerîc diphtheria toxoid further used for formulation of multivalent vaccinc(s). Further PLgel, Sephacryl S-200HR, Sephadex, Bio-Gel (cross linked polyacrylamide), agarose gel and/or Styragel may also be used for the purpose of purification using Gel 10 penneation chromatography. The purified diphtheria toxoid is stabilizcd by addition of

Histidine (200mM) amino acid buffer solution.

In third aspect of the ninth embodiment, diphtheria toxoid may be adsorbed on to adjuvant selected from the group of aluminium sait (Al³⁰ such as aluminium hydroxide (Al(OH)s) or aluminium phosphate (AIPO4), alum, calcium phosphate, MPLA, 3D-MPL, QS21, a CpG15 containing oligodeoxynucleotide adjuvant, liposome, or oil-in-water émulsion or a combination thereof.

Yct preferably diphtheria toxoid may be adsorbed on to aluminium sait including aluminium hydroxide and aluminium phosphate, preferably on Alum phosphate.

Yet preferably the Diphtheria toxoid (D) antigen may be adsorbed on to aluminium 20 phosphate having percentage adsorption of at least 50%.

• Tctanus toxoid (T)

Tetanus is an acute infections disease caused by toxîgenic strains of the bacterium Clostridium tetani (C. tetani), a gram-positive, spore-forming, strictly anaérobie bacterium, Tetanus toxin is an exotoxin secreted by Clostridium tetani, possesses antigenic properties 25 and is toxic in nature. To reduce toxicity, the toxin is convertcd to the inactive toxoid by subjecting it to inactivation. The inactivation process may be selected from one or more of treatment with Heat, UV, Formai in /Formaldéhyde, Acetylethyleneimine, etc. To increase immunogcnicity, the toxoid is adsorbed to an adjuvant. The toxoid thus formed is able to induce anti toxin antibodies against Clostridium tetani. Existence of dimers can lead to 30 adverse réactions.

In tenth embodiment of the présent invention, Tetanus toxoid was prepared.

ln first aspect of the tenth embodiment, Tetanus toxin may be obtained from Clostridium tetani and detoxified using a suitable inactivatîng agent. The example of suitable înactivating agent includes Formaldéhyde.

In second aspect of the tenth embodiment, Tetanus toxoid obtained may be purified using Gel 5 filtration chromatography with Sephacryl S-300 HR as resin with Iinear flow rate of 2 - 5 ml/min and stabilized by addition of an amino acid buffer solution (Histidine, lysine, glycine, arginine) or polysorbatc solution at a final concentration of 5 - 300 mM, and stored at température -20 to +40 °C till further use. The purified T thus obtained was a homogenous fraction devoid of undesirable aggregates with at least 80 — 90 % monomeric tetanus toxoid 10 further used for formulation of multivalent vaccine. Further PLgel, Sephacryl S-200HR,

Sephadcx, Bio-Gel (cross-linked polyacrylamide), agarose gel and Styragel may also be used for the purpose of purification using Gel perméation chromatography. The purified tetanus toxoid is stabilized by addition of Histidine (200mM) amino acid buffer solution.

In third aspect of the tenth embodiment, Tetanus toxoid may be adsorbed on to adjuvant 15 selected from the group of aluminium sait (Α1³⁺⁾ such as aluminium hydroxide (Al(OH)r) or aluminium phosphate (AIPO4), alum, calcium phosphate, MPLA, 3D-MPL, QS21, a CpGcontaining oligodeoxynuclcotidc adjuvant, liposome, or oil-in-water émulsion or a combination thereof.

Yet preferably tetanus toxoid may be adsorbed on to aluminium sait including aluminium 20 hydroxide and Aluminium phosphate, preferably on Alum phosphate.

Yet preferably the tetanus toxoid (T) antigen may be adsorbed on to aluminium phosphate having percentage adsorption of at least 40%.

• Pertussis Antigen

Pcrtussis (whooping cough) is caused by Bordetella pertussis, a small Gram-ncgative 25 eoccobacillus that infects the mucosal layers of the human respiratory tract. Two forms of vaccine are in use, the whole-cell pertussis vaccine (wP), and the acellular pertussis vaccine (aP). Whole-cell pertussis vaccines are suspensions of the entire B. pertussis organisai that has been inactivated, usually with formalin. Immunization with wP vaccine is relative! y tnexpensive and highly effective. Also, presence of wP in combination vaccines acts as an 30 adjuvant for many other antigenic componcnt.

Acellular pertussis (aP) vaccines contain purified components of B. pertussis such as inactivated pertussis toxin either alone or in combination with other B. pertussis components such as fiiamentous haemagglutinin, fimbrial antigens, pertaetin, and modified adenylate cyelase more particularly a non-cytotoxic polypeptide, derived from the adenylate eyclase protein (CyaA-derived polypeptide) of a Bordetella pertussis. Acellular pertussis vaccine offers less adverse réaction as compared to wP vaccine.

In eleventh embodiment of the présent invention, the pertussis vaccine is pertussis antigen selected from one or more of whole cell pertussis or acellular pertussis.

In first aspect ofthe eleventh embodiment, pertussis vaccine is an acellular pertussis antigen selected from one or more of fiiamentous haemagglutinin, fimbrial antigens, pertaetin, and modified adenylate eyclase more particularly a non-cytotoxic polypeptide, derived from the adenylate eyclase protein (CyaA-derived polypeptide) of a Bordetella pertussis. Acellular pertussis antigens may be expressed in suitable host using recombîuant DNA technology.

Preferably acellular pertussis antigen may be selected from - Bordetella toxin in detoxified form (in particular either genctically or chemically detoxified), in particular Pertussis toxoid; Fiiamentous Haemagglutinin; Pertaetin; or Fimbriac. Particularly Pertussis toxoid: 1 to 50 mîcrograms (More particularly 8pg); - Fiiamentous Haemagglutinin: 1 to 50 micrograins (More particularly 8pg); - Pertaetin: 1 to 20 mîcrograms (More particularly 2.5pg); Optionally, Fimbriae: 2 to 25 mîcrograms; per 0.5 ml.

In second aspect of the eleventh embodiment, pertussis vaccine is a whole cell pertussis comprising of Bordetella pertussis strains 134, 509, 25525 and 6229 in a spécifie ratio and subsequently inactivated by utilizing improved methods of inactivation devoid of thimcrosal; hence leading to reduced reactogenicity and increased potency. Preferably, wP antigen is made from Bordetella pertussis strains 134, 509, 25525 and 6229 mixed in a ratio of 1:1:0.25:0.25.

In third aspect of the eleventh embodiment, wP inactivation process includes heat inactivation at 56±2°C for 10 to 15 minutes in presence of formaldéhyde; wherein wP bulk remains non-clumpy and easily homogenized thereby leading to reduced reactogenicity and giving better wP potency for a longer duration.

• Hepatitis B surface antigen (HBsAg)

Hepatitis B is a potentially life-threatening liver infection causée! by the Hepatitis B virus (HBV). Hepatitis B surface antigen (HBsAg) is a surface protcîn that also acts as an immunogen in highly effective vaccines for prévention of HBV infection. HBsAg protein can be recombinantly expressed in a suitable host microorganism; or can be isolated from the blood plasma of a chronic Hepatitis B patient/carrier.

In twelfth embodiment of the present invention, Hepatitis B surface antigen (HBsAg) was prepared.

In one of the aspect of twelfth embodiment, HBsAg was expressed in Hanscnula polymorpha yeast cells using recombinant DNA technology. Other yeasts such as Saccharomyces cerevisiae may also be used as host cell for recombinant expression of HBsAg.

In one of the aspect of twelfth embodiment, Hepatitis B antigen (HBsAg) may be adsorbed on to adjuvant selected from the group of aluminium sait (Al³fl such as aluminium hydroxidc iAl(OHh) or aluminium phosphate (AIPO4), alum, calcium phosphate, MPLA, 3D-MPL, QS21, a CpG-containing oligodeoxynucleotide adjuvant, liposome, or oil-in-water émulsion or a combination thereof.

Yet preferably Hepatitis B antigen (HBsAg) may be adsorbed on to Aluminium sait including Aluminium hydroxide and Aluminium phosphate, preferably on Alum phosphate.

Yet preferably the Hepatitis B surface antigen (HBsAg) may be adsorbed on to aluminium phosphate having percentage adsorption of at least 70%.

In thirteenth embodiment of the present disclosure, the process for préparation of combination vaccine composition/formulation comprising Dose reduced IPV is disclosed.

One of the preferred aspects of thirteenth embodiment, wherein the process for préparation of Combination Vaccine Compositions comprising Dose reduced IPV, HBs, D, T, wP. and Hib PRP - Protein conjugate is as given below:

a) adsorbing IPV (Sabîn/Salk strain) bulk individually on Aluminium hydroxide, followed by pH adjustment to 6.2 - 6.6, more preferably 6.5.

b) adsorbing D on Aluminium phosphate, followed by pli adjustment to 5.5 - 6.5

c) adsorbing T on Aluminium phosphate, followed by pH adjustment to 5.5 - 6.5 cl) adsorbîng HBsAg on Aluminium phosphate, followed by pH adjustment to 6.0 — 6.5.

e) blending the mixture as obtained in step (b), (c), (d) by agitation at room température for 18-24 hours.

f) Blending the mixtures as obtained in step (a) and (e), followed by pH adjustment to 6.4 -

6.6 and agitation at room température for 60 minutes.

g) adding inactivated wP antigen / acellular pertussis antigen and a stabiliser more preferably amino acid buffer solution (Histidine 100 -300 mM) to the above mixture in step (1), followed by agitation for 60 minutes and icft in static condition for overnight at 2 - 8 °C.

h) adding Hib antigen to the mixture obtained in step (g) at 2 — 8 °C, followed by pli 10 adjustment to 6.4 — 6.6.

i) Adjusting pH to 6.0 to 7.0 with Sodium Hydroxide / Sodium Carbonate and adding normal saline (0.9% NaCI) or WFI (q.s.) to make up the volume of the mixture obtained in step b, followed by agitation for 2 hours.

j) The process may further comprise of adding preservative to the mixture obtained in step (h) 15 selected from:

i. 2-Phenoxy éthanol in an amount of 1 to 6 mg per 0.5 ml (v/v) and methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v); or ii. 2-Phenoxyethanol in an amount of 1 to 6 mg per 0.5 ml(v/v)and propylparabcn in an amount of 0.05 - 0.2 mg per 0.5 ml (w/v); or iii. 2-Phcnoxycthanol in an amount of 1 to 6 mg per 0.5 ml(v/v), methylparaben in an amount of 0,1 — 1.5 mg per 0.5 ml (w/v) and propylparaben in an amount of 0.05 — 0.2 mg per 0.5 ml (w/v);

iv. 2-phenoxy éthanol in an amount of 1 to 6 mg (v/v) per 0.5ml as preservative.

In fourteenth embodiment of the present disclosure, the combination vaccine 25 composition/formulation comprising Dose reduced IPV is an ail liquid hexavalent vaccine formulation comprising of:

Table 1
Sr. No.	Formulation Components	Antigen Unit/0.5ml Dose	Prefcrred Antigen Unit/0.5mi Dose (In any of the combination)
1	Diphtheria Toxoid (D)	1-50 Lf	Preferably one of 10 or 22.5 or 25

			Lf
2	Tetanus toxoid (T)	1-30 Lf	Preferably one of 2 or 7.5 or 10 Lf
3	Inactivated B, pertussis antigen (wP)	1-50 IOU	Preferably one of 12 or 15 or 16 IOU
4	IIBs antigen	1-20 pg	Preferably one of 8 or 10 or 12.5 pg
5	Hib PRP-ΤΤ conjugate antigen	1-20 pg ofPRP	Preferably one of 8 or 10 or 13 pg ofPRP
6	Inactivated Polio Virus (IPV) Sabin Serotype
Type 1 (D antigen units)	1 -20 DU	5 DU
Type 2 (D antigen units)	1 - 20 DU	16 DU
Type 3 (D antigen units)	1 -20 DU	10 DU
7	Total Aluminium Content (AP+)	0.1 - 1.2 mg	Preferably NMT 0.3 or NMT 0.55 or NMT 0.9
8	2-Phenoxyethanol	1 - 6 mg	Preferably onc of 2 or 2.5 or 3.25 mg
9	L-Histidine	0.1-5 mg	Preferably one of0.7 or 1 or 1.55 mg

NMT — Not more than

In fifteentli embodiment of the présent disclosure, the combination vaccine composition/formulation comprising Dose reduced IPV is an ail liquid hexavalent vaccine formulation comprising of:

Table 2
Sr. No.	Formulation Components	Antigen Unit/0.5ml Dose	Prcfcrred Antigen Unit/0.5ml Dose (In any of the combination)
1	Diphtheria Toxoid (D)	1-50 Lf	Preferably one of 10 or 22.5 or 25 Lf
2	Tetanus toxoid (T)	1-30 Lf	Preferably one of 2 or 7.5 or 10 Lf
3	Inactivated B. pertussis antigen (wP)	1-50 IOU	Preferably one of 12 or 15 or 16 IOU
4	HBs antigen	1-20 pg	Preferably one of 8 or 10 or 12.5 pg
5	Hib PRP-ΤΤ conjugate antigen	1-20 pg ofPRP	Preferably one of 8 or 10 or 13 pg ofPRP
6	Inactivated Polio Virus (IPV) Sabin Serotype
Type 1 (D antigen units)	1 -20 DU	2.5 DU
Type 2 (D antigen units)	1 -20 DU	8 DU
Type 3 (D antigen units)	1 -20 DU	5 DU
7	Total Aluminium Content (Al3+)	0.1-1.2 mg	Preferably NMT 0.3 or NMT 0.55 or NMT 0.9
8	2-Phenoxyethanol	1 - 6 mg	Preferably one of 2 or 2.5 or 3,25 ___ mg

9

L-Histidine

0.1-5 mg

Preferably one of 0.7 or 1 or 1.55 mg

N MT - Not more than

In sixteenth embodiment of the présent disclosure, the combination vaccine composition/formulation comprising Dose reduced IPV is an ail liquid hexavalcnt vaccine formulation comprising of:

Table 3
Sr. No.	Formulation Components	Antigen Unit/0.5ml Dose	Preferred Antigen Unit/0.5ml Dose (In any ofthe combination)
1	Diphtheria Toxoid (D)	1-50 Lf	Preferably one of 10 or 22.5 or 25 Lf
2	Tctanus toxoid (T)	1-30 Lf	Preferably one of2 or 7.5 or 10 Lf
3	Inactivated B. pcrmssis antigen (wP)	1-50 IOU	Preferably onc of 12 or 15 or 16 IOU
4	HBs antigen	1-20 pg	Preferably one of 8 or 10 or 12.5 pg
5	Hib PRP-TT conjugale antigen	1-20 pg ofPRP	Preferably one of 8 or 10 or 13 pg ofPRP
6	Inactivated Polio Virus (IPV) Sabin Serotype
Type 1 (D antigen units)	1 -20 DU	7.5 DU
Type 2 (D antigen units)	1 - 20 DU	16 DU
Type 3 (D antigen units)	1 - 20 DU	10 DU
7	Total Aluminium Content (AP+)	0.1 — 1.2 mg	Preferably NMT 0.3 or NMT 0.55 or NMT 0.9
8	2-Phenoxy éthanol	1 - 6 mg	Preferably one of 2 or 2.5 or 3.25 mg
9	L-Histidine	0.1-5 mg	Preferably one of 0.7 or 1 or 1.55 mg

NMT - Not more than

In seventeenth embodiment of the présent disclosure, the combination vaccine composition/formulation comprising Dose reduced IPV is an ail liquid hexavalent vaccine formulation comprising of:

Table 4

Sr. No.	Formulation Components	Antigen Unit/0.5ml Dose	Preferred Antigen Unît/0.5m] Dose (In any ofthe combination)
1	Diphtheria Toxoid (D)	1-50 Lf	Preferably one of 10 or 22.5 or 25 Lf
2	Tetanus toxoid (T)	1-30 Lf	Preferably one of 2 or 7.5 or 10 Lf
3	Inactivated B. pertussis antigen	1-50 IOU	Preferably one of 12 or 15 or î 6

	(wP)		IOU
4	HBs antigen	1-20 pg	Preferably one of 8 or 10 or 12.5 pg
5	Hib PRP-TT conjugale antigen	1-20 pg ofPRP	Preferably one of 8 or 10 or 13 pg ofPRP
6	Inactivated Polio Virus (IPV) Salk Serotype
Mahoncy Type 1 (D antigen units)	1 -20 DU	7.5 DU
MEF-i Type 2 (D antigen units)	1 - 20 DU	16 DU
Saukett Type 3 (D antigen units)	1 -20 DU	10 DU
7	Total Akiminium Content (Al3*)	0.1-1.2 mg	Preferably NMT 0.3 or NMT 0.55 or NMT 0.9
8	2-Phenoxyelhanol	.1 - 6 mg	Preferably one of 2 or 2.5 or 3.25 mg
9	L-Histidine	04-5 mg	Preferably one of 0.7 or 1 or 1.55 mg

NMT - Not more than

In eighteenth embodiment of the présent disclosure, the combination vaccine composition/formulation comprising Dose reduced IPV is an ali liquid hexavalent vaccine formulation comprising of:

Table 5
Sr. No.	Formulation Components	Antigen Unit/0.5ml Dose	Preferred Antigen Unit/0.5ml Dose (In any of the combination)
1	Diphtheria Toxoid (D)	1-50 Lf	Preferably one of 10 or 22.5 or 25 Lf
2	Te tenus toxoid (T)	1-30 Lf	Preferably one of 2 or 7.5 or 10 Lf
3	Inactivated B. pertussis antigen (wP)	1-50 IOU	Preferably one of 12 or 15 or 16 IOU
4	HBs antigen	1-20 pg	Preferably one of 8 or 10 or 12,5 pg
5	Hib PRP-TT conjugale antigen	1-20 pg of PRP	Preferably one of 8 or 10 or 13 pg ofPRP
6	Inactivated Polio Virus (IPV) Salk Serotype
Mahoncy Type 1 (D antigen units)	1 -20 DU	8 DU
MEF-1 Type 2 (D antigen units)	1 - 20 DU	2 DU
Saukett Type 3 (D antigen units)	1 - 20 DU	5 DU
7	Total Aluminium Content (AF+)	0.1 - 1.2 mg	Preferably NMT 0.3 or NMT 0.55 or NMT 0.9
8	2-PhenoxyethanoI	1-6 mg	Preferably one of 2 or 2.5 or 3.25 mg
9	L-Histidine	0.1—5 mg	Preferably one of0.7 or 1 or 1.55

mg

ΝΜΤ - Not more than

In nineteenth embodiment of the présent disclosure, the combination vaccine composition/formulation comprising Dose reduced IPV is an ail liquid hexavalent vaccine formulation comprising of:

Table 6
Sr. No.	Formulation Components	Antigen Unit/0.5ml Dose	Preferred Antigen Un.it/0.5ml Dose (In any of the combination)
I	Diphtheria Toxoid (D)	1-50 Lf	Preferably one of 10 or 22.5 or 25 Lf
2	Tetanus toxoid (T)	1-30 Lf	Preferably one of 2 or 7.5 or 10 Lf
3	Inactivated B, pertussis antigen (wP)	1-50 IOU	Preferably one of 12 or 15 or 16 IOU
4	HBs antigen	1-20 gg	Preferably one of 8 or 10 or 12.5 gg
5	Hib PRP-TT conjugale antigen	1-20 gg ofPRP	Preferably one of 8 or 10 or 13 gg ofPRP
6	Inactivated Polio Virus (IPV) Salk Serotype
Mahoney Type I (D antigen units)	1 -20 DU	5 DU
MEF-1 Type 2 (D antigen units)	1 - 20 DU	2 DU
Saukett Type 3 (D antigen units)	1 - 20 DU	5 DU
7	Total Aluminium Content (Alî+)	0.1 -1.2 mg	Preferably NMT 0.3 or NMT 0.55 or NMT 0.9
8	2-Phenoxyethanol	1 - 6 mg	Preferably one of 2 or 2.5 or 3.25 mg
9	L-Histidine	0.1 -5 mg	Preferably one of 0.7 or 1 or 1.55 mg

NMT - Not more than

In twentieth embodiment of the présent disclosure, the combination vaccine composition/formulation comprising Dose reduced IPV is an ail liquid hexavalent vaccine formulation comprising of:

Table 7
Sr. No.	Formulation Components	Antigen Unit/0.5ml Dose	Preferred Antigen Unit/0.5ml Dose (In any ofthe combination)
1	Diphtheria Toxoid (D)	1-50 Lf	Preferably one of 10 or 22,5 or 25 Lf
2	Tetanus toxoid (T)	1-30 Lf	Preferably one of 2 or 7.5 or lOLf
3	Inactivated B. pertussis antigen	1-50 IOU	Preferably one of 12 or 15 or ] 6

	(wP)		IOU
4	HBs antigen	1-20 pg	Preferably one of 8 or 10 or 12.5 pg
5	Hib PRP-TT conjugale antigen	1-20 pg ofPRP	Preferably one of 8 or 10 or 13 pg ofPRP
6	Inaetivated Polio Virus (IPV) Salk Serotype
Mahoney Type 1 (D antigen units)	I -20 DU	10 DU
MEF-1 Type 2 (D antigen units)	1 - 20 DU	2 DU
Saukett Type 3 (D antigen units)	1 - 20 DU	10 DU
7	Total Aluminium Content (Al3+)	0J - 1,2 mg	Preferably NMT 0.3 or NMT 0.55 or NMT 0.9
8	2-Phenoxyelhanol	1 - 6 mg	Preferably one of 2 or 2.5 or 3,25 mg
9	L-Histîdine	0.1-5 mg	Preferably one of 0.7 or 1 or 1.55 mg

NMT — Not more than

In twenty first embodiment οΓ the présent disclosure, the combination vaccine composition/formulation comprising Dose reduced IPV is an ali liquid hexavalent vaccine formulation comprising of:

Table 8
Sr. No.	Formulation Components	Antigen Unit/0.5ml Dose	Preferred Antigen Unit/0.5ml Dose (Jn any of the combination)
1	Diphtheria Toxoid (D)	1-50 Lf	Preferably one of 10 or 22.5 or 25 Lf
2	Tetanus toxoid (T)	J-30 Lf	Preferably one of 2 or 7.5 or 10 Lf
3	Inaetivated B. pertussis antigen (wP)	1-50 IOU	Preferably one of 12 or 15 or 16 IOU
4	HBs antigen	1-20 pg	Preferably one of 8 or 10 or 12,5 pg
5	Hib PRP-TT conjugale antigen	1-20 pg ofPRP	Preferably one of 8 or 10 or 13 pg ofPRP
6	Inaetivated Polio Virus (IPV) Salk Serotype
Mahoney Type 1 (D antigen units)	1 -20 DU	10 DU
MEF-1 Type 2 (D antigen units)	1 - 20 DU	2 DU
Saukett Type 3 (D antigen units)	1 - 20 DU	5 DU
7	Total Aluminium Content (Al3+)	0.1 - 1.2 mg	Preferably NMT 0.3 or NMT 0.55 or NMT 0.9
8	2-Phenoxyethanol	1 - 6 mg	Preferably one of 2 or 2.5 or 3.25 mg
9	L-Histidine	0.1-5 mg	Preferably one of 0.7 or I or 1.55

mg

NMT - Not more than

In twenty first embodiment of the présent disclosure, the combination vaccine composition/formulation comprising Dose reduced IPV is an ail liquid hexavalcnt vaccine formulation comprising of:

Table 9
Sr. No,	Formulation Components	Antigen Unît/0.5ml Dose	Preferred Antigen Unit/0.5mI Dose (In any ofthe combination)
1	Diphtheria Toxoid (D)	1-50 Lf	Preferably onc of 10 or 22.5 or 25 Lf
2	Tetanus toxoid (T)	1-30 Lf	Preferably one of 2 or 7.5 or 10 Lf
3	Inactivated B. pertussis antigen (wP)	1-50 IOU	Preferably onc of 12 or 15 or 16 IOU
4	HBs antigen	1-20 Mg	Preferably one of 8 or 10 or 12.5 pg
5	Hib PRP-TT conjugate antigen	1-20 pg ofPRP	Preferably one of 8 or 10 or 13 pg ofPRP
6	Inactivated Polio Virus (IPV) Salk Serotype
Mahoney Type 1 (D antigen units)	1 -20 DU	10 DU
MEF-1 Type 2 (D antigen units)	1 - 20 DU	2 DU
Saukett Type 3 (D antigen units)	1 - 20 DU	12 DU
7	Total Aluminium Content (AP4)	0.1 - 1.2 mg	Preferably NMT 0.3 or NMT 0.55 or NMT 0.9
8	2-I’henoxyethanol	1 - 6 mg	Preferably one of 2 or 2.5 or 3.25 mg
9	L-Histidine	0.1-5 mg	Preferably one of 0.7 or 1 or 1.55 mg

NMT - Not more than

In twenty second embodiment of the présent disclosure, the combination vaccine composition/formulation comprising Dose reduced IPV is an ail liquid hexavalent vaccine formulation comprising of:

Table 10
Sr. No.	Formulation Components	Antigen Unit/0.5ml Dose	Preferred Antigen U ni 170.5 ml Dose (In any of the combination)
1	Diphtheria Toxoid (D)	1-50 Lf	Preferably one of 10 or 22.5 or 25 Lf
2	Tetanus toxoid (T)	1-30 Lf	Preferabiy one of2 or 7.5 or 10 Lf
3	Inactivated B. pertussis antigen	1-50 IOU	Preferably one of 12 or 15 or 16

	(wP)		IOU
4	HBs antigen	1-20 pg	Preferably one of 8 or 10 or 12.5 pg
5	Hib PRP-TT conjugale antigen	1-20 pg ofPRP	Preferably one of 8 or 10 or 13 pg ofPRP
6	Inactivated Polio Virus (IPV) Salk Serotype
Mahoncy Type 1 (D antigen units)	1 -20 DU	10 DU
MEF-1 Type 2 (D antigen units)	1 - 20 DU	2 DU
Saukett Type 3 (D antigen units)	1 - 20 DU	16 DU
7	Total Aluminium Content (Al3+)	0.1 -1.2mg	Preferably NMT 0.3 or NMT 0.55 or NMT 0.9
8	2-P h en oxy éthanol	1 - 6 mg	Preferably onc of 2 or 2.5 or 3.25 mg
9	L-Histidine	0.1-5 mg	Preferably one of 0.7 or 1 or 1.55 mg

NMT - Not more than

In twenty third embodiment of the présent disclosure, one or more antigens of the final combination vaccine compositîon/formulation comprising Dose reduced IPV may not be substantially adsorbed on to any adjuvant.

In twenty fourth embodiment of the présent disclosure, the pH of the compositîon/formulation may be in the range of pH 6.0 to pH 8.0; more preferably in the range of pli 6.0 to pH 7.5; stifl more preferably in the range of pH 6.2 to pH 7.2; and most preferably in the range of pH 6.3 to pH 6.8.

in twenty fifth embodiment of the présent disclosure, WFI or 0.9% saline (NaCl) may be 10 added to the final combination vaccine composition to make up the volume.

In twenty sixth embodiment of the présent disclosure, the compositîon/formulation may addîtionally comprise of a buffering agent selected from the group consistîng of carbonate, phosphate, acetate, succinate, borate, citrate, lactate, gluconate and tartrate, as well as more complcx organic buffering agents including a phosphate buffering agent that conta ins sodium 15 phosphate and/or potassium phosphate in a ratio selected to achieve the desired pH. In another example, the buffering agent contains Tris (hydroxymethyl) aminomethane, or Tris, formulated to achieve the desired pH. Yet in another exampie, the buffering agent could be the minimum essential medium with Hanks salts. Other buffers, such as HEPES, piperazine-N, N'-bis (PIPES), and 2-ethanesulfonic acid (MES) are also envisaged. by the 20 présent disclosure. The buffer aids in stabilizîng the immunogenic composition of the présent disclosure. The amount of the buffer may be m the range of 0.1 mM to 100 mM, preferably selected from 5mM, 6mM, 7mM, 22 mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM and 30mM.

Yet another aspect of the embodiment, the composition/formulation may additionally comprise of pharmaceutically acceptable excipients selected from the group consisting of surfactants, polymers and salts. Examples of Surfactants may include non-ionic surfactants such as polysorbate 20, polysorbate 80, etc. Examples of the polymers may include dextran, carboxymethyl cellulose, hyakironic acid, cyclodextrin, etc. Examples of the salts may include NaCl, KC1, KH2PO4, Na2HPO4.2H2O, CaCh, MgCh, etc.Preferably, the sait may be NaCl. Typically the amount of the sait may be in the range of 100 mM to 200 mM.

Amino acids, such as Histidine, glycine, arginine and lysine may be added to stabilize the immunogenic composition.

In twenty seventh embodiment of the présent disclosure, the composition/formulation may additionally comprise of one or more adjuvant selected from the group of aluminium sait (A13+) such as aluminium hydroxide (A1(OH)3) or aluminium phosphate (AIPO4), alum, calcium phosphate, MPLA, 3D-MPL, QS21, a CpG-containing oligodcoxynucleotide adjuvant, liposome, or oil-in-water émulsion.

Yet preferably the composition comprises aluminium phosphate (A1PO₄) as adjuvant.

Yet preferably the composition comprises aluminium hydroxide (AIOH3) as adjuvant.

In one of the aspects of the twenty seventh embodiment, antigens of the final formulation may be adsorbed on to in situ aluminium phosphate gel or readymade Aluminium phosphate gel or a combination thereof.

In one of the preferred aspects of the twenty seventh embodiment, the composition of the présent disclosure may contain the adjuvant in an amount of 2.5 mg/0.5 ml or less, and specifically, in an amount of 1.5 mg/0.5 ml to 0.1 mg/0.5 ml.

In twenty cighth embodiment of the présent disclosure, the composition may additionally comprise of an immunostimulatory component selected from the group consisting of an oil and water émulsion, MF-59,a liposome, a lipopolysaccharide, a saponin, lipid A, lipid A dérivatives, Monophosphoryl lipid A, 3—deacylatcd monophosphoryl lipid A, AS01, AS03, an oligonucleotide, an oligonucleotide comprising at Least one unmethylated CpG and/or a liposome, Freund s adjuvant, Freund s complote adjuvant, Freund’s incomplète adjuvant, polymcrs, co-polymcrs such as polyoxyethylene-polyoxypropylene copolymers, including block co-polymcrs, polymer p 1005, CRL-8300 adjuvant, muramyl dipcptide, TLR-4 agonists, flagellin, flagellins derived from grain négative bacteria, TLR-5 agonists, fragments of flagellins capable of binding to TLR-5 rcceptors, Alpha-C-galactosylceramide, Chitosan, Interleukin-2, QS-21, ISCOMS, squalene mixtures (SAF-1), Quil A, choiera toxin B subunit, polyphosphazcnc and dérivatives, mycobacterium cell wall préparations, mycolic acid dérivatives, non-ionic block copolymer surfactants, OMV, fllbp, saponin combination with sterols and lipids.

In twenty ninth embodiment of the présent disclosure, the composition may additionally comprise of preservative selected from the group consisting of metbylparaben, propylparaben, Benzéthonium chloridc (Phcmerol), Phénol, m-cresol, ThiomcrsaL Formaldéhyde, benzalkonium chloridc, bcnzyl alcohol, chlorobutanol, p-chlor-m-crcsol, or benzyl alcohol or a combination thereof A vaccine composition may include preservative for a single immmiization, or may include preservative for multiple immunizations (i.e. a ‘multidose’ kit). The inclusion of a preservative is prcferrcd in multidose arrangements. As an alternative (or in addition) to including a preservative in multidose compositions, the compositions may be contained in a container having an aseptie adapter for removal of material. Typically the amount of the preservative may be in the range of 0.1 mg to 50 mg.

Yet according to a preferred aspect of the twenty ninth embodiment, the composition may altematively comprise of preservative combination selected from:

i. 2-Phenoxyéthanol in an amount of 1 to 6 mg per 0.5 ml (v/v); more preferably one of 2 or 2.5 or 3.25 mg per 0.5 ml (v/v) and methylparaben in an amount of 0.1 — 1.5 mg per 0.5 ml (w/v); more preferably onc of 0.7 or 0.9 or 1 mg per 0.5 ml (w/v) or ii. 2-Phenoxycthanol in an amount of 1 to 6 mg per 0.5 ml(v/v); more preferably one of 2 or 2.5 or 3.25 mg per 0.5 ml (v/v) and propylparaben în an amount of 0.05 - 0.2 mg per 0.5 ml (w/v); more preferably one of 0.05 or 0.1 or 0.15 mg per 0.5 ml (w/v) or iii. 2-Phenoxyethanol in an amount of 1 to 6 mg per 0.5 ml(v/v); more preferably onc of 2 or 2.5 or 3.25 mg per 0.5 ml (v/v), methylparaben in an amount of 0.1 — 1.5 mg per 0.5 ml (w/v); more preferably one of 0.7 or 0.9 or 1 mg per 0.5 ml (w/v) and propylparaben in an amount of 0.05 - 0.2 mg per 0.5 ml (w/v); more preferably one of 0.05 or 0.1 or 0.15 mg per 0.5 ml (w/v).

In thirtieth embodiment of the présent disclosure, the composition may additionally comprise of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosîty enhancing additivcs, flavoring agents, colors, and the like, depending upon the route of administration and the préparation desired.

In thirty first embodiment of the présent disclosure, the composition may be fully liquid but is not limited thereto. Suitable forms of liquid préparation may include solutions, suspensions, émulsions, syrups, isotonie aqueous solutions, viscous compositions and élixirs that are buffered to a desired pH.

The composition of the présent disclosure may be in the form of transdermal préparations 10 including lotions, gels, sprays, ointments or other suitable techniques. If nasal or respiratory (mucosal) administration is desired (e.g., aérosol inhalation or insufflation), compositions can be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aérosol dispenser. Aérosols are usually under pressure by means of a hydrocarbon. Pump dispensers can preferably dispense a metered dose or a dose having a particular particle size. When in 15 the form of solutions, suspensions and gels, in some embodiments, the immunogenic compositions contain a major amount of water (preferably purified water) in addition to the active ingredicnt(s).

In thirty second embodiment of the présent disclosure, the said vaccine composition may be stable at 2-8 deg C for 12 to 36 months; at 25 deg C for 2 to 6 months; at 37 deg C for 1 week 20 to 4 weeks.

In thirty third embodiment of the présent disclosure, the composition may be formulated for use in a method for reducing the onset of or preventing a health condition comprising diphtheria, tetanus, pertussis, hepatitis B viras, Haemophilus influenzae type b, polio virus infection involving administration of an immunologically effective amount of the 25 immunogenic composition to a human subject via parentéral or subeutaneous or intradermal, intramuscular or intraperitoneal or intravenous administration or injectable administration or suslained release from implants or administration by eye drops or nasal or rectal or buccal or vaginal, pérorai or intragastric or mucosal or perlinqual, alveolar or gingival or oifactory or respiratory mucosa administration or any other routes of immunization.

In thirty fourth embodiment of the présent disclosure, tire composition coukl be formulated as single dose vials or muitidose vials (2 Dose or 5 Dose or 10 Dosevials) or multidose kit or as pre-filled syringes wherein the said immunogenic composition may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of vaccination is foilowed by 1-3 separate doses given at subséquent time intcrvals after 1-3 years if nceded. The dosage regimen will also, at least in part, be determined on the need of a booster dose 5 required to confer protective immunity.

Yet preferably the composition may be formulated for administration to a human subject or children 2 years of âge or below according to a two dose regimens consisting of a first dose, and second dose at subséquent time intervals after 1-3 years.

Yet preferably the composition may be administered concomitantly with other drugs or any 10 other vaccine.

Compositions may be presented in vials, or they may be presented in ready fiiled syringes.

The syringes may be supplied with or without nccdlcs. A syringe will include a single dose of the composition, whereas a via] may include a single dose or multiple doses (e.g. 2 doses). In one embodiment the dose is for human. In a further embodiment the dose is for an adult, 15 adolescent, toddler, infant or less than one year old human and may be administered by injection.

Vaccines ofthe invention may be packaged in unit dose form or in multiple dose form (e.g. 2 doses). The said multidose composition can be selected from a group consisting of 2 dose, 5 dose and 10 dose .For multiple dose forms, vials are preferred to pre-filled syringes. Effective 20 dosage volumes can be routinely established, but a typical human dose of the composition for injection has a volume of Ü.5mL.

Biological Source of Strains used in Combination Vaccine:

DIPHTHERIA TOXOID:

The strain Corynebacterium diphtheriae PW8 CN2000 was obtained from the Wellcome 25 Research Laboratory, London, United Kîngdom by the National Control Authority Central

Research Institute (C.R.I.) Kasauli, Himacbal Pradesh, India in lyophilized form in the year 1973. The strain was revived and further lyophilized under Master Seed Lot- C. diphtheriae CN2000 AI at C.R.I. Kasauli.

TETANUS TOXOID:

fhe strain Clostridium tetani Harvard Strain No.49205 was obtained from The Rijks Institute Voor de Volksgezondheid (Netherlands) by the National Control Authority C.R.I. Kasauli, in Lyophilized form.

PERTUSSIS:

Manufacturing of Pertussis vaccine bulk at SIIPL involves usage of four strains of Bordetella pertussis viz. Strains 134, 509, 6229 and 25525.The Master Seed of Strains 134 and 509 are origînally from Rijks Institute, The Netherlands, obtained through National Control Authority, Central Research Institute. Kasauli, Himachal Pradesh, India. The Master Seed of Strains 6229 and 25525 are origînally from Lister Institute, Engl and.

HEPATITIS B:

Rhcin Biotcch (Germany) constructed the recombinant Hanscnulapolymorpha strain containing the HBsAg surface antigen gene, Rhein Biotech also made the Master Cell Bank (MCB Hanscnulapolymorpha K3/8-1 strain ADW, 12/94) and performed ali the characterization tests on this bank.

HAEMOPHILUS INFLUENZAE TYPE b:

The source organism for génération of cell substrate is Haemophilus influenzae type b, strain 760705. The strain was origînally isolated from a 2 year and 2 months old baby boy (born on 14-8-74)in Novcmber 1976.Three passages of the strain took place before storage at -70 °C at the Academie Medical Centre (AMC), University of Amsterdam. This strain was transferred to SIIPL as a part of collaboration between SIIPL and Netherlands Vaccines Institute (NVI, The Netherlands).

IPV:

The strain and source of Salk poliovirus is given below.

Poliovirus type 1:

Salk

Strain: Mahoney

Source: Biltboven Biologicals, Netherlands

Sabin

Source: PT Bio Farma, Indonesia

Poliovirus type 2:

Salk

Strain: M.EF1

Source: Bilthoven Biologicals, Netherlands

Sabin

Source: PT Bio Farina, Indonesia

Poliovirus type 3:

Salk

Strain: Saukett

Source: Bilthoven Biologicals, Netherlands

Sabin

Source: PT Bio Farina, Indonesia

Throughout this spécification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integcr or step, or group of éléments, îmegers or steps, but not the exclusion of any other element, integer or step, or group of éléments, integers or steps.

The use of the expression “at least” or “at least one” suggests the use of one or more éléments 20 or ingrédients or quantitics, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results. While certain embodiments of the inventions hâve been described, these embodiments hâve been presented by way of cxample only, and are not intended to limit the scope of the inventions. Variations or modifications to the formulation of this invention, within the scope of the invention, may occur to those skilled in 25 the art upon reviewing the dîsclosure herein. Such variations or modifications are well within the spirit of this invention.

The numerical values given for various physical parameters, dimensions and quantités are only approximate values and it is envisaged that the values higher than the numerical value assigned to the physical parameters. dimensions and quantifies fall within the scope of the invention unless there is a statement in the spécification to the contrary.

While considérable emphasis has been placed herein on the spécifie features of the preferred embodiment, it will bc apprcciated that many additional feanires can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those ski lied in the art from the disclosure herein, whereby it is to be distinctiy understood that the foregoing descriptive matter is to be înterpreted merely as illustrative of the disclosure and not as a limitation.

Examplcs

The following examples are included to demonstrate preferred embodiments ofthe invention. It should be apprcciated by those of skill in the art that the compositions and techniques disclosed in the examples which follow represent techniques discovered by the inventor to fonction weli in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in iight of the présent disclosure, appreciate that many changes can bc made in the spécifie embodiments which are disclosed and still obtain a like or similar resuit without departing from the spirit and scope of the invention.

Example 1 - Purification of Sabin IPV (sIPV)

1. Tangential flow filtration (TFF):

Clarified harvest pool was concentrated to I0X using tangential flow filtration system wîth lOOKda cassettes(0.5m³) and then diafiltered 3 times of harvest volume with phosphate buffer (30mM-70mM, pH : 7.0)

2. Column Chromatography: Ion Exchange Chromatography (IEC).

10X TFF concentrate was passed through DEAE Sepharose fast flow (Weak- Anion cxchanger) packcd in column xk-26 using Akta explorer (GE Hcalthcarc).Negativcly charged impurities was found to bind to the column whereas polio virus was collected in flow through with phosphate buffer (30mM-70mM, pli : 7.0)

3. Non-Phosphate Buffer (TRIS, TBS, MOPS, HEPES, and bicarbonate) exchange:

To minimize the loss of antigen in a quîte cumbersome inactivation procedure (13days), purified virus pool was buffer exchanged from phosphate buffer to Non-Phosphate Buffer (TRIS, TBS, MOPS, ITEPES, and bicarbonate) (30mM-70mM, pH: 7) with TFF system (100 KDa, 0.1 m2). The purified virus pool was exchanged with three volumes of Non-Phosphate 5 Buffer (TRIS, TBS, MOPS. FIEPES, and bicarbonate).

Example 2 - Inactivation of sIPV in presence of Non-Phosphate Buffers (TRIS, TBS, MOPS, HEPES, and bicarbonate) and Phosphate buffer

10X conccntrate diluted 10 times with M - 199 (with 0.5% glycine) so as to achieve final concentration ix. Inactivation agent formalin (0.025%) was added into purified virus bulk 10 while constant mixing. Inactivation was carried out at 37°C while continuons stirring for 13 days containing 0.22μ filtration on 7th day and 13th day.

Table 11: M - 199 composition
Component	Concentration (g/L)
Calcium Chloride	0.2
Ferrie Nitrate · 9H?O	0.00072
Magnésium Sulfate (anhydrous)	0.09767
Potassium Chloride	0.4
Sodium Aeetate (anhydrous)	0.05
Sodium Chloride	6.8
Sodium Phosphate Monobasic (anhydrous)	0.122
L-Alan inc	0.025
L-Arginine · HCL	0,07
L-Aspartic Acid	0.03
L-Cysteine · HCl · ICO	0.00011
L-Cystine · 2HC!	0.026
L-OJutamic Acid	0.0668
L-Glutamine	0.1
Glycine	0.05
L-Histidine · HCl · H2O	0.02188
Hydroxy-L-Proline	0.01
L-Isoleueinc	0.02
L-Leucine	0.06
L-Lysine · HCI	0.07
L-Methionine	0.015
L-Phenyl alanine	0.025
L-Proline	0.04
L-Serine	0.025
L-Thréonine	0.03
L-Tryptophan	0.01
L-Tyrosine · 2Na · 2H3O	0.05766
L-Valine	0.025
Ascorbic Acid · Na	0.0000566
D-Biotin	0.00001
Calciferol	0.0001

Choline Chloride	0.0005
Folie Acid	0.00001
Menadione (sodium bisulfite)	0.000016
myo-Inositol	0.00005
Niacinamide	0.000025
Nicotinic Acid	0.000025
p-Aminobenzoic Acid	0.00005
D-Pamothenic Acid (hemicalcium)	0.00001
Pyridoxal · HCl	0.000025
Pyridoxine · HCl	0.000025
Retinol Acetate	0.00014
Riboflavin	0.00001
DL-a-Tocophcrol Phosphate · Na	0.00001
Thiamine · HCl	0.00001
Adcuine Sulfate	0.01
Adcnosiiic Triphosphate · 2Na	0.001
Adcnosinc MonophosphaLc · Na	0.0002385
Cholestérol	0.0002
Deoxyribose	0.0005
Glucose	1
Glutathione (reduced)	0.00005
Guanine · HCl	0.0003
Hypoxanthine	0.0003
Phénol Red * Na	0.0213
Polyoxyethylenesorbi tan Monooleate (TWEEN 80)	0.02
Ribosc	0.0005
Thymine	0.0003
Uracil	0.0003
Xanthine · Na	0.000344
Sodium Bicarbonate	2.2

A. Effect of non-phosphate buffets (TRIS) on D antigen loss compared to phosphate buffet:

Table 12: D-Antigen Units (DU/ml) before Formalin inactivation and after Fonnalin inactivation in presence of TRIS buffer (30 inM at pH 7.0)
	IPV 1	IPV 2	IPV 3
Before Formalin inactivation	607.3	193.9	40.7
After Formalin inactivation	408.1	181.4	34.1

Table 13: D-Antigen Units (DU/ml) before Formalin inactivation and after Formalin inactivation in presence of TRIS (40 mM at pH 7.0)
	IPV l	IPV 2	IPV 3
Before Formalin inactivation	607.3	193.9	40.7
After Formalin inactivation	487.1	185.2	37.9

Table 14: D-Antigen Units (DU/ml) before Formalin inactivation and after Formalin inactivation in presence of TRIS buffer (50 mM at pH 7.0)

	IPV 1	IPV 2	IPV 3
Before Formalin inactivation	607.3	193.9	40.7
After Formalin inactivation	451.9	175.9	31.0

When formaldéhyde inactivation mctiods were particularly carried out in presence of phosphate buffer, significant D-antigen lusses were observed, whereas it was found that formaldéhyde inactivation in presence of Non-Phosphate Buffer (TRIS) resulted in minimum loss of D-antigen. Further, TRIS Buffer at a concentration of 40mM was found to be most 5 efficient in terms of D-Antigen content préservation for sIPV 1,2 and 3.

B. Effect ol non-phosphate buffers other than TRIS (TBS, MOPS, HEPES, and bicarbonate) on D antigen loss compared to phosphate buffer:

Table 15: D-Antigen Units (DU/ml) before Formalin inactivation and after Formalin inactivation in presence of phosphate buffer (40 mM at pH 7.0)
	IPV 1	IPV 2	IPV 3
Before Formalin inactivation	607.3	193.9	40.7
After Formalin inactivation	52.7	22.63	4.21

Table 16: D-Antigen Units (DU/ml) before Formalin inactivation and after Formalin inactivation in presence of TBS (40 mM at pH 7.0)
	IPV 1	IPV 2	IPV 3
Before Formalin inactivation	607,3	193.9	40.7
After Formalin inactivation	399.15	170.5	22

Table 17: D-Antigen Units (DU/ml) before Formalin inactivation and after Formalin inactivation in presence of MOPS (40 mM at pH 7.0)
	IPV 1	IPV 2	IPV 3
Before Formalin inactivation	607.3	193.9	40.7
After Formalin inactivation	400.21	165	20.1

Table 18: D-Antigen Units (DU/ml) before Formalin inactivation and after Formalin inactivation in presence of HEPES (40 mM at pH 7.0)
	IPV 1	IPV 2	IPV 3
Before Formalin inactivation	607.3	193.9	40.7
After Formalin inactivation	385.45	172	19.9

Table 19: D-Antigen Units (DU/ml) before Formalin inactivation and after

Formalin inactivation in presence of bicarbonate (40 mM at pH 7.0)

	IPV l	IPV 2	IPV 3
Before Formalin inactivation	607.3	193.9	40.7
After Formalin inactivation	395.6	179	21.3

When formaldéhyde inactivation methods were particularly carried out in presence of phosphate buffer, significant D-antigen losses were observed, whereas it was found that formaldéhyde inactivation in presence of Non-Phosphate Buffer other than TRIS (TBS, MOPS, FIEPES, and bicarbonate) resulted in minimum loss of D-antigen.

Method: D-antigen content détermination by EL1SA.

Day 1 : Plate coating:

1. 100 pl of spécifie bovine anti polio was pipetted in PB S per well

2. Microtiter plate was sealcd and incubated overnight at room température.

Day 2: Blocking:

1. The plates were washed (Washing/dilution buffer -0.05% tween 20 in Ix PBS)3 times.

2. 300pl block buffer (1% BSA in PB S) was pipetted per well.

3. The plate was sealed and incubated for 45minutes al 37±1°C.

Sample addition:

]. The plate was washed 3 times.

2. lOOpl of sample diluent was added in ail wells except well of row A.

3. 100 pl standard was added to first two wells of column 2 and 3.

4. 100pl sample was added to first two wells of column 4-12.

5. Prediluling sample to a suitable concentration.

6. 100p] sample diluents was added to first two wells of column l.

7.

Serial two fold dilution were made down the column by transfening lOOul from each well to adjacent wel! of the same column and discardmg lOOul from the last well.

8. Incubating al 37°c for 2 hr.

9. Plates were kept overnight at 4°C.

Day 3: Monocional antibody addition:

1. The plate was washed 3 times.

2. ΙΟΟμΙ diluted( 1:240) type spécifie monoclonal antibodies were added.

3. The plates were sealed and incubated for 2 honrs at 37°C.

Conjugale:

1. The plate were washed 3 times

2. 100μ] diluted conjugale ( Typel- 1:2400 ,Type2- 1:1500, Type3 - 1: 4800) was added.

3. The plate was sealed and incubated for I hour at 37°C.

Substrate addition:

1. ΙΟΟμΙ TM B substrate was added to ail wells.

2. Mixture incubated at room température for 10 minutes.

3. Réaction was stopped by adding ΙΟΟμΙ 2M H2SO4.

4. Plate was read at 450/630nm.

5. D antigen concentration was calculated using KC4 software.

Example 3 - Adsorption of slPV:

1. Autoclaved 1 % stock of Al(OH).i and AlPCfi was used for the préparation of formulations.

2. Desired volume of A1(OH)VA]PO4 was taken to get the required concentration of aluni ni a 100 ml glass bottle.

3. Inactivated polio virus bulk with known D-Ag Unit was added and volume make up was donc with diluent.

4, Final formulation pH was adjusted to 6.5 with 1 N HCl / NaOH .

5. The formulation bulk was kept on magnetic stirrer ovemight at 2-8°C.

Exampie 4 - Preforniulation Studies

Different concentrations of Al(OH)j & AlPOi were prepared in 0.9% saline and in WF1 to check size and zêta potential with respect to change in pH.

It was observed that zêta potential of ΑΙΡΟλ decreases (negativity) with increase in pH from 5 to 7.5 in presence of WFI as well as in saline (Refer Figure 1 and 2).

Whereas, zêta potential of Al(OH)j in saline remains constant, independent of pH and A1(OH)3 sait concentration (Refer Figure 3 and 4).

Exampie 5 - Adsorption studies of sIPV on Aluni phosphate and Aluni hydroxide

Table 20: Sabin Type 1, 2 & 3 (Titer 10⁶⁰/dose) adsorption on aluni (Aluni phosphate and

Aluni Hydroxide)

	Sample	Titer (per does)	Virus Particles (in K)	% free în SUP	% adsorbed on gel
Type 1, AlOHj	Control	5.45	284	NA
125ug/dosc	4.15	14	4.98	95.02
A1+++ 250ug/dose	3.85	7	2.49	97.51
A1+++ 500tig/dose	3.8	6.3	2.24	97.78
Type i,	Control	5.84	691	__NA

AIPO4	A1+++ 125ug/dose	3.49	3	0.43	99.57
A1+++ 250ug/dose	3.09	1.2	0.17	99.83
Α1+Ή500ug/dose	2.94	0.87	0.12	99.87
Type 2, ΑΙΟΗλ	Control	5.49	309	NA
A1+++ ] 25ug/dosc	3.59	3.89	1.25	98.75
A1+-H250ug/dose	3.49	3.09	1	99
A1+++ 500ug/dose	3.49	3.09	1	99
* ype 2, AIPO4	Control	5.49	309	NA
A1+++ 125ug/dose	3.15	1.41	0.45	99.5
AI+++ 250ug/dosc	3.09	1.23	0.39	99.6
A1+++ 500ug/dose	3.09	1.23	0.39	99.6
Type 3, AIOH3	Control	5.59	389	NA
A1++T 125ug/dosc	4.14	13.8	3.54	96.47
A J++-r 250ug/dose	3.94	8.7	2.23	97.77
A1+++ 500ug/dose	3.54	3.4	0.87	99.13
Type 3, AIPO4	Control	5.59	389	NA
Α1+-ί·+ 125ug/dose	5.34	218	56.04	43.96
A1+++ 250ug/dose	5.24	173	44.47	55.53
A1+++ 500ug/dose	5.16	144	37.01	62.9

It was found that Sabin polio virus type-3 shows only 50-60% adsorption with aluminium phosphate (AlPOO-Whereas, Sabin polio virus type-3 shows at least 90% adsorption with Al(OH)i. Thus, Alum hydroxide was found to be more efficient as compared to Alum 5 phosphate with respect to adsorption of Sabin Type 1, 2 and 3.

Example 6 - Iminunogenicity studies of Alum Adsorbed slPV

To check immune response of adjuvanted sIPV in rat (Sera Neutralization Test) SNT test was carried out. Sera was separated and used to test the presence of neutralizîng antîbodies for type spécifie polio virus. Control sera used to validatc the test. Virus back-titration was also performed to get the number of challenge virus particles added.

Animal Model: Wîstar rat (8 weeks, approx 200 gm) 50% male and 50 % female per group.

Route of Inoculation: Intra Muscular.

Volume: 0.5 ml

Blood withdrawal: on day21.

Site of bleeding: Rétro-Orbital plexus.

Table 21: Immunogenkiry results of Alum Adsorbed sIPVType 1

Rat No

Group 1

Group 2

Group 3

Group 4

Group 5

Groap $

Group 7

Group 13

Cornu·. IPX

5 0V IJSmgOH

2.mu l.lSmgOH

1DU l.IimgOH

5DU l.SmgPOl

2.SDU L3ïtigPQ4

1DU LSx»gPO4

-ve control

SNT

Sera Titei

SNT *ve

Sera Titei

SNT -ve

Sera Tifer

SNT +ve

Sera Tïtea-

SN T rve

Sent Titer

SN T

S sra Ti«r

SN T -Te

Sera TÆer

SNT -ΤΈ

Sera Ther

1

1

(17)

$

(1:256)

1

(1:2)

4

(1:16)

5

(1:32)

(1:32)

2

(1:4)

0

(<17)

2

I

(1:2)

5

(1:32)

1

(1:2)

7

(1:128)

s

¢1:256)

4

(1:16)

1

(1:2)

D

(<17}

3

G

(<17)

(1:128)

3

(1:8)

9

(<1:2)

4

(1:16)

6

(1:64)

0

(<1:2)

0

(<17}

4

0

(<17)

n

(12W!)

(14)

2

(1:4)

1

(1:2)

5

(1:32)

0

(<1;2Ί

0

(<i:2)

3

(1:128)

3

(1:8)

7

(1:128)

5

(1:32)

6

(1:64)

4

(1:16)

1

(1:2)

0

(<1:2>

6

4

(1:15)

7

(1:12B)

i

(1:123)

1

(1:2)

X

(1:32)

6

(1:64)

3

(1:8)

0

(<1:2)

7

3

(1:8)

5

(1:32)

4

(1:16)

1

(1:2)

8

(1:256)

7

(1:128)

0

CR2)

0

«7}

g

1

(1:2)

7

¢1:128)

3

(1:8)

2

(1:4)

6

(1:64)

9

(<17)

0

(<1:2)

0

(<17)

9

3

(1:8)

s

(1:256)

2

(1:4)

3

(1:8)

8

(1:256)

4

(1:16)

4

(1:16)

0

(<17}

10

3

(1:8)

7

(1:128)

4

(1:16)

5

(1:32)

6

(1:64)

2

(1:4)

2

(1:4)

0

(<17)

Interprétation; It was surprisingly found that Alum hydroxide adjuvanted Type 1 Sabin IPV having 5 DU/dose gave better séroconversion as compared to Salk IPV vaccine with 40DU/dose and Alum phosphate adjuvanted Sabin IPV having 5 DU/dose.

Table 22: Immunogenicity results of Alum Adsorbed sTPV Type 2
Rat No	Group 1	Group 2	Group 3
4DU( O.ômgOH)	8DLJ( O.ômgOH)	16DU O.ômgOH

	SNT +ve	Sera Titer	SNT +ve	Sera Titer	SNT +ve	Sera Titer
1	3	(!:8)	4	(1:16)	7	(1:128)
2	4	(1:16)	6	(1:64)	5	(1:32)
3	0	(<1:2)	3	(1:8)	5	(1:32)
4	3	(1:8)	4	(1:16)	6	(1:64)
5	5	(1:32)	7	(1:128)	6	(1:64)
6	6	(1:64)	4	(1:16)	9	(1:512)
7	4	(1:16)	7	(1:128)	4	(1:16)
8	5	(1:32)	3	(1:8)	8	(1:256)
9	7	(1:128)	8	(1:256)	8	(1:256)
10	5	(1:32)	3	(1:8)	8	(1:256)

Interprétation: Type 2 slPV having 8 DU/dose with adjuvant gave équivalent séroconversion as comparée! to Salk IPV vaccine with 8DU/ eiose.

Table 23: Immunogenicity resuits of Alum Adsorbed slPV Type 3

Rat No	Group 1	Group 2	Group 3
10 DU O.ômgOH	5DU O.ômgOH	2.5DU O.ômgOH
SNT +ve	Sera Titer	SNT +vc	Sera Titer	SNT +vc	Sera Titer
1	3	(1:8)	2	(1:4)	0	(<1:2)
2	0	(<1:2)	5	(1:32)	1	(1:2)
3	2	(1:4)	3	(1:8)	1	(1:2)
4	4	(1:16)	2	(1:4)	0	(<1:2)
5	4	(1:16)	2	(1:4)	1	(1:2)
6	4	(1:16)	1	(1:2)	1	(1:2)
7	9	(1:512)	0	(<1:2)	2	(1:4)
8	7	(1:128)	2	(1:4)	2	(1:4)
9	1	(1:2)	0	(<1:2)	1	(1:2)
10	5	(1:32)	7	(1:128	1	(1:2)

Interprétation: It was found that Type 3 slPV having LODU/dose with adjuvant gave équivalent sero conversion as compared to Salk IPV vaccine with 32DU/ dose.

Table 24: Maximum elose réduction observed for individual Sabm Type 1, 2 & 3 after studies
slPV	Standard dose	*SI1L Dose	Dose réduction
Type 1	40DU	5DU	~8 Folds

Type 2	8DU	8DU	Equivalent
Type 3	32DU	10DU	~3 Folds

*SIIL: Sérum Institute of India In House dose reduced IPV préparation.

Table 25: Immune response study of the adjuvanted SABIN poliovirus

	Type 1	Type 2	Type 3
D-Antigen units of Sabin’s strain	5	16	10
2.5	8	5
7.5	16	10

Interprétation:

We observed that if the viruses are adjuvanted with Al (OH)i shows excellent dose sparing.

If we consider single dose regimen for immunization then 5-16-10 D-Ag arc bcst combination for Sabin’s polio type 1, 2 and 3 respectively.

If we consider two doses for immunization then 2.5-8-5 gives excellent immunily.

Table 26: Immune response study of the adjuvanted SALK poliovirus

D-antigen units for Salk Strain	Type 1	Type 2	Type 3
8	2	5
5	2	5
10	2	10
7.5	16	10
10	2	5
10	2	12
10	2	16

Interprétation:

We observed tirai if the viruses are adjuvanted with Al (O1T)3 shows excellent dose sparing.

If we consider single dose regimen for immunization then 8-2-5 D-Ag are best combination for Salk’s polio type 1, 2 and 3 respectively.

lf we consider two doses for immunization then 5-2-5 gives excellent immunity.

Example 7: Purification of Diphtheria toxoid (D) and Tetanus Toxoid (T)

Tetanus Toxoid and Diphtheria toxoid was purified using Gel filtration chromatography and 5 stabilîzed by addition of an amino acid buffer solution (Histidine, lysine, glycine, arginine) or polysorbate solution at a final concentration of 5 - 300 mM, and stored at température -20 to -1-40 °C till further use.

Table 27: Gel filtration chromatography Process Paramctcrs
Sr. No.	Parameter	Details
1	Method of Purification	Gel Filtration Chromatography (GFC)
2	Resin	Sephacryl S-300 HR
3	Column Used	XK 26/70cm
4	Column Packed bed height	~50cm
5	Linear Flow rate	1 - 3 ml/min
6	Sample (T ) loading	4% of total bed volume
7	Fraction Collection	5 ml each (2min)
8	Analysis	Lowry’s assay, LF estimation, % Monomer
8	Buffer	0.85% NaCl pH 6-7

Refer Fig 5: Diphtheria Toxoid Purification - Chromatogram of Gel Filtration Chromatography

Table 28: % Rccovery & Monomer content of purified Diphtheria toxoid
No.	Sample	Protein Cône. (mg/mL)	% Mono mer	% Recovery
1	Native DT	___	67.19
2	Native CRM	___	86.96	--
3	GFC DT FR 8	2.03	83.21	Injected DT Qty 120mg
4	GFC DT FR 9	3.38	87,15

5	GFC DT FR 10	4.08	87.24	Obtained Qty 75.1mg (62.5%)
6	GFC DT FR 11	3.52	86.60
7	GFC DT FR 12	2.01	89.97
8	GFC DT FR 10-1 1	3.83	86.03	31.6%
9	GFC DT FR 9-12	2.96	87.42	48.9%
10	GFC DT FR 7-13	2.19	86.24	63.3%

Results:

The percent nionomer content was found to be in the range of 80 - 90 %.

Example 8: Préparation of Hib PRP- Protein conjugate

a) PRP polysaccharide was produced as follows:

H. Influenzae type-b bacteria was grown in semi synthetic media under certain conditions of température, agitation and optical density etc. PRP is an outer membrane bound polysaccharide, gets released into the medium during the fermentation under agitation condition. Fermented biomass separated broth contains crude PRP, which is again purified by 10 précipitation using a détergent N, N, N-trimethyl-1 -hexadecanaminium bromide, followed by éthanol gradient précipitation and filtration. Final purified PRP polysaccharide was tested foimeeting the spécifications like endotoxin, nucleic acid and protein as per the WHO, BP, EP, IP etc.

b) Hib PRP - CRM197 carrier protein conjugate was prepared as follows:

The polysaccharide conjugate was prepared by coupling of PRP polysaccharide with a CRM 197 carrier protein. The input ratio of reactants i.e. PRP polysaccharide, CD AP and CRM197 was selected at 1:1.5:1 ratio for conjugation reaction. During eonjugation, purified PRP polysaccharide was depolymerized using an alkaline buffer (0.4M Carb-Bicarbonate buffer, pH 10.5 ±0.1) to achieve size reduced PRP. Size reduced PRP is treated for 20 cyanylation using CDA P (l-cyano-4-dimethylamino pyridinium tetrafluoroborate) chemistry to form a cyanate ester. The activated cyanylatcd polysaccharide may thus be coupled directly with amino group on the carrier protein CRM 197. The degree of conversion of Hib conjugate was confirmed by the HP LC. The conjugation reaction was quenched by achieving the desired level of conversion of conjugate with the spécification of not less than 65% conversion of Hib conjugate, and then conjugale reaction was neutralized by Glycine (2M) addition. The Hib PRP-CRM197 Conjugate is purified on ultra-filtration membrane filters (SOOkDa and lOOkDa) to remove nonreactive rcagents and by-products. Final conjugate bulk was 0.22 pm filtered and stored at 2-8°C.

Quality characteristics of Hib PRP-CRM197 conjugate antigen obtained were as follow:

PRP content (mg/mL) : 1.49

Protein content (mg/mL): 2.98

Ratio (Ps : Protein) :0.52

Free PRP (%) :1.77

PMW (kD) :983

AvgMW(kD) :752

c) Hib PRP - TT carrier protein conjugate was prepared as follows:

The concentrated polysaccharide is depolymerized under mild alkalînc conditions using carbonate-bicarbonate buffer. After target polysaccharide size is reachcd, the depolymerized polysaccharide is activated using Cyanogen Bromide. This activation is donc under nitrogen environment. Freshly prepared adîpic aeîd dihydrazide (ADH) solution is added within 6-10 minutes to the reaction mixture obtained. The reaction is carried out for NLT 16 hours at 2-10 °C. The réaction mixture obtained is concentrated and diafiltered volume by volume with phosphate buffer saline (PBS) using 10 kD NMWCO UF membrane to remove free ADH. The temoval of ADH is monitored on HPLC and diafillration is continued till free ADH level reaches below 5%. The resulting retentatc is further diafiltered with NLT 5X MES-NaCl buffer. Thîs is further concentrated to achieve a concentration of NLT 20 mg/mL. The refentate is passed through a 0.22 pm filter, which serves as a clarification step. The filtered activated polysaccharide is collected, sampled, aliquoted and stored at 2-8 °C till further processing. A sample is drawn from the processed polysaccharide pool for analysis, which incîudes PRP molecular size (kD), PRP content, and PRP degree of activation. The conjugation reaction requires two components viz. processed polysaccharide and the carrier protein (TT). The carrier protein is concentrated and diafiltered with MES-NaCl buffer using 10 kD UF NMWCO membrane. This diafiltered carrier-protein is then further concentrated to NLT 20 mg/mL using tire same membrane. The two components are mixed in appropriate quantifies in die ratio of PRP: TT = 1:1 (w/w) in presence of EDC under stirring. The conjugation reaction is quenched using phosphate EDTA buffer. The conjugation reaction is monitored on HP LC and is continued till > 85% conversion of protein (based on the free protein conversion to conjugate) is reached.

Quality characteristics of Hib PRP-TT conjugate antigen obtained were as follow:

PRP content (pg/0.5ml)	: 8.1
Ratio (PRP:TT)	: 0.5
Free PRP (%)	: 4.8%
PMW (kD)	: 983
Avg MW (kD)	: 752

Example 9: Process of manufacturing inactivated wP antigen

Inactivation method of Whole cell pertussis (wP) antigen:

Inactivation method optimization is doue after performîng varions experiments which include inactivation at 56°C for 10min in presence of formaldéhyde, 56°C for 15min in presence of formaldéhyde, 56°C for 10min in presence of hymine, 56°C for 15min in presence of hymine and only heating at 56°C for 30min. No significant différence in potency is observed with these methods. Out of these methods, 56°C for 10min in presence of formaldéhyde is selected because pertussis cell mass produced using this method is more homogeneous as comparcd to other methods mentioned above.

Process of manufacturing inactivated wP antigen comprises the following steps:

a), inactivation at 56°C for 10 - 15 minutes pertussis strains 134

b), inactivation at 56°C for 10 - 15 minutes pertussis strains 509

e). inactivation at 56°C for 10-15 minutes pertussis strains 25525 and 6229

c). inactivation at 56°C for 10 — 15 minutes pertussis strains 6229 in presence of formaldéhyde of Bordetella in presence of formaldéhyde of Bordetella in presence of formaldéhyde of Bordetella in presence of formaldéhyde of Bordetella

d) . subscquently mixing inactivated Bordetella pertussis strains 134, 509, 25525 and 6229 in a ratio of 1:1:0.25:0.25.

e) . optionally adsorbed onto aluminium based adjuvant,

The process is devoid of thiomcrsal and inactivatcd whole cell pertussis antigen romains nonclumpy and homogeneous thereby leading to reduced reactogcnicity and giving better potcncy for a longer duration.

Example 10: Hexavalent Combination Vaccine Compositions comprising Dose reduced IPV, D, T, HepB, wP, and Hib antigen are as given below:

Table - 29: Combination Vaccine comprising IPV (Salk Strain type i(Malioney) or type 2(MEF) or type 3(Saukett))
S. No,	Formulation Components	Combination composition in accordance with the présent disclosure [per 0.5ml Dose|
1	2	3	4	5	6	7
l	Diphthcria Toxoid (D)	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf
2	Tctanus toxoid (T)	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf
3	hiactivated B. pertussis antigen (wP)	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU
4	HBs antigen	12.5 pg	12.5 pg	12.5 hg	12.5 bg	12.5 pg	12.5 bg	12.5 bg
5	Hib PRP-TT conjugale antigen	10 pg of PRP	10 pg of PRP	10 pg of P RJ’	10 bg of PRP	10 bg of PRP	10 bg of PRP	10 bg of P RI’
6	Inactivated Polio Virus (IPV)
Type 1(D antigen units)	7.5	8	5	10	10	10	10
Type 2 (D antigen units)	16	2	2	2	2	2	2
Type 3(D antigen units)	10	5	5	10	5	12	16
7	Total Aluminium Content (ap+)	Not more than 0.9 mg	Not more than 0.9 mg	Not more than 0.9 mg	Not more than 0.9 mg	Not more than 0.9 mg	Not ni oie than 0.9 mg	Not more than 0.9 mg
8	L-Histidinc	1.55 mg	1.55 mg	1,55 mg	1.55 mg	1,55 mg	1.55 mg	1.55 mg

8

2-Phenoxy éthanol

3.25 mg

3.25 mg

3.25 mg

3.25 mg

3.25 mg

3.25 mg

3.25 mg

Additionaîly adjusting the pH of the composition as disclosed above to about 6.0 to 7.0 with Sodium Hydroxîde / Sodium Carbonate and make tip the volume by adding normal saline (0.9%).

The composition may be devoid of IPV Type 2

May additionaîly comprise of one of the prescrvative combination iv. 2-Phenoxyethanol in an amount of 1 to 6 mg per 0.5 ml (v/v) and methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v); or

v. 2-Phenoxy éthanol in an amount of 1 to 6 mg per 0.5 ml(v/v)and propylparaben in an amount of 0.05 - 0.2 mg per 0.5 ml (w/v); or vi. 2-Phcnoxycthanol in an amount of 1 to 6 mg per 0.5 ml(v/v), methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v) and propylparaben in an amount of 0.05 0.2 mg per 0.5 ml (w/v).

The Hexavalent Combination Vaccine Compositions comprising Dose reduced IPV, D, 15 T, HepB, acellular pertussis, and Hib antigen may comprise of acellular pertussis antigen selected from - Bordetella toxin in detoxifïed form (in particular cithcr genetically or chemically detoxifïed), in particular Pertussis toxoid; Filamcntous Hacmagglutinin; Pertactin; or Fimbriae. Particularly Pertussis toxoid: 1 to 50 micrograms (More particularly 8pg); - Filamentous Haemagglutinin: 1 to 50 micrograms (More particularly 8pg); 20 Pertaclin: 1 to 20 micrograms (More particularly 2.5gg); - Optionally, Fimbriae: 2 to 25 micrograms; per 0.5 ml.

Table - 30: Combination Vaccine comprising IPV (Sabin Strain type 1 or type 2 or type 3) ___________________________________________________
S. No.	Formulation Components	Combination composition în accordance with the présent disclosure |pcr 0.5ml Dose|
1	2	3
1	Diphtheria Toxoid (D)	22.5 Lf	22.5 Lf	22.5 Lf
2	Tetanus toxoid (T)	7.5 Lf	7.5 Lf	7.5 Lf
3	JnaclivaLcd B. pertussis	15 IOU	15 IOU	15 IOU

antigen (wP)

4	HBs antigen	12.5 pg	12.5 pg	12.5 pg
5	Hib PRP-TT conjugale antigen	10 pg ofPRP	10 pg ofPRP	10 pg ofPRP

Inactivated Polio Virus

6	(IPV)
Type 1(D antigen units)	5	2,5	7.5
Type 2 (D antigen units)	16	8	16
Type 3(D antigen units)	10	5	10
7	Total Aluminium Content (Al3+)	Not more than 0.9 mg	Not more than 0.9 mg	Not more than 0.9 ______mg
8	L-Histidine	1.55 mg	1.55 mg	1.55 mg
8	2-Phenoxyethanol	3,25 mg	3.25 mg	3.25 mg
Additionally adjusting the pH of the composition as disclosed above to about 6.0 to 7.0 with
Sodium Hydroxide / Sodium Carbonate and make up the volume by adding normal saline

(0.9%).

The composition may be devoid of IPV Type 2

May additionally comprise of one of the preservative combination

i. 2-Phenoxyéthanol in an amount of 1 to 6 mg per 0.5 ml (v/v) and methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v); or ii. 2-Phenoxyethanol in an amount of 1 to 6 mg per 0.5 ml(v/v)and propylparaben in an amount of 0.05 - 0.2 mg per 0.5 ml (w/v); or iii. 2-Phenoxyethanol in an amount of 1 to 6 mg per 0.5 ml(v/v), methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v) and propylparaben in an amount of 0.05 0.2 mg per 0.5 ml (w/v).

The Hexavaleiit Combination Vaccine Compositions comprising Dose reduced IPV, D, T, Hep B, acellular pertussis, and Hib antigen may comprise of acellular pertussis antigen selected from - Bordetella toxin in detoxified form (in particular eîther genetically or Chemical ly detoxified), in particular Pertussis toxoid; Fi lamentons Haemagglutinin; Pertactin; or Fimbriae. Particularly Pertussis toxoid: 1 to 50 micrograms (More particularly 8pg); - Filamentous Haemagglutinin: 1 to 50 micrograms (More particularly 8pg); Pertactin: 1 to 20 micrograms (More particularly 2.5pg); - Optionally, Fimbriae: 2 to 25 micrograms; per 0.5 ml.

Example 11: Process for préparation of Hexavalent Combination Vaccine Compositions comprising Dose reduced IPV, HBs, D, T, wP, and Hib PRP - Protein conjugate is as given below;

a) adsorbing IPV (Sabin/Saik strain) bulk individually on Aluminium hydroxide, followed by pH adjustment to 6.2 - 6.6, more preferably 6.5.

b) adsorbing D on Aluminium phosphate, followed by pl-1 adjustment to 5.5 - 6.5

c) adsorbing T on Aluminium phosphate, followed by pH adjustment to 5.5 — 6.5

d) adsorbing HBsAg on Aluminium phosphate, followed by pH adjustment to 6.0 - 6.5.

e) blending the mixture as obtained in step (b), (c), (d) by agitation at room température for 18-24 hours.

I) Blending the mixtures as obtained in step (a) and (e), followed by pH adjustment to 6.4 6.6 and agitation at room température for 60 minutes.

g) adding inactivated wP antigen l acellular pertussis antigen and a stabilîzer (Histidinc amino acid buffer solution 200 mM) to the above mixture in step (f), followed by agitation for 60 minutes and left in static condition for overnight at 2 - 8 °C.

h) adding Hib antigen to the mixture obtained in step (g) at 2 - 8 °C, followed by pH adjustment to 6.4 - 6.6.

i) Adjusting pH to 6.0 to 7.0 with Sodium Hydroxide / Sodium Carbonate and adding normal saline (0.9% NaCl) or WFI (q.s.) to make up the volume of the mixture obtained in step h, followed by agitation for 2 hours.

j) The process further comprises adding preservative lo the mixture obtained in step (h) selected from:

v. 2-Phenoxyethanol in an amount of 1 to 6 mg per 0.5 ml (v/v) and methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v); or vi. 2-Phenoxyethanol in an amount of l to 6 mg per 0.5 ml(v/v)and propylparaben in an amount of 0.05 - 0.2 mg per 0.5 ml (w/v); or vii. 2-Phenoxyethanol in an amount of l to 6 mg per 0.5 ml(v/v), methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v) and propylparaben in an amount of 0.05 0.2 mg per 0.5 ml (w/v);

viii. 2-phenoxyethanoi in an amount of 1 to 6 mg (v/v) per 0.5ml as preservative.

Example 12: Absorption and Potency profile of Individual Antigens

Table 31 : This table gives a brief on the percentage adsorption of individual antigens, and Potency profile of individual antigens in SIIPL Combination vaccine
Test	Range	0 Day
Dose		0.5 ml
Hepatitîs B In-Vivo Potency R..P (95% CL)	(0.77-1.72)	1.14
Kib PRP Content (pg/0.5 ml) (Total PRP)	Actual value.	11.32 pg/0.5ml
Free PRP (%)	Actual value.	1.7
Diphthcria component potency (Lf/ml)	Actual value.	42
Tetanus component potency (Lf/ml)	Actual value.	16.7
Pertussis component potency ( lU/dose)	NLT 4 JU/dosc	6.16 (3,34-11,41)
Adsorption Hepatitis-B (%)	AcUial value.	99.71
Adsorption: Tetanus Component (%)	Actual value.	100%
Adsorption; Diphthcria Component (%)	Actual value.	105%
IPV Salk Strain D Antigen unit (DU/0.5 ml) EL1SA	Type 1= 10 DU /0.5 ml	5.98
Type 2= 2 DU i 0.5 ml	1.6
Type 3= 10 DU/0.5 ml	7.87
IPV Salk Strain Invivo Efficacy	Type 1 (LL-UL)	88.9% (25.4 — 266.9)
Type 3 (LL-UL)	93.9% (38.7-245.0)

Table 32; This table gives a brief on the percentagc adsorption of individual antigens, and Potency profile of individual antigens in SIIPL Combination vaccine

Test	Range	0 Day
Dose		0.5 ml
Hepatitis B Ιπ-Vivo Potcncy R.P (95% CL)	(0.77-1.72)	1.21
Hib PRP Content (pg/0,5 ml) (Total PRP)	Actual value.	11.87 gg/0.5ml
Free PRP (%)	Actual value.	Not détectable
Diphtheria component potenev (Lf/ml)	Actual value.	42
Tetanus component potcncy (Lf/ml)	Actual value.	16.8
Pertussis component potcncy ( HJ/dose)	NLT 4 lU/dosc	7.13 (3.45-15.85)
Adsorption Hcpatitis-B (%)	Actual value.	99.8
Adsorption: Tetanus Component (%)	Actual value.	100%
Adsorption: Diphtheria Component (%)	Actual value.	105%
IPV Sabin Strain D Antigen unit (DU/0.5 ml) ELISA	Type 1=5 DU/0.5 ml	3.84
Type 2= 16 DU /0.5 ml	8.66
Type 3= 10 DU ! 0.5 ml	14.58
IPV Sabin Strain Invivo Efficacy	Type 1	>100.0%
Type 2	>100.0%
Type 3	>100.0%

Claims (31)

1. A dose reduced Inactivated Polio vaccine (IPV) composition comprising:

an inactivated polio virus antigen selected from the group consisting of IPV Type 1 at a dose

5 between 5 D-antigen units and 10 D-antigen units (DU) IPV Type 2 at a dose between 1 Dantigen units and 16 D-antigen units (DU), and IPV Type 3 at a dose between 1 D-antigen units and 16 D-antigen units (DU), wherein the inactivated polio virus antigen is per 0.5 ml.

2. The composition as claîmed in claim 1, wherein the composition is a combination vaccine

10 containing dose reduced Inactivated Polio vaccine (IPV) and a one or more non-IPV antigen selected from the group consisting of Diphtheria toxoid (D), Tetanus toxoid (T), Whole cell pertussis (wP), hepatitis B virus surface antigen (HBsAg), Haemophilus influenzae b PRPCarrier protein conjugale (Hib), Haemophilus influenzae(a, c, d, e, / serotypes and the unencapsulated strains),Neisseria meningitidis A antigen(s), Neisseria meningitidis C

15 antigen(s), Neisseria meningitidis W-135 antigen(s), Neisseria meningitidis T 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, D, E, F and G strains) antigen(s), Human papilloma virus, HIV, Salmonella typhi antigen(s) , acelltdar pertussis, modified adenylate cyclase, Malaria

20 Antigen (RTS,S), Measles, Mumps, Rubella, Dengue, Zika, Ebola, Chikungunya, Japanese encephalitis, rotavims, Diarrheal antigens, Flavivirus, smallpox, yellow fever, Shingles, and Varicella virus antigens.

3. The composition as claimed in claim 1, wherein the IPV Type 1 antigen is the Mahoney 25 strain (Salk Type 1) or Sabin strain; and/or wherein the IPV Type 2 antigen is the MEF-1 strain (Salk Type 2) or Sabin strain; and/or wherein the IPV Type 3 antigen is the Saukett strain (Salk Type 3) or Sabin strain.

4. The composition as claimed in claim 1, wherein the IPV Type 1, 2 and 3 antîgens are 30 adsorbed onto aluminium sait of hydroxide (A1(OH)3) or phosphate (A1PO₄) or a mixture of both having percentage adsorption of at least 90% and total aluminium content (Ar⁺) in the composition is in an amount of 0.1 to 1.2 mg per 0.5 ml.

5. The composition as claimed in claim 1, wherein 0.5 ml of the composition comprises: the D antigen in an amount of 1 to 50 Lf; the T antigen in an amount of 1 to 30 Lf; the wP antigen in an amount of 1 to 50 1OU; the HBsAg antigen in an amount of 1 to 20 pg; the Hib antigen in an amount of 1 to 20 pg; the IPV Type 1 antigen in an amount of 5-10 DU, the IPV Type 2 antigen in an amount of 1 - 16 DU, and the IPV Type 3 antigen în an amount of 1 - 16 DU.

6. The composition as claimed in claim 1, wherein the inactivated polio virus antigen dose is selected from the group consisting of:

i) IPV Type 1 (Sabin straîn) in an amount of 2.5 DU, IPV Type 2 (Sabin strain) in an amount of 8 DU, and IPV Type 3 (Sabin s Pain) in an amount of 5 DU, per 0.5 ml;

ίί) IPV Type 1 (Sabin strain) in an amount of 5 DU, IPV Type 2 (Sabin strain) in an amount of 16 DU, and IPV Type 3 (Sabin strain) in an amount of 10 DU, per 0.5ml;

iii) IPV Type 1 (Sabin strain) în an amount of 5 DU, IPV Type 2 (Sabin strain) in an amount of 8 DU, and IPV Type 3 (Sabin strain) in an amount of 10 DU, per 0.5ml;

iv) IPV Type 1 (Mahoney strain) in an amount of 7.5 DU, IPV Type 2 (MEF-1 strain) in an amount of 16 DU, and IPV Type 3 (Sauketl strain) în an amount of 10 DU, per 0.5ml;

v) IPV Type 1 (Mahoney strain) in an amount of 8 DU, IPV Type 2 (MEF-1 strain) in an amount of 2 DU, and IPV Type 3 (Saukett strain) in an amount of 5 DU, per 0.5ml;

vi) IPV Type 1 (Mahoney strain) în an amount of 10 DU, IPV Type 2 (MEF-1 strain) in an amount of 2 DU, and Type 3 (Saukett strain) in an amount of 5 DU, per 0.5ml;

vii) IPV Type 1 (Mahoney strain) in an amount of 10 DU, IPV Type 2 (MEF-1 strain) in an amount of 2 DU, and IPV Type 3 (Saukett strain) in an amount of 10 DU, per 0.5ml;

viii) IPV Type 1 (Mahoney strain) in an amount of 10 DU, IPV Type 2 (MEF-1 strain) in an amount of 2 DU, and IPV Type 3 (Saukett strain) in an amount of 12 DU, per 0.5ml;

ix) IPV Type 1 (Mahoney strain) in an amount of 10 DU, IPV Type 2 (MEF-1 strain) in an amount of 2 DU, and IPV Type 3 (Saukett strain) in an amount of 16 DU, per 0.5ml;

x) IPV Type 1 (Mahoney strain) in an amount of 7.5 DU, IPV Type 2 (MEF-1 strain) in an amount of 16 DU, and IPV Type 3 (Saukett strain) in an amount of 10 DU, per 0.5ml; and xi) IPV Type 1 (Mahoney strain) in an amount of 5 DU, IPV Type 2 (MEF-1 strain) in an amount of 2 DU, and IPV Type 3 (Saukett strain) in an amount of 5 DU, per 0.5ml.

7. The composition as claimed in claim 1, wherein the composition does not comprise the IPV Type 2 antigen.

8. The composition as claimed in claims 2 and 5, wherein the whole cell pertussis antigen in the composition is of Bordetella pertussis strains 134, 509, 25525 and 6229 in a ratio of 1:1:0.25:0.25.

9. The composition as claimed in claims 2 and 5, wherein the acellular pertussis antigen comprises one or more anligens selected from modified adenyiate cyclase, Pertussis toxoid (PT) l-50pg, Filamentous hemagglutînin (FHA) l-50pg, Pertactin (P69 or PRN) l-20pg or Fimbrial proteins (FIM 1,2 and 3) 2-25 pg; per 0.5ml.

10. The composition as claimed in claims 2 and 5, wherein the Hib antigen is a Hib polyribosylribitol phosphate (PRP) polysaccharide conjugated to a carrier protein using a cyanylation conjugation chemistry reagent or reductive amination conjugation chemistry, wherein the cyanylation reagent is selected from Cyanogen Bromîde, l-cyano-4dimethylaminopyridinium tetrafluoroborate (CDAP)l-cyano-4-pyrrolidinopyridinium tetrafluorborate (CPPT), 1-cyanoimidazole namely (1-CI), l-cyanobenzoiriazole( 1-CBT) or 2-cyanopyridazine-3(2H)one (2-CPO); and the carrier protein is selected from a group comprising of tetanus toxoid,CRM 197, Diphtheria toxoid, Neisseria meningitidis outer membrane complex, fragment C of tetanus toxoid, pertussis toxoid, protein D of H. influenzae, E.colî LT, E. coli ST, and exotoxin A from Pseudomonas aemginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and léthal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human sérum albumin, bovine sérum albumin (BSA), purified protein dérivative of tuberculin (PPD), synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins comprising multiple human CD4+ T cell epitopes from various palhogen-derived antigens such as N 19, iron-uptake proteins, toxin A or B from C. difficile and S.agalactiae proteins with or without linker.

IL The composition as claimed în claim 10, wherein a percentage adsorption of lhe Hib antigen onto the adjuvant is less than 20%.

12. The composition as claimed in claims 2 and 5, wherein the D, T and HBsAg antigens are individuaily adsorbed onto an adjuvant, the adjuvant selected from the group consisting of: aluminium sait (Al³⁺) such as aluminium hydroxide (A1(OH)₃) or aluminium phosphate (AIPO4).

13. The composition as claimed in claim 12, wherein the adjuvant is aluminium phosphate (AIPO₄).

14. The composition as claimed in claim 12, wherein the D antigen is adsorbed onto the aluminium sait having percentage adsorption of at least 50%.

15. The composition as claimed in claim 12, wherein the T antigen is adsorbed onto lhe aluminium sait having a percentage adsorption of at least 40%.

16. Ί he composition as claimed in claim 12, wherein the HBsAg antigen is adsorbed onto the aluminium sait having a percentage adsorption of al least 50%.

17. The composition as claimed in claim 1, wherein the composition is a single dose free of preservatives.

18. The composition as claimed in claim 1, wherein the composition is a multi-dose composition comprising one or more preservatives, wherein the one or more preservatives is selected from the group consisting of 2-phenoxyethanol, methylparaben, propylparaben, and combinations thereof.

19. The composition as claimed in claim 18, wherein the one or more preservatives is:

i. 2-Phenoxy éthanol in an amount of 1 to 6 mg per 0.5 ml (v/v) and methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v);

ii. 2-Phenoxyethanol in an amount of 1 to 6 mg per 0.5 ml(v/v)and propylparaben in an amount of 0.05 - 0.2 mg per 0.5 ml (w/v);

îii. 2-Phenoxyethanol in an amount of 1 to 6 mg per 0.5 ml(v/v), methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v) and propylparaben in an amount of 0.05 0.2 mg per 0.5 ml (w/v); and iv. 2-phenoxyethanol in an amount of 1 to 6 mg (v/v) per 0.5ml as preservative.

20. The composition as claimed in claim 1, wherein the composition comprises a buffer selected from the group consisting of carbonate, phosphate, acetate, succinate, borate, citrate, lactate, gluconate, tartrate, MEM, TRIS, HEPES, piperazine-N, Ν'-bis (PIPES), 2-ethanesulfonic acid (MES) and sodium chloride in a concentration of 0.5% to 1.5%.

21. The composition as claimed in claim 1, wherein the composition further comprises one or more of a pharmaceutically acceptable transporter, excipient, binder, carrier, isotonie agent, emulsifier or humectant.

22. The composition as claimed in claim 21, wherein the pharmaceutically acceptable excipient is selected from the group consisting of sugars, polyols, surfactants, polymers, salts, amino acids and pH modifiers.

23. The composition as claimed în claim 22, wherein the amino acid is L-Histidine, in a concentration of 0.5 - 5 mg per 0.5ml.

24. The composition as claimed in claims 2 and 5, wherein the 0.5 ml of the composition comprises: the D antigen in an amount of 22.5 Lf; the T antigen in an amount of 7.5 Lf; the wP antigen in an amount of 15 IOU; the HBsAg in an amount of 12.5 pg; the Hib antigen in an amount of 10 pg; IPV Type 1 antigen (Mahoney strain) in an amount of 5, 7.5, 8 or 10 DU, IPV Type 2 antigen (MEF-1 strain) in an amount of 2 or 16 DU, and IPV Type 3 antigen (Saukett strain) in an amount of 5,10, 12 or 16 DU; a total aluminum content (AP⁺) not more than 0.9 mg; 2-Phenoxyethanol in an amount of 3.25 mg; and L-Histidine in an amount of 1.55 mg.

25. The composition as claimed in claim 24, wherein the IPV Type 1 antigen (Mahoney strain) is in an amount of 10 DU, the IPV Type 2 antigen (MEF-1 strain) is in an amount of 2 DU, and the IPV Type 3 antigen (Saukett straîn) îs in an amount of 10 DU.

26. The composition as claimed in claim 24, wherein the IPV Type 1 antigen (Mahoney straîn) is in an amount of 10 DU and the IPV Type 3 antigen (Saukett strain) is in an amount of 10 DU.

27. The composition as claimed in claims 2 and 5, wherein the 0.5 ml of the composition comprises: the D antigen in an amount of 22.5 Lf; the T antigen in an amount of 7.5 Lf; the wP antigen in an amount of 15 IOU; the HBsAg in an amount of 12.5 pg; the Hib antigen in an amount of 10 pg; IPV Type 1 antigen (Sabin strain) in an amount of 2.5 or 5 or 7.5 DU, IPV Type 2 antigen (Sabin strain) in an amount of 8 or 16 DU, and IPV Type 3 antigen (Sabin strain) in an amount of 5 or 10 DU; a total aluminum content (Al³⁺) not more than

0.9 mg;2-Phenoxyethanol in an amount oi 3.25 mg; and L-Histidine in an amount of 1.55 mg.

28. The composition as claimed in claim 27, wherein the IPV Type 1 antigen (Sabin strain) is in an amount of 5 DU, the IPV Type 2 antigen (Sabin strain) is in an amount of 16 DU, and the IPV Type 3 antigen (Sabin strain) is in an amount of 10 DU.

29. The composition as claimed in claim 27, wherein the IPV Type 1 antigen (Sabin strain) is in an amount of 5 DU and the IPV Type 3 antigen (Sabin strain) is in an amount of 10 DU.

30. A process of manufacturing a dose reduced Inactivated Polio vaccine (IPV) composition comprising:

(i) an inactivated polio virus antigen selected from the group consisting of IPV Type 1 at a dose less than 15 D-antigen units (DU), IPV Type 2 at a dose less than 18 D-antigen unit (DU), and IPV Type 3 at a dose less than 17 D-antigen unît (DU), per 0.5 ml;

(ii) a diphtheria toxoid, (D) antigen in an amount of 1 to 50 Lf per 0.5 ml;

(iii) a tetanus toxoid, (T) antigen in an amount of 1 to 30 Lf per 0.5 ml;

(iv) a whole cell pertussîs, (wP) antigen in an amount of 1 to 50 IOU per 0.5 ml or acellular pertussis, (aP) comprising one or more of modified adenylate cyclase, Pertussîs toxoid (PT) l-50pg, Filamentous hemagglutînin (FHA) l-50pg, Pertactin (P69 or PRN) l-20pg or Fimbrial proteins (FIM 1,2 and 3) 2-25pg; per 0.5ml;

(v) a hepatîtis B virus surface antigen, (HBsAg) în an amount of 1 to 20 pg per 0.5 ml; and (vi) a Haemophilus influenzae type b antigen, (Hib) in an amount of 1 to 20 pg per 0.5 ml; the process comprising the steps of:

a) adsorbing the IPV (Sabin/Salk strain) bulk indîvîdually on aluminium hydroxide, followed by pH adjustment to 6.2 - 6.6, more preferably 6.5;

b) adsorbing the D on aluminium phosphate, followed by pH adjustment to 5.5 - 6.5;

c) adsorbing the T on aluminium phosphate, followed by pH adjustment to 5.5 - 6.5;

d) adsorbing the HBsAg on aluminium phosphate, followed by pH adjustment to 6.0 6.5;

e) blending a mixture obtained from steps b), c), and d) by agitation at room température for 18 - 24 hours;

f) blending a content of step a) with the mixture obtained in step e), followed by pH adjustment to 6.4 - 6.6 and agitation at room température for 60 minutes;

g) adding an inactivated wP antigen/acellular pertussis antigen and a stabilizer (Histidine amino acid buffer solution 100-300mM) to the mixture in step f), followed by agitation for 60 minutes and left in static condition for overnight at 2 - 8 °C;

h) adding the Hib antigen to lhe mixture obtained in step g) at 2 - 8 °C, followed by pH adjustment to 6.4 - 6.6; and

i) adjustîng the pH to 6.0 to 7.0 with sodium hydroxide / sodium carbonate and adding normal saline (0.9% NaCI) or WFI (q.s.) to inake up a volume of the mixture obtained in step h), followed by agitation for 2 hours.

31. The process as claimed in claim 30, wherein the process further comprises adding a preservalive to the mixture obtained in step h) selected from the group consisting of:

i. 2-Phenoxyethanol in an amount of 1 to 6 mg per 0.5 ml (v/v) and methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v);

ii. 2-Phenoxyéthanol in an amount of 1 to 6 mg per 0.5 ml(v/v)and propylparaben in an amount of 0.05 — 0.2 mg per 0.5 ml (w/v);

iii. 2-Phenoxyéthanol in an amount of 1 to 6 mg per 0.5 ml(v/v), methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v) and propylparaben in an amount of 0.05 0.2 mg per 0.5 ml (w/v); and iv. 2-phenoxyethanol in an amount of 1 to 6 mg (v/v) per 0.5ml as preservatîve.

⁶⁴

32. The process as claimed in claim 30, wherein i) the purified diphtheria toxoid and the tétanos toxoid is obtained using Gel Perméation chromatography and stabiiized using an amino acid buffer solution (histidine 100- 300mM)have a monomeric content of at least 80%, ii) the Hib PRP - carrier protein conjugate is prepared using a cyanylation conjugatîon 5 process and subsequently blended at low température in presence of an excipient shows greater stability with minimum free PRP release and improved immunogenicity, and iii) the whole cell pertussis antigen is added at a later stage in a blend minimizes hydrolysis based dégradation and provides a stable and immunogenic wP antigen.