FIELD OF THE INVENTION

The present invention relates to a combination of preservatives for vaccine composition or formulation, more particularly a combination of 2-phenoxyethanol and formaldehyde.

BACKGROUND OF THE INVENTION

Preservatives are generally used in multi-dose presentations of vaccines to prevent contamination due to repeated withdrawal of the vaccine doses from the container. However, it does not preclude the use of preservative in the single dose presentation of the vaccine.

A preservative which is commonly used in pharmaceutical preparations, including vaccines, is 2- phenoxyethanol or 2-PE {Meyer, Brian K. et al. Journal of Pharmaceutical Sciences (2007) 96: 3155- 3167). One of the reasons prompting the use of 2-PE in several of the DTP combination vaccines probably has to do with the presence of IPV antigen in them.

W02008020322 describes a process of making a combination vaccine comprising diphtheria and tetanus toxoid with 2-PE and mixing these components with another antigen.

While 2-PE has been found to be a better alternative to thimerosal, particularly when polio antigen is one of the components, because of the deleterious effect of thimerosal on the polio antigen {Sawyer, Leigh A. et al. Vaccine (1994) 12: 851-856), but 2-PE alone has been found to be a weak preservative.

WO1998034594 discloses a preservative combination having 2-PE along with methyl and propyl paraben.

W02020021416 discloses a preservative system which is free of thimerosal and comprising 2-PE and at least one other preservative selected form m-cresol, benzyl alcohol, phenol and benzoic acid.

Hekkens, F.E.N. et al. (Journal of Biological Standardization (1981) 9: 277-285) discloses a preservative combination having 2-PE and formaldehyde. Hekkens, F.E.N. et. al. used 0.5% 2-PE in combination with formaldehyde at either 25, 50, 75 pg/mL and recommends such a combination to be employed for polio antigen containing combination vaccines. W02017048038 discloses a preservative combination having 2-PE along with formaldehyde at a concentration of at least 7 mg/mL and 120 pg/mL respectively as an effective preservative concentration.

IN201917040656 discloses a polyvalent pneumococcal vaccine composition comprising a preservative combination having at least 4 mg/mL 2-PE and at least 90 pg/mL formaldehyde.

In the prior art there has been a trend to increase the concentration of 2-PE or formaldehyde, more particularly formaldehyde, for the combination to act as effective preservative.

However, increasing the concentration of preservatives could have negative impact on the antigens present in the vaccine composition. Formaldehyde has been known to cross link proteins, and increasing the concentration of formaldehyde may compromise the stability and immunogenicity of the antigens contained in the vaccine composition.

Therefore, there is a need to have a preservative combination at concentration which is effective and which also does not adversely affect the stability and/or immunogenicity of the antigens.

SUMMARY OF THE INVENTION

Accordingly, the invention relates to a vaccine composition comprising 2-phenoxyethanol (2-PE) and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025%.

In one of the embodiments, the invention relates to a vaccine composition comprising 2-phenoxyethanol (2-PE) and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%.

In one of the embodiments, the invention relates to a vaccine composition comprising 2-phenoxyethanol (2-PE) and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0024%, and the vaccine composition is presented either in a single dose container or a multi dose container.

In one of the embodiments, the invention relates to a vaccine composition comprising 2-phenoxyethanol (2-PE) and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, and wherein the vaccine composition comprises one or more antigens. In one of the embodiments, the invention relates to a vaccine composition comprising 2-phenoxythanol (2-PE) and formaldehyde, wherein the 2-PE is present in an amount of 0.4% to 0.7% and the formaldehyde is present in an amount from 0.0002% to 0.0024%.

In one of the embodiments, the invention relates to a vaccine composition comprising 2-phenoxy ethanol (2-PE) and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0024%, and wherein the vaccine composition comprises at least one antigen selected from the group comprising of diphtheria (D), tetanus (T), pertussis (P), hepatitis B (HepB), Haemophilus influenzae type b (Hib), polio (IPV), hepatitis A virus (HAV), Streptococcus pneumoniae (SP), Neisseria meningitidis (NM), rotavirus (RV), flavivirus (FV), human papillomavirus (HPV).

In one of the embodiments, the invention relates to a vaccine composition comprising 2-phenoxy ethanol (2-PE) and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0024%, and wherein the vaccine composition comprises antigens selected from the group comprising of diphtheria (D), tetanus (T), pertussis (P), hepatitis B (HepB), Haemophilus influenzae type b (Hib), and polio (IPV).

In one of the embodiments, the invention relates to a vaccine composition comprising 2-phenoxy ethanol (2-PE) and formaldehyde, wherein the 2-phenoxyethanol is present in an amount from 0.4% to 0.7% and the formaldehyde is present in an amount from 0.0002% to 0.0024%, and wherein the vaccine composition comprises antigens selected from the group comprising of diphtheria (D), tetanus (T), pertussis (P), hepatitis B (HepB), Haemophilus influenzae type b (Hib), and polio (IPV).

In one of the embodiments, the invention relates to a vaccine composition comprising 2-phenoxy ethnaol and formaldehyde, wherein the formaldehyde is present in an amount of 0.0002% to 0.0014% and the 2-phenoxyethanol is present in an amount from 0.5% to 0.6%, and wherein the vaccine composition comprises antigens selected from the group comprising of diphtheria (D), tetanus (T), whole cell pertussis (wP), hepatitis B (HepB), Haemophilus influenzae type b (Hib), and polio (IPV).

The invention also relates to a method of preparing a vaccine composition comprising 2- phenoxyethanol, formaldehyde in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, wherein the method comprises a step of adding 2-phenoxyethanol, formaldehyde, or both, exogenously to the vaccine composition.

The invention also relates to a method of preparing a vaccine composition comprising 2- phenoxyethanol, formaldehyde in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, wherein the method comprises a step of adding 2-phenoxyethanol, formaldehyde, or both, endogenously to the vaccine composition.

In one of the embodiments, the invention relates to a method of preparing a vaccine composition comprising 2-phenoxyethnaol, formaldehyde in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, wherein the method comprises a step of adding partial amount of 2-phenoxyethanol, formaldehyde, or both, endogenously to the vaccine composition.

In one of the embodiments, the invention relates to a method of preparing a vaccine composition comprising 2-phenoxyethanol, formaldehyde in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, wherein the method comprises a step of adding partial amount of 2-phenoxyethnaol, formaldehyde, or both, exogenously to the vaccine composition.

The invention also relates to a method of preparing a vaccine composition comprising 2- phenoxyethanol, formaldehyde in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, wherein the method comprises a step of adding 2-phenoxyethanol, formaldehyde, or both, either exogenously or endogenously, to the vaccine composition, wherein the method further comprises a step of adding at least one antigen selected from the group comprising of diphtheria (D), tetanus (T), pertussis (P), hepatitis B (HepB), Haemophilus influenzae type b (Hib), polio (IPV), hepatitis A virus (HAV), Streptococcus pneumoniae (SP), Neisseria meningitidis (NM), rotavirus (RV), flavivirus (FV), and human papillomavirus (HPV).

The invention also relates to a method of preparing a vaccine composition comprising 2- phenoxyethanol, formaldehyde in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, wherein the method comprises a step of adding partial amount of 2-phenoxyethanol, formaldehyde, or both, either exogenously or endogenously, to the vaccine composition, wherein the method further comprises a step of adding at least one antigen selected from the group comprising of diphtheria (D), tetanus (T), pertussis (P), hepatitis B (HepB), Haemophilus influenzae type b (Hib), polio (IPV), hepatitis A virus (HAV), Streptococcus pneumoniae (SP), Neisseria meningitidis (NM), rotavirus (RV), flavivirus (FV), and human papillomavirus (HPV). DETAILED DESCRIPTION OF THE INVENTION

The singular forms "a", "an" and "the" as used in the specification also include plural aspects unless the context dictates otherwise. Similarly, any singular term used in the specification also mean plural or vice versa unless the context dictates otherwise.

It must be noted that the words “comprising” or any of its forms such as “comprise” or “comprises”, “having” or any of its forms such as “have” or “has”, “including” or any of its forms such as “include” or includes”, or “containing” or any of its forms such as “contain” or “contains” are open-ended and do not exclude additional unrecited elements or method steps.

Wherever any quantity or range is stated one skilled in the art will recognize that quantity or range within 10 or 20 percent of the stated values can also be expected to be appropriate and reasonable and included within the scope of the invention.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, protein, adjuvant, pharmaceutical biotechnology, and biopharmaceutical manufacturing described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed or as described throughout the present specification.

The term “formulation” or “composition” has been used interchangeably to mean a composition comprising one or more antigens and/or one or more excipients.

The term “antigen” as used herein means a component of a formulation or composition which is responsible for stimulating an immune response i.e., generation of antibodies against it when administered to an animal or human.

The term “excipient” as used herein means a component of a composition or formulation other than an antigen.

The term “container” as used herein means a receptacle for holding the vaccine composition, for example, a vial or a syringe or an analogous device. The amount of 2-phenoxyethanol and/or formaldehyde present in the vaccine composition or formulation is typically the amount present in a dose of the vaccine composition. The term “dose” as used herein means a volume of dose that is targeted to be administered to a subject such as an animal or human subject, and does not include any excess fill volume or overages that may be added to the targeted dose. In accordance with the invention, suitable dosage volumes may be in a range between 0.1 mL to 1.0 mL, 0.2 to 0.8 mL, 0.3 to 0.6 mL or preferably 0.5 mL.

The term “not adsorbed” or “un-adsorbed” or any variant thereof as used herein means there is no specific attempt made to adsorb an agent, such as an antigen, onto an adjuvant. However, it does not exclude any adsorption of an antigen onto an adjuvant that may happen without any deliberate steps being taken i.e., when the antigens are added to the formulation not intended to be adsorbed, or during the storage of the vaccine composition.

The term “endogenous” or “endogenously” or any variant thereof as used herein in the context of preservatives (i.e., 2-phenoxyethanol or formaldehyde) denotes the presence or inclusion or addition of one or both of the preservatives during the manufacturing process of one or more antigens or their addition to one or more of the bulk antigens in total or partial amounts in accordance with the invention. For example, one or both of the preservatives may be totally or partially contributed by the manufacturing process of one or more antigens or present with one or more of the bulk antigens, and consequently may or may not require, depending upon the amount present, exogenous addition of one or both of the preservatives to the vaccine composition.

The term “exogenous” or “exogenously” or any variant thereof as used herein in the context of one or both of the preservatives (i.e., 2-phenoxyethanol or formaldehyde) denotes the addition of one or both of the preservatives to one or more antigens or mixture of antigens at the time of preparing the vaccine composition.

The term “addition” “inclusion” “adding” or “added” or any variant thereof as used herein means the step of combining or mixing one component with another component of the vaccine composition i.e., combining or mixing one or both of the preservatives, one or more antigens, or any other excipient(s) in no particular order. For example, when component “X” is added to component “Y” it not only means addition of component “X” to “Y”, but also vice versa.

The amount of preservative contained in the composition or formulation is expressed either as v/v or w/v and is apparent to a person skilled in the art. Vaccine Composition

The invention provides a vaccine composition comprising 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025%.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0024%.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.002%.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0018%.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0016%.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0014%.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0012%.

The invention provides a vaccine composition comprising 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025%.

In an embodiment, the formaldehyde is present in an amount of less than 0.0025%, equal to or less than 0.0018%, equal to or less than 0.0016%, equal to or less than 0.0014%, equal to or less than 0.0013%, or equal to or less than 0.00125%.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0024%.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.002%.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0018%. In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0016%.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0014%.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0012%.

In an embodiment, the 2-phenoxyethanol is present at a concentration of 0.5-0.6%.

The vaccine composition comprising 2-phenoxyethanol and formaldehyde of the invention may be used for a variety of vaccine compositions, viz., monovalent or multivalent (combination) vaccines. The term “monovalent” herein means a vaccine composition comprising one or more antigens derived from a single species or targeting the same disease, whereas the term “multivalent’ herein means a vaccine composition comprising two or more antigens derived from different species or targeting two or more diseases.

Such monovalent and multivalent vaccines, and their method of preparation is well known in the art. A variety of antigens may be included in the vaccine composition in accordance with the invention.

Diphtheria (D)

Diphtheria antigen is typically diphtheria toxoid which can be obtained from Corynebacterium diphtheriae. The method of culturing Corynebacterium diphtheriae, purification of diphtheria toxin and its inactivation or detoxification to obtain diphtheria toxoid are well known to a person skilled in the art (Manual for the production and control of vaccines: diphtheria toxoid, World Health Organization, B LG/UNDP/77.1 Rev.l (1977); US3135662; W02006100108). In brief, diphtheria toxoid is prepared by growing a strain of Corynebacterium diphtheriae in a fermenter containing suitable medium. The diphtheria toxin produced by the bacterium is extracted and purified, followed by its inactivation by treating with chemicals such as formaldehyde or glutaraldehyde to obtain diphtheria toxoid. In an alternative, the diphtheria toxin after extraction may be first inactivated and then purified to obtain the diphtheria toxoid.

In one of the embodiments, diphtheria toxoid is present at an amount of 5-50 Lf, 10-30 Lf, or 10-20 Lf per dose, preferably as a 0.5 mL dosage volume. In one of the embodiments, diphtheria toxoid is adsorbed onto an aluminium adjuvant, either aluminium hydroxide or aluminium phosphate or a mixture of both. In one of the embodiments, diphtheria toxoid is a component of the monovalent or multivalent vaccine composition.

Tetanus (T)

Tetanus antigen is typically tetanus toxoid which can be obtained from Clostridium tetani. The method of culturing Clostridium tetani, purification of tetanus toxin and its inactivation or detoxification to obtain tetanus toxoid are well known to a person skilled in the art (Manual for the production and control of vaccines: tetanus toxoid, World Health Organization BLG/UNDP/77.2 Rev.l (1977); Fratelli, Fernando et al. Biotechnology Progress (2010) 26: 88-92; US6060067). In brief, tetanus toxoid is prepared by growing a strain of Clostridium tetani in a fermenter containing suitable medium. The tetanus toxin produced by the bacterium is extracted and purified, followed by its inactivation by treating with chemicals such as formaldehyde or glutaraldehyde to obtain tetanus toxoid. In an alternative, the tetanus toxin after extraction may be first inactivated and then purified to obtain the tetanus toxoid.

In one of the embodiments, tetanus toxoid is present at an amount of 2-30 Lf, 5-20, 5-15 Lf or 5-10 Lf per dose, preferably as a 0.5 mL dosage volume. In one of the embodiments, tetanus toxoid is adsorbed onto an aluminium adjuvant, either aluminium hydroxide or aluminium phosphate or a mixture of both. In one of the embodiments, tetanus toxoid is a component of the monovalent or multivalent vaccine composition.

Pertussis (P)

Pertussis antigen is obtained from Bordetella pertussis and can either be cellular (whole cell pertussis - wP) or acellular (purified pertussis components or acellular pertussis - aP). Preparation of whole cell pertussis (wP) is well known in the art. It is generally produced by culturing a strain of Bordetella pertussis and inactivating or detoxifying the wP by heat or chemicals or a combination of both (Manual for the production and control of vaccines: pertussis vaccine, World Health Organization, B LG/UNDP/77.3 Rev.l (1977); Gupta, R.K. et al. Vaccine (1986) 4: 185-190; J Biol Stand vl7 p87-98 1987; Gupta, R.K. et al. Vaccine (1988) 6: 491-496; Thalen, Marcel et al. Vaccine (2008) 26: 653-663; W02006002502). If chemicals are used to inactivate wP, it is preferable that formaldehyde, glutaraldehyde, acetone or their combination is used. Thimerosal is recommended to be avoided. However, if thimerosal is used for inactivation of wP, the residual thimerosal may be removed by treating the suspension of wP with compounds such as EDTA, or cysteine.

In one of the embodiments, wP is inactivated by heat. In another embodiment, wP is inactivated by chemicals, preferably formaldehyde. In one of the embodiments, wP is inactivated preferably by glutaraldehyde. In one of the embodiments, wP is inactivated by a combination of heat and chemicals. In one of the embodiments, wP is present at an amount of 5-50 IOU, 10-40 IOU, or 10-25 IOU per dose, preferably as a 0.5 mL dosage volume. In one of the embodiments, whole cell pertussis (wP) is a component of the monovalent or multivalent vaccine composition.

Acellular pertussis (aP) antigen may comprise one or more immunogenic components from Bordetella pertussis such as pertussis toxoid (PT), filamentous haemagglutinin (FHA), pertactin (PRN), fimbrial antigens such as Fim 2, & 3. Acellular pertussis components may be obtained by culturing a strain of Bordetella pertussis, followed by their purification and inactivation. Such methods are well known in the art (W098/00167).

In one of the embodiments, pertussis toxoid is present at an amount of 2-50, 5-40, 10-30 microgram per dose, preferably as a 0.5 mL dosage volume. In another embodiment, FHA is present at an amount of 2-50, 5-40, 10-30 microgram per dose, preferably as a 0.5 mL dosage volume. In one of the embodiments, PRN is present at an amount of 1-10, 2-8, 2-6, microgram per dose, preferably as a 0.5 mL dosage volume. In one of the embodiments, Fim2 and Fim3 are present at an amount of 1-10, 2-8, 2-7 microgram per dose, preferably as a 0.5 mL dosage volume. In one of the embodiments, pertussis components may be adsorbed onto aluminium adjuvant, either aluminium hydroxide or aluminium phosphate or a mixture of both. In one of the embodiments, one or more aP components are part of the monovalent or multivalent vaccine composition.

Haemophilus influenzae type b (Hib)

Hib antigen used in the present invention is a capsular polysaccharide of Haemophilus influenzae type b conjugated to carrier protein. Methods of preparing such polysaccharide protein conjugates are well known to a person skilled in the art. The polysaccharide protein conjugate may be prepared by covalently attaching purified capsular polysaccharides to carrier protein using a variety of chemical methods. For example, the polysaccharide protein conjugate may be prepared as described by Schneerson, R. et al. (1980) J. Exp. Med. 152: 361-476; Chit, chiayung et al. (1983) Infection and Immunity 40(1): 245-256. The polysaccharide (PS) is first activated in the presence of cyanogen bromide to generate a cyanate ester. The activated polysaccharide is then linked (derivatized) to the spacer, for example, adipic dihydrazide (AH). It is also possible to directly link spacer without activation of the polysaccharide. The derivatized polysaccharide (PS-AH) is conjugated to the carrier protein using carbodiimides such as l-ethyl-3-(3-dimethylaminopropyl (EDAC or EDC), N,N- Dicyclohexyl carbodiimide (DCC), or N,N-Diisopropyl carbodiimide (DIC). It is also possible to first derivatize the protein, and such derivatized protein may then be conjugated to the polysaccharide as described by Laferriere, Craig (2011) Glycoconjugate Journal 28: 463-472; Silveira, I.A. et al (2007) Vaccine 25: 7261-7270; US 4,496,538). Activation of polysaccharide may also be achieved by organic cyanylating agents such as l-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP), N- cyanotriethylammonium tetrafluoroborate (CTEA), and p_Nitrophenylcyanate (pNPC) as described by Lees, Andrew US5693326; Lees, Andrew et al. (1996) Vaccine 14(3): 190-198. Alternatively, polysaccharides, or fragments thereof, may be activated selectively at their terminal reducing ends by introducing aldehydes which may be directly or indirectly (through a linker or spacer) coupled to carrier protein by reductive amination to obtain the conjugate ( Jennings , H. J. US4356170; P. W. Anderson, et al. (1986) J. Immunol. 137: 1181-1186; Gray GR. (1978) Methods Enzymology; 50: 155-160).

In one of the embodiments, Hib is present in an amount of 2-20, 5-18, 7-15 microgram per dose, preferably as a 0.5 mL dosage volume. In one of the embodiments, Hib may be adsorbed onto an aluminium adjuvant. In another embodiment, Hib is not adsorbed onto an aluminium adjuvant. It is preferable that Hib is not adsorbed onto any adjuvant or remains un-adsorbed onto any adjuvant. However, it is entirely possible that some amount of Hib may get adsorbed onto the adjuvant passively i.e., without any deliberate attempt of adsorbing the Hib antigen. In one of the embodiments, Hib is a component of the monovalent or multivalent vaccine composition.

Hepatitis B (HepB)

Hepatitis B antigen is obtained from Hepatitis B virus and is generally the surface protein of Hepatitis B Virus viz., Hepatitis B surface Antigen (HBsAg). Methods of preparing HBsAg are well known to a person skilled in the art. HBsAg may be obtained by purifying the hepatitis b particle from the plasma of chronic hepatitis B carriers. More commonly, HBsAg is obtained by recombinant DNA technology by expressing the HBsAg gene in an expression system (bacterial, yeast or mammalian expression system). The bacterium or yeast or mammalian cells carrying the HBsAg gene is grown in culture medium and HBsAg is purified through a series of downstream steps viz., precipitation, filtration, chromatography etc ( Miyanohara , A et al. PNAS (1983) 80(1): 1-5.; Cregg J. M. et al. Biotechnology (1987) 5:479-485; Gurramkonda, Chandrasekhar et al. (2009) Microbial Cell Factories 8:13).

In one of the embodiments, HBsAg is present at an amount of 2-20, 5-18, 7-15 microgram per dose, preferably as a 0.5 mL dosage volume. In one of the embodiments, HBsAg is adsorbed onto aluminium adjuvant, either aluminium hydroxide or aluminium phosphate or a mixture of both. In one of the preferred embodiments, HBsAg is adsorbed onto aluminium phosphate adjuvant. In one of the embodiments, HBsAg is a component of the monovalent or multivalent vaccine composition.

Polio (IPV)

Polio antigen is typically inactivated poliomyelitis virus. The poliomyelitis (polio) virus may either be derived from Salk strain (wild strain) or Sabin strain (attenuated strain). Generally, IPV antigen includes three wild strains of Salk polioviruses viz., salk type 1 (Mahoney), salk type 2 (MEF) and salk type 3 (Saukett) or the Sabin strains viz., sabin type 1, sabin type 2 and sabin type 3. These viruses are grown on culture medium, harvested, purified and inactivated individually before being combined as an IPV standalone vaccine. Methods of producing inactivated poliomyelitis virus vaccine are well known in the art. Typically polioviruses are grown in suitable cell lines (VERO or MRC5) on microcarriers in bioreactors containing the appropriate cell culture medium for the growth of the virus. The virus is harvested and purified by means of a several downstream steps such as ultrafiltration, diafiltration, one or more chromatography step, followed by their inactivation. Inactivation of viruses is generally achieved by treatment with formaldehyde, however other agents may also be used. The individual viruses (salk or sabin viruses types) are concentrated and mixed to obtain IPV bulk which may be used to prepare a standalone IPV vaccine or combined with other antigens to prepare a multivalent or combination vaccine (Vidor E. and Plotkin, S.A. (2013) Poliovirus Vaccine (Inactivated), 573-797, In Stanley A. Plotkin, Walter A. Orenstein and Paul A. Offit (ed.), Vaccines, 6 ^th edition, Saunders, ISBN 978-1-4557-0090-5; W098/00167).

In one of the embodiments, the polio antigen comprises salk polioviruses viz., salk type 1 (Mahoney), salk type 2 (MEF) and salk type 3 (Saukett). In another embodiment, the polio antigen comprises sabin polioviruses viz., sabin type 1, sabin type 2, and sabin type 3. In one of the embodiments, salk type 1 poliovirus is present in an amount of 10-50, 15-45, 20-40, 30-40, or more typically 40 DU (D antigen units) per dose, preferably as a 0.5 mL dosage volume. In one of the embodiments, salk type 2 poliovirus is present in an amount of 2-15, 5-10, or more typically 8 DU per dose, preferably as a 0.5 mL dosage volume. In one of the embodiments, salk type 3 poliovirus is present in an amount of 8-40, 15-35, 25- 35 or more typically 32 DU per dose, preferably as a 0.5 mL dosage volume. In one of the embodiments, the salk polioviruses viz., salk type 1, salk type 2 and salk type 3 are present in an amount of 40-8-32 DU respectively per dose, preferably as a 0.5 mL dosage volume.

In one of the embodiments, polioviruses are not adsorbed onto any adjuvant. In another embodiment, the polioviruses are adsorbed onto aluminium adjuvant, either aluminium hydroxide, aluminium phosphate or a mixture of both. In one of the embodiments, IPV is a component of the monovalent or multivalent vaccine composition.

Additional antigens

Additional antigens that may be part of the composition in accordance with the invention are: a hepatitis A virus (HAV) antigen, such as an inactivated hepatitis A virus; a Streptococcus pneumoniae (SP) antigen, such as a saccharide of S. pneumoniae serotype conjugated to carrier protein; a Neisseria meningitidis antigen (NM) antigen, such as a saccharide of N. meningitidis serotype conjugated to carrier protein; a rotavirus (RV) antigen, such as an inactivated rotavirus or an immunogenic protein of rotavirus; a flavivirus (FV) antigen, such as a Japanese encephalitis virus, dengue virus, west nile virus a human papillomavirus virus (HPV) antigen, such as a virus like particle (VLP) of one or more HPV types or an immunogenic protein of HPV ;

In an embodiment, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, and one or more antigens, wherein the formaldehyde is present in an amount of less than 0.0025%.

In an embodiment, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, and one or more antigens selected from the group comprising of diphtheria (D), tetanus (T), pertussis (P), hepatitis B (HepB), Haemophilus influenzae type b (Hib), polio (IPV), hepatitis A virus (HAV), Streptococcus pneumoniae (SP), Neisseria meningitidis (NM), rotavirus (RV), flavivirus (FV), and human papillomavirus (HPV), wherein the formaldehyde is present in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, 0.0002% to 0.0016%, 0.0002% to 0.0014%, or 0.0002% to 0.0012%.

In an embodiment, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, and one or more antigens selected from the group comprising of diphtheria (D), tetanus (T), pertussis (P), hepatitis B (HepB), Haemophilus influenzae type b (Hib), and polio (IPV), wherein the formaldehyde is present in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, 0.0002% to 0.0016%, 0.0002% to 0.0014%, or 0.0002% to 0.0012%.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and a diphtheria (D) antigen, preferably diphtheria toxoid.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and a tetanus (T) antigen, preferably tetanus toxoid.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and a pertussis (P) antigen. In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and a pertussis (P) antigen, preferably whole cell pertussis (wP).

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and a pertussis (P) antigen, preferably one or more components of pertussis i.e., acellular pertussis (aP).

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethnaol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and a Hib antigen, preferably Haemophilus influenzae type b polysaccharide conjugated to a carrier protein.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and a hepatitis B (HepB) antigen, preferably the surface protein of hepatitis B virus such as Hepatitis B surface Antigen (HBsAg).

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and a polio (IPV) antigen, preferably, an inactivated poliomyelitis virus derived from salk strains or sabin strains.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and at least two antigens selected from the group comprising of D, T, P, HepB, Hib, IPV, HAV, SP, NM, RV, FV and HPV.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and at least three antigens selected from the group comprising of D, T, P, HepB, Hib, IPV, HAV, SP, NM, RV, FV and HPV.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and at least four antigens selected from the group comprising of D, T, P, HepB, Hib, IPV, HAV, SP, NM, RV, FV and HPV.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and at least five antigens selected from the group comprising of D, T, P, HepB, Hib, IPV, HAV, SP, NM, RV, FV and HPV.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and at least six antigens selected from the group comprising of D, T, P, HepB, Hib, IPV, HAV, SP, NM, RV, FV and HPV.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and at least seven antigens selected from the group comprising of D, T, P, HepB, Hib, IPV, HAV, SP, NM, RV, FV and HPV.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and at least eight antigens selected from the group comprising of D, T, P, HepB, Hib, IPV, HAV, SP, NM, RV, FV and HPV.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and at least nine antigens selected from the group comprising of D, T, P, HepB, Hib, IPV, HAV, SP, NM, RV, FV and HPV.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and at least ten antigens selected from the group comprising of D, T, P, HepB, Hib, IPV, HAV, SP, NM, RV, FV and HPV.

In one of the embodiments, a vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and at least eleven antigens selected from the group comprising of D, T, P, HepB, Hib, IPV, HAV, SP, NM, RV, FV and HPV. In one of the embodiments, the vaccine composition of the invention comprises 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and D, T, P, HepB, Hib, IPV, HAV, SP, NM, RV, FV and HPV antigens.

In one of the preferred embodiments, a vaccine composition of the invention comprises 2- phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and an antigen selected from the group comprising of diphtheria (D), tetanus (T) and whole cell pertussis (wP).

In one of the preferred embodiments, a vaccine composition of the invention comprises 2- phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and an antigen selected from the group comprising of diphtheria (D), tetanus (T), whole cell pertussis (wP), and hepatitis B (HepB).

In one of the preferred embodiments, a vaccine composition of the invention comprises 2- phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and an antigen selected from the group comprising of diphtheria (D), tetanus (T), whole cell pertussis (wP), hepatitis B (HepB), and Haemophilus influenzae type b (Hib).

In one of the preferred embodiments, a vaccine composition of the invention comprises 2- phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025% or in an amount from 0.0002% to 0.0024% and an antigen selected from the group comprising of diphtheria (D), tetanus (T), whole cell pertussis (wP), hepatitis B (HepB), Haemophilus influenzae type b (Hib), and polio (IPV).

In one of the preferred embodiments, a vaccine composition of the invention comprises 2- phenoxyethnaol and formaldehyde, wherein the formaldehyde is present in an amount of 0.0002% to 0.0014% and the 2-phenoxyethanol is present in an amount from 0.5% to 0.6%, and an antigen selected from the group comprising of diphtheria (D), tetanus (T), whole cell pertussis (wP), hepatitis B (HepB), Haemophilus influenzae type b (Hib), and polio (IPV).

Methods of preparing vaccine compositions according to the invention

The invention is not limited to any specific method of preparation of the vaccine compositions. Methods of preparation of vaccine compositions are well known to a person skilled in the art. There are several ways in which the antigens may be formulated or combined. Some antigens may be pre-adsorbed (separate adsorption), some antigens may be adsorbed during the steps of addition of one or more antigens (sequential adsorption), while some antigens remain un-adsorbed or not adsorbed to an adjuvant. Compositions comprising different antigens may be prepared by any of the methods described in the prior art, for example, the methods described in W093/24148; W02005/089794; W02008/028956;W098/00167; IN 524/KOL/2003; W02010046934; W02010046935;

WO2013107988.

In one of the embodiments, the invention relates to a method of preparing a vaccine composition comprising 2-phenoxyethanol, formaldehyde in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, wherein the method comprises a step of adding 2-phenoxyethanol, formaldehyde, or both, exogenously to the vaccine composition.

In one of the embodiments, the invention relates to a method of preparing a vaccine composition comprising 2-phenoxyethanol, formaldehyde in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, wherein the method comprises a step of adding 2-phenoxyethanol, formaldehyde, or both, endogenously to the vaccine composition.

In one of the embodiments, the invention relates to a method of preparing a vaccine composition comprising 2-phenoxyethnaol, formaldehyde in an amount from 0.0002% to 0.0024%, %, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, wherein the method comprises a step of adding partial amount of 2-phenoxyethanol, formaldehyde, or both, endogenously to the vaccine composition.

In one of the embodiments, the invention relates to a method of preparing a vaccine composition comprising 2-phenoxyethanol, formaldehyde in an amount from 0.0002% to 0.0024%, %, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, wherein the method comprises a step of adding partial amount of 2-phenoxyethnaol, formaldehyde, or both, exogenously to the vaccine composition.

In one of the embodiments, the method comprises a step of adding 2-phenoxyethanol, formaldehyde in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, or both, after adding all the antigens or before adding any of the antigens of the vaccine composition. In another embodiment, the method comprises a step of adding 2-phenoxyethanol, formaldehyde in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, or both, at any intermediate step during the addition of any one of the antigens of the vaccine composition. In yet another embodiment, the method comprises a step of adding 2-phenoxyethanol, formaldehyde in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, or both, pre-mixed with one or more of the antigens of the vaccine composition.

In one of the embodiments, the method comprises a step of adding 2-phenoxyethanol, formaldehyde in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, or both, after the addition of the first antigen or pre-mixed with the first antigen.

Some of the embodiments of the present invention are set out in the following numbered paragraphs:

1. A vaccine composition comprising 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount of less than 0.0025%.

2. A vaccine composition comprising 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.0024%.

3. A vaccine composition comprising 2-phenoxyethanol and formaldehyde, wherein the formaldehyde is present in an amount from 0.0002% to 0.002%.

4. The vaccine composition according to any of the preceding paragraphs, wherein the formaldehyde is present in an amount from 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%.

5. The vaccine composition according to any of the preceding paragraphs, wherein the 2- phenoxyethanol is present in an amount from 0.4% to 0.7%, or preferably 0.5% to 0.6%.

6. The vaccine composition according to any of the preceding paragraphs, wherein the vaccine composition comprises at least one antigen selected from the group comprising of diphtheria (D), tetanus (T), pertussis (P), hepatitis B (HepB), Haemophilus influenzae type b (Hib), polio (IPV), hepatitis A virus (HAV), Streptococcus pneumoniae (SP), Neisseria meningitidis (NM), rotavirus (RV), flavivirus (FV), and human papillomavirus (HPV).

7. The vaccine composition according to paragraph 6, wherein the diphtheria antigen is a diphtheria toxoid in an amount from 5 to 50, 10 to 30, or 10 to 20 Lf.

8. The vaccine composition according to paragraph 6, wherein the tetanus antigen is a tetanus toxoid in an amount from 2 to 30, 5 to 20, 5 to 15, or 5 to 10 Lf.

9. The vaccine composition according to paragraph 6, wherein the pertussis antigen is either whole cell pertussis (wP) or acellular pertussis (aP).

10. The vaccine composition according to paragraph 9, wherein the whole cell pertussis is present in an amount from 5 to 50, 10 to 40, or 10 to 25 IOU.

11. The vaccine composition according to paragraph 6, wherein the hepatitis B antigen is present in an amount from 2 to 20, 5 to 18, or 7 to 15 pg. 12. The vaccine composition according to paragraph 6, wherein the Haemophilus influenzae type b antigen is present in an amount from 2 to 20, 5 to 18, or 7 to 15 pg.

13. The vaccine composition according to paragraph 6, wherein the polio antigen is an inactivated poliomyelitis virus derived from either a salk strain or a sabin strain.

14. The vaccine composition according to paragraph 13, wherein the salk strains are salk type 1 (Mahoney), salk type 2 (MEF), and salk type 3 (Saukett).

15. The vaccine composition according to paragraph 14, wherein the salk type 1 (Mahoney), salk type 2 (MEF) and salk type 3 (Saukett) are present in an amount from 10 to 50 DU, 2 to 15 DU, and 8 to 40 DU respectively.

16. The vaccine composition according to paragraph 15, where in the salk type 1 (Mahoney), salk type 2 (MEF), and salk type 3 (Saukett) are present in an amount of 40 DU, 8 DU and 32 DU respectively.

17. The vaccine composition according to any of the preceding paragraphs, wherein the vaccine composition comprises the diphtheria antigen in an amount from 10 to 20 Lf, the tetanus antigen in an amount from 5 to 10 Lf, the whole cell pertussis antigen in an amount from 10 to 25 IOU, the hepatitis B antigen in an amount from 7 to 15 pg, the haemophilus influenzae type b antigen in an amount from 7 to 15 pg, and the polio antigen comprising salk type 1 (Mahoney), salk type 2 (MEF), and salk type 3 (Saukett) in an amount of 10 to 50 DU, 2 to 15 DU and 8 to 40 DU respectively.

18. A method of preparing a vaccine composition comprising 2-phenoxyethanol, formaldehyde in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, wherein the method comprises a step of adding 2-phenoxyethanol, formaldehyde, or both, endogenously to the vaccine composition.

19. A method of preparing a vaccine composition comprising 2-phenoxyethanol, formaldehyde in an amount from 0.0002% to 0.0024%, 0.0002% to 0.002%, 0.0002% to 0.0018%, preferably 0.0002% to 0.0016%, or more preferably 0.0002% to 0.0014%, wherein the method comprises a step of adding 2-phenoxyethanol, formaldehyde, or both, exogenously to the vaccine composition.

20. The method of preparing a vaccine composition comprising 2-phenoxyethnaol and formaldehyde according to paragraph 18, wherein the method comprises a step of adding partial amount of 2-phenoxyethanol, formaldehyde, or both, endogenously to the vaccine composition.

21. The method of preparing a vaccine composition comprising 2-phenoxyethnaol and formaldehyde according to paragraph 19, wherein the method comprises a step of adding partial amount of 2-phenoxyethnaol, formaldehyde, or both, exogenously to the vaccine composition.

22. The method according to paragraphs 18 to 21, wherein the method further comprises a step of adding at least one antigen selected from the group comprising of diphtheria (D), tetanus (T), pertussis (P), hepatitis B (HepB), Haemophilus influenzae type b (Hib), polio (IPV), hepatitis A virus (HAV), Streptococcus pneumoniae (SP), Neisseria meningitidis (NM), rotavirus (RV), flavi virus (FV), human papillomavirus (HPV). Examples Example - 1

Antimicrobial Effectiveness Test (AET)

The procedure described in European Pharmacopoeia (Ph Eur) chapter 5.1.3 was followed for testing the preservative efficacy. In brief, the preparation was challenged with lxlO ⁵ to lxlO ⁶ CFU/mL of each microorganism -Escherichia coli (ATCC No. 8739), Pseudomonas aeruginosa (ATCC No. 9027), Staphylococcus aureus (ATCC No. 6538), Candida albicans (ATCC No. 10231) and Aspergillus brasiliensis (ATCC No. 16404). Challenged preparations were incubated at 22.5+2.5°C and viable counts were measured at 24 hours, 7 ^th day, 14 ^th day and 28 ^th day after incubation.

Test results were evaluated against the acceptance criteria described in Ph Eur 0153 -

• Bacteria - no increase at 24 h and 7 days, 3 logio reduction at 14 days, no increase at 28 days;

• Fungi - no increase at 14 days and 28 days. Table 1: AET test data for the batches manufactured with different concentration of formaldehyde and 2-phenoxyethanol (2-PE)

Example - 2

Stability tests on Hexavalent vaccine composition (0.5 mL)

Acceptance criteria for test parameters

1. The lower confidence limit (P=0.95) of the estimated potency is not less than 30 IU/SHD (Ref: IP2018/T7z Eur 2.7.6/WHO TRS 980 Annex 4).

2. The lower confidence limit (P=0.95) of the estimated potency is not less than 60 IU/SHD (Ref: IP2018/P/Z Eur 2.7.8/WHO TRS 980 Annex 5).

3. The estimated potency is not less than 4.0 IU/SHD (Ref: IP2018// ^J /z Eur 2.7.7/WHO TRS 941 Annex 6).

4. The upper confidence limit (P= 0.95) of the estimated relative potency is not less than 1.0 (Ref: IP2018/P/Z. Eur. 2.7.15/WHO TRS 978 Annex 4).

5. At least 50% of mice should show seroconversion (Ref: Ph Eur 01/2019:1219)

6. Type 1: 40 DU/dose; Type 2: 8 DU/dose; Type 3: 32 DU/dose when measured through a suitable immunochemical assay (Ref: IP2018// ^J /z Eur 2.7.1/WHO TRS 910 Annex 2). The variability between the theoretical D antigen units (DU) 40:8:32 for type 1, type 2, and type 3 respectively and the reported DU for three IPV types in the table above are within in the analytical variability for D antigen ELISA and are considered indicative of stable composition. Note:

NA = Not Applicable / Not Available IU = International Units SHD = Single Human Dose

UCL = Upper Confidence Limit