EA043311B1 - COMBINED VACCINE COMPOSITION CONTAINING A REDUCED DOSE OF INACTIVATED POLIO VIRUS AND METHOD FOR ITS OBTAINING - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to the field of biotechnology, more specifically it relates to a method for producing a multi-dose combination vaccine composition containing a group of antigens/immunogens and a preservative. The present invention also relates to an improved methodology in the field of combination vaccine production.

State of the art

A combination vaccine that can provide immunogenicity against a large number of diseases always has an advantage over a monovalent vaccine because it reduces the number of injections administered, reduces complications associated with multiple intramuscular injections, reduces administration and production costs, reduces storage costs, and reduces the risk of delayed or missed vaccinations and improves patient compliance with vaccination by reducing the number of individual vaccinations. In addition, fully liquid combination vaccine preparations have clear advantages over formulations that require reconstitution. The average preparation time for a completely liquid vaccine was found to be almost half that of a non-full liquid vaccine. Almost all medical personnel (97.6%) stated that they would prefer to use a completely liquid vaccine in their daily practice (see Soubeyrand B. et al., Assessment of preparation time with fully-liquid versus non-fully liquid pediatric hexavalent vaccines A time and motion study; Vaccine 2015;33:3976-82).

Currently known and available combination vaccines may not contain suitable formulations of the respective antigens in the appropriate immunogenic forms to achieve the desired levels of safety, efficacy and immunogenicity in a susceptible human population for a range of diseases in a single administration. The number of different vaccine combinations that can be created with just a few additional antigens is significant. By adding 1 to 4 other antigenic components (eg, HIB (lyophilized or liquid), HBV, IPV, HAV) to DTwP or DTaP, 44 possible different vaccine combinations can be obtained. This number would increase into the thousands if individual components from different manufacturers were considered. Since each individual new combination vaccine (given the differences in components depending on the source) must be developed separately to demonstrate safety, stability, compatibility and effectiveness, the development of all these vaccines becomes a complex task.

Antigens of the combined vaccine Antigens of diphtheria and tetanus

Diphtheria and tetanus are acute infections caused by Corynebacterium diphtheriae and Clostridium tetani, respectively. In both cases, the cause of clinical disease is a powerful exotoxin from these bacteria. Vaccines that provide protection against these bacteria contain toxins that are chemically modified to form toxoids, a chemically modified toxin that is no longer toxic but is still antigenic. Diphtheria and tetanus toxins are produced when Corynebacterium diphtheriae and Clostridium tetani are grown in a medium containing bovine extract. Toxins are inactivated using the following treatments, which include heat, UV, formaldehyde/formaldehyde, glutaraldehyde, acetylethylenimine, etc. to produce toxoids [diphtheria toxoid (D) and tetanus toxoid (T)]. Concerns regarding bovine spongiform encephalopathy (BSE), transmissible spongiform encephalopathy (TSE), Creutzfeldt-Jakob disease (CJD and variant CJD) may arise because the animal components used in the culture media containing bovine extract spread through vaccine (see WHO Guidelines on Transmissible Spongiform Encephalopathies in relation to Biological and Pharmaceutical Products; 2003 &EMEA/CPMP/BWP/819/01; 24 April 2001).

Whooping cough antigens

Introduction in the 1940s whole-cell vaccines consisting of chemically and heat-inactivated Bordetella pertussis organisms have led to a dramatic reduction in the incidence of whooping cough caused by B. pertussis.

Whole cell DPT vaccines are generally associated with several local side effects (eg, erythema, swelling, and pain at the injection site), fever, and other mild systemic effects (eg, somnolence, irritability, and anorexia) (see Cody CL, Baraff LJ, Cherry JD, Marcy SM, Manclarck CR; The nature and rate of adverse reactions associated with DTP and DT immunization in infants and children. Paediatrics 1981;68:650-60) and Long SS, DeForest A, Pennridge Pediatric Associates, et al., Longitudinal study of adverse reactions following Diphtheria-tetanus-pertussis vaccine in infant. Paediatrics 1990; 85:294-302). More serious systemic manifestations (eg, seizures {with or without fever} and hypotensive-hyporesponsive episodes) are less common (rate of one per 1,750 doses administered) in children receiving whole-cell DTP vaccine (see Cody CL, Baraff LJ, Cherry JD , Marcy SM, Manclarck CR; The nature and rate of adverse reactions associated with DTP and DT immunization in infants and children. Paediatrics 1981; 68:650-60). Acute encephalopathy is even less common (ratio 0-10.5 cases per million doses administered). Experts agree that

- 1 043311 whole cell pertussis vaccine causes permanent brain damage in some rare cases (see Institute of Medicine; DPT vaccine and chronic nervous system dysfunction, a new analysis;

Washington D.C., National Academy Press, 1994).

Several reports citing the relationship between whole-cell pertussis vaccination, reactogenicity, and serious side effects have led to decreased vaccine acceptance and subsequent resurgence of epidemics (Miller, D.L., Ross, E.M., Alderslade, R., Bellman, M.H., and Brawson, N. S. B. (1981) Pertussis immunization and serious acute neurological illness in children: Brit Med. J. 282: 1595-1599).

Adverse reactions associated with whole-cell pertussis (wP) vaccines are a barrier to their continued use worldwide, so wP-based combination vaccines have gradually been replaced by acellular pertussis combination vaccines in industrialized countries.

More recently, pertussis vaccines with specific components have been developed. Fully liquid hexavalent acellular pertussis vaccines (DTaP IPV PRP-T-HBsAg) have been previously reported (EP 1028750).

Infanrix® Hexa (GSK) is currently the only hexavalent combination vaccine for children containing Salk IPV available on the global market. This product (DTaP3-IPVHBV//Hib) is sold as a syringe pre-filled with the pentavalent product, packaged with lyophilized Hib PRP-T antigen conjugate in a separate vial for reconstitution with the rest of the vaccine before use.

The second hexavalent vaccine, Hexyon® (also called Hexacima® and Hexaxim®), is an all-liquid hexavalent vaccine from Sanofi Pasteur; however, it is also an acellular vaccine. This vaccine will likely be aimed at private markets in Europe and around the world.

Bharat Biotech International is developing a heptavalent combination vaccine which consists of DT, acellular pertussis, Sabin IPV (type 1: 40 DU, type 2: 8 DU, type 3: 32 DU), one strain of inactivated rotavirus (strain G9 i.e. . Strain 116E), conjugate of Haemophilus influenza type b PRP with TT and recombinant vaccine against hepatitis B.

However, there are concerns about the long-term effectiveness of acellular pertussis (aP) vaccines, especially in developing country settings. Recent research suggests that immunity to whooping cough wanes during adolescence and that this is responsible for the increase in cases in infants under six months of age before they are fully vaccinated. Vaccine effectiveness was estimated at 24% in children aged 8 to 12 years immunized in infancy with the aP vaccine. An observational study from Australia also found higher incidence rates among adolescents who received the aP vaccine in infancy compared with those who received the wP vaccine (relative risk 3.3, 95 percent confidence interval 2.4 to 4.5) .

In terms of cost, aP antigens have historically been 10 to 30 times higher than wP antigens due to differences in production and royalty costs and therefore represent an economic burden for developing countries. As a result, the cost of hexavalent wP-based vaccines is better suited for public sector use in low-resource countries.

Consequently, the use of whole-cell pertussis (wP) vaccines in hexavalent vaccines intended for developing countries has become important due to cost and emerging concerns about the long-term effectiveness of aP vaccines, especially in developing country settings.

Compared to the best whole-cell pertussis (wP) vaccines, aP vaccines are not as effective in mass immunization programs (Vickers et al. 2006; Cherry 2012).

Recent studies of outbreaks in highly immunized populations have shown that the duration of protection of aP vaccines is too short (Klein et al., 2012; Misegades et al., 2012), resulting in decreased immunity in older children and adolescents and, consequently, an increase in the number of cases in this age group (Skowronski et al., 2002; Klein et al., 2012). This sharply distinguishes aP vaccines from wP vaccines, which provide protection even into adolescence (Klein et al., 2012). As a result of these shortcomings in countries that switched to the aP vaccine in the 1990s, we now have a generation of children who are not only less protected from whooping cough, but may also be less responsive to booster immunizations because of the vaccine with which the child was primed , may determine its immune response to a later booster vaccination (Podda et al., 1995; Mascart et al., 2007; Sheridan et al., 2012; Liko, Robison and Cieslak 2013; Smits et al., 2013).

One of the most important factors influencing the reactogenicity of wP is the presence of lipooligosaccharide (LOS), an outer membrane endotoxin of bacteria.

Inactivation of toxins in wP vaccines can be carried out by various methods, but no active heat-labile toxin should be detected in the final product. In the mass production process of whole cell pertussis (wP) vaccine, many manufacturers use heat/formaldehyde treatment to inactivate wP toxins. Several reports mention the use of thimerosal to inactivate wP. However, the use of thimerosal causes loss of antigenicity of IPV (Vaccine 1994 Volume 12, No. 9 851-856. Deleterious effect of thimerosal on the potency of inactivated poliovirus vaccine), and therefore in the case of a combination vaccine containing IPV, it may be necessary to To maintain its effectiveness over time, IPV was kept in a separate ampoule from the wP ampoule containing thimerosal, or a change in the method of general initial inactivation of the pertussis pathogen. Some antigens, such as active RT, can also serve as modifiers of the immune response, and significant differences in immune responses to various antigens between different vaccines have been observed (WHO, 1993).

Chemical extraction of LOS resulted in a significant reduction in endotoxin content (20%) and a dramatic reduction in endotoxin-related toxicity (up to 97%), depending on the in vitro or in vivo test used. LOS extraction did not affect product integrity and, more importantly, did not affect the effectiveness and/or stability of DTP. Moreover, almost no differences in antibody and T-cell responses were observed (see Waldely Oliveira Dias et al., An improved whole cell pertussis vaccine with reduced content of endotoxin; Human Vaccines & Immunotherapeutics 9:2, 339-348; February 2012 ).

Hepatitis B antigens

There are different strains of the hepatitis virus. Hepatitis B is a disease caused by the hepatitis B virus (HepB), which attacks the human liver and causes inflammation called hepatitis. The vaccine against this disease contains one of the viral envelope proteins, hepatitis B surface antigen (HBsAg). Vaccines that have been used for mass immunization are now available, such as the Recombivax HB® and Comvax® product from Merck, Engerix-B® and Pediarix® from Glaxo SmithKline Biologicals. A combination vaccine containing a hepatitis B component has been associated with both higher efficacy and higher compliance compared with the single antigen HepB vaccine (see Kurosky et al., Effect of Combination Vaccines on Hepatitis B Vaccine Compliance in Children in the United States; The Pediatric Infectious Disease Journal. 36(7):e189-e196, JUL 2017). Several sources mention the adsorption of hepatitis B surface antigen on aluminum phosphate in combination with other antigens. Hexavac® is a combination vaccine that was withdrawn from the market due to the low immunogenicity of the hepatitis B component. Therefore, there is a need for a combination vaccine composition containing hepatitis B antigen with adequate or enhanced immunogenicity.

Haemophilus influenzae (Hib) antigens

Haemophilus influenzae is a Gram-negative coccobacilli that is a normal part of the flora of the upper respiratory tract. Haemophilus influenzae type b (Hib b) is a leading cause of invasive blood-borne infections in young children and a leading cause of meningitis in the first two years of life. Immunization against Haemophilus influenzae began in Canada in 1987 with a polysaccharide vaccine [polyriboserybitol phosphate (PRP)] Polyriboserybitol phosphate (PRP) capsule Hib is a major virulence factor for the organism. Anti-PRP antibodies are a major contributor to serum bactericidal activity, and increased antibody levels are associated with a reduced risk of invasive disease. PRP is a T cell independent antigen and is therefore characterized by:

a) induction of a weak antibody response in infants and children under 18 months of age,

b) a variable and quantitatively smaller antibody response than that of T-cell-dependent antigens,

c) production of a higher proportion of immunoglobulin M (IgM) and

d) failure to elicit a booster response.

Initial vaccines based only on the PRP component were ineffective in infants. Further efforts have been made to create a PRP conjugate vaccine, in which PRP is conjugated to proteins called carrier proteins, such as Neisseria meningitides outer membrane protein, diphtheria toxoid, tetanus toxoid, and CRM 197. The inclusion of Hib conjugate components in combination vaccines has been associated with decreased immunogenicity of Hib. In addition, Hib conjugates are unstable in an aqueous environment and cannot withstand long-term storage in this form. Consequently, Haemophilus influenzae b (Hib) PRP polysaccharide is often prepared as a dry solid that is reconstituted with a liquid formulation of other antigens before administration. For example, in Infanrix® hexa (WO 99/48525).

Polio antigen

There are different types of vaccine available:

Live attenuated oral polio vaccine (OPV), developed by Dr. Albert Sabin in 1961. OPV containing Sabin strains is administered orally.

Inactivated (killed) polio vaccine (IPV), developed in 1955 by Dr. Jonas Salk. IPV containing Salk strains is given by injection.

Recently, inactivated Sabin poliovirus, which was obtained by inactivating Sabin strains of poliovirus with formaldehyde, was created for injection and has also become available in commercial products.

Both live attenuated (OPV) and inactivated (IPV) polio vaccines have proven effective in controlling polio worldwide. The polio vaccine may contain the Salk or Sabin strains.

In 1955, Dr. Jonas Salk was able to inactivate the wild-type poliovirus, allowing it to be used in an injectable dosage form, and he named it the Salk strain, which includes type 1 Mahoney, type 2 MEF, and type 3 Saukett, which was used in the polio vaccine . Sabin strains include Sabin 1 and Sabin 2 strains.

The currently accepted standard dose of polio vaccine(s) contains 40 units of inactivated poliovirus type 1 D antigen (Mahoney), 8 units of inactivated poliovirus type 2 D antigen (MEF-I), and 32 units of inactivated poliovirus type 3 D antigen (Saukett) e.g. Infanrix -hexa® (WO 99/48525).

IPV is currently available either as a single product without an adjuvant or in various combinations, including DT-IPV (with diphtheria and tetanus toxoids) and hexavalent IPV vaccines (additionally with pertussis, hepatitis B, Haemophilus influenzae b and adjuvant), e.g. Infanrix® hexa (WO 99/48525).

However, compared with OPV, the overall production cost of IPV is significantly higher. This is mainly due to the following reasons:

(i) the need for more virus per dose;

(ii) the need for additional downstream processing (i.e. concentration, purification and inactivation) and associated QC testing;

(iii) loss of antigen or poor recovery during downstream processing and iv) containment.

Until now, financial challenges have been a major barrier to innovation and adoption of IPV in low- and middle-income countries.

In the future, global demand for IPV following poliovirus eradication could increase from the current level of 80 million doses to 450 million doses per year. Therefore, approaches to stretching the supply of IPV are likely to be required.

The present inventors have surprisingly discovered that a reduced dose of IPV provides non-inferior/equivalent protection against polio compared to a standard dose of IPV antigen. Effective reduced-dose vaccine compositions that provide protection against infection using a lower dose of IPV antigen are desirable in situations where the supply of conventional vaccine is insufficient to meet global needs or where the cost of producing a conventional vaccine prevents the vaccine from being marketed at a price affordable to developing countries . Also, exposure to a lower dose of IPV, compared to existing marketed compositions, may be safer. Thus, there is a need to evaluate different strategies to make IPV available at more affordable prices. Therefore, a combination vaccine containing a reduced dose of IPV could make it even cheaper and easier to administer.

In the case of pandemic influenza vaccines, the use of adjuvants has reduced the dose, increased availability, and reduced the cost of the vaccine. It has therefore been hypothesized that an adjuvanted IPV vaccine formulation would reduce the cost as well as increase the number of IPV doses available worldwide.

In addition, aluminum salts, considered safe and already used in combination IPV vaccines, have the least development hurdles and are inexpensive to produce. However, it is known that aluminum adjuvants do not allow significant dose reduction.

In addition, the whole cell pertussis antigen present in the hexavalent vaccine has proven to be a potent immunostimulant. Due to the immunostimulatory effect of aluminum phosphate adjuvant and whole cell pertussis vaccine, the present inventors expect to obtain a good immune response by reducing the dose of IPV.

Other antigens

Other antigens that may be included in the combination vaccine include Haemophilus influenzae (serotypes a, c, d, e, f and non-encapsulated strains), hepatitis (strains A, C, D, E, F and G), Neisseria meningitis A , B, C, W, X, Y, influenza, pneumococcus, streptococcus, anthrax, dengue, malaria, measles, mumps, rubella, BCG, Japanese encephalitis, rotavirus, smallpox, yellow fever, typhoid, shingles, virus chickenpox and others.

The range and type of antigens used in a combination vaccine depend on the age of the target population, such as infants, children under three years of age, children, adolescents and adults. Earliest known combination vaccine that can prevent infection by Bordetella pertussis, Clostridium tetani, Corynebacterium Diphthehae and possibly inactivated poliovirus

- 4 043311 (IPV), and/or hepatitis B virus, and/or Haemophilus influenzae type B infection (see, for example, WO

93/24148, WO 97/00697, WO 2000/030678, WO 2008/028956, US 6013264 and WO 2005089794).

However, the well-documented phenomenon of antigenic competition has complicated and complicated the development of multivalent vaccines. This phenomenon refers to the observation that administration of multiple antigens together often results in a decreased response to certain antigens compared to the immune response to those antigens when administered separately.

Meanwhile, a multi-dose vaccine must contain a preservative to avoid contamination by harmful microorganisms. For vaccines exported to less developed countries, multi-dose preservative-containing vaccines are preferred, taking into account the conditions in the countries where the vaccines will be used, distribution methods, etc. Examples of preservatives used include benzethonium chloride (femerol), thiomersal, phenol, formaldehyde and 2-phenoxyethanol (2-PE), known in the art. Preservatives suitable for vaccines must be environmentally friendly, effective against bacteria, yeasts and other fungi, and must not adversely affect the immunogenic effect of the vaccine.

Thiomersal is an ethylmercury derivative that is widely used as a preservative in many vaccines. Thimerosal is known to prevent the growth of contaminating microorganisms and maintain sterile conditions during vaccine storage or use, and many WHO prequalified (PQ) combination vaccines contain thimerosal as a preservative. However, there have been reports of some allergic reactions (in approximately 16% of the population) to thiomersal, mainly in the form of local delayed-type hypersensitivity reactions, including redness and swelling at the injection site.

In addition, 2-PE is commonly used as a preservative in inactivated polio vaccine instead of thiomersal because the use of thiomersal as a preservative in inactivated polio vaccine is known to reduce the effectiveness of the vaccine by 50% or more within a week, even when refrigerated. (Vaccine 1994 Volume 12 No.9 851 - 856. Deleterious effect of thimerosal on the potency of inactivated poliovirus vaccine).

Combination vaccines (including D, T, wP, Hib, HBsAg and IPV) also use 2-PE at a concentration of 5 mg/ml (WO 2010046934, WO 2008020322 and WO 2012093406).

However, 2-PE was found to have weaker antimicrobial activity than thimerosal against yeasts and fungi in a DPT-based combination vaccine at 2-8°C. Increasing the preservative efficacy of the combination vaccine by increasing the amount of 2PE to meet the required criteria is one possible option. However, increasing concentrations of 2-PE may raise safety concerns in young children who will receive combination vaccines and thus lead to regulatory barriers to the approval of such vaccine(s).

Therefore, it would be advantageous to improve the preservative efficacy of a combination vaccine by combining 2-PE with at least one other preservative that meets safety and regulatory criteria. Examples of preservatives other than 2-PE that can be used include benzethonium chloride (femerol), paraben esters, phenol, formaldehyde, known in the art.

It was found that methyl and propyl parabens, benzyl alcohol passed antimicrobial tests in accordance with USP, BP and EP. Moreover, these preservatives are non-toxic but effective. The toxicity of parabens is relatively low due to the ease and speed with which the body eliminates these drugs. The LD ₅₀ of methylparaben in mice when administered intraperitoneally is 1 g/kg. A mixture of methyl and propyl parabens has never been used in commercial vaccines.

The inventors of the present invention have found that the preservative effectiveness of a mixture of 2-phenoxyethanol and paraben esters (eg, methyl-, propylparabens) is relatively more effective compared to 2-phenoxyethanol alone.

In addition, the present inventors have discovered that the immunogenicity, reactogenicity, stability and maintenance of the correct form of the antigens in the combination vaccine composition depend on the method of formulation, which includes:

a) the process of obtaining individual antigens;

b) sequence of addition of antigens;

c) the use of certain adjuvants in certain quantities for certain antigens;

d) individual adsorption or combined adsorption of antigens on adjuvants, where combined adsorption has its advantages in terms of simplicity of the process, but the disadvantages include that the first pre-adsorbed antigens may be desorbed partially or completely during the addition of subsequent antigens. Antigens added at the last stage may not be completely adsorbed, since previous antigens may saturate the adsorption capacity. Weakly adsorbed antigens may desorb during storage;

- 5 043311

e) degree of antigen adsorption to adjuvants;

f) use of a minimum concentration of alum;

g) use of the optimal concentration and type of preservative;

h) use of various parameters including agitation, temperature and pH.

Objectives of the invention

Some of the objects of the present invention that are satisfied by at least one embodiment are as follows:

The object of the present invention is to solve one or more problems of the prior art, or at least provide a useful alternative.

Another object of the present invention is to provide an all-liquid combination vaccine suitable for preventing and preventing infections caused by diphtheria, tetanus, pertussis, poliomyelitis, Haemophilus influenzae and hepatitis B, or for preventing, ameliorating or delaying the onset or progression of their clinical manifestations.

Another object of the present invention is to provide a fully liquid combination vaccine containing various inactivated poliovirus (IPV) antigens in reduced doses that demonstrates noninferior/equivalent protection against polio compared to a standard dose of IPV antigen.

It is yet another object of the present invention to provide an all-liquid combination vaccine containing at least one paraben, that is, a methyl or propylparaben preservative and 2-phenoxyethanol (2-PE), to improve the preservative efficacy of a multi-dose combination vaccine.

It is yet another object of the present invention to provide an improved method for producing such a combination vaccine composition/formulation wherein the vaccine exhibits improved immunogenicity, reduced reactogenicity, improved stability and furthermore meets the seroprotection criterion for each of said immunogenic components.

Other objects and advantages of the present invention will become more apparent from the following description, which is not intended to limit the scope of the present invention.

The essence of the invention

A combination vaccine composition containing a reduced dose of inactivated poliovirus (IPV) antigen in combination with other antigens/immunogens and at least one paraben ester, i.e. methyl or propylparaben and 2-phenoxyethanol (2-PE), used as a preservative, wherein the preservative efficacy of the multi-dose combination vaccine is improved, and the method for its preparation.

The present invention relates to a combination vaccine composition containing:

a) Highly purified diphtheria toxoid (D) and tetanus toxoid (T), obtained using a semi-synthetic medium, subsequently detoxified and individually adsorbed on an aluminum phosphate adjuvant, resulting in enhanced immunogenicity.

b) Inactivated whole cell component of B. pertussis (wP), obtained using a combination of heat and chemical inactivation, specific strains of Bordetella pertussis in a specific ratio, resulting in reduced reactogenicity and increased effectiveness.

c) Capsular polysaccharide antigen (PRP) of Haemophilus influenzae type b (Hib) conjugated to carrier protein (CP)

d) A reduced dose of IPV (inactivated poliovirus) Salk or Sabin demonstrating comparable efficacy to the standard dose, obtained using improved formaldehyde inactivation techniques and, if necessary, adsorption to an aluminum phosphate adjuvant.

e) Hepatitis B surface antigen (HepB) is individually adsorbed onto an aluminum phosphate adjuvant, resulting in enhanced immunogenicity.

f) Minimum amount of alum, which ensures reduced reactogenicity.

g) At least one paraben ester, i.e. methyl or propyl paraben other than 2-phenoxyethanol (2-PE), as a preservative.

Detailed Description of the Invention

Although the present invention may be embodied in various embodiments, certain embodiments of the invention are set forth in the following detailed discussion with the understanding that the present discussion is intended to be exemplary of the principles of the invention and is not intended to limit the scope of the present invention to that which is illustrated and disclosed in this specification . Embodiments of the invention are provided in order to fully and comprehensively convey the scope of the present invention to one skilled in the art. Numerous details regarding specific components and methods are disclosed to provide a thorough understanding of the embodiments of the present invention. It will be apparent to one skilled in the art that the details presented in the embodiments of the invention should not be construed as limiting the scope of the present invention. In some variants

- 6 043311 implementation of the invention, the well-known composition, well-known methods and well-known techniques are not described in detail.

The terminology used in the present invention is intended only for the purpose of explaining a particular embodiment, and such terminology should not be construed as limiting the scope of the present invention. As used herein, singular forms may also include plural forms unless the context clearly dictates otherwise.

Terms first, second, third, etc. should not be construed as limiting the scope of the present invention, since the above terms can only be used to distinguish one element, component, region, layer or section from another component, region, layer or section. Terms such as first, second, third, etc., when used herein, do not imply a particular sequence or order unless explicitly stated in the present invention. The present invention relates to an immunogenic composition and a method for its preparation.

The term vaccine may optionally be replaced by the term immunogenic composition and vice versa.

D-antigen units (also called international units or ME):

Antigenic form D of poliovirus induces protective neutralizing antibodies. The D-antigen units referred to herein (e.g., in the vaccines of the present invention) are the measured total D-antigen units of each unadsorbed IPV antigen type in the total pre-formulation of the final vaccine that are added to each dose of the formulated human vaccine (typically 0.5 final volume ml). Reliable methods for measuring D-antigen units are well known in the art and are published, for example, by the European Pharmacopoeia. For example, D-antigen units can be measured using an ELISA test as described below in Example 1 (Quantification of D-antigen by ELISA). The European Pharmacopoeia provides a test sample (European Pharmacopoeia biological reference preparation, available from the European Pharmacopoeia Secretariat, e.g. under code P 2160000) to standardize such methods between manufacturers (Pharmeuropa Special Issue, Bio 96-2). Thus, the unit size of the D antigen is well known in the art.

As used herein, the term dose generally means one administration of the vaccine of the present invention, which is usually one injection. The typical human dose is 0.5 ml. Of course, different doses can be included in the vaccine administration regimen.

As used herein, the term IPV or an immunogenic composition containing these components is intended to refer to inactivated poliovirus type 1 (eg, Mahoney, which is preferably used), type 2 (eg, MEF-1) or type 3 (eg, Saukett), or Sabin serotypes 1, 2, 3 - combinations of two or all three of these types. An example of a full (or standard) dose (40-8-32 units of IPV D-antigen based on Salk types 1, 2 and 3, respectively) of an IPV immunogenic composition for the purposes of the present invention is Poliovac® (Serum Institute of India Pvt. Ltd.) . Thus, if it is stated here that in the immunogenic composition of the present invention there is a one-, two-, three-fold dose reduction (reduction) compared to the standard dose of Salk-based IPV, then this means that each dose of the specified vaccine contains units of D -antigen, equal to X% dose reduction of 40, 8 and/or 32 units of IPV types 1, 2 and/or 3 D antigen, respectively (when measured for each IPV antigen type in the total mass).

As used herein, the term saccharide can refer to a polysaccharide or an oligosaccharide and includes both of these types of saccharides. The capsular saccharide antigen may be a full-length polysaccharide or may be bacterial saccharides of a certain size and oligosaccharides (which naturally have a small number of repeating units, or which are polysaccharides reduced in size for ease of manipulation but still capable of inducing protective immune response in the host).

In accordance with a first embodiment of the present invention, the combination vaccine composition includes a group of antigens/immunogens selected from, but not limited to, diphtheria toxoid (D), tetanus toxoid (T), whole cell B. pertussis (wP), Haemophilus influenzae type b (Hib), a PRP-CP conjugate, hepatitis B (HepB), a reduced dose of inactivated poliovirus (IPV), and further contains a combination of 2-phenoxyethanol and at least one paraben ester preservative.

In accordance with a second embodiment of the present invention, the combination vaccine composition may further comprise one or more antigens selected from the group consisting of, but not limited to, Haemophilus influenzae antigens (serotypes a, c, d, e, f and non-encapsulated strains), hepatitis (strains A, C, D, E, F and G), Neisseria meningitidis A, B, C, Y, W135 or X, influenza, Staphylococcus aureus, Salmonella typhi antigen(s), acellular pertussis antigen, modified adenylate cyclase, malaria antigen (RTS, S), antigens of pneumococci, streptococci, anthrax, dengue, malaria, measles, mumps, rubella, BCG, human papillomavirus, Japanese encephalitis, dengue, Zika fever, Ebola, chikungunya, rotavirus,

- 7 043311 smallpox, yellow fever, flavivirus, herpes zoster, varicella zoster virus, respectively.

In accordance with a third embodiment of the present invention, the IPV strains used in the combination vaccine composition include inactivated Sabin strains selected from the group type 1, type 2 and type 3, or inactivated Salk strains selected from the group Mahoney type 1, type 2 MEF and type 3 Saukett.

In one aspect of the third embodiment, poliovirus can be grown in the following manner:

The cell line CCL81-VERO (monkey kidney) is used as host cells for growing polioviruses, i.e. Sabin and Salk strains.

After infection of host cells with the desired strain of poliovirus and incubation for 72 hours, the medium containing the virus and cell debris is pooled and collected in one container.

The filtrate is subjected to tangential flow filtration with a 100 kDa cassette; diafiltered using phosphate buffer and purified using anion exchange chromatography.

Viruses must be inactivated using appropriate inactivation methods before being administered to patients.

However, the present inventors have unexpectedly discovered that the high percentage loss of D-antigen after formaldehyde inactivation may be due to the presence of a phosphate buffer, which unexpectedly causes undesirable aggregation of poliovirus particles.

Therefore, an important aspect of the present invention is an improved formalin inactivation method comprising the following steps:

a) the purified virus pool is buffer exchanged from phosphate buffer to Tris buffer in the range (30 to 50 mM) with a pH of 7 to 7.5;

b) M-199 medium containing glycine (5 g/l) is added to the above mixture;

c) add 0.025% formaldehyde and then mix;

d) then the mixture is incubated at a temperature of 37°C for 5-13 days with continuous stirring of the virus mass on a magnetic stirrer;

e) the mixture after incubation is subjected to intermediate tangential flow filtration (100 kDa, 0.1 m ² ) on day 7 and final filtration after inactivation;

f) the filtered volume is then stored at a temperature of 2-8°C;

g) perform a D-Ag ELISA to determine D-antigen units.

In accordance with a fourth embodiment of the present invention, the IPV strains used in the combination vaccine composition include a reduced dose of inactivated Sabin strains selected from the group of type 1, type 2 and type 3, or inactivated Salk strains selected from the Mahoney group of type 1, type 2 MEF and type 3 Saukett.

According to a fifth embodiment of the present invention, IPV (Sabin or Salk strains) cannot be adsorbed individually to any adjuvant and subsequently added to the final combination vaccine composition.

In accordance with a preferred aspect of the fifth embodiment of the invention, IPV (Sabin or Salk strains) may be adsorbed to an adjuvant, more preferably an aluminum salt in the form of aluminum phosphate or aluminum hydroxide, present in a combination vaccine, wherein the percentage of IPV antigen adsorption for IPV type 1 may be in the range 10-30%, Type 2 IPV can range from 60-100%, and Type 3 IPV can range from 0-25%.

In accordance with a sixth embodiment of the present invention, the IPV component(s) (Sabin or Salk strains) can be individually adsorbed to an adjuvant selected from the group consisting of an aluminum salt (Al ³⁺ ), such as aluminum hydroxide (Al(OH) ₃ ) or aluminum phosphate (AlPO ₄ ), alum, calcium phosphate, MPLA, 3D-MPL, QS21, CpG-containing oligodeoxynucleotide adjuvant, liposomes or oil-in-water emulsion, or combinations thereof (for example, before or after mixing with other components, if they are). In the case of adsorption, one or more IPV components may be adsorbed separately or together as a mixture onto aluminum hydroxide (Al(OH) ₃ ) or aluminum phosphate.

IPV component(s) (Sabin or Salk strains) can be adsorbed onto aluminum salts using the following procedure:

Taking the desired volume of autoclaved Al(PO) ₄ or Al(OH) ₃ to obtain a final alum (Al ³⁺ ) concentration of 0.1 to 0.8 mg per dose in a 50 ml container.

Adding the total mass of IPV with a certain number of D-antigen units and adjusting the volume with a diluent (10xM-199 + 0.5% glycine),

Adjusting the pH of the final composition and obtaining a final composition with a pH of 6 to 7.5.

In one aspect of the sixth embodiment, adsorption of formaldehyde-inactivated IPV can be carried out on alum (Al ³⁺ ) at a concentration selected from 0.1 mg per dose, 0.2 mg per dose, 0.3 mg per dose, 0 .4 mg per dose, 0.5 mg per dose, 0.6 mg per dose, 0.7 mg per dose and 0.8 mg per dose, preferably 0.1 mg per dose to 1.25 mg per dose for each serotype and at a pH value selected from 6.2, 6.3, 6.4, 6.5, 6.6, 6.7 and 6.8, preferably 6.5.

- 8 043311

In yet another aspect of the sixth embodiment of the invention, the percentage of D antigen recovery after formalin inactivation in the presence of Tris may be 50, 60, 70, or 80%, and the percentage of adsorption after adsorption on aluminum phosphate may be 70 to 80%, 80 to 90%, or from 90 to 99% or from 95 to 99%.

According to a seventh embodiment of the present invention, diphtheria toxin (exotoxin) and tetanus toxin (exotoxin) were obtained from Corynebacterium diphtheria and Clostridium tetani, respectively, and subsequently detoxified using a suitable inactivation method. The diphtheria toxoid (D) and tetanus toxoid (T) thus obtained can be purified by gel filtration chromatography. The purified DT obtained in this way was subsequently used to prepare a combination vaccine.

In one aspect of the seventh embodiment, diphtheria toxin is produced by growing Corynebacterium diphtheriae in a semi-synthetic medium consisting of the following ingredients at optimal concentrations in any of the following combinations:

Combination 1:

Casein hydrolyzate, maltose monohydrate, glacial acetic acid, sodium lactate, magnesium sulfate, β-alanine, pimelic acid, niacin, copper sulfate, zinc sulfate, manganese chloride, L-cystine, calcium chloride dihydrate, potassium dihydrogen orthophosphate, dipotassium hydrogen phosphate, sulfate iron and water for injection.

Combination 2:

Casein hydrolyzate, maltose monohydrate, glacial acetic acid, sodium lactate, magnesium sulfate, β-alanine, pimelic acid, niacin, manganese chloride, L-cystine, calcium chloride dihydrate, potassium dihydrogen orthophosphate, dipotassium hydrogen phosphate, ferrous sulfate and water for injection.

Combination 3:

Casein hydrolyzate, maltose monohydrate, glacial acetic acid, sodium lactate, β-alanine, pimelic acid, niacin, copper sulfate, zinc sulfate, manganese chloride, L-cystine, calcium chloride dihydrate, potassium dihydrogen orthophosphate, dipotassium hydrogen phosphate and water for injection.

Combination 4:

Yeast extract, maltose monohydrate, glacial acetic acid, sodium lactate, magnesium sulfate, β-alanine, pimelic acid, niacin, copper sulfate, zinc sulfate, manganese chloride, L-cystine, calcium chloride dihydrate, potassium dihydrogen orthophosphate, dipotassium hydrogen phosphate, sulfate iron and water for injection.

In accordance with the second aspect of the seventh embodiment of the invention, tetanus toxin is produced by growing Clostridium tetanus in a semi-synthetic medium consisting of the following ingredients in optimal concentrations in any of the following combinations:

Combination 1:

Casein hydrolysate, calcium chloride, dipotassium hydrogen phosphate, dextrose anhydrous, sodium chloride, magnesium sulfate, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride, calcium pantothenate, niacin, L-cystine, ferric chloride, vitamin B12 solution, biotin, conc. HCl, NaOH, absolute ethanol and water for injection.

Combination 2:

Hydrolyzed casein, calcium chloride, β-alanine dipotassium hydrogen phosphate, anhydrous dextrose, sodium chloride, magnesium sulfate, ferrous sulfate, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride, calcium pantothenate, niacin, L-cystine, ferric chloride, vitamin B12 solution, biotin , conc. HCl, NaOH, absolute ethanol and water for injection.

Combination 3:

Casein hydrolyzate, calcium chloride, dipotassium hydrogen phosphate, dextrose anhydrous, sodium chloride, zinc sulfate, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride, calcium pantothenate, niacin, L-cystine, ferric chloride, vitamin B12 solution, biotin, conc. HCl, NaOH, absolute ethanol and water for injection.

Combination 4:

Hydrolyzed casein, calcium chloride, dipotassium hydrogen phosphate, anhydrous dextrose, sodium chloride, magnesium sulfate, manganese chloride, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride, calcium pantothenate, niacin, L-cystine, ferric chloride, vitamin B12 solution, biotin, conc. HCl, NaOH, absolute ethanol and water for injection.

In yet another aspect of the seventh embodiment, the diphtheria and tetanus toxin are detoxified using one or a combination of the following inactivation methods, which include heat, UV, formalin/formaldehyde, acetyleneimine, etc.

According to an eighth embodiment of the present invention, the hepatitis antigen (Hep) used in the combination vaccine composition includes hepatitis antigens derived from the surface of a hepatitis B strain (HBsAg).

- 9 043311

In one aspect of the ninth embodiment, HBsAg can be produced by one of the following methods:

By purifying particulate antigen from the plasma of chronic hepatitis B carriers, as large amounts of HBsAg are synthesized in the liver and enter the bloodstream during HBV infection.

By expressing the protein using recombinant DNA techniques.

According to a ninth embodiment of the present invention, diphtheria toxoid (D), tetanus toxoid (T), hepatitis B surface antigen (HBsAg) are individually adsorbed to an adjuvant selected from the group consisting of an aluminum salt (Al ³⁺ ), such as aluminum hydroxide (Al(OH) ₃ ) or aluminum phosphate (AlPO ₄ ), alum, calcium phosphate, MPLA, 3D-MPL, QS21, CpG-containing oligodeoxynucleotide adjuvant, liposomes or oil-in-water emulsion, or combinations thereof.

Preferably, diphtheria toxoid (D), tetanus toxoid (T) and hepatitis B surface antigen (HBsAg) are individually adsorbed onto the aluminum phosphate.

In one aspect of the ninth embodiment, the diphtheria toxoid antigen (D) adsorbed on aluminum phosphate has an adsorption percentage of at least 50%.

In another aspect of the ninth embodiment, the tetanus toxoid (T) antigen adsorbed on aluminum phosphate has an adsorption percentage of at least 40%.

In yet another aspect of the ninth embodiment, the hepatitis B surface antigen (HBsAg) adsorbed on aluminum phosphate has an adsorption percentage of at least 70%.

According to a tenth embodiment of the present invention, the Hib antigen used in the combination vaccine of the present invention is derived from the capsular polysaccharide of Haemophilus influenzae type B (Hib) strain 760705.

In accordance with one aspect of the tenth embodiment, the PRP Hib antigen is conjugated to a carrier protein selected from the group of carrier proteins consisting of, but not limited to, CRM ₁₉₇ , diphtheria toxoid, Neisseria meningitidis outer membrane complex, tetanus toxoid fragment C, pertussis toxoid, H. influenzae protein D, E. coli LT, E. coli ST and exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMC), 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 lethal factor (LF) of Bacillus anthracis, ovalbumin, fisurella hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA) and purified tuberculin derivative (PPD), synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins containing multiple CD4+ T cells human epitopes for various pathogenic antigens, such as N 19, iron uptake proteins, toxin A or B from C. difficile and S. agalactiae proteins.

Preferably, PRP Hib is conjugated to tetanus toxoid (TT) using CNBr chemistry, reductive amination chemistry, cyanylation chemistry, or any other chemistry already disclosed in Kniskern et al., Conjugation: design, chemistry, and analysis in Ellis et al., Development and analysis clinical uses of Haemophilus influenzae type B conjugate vaccines. New York: Marcel Dekker, 1994: 37-69.

According to the second aspect of the tenth embodiment, the carrier protein is present in the composition of the present invention in both free and conjugated form, the unconjugated form preferably comprising no more than 20% of the total amount of carrier protein in the composition as a whole and more preferably in an amount of less than 5 % by weight.

In accordance with the third aspect of the tenth embodiment of the invention, the Hib antigen is substantially not adsorbed to any adjuvant.

In accordance with the fourth aspect of the tenth embodiment of the invention, the Hib antigen does not undergo planned or deliberate adsorption to any adjuvant.

According to the fifth aspect of the tenth embodiment of the invention, the percentage of adsorption of Hib antigen to any adjuvant is less than 20%.

According to an eleventh embodiment of the present invention, the whole cell pertussis antigen (wP) preparation used in the combination vaccine composition of the present invention is preferably obtained from Bordetella pertussis strains 134, 509, 25525 and 6229, mixed in a certain ratio and subsequently inactivated using improved inactivation methods , which do not use thiomersal, resulting in reduced reactogenicity and increased activity, and the wP antigen may or may not be adsorbed to aluminum-based adjuvants.

In accordance with one aspect of the eleventh embodiment of the invention, the whole cell pertussis antigen (wP) preparation used in the combination vaccine composition of the present invention is preferably obtained from Bordetella pertussis strains 134, 509, 25525 and 6229 mixed in a ratio of 1:1:0.25: 0.25.

According to the second aspect of the eleventh embodiment of the invention, the preparation

- 10 043311 whole cell pertussis antigen (wP) used in the combination vaccine composition is inactivated by one or more of the following inactivation methods, which include heat, UV, formaldehyde/formaldehyde, acetylethylenimine, etc.

Preferably, the whole cell pertussis antigen (wP) preparation used in the combination vaccine composition is inactivated using a combination of heat and chemical treatment. However, it is preferable to carry out inactivation by heating at a temperature of 56±2°C, from 10 to 15 minutes in the presence of formaldehyde, while the bulk of wP remains non-lumpy and is easily homogenized, which leads to a decrease in reactogenicity and gives better wP efficiency for more for a long time.

In accordance with a third aspect of the eleventh embodiment, the whole cell pertussis antigen (wP) preparation used in the combination vaccine composition may or may not be adsorbed to an aluminum-based adjuvant such as aluminum hydroxide, aluminum phosphate, or a combination thereof (for example, before or after mixing with other components, if any). In the case of adsorption, one or more wP strains (i.e., 134, 509, 25525, and 6229) may be adsorbed individually or together as a mixture.

According to a twelfth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

Table 1

Item no. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf* Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 IOU** Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Sabin Type 1 (D-antigen units) 1-50 DU*** Preferably one of 5 or 10 or 20 DU Type 2 (D-antigen units) 1 - 50 DU Preferably one of 8 or 4 or 16 DU Type 3 (D-antigen units) 1 - 50 DU Preferably one of 10 or 16 or 32 DU 7 Total aluminum content (AP ⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg 9 Methylparaben 0.1 -1.5 mg Preferably one of 0.7 or 0.9 or 1 mg 10 Propylparaben 0.05 - 0.2 mg Preferably one of 0.05 or 0.1 or 0.15 mg

*Lf - Flocculation Units **IOU - International Opacity Units ***DU - D-Antigen Units

According to a thirteenth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

- 11 043311

table 2

Item No. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 lOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Salk Mahoney Type 1 (Dantigen units) 1 -50 DU Preferably one of 7.5 or 10 or 20 or 40 DU MEF-1 Type 2 (Dantigen units) 1 - 50 DU Preferably one of 1.5 or 2 or 4 or 8 DU Saukett Type 3 (Dantigen units) 1 - 50 DU Preferably one of 6 or 10 or 16 or 32 DU 7 Total aluminum content (AP ⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg 9 Methylparaben 0.1 -1.5 mg Preferably one of 0.7 or 0.9 or 1 mg 10 Propylparaben 0.05 - 0.2 mg Preferably one of 0.05 or 0.1 or 0.15 mg

According to a fourteenth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

Table 3

Item no. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 IOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Conjugated antigen Hib PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Sabin Type 1 (D-antigen units) 1-50 DU Preferably one of 5 or 10 or 20 DU Type 3 (D-antigen units) 1 - 50 DU Preferably one of 10 or 16 or 32 DU 7 Total aluminum content (Al ³⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg 9 Methylparaben 0.1 - 1.5 mg Preferably one of 0.7 or 0.9 or 1 mg 10 Propylparaben 0.05 - 0.2 mg Preferably one of 0.05 or 0.1 or 0.15 mg

According to a fifteenth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

- 12 043311

Table 4

Item No. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 lOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Salk Mahoney Type 1 (Dantigen units) 1 -50 DU Preferably one of 7.5 or 10 or 20 or 40 DU Saukett Type 3 (Dantigen units) 1 - 50 DU Preferably one of 6 or 10 or 16 or 32 DU 7 Total aluminum content (AP ⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg 9 Methylparaben 0.1 - 1.5 mg Preferably one of 0.7 or 0.9 or 1 mg 10 Propylparaben 0.05 - 0.2 mg Preferably one of 0.05 or 0.1 or 0.15 mg

According to a sixteenth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

Table 5

Item No. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 IOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Sabin Type 1 (D-antigen units) 1-50 DU Preferably one of 5 or 10 or 20 DU Type 2 (D-antigen units) 1 -50 DU Preferably one of 8 or 4 or 16 DU Type 3 (D-antigen units) 1 -50 DU Preferably one of 10 or 16 or 32 DU 7 Total aluminum content (AP ⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg 9 Methylparaben 0.1 -1.5 mg Preferably one of 0.7 or 0.9 or 1 mg

According to a seventeenth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

- 13 043311

Table 6

Item No. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 lOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Salk Mahoney Type 1 (Dantigen units) 1 -50 DU Preferably one of 7.5 or 10 or 20 or 40 DU MEF-1 Type 2 (Dantigen units) 1 -50 DU Preferably one of 1.5 or 2 or 4 or 8 DU Saukett Type 3 (Dantigen units) 1 -50 DU Preferably one of 6 or 10 or 16 or 32 DU 7 Total aluminum content (AP ⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg 9 Methylparaben 0.1 -1.5 mg Preferably one of 0.7 or 0.9 or 1 mg

According to an eighteenth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

Table 7

Item No. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 IOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP Inactivated Sabin poliovirus (IPV) serotype 6 Type 1 (D-antigen units) 1-50 DU Preferably one of 5 or 10 or 20 DU Type 3 (D-antigen units) 1 -50 DU Preferably one of 10 or 16 or 32 DU 7 Total aluminum content (Al ³⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg 9 Methylparaben 0.1 - 1.5 mg Preferably one of 0.7 or 0.9 or 1 mg

According to a nineteenth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

- 14 043311

Table 8

Item No. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 lOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Salk Mahoney Type 1 (Dantigen units) 1 -50 DU Preferably one of 7.5 or 10 or 20 or 40 DU Saukett Type 3 (Dantigen units) 1 -50 DU Preferably one of 6 or 10 or 16 or 32 DU 7 Total aluminum content (Al ³⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg 9 Methylparaben 0.1 - 1.5 mg Preferably one of 0.7 or 0.9 or 1 mg

According to the twentieth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

Table 9

Item No. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 IOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Sabin Type 1 (D-antigen units) 1-50 DU Preferably one of 5 or 10 or 20 DU Type 2 (D-antigen units) 1 -50 DU Preferably one of 8 or 4 or 16 DU Type 3 (D-antigen units) 1 -50 DU Preferably one of 10 or 16 or 32 DU 7 Total aluminum content (Al ³⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg 9 Propylparaben 0.05 - 0.2 mg Preferably one of 0.05 or 0.1 or 0.15 mg

In accordance with the twenty-first embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

-15043311

Table 10

Item no. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 lOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Salk Mahoney Type 1 (Dantigen units) 1 -50 DU Preferably one of 7.5 or 10 or 20 or 40 DU MEF-1 Type 2 (Dantigen units) 1 -50 DU Preferably one of 1.5 or 2 or 4 or 8 DU Saukett Type 3 (Dantigen units) 1 -50 DU Preferably one of 6 or 10 or 16 or 32 DU 7 Total aluminum content (AP ⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg 9 Propylparaben 0.05 - 0.2 mg Preferably one of 0.05 or 0.1 or 0.15 mg

In accordance with the twenty-second embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

Table 11

Item no. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 IOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Sabin Type 1 (D-antigen units) 1-50 DU Preferably one of 5 or 10 or 20 DU Type 3 (D-antigen units) 1 -50 DU Preferably one of 10 or 16 or 32 DU 7 Total aluminum content (Al ³⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg 9 Propylparaben 0.05 - 0.2 mg Preferably one of 0.05 or 0.1 or 0.15 mg

In accordance with the twenty-third embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

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Table 12

Item no. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 lOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Salk Mahoney Type 1 (Dantigen units) 1 -50 DU Preferably one of 7.5 or 10 or 20 or 40 DU Saukett Type 3 (Dantigen units) 1 -50 DU Preferably one of 6 or 10 or 16 or 32 DU 7 Total aluminum content (AP ⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg 9 Propylparaben 0.05 - 0.2 mg Preferably one of 0.05 or 0.1 or 0.15 mg

According to a twenty-fourth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

Table 13

Item no. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 IOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Sabin Type 1 (D-antigen units) 1-50 DU Preferably one of 5 or 10 or 20 DU Type 2 (D-antigen units) 1 -50 DU Preferably one of 8 or 4 or 16 DU Type 3 (D-antigen units) 1 -50 DU Preferably one of 10 or 16 or 32 DU 7 Total aluminum content (Al ³⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 Methylparaben 0.1 -1.5 mg Preferably one of 0.7 or 0.9 or 1 mg 9 Propylparaben 0.05 - 0.2 mg Preferably one of 0.05 or 0.1 or 0.15 mg

In accordance with the twenty-fifth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

- 17 043311

Table 14

Item no. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 lOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Salk Mahoney Type 1 (Dantigen units) 1 -50 DU Preferably one of 7.5 or 10 or 20 or 40 DU MEF-1 Type 2 (Dantigen units) 1 -50 DU Preferably one of 1.5 or 2 or 4 or 8 DU Saukett Type 3 (Dantigen units) 1 -50 DU Preferably one of 6 or 10 or 16 or 32 DU 7 Total aluminum content (AP ⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 Methylparaben 0.1 -1.5 mg Preferably one of 0.7 or 0.9 or 1 mg 9 Propylparaben 0.05 - 0.2 mg Preferably one of 0.05 or 0.1 or 0.15 mg

In accordance with the twenty-sixth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

Table 15

Item no. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 IOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP Inactivated Sabin poliovirus (IPV) serotype 6 Type 1 (D-antigen units) 1-50 DU Preferably one of 5 or 10 or 20 DU Type 3 (D-antigen units) 1 - 50 DU Preferably one of 10 or 16 or 32 DU 7 Total aluminum content (Al ³⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 Methylparaben 0.1 - 1.5 mg Preferably one of 0.7 or 0.9 or 1 mg 9 Propylparaben 0.05 - 0.2 mg Preferably one of 0.05 or 0.1 or 0.15 mg

According to a twenty-seventh embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

- 18043311

Table 16

Item No. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 lOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Salk Mahoney Type 1 (Dantigen units) 1 -50 DU Preferably one of 7.5 or 10 or 20 or 40 DU Saukett Type 3 (Dantigen units) 1 -50 DU Preferably one of 6 or 10 or 16 or 32 DU 7 Total aluminum content (AP ⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 Methylparaben 0.1 - 1.5 mg Preferably one of 0.7 or 0.9 or 1 mg 9 Propylparaben 0.05 - 0.2 mg Preferably one of 0.05 or 0.1 or 0.15 mg

In accordance with the twenty-eighth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

Table 17

Item no. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 IOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Sabin Type 1 (D-antigen units) 1-50 DU Preferably one of 5 or 10 or 20 DU Type 2 (D-antigen units) 1 - 50 DU Preferably one of 8 or 4 or 16 DU Type 3 (D-antigen units) 1 - 50 DU Preferably one of 10 or 16 or 32 DU 7 Total aluminum content (Al ³⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg

In accordance with the twenty-ninth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

- 19 043311

Table 18

Item no. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 lOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Salk Mahoney Type 1 (Dantigen units) 1 -50 DU Preferably one of 7.5 or 10 or 20 or 40 DU MEF-1 Type 2 (Dantigen units) 1 -50 DU Preferably one of 1.5 or 2 or 4 or 8 DU Saukett Type 3 (Dantigen units) 1 -50 DU Preferably one of 6 or 10 or 16 or 32 DU 7 Total aluminum content (Al ³⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg

In accordance with the thirtieth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

Table 19

Item no. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 IOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Sabin Type 1 (D-antigen units) 1-50 DU Preferably one of 5 or 10 or 20 DU Type 3 (D-antigen units) 1 - 50 DU Preferably one of 10 or 16 or 32 DU 7 Total aluminum content (Al ³⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg

In accordance with the thirty-first embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

- 20 043311

Table 20

Item no. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 3 4 5 Tetanus toxoid (T) Inactivated B. pertussis antigen (wP) HBs antigen Hib conjugated antigen PRP-TT 02-10 Lf 12-16 lOU 7-15 mcg 7-13 mcg PRP Preferably one of 2 or 4 or 10 Lf Preferably one of 12 or 14 or 16 IOU Preferably one of 8 or 10 or 15 mcg Preferably one of 8 or 10 or 13 µg PRP Inactivated poliovirus (IPV) serotype Salk 6 Mahoney Type 1 (Dantigen units) 1 -50 DU Preferably one of 7.5 or 10 or 20 or 40 DU Saukett Type 3 (Dantigen units) 1 -50 DU Preferably one of 6 or 10 or 16 or 32 DU 7 Total aluminum content (AP ⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63 8 2-phenoxyethanol 1 - 6 mg Preferably one of 2 or 2.5 or 3 mg

In accordance with the thirty-second embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

Table 21

Item no. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf h Inactivated antigen B. 12-16 IOU Preferably one of 12 pertussis (wP) or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen 7-13 mcg PRP Preferably one of 8 PRP-TT or 10 or 13 mcg PRP Inactivated Sabin poliovirus (IPV) serotype 6 Type 1 (D-antigen units) 1-50 DU Preferably one of 5 or 10 or 20 DU Type 2 (D-antigen units) 1 -50 DU Preferably one of 8 or 4 or 16 DU Type 3 (D-antigen units) 1 -50 DU Preferably one of 10 or 16 or 32 DU Total aluminum content 0.1 - 0.6 mg Preferably no more 7 (Al3 ⁺ ) 0.3 or not more than 0.55 or not (as aluminum phosphate) more than 0.63

In accordance with the thirty-third embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

- 21 043311

Table 22

Item no. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 lOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Salk Mahoney Type 1 (Dantigen units) 1 -50 DU Preferably one of 7.5 or 10 or 20 or 40 DU MEF-1 Type 2 (Dantigen units) 1 -50 DU Preferably one of 1.5 or 2 or 4 or 8 DU Saukett Type 3 (Dantigen units) 1 -50 DU Preferably one of 6 or 10 or 16 or 32 DU 7 Total aluminum content (AP ⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63

In accordance with the thirty-fourth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

Table 23

Item no. Composition components Antigen unit per 0.5 ml dose Preferred number of antigen units per 0.5 ml dose (In any combination) 1 Diphtheria toxoid (D) 10-25 Lf Preferably one of 10 or 20 or 25 Lf 2 Tetanus toxoid (T) 02-10 Lf Preferably one of 2 or 4 or 10 Lf 3 Inactivated B. pertussis antigen (wP) 12-16 IOU Preferably one of 12 or 14 or 16 IOU 4 HBs antigen 7-15 mcg Preferably one of 8 or 10 or 15 mcg 5 Hib conjugated antigen PRP-TT 7-13 mcg PRP Preferably one of 8 or 10 or 13 µg PRP 6 Inactivated poliovirus (IPV) serotype Sabin Type 1 (D-antigen units) 1-50 DU Preferably one of 5 or 10 or 20 DU Type 3 (D-antigen units) 1 -50 DU Preferably one of 10 or 16 or 32 DU 7 Total aluminum content (AP*) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63

In accordance with the thirty-fifth embodiment of the present invention, the multi-dose combination vaccine composition/formulation includes the following.

- 22 043311

Table 24

Item no. 1 2 3 4 5 6 p Preferred d h number of antigen units Components of the antigen composition per _p ** _s per 0.5 ml dose (In any dose of 0.5 ml ", ^} combinations) _p . - - 10-25 Lf Preferably one of 10 Diphtheria toxoid (D) pl , ^{n k v} ' or 20 or 25 Lf „ _ „ 02-10 Lf Preferably one of 2 Tetanus toxoid (T) 7 , ^v ' or 4 or 10 Lf Inactivated antigen B. 12-16 IOU Preferably one of 12 pertussis (wP) or 14 or 16 IOU. _ip 7-15 µg Preferably one of 8 HBs antigen _{or 10 or 15 µg} Hib conjugate antigen 7-13 µg PRP Preferably one of 8 PRP-TT or 10 or 13 µg PRP Inactivated poliovirus (IPV) serotype Salk Mahoney Type 1 (Dantigen units) 1 -50 DU Preferably one of 7.5 or 10 or 20 or 40 DU Saukett Type 3 (Dantigen units) 1 - 50 DU Preferably one of 6 or 10 or 16 or 32 DU 7 Total aluminum content (AP ⁺ ) (as aluminum phosphate) 0.1 - 0.6 mg Preferably no more than 0.3 or no more than 0.55 or no more than 0.63

In accordance with the thirty-sixth embodiment of the present invention, one or more antigens of the final combination vaccine composition may not be significantly adsorbed to any adjuvant.

According to the thirty-seventh embodiment of the present invention, the pH value of the immunogenic composition may be in the range of pH 6.0 to pH 8.0; more preferably in the range of pH 6.0 to pH 7.5; even 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 accordance with a thirty-eighth embodiment of the present invention, the immunogenic composition may further comprise a buffering agent selected from the group consisting of carbonate, phosphate, acetate, succinate, borate, citrate, lactate, gluconate and tartrate, as well as more complex organic buffering agents including a phosphate buffering agent that contains sodium phosphate and/or potassium phosphate in a ratio selected to achieve the desired pH value. In another example, the buffering agent contains tris(hydroxymethyl)aminomethane or Tris included in the formulation to achieve the desired pH. In yet another example, the buffering agent may be Hank's salts minimal growth medium. Other buffers such as HEPES, piperazine-N,N'-bis (PIPES) and 2-ethanesulfonic acid (MES) are also contemplated by the present invention. The buffer helps stabilize the immunogenic composition of the present invention. The amount of buffer may range from 0.1 to 100 mM, preferably selected from 5 mM, 6 mM, 7 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM and 30 mM.

In yet another aspect of the embodiment of the invention, the immunogenic composition may further comprise pharmaceutically acceptable excipients selected from the group consisting of surfactants, polymers and salts. Examples of surfactants may include nonionic surfactants such as polysorbate 20, polysorbate 80, etc. Examples of polymers may include dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin, etc. Examples of salts may include NaCl, KCl, KH ₂ PO ₄ , Na ₂ HPO ₄ -2H ₂ O, CaCl ₂ , MgCl _{2 ,} etc. Preferably, the salt may be NaCl. Typically, the amount of salt may be in the range of 100 to 200 mM.

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

According to a thirty-ninth embodiment of the present invention, the immunogenic composition may further comprise one or more adjuvants selected from the group consisting of aluminum salt (Al ³⁺ ), such as aluminum hydroxide (Al(OH) ₃ ) or aluminum phosphate (AlPO ₄ ), alum, calcium phosphate, MPLA, 3D-MPL, QS21, CpG-containing oligodeoxynucleotide adjuvant, liposome or oil-in-water emulsion.

Preferably the composition contains aluminum phosphate (AlPO ₄ ) as an adjuvant.

Preferably the composition contains aluminum hydroxide (AlOH ₃ ) as an adjuvant.

In one aspect of the thirty-ninth embodiment of the invention, the antigens of the final composition can be adsorbed onto an in situ aluminum phosphate gel or a preformed aluminum phosphate gel, or a combination thereof.

In one preferred aspect of the thirty-ninth embodiment, the composition of the present invention may contain an adjuvant in an amount of 2.5 mg per 0.5 ml or less, and specifically in an amount of 1.5 mg per 0.5 ml to 0.1 mg by 0.5 ml.

According to the fortieth embodiment of the present invention, the immunogenic

- 23 043311 the composition may additionally contain an immunostimulating component selected from the group consisting of oil and water emulsion, MF-59, liposome, lipopolysaccharide, saponin, lipid A, lipid A derivatives, monophosphoryl lipid A, 3-deacylated monophosphoryl lipid A, AS01, AS03 , oligonucleotide, oligonucleotide containing at least one unmethylated CpG and/or liposome, Freund's adjuvant, complete Freund's adjuvant, incomplete Freund's adjuvant, polymers, copolymers, such as polyoxyethylene-polyoxypropylene copolymers, including block copolymers, polymer p 1005, adjuvant CRL-8300 , muramyl dipeptide, TLR-4 agonists, flagellin, flagellins derived from gram-negative bacteria, TLR-5 agonists, flagellin fragments capable of binding to TLR-5 receptors, alpha-C-galactosylceramide, chitosan, interleukin-2, QS-21, ISCOMS , mixtures of squalene (SAF-1), Quil A, cholera toxin subunit B, polyphosphazene and derivatives, mycobacterial cell wall preparations, mycolic acid derivatives, nonionic surfactant block copolymers, OMV, fHbp, combinations of saponin with sterols and lipids.

In accordance with the forty-first embodiment of the present invention, the immunogenic composition may further comprise a preservative selected from the group consisting of benzethonium chloride (femerol), phenol, m-cresol, thiomersal, formaldehyde, benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chloro- m-cresoal or benzyl alcohol, or combinations thereof. The vaccine composition may contain a preservative for a single immunization or may contain a preservative for multiple immunizations (ie, a multi-dose kit). In multi-dose regimens, the inclusion of a preservative is preferred. As an alternative (or in addition) to including a preservative in multi-dose compositions, the compositions can be placed in a container having an aseptic adapter for removing the material. Typically, the amount of preservative may range from 0.1 to 50 mg.

According to a forty-second embodiment of the present invention, the immunogenic composition may further contain auxiliary agents such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing agents, flavoring agents, coloring agents and the like, depending on the route of administration and the desired formulation.

In accordance with the forty-third embodiment of the present invention, the immunogenic composition may be completely liquid, but is not limited to this. Suitable liquid formulation forms may include solutions, suspensions, emulsions, syrups, isotonic aqueous solutions, viscous compositions and tinctures that are buffered to the desired pH.

The immunogenic composition of the present invention may be in the form of transdermal preparations, including lotions, gels, sprays, ointments, or other suitable forms. If nasal or mucosal administration is desired (eg, aerosol inhalation or insufflation), the compositions may be formulated and dispensed using a compressible nebulizer, pump-type nebulizer, or aerosol nebulizer. Aerosols are usually pressurized using a hydrocarbon. Pump-type nebulizers may preferably dispense a metered dose or a dose having a specific particle size. When the compositions are in the form of solutions, suspensions and gels, in some embodiments, the immunogenic compositions contain a large amount of water (preferably purified water) in addition to the active ingredient(s).

According to the forty-fourth embodiment of the present invention, the combination vaccine can be stable at a temperature of 2-8°C for 12 to 36 months; at a temperature of 25°C from 2 to 6 months; at a temperature of 37°C from 1 week to 4 weeks.

In accordance with a forty-fifth embodiment of the present invention, an immunogenic composition can be formulated for use in a method of reducing the onset or preventing a pathological health condition including diphtheria, tetanus, whooping cough, hepatitis B virus infection, Haemophilus influenzae type b, polio virus, including itself administering an immunologically effective amount of an immunogenic composition to a human subject by parenteral, subcutaneous or intradermal, intramuscular or intraperitoneal or intravenous administration, or injection, or sustained release from implants, or administration as eye drops or nasal, rectal, buccal or vaginal, oral or intragastric, or transmucosal, or sublingual, alveolar or gingival, or olfactory, or into the mucous membrane of the respiratory tract, or any other route of immunization.

According to the forty-sixth embodiment of the present invention, the immunogenic composition can be prepared in the form of single dose ampoules or multi-dose ampoules (2 doses, 5 doses or 10 doses per ampoule), or a multi-dose kit, or in the form of pre-filled syringes, where said immunogenic composition may be administered in a single-dose regimen or, preferably, in a multiple-dose regimen in which the primary course of vaccination is followed by 1-3 separate doses administered at subsequent intervals of 1-3 years, if necessary. The dosage regimen will also be determined, at least in part, by the need for a booster.

- 24 043311 dose required to create protective immunity.

Preferably, the immunogenic composition may be formulated for administration to human subjects or children 2 years of age or younger according to a two-dose regimen consisting of a first dose and a second dose followed by 1-3 year intervals.

Preferably, the immunogenic composition can be administered simultaneously with other drugs or any other vaccine.

In accordance with the forty-seventh embodiment of the present invention, the inventors have discovered that a multi-dose all-liquid combination vaccine with improved immunogenicity and reduced reactogenicity can be obtained when the vaccine is produced by the method described below, taking into account:

i) the process of obtaining individual antigens, ii) the sequence of addition of antigens, iii) the use of specific adjuvants in a certain amount for specific antigens, iv) individual adsorption or combined adsorption of antigens on adjuvants,

v) extent of antigen adsorption to adjuvants, vi) use of minimum concentration of alum, vii) use of optimum concentration and type of preservative and viii) use of various parameters including agitation, temperature and pH.

Biological source of the strains used in the SIIPL combination vaccine

Diphtheria toxoid:

Corynebacterium diphtheriae strain PW8 CN2000 was obtained from the Wellcome Research Laboratory, London, United Kingdom by the Central Research Institute of the National Inspectorate (C.R.I.), Kasauli, Himachal Pradesh, India, in lyophilized form in 1973.

Tetanus toxoid:

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

Whooping cough:

The production of a batch of SIIPL pertussis vaccine involves the use of four strains of Bordetella pertussis, namely strains 134, 509, 6229 and 25525. The source material of strains 134 and 509 was originally obtained from the Rijks Institute, the Netherlands and was obtained through the National Control Authority of the Central Research Institute, Kasauli , Himachal Pradesh, India. The source material for strains 6229 and 25525 was originally obtained from the Lister Institute, England.

Hepatitis B:

Rhein Biotech (Germany) has constructed a recombinant Hansenulapolymorpha strain containing the HBsAg surface antigen gene. Rhein Biotech also established a core cell bank (MSB Hansenulapolymorpha K3/8-1, strain ADW, 12/94) and performed all characterization tests in this bank.

Haemophilus influenzae type b:

The source organism for obtaining the cell substrate is Haemophilus influenzae type b, strain 760705. The strain was initially isolated from a boy aged 2 years and 2 months (born 14-8-74) in November 1976. Three subcultures of this strain were carried out before placement it was stored at -70°C at the Academic Medical Center (AMC) of the University of Amsterdam. This strain was provided to SIIPL as part of a collaboration between SIIPL and the Netherlands Vaccine Institute (NVI, the Netherlands).

IPV:

The strain and source of Salk poliovirus are listed below.

Poliovirus type 1:

Strain: Mahoney

Source: Bilthoven Biologicals, The Netherlands.

Poliovirus type 2:

Strain: MEF1

Source: Bilthoven Biologicals, The Netherlands.

Poliovirus type 3:

Strain: Saukett

Source: Bilthoven Biologicals, The Netherlands.

As used herein, the term contain or variants thereof, such as contains or containing, should be understood to mean the inclusion of the specified element, integer or step, or group of elements, integers or stages, but not the exclusion of any other element, integer or step

- 25 043311 or stage, or group of elements, integers or stages.

Use of the term at least or at least one implies the use of one or more elements, ingredients or amounts, as the use may be in an embodiment of the invention to achieve one or more desired objectives or results. Although certain embodiments of the invention have been described, these embodiments have been presented by way of example only and are not intended to limit the scope of the invention. Variations or modifications in the composition of this invention within the scope of the invention may be made by those skilled in the art after reading this description. Such variations or modifications are entirely within the spirit of the present invention.

The numerical values given for the various physical parameters, dimensions and quantities are approximate values only, and values greater than the numerical value assigned to the physical parameters, dimensions and quantities are intended to be included within the scope of the invention unless otherwise stated in the specification.

Although this description places considerable emphasis on the features of the preferred embodiment of the invention, it should be understood that many additional features can be added and that many changes can be made to the preferred embodiment without departing from the principles of the present invention. These and other changes in the preferred embodiment of the present invention will be apparent to those skilled in the art from the present description, it being clearly understood that the foregoing description is to be interpreted only as an illustration of the invention and not as a limitation thereof.

Advantages of the invention

The present invention described above has several technical advances and advantages, including, but not limited to, the creation of a combination vaccine composition containing D, T, wP, HBsAg, PRP-TT Hib conjugate and IPV, as well as a method for its preparation. Compared to other combination vaccine composition, the present invention provides the following advantages:

1. Fully liquid combination vaccine.

2. Reduced dose of IPV antigen compared to the standard dose, demonstrating comparable effectiveness compared to the standard dose (40-8-32 DU).

3. Increased immunogenicity of antigens D, T, wP, HepB, Hib, IPV.

4. Increased stability at 2-8°C and room temperature, when tested for 12 months.

5. Highly purified diphtheria toxoids (D) and tetanus toxoids (T) produced using semi-synthetic media free of transmissible spongiform encephalopathy (TSE) or bovine spongiform encephalopathy (BSE).

6. B. pertussis whole cell antigen (wP) includes Bordetella pertussis strains 134, 509, 25525 and 6229 in a ratio of 1:1:0.25 0.25, thereby improving the effectiveness and immunogenicity against B. pertussis.

7. An improved method for inactivating the whole cell component of B. pertussis (wP) using a combination of heat and formaldehyde inactivation. The method does not use thiomersal, and the whole cell pertussis antigen remains homogeneous rather than lumpy, resulting in reduced reactogenicity and better efficacy over a longer period of time.

8. Low content of free PRP (less than 7%) in the total mass of the PRP-TT conjugate Haemophilus influenzae type b.

9. The percentage of Hib antigen adsorption to any adjuvant is less than 20%.

10. Improved adsorption profile of diphtheria toxoid antigen (D), tetanus toxoid antigen (T) and hepatitis B surface antigen (HepB) adsorbed individually to aluminum phosphate adjuvant, thereby increasing potency and immunogenicity.

11. Minimum total alum content (Al ³⁺ ), which ensures reduced reactogenicity.

12. An optimized concentration of 2-phenoxyethanol (2-PE) and at least one paraben ester (methylparaben or propylparaben) as a preservative to effectively maintain the antimicrobial capacity of a multi-dose all-liquid combination vaccine.

Examples

The following examples are included to demonstrate preferred embodiments of the present invention. It will be understood by those skilled in the art that the compositions and techniques disclosed in the following examples are techniques proposed by the present inventors that have worked well in the practice of the invention, and thus may be considered preferred methods for its implementation. implementation. However, those skilled in the art should, in light of the present disclosure, appreciate that many changes may be made to the specific disclosed embodiments of the invention to achieve the same or a similar result without departing from the spirit and scope of the invention.

- 26 043311 em.

Example 1 Various combinations of vaccine compositions according to the present invention

Table 25

Combination vaccine containing IPV (Salk strain type 1 (Mahoney), or type 2 (MEF), _________________________, or type 3 (Saukett))_________________________

Item No. Composition components Combined composition by invention h in accordance with this per dose of 0.5 ml] 1 2 3 4 5 6 7 8 9 10 eleven 12 1 Diphtheria toxoid (D) 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf 20 Lf 20 Lf 20 Lf 20 Lf 2 Tetanus toxoid (T) 02 Lf 02 Lf 02 Lf 02 Lf 02 Lf 02 Lf 02 Lf 02 Lf 4 Lf 4 Lf 4 Lf 4 Lf 3 Inactivated B. pertussis antigen (wP) 12 IOU 12 IOU 12 IOU 12 IOU 12 IOU 12 IOU 12 IOU 12 IOU 14 IOU 14 IOU 14 IOU 14 IOU 4 HBs antigen 8 mcg 8 mcg 8 mcg 8 mcg 8 mcg 8 mcg 8 mcg 8 mcg 15 mcg 15 mcg 15 mcg 15 mcg 5 Hib conjugated antigen PRP-TT 8 mcg PRP 8 µg PR R 8 µg PR R 8 µg PR R 8 µg PR R 8 µg PR R 8 µg PR R 8 µg PR R 10 µg PR R 10 µg PR R 10 µg PR R 10 µg PR R 6 Inactivated poliovirus (IPV) Type 1 (Dantigen units) 7.5 or 10 or 20 or 40 7.5 or 10 or 20 or 40 7.5 or 10 or 20 or 40 7.5 or 10 or 20 or 40 7.5 or 10 or 20 or 40 7.5 or 10 or 20 or 40 7.5 or 10 or 20 or 40 7.5 or 10 or 20 or 40 7.5 or 10 or 20 or 40 7.5 or 10 or 20 or 40 7.5 or 10 or 20 or 40 7.5 or 10 or 20 or 40 Type 2 (Dantigen units) 1.5 or 2 or 4 or 8 - 1.5 or 2 or 4 or 8 - 1.5 or 2 or 4 or 8 - 1.5 or 2 or 4 or 8 - 1.5 or 2 or 4 or 8 - 1.5 or 2 or 4 or 8 - Type 3 (Dantigen units) 6 or 10 or 16 or 32 6 or 10 or 16 or 32 6 or 10 or 16 or 32 6 or 10 or 16 or 32 6 or 10 or 16 or 32 6 or 10 or 16 or 32 6 or 10 or 16 or 32 6 or 10 or 16 or 32 6 or 10 or 16 or 32 6 or 10 or 16 or 32 6 or 10 or 16 or 32 6 or 10 or 16 or 32 7 Total aluminum content (AP ⁺ ) (as aluminum phosphate) Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg 8 2-phenoxyethanol 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 9 Methylparaben 0.9 mg 0.9 mg 0.9 mg 0.9 mg - - - - 0.9 mg 0.9 mg 0.9 mg 0.9 mg 10 Propylparaben 0.1 mg 0.1 mg - - 0.1 mg 0.1 mg - - 0.1 mg 0.1 mg - -

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Item No. Composition components Combination composition according to the present invention [per 0.5 ml dose] 13 14 15 16 17 18 19 20 21 22 23 24 Ί Diphtheria 20 Lf 20 20 20 25 25 25 25 25 25 25 25 toxoid (D) Lf Lf Lf Lf Lf Lf Lf Lf Lf Lf Lf 2 Tetanus toxoid (T) 4 Lf 4 Lf 4 Lf 4 Lf 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf Inactivated bathroom 14 14 14 14 16 16 16 16 16 16 16 16 B. pertussis antigen (wP) IOU IOU IOU IOU IOU IOU IOU IOU IOU IOU IOU IOU 4 HBs antigen 15 15 15 15 15 15 15 15 15 15 15 15 mcg mcg mcg mcg mcg mcg mcg mcg mcg mcg mcg mcg Conjugiro- 10 10 10 10 13 13 13 13 13 13 13 13 bathroom mcg mcg mcg mcg mcg mcg mcg mcg mcg mcg mcg Hib antigen mcg PRP PR PR PR PR PR PR PR PR PR PR PR PRP-TT Inactivated poliovirus R R R R R R R R R R R (IPV) 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 or or or or or or or or or or or or Type 1 10 10 10 10 10 10 10 10 10 10 10 10 (units D- or or or or or or or or or or or or antigen) 20 20 20 20 20 20 20 20 20 20 20 20 or or or or or or or or or or or or 40 40 40 40 40 40 40 40 40 40 40 40 1.5 1.5 1.5 1.5 1.5 1.5 6 or or or or or or Type 2 2 2 2 2 2 2 (units D- or — or — or — or or — or — antigen) 4 4 4 4 4 4 or or or or or or 8 8 8 8 8 8 6 6 6 6 6 6 6 6 6 6 6 6 or or or or or or or or or or or or Type 3 10 10 10 10 10 10 10 10 10 10 10 10 (units D- or or or or or or or or or or or or antigen) 16 16 16 16 16 16 16 16 16 16 16 16 or or or or or or or or or or or or 32 32 32 32 32 32 32 32 32 32 32 32 General content Not Not Not Not Not Not Not Not Not Not Not Not aluminum bo- bo- bo- bo- bo- bo- bo- bo- bo- bo- bo- bo- 7 (ARD more more more more more more more more more more more more (as 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 aluminum phosphate) mg mg mg mg mg mg mg mg mg mg mg mg h 2-phenoxy- 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 ethanol mg mg mg mg mg mg mg mg mg mg mg mg 9 Methylparaben - - - - 0.9 mg 0.9 mg 0.9 mg 0.9 mg - - - - 10 Drank through 0.1 0.1 0.1 0.1 0.1 0.1 paraben mg mg mg mg mg mg

Additionally, adjust the pH of the composition as described above to approximately 6.0-7.0 with sodium hydroxide/sodium carbonate and adjust the volume by adding saline (0.9%). The vaccine may contain traces of glutaraldehyde, formaldehyde, neomycin, streptomycin and polymyxin B, which are used in the manufacturing process.

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Table 26

Combination vaccine containing IPV (Sabin strain: type 1, type 2 and type 3)

Item No. Composition components Combination composition according to the invention I am in accordance with the present at a dose of 0.5 ml] 1 2 3 4 5 6 7 8 9 10 eleven 12 1 Diphtheria toxoid (D) 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf 20 Lf 20 Lf 20 Lf 20 Lf 2 Tetanus toxoid (T) 02 Lf 02 Lf 02 Lf 02 Lf 02 Lf 02 Lf 02 Lf 02 Lf 4 Lf 4 Lf 4 Lf 4 Lf 3 Inactivated antigen B. pertussis (wP) 12 IOU 12 IOU 12 IOU 12 IOU 12 IOU 12 IOU 12 IOU 12 IOU 14 IOU 14 IOU 14 IOU 14 IOU 4 HBs antigen 8 mcg 8 mcg 8 mcg 8 mcg 8 mcg 8 mcg 8 mcg 8 mcg 15 mcg 15 mcg 15 mcg 15 mcg 5 Hib conjugated antigen PRP-TT 8 mcg PRP 8 mcg PRP 8 mcg PRP 8 mcg PRP 8 mcg PRP 8 mcg PRP 8 mcg PRP 8 mcg PRP 10 mcg PRP 10 mcg PRP 10 mcg PRP 10 µg PR R 6 Inactivated poliovirus (IPV) Type 1 (D-antigen units) 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 Type 2 (D-antigen units) 4 or 8 or 16 - 4 or 8 or 16 - 4 or 8 or 16 - 4 or 8 or 16 - 4 or 8 or 16 - 4 or 8 or 16 - Type 3 (D-antigen units) 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 7 Total aluminum content (AP ⁺ ) (as aluminum phosphate) Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.5 5 mg 8 2phenoxyethanol 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 9 Methylparaben 0.9 mg 0.9 mg 0.9 mg 0.9 mg - - - - 0.9 mg 0.9 mg 0.9 mg 0.9 mg 10 Propylparaben 0.1 mg 0.1 mg - - 0.1 mg 0.1 mg - - 0.1 mg 0.1 mg - -

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Item No. Composition components Combination composition according to the present invention [per 0.5 ml dose] 13 14 15 16 17 18 19 20 21 22 23 24 1 Diphtheria toxoid (D) 20 Lf 20 Lf 20 Lf 20 Lf 25 Lf 25 Lf 25 Lf 25 Lf 25 Lf 25 Lf 25 Lf 25 Lf 2 Tetanus toxoid (T) 4 Lf 4 Lf 4 Lf 4 Lf 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf 10 Lf 3 Inactivated antigen B. pertussis (wP) 14 IOU 14 IOU 14 IOU 14 IOU 16 IOU 16 IOU 16 IOU 16 IOU 16 IOU 16 IOU 16 IOU 16 IOU 4 HBs antigen 15 mcg 15 mcg 15 mcg 15 mcg 15 mcg 15 mcg 15 mcg 15 mcg 15 mcg 15 mcg 15 mcg 15 mcg 5 Hib conjugated antigen PRP-TT 10 mcg PRP 10 mcg PRP 10 mcg PRP 10 mcg PRP 13 mcg PRP 13 mcg PRP 13 mcg PRP 13 mcg PRP 13 mcg PRP 13 mcg PRP 13 mcg PRP 13 mcg PRP 6 Inactivated poliovirus (IPV) Type 1 (D-antigen units) 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 5 or 10 or 20 Type 2 (D-antigen units) 4 or 8 or 16 - 4 or 8 or 16 - 4 or 8 or 16 - 4 or 8 or 16 4 or 8 or 16 - 4 or 8 or 16 - Type 3 (D-antigen units) 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 10 or 16 or 32 7 Total aluminum content (Al ³⁺ ) (as aluminum phosphate) Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg Not more than 0.55 mg 8 2phenoxyethanol 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 2.5 mg 9 Methylparaben - - - - 0.9 mg 0.9 mg 0.9 mg 0.9 mg - - - - 10 Propylparaben 0.1 mg 0.1 mg - - 0.1 mg 0.1 mg - - 0.1 mg 0.1 mg - -

Additionally, adjust the pH of the composition as described above to approximately 6.0-7.0 with sodium hydroxide/sodium carbonate and adjust the volume by adding saline (0.9%). The vaccine may contain traces of glutaraldehyde, formaldehyde, neomycin, streptomycin and polymyxin B, which are used in the manufacturing process.

Example 2. Method for obtaining the total mass of Haemophilus influenzae type b conjugate

A general view of the production stages is presented in the block diagram in Fig. 1. Each of the 53 steps of the method is briefly described below:

Stage 1. Seed crop stage I.

Shake flask (S1):

A test tube with a working batch of inoculum is used to inoculate a shake flask at the inoculum stage, the flask contains inoculum medium filtered through a filter with a pore size of 0.22 microns. Use a 125 ml disposable PETG flask with a working volume of 25 ml. This stage is carried out in a shaker-incubator with controlled mixing (200±50 rpm) and temperature (36±2°C). Once adequate bacterial growth has been achieved (OD ₅₉₀ >1.0), the culture is transferred to the next seed culture step (step S2), which is described in step 2. To ensure the purity of the culture (Gram-negative coccobacilli), Gram staining is performed as a production control.

Stage 2: Seed crop stage II.

Shake flask (S2):

The seed stage S2 consists of 2-liter Fernbach flasks (S2A and S2B) with a working volume of 800 ml. Flask S2A is used to measure OD ₅₉₀ until OD ₅₉₀ meets the acceptance criteria, and flask S2B is used for inoculation in step S3. Both flasks are loaded with filter-sterilized medium, which is identical to the medium from the seed culture stage S1. The Stage S1 flask is used to inoculate both Stage II shake flasks. This stage is carried out in a shaker incubator with controlled mixing (200±50 rpm) and temperature (36±2°C). Once adequate bacterial growth has been achieved (OD ₅₉₀ >1.0), the culture is transferred to the next seed culture step (step S3), which is described in step 3. To ensure the purity of the culture (Gram-negative coccobacilli), Gram staining is performed as a production control.

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Stage 3: Seed crop stage III.

Fermenter:

The seed stage S3 consists of a 120 L fermenter with a working volume of 35 L. The fermenter is loaded with a medium identical to the previous seed culture steps. The S2 stage flask is used to inoculate the seed culture fermenter. Growth is carried out at temperature (36±2°C), DO (dissolved oxygen) (set point 10%), stirring (300-600 rpm), aeration (1-5 l/min) and back pressure (0.2 bar ) in the seed culture fermenter. Once adequate bacterial growth has been achieved (OD ₅₉₀ >1.0), the culture is transferred to the next production step (step S4), which is described in step 4. To ensure the purity of the culture (Gram-negative coccobacilli), Gram staining is performed as a production control.

Stage 4: Fermentation in a volume of 1200 l

The 1200 liter production fermenter has a working volume of 800 liters. It is loaded with the main components of the medium and sterilized in place with steam. Various additives are then added after passing through a 0.22 micron filter. The fermenter is inoculated with the stage S3 culture obtained in stage 3. Fermentation is carried out at a controlled level of dissolved oxygen (20% - set value), temperature (36±2°C), pH (7.1-7.4), stirring (40- 400 rpm), aeration (50-300 l/min) and back pressure (0.2 bar). Nutrients are added twice during the fermentation process. Growth is monitored by measuring OD ₅₉₀ (OD ₅₉₀ > 3.5) and fermentation is considered complete once the stationary phase is reached. During growth and stationary phase, the polysaccharide product is secreted and accumulates in the culture fluid. To ensure the purity of the culture (Gram-negative coccobacilli), Gram staining is performed as a production control.

Stage 5: Formalin treatment

Reduction of bioburden is achieved at this stage through the use of a chemical agent (formalin). 0.1% formaldehyde is added and the fermented broth is incubated for 2 hours at 37°C. After treatment with formalin, the container is quickly cooled to a temperature of <15°C. The addition of formaldehyde has been approved to reduce bioburden. This is confirmed by plating on culture dishes after the incubation period. The reduced bioburden broth is ready for collection as described in step 6.

Step 6: Culture collection by continuous centrifugation

Continuous centrifugation is used as the main step in culture collection. This step is performed to separate the crude broth containing the polysaccharide from the inactivated biomass. A continuous centrifuge is used to remove >90% of the biomass as measured by OD ₅₉₀ reduction. The centrifuge operates at approximately 15,000 g and a liquid flow of 200-500 l/h. The centrifuged supernatant is further processed as described in step 7.

Stage 7: Depth filtration through 50LP filter

The centrifuged supernatant is passed through a 50LP depth filter to remove coarse material such as cell debris. This step allows the product to pass through the filtrate and is complemented by an additional depth filter as described in step 8.

Stage 8: Depth filtration through 90LP filter

The filtrate from the 50LP depth filter is then passed through a 90LP depth filter (0.22 µm nominal) to further remove any insoluble material that may not have been retained by the previous depth filter. This step ensures that the filtrate is virtually free of cell debris and can reliably pass through a 0.22 µm filter. The subsequent filtering step is described in step 9.

Steps 9 and 10: Filtration through 0.22 µm filter:

The filtrate from the 90LP depth filter is then passed through a 0.22 µm pore size filter and the filtrate is collected in a collection tank.

Steps 11 and 12: Concentration of the fraction with a mass of more than 100 kDa and diafiltration:

This step is carried out to remove medium components and low molecular weight impurities. In addition, concentration is carried out to reduce the working volume. The molecular weight cutoff of 100 kDa was chosen because the molecular weight of Hib polysaccharide (PRP) is >500 kDa. The broth is concentrated approximately 10-fold and then diafiltered with at least 5 volumes of 0.01 M PBS buffer (pH 7.2). The resulting product in the retentate is called crude PRP and is further processed as described in step 13. The concentrated broth is transferred to the further processing area through a transfer port through a 0.22 µm pore size filter to ensure that no bacteria enter the area further processing.

Step 13: Precipitation of STAV

CTAB (cetyl trimethylammonium bromide) is a cationic detergent that is used to precipitate polysaccharides. CTAB consists of a hydrophilic region as well as a hydrophobic part and precipitates proteins, nucleic acids and polysaccharides. The crude PRP obtained in step 12 is precipitated at a CTAB concentration of 1% and incubated for >2 hours. Collection of the CTAB precipitate is described in step 14.

Steps 14, 15 and 16: Centrifugation, collection and storage of CTAB sediment

The CTAB sediment is centrifuged in a continuous centrifuge at 15,000 rpm in SEZ-3,

FF. The CTAB precipitate is collected, weighed, aliquoted and stored at <-20°C for further processing. This is the first stop in the process.

Steps 17 and 18: Thawing and dissolving STAV paste

Thaw frozen CTAB paste to room temperature. The thawed sediment is dissolved in a 5.85% NaCl solution. Dissolution is carried out in a stirred tank and the polysaccharide product is dissolved in the aqueous phase. The reservoir contains undissolved material consisting of precipitated proteins and nucleic acids. This suspension is further processed as described in step 19.

Step 19: Centrifugation

The material obtained in step 18 is centrifuged at a temperature of 2-8°C, 5000-6500 rpm for 20-30 minutes to remove undissolved material. The centrifuged supernatant is collected and processed as described in step 20.

Step 20: Precipitation with 72% ethanol

72% ethanol is used to precipitate PRP. 96% ethanol is used to obtain a final ethanol concentration of 72% in the supernatant obtained in step 19.

This precipitation is carried out at a temperature of 2-8°C overnight. The resulting precipitate is collected as described in step 21.

Steps 21 and 22: Centrifugation and sediment dissolution:

The precipitate obtained by the action of 72% ethanol is collected by centrifugation at a temperature of 28°C, 5000-6500 rpm for 20-30 minutes. The resulting precipitate is dissolved in water for injection until visually transparent. Subsequent processing of the solubilized sediment is described in step 23.

Step 23: Precipitation of DOC and 32% ethanol

6% sodium acetate and 1% sodium deoxycholate (DOC) are added to the material obtained in step 22. 96% ethanol is used to obtain a final ethanol concentration of 32%. Both DOC and 32% alcohol cause protein impurities to precipitate, allowing the polysaccharide to remain in the liquid phase. Precipitation is carried out at a temperature of 2-8°C overnight (at least 8 hours).

Step 24: Centrifugation

The material obtained in step 23 is centrifuged at a temperature of 2-8°C, 5000-6500 rpm for 20-30 minutes to remove sediment. The centrifuged supernatant is collected and processed as described in step 25.

Stage 25: Depth and carbon filtration

The supernatant solution obtained in step 24 contains soluble PRP and is subjected to depth filtration followed by carbon filtration to remove nucleic acids and colorants. The removal of nucleic acids is monitored by periodically measuring the optical density at a wavelength of 260 nm (A ₂₆₀ ). Once the target A ₂₆₀ is reached, the solution is filtered through a 0.22 μm filter and the filtered solution is further processed as described in step 26.

Step 26: Precipitation with 64% ethanol

The filtered material obtained in step 25 is further precipitated with 96% ethanol to a final concentration of 64% ethanol. This precipitation is carried out at a temperature of 2-8°C overnight. The resulting precipitate is collected by centrifugation and processed as described in step 27.

Step 27: Collection and dissolution of sediment:

The supernatant is decanted and removed to collect the sediment. The sediment is dissolved in water for injection at room temperature.

Step 28: Concentration of the fraction with a mass greater than 300 kDa and diafiltration

The precipitate solution is concentrated using a 300 kDa nominal molecular weight cutoff (NMWCO) membrane. It is additionally subjected to diafiltration with at least 8 volumes of water for injection. The resulting retentate is further processed as described in step 29.

Steps 29 and 30: Filtration through 0.22 µm filter and storage of purified PRP

The ultrafiltration (UF) NMWCO 300 kDa retentate is passed through a 0.22 μm pore size filter during the purification step to minimize bioburden. The resulting purified PRP is aliquoted and stored at <-20°C until further use as described in step 31. A sample of the purified PRP is sent for quality control analysis.

Step 31: Thaw and Combine:

Depending on the batch size of the conjugate, an appropriate amount of the native polysaccharide obtained in step 30 is thawed. The combined material is analyzed for PRP content, which is required for further processing as described in step 32.

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Step 32: Concentration through UF membrane with NMWCO 100 kDa

The combined purified polysaccharide must have a minimum concentration (8-12 mg/ml) for further processing. If the polysaccharide concentration in the pooled solution is below the target value, the pooled polysaccharide solution is concentrated using a 100 kDa NMWCO UF membrane. A sample is taken after concentration to ensure that the minimum concentration is achieved in subsequent steps (step 33).

Step 33: Alkaline depolymerization

The concentrated polysaccharide (74 g/110 g equivalent) obtained in step 32 is depolymerized under mild alkaline conditions using a carbonate-bicarbonate buffer. Once the target polysaccharide size is reached, the depolymerized polysaccharide is activated as described in step 34.

Step 34: Polysaccharide Activation

The depolymerized polysaccharide obtained in step 33 is activated with cyanogen bromide. Activation is carried out in a nitrogen environment. Cyanogen bromide is a highly toxic chemical and appropriate precautions should be taken when handling it.

Step 35: Attaching the Linker

A freshly prepared adipic acid dihydrazide (ADH) solution is added over 6-10 minutes to the reaction mixture obtained in step 34. The reaction is carried out for 16 hours at a temperature of 210°C. The role of the ADH linker is to create amino groups in the polysaccharide necessary for the conjugation reaction.

Step 36: Concentration and Diafiltration

The reaction mixture obtained in step 35 is concentrated and volumetric diafiltered with phosphate-buffered saline (PBS) using a 10 kDa NMWCO UF membrane to remove free ADH. ADH removal is monitored by HPLC and diafiltration is continued until free ADH levels are below 5%. The resulting retentate is additionally subjected to diafiltration with at least 5 volumes of MES-NaCl buffer. It is further concentrated to achieve a concentration of at least 20 mg/ml. This concentrated treated PRP is stored at 2-8°C until further use as described in step 37.

Steps 37 and 38: Filtration through 0.22 µm filter and storage of treated PRP

The retentate from step 36 is passed through a 0.22 µm filter which is used in the purification step. This also ensures that bioburden levels are controlled during the process, which is carried out in a Class C area. The filtered activated polysaccharide is collected, sampled, aliquoted and stored at 2-8°C until further processing. A sample is taken from the pool of processed polysaccharides for analysis, which includes determination of PRP molecular size (kDa), PRP content, and degree of PRP activation. Further processing of the treated PRP is described in step 40.

Step 39: TT concentration through UF membrane with NMWCO 10 kDa and diafiltration

The conjugation reaction requires two components, a processed polysaccharide and a carrier protein (TT). The carrier protein is concentrated and diafiltered with MES-NaCl buffer using a NMWCO 10 kDa UF membrane. This diafiltered carrier protein is then further concentrated to a concentration of at least 20 mg/ml using the same membrane.

Stage 40: Conjugation

The conjugation reaction requires two components, a processed polysaccharide and a carrier protein (TT). The activated polysaccharide component is prepared in step 38. The carrier protein is prepared in step 39. The two components are mixed in appropriate amounts in the ratio PRP:TT=1:1 (w/w) in the presence of 1-ethyl-3-3-dimethylaminopropyl- carbodiimide (EDC) with stirring. The conjugation reaction is monitored by HPLC and continued until >85% protein conversion (conversion of free protein into conjugate) is achieved.

Step 41: Stopping the Reaction

After the conjugation reaction reaches the acceptance criteria for conversion (step 40), the reaction is stopped by quenching. The conjugation reaction is quenched using EDTA phosphate buffer. The conjugation reaction product is then processed as described in step 42.

Step 42: Filtration through 30SP and 0.22 micron filter

The conjugate obtained in step 41 is filtered through a 30SP filter followed by filtration through a 0.22 μm pore size filter. This will ensure that any large aggregates are removed. The filtered conjugate is processed as described in step 43.

Step 43: Ultrafiltration through UF membrane with NMWCO 300 kDa and diafiltration

The conjugation reaction mixture obtained in step 42 is diafiltered with 0.05% saline using a 300 kDa NMWCO UF membrane. Diafiltration is carried out to remove conjugation reagents and unreacted TT. The resulting retentate is further processed as described in step 44.

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Steps 44 and 45: Filtration through 0.22 µm filter and storage of crude conjugate

The retentate from step 43 is passed through a 0.22 µm filter which is used in the purification step. This also ensures that bioburden levels are controlled during the process, which is performed in a Class C area. The filtered crude conjugate is collected, sampled and stored at 2-8°C until further processing. Further processing of the crude conjugate is described in step 46.

Step 46: Dilution of the crude conjugate

The crude conjugate from step 45 is diluted with water for injection to a target concentration of 4 ± 1 mg/ml, if required, and further processed using the precipitation steps described in step 47.

Step 47: Ammonium sulfate precipitation

The diluted conjugate reaction mixture is further treated to remove free PRP using ammonium sulfate (50 w/v% stock solution). The precipitation stage is carried out at a temperature of less than 15°C with stirring. During the precipitation step, the conjugate precipitates and free PRP remains in the supernatant. After adding ammonium sulfate, the resulting suspension is stored at a temperature below 15°C without stirring for at least 12 hours.

Step 48: Collection and dissolution of sediment

The suspension obtained at step 47 is centrifuged at ~7000 g at a temperature of 2-8°C for 40±10 minutes. The supernatant is removed by decanting, and the resulting precipitate is dissolved in triphysiological solution.

Step 49: Diafiltration through NMWCO 300 kDa membrane

The solution obtained in step 48 is filtered through a 30SP depth filter and diafiltered with 20 mM Tris saline using a 300 kDa NMWCO membrane.

Step 50: Purification by Gel Filtration Chromatography

The solution obtained in step 49 is loaded onto an approximately 70 L GFC column containing Toyopearl HW-65F hydroxylated methacrylic polymer beads for size exclusion chromatography. The use of gel filtration chromatography on the treated conjugate (after ammonium sulfate precipitation) reduces the levels of free PRP in the resulting material. The column is eluted with 20 mM Tris 0.9% NaCl and fractions are collected based on absorbance A ₂₈₀ . Fractions meeting the acceptance criteria for free PRP, ratio and molecular size are pooled and the pooled pool is further processed as described in step 51.

Step 51: Diafiltration through NMWCO 300 kDa membrane

The pooled conjugate eluate obtained in step 50 is diafiltered with 20 mM Tris using a 300 kDa NMWCO UF membrane. This volume of retentate is designed to contain approximately 1 mg/ml PRP.

Steps 52 and 53: Filtration through a 0.22 µm filter

The total mass of conjugate obtained in step 51 is filtered through a 0.22 μm pore size filter in a Class A zone to ensure sterility. The 0.22 µm filter was tested for integrity. A sample of the filtered conjugate is sent to quality control for full analysis. The filtered conjugate is labeled Sterile General Hib Conjugate and stored at 2-8°C. The general conjugate is stored at 2-8°C for a maximum of 3 months, after which, if not in use, it can be stored at -70°C for a maximum period of 1 year.

The qualitative characteristics of the resulting Hib PRP-TT conjugated antigen were as follows:

PRP content (µg per 0.5 ml): 8.1

Ratio (PRP:TT): 0.5

Free PRP (%): 4.8%

PMW (kDa): 983

Average MW (kDa): 752

Example 3. Method for obtaining inactivated wP antigen

Method for inactivating whole cell pertussis antigen:

Optimization of the inactivation method is carried out after conducting various experiments, which include inactivation at a temperature of 56°C for 10 minutes in the presence of formaldehyde, inactivation at a temperature of 56°C for 15 minutes in the presence of formaldehyde, inactivation at a temperature of 56°C for 10 min in the presence of hymin, inactivation at 56°C for 15 min in the presence of hymin, and heating only at 56°C for 30 min. There is no significant difference in the effectiveness of these methods. Of these methods, inactivation at 56°C for 10 min in the presence of formaldehyde was selected because the pertussis cell mass obtained using this method is more uniform compared to the other methods mentioned above.

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The method for obtaining inactivated wP antigen includes the following steps:

a) inactivation at a temperature of 56°C for 10-15 minutes in the presence of strain formaldehyde

Bordetella pertussis 134

b) inactivation at a temperature of 56°C for 10-15 minutes in the presence of formaldehyde of the strain

Bordetella pertussis 509

c) inactivation at a temperature of 56°C for 10-15 minutes in the presence of formaldehyde of Bordetella pertussis strains 25525 and 6229

d) inactivation at a temperature of 56°C for 10-15 minutes in the presence of formaldehyde of Bordetella pertussis strain 6229

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

f) optional adsorption to an aluminum-based adjuvant.

This method does not use thiomersal and the whole cell pertussis antigen remains homogeneous rather than lumpy, which leads to reduced reactogenicity and provides better effectiveness over a longer period of time.

Example 4: Method for producing inactivated poliovirus (IPV)

1. Polyvirus can be grown in the following way:

a) The cell line CCL81-VERO (monkey kidney) is used as host cells for growing polioviruses, i.e. Sabin and Salk strains.

b) After infection of host cells with the desired strain of poliovirus and incubation for 72 hours, the medium containing the virus and cell debris is combined and collected in one container.

c) The filtrate is subjected to tangential flow filtration with a 100 kDa cassette; diafiltered using phosphate buffer and purified using anion exchange chromatography.

d) Viruses must be inactivated by appropriate inactivation methods before administration to patients.

2. Inactivation with formalin includes the following steps:

a) The purified pooled virus pool is buffer exchanged from phosphate buffer to Tris buffer in the range (30 to 50 mM) with a pH of 7 to 7.5;

b) To the above mixture add M-199 medium containing glycine (5 g/l);

c) Add 0.025% formaldehyde and then mix;

d) Then the mixture is incubated at a temperature of 37°C for 5-13 days with continuous stirring of the virus mass on a magnetic stirrer;

e) The mixture after incubation is subjected to intermediate tangential flow filtration (100 kDa, 0.1 m ² ) on day 7 and final filtration after inactivation;

f) The filtered volume is then stored at a temperature of 2-8°C;

g) Perform D-Ag determination using ELISA to determine D-antigen units;

h) The total monovalent pool of type 1, type 2 and type 3 IPVs is then mixed to form trivalent or divalent IPV (serotype Salk or Sabin);

i) Adjust the pH of the final composition to obtain a final composition with a pH of 6 to 6.8;

j) IPV antigen (Sabin or Salk strains) is then added to the final combination vaccine composition adsorbed on an adjuvant (aluminum phosphate) (the adjuvant is present in the combination vaccine), where the percentage of IPV antigen adsorption for IPV type 1 has been found to be range 10-30%, IPV type 2 in the range 60-100% and IPV type 3 in the range 0-25%.

3. Procedure for obtaining IPV (strains Sabin and Salk) by individual adsorption on aluminum salts:

a) Take the desired volume of autoclaved AlPO ₄ to obtain a final alum (Al ³⁺ ) concentration of 0.1 to 0.8 mg per dose in a 50 ml container;

b) Add the bulk of IPV with a certain number of D-antigen units and adjust the volume with a diluent (10xM-199 + 0.5% glycine);

c) Adjust the pH of the final composition to obtain a final composition with a pH of 6 to 6.8.

4. Monovalent pool adsorbed on alum, respectively, in a trivalent or divalent IPV composition (Salk or Sabin serotype)

Results:

At the same time, the percentage adsorption of IPV types 1, 2 and 3 (Sabin and Salk) on aluminum phosphate salts (AlPO ₄ ) was at least 90%.

The authors of the present invention were able to achieve a twofold reduction in the dose of poliovirus antigens (whereas the standard dose of poliovirus antigens for type 1 is 40 DU, for type 2 - 8DU, for type 3 - 32DU).

- 35 043311

Table 27

Adsorption studies of IPV strain Sabin on aluminum phosphate

Sample Titer (per dose) Viral particles (in thousands) % free in supernatant % Adsorbed on gel Type 1, AIPO4 Control 5.84 691 Not applicable Al ³⁺ 125 mcg/dose 3.49 3 0.43 99.57 Al ³⁺ 250 mcg/dose 3.09 1.2 0.17 99.83 Al ³⁺ 500 mcg/dose 2.94 0.87 0.12 99.87 Type 2, AIPO4 Control 5.49 309 Al ³⁺ 125 mcg/dose 3.15 1.41 0.45 99.5 Al ³⁺ 250 mcg/dose 3.09 1.23 0.39 99.6 Al ³⁺ 500 mcg/dose 3.09 1.23 0.39 99.6 Type 3, AIPO4 Control 5.59 389 Not applicable Al ³⁺ 125 mcg/dose 5.34 218 56.04 43.94 Al ³⁺ 250 mcg/dose 5.24 173 44.47 55.53 Al ³⁺ 500 mcg/dose 5.16 144 37.01 63.9

Example 5. Method for producing a combination vaccine

This example provides a brief description of the method for preparing a combination vaccine composition containing D, T, wP, HBsAg, Hib PRP-TT conjugate, IPV and preservative:

Component I - diphtheria toxoid adsorbed on alum

Component II - tetanus toxoid adsorbed on alum

Component III - wP antigen (as described in example 3)

Component IV - hepatitis B surface antigen adsorbed on alum

Component V - Hib PRP conjugate (as described in example 2)

Component VI - IPV antigen (as described in example 4)

1. Preparation of component I containing diphtheria toxoid adsorbed on alum:

a) Transferring aluminum phosphate to a container/container;

b) Reduction of diphtheria toxoid;

c) Adjust the pH to between 4.5 and 5.5 using acetic acid/sodium hydroxide;

d) Waiting for stabilization;

e) Adjust the pH value to the range of 5.5 to 6.5 using sodium hydroxide/sodium carbonate;

f) Waiting for stabilization.

2. Preparation of component II containing tetanus toxoid adsorbed on alum:

a) Transferring aluminum phosphate to a container/container;

b) Addition of tetanus toxoid;

c) Adjust the pH to between 4.5 and 5.5 using acetic acid/sodium hydroxide;

d) Waiting for stabilization;

e) Adjust the pH value to the range of 5.5 to 6.5 using sodium hydroxide/sodium carbonate;

f) Waiting for stabilization.

3. Preparation of component IV containing hepatitis B surface antigen adsorbed on alum:

a) Transferring aluminum phosphate to a container/container;

b) Addition of hepatitis B surface antigen;

c) Adjust the pH to between 4.5 and 5.5 using acetic acid/sodium hydroxide;

d) Waiting for stabilization;

e) Adjust the pH value to the range of 5.5 to 6.5 using sodium hydroxide/sodium carbonate;

f) Waiting for stabilization.

4. A method for producing a combination vaccine composition containing D, T, wP, HBsAg, Hib PRP-TT conjugate, IPV and a preservative.

- 36 043311

1. Add saline solution to the mixing container/container;

2. Adding component I;

3. Mixing component II with component I and stirring at room temperature for 30-45 minutes;

4. Adding component III to the above mixture, followed by stirring at room temperature for 30-60 minutes;

5. Adding component IV to the mixture obtained in step 4, followed by stirring at room temperature for 30-60 minutes;

6. Adding component V to the mixture obtained in step 5, followed by stirring at a temperature of 6-16°C for 30-60 minutes;

7. Adding component VI to the mixture obtained in step 6, followed by stirring at a temperature of 6-16°C;

8. Add one of the combinations of preservatives described below to the mixture obtained in step 7 at a temperature of 6-16°C.

a) 2-phenoxyethanol in an amount of from about 1 mg per 0.5 ml to 6 mg per 0.5 ml (v/v); or

b) 2-phenoxyethanol at about 1 mg per 0.5 ml to 6 mg per 0.5 ml (v/v) and methylparaben used at a concentration of 0.1-1.5 mg per 0.5 ml (w/v); or

c) 2-phenoxyethanol at about 1 mg per 0.5 ml to 6 mg per 0.5 ml (v/v) and propylparaben used at a concentration of 0.05-0.2 mg per 0.5 ml (w/v); or

d) 2-phenoxyethanol in amounts ranging from about 1 mg per 0.5 ml to 6 mg per 0.5 ml (v/v), methylparaben used at a concentration of 0.1-1.5 mg per 0.5 ml (w/v), and propylparaben used at a concentration of 0.05-0.2 mg per 0.5 ml (w/v).

9. Check the pH value, if necessary adjust the pH value to the range of 6.0 to 7.5 using sodium hydroxide/sodium carbonate.

10. Bringing the mixture obtained at stage 9 to the final volume with physiological solution (0.9%), followed by stirring for 3 hours.

Example 6. Antigen adsorption, activity and stability profile.

Table 28

This table provides a summary of the percentage of adsorption of individual antigens, activity profile and stability of individual antigens in the SIIPL combination vaccine at 2-8°C for 12 months

Index Limits/Characteristics Day 0 6 months 12 months Hepatitis B in-vivo activity *R.P (95% **CL) (0.61-1.12) 0.83 N.d. Compliant Contents Hib PRP Actual 9.3 8.46 (µg/0.5 ml) (Total PRP) meaning µg/0.5 ml µg/0.5 ml 10.03 Free PRP (%) 8 n.d. n.d. 98.5120 Diphtheria activity No less ME on N.D. 95.8463 IU per dose component (ME per dose) 30 IU per dose. D ⁰³ U (69.9650137.247) 139.030 Tetanus activity No less ME on N.d. 382,079 ME per dose component (ME per dose) 40 IU per dose dose (88.2850208.688) 4.6749 4.8410 Whooping cough activity No less ME on ME on 5.131 component (ME per dose) 4 IU per dose dose (2.6492- dose (2.7331- 8.2763) 8.6081) Hepatitis B adsorption (%) Actual value 89.44 82.65 75.52 Adsorption of tetanus Actual 63.0 59.0 n.d. component(%) meaning Adsorption of diphtheria Actual 81.0 72.0 n.d. component (%) meaning D-antigen (DU per 0.5 ml) (= 75% of nominal Type 1= 20 DU per 0.5 ml 22,414 Corresponding Corresponding values are acceptable) welcomes welcomes Type 2= 4 DU per 0.5 ml 4,692 Corresponding Corresponding welcomes welcomes Type 3= 16 DU per 0.5 ml 22,084 Corresponding Corresponding welcomes welcomes General content No more 0.2768 n.d. n.d. aluminum 0.6 mg per 0.5 ml

*R.P - Relative activity **CL - Confidence level n.d. - No data

- 37 043311

Table 29

Brief description of the percentage of adsorption of individual antigens, activity profile and stability of individual antigens in a combination vaccine at a temperature of 25±2°C for 12 months

Index Limits/Characteristics Day 0 6 months 12 months Hepatitis B invivo activity *R.P (95% **CL) At least 1.0 Compliant n.d. Compliant Hib PRP content (µg/0.5 ml) (Total PRP) Actual value 8.6 mcg/0.5 ml 8.20 mcg/0.5 ml n.d. Activity of the diphtheria component (ME per dose) At least 30 IU per dose. 98.5120 IU per dose (69.9650137.247) n.d. 96.5482 IU per dose (65.9292137.687) Activity of the tetanus component (ME per dose) At least 40 IU per dose 139.030 IU per dose (88.2850208.688) n.d. n.d. Activity of the pertussis component (ME per dose) At least 4 IU per dose 4.6749 ME per Dose (2.64928.2763) 4.5170 ME per Dose (2.48948.2672) 3.4899 ME per Dose (1.8699*6.4750) Hepatitis B adsorption (%) Actual value 89.44 83.92 83.00 Adsorption of tetanus component (%) Actual value 59.0 31.0 40.0 Adsorption of diphtheria component (%) Actual value 79.0 72.0 69.0 D-antigen (DU per 0.5 ml) (= 75% of nominal value acceptable) Type 1=20 DU per 0.5 ml 22.414 Compliant Compliant Type 2= 4 DU per 0.5 ml 4.692 Compliant Compliant Type 3= 16 DU per 0.5 ml 22.082 Compliant Compliant Total aluminum content Not more than 0.6 mg per 0.5 ml 0.2846 n.d. n.d.

n.d. - No data

Table 30 Efficacy of reduced-dose and standard-dose IPV combination vaccine in vivo

Item No. Description Poliovirus type 1 Poliovirus type 3 Result Efficiency lower limit Upper limit Efficiency lower limit Upper limit 1 Hexavalent with 408-32 DU IPV 253.3% 124.9% 705.6% 212.2% 95.3% 755.5% Acceptable 2 Hexavalent with 204-16 DU IPV 164.4% 63.9% 571.3% 143.2% 64.3% 418.6% Acceptable 3 Hexavalent with 204-16 DU IPV 170.0% 76.4% 472.5% 132.3% 62.6% 340.5% Acceptable 4 Poliovac with full dose IPV 98.5% 30.9% 279.4 122.8 57.3% 269.8% Acceptable

results

Batches of the hexavalent vaccine made with half the strength of IPV have shown promising results in trials.

The in vivo efficacy of the IPV hexavalent vaccine produced with half the IPV strength was found to be comparable to the currently available vaccine (Poliovac in the market manufactured by SIIPL) with the full dose of IPV.

Example 7 Antimicrobial Test

The present inventors, in developing multi-dose combination vaccines containing D, T, wP, Hib, HBsAg and IPV vaccines, tested their antimicrobial properties by first adding 2-phenoxyethanol (2-PE), which is commonly used as a preservative in the art. at a concentration of 2.5 mg per 0.5 ml dose. However, 2-PE was found to have weaker antimicrobial activity than thiomersal against yeasts and fungi in a DPT-based combination vaccine.

Increasing the amount of 2-PE (preservative) to meet the required criteria may cause safety concerns in young children receiving the vaccine and may also affect the stability of the final products. In addition, the amount of preservative(s) contained in vaccines must meet the requirements defined in the US Pharmacopoeia, the European Pharmacopoeia, the WHO Pharmacopoeia, or a combination thereof, regarding vaccine safety.

In this regard, the authors of the present invention conducted experiments to develop a new

- 38 043311 a composition that can meet the requirements for antimicrobial activity by combining 2-PE with another preservative, such as paraben, in a multi-dose combination vaccine that meets the criteria for safety and antimicrobial activity. In the present invention, the test for antimicrobial activity was carried out in accordance with the criteria of the European Pharmacopoeia category

B (EP-B) requested by WHO for vaccine products.

Table 31

Details of the different combinations and concentrations of preservatives __________tested with the combination vaccine_____

Combinations 1 2 3 4 5 6 Methylparaben (MP) - 0.18% 0.18% 0.18% - 0.18% Propylparaben(PP) - 0.02% 0.02% - 0.02% 0.02% 2-phenoxyethanol (PE) 0.5% 0.5% 0.4% 0.5% 0.5% -

Antimicrobial efficacy screening

The hexavalent combination vaccine preparations described in Example 1 were inoculated with a total of six microorganisms, including four different species of bacteria - Staphylococcus aureus (ATCC NO.- 6538), Pseudomonas aeruginosa (ATCC NO.- 9027), Escherichia coli (ATCC NO.- 9027), Escherichia coli (ATCC NO.- 6538). 8739) and Staphylococcus arlettae (environmental isolate EMI); one yeast fungus - Candida albicans (ATCC NO.- 10231) and one fungus - Aspergillus brasiliensis (ATCC NO.- 16404) in an amount of 10 ⁵ to 10 ⁶ CFU/ml in vaccine preparations at 0 h, respectively. Bacterial, fungal, and yeast samples were then collected at 0, 24 h, 7, 14, and 28 days, cultured in solid media, colony counts between 3 and 5 days, and logarithmically reduced colony counts. The results are shown in the table below. 38.

Table 32

Antimicrobial efficacy testing results

Item no. Culture CFU highlighted! ml positive control (0 hours) Number of isolated CFU/ml results 24 hours ^7th day ^14th day ^Day 28 Combination 1 0.5% 2PE S. aureus 2ХУ ⁵ /ml 87000 900 0 - Acceptable P. aeruginosa 1.8 X 10 ⁵ /ml 28000 100 0 - E.coli YUH Yu ⁵ /ml 9000 0 - - S.arlettae (EMI) 2.8 X Yu ⁵ /ml 114000 1600 100 0 C.albicans 1.9 X 10 ⁵ /ml N.D. 400 0 - A. brasiliensis 2.4 X ¹⁵ /ml n.d. 1600 100 0 Combination 2 0.5% 2PE+0.18%MR+0.0 2%PP S. aureus 2ХУ ⁵ /ml 0 - - - Acceptable P. aeruginosa 1.8 X Yu ⁵ /ml 0 - - - E.coli YUH Yu ⁵ /ml 0 - - - S.arlettae (EMI) 2.8 X Yu ⁵ /ml 0 0 - - C.albicans 1.9X10 ⁵ /ml n.d. 0 - - A. brasiliensis 2.4 X ¹⁵ /ml n.d. 0 - - Combination 3 0.4% 2PE+0.18%MR+0.0 2%PP S. aureus 2ХУ ⁵ /ml 10 0 - - Acceptable P. aeruginosa 1.8Х Yu ⁵ /ml 0 - - - E.coli YUH Yu ⁵ /ml 0 - - - S.arlettae (EMI) 2.8 X Yu ⁵ /ml 10 0 - - C.albicans 1.9Х Yu ⁵ /ml n.d. 0 - - A. brasiliensis 2.4 X ¹⁵ /ml n.d. 0 - - Combination 4 0.5% 2PE+0.18%MR S. aureus 2ХУ ⁵ /ml 10 0 - - Acceptable P. aeruginosa 1.8 X Yu ⁵ /ml 170 10 0 - E.coli YUH Yu ⁵ /ml 0 - - - S.arlettae (EMI) 2.8 X Yu ⁵ /ml 20 0 - - C.albicans 1.9Х Yu ⁵ /ml n.d. 0 - - A. brasiliensis 2.4 X ¹⁵ /ml n.d. 0 - - Combination 5 0.5% 2PE + 0.02% PP S. aureus 2 X 10 ⁵ /ml 20 0 - - Acceptable P. aeruginosa 1.8Х Yu ⁵ /ml 0 - - - E.coli YUH Yu ⁵ /ml 10 0 - - S.arlettae (EMI) 2.8 X Yu ⁵ /ml 100 0 - - C.albicans 1.9X10 ⁵ /ml n.d. 0 - - A. brasiliensis 2.4 X ¹⁵ /ml n.d. 10 0 - Combination 6 0.18%MR+0.02%RR S. aureus 2ХУ ⁵ /ml 70000 700 0 - Acceptable P. aeruginosa 1.8 X Yu ⁵ /ml 21000 0 0 - E.coli YUH Yu ⁵ /ml 6000 0 0 - S.arlettae (EMI) 2.8 X Yu ⁵ /ml 101000 900 0 - C. albicans 1.9 X Yu ⁵ /ml n.d. 100 0 - A. brasiliensis 2.4 X ¹⁵ /ml n.d. 700 0 -

n.d. - No data; 0.5% 2PE - 2.5 mg/0.5 ml dose; 0.4% 2PE - 2 mg/0.5 ml dose; 0.18%MR - 0.9 mg/0.5 ml dose; 0.02% PP - 0.1 mg/0.5 ml dose; CFU - colony-forming unit.

-

Claims (26)

results It was found that all hexavalent vaccines produced in different combinations met the preservative efficacy according to European Pharmacopoeial Category B. However, it was found that the effectiveness varied between different combinations. The preservative efficacy of the hexavalent vaccine containing 2PE, MR and PP was very effective compared to other combinations of preservatives, namely 2PE alone, 2PE with PP, 2PE with MR and PP with MR. It was also noted that the preservative effectiveness of the hexavalent vaccine containing 0.5% 2PE with PP and MR was more effective compared to the same combinations, but with 0.4% 2PE. Example 8: Comparison of SIIPL Reduced Dose Combination Vaccine with Easy Six Vaccine (Panacea) Table 33 This table provides a comparison of the percentage of individual antigen adsorption, activity, free PRP content between the reduced dose combination vaccine SIIPL and the Easy Six vaccine (Panacea) Description of testA Hexavalent vaccine SIIPL with reduced dose IPV Panacea Easy-Six™ Combination Vaccine with Full Dose IPV NV activity in vitro (µg/ml) 46,969 23,167 NV adsorption (%) 91.8 More than 90.0 NV activity in vivo 1.18 0.71(0.42-1.13) Total PRP content (µg/0.5 ml) 9.18 13.20 Free PRP (%) 9.0 19.45 Free formaldehyde (% w/v) 0.0011 0.0011 2-PE (% w/v) 0.499 0.660 Diphtheria adsorption (%) 82 38 Tetanus adsorption (%) 63 thirty Type 1 (DU/0.5 ml) 22,414 43,504 Type 2 (DU/0.5 ml) 4,692 8,056 Type 3 (DU/0.5 ml) 22,084 39.84 Aluminum (mg/dose) 0.2846 0.6034 In vivo efficacy of IPV Type-1 170.0% Not verified IPV Type-3 132.3% Activity of the diphtheria component 98.5120 IU/dose (69.9650-137.247) More than 40 Activity of the tetanus component 139.030 IU/dose (88.2850-208.688) More than 50 Activity of the pertussis component 4.6749 IU/dose (2.6492-8.2763) 3.2221(1.8032-5.7706) CLAIM 1. A completely liquid multi-dose immunogenic composition, in which 0.5 ml of the composition contains: (i) diphtheria toxoid (D) in an amount of 10 to 25 Lf, adsorbed on an aluminum salt adjuvant having an adsorption percentage of at least 50%; (ii) tetanus toxoid (T) in an amount of 2 to 10 Lf, adsorbed on an aluminum salt adjuvant having an adsorption percentage of at least 40%; (iii) inactivated whole cell pertussis (wP) containing inactivated Bordetella pertussis strains 134, 509, 25525 and 6229 in a ratio of 1:1:0.25:0.25 in an amount of 12 to 16 IOU; (iv) hepatitis B virus surface antigen (HBsAg) in an amount of 7 to 15 μg, adsorbed on an aluminum salt adjuvant having an adsorption percentage of at least 50%; (v) Haemophilus influenzae type b antigen (Hib) in an amount of 7 to 13 μg; (vi) inactivated poliovirus (IPV) antigen containing IPV type 1 in an amount of 1-25 DU, IPV type 2 in an amount of 1-10 DU and IPV type 3 present in an amount of 1-20 DU; and (vii) the total aluminum (Al ³⁺ ) content of the composition is from 0.1 to 0.6 mg and (viii) a preservative containing 2-phenoxyethanol in an amount of 1-6 mg (v/v); methylparaben in an amount of 0.1-1.5 mg (wt./vol.) and propylparaben in an amount of 0.05-0.2 mg (wt./vol.); or 2-phenoxyethanol in an amount of 1-6 mg (v/v) and methylparaben in an amount of 0.1-1.5 mg per 0.5 ml (w/v); or 2-phenoxyethanol in an amount of 1-6 mg (v/v) and propylparaben in an amount of 0.05-0.2 mg (w/v). 2. The immunogenic composition according to claim 1, wherein the Hib antigen is a Hib polyribosylribitol phosphate (PRP) polysaccharide conjugated to a carrier protein using cyanylation conjugation chemistry or reductive amination conjugation chemistry, wherein - 40 043311 the specified cyanylation reagent is selected from cyanogen bromide, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP), 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT), 1-cyanoimidazole, namely (1-CI), 1-cyanobenzotriazole (1-CBT ) or 2-cyanopyridazin-3(2H)-one (2CPO); and the carrier protein is selected from the group consisting of tetanus toxoid, CRM ₁₉₇ , diphtheria toxoid, Neisseria meningitidis outer membrane complex, tetanus toxoid fragment C, pertussis toxoid, H. influenzae protein D, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMC), 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 lethal factor (LF) of Bacillus anthracis, ovalbumin, fisurella lymph hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA) and purified tuberculin derivative (PPD), synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins containing multiple human CD4+ T cell epitopes for various pathogenic antigens such as N 19, iron uptake proteins, toxin A or B from C. difficile and S. agalactiae proteins with or without a linker. 3. The immunogenic composition according to claim 1, wherein the IPV antigens are Salk strains selected from the group consisting of Mahoney, MEF-1, Saukett strains or Sabin strains selected from the group consisting of Sabin type 1, Sabin type 2 strains or Sabin type 3. 4. The immunogenic composition according to claim 1, wherein D, T and HBsAg are individually adsorbed on an aluminum salt (Al ³⁺ ) adjuvant selected from aluminum hydroxide (Al(OH) ₃ ) and aluminum phosphate (AlPO4). 5. The immunogenic composition according to claim 1, wherein the IPV antigens are adsorbed on aluminum phosphate salt (AlPO ₄ ), and the percentage of adsorption of IPV type 1 is in the range from 10 to 100%, IPV type 2 is in the range from 60 to 100%, and Type 3 IPV ranges from 10 to 100%. 6. The immunogenic composition according to claim 1, where the percentage of adsorption of the Hib antigen on the adjuvant is less than 20%. 7. The immunogenic composition according to claim 1, where the composition further contains a diluent medium or buffer selected from sodium chloride or phosphate-buffered saline. 8. The immunogenic composition according to claim 7, where the composition contains sodium chloride as a diluent or as a buffer in a concentration of 0.5 to 1.5%. 9. Immunogenic composition according to claim 1, where 0.5 ml of the composition contains one of the following compositions: a) antigen D in the amount of 10 Lf; T antigen in the amount of 2 Lf; wP antigen in the amount of 12 IOU; HBsAg in the amount of 8 μg; Hib antigen in an amount of 8 μg; IPV antigen, type 1 (Mahoney strain) in the amount of 20 DU and type 3 (Saukett strain) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); or b) antigen D in the amount of 20 Lf; T antigen in the amount of 4 Lf; wP antigen in the amount of 14 IOU; HBsAg in the amount of 15 μg; Hib antigen in an amount of 10 μg; IPV antigen, type 1 (Mahoney strain) in the amount of 20 DU and type 3 (Saukett strain) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); or c) antigen D in the amount of 25 Lf; T antigen in the amount of 10 Lf; wP antigen in the amount of 16 IOU; HBsAg in the amount of 15 μg; Ib-antigen Hib in the amount of 13 μg; IPV antigen, type 1 (Mahoney strain) in the amount of 20 DU and type 3 (Saukett strain) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (w/v). 10. Immunogenic composition according to claim 1, where 0.5 ml of the composition contains one of the following compositions: a) antigen D in the amount of 10 Lf; T antigen in the amount of 2 Lf; wP antigen in the amount of 12 IOU; HBsAg in the amount of 8 μg; Hib antigen in an amount of 8 μg; IPV antigen, type 1 (strain Mahoney) in the amount of 20 DU, type 2 (strain MEF-1) in the amount of 4 DU and type 3 (strain Saukett) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); or b) antigen D in the amount of 20 Lf; T antigen in the amount of 4 Lf; wP antigen in the amount of 14 IOU; HBsAg in the amount of 15 μg; Hib antigen in an amount of 10 μg; IPV antigen, type 1 (strain Mahoney) in the amount of 20 DU, type 2 (strain MEF-1) in the amount of 4 DU and type 3 (strain Saukett) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); or c) antigen D in the amount of 25 Lf; T antigen in the amount of 10 Lf; wP antigen in the amount of 16 IOU; HBsAg in the amount of 15 μg; Hib antigen in the amount of 13 μg; IPV antigen, type 1 (strain Mahoney) in the amount of 20 DU, type 2 (strain MEF-1) in the amount of 4 DU and type 3 (strain Saukett) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 - 41 043311 mg (v/v); methylparaben in the amount of 0.9 mg (w/v). 11. Immunogenic composition according to claim 1, where 0.5 ml of the composition contains one of the following compositions: a) antigen D in the amount of 10 Lf; T antigen in the amount of 2 Lf; wP antigen in the amount of 12 IOU; HBsAg in the amount of 8 μg; Ib-antigen Hib in the amount of 8 μg; IPV antigen, type 1 (Mahoney strain) in the amount of 20 DU and type 3 (Saukett strain) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); propylparaben in an amount of 0.1 mg (w/v); or b) antigen D in the amount of 20 Lf; T antigen in the amount of 4 Lf; wP antigen in the amount of 14 IOU; HBsAg in the amount of 15 μg; Hib antigen in an amount of 10 μg; IPV antigen, type 1 (Mahoney strain) in the amount of 20 DU and type 3 (Saukett strain) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); propylparaben in an amount of 0.1 mg (w/v); or c) antigen D in the amount of 25 Lf; T antigen in the amount of 10 Lf; wP antigen in the amount of 16 IOU; HBsAg in the amount of 15 μg; Hib antigen in the amount of 13 μg; IPV antigen type 1 (strain Mahoney) in the amount of 20 DU and type 3 (strain Saukett) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); propylparaben in an amount of 0.1 mg (w/v). 12. Immunogenic composition according to claim 1, where 0.5 ml of the composition contains one of the following compositions: a) antigen D in the amount of 10 Lf; T antigen in the amount of 2 Lf; wP antigen in the amount of 12 IOU; HBsAg in the amount of 8 μg; Hib antigen in an amount of 8 μg; IPV antigen, type 1 (strain Mahoney) in the amount of 20 DU, type 2 (strain MEF-1) in the amount of 4 DU and type 3 (strain Saukett) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); propylparaben in an amount of 0.1 mg (w/v); or b) antigen D in the amount of 20 Lf; T antigen in the amount of 4 Lf; wP antigen in the amount of 14 IOU; HBsAg in the amount of 15 μg; Hib antigen in an amount of 10 μg; IPV antigen, type 1 (strain Mahoney) in the amount of 20 DU, type 2 (strain MEF-1) in the amount of 4 DU and type 3 (strain Saukett) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); propylparaben in an amount of 0.1 mg (w/v); or c) antigen D in the amount of 25 Lf; T antigen in the amount of 10 Lf; wP antigen in the amount of 16 IOU; HBsAg in the amount of 15 μg; Hib antigen in the amount of 13 μg; IPV antigen, type 1 (strain Mahoney) in the amount of 20 DU, type 2 (strain MEF-1) in the amount of 4 DU and type 3 (strain Saukett) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); propylparaben in an amount of 0.1 mg (w/v). 13. Immunogenic composition according to claim 1, where 0.5 ml of the composition contains one of the following compositions: a) antigen D in the amount of 10 Lf; T antigen in the amount of 2 Lf; wP antigen in the amount of 12 IOU; HBsAg in the amount of 8 μg; Hib antigen in an amount of 8 μg; IPV antigen, type 1 (Mahoney strain) in the amount of 20 DU and type 3 (Saukett strain) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); propylparaben in an amount of 0.1 mg (w/v); or b) antigen D in the amount of 20 Lf; T antigen in the amount of 4 Lf; wP antigen in the amount of 14 IOU; HBsAg in the amount of 15 μg; Hib antigen in an amount of 10 μg; IPV antigen, type 1 (Mahoney strain) in the amount of 20 DU and type 3 (Saukett strain) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); propylparaben in an amount of 0.1 mg (w/v); or c) antigen D in the amount of 25 Lf; T antigen in the amount of 10 Lf; wP antigen in the amount of 16 IOU; HBsAg in the amount of 15 μg; Hib antigen in the amount of 13 μg; IPV antigen type 1 (strain Mahoney) in the amount of 20 DU and type 3 (strain Saukett) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); propylparaben in an amount of 0.1 mg (w/v). 14. Immunogenic composition according to claim 1, where 0.5 ml of the composition contains one of the following compositions: a) antigen D in the amount of 10 Lf; T antigen in the amount of 2 Lf; wP antigen in the amount of 12 IOU; HBsAg in the amount of 8 μg; Hib antigen in an amount of 8 μg; IPV antigen, type 1 (strain Mahoney) in an amount of 7.5 DU, type 2 (strain MEF-1) in an amount of 1.5 DU and type 3 (strain Saukett) in an amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); propylparaben in an amount of 0.1 mg (w/v); or b) antigen D in the amount of 20 Lf; T antigen in the amount of 4 Lf; wP antigen in the amount of 14 IOU; HBsAg in the amount of 15 μg; Hib antigen in an amount of 10 μg; IPV antigen, type 1 (strain Mahoney) in the amount of 20 DU, type 2 (strain MEF-1) in the amount of 4 DU and type 3 (strain Saukett) in the amount of 16 DU - 42 043311 respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); propylparaben in an amount of 0.1 mg (w/v); or c) antigen D in the amount of 25 Lf; T antigen in the amount of 10 Lf; wP antigen in the amount of 16 IOU; HBsAg in the amount of 15 μg; Hib antigen in the amount of 13 μg; IPV antigen, type 1 (strain Mahoney) in the amount of 20 DU, type 2 (strain MEF-1) in the amount of 4 DU and type 3 (strain Saukett) in the amount of 16 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); propylparaben in an amount of 0.1 mg (w/v). 15. Immunogenic composition according to claim 1, where 0.5 ml of the composition contains one of the following compositions: a) antigen D in the amount of 10 Lf; T antigen in the amount of 2 Lf; wP antigen in the amount of 12 IOU; HBsAg in the amount of 8 μg; Hib antigen in an amount of 8 μg; IPV antigen type 1 (strain Mahoney) in the amount of 7.5 DU and type 3 (strain Saukett) in the amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); or b) antigen D in the amount of 20 Lf; T antigen in the amount of 4 Lf; wP antigen in the amount of 14 IOU; HBsAg in the amount of 15 μg; Hib antigen in an amount of 10 μg; IPV antigen, type 1 (Mahoney strain) in an amount of 7.5 DU and type 3 (Saukett strain) in an amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); or c) antigen D in the amount of 25 Lf; T antigen in the amount of 10 Lf; wP antigen in the amount of 16 IOU; HBsAg in the amount of 15 μg; Hib antigen in the amount of 13 μg; IPV antigen, type 1 (Mahoney strain) in an amount of 7.5 DU and type 3 (Saukett strain) in an amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (w/v). 16. An immunogenic composition according to any of the preceding paragraphs, where 0.5 ml of the composition contains one of the following compositions: a) antigen D in the amount of 10 Lf; T antigen in the amount of 2 Lf; wP antigen in the amount of 12 IOU; HBsAg in the amount of 8 μg; Hib antigen in an amount of 8 μg; IPV antigen, type 1 (strain Mahoney) in an amount of 7.5 DU, type 2 (strain MEF-1) in an amount of 1.5 DU and type 3 (strain Saukett) in an amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); or b) antigen D in the amount of 20 Lf; T antigen in the amount of 4 Lf; wP antigen in the amount of 14 IOU; HBsAg in the amount of 15 μg; Hib antigen in an amount of 10 μg; IPV antigen, type 1 (strain Mahoney) in an amount of 7.5 DU, type 2 (strain MEF-1) in an amount of 1.5 DU and type 3 (strain Saukett) in an amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); or c) antigen D in the amount of 25 Lf; T antigen in the amount of 10 Lf; wP antigen in the amount of 16 IOU; HBsAg in the amount of 15 μg; Hib antigen in the amount of 13 μg; IPV antigen, type 1 (strain Mahoney) in an amount of 7.5 DU, type 2 (strain MEF-1) in an amount of 1.5 DU and type 3 (strain Saukett) in an amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (w/v). 17. Immunogenic composition according to claim 1, where 0.5 ml of the composition contains one of the following compositions: a) antigen D in the amount of 10 Lf; T antigen in the amount of 2 Lf; wP antigen in the amount of 12 IOU; HBsAg in the amount of 8 μg; Hib antigen in an amount of 8 μg; IPV antigen, type 1 (Mahoney strain) in an amount of 7.5 DU and type 3 (Saukett strain) in an amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); propylparaben in an amount of 0.1 mg (w/v); or b) antigen D in the amount of 20 Lf; T antigen in the amount of 4 Lf; wP antigen in the amount of 14 IOU; HBsAg in the amount of 15 μg; Hib antigen in an amount of 10 μg; IPV antigen, type 1 (Mahoney strain) in an amount of 7.5 DU and type 3 (Saukett strain) in an amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); propylparaben in the amount of 0.1 mg (wt./vol.), c) antigen D in the amount of 25 Lf; T antigen in the amount of 10 Lf; wP antigen in the amount of 16 IOU; HBsAg in the amount of 15 μg; Hib antigen in the amount of 13 μg; IPV antigen type 1 (strain Mahoney) in the amount of 7.5 DU and type 3 (strain Saukett) in the amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); propylparaben in an amount of 0.1 mg (w/v). 18. Immunogenic composition according to claim 1, where 0.5 ml of the composition contains one of the following compositions: a) antigen D in the amount of 10 Lf; T antigen in the amount of 2 Lf; wP antigen in the amount of 12 IOU; - 43 043311 HBsAg in the amount of 8 μg; Hib antigen in an amount of 8 μg; IPV antigen, type 1 (strain Mahoney) in an amount of 7.5 DU, type 2 (strain MEF-1) in an amount of 1.5 DU and type 3 (strain Saukett) in an amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); propylparaben in an amount of 0.1 mg (w/v); or b) antigen D in the amount of 20 Lf; T antigen in the amount of 4 Lf; wP antigen in the amount of 14 IOU; HBsAg in the amount of 15 μg; Hib antigen in an amount of 10 μg; IPV antigen, type 1 (strain Mahoney) in an amount of 7.5 DU, type 2 (strain MEF-1) in an amount of 1.5 DU and type 3 (strain Saukett) in an amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); propylparaben in an amount of 0.1 mg (w/v); or c) antigen D in the amount of 25 Lf; T antigen in the amount of 10 Lf; wP antigen in the amount of 16 IOU; HBsAg in the amount of 15 μg; Hib antigen in the amount of 13 μg; IPV antigen, type 1 (strain Mahoney) in the amount of 40 DU, type 2 (strain MEF-1) in the amount of 8 DU and type 3 (strain Saukett) in the amount of 32 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); propylparaben in an amount of 0.1 mg (w/v). 19. Immunogenic composition according to claim 1, where 0.5 ml of the composition contains one of the following compositions: a) antigen D in the amount of 10 Lf; T antigen in the amount of 2 Lf; wP antigen in the amount of 12 IOU; HBsAg in the amount of 8 μg; Hib antigen in an amount of 8 μg; IPV antigen, type 1 (Mahoney strain) in an amount of 7.5 DU and type 3 (Saukett strain) in an amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); propylparaben in an amount of 0.1 mg (w/v); or b) antigen D in the amount of 20 Lf; T antigen in the amount of 4 Lf; wP antigen in the amount of 14 IOU; HBsAg in the amount of 15 μg; Hib antigen in an amount of 10 μg; IPV antigen, type 1 (Mahoney strain) in an amount of 7.5 DU and type 3 (Saukett strain) in an amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); propylparaben in an amount of 0.1 mg (w/v); or c) antigen D in the amount of 25 Lf; T antigen in the amount of 10 Lf; wP antigen in the amount of 16 IOU; HBsAg in the amount of 15 μg; Hib antigen in the amount of 13 μg; IPV antigen type 1 (strain Mahoney) in the amount of 7.5 DU and type 3 (strain Saukett) in the amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); propylparaben in an amount of 0.1 mg (w/v). 20. Immunogenic composition according to claim 1, where 0.5 ml of the composition contains one of the following compositions: a) antigen D in the amount of 10 Lf; T antigen in the amount of 2 Lf; wP antigen in the amount of 12 IOU; HBsAg in the amount of 8 μg; Hib antigen in an amount of 8 μg; IPV antigen, type 1 (strain Mahoney) in an amount of 7.5 DU, type 2 (strain MEF-1) in an amount of 1.5 DU and type 3 (strain Saukett) in an amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); propylparaben in an amount of 0.1 mg (w/v); or b) antigen D in the amount of 20 Lf; T antigen in the amount of 4 Lf; wP antigen in the amount of 14 IOU; HBsAg in the amount of 15 μg; Hib antigen in an amount of 10 μg; IPV antigen, type 1 (strain Mahoney) in an amount of 7.5 DU, type 2 (strain MEF-1) in an amount of 1.5 DU and type 3 (strain Saukett) in an amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); propylparaben in an amount of 0.1 mg (w/v); or c) antigen D in the amount of 25 Lf; T antigen in the amount of 10 Lf; wP antigen in the amount of 16 IOU; HBsAg in the amount of 15 μg; Ib-antigen Hib in the amount of 13 μg; IPV antigen, type 1 (strain Mahoney) in an amount of 7.5 DU, type 2 (strain MEF-1) in an amount of 1.5 DU and type 3 (strain Saukett) in an amount of 6 DU, respectively; total aluminum content (Al ³⁺ ) not more than 0.55 mg; 2-phenoxyethanol in an amount of 2.5 mg (v/v); methylparaben in the amount of 0.9 mg (wt./vol.); propylparaben in an amount of 0.1 mg (w/v). 21. A method for producing a completely liquid multi-dose immunogenic composition, in which 0.5 ml of the composition contains: (i) diphtheria toxoid (D) in an amount of 10 to 25 Lf, adsorbed on an aluminum salt adjuvant having an adsorption percentage of at least 50%; (ii) tetanus toxoid (T) in an amount of 2 to 10 Lf, adsorbed on an aluminum salt adjuvant having an adsorption percentage of at least 40%; (iii) inactivated whole cell pertussis (wP) containing inactivated Bordetella pertussis strains 134, 509, 25525 and 6229 in a ratio of 1:1:0.25:0.25 in an amount of 12 to 16 IOU; (iv) hepatitis B virus surface antigen (HBsAg) in an amount of 7 to 15 μg, adsorbed on an aluminum salt adjuvant having an adsorption percentage of at least 50%; (v) Haemophilus influenzae type b antigen (Hib) in an amount of 7 to 13 μg; (vi) inactivated poliovirus (IPV) antigen containing IPV type 1 in an amount of 1-25 DU, IPV type 2 in an amount of 1-10 DU and IPV type 3 present in an amount of 1-20 DU; - 44 043311 (vii) the total aluminum content (Al ³⁺ ) in the composition is from 0.1 to 0.6 mg and (viii) a preservative containing 2-phenoxyethanol in an amount of 1-6 mg (v/v); methylparaben in an amount of 0.1-1.5 mg (wt./vol.) and propylparaben in an amount of 0.05-0.2 mg (wt./vol.); or 2-phenoxyethanol in an amount of 1-6 mg (v/v) and methylparaben in an amount of 0.1-1.5 mg per 0.5 ml (w/v); or 2-phenoxyethanol in an amount of 1-6 mg (v/v) and propylparaben in an amount of 0.05-0.2 mg (w/v); This method includes the following steps: a) adding 80% of the total mass of physiological solution (NaCl) to the mixing container/container; b) adding component I containing diphtheria toxoid to the mixing container/container; c) adding component II containing tetanus toxoid to component I in step (b) with stirring at room temperature; d) adding component III containing inactivated whole cell pertussis antigen to the mixture obtained in step (c) with stirring at room temperature; e) adding component IV containing hepatitis B surface antigen to the mixture obtained in step (d) at room temperature; f) adding component V containing the Hib antigen to the mixture obtained in step (e) at 616°C; g) adding component VI containing the IPV antigen to the mixture obtained in step (f) at 616°C; h) adding a combination of preservatives to the mixture obtained in step (g) at 6-16°C selected from 2-phenoxyethanol in an amount from 1 to 6 mg per 0.5 ml (v/v) and methylparaben in an amount from 0.1 to 1.5 mg per 0.5 ml (w/v); or 2-phenoxyethanol in an amount from 1 to 6 mg per 0.5 ml (v/v) and propylparaben in an amount of 0.050.2 mg per 0.5 ml (w/v); or 2-phenoxyethanol in an amount from 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 0.05-0.2 mg per 0.5 ml (w/v); i) adjusting the pH of the mixture obtained in step (h) to 6.0-7.0 using sodium hydroxide/sodium carbonate and j) adding the remaining 20% of the total saline solution (NaCl) to adjust the volume while mixing. 22. The method according to claim 21, characterized in that the production of component I includes the following steps: a) transferring aluminum phosphate into a container/container; b) adding diphtheria toxoid; c) adjusting the pH to 4.5-5.5 using acetic acid/sodium hydroxide; d) stabilization; f) adjusting the pH value to 5.5-6.5 using sodium hydroxide/sodium carbonate and f) stabilization with histidine buffer. 23. The method according to claim 21, characterized in that the production of component II includes the following steps: a) transferring aluminum phosphate into a container/container; b) adding tetanus toxoid; c) adjusting the pH to 4.5-5.5 using acetic acid/sodium hydroxide; d) stabilization; f) adjusting the pH value to 5.5-6.5 using sodium hydroxide/sodium carbonate and f) stabilization with histidine buffer. 24. The method according to claim 21, characterized in that the production of component III includes the following steps, such as: a) inactivation of the Bordetella pertussis 134 strain at a temperature of 56°C for 10-15 minutes in the presence of formaldehyde; b) inactivation of the Bordetella pertussis 509 strain at a temperature of 56°C for 10-15 minutes in the presence of formaldehyde; c) inactivation of the Bordetella pertussis strain 25525 at a temperature of 56°C for 10-15 minutes in the presence of formaldehyde; d) inactivation of the Bordetella pertussis strain 6229 at a temperature of 56°C for 10-15 minutes in the presence of formaldehyde; f) subsequent mixing of inactivated Bordetella pertussis strains 134, 509, 25525 and 6229 in a ratio of 1:1:0.25:0.25 and - 45 043311 f) optional adsorption to an aluminum-based adjuvant; At the same time, the method does not use thiomersal and the inactivated whole-cell pertussis antigen remains homogeneous rather than lumpy, which leads to a decrease in reactogenicity and provides better effectiveness over a longer period of time. 25. The method according to claim 21, characterized in that the production of component IV includes the following steps: a) transferring aluminum phosphate into a container/container; b) addition of hepatitis B surface antigen; c) adjusting the pH to 4.5-5.5 using acetic acid/sodium hydroxide; d) stabilization; f) adjusting the pH value to 5.8-6.8 using sodium hydroxide/sodium carbonate and I) stabilization. 26. The method according to claim 21, characterized in that the production of component V includes the following steps: a) fermentation of Haemophilus influenzae type b; b) inactivation at a temperature of 37°C for 2 hours in the presence of 0.1% formaldehyde; c) purifying Hib polyribosyl ribitol phosphate (PRP) polysaccharide; d) conjugating the purified product of the step with tetanus toxoid (TT) using cyanogen bromide cyanylation conjugation chemistry in the presence of an adipic acid dihydrazide (ADH) linker; e) purifying the conjugate of step d and f) filtering the purified conjugate preferably through a filter with a pore size of 0.22 μm; in this case, the percentage of free PRP is no more than 5% of the total amount of purified Hib conjugate. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2