A PROCESS FOR ENTEROVIRUS PURIFICATION AND INACTIVATION AND VACCINE COMPOSITIONS OBTAINED THEREOF

Abstract

The present invention pertains to the field of industrial scale inactivation of various enteroviruses and large and industrial scale production of enterovirus vaccine compositions and combinations of various enteroviruses so obtained.

Claims

1. A method for producing Enteroviral particles useful for preparation of vaccine compositions, wherein the method comprises the steps of: a. producing Enteroviral particles in Vero cells; b. purification of the Enteroviral particles either by Single Step Chromatography or Two Step Chromatography; c. stabilization of purified Enteroviral particles; and d. inactivation of the Enteroviral particles to obtain Enteroviral particles; wherein the said method comprises addition of one or more excipient including stabilizers and adjuvants before or after the inactivation step, and wherein the D-antigen recovery is in the range of 80% to 99%.

2. The method as claimed in claim 1, wherein the Single Step Chromatography is carried by using a single column prepared by mixing one or more ion exchange resin with one or more hydrophobic resin.

3. The method as claimed in claim 1, wherein the Two Step Chromatography is carried by first running Size Exclusion Chromatography or Gel Filtration Chromatography followed by ion exchange chromatography.

4. The method as claimed in claim 1, wherein the inactivation process is selected from heat inactivation, chemical inactivation, irradiation or UV treatment.

5. The method as claimed in claim 4, wherein the chemical inactivation is carried by using one or more chemicals selected from a group of formalin, beta-propiolactone, hydrogen peroxide, glutaraldehyde, N-acetyl ethyleneimine, binary ethyleneimine, tertiary ethyleneimine, ascorbic acid, caprylic acid, psolarens or a detergent; and an inactivation buffer.

6. The method as claimed in claim 5, wherein formalin inactivation involves addition of inactivation buffer selected from a group comprising phosphate buffer, TRIS, Sodium Acetate, TBS, MOPS, HEPES or bicarbonate buffer in presence or absence of salt.

7. The method as claimed in claim 6, wherein the inactivation buffer comprises a buffer selected from a group of phosphate buffer, combinations of phosphate buffer and TRIS, sodium acetate, salts with sodium acetate, TBS, MOPS, HEPES and bicarbonate buffers; M-199; glycine and tween 80.

8. The method as claimed in claim 7, wherein the phosphate buffer is used at a concentration in the range of 1 mM-10 mM, nad 2 mM with or without NaCl in the pH range of one of 6.0 to 7.5, and at 7.2.

9. The method as claimed in claim 7, wherein the phosphate buffer is used at a concentration in the range of one of 1 mM-10 mM, and 2 mM along with saline at a concentration of 10 mM in the pH range of one of 6.0 to 7.5, and at 7.2.

10. The method as claimed in claim 7, wherein the sodium acetate buffer is used at a concentration in the range of one of 20 mM-50 mM, and 25 mM with or without NaCl in the pH range of one of 6.0 to 7.5, and at 7.2.

11. The method as claimed in claim 10, wherein the sodium acetate buffer is used at a concentration of 25 mM having the pH of about 7.2.

12. The method as claimed in claim 1, wherein the stabilizer is selected from the group of sucrose, maltose, trehalose, human serum albumin, and M199.

13. The method as claimed in claim 1, wherein the excipient is added before the inactivation step.

14. The method as claimed in claim 1, wherein inactivation is carried by an inactivation buffer comprising a buffer selected from a group of phosphate buffer, combinations of phosphate buffer and TRIS, sodium acetate, salts with sodium acetate, TBS, MOPS, HEPES or bicarbonate buffers; M-199 or M-199 in combination with HEPES; glycine and tween 80.

15. The method as claimed in claim 14, wherein the phosphate buffer is used at a concentration in the range of one of 1 mM-10 mM, and 2 mM at the pH range of one of 6.0 to 7.5, and at 7.2 with one or more stabilizer.

16. The method as claimed in claim 15, wherein the stabilizer is selected from a group comprising sucrose in 5% w/v concentration, M199 in 9.9 g/L concentration, trehalose in 5% w/v concentration, maltose in 5% w/v concentration and human serum albumin in 5% w/v concentration.

17. The method as claimed in claim 15, wherein stabilizer is M199 in 9.9 g/L concentration and trehalose in 5% w/v concentration.

18. The method as claimed in claim 1, wherein the Enteroviral particles are of polioviruses.

19. The method as claimed in claim 18, wherein the polioviruses are Sabin serotypes 1, 2 and 3.

20. The method as claimed in claim 19, wherein the polioviruses are one of Sabin serotypes IPV type 1, IPV type 2, and IPV type 3.

21. A method for preparing a dose reduced inactivated Polio vaccine containing Sabin polioviruses, said method comprising the steps of: a. producing polioviruses in Vero cells; b. purification of the polioviruses; c. stabilization of purified polioviruses by addition of M-199 medium containing glycine and tween 80; d. inactivation of the polio viruses by using formaldehyde 0.025% at 37 C. for 13 days in presence of Phosphate buffer with saline at a concentration between 2 mM to 10 mM or Sodium acetate to reduce aggregation of the poliovirus particles thereby reducing the D-antigen losses post inactivation by greater than 90% as compared to other buffers; and e. formulating the said polio vaccine.

Description

BRIEF DESCRIPTION

[0049] Some of the embodiments will be described in detail, with references to the following Figures, wherein like designations denote like members, wherein:

[0050] FIG. 1: Quality of the Poliovirus-1 protein by SDS PAGE after single chromatography: Column 1-10;

[0051] FIG. 2: Chromatogram Profile showing Poliovirus-1 Protein Peak (Indicated by Arrow) after single chromatography;

[0052] FIG. 3: Quality of the Poliovirus-3 protein by SDS PAGE after single chromatography: Column 1-10;

[0053] FIG. 4: Chromatogram Profile showing Poliovirus-3 Protein Peak (Indicated by Arrow) after single chromatography;

[0054] FIG. 5: SDS PAGE showing the quality of Poliovirus-1 protein for two-step chromatography;

[0055] FIG. 6: Chromatogram showing the peak of Poliovirus-1 protein for two-step chromatography;

[0056] FIG. 7: SDS PAGE showing the quality of Poliovirus-2 protein for two-step chromatography;

[0057] FIG. 8: SDS PAGE showing the quality of Poliovirus-3 protein for two-step chromatography;

[0058] FIG. 9: Chromatogram showing the peak of Poliovirus-3 protein for two-step chromatography;

[0059] FIG. 10: Effect of buffer concentration in D-Antigen recoveries;

[0060] FIG. 11: D-Antigen Recovery during Inactivation Stages; and

[0061] FIG. 12: Effect of stabilizers in D-Antigen Recovery.

DETAILED DESCRIPTION

[0062] Embodiments of the present invention disclose a novel process for large and industrial scale production of stable and effective immunogenic compositions for conferring protection against various Enteroviruses, that cause paralytic poliomyelitis, aseptic meningitis, febrile illness, rash, myopericarditis, epidemic pleurodynia, hand foot and mouth disease (by EV71), epidemic conjunctivitis (by EV70), Polio-like illness, herpangina, acute hemorrhagic conjunctivitis, severe neonatal disease, Bornholm disease, and encephalitis. Combination vaccine(s) comprising of two or more immunogenic elements selected from various Enteroviruses can be included against wide protection against Enteroviruses. All the methods mentioned in embodiments of the the invention are applicable to any genotype or genotypic variants/serotypes/strains of Enteroviruses.

[0063] In one aspect of embodiments of the the invention, there is provided a method for producing Enteroviral (D-Antigen) particles useful for preparation of vaccine compositions, wherein the method comprises the steps of: [0064] a. producing Enteroviral particles in Vero cells; [0065] b. purification of the Enteroviral particles either by Single Step Chromatography or Two Step Chromatography; [0066] c. stabilization of purified Enteroviral particles; and [0067] d. inactivation of the Enteroviral particles to obtain Enteroviral (D-Antigen) particles; wherein the said method comprises addition of one or more excipient including stabilizers and adjuvants before or after the inactivation step, and wherein the D-antigen recovery is in the range of 80% to 99%.

[0068] In some embodiments of the invention, in the above described method for producing Enteroviral (D-Antigen) particles, the Single Step Chromatography is carried by using a single column prepared by mixing one or more ion exchange resin with one or more hydrophobic resin. In some embodiment, the ion exchange resin is anion or/and cation exchanger resins selected from DEAE, QEAE, SP Sepharose, Poly CSX-35 or CM Sepharose; the hydrophobic resin is selected from Butyl 650, Phenyl 650, Butyl Sepharose, phenyl Sepharose or octyl Sepharose.

[0069] In other embodiments of the invention, in the above described method for producing Enteroviral (D-Antigen) particles, the Two Step Chromatography is carried by first running Size Exclusion Chromatography or Gel Filtration Chromatography followed by ion exchange chromatography. In some embodiment, purification is done by two step chromatography involving SEC (Size Exclusion Chromatography) using Sepharose CL-6B and (Ion-Exchange Chromatography) IEC-DEAE 650M. In some embodiments, two step column chromatography is carried by running GFC (Gel Filtration Chromatography) containing Sepharose CL-6B followed by Ion exchange containing DEAE 650 Matrix resins.

[0070] In yet other embodiments of the invention, in the above described method for producing Enteroviral (D-Antigen) particles, the inactivation process is selected from heat inactivation, chemical inactivation, irradiation or UV treatment. In some embodiments, the chemical inactivation is carried by using one or more chemicals selected from a group of formalin, beta-propiolactone (BPL), hydrogen peroxide, glutaraldehyde, N-acetyl ethyleneimine, binary ethyleneimine, tertiary ethyleneimine, ascorbic acid, caprylic acid, psolarens or a detergent; and an inactivation buffer.

[0071] In some embodiment, the inactivation is carried by addition of formalin and inactivation buffer selected from a group comprising phosphate buffer, TRIS, Sodium Acetate, TBS, MOPS, HEPES or bicarbonate buffer in presence or absence of salt. In some embodiment, the inactivation buffer comprises a buffer selected from a group of phosphate buffer, combinations of phosphate buffer and TRIS, Sodium Acetate, salts with sodium acetate, TBS, MOPS, HEPES and bicarbonate buffers; M-199; glycine and tween 80.

[0072] In some embodiment, in the formalin inactivation the phosphate buffer is used at a concentration in the range of 1 mM-10 mM, in particular 2 mM with or without NaCl in the pH range of 6.0 to 7.5, in particular at 7.2. In some other embodiment, the phosphate buffer is used at a concentration in the range of 1 mM-10 mM, in particular 2 mM along with saline at a concentration of 10 mM in the pH range of 6.0 to 7.5, in particular at 7.2.

[0073] In some embodiment, in the formalin inactivation, sodium acetate buffer is used at a concentration in the range of 20 mM-50 mM, in particular 25 mM with or without NaCl in the pH range of 6.0 to 7.5, in particular at 7.2. In some embodiment, sodium acetate buffer is used at a concentration of 25 mM having the pH of about 7.2.

[0074] In some embodiment, in the above described method for producing Enteroviral (D-Antigen) particles, the used stabilizers are selected from the group of sucrose, maltose, trehalose, human serum albumin, and M199.

[0075] In some embodiment, in the above described method for producing Enteroviral (D-Antigen) particles, the excipient is added before the inactivation step.

[0076] In some embodiment, in the above described method for producing Enteroviral (D-Antigen) particles, the inactivation is carried by an inactivation buffer comprising a buffer selected from a group of phosphate buffer, combinations of phosphate buffer and TRIS, sodium acetate, salts with sodium acetate, TBS, MOPS, HEPES or bicarbonate buffers; M-199 or M-199 in combination with HEPES; glycine and tween 80. In some embodiment, the phosphate buffer is used at a concentration in the range of 1 mM-10 mM, in particular 2 mM at the pH range of 6.0 to 7.5, in particular at 7.2 with one or more stabilizer.

[0077] In some embodiments of the invention the inactivation buffer is selected from any one of the following: [0078] a. Base 1+2 mM PB; [0079] b. Base 1+2 mM PB+50 mM NaCl; [0080] c. Base 1+2 mM PB+100 mM NaCl; [0081] d. Base 1+25 mM Sodium acetate; [0082] e. Base 1+25 mM Sodium acetate+50 mM NaCl; [0083] f. Base 1+25 mM Sodium acetate+100 mM NaCl; [0084] g. Base 1+50 mM Sodium acetate; [0085] h. Base 1+50 mM Sodium acetate+50 mM NaCl; [0086] i. Base 1+50 mM Sodium acetate+100 mM NaCl; [0087] j. Base 1+10 mM PBS; [0088] k. Base 1+10 mM PBS (5 Diluted) or [0089] l. Base 1+40 mM Tris; [0090] wherein Base 1 is 1 (9.9 g/L) concentrated M-199 with 0.5% glycine and 0.01% tween 80.

[0091] In some embodiment, in the above described method for producing Enteroviral (D-Antigen) particles, the stabilizer is selected from a group comprising sucrose in 5% w/v concentration, M199 in 9.9 g/L concentration, trehalose in 5% w/v concentration, maltose in 5% w/v concentration and human serum albumin in 5% w/v concentration. In some embodiment, the stabilizer is M199 in 9.9 g/L concentration and trehalose in 5% w/v concentration.

[0092] In some embodiment, there is provided a method for producing Enteroviral (D-Antigen) particles useful for preparation of vaccine compositions, wherein the Enteroviral particles are of polioviruses. In some embodiment, the polioviruses are Sabin serotypes 1, 2 and 3. In some other embodiment, the polioviruses are Sabin serotypes IPV type 1 (Sabin-1 strain), IPV type 2 (Sabin-2 strain), and/or IPV type 3 (Sabin-3strain).

[0093] In another aspect of embodiments of the invention, there is provided a method for preparing a dose reduced inactivated Polio vaccine containing Sabin polioviruses, said method comprises the steps of: [0094] a. producing polioviruses in Vero cells; [0095] b. purification of the polioviruses; [0096] c. stabilization of purified polioviruses by addition of M-199 medium containing glycine and tween 80; [0097] d. inactivation of the polio viruses by using formaldehyde 0.025% at 37 C. for 13 days in presence of Phosphate buffer with saline at a concentration between 2 mM to 10 mM or Sodium acetate (20 mM-30 mM) to reduce aggregation of the poliovirus particles thereby reducing the D-antigen losses post inactivation by greater than 90% as compared to other buffers; and [0098] e. formulating the said polio vaccine.

[0099] Enteroviruses can be grown in cell cultures. The cell culture used for enterovirus growth is VERO cell line which is a continuous cell line derived from kidney epithelial cells of African green monkey. Vero cells have received worldwide regulatory acceptance and are preferred to be production cell substrates that are used to manufacture the bulk product (vaccine production). VERO cells can conveniently be cultured on cell factories, polyester non woven fabrics or cellulose based beads for increasing the yield of cells. After growth, virions are purified by ultrafiltration and chromatography. Prior to administration to patients, the viruses must be inactivated, and this can be achieved by treatment with formaldehyde. An important aspect of embodiments of the instant invention is that said improved process of purification and inactivation of Enterovirus consists of following steps:

[0100] A. Cell Culture

[0101] Viruses used in embodiments of the present invention are grown in cell factories or fermenters. After attaining the required cell densities, virus is infected with 0.01 MOI (Multiplicity of Infection) of three serotypes of Enterovirus.

[0102] Vero cells derived from African green monkey are used as host cells for the production of Enteroviral antigens. Sabin type polioserotypes-1, 2, 3 were received from National Institute for Biological Standards and Control (NIB SC), UK. The poliovirus Sabin strains is used for infection on Vero cells with 0.01 MOI for the production of Enteroviral virions. Infected Vero Cells are then grown in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 5% New Born Calf Serum. Once the confluency is attained in cell factories, the control cell factory is trypsinized with recombinant trypsin for the cell count. Cell count is performed on Neubauer's heamocytometer and total numbers of cells are calculated. Enteroviral inoculum is calculated using the formula:

Required inoculum=Total Number of CellsMOI/Virus titer

[0103] Viral inoculums is then prepared in DMEM and added to the cell factories for viral adsorption and incubated at 32.5 C. for 60 minutes. After the adsorption, the cell factories are topped up with DMEM and incubated at 32.5 C. for the cytopathic effect to be observed. Once the 90-95% cytopathic effect is observed, the viral harvest is collected and clarified using 0.8+0.45 capsule filter. The clarified viral harvest further used for downstream processing like concentration, purification and further inactivated with formalin.

[0104] B. Two-Step Chromatography (TSC): Purification of the viral harvest by using two step chromatography (Size Exclusion Chromatography) SEC using Sepharose CL-6B and (Ion-Exchange Chromatography) IEC-DEAE 650M.

[0105] In some embodiment, the purification of the viral antigens is carried out by two step column chromatography by running GFC (Gel Filtration Chromatography) containing Sepharose CL-6B followed by Ion exchange containing DEAE 650 Matrix resins.

[0106] Clarified harvest pool is concentrated to 400-700 using 2 step tangential flow filtration system with 0.1 m.sup.2 and 50 cm.sup.2 100 KDa cassettes and collected the retentate. Cassette is washed with phosphate buffered Saline (10 mM, pH: 7.2); then added the wash sample to retentate and used as load for column chromatography.

[0107] The purification is done by GFC using Sepharose CL-6B and then Ion Exchange Chromatography (IEC). Virus peak is collected after passing the 400-700 TFF concentrate through gel filtration column and collected virus containing peak pool. This is used as a load for DEAE 650M (Weak-Anion exchanger) using Akta purification system (GE Healthcare). Impurities are bound to the column whereas poliovirus are collected in flow-through containing 10 mM phosphate buffer saline. The product quality is high with consistency.

[0108] Table 1 below depicts the parameters like height, volume, type of matrix, flow rate and buffers used for the purification of polioviruses:

TABLE-US-00001 TABLE 1 Chromatography Column Parameters Column Parameters SEC IEC Column XK26/100 XK16/40 Height 90-100 cm 10 cm Bed Volume 480 mL 20 mL Matrix Sepharose CL-6B DEAE 650M/DEAE Sepharose Linear Flow 20-30 cm/hr 150 cm/hr Equilibration 10 mM phosphate Buffer + 10 mM phosphate Buffer + Buffer 150 mM NaCl (pH-7.2) 150 mM NaCl (pH-7.2)

[0109] A Stable Bulk Concentrate is obtained, which is mixed with Inactivation Buffer for the process of inactivation.

[0110] C. Single Step Chromatography (SSC)

[0111] In some embodiment, the purification of the viral harvest is done by single chromatographic column using mixture of two different manufacturer resins that have high salt tolerance. Different anion and cation exchanger resins like DEAE, QEAE, SP Sepharose, Poly CSX-35, CM Sepharose were used in combination with hydrophobic resins like Butyl 650, Phenyl 650, Butyl Sepharose, phenyl Sepharose and octyl Sepharose.

[0112] The unique functionality of mixed mode is that, resins have functional surface chemistry synthesized by covalently binding polyethylene imine (PEI) to polymethacrylate beads. The PEI is modified to provide carboxylic acid groups and sulfurous acid groups with strong cation-anion exchange sites similar to sulfurous ion exchange resins which offer enhanced performance and better selectivity than conventional ion exchange media with equivalent capacities. Proteins having similar isoelectric points can be easily separated. The efficiency for removal of impurities is greater in a mixed-mode chromatography with fixed amounts of two different resins there by maintaining uniform pH and solubility throughout the column thus saving the process time. The product recoveries and yields are more in SSC.The single column XK16/40 is prepared by packing the column with hydrophobic resin with cation exchanger. The column height was 5 cm with a bed volume of 10 mL. Column was equilibrated with 0.2 M Sodium acetate buffer (pH 4.88) and purified the viral harvest using the Akta purification system. As the column contains cation exchanger, the enteroviral particles are bound to the column in the pH range of 4.8-5.2. Later they are eluted out in the washes with pH range of 5.2-6.0.

[0113] The purified bulk is further concentrated and buffer exchanged using tangential flow filtration using 100 KDa cassette attached to a low shear pump. The concentrated bulk is then stabilized [Stable Concentrated Bulk] using M199 and inactivated.

[0114] D. Preparation of Inactivation Buffer

TABLE-US-00002 TABLE 2 Inactivation buffer composition Composition Concentration M199 1X (9.9 g/L) Glycine 0.5% Tween 80 0.01% Phosphate Buffer 2 mM PB Phosphate Buffer with saline 2 mM PBS Sodium Acetate 25 mM NaOAc

[0115] In some embodiment, 10 concentrated M-199 with 0.5% glycine and 0.01% tween 80 is added to make the final inactivation buffer containing 2 mM PB, 2 mM PBS or 25 mM NaOAc, so as to achieve final concentration of 1 M199. Adding M-199 medium containing glycine (5 gm/L) to mixture of the Stable Concentrated Bulk.

[0116] Inactivation buffer to be used during formaldehyde inactivation can be selected from the group of PB, PB with salt, Combination of PB and TRIS, Sodium acetate and various concentrations of salt with sodium acetate. Other buffers used for inactivation of viruses include TBS, MOPS, HEPES and bicarbonate buffers within a range of 1 mM-100 mM.

[0117] E. Preparation of Incubating Bulk Mixture

[0118] Stable Concentrated Bulk of Enterovirus such as Sabin IPV Type-1 or Type-2 or Type-3 purified bulk is mixed with 2 mM Phosphate buffer having pH 7.2, 10 mM Phosphate Buffer with Saline pH 7.2 (5 diluted in inactivation) and 25 mM Sodium Acetate pH 7.2 to obtain Incubating Bulk Mixture.

[0119] F. Inactivation

[0120] In some embodiment, the Incubating Bulk Mixture is then treated with 0.025% formaldehyde while mixing, at 37 C. from 0 to 13 days on magnetic stirrer. The formalin inactivation with PB buffer occurs in the presence of saline. Post-incubation mixture is thereafter subjected to intermediate 0.22 filtration on day 6 and final filtration on day 13, storing the Final Bulk so obtained at 20 C.

[0121] Reports and literature are available that poliovirus gets completely inactivated with formaldehyde at the concentration of 1:4000 at 37 C. in 12 days. It is also observed that mice showed no paralysis after inactivation with formalin after 12 days. The absence of infectious virus in sequential aliquots and inactivated samples was confirmed by virus titration assays in Vero cells and by the addition and passage of treated virus samples on Vero cell monolayers for period upto 21 days.

[0122] The formalin inactivation and formulation with excipients results in D-Antigen recovery post-inactivation more than 90%. Different excipients like sucrose, maltose, trehalose, M199, HSA and PEG were used in combinations at 2-8 C., RT and 37 C. Combination of sucrose and trehalose was found to show stable D-antigen at 37 C.

[0123] This inactivation process used in embodiments of the present invention gives higher D-Antigen recoveries due to lower salt concentration. When higher salt concentrations are used in buffers, the viral particle aggregates are formed decreasing the stability of D-Antigen during the inactivation period.

[0124] In some embodiment, the viruses are inactivated either by heat, gamma irradiation, and ultra violet light or by chemical means. Chemical inactivating agents are selected from the following list which includes but is not limited to: formalin, beta-propiolactone, hydrogen peroxide, glutaraldehyde, N-acetylethyleneimine, binary ethyleneimine, tertiary ethyleneimine, ascorbic acid, caprylic acid, psolarens, detergents including non-ionic detergents etc. is added to a virus suspension to inactivate the Enteroviruses. Enteroviruses can also be inactivated by using gamma irradiation.

[0125] G. Vaccine Formulations

[0126] Various vaccine formulations are prepared from the antigens recovered from the process as disclosed under embodiments of theinvention. In some embodiment, the vaccine formulations prepared according to embodiments of the invention include pharmaceutical excipients such as stabilizers, adjuvants and the like. The vaccine formulations are prepared with or without stabilizers. Stabilizers may be selected from a range of stabilizers acceptable for the use in vaccine for human use. The vaccine formulations can be prepared with or without the use of adjuvants. The vaccine formulations can be a single dose or multi dose vaccine formulations.

[0127] Further, a combination of vaccine formulations can be prepared, where in IPV may be used in combination with two or more vaccine antigens obtained from the process as disclosed under embodiments of the invention. The vaccine formulations may be used with or without adjuvant(s). The vaccine formulations can be a single dose or multi dose vaccine formulations.

[0128] Poliovirus type 2 is virulent when compared to Type 1 and 3. However, capsid proteins of Type 2 are similar to that of Type 1 and 3; hence the same strategies can be followed for Type 2 as applicable to the other poliovirus serotypes for purification and inactivation. The Enterovirus is grown on Vero cell lines, in the presence of DMEM supplemented with 5% New Born Calf Serum. The viral harvest thus obtained is then clarified, filtered and purified by using two-step chromatography; SEC using Sepharose CL-6B and IEC-DEAE 650M. Alternatively, purification of the viral harvest can also be done by single chromatographic column using mixture of two different manufacturer resins that have high salt tolerance. Different anion exchanger resins like DEAE, QEAE, SP Sepharose, Poly CSX-35, CM Sepharose were used in combination with Butyl 650, Phenyl 650, Butyl Sepharose, phenyl Sepharose and aktyl Sepharose.

[0129] The concentrated bulk is then stabilized using M199 and inactivated. Sabin IPV Type-1 or Type-2 or Type-3 purified bulk is added to 2 mM Phosphate buffer having pH 7.2, 10 mM Phosphate Buffer with Saline pH 7.2 (5 diluted in inactivation) and 25 mM Sodium Acetate pH 7.2. Inactivation of the virus bulk and buffer mixture is carried out by addition of 0.025% formaldehyde at 37 C. from 0 to 13 days on magnetic stirrer.

[0130] Various vaccine formulations can be prepared with or without adjuvants. Two or more antigens obtained by the process disclosed under embodiments of the invention can be used to prepare combination vaccine compositions.

[0131] In some embodiments of the invention, the vaccine formulation prepared comprises any one of the following: [0132] a. Base 1+D-Antigen in 2 mM PB; [0133] b. D-Antigen in 2 mM PB+Sucrose 5% (w/v); [0134] c. D-Antigen in 2 mM PB+Trehalose 5% (w/v); [0135] d. D-Antigen in 2 mM PB+Maltose 5% (w/v); [0136] e. Base 1+D-Antigen in 2 mM PB+Sucrose 5% (w/v); [0137] f. Base 1+D-Antigen in 2 mM PB+Trehalose 5% (w/v); [0138] g. Base 1+D-Antigen in 2 mM PB+Maltose 5% (w/v); [0139] h. D-Antigen in 2 mM PB+Sucrose 5% (w/v)+Trehalose 5% (w/v); [0140] i. D-Antigen in 2 mM PB+Sucrose (5% (w/v)+Maltose 5% (w/v); [0141] j. D-Antigen in 2 mM PB+Trehalose 5% (w/v)+Maltose 5% (w/v); [0142] k. Base 1+D-Antigen in 2 mM PB+Sucrose 5% (w/v)+Trehalose 5% (w/v); [0143] l. Base 1+D-Antigen in 2 mM PB+Sucrose 5% (w/v)+Maltose 5% (w/v); or [0144] m. Base 1+D-Antigen in 2 mM PB+Sucrose 5% (w/v)+Trehalose 5% (w/v)+Maltose 5% (w/v), wherein Base 1 is 1 (9.9 g/L) concentrated M-199 with 0.5% glycine and 0.01% tween 80.

EXAMPLES

Example 1

[0145] Vero cells were revived and grown in a tissue culture flask at 37 C. The cells were passaged step by step to a higher surface area culture flask by trypsinizing the confluent monolayer. Dulbecco's Essential medium along with 5% New Born Calf Serum was used for culturing of cells. Phosphate buffered saline was used during cell expansion to remove debris, dead cells and traces of serum. After the required cells were grown, the cells were infected with enteroviruses with 0.01 MOI and incubated at 32.5 C. till complete cytopathic effect was observed. Once the cytopathic effect was observed, the harvest was collected and clarified using 0.8+0.45 u capsule filter and purified by column chromatography.

Example 2

[0146] In one embodiment of the present invention, purification is done by Single Column Purification Process in the following manner:

[0147] The purification of the viral antigens with high quality was done by using single chromatography column containing mixture of two different manufacturer resins which was tolerant to high salt concentrations there by recovering the antigenic particles with very high quality and quantity. The single column XK16/40 was prepared by packing the column with hydrophobic resin with cation exchanger. The column height was 5cm with a bed volume of 10 mL. Column was equilibrated with 0.2M Sodium acetate buffer (pH 4.88) and purified the viral harvest using the Akta purification system. As the column contains cation exchanger, the enteroviral particles were bound to the column in the pH range of 4.8-5.2. Later they were eluted out in the washes with pH range of 5.2-6.0.

[0148] The purified bulk was further concentrated and buffer exchanged by tangential flow filtration using 100 KDa cassettes attached to a low shear pump. The concentrated bulk was then stabilized using M199 and inactivated.

[0149] FIG. 1 to FIG. 4 depicts the quality of Poliovirus-1 and Poliovirus-3 proteins in SDS PAGE after single column chromatography and chromatogram profile showing Poliovirus-1 and Poliovirus-3 protein peaks after single column chromatography. Purity of the D-Antigen obtained is very high as evident from the above mentioned figures.

Example 3

[0150] In another embodiment of the present invention, following process was carried out to obtain high yield of the D-antigen:

[0151] 1.1 Purification of Sabin IPV (sIPV)

[0152] 1) Tangential flow filtration (TFF):

[0153] The clarified harvest can be quantified for D-Antigen at harvest level by ELISA. Then the clarified harvest pool was concentrated to 400-700 using tangential flow filtration system with 100 kda cassettes and collected the retentate. Cassette was washed with phosphate buffered Saline (10 mM, pH: 7.2); then added the wash sample to retentate and used as load for column chromatography.

[0154] 1.2 Two step Column Chromatography:

[0155] The purification was done by GFC using Sepharose CL-6B and then Ion Exchange Chromatography (IEC). Virus peak was collected after passing the 400-700 TFF concentrate through gel filtration column and used as a load for DEAE 650M (Weak-Anion exchanger) using Akta purification system (GE Healthcare). Impurities were bound to the column whereas polio virus was collected in flow through with 10 mM phosphate buffer saline.

[0156] During column chromatography, peak fractions were collected and quality was assessed by SDS-PAGE analysis. Based on the SDS gel results, pure fractions were pooled as flow through wash and D-Ag will be tested by ELISA. Hence, during inactivation with various buffers different volumes of samples were used based on the volume of flow through wash obtained.

[0157] FIG. 5 to FIG. 9 depicts the quality of PV-1, PV-2 and PV-3 proteins in SDS PAGE after two-column chromatography and chromatogram profile showing Poliovirus-1 and Poliovirus-3 protein peaks after two-step column chromatography. Yields of the D-Antigen obtained are given in the Tables 4 and 5.

Example 4

[0158] Inactivation of sIPV:

[0159] Inactivation buffer: 1 (9.9 g/L) concentrated M-199 with 0.5% glycine and 0.01% tween 80 (Base 1) was added to make the final inactivation buffer containing 2 mM PB, 2 mM PBS or 25 mM NaOAc, so as to achieve final concentration of 1. The final inactivation buffer containing D-Antigen was thereafter subjected to inactivation step.

[0160] An aspect of formaldehyde inactivation can occur in presence of PB buffer or PB buffer with saline or sodium acetate having concentration selected from 1 mM-100 mM, in particular 2 mM for PB and 25 mM for sodium acetate at a pH selected from (6.0-7.5) in particular between (6.9 to 7.3) and in particular at pH 7.2 adjusted by using hydrochloric acid, di-sodium hydrogen phosphate dihydrate, sodium hydroxide, acetic acid, phosphoric acid, sodium acetate trihydrate; wherein said inactivation utilizes phosphate buffer and sodium acetate. Different inactivation buffers used are given below:

TABLE-US-00003 TABLE 3 Inactivation Buffers Inactivation Buffers Composition Base 1 1X (9.9 g/L) concentrated M-199 with 0.5% glycine and 0.01% tween 80 Inactivation Buffer 1 Base 1 + 2 mM PB Inactivation Buffer 2 Base 1 + 2 mM PB + 50 mM NaCl Inactivation Buffer 3 Base 1 + 2 mM PB + 100 mM NaCl Inactivation Buffer 4 Base 1 + 25 mM Sodium acetate Inactivation Buffer 5 Base 1 + 25 mM Sodium acetate + 50 mM NaCl Inactivation Buffer 6 Base 1 +25 mM Sodium acetate + 100 mM NaCl Inactivation Buffer 7 Base 1 + 50 mM Sodium acetate Inactivation Buffer 8 Base 1 + 50 mM Sodium acetate + 50 mM NaCl Inactivation Buffer 9 Base 1 + 50 mM Sodium acetate + 100 mM NaCl Inactivation Buffer 10 Base 1 + 10 mM PBS Inactivation Buffer 11 Base 1 + 10 mM PBS (5X Diluted) Inactivation Buffer 12 Base 1 + 40 mM Tris

[0161] Inactivation agent formalin (0.025%) was added into purified virus bulk while constant mixing. Inactivation was carried out at 37 C. while continuous stirring for 13 days containing 0.22 filtration on 6.sup.th and 13.sup.th day. When formaldehyde inactivation methods were particularly carried out in presence of phosphate buffer with saline and sodium acetate, significant D-antigen recoveries were observed for Sabin strains type-1, 2, 3.

[0162] Effects of buffer concentration in D-Antigen recoveries, for Type-I poliovirus, are depicted in FIG. 10 Results obtained are tabled in Table 4 below:

TABLE-US-00004 TABLE 4 Poliovirus Type-1 D-Antigen recovery after Inactivation with inactivation buffers D D D antigen antigen antigen Volume at 0.sup.thday at 6.sup.th day at13.sup.th day used (DU/ (DU/ (DU/ 0% of Buffers (mL) mL) mL) mL) Recovery Inactivation Buffer 1 20 1933 1870 1658 85.7 Inactivation Buffer 2 20 1894 1649 1720 90.8 Inactivation Buffer 3 20 2255 1392 1273 56.4 Inactivation Buffer 4 20 1572.5 1140 1548 98.4 Inactivation Buffer 5 20 1722 1059 952.1 54.3 Inactivation Buffer 6 20 1536 1454 996 64.1 Inactivation Buffer 7 20 1743 1271 1203 69 Inactivation Buffer 8 20 1612 1377 1397 86.6 Inactivation Buffer 9 20 2083 903.8 951.9 45.6 Inactivation Buffer 10 15 1994 1313 1051 52.7 Inactivation Buffer 11 75 1880 1519 1595 84.8

[0163] Table 4 above depicts the percentage of recovery in D-Antigen after inactivation from Day 0 to Day 13. It was observed that more than 90% recovery was observed in 2 mM PB, 2 mM PB with 50 mM NaCl, 25 mM Sodium Acetate, 50 mM Sodium Acetate with 50 mM NaCl and 10 mM PBS (5 times diluted) for Poliovirus Type-1 inactivated bulk.

[0164] Observation of Poliovirus Type-3 D-Antigen after Inactivation:

[0165] Inactivation Buffer 12 was selected to study the comparative inactivation as compared to the best of Inactivation Buffer obtained for Type-I poliovirus. Inactivation study was also performed for Poliovirus type-3 by choosing the best inactivation buffers based on the higher recoveries obtained for Type-1 along with comparison with Tris buffer as depicted in Table 5 below:

TABLE-US-00005 TABLE 5 Poliovirus Type-3 D-Antigen recovery after Inactivation with inactivation buffers % of D D D Recovery antigen antigen antigen D-Ag at 0 at 6.sup.th at 13.sup.th after Volume day day day 13.sup.th day used (DU/ (DU/ (DU/ (DU/ Buffers (mL) mL) mL) mL) mL) Inactivation Buffer 1 55 1391.82 1257 1266 91.01 Inactivation Buffer-12 1366 1299 93.3 Inactivation Buffer -4 1392 1324 95.1

[0166] Table 5 above depicts the percentage of D-Antigen recovery after inactivation from Day 0 to Day 13. It is reconfirmed that the D-Antigen recovery is more than 90% in 2 mM PB and 25 mM Sodium acetate in comparison to 40 mM Tris which is already claimed. D-Antigen Recovery during Inactivation Stages is depicted in FIG. 11.

Example 5

[0167] A key challenge in developing a purified antigenic vaccine is ensuring the stability and its efficacy during the storage of inactivated bulk. Therefore, the present study shows various excipients and its combinations for assessing the stability of inactivated D-Antigen till 30 days at 37 C. Following are the list of formulations along with its composition at the pH range of 6.0 to 7.5, at in particular 7.2.

TABLE-US-00006 TABLE 6 D-Antigen with various excipient Control D-Antigen in 2 mM PB (Without stabilizers) Formulation 1 Base 1 + D-Antigen in 2 mM PB Formulation 2 D-Antigen in 2 mM PB + Sucrose 5%(w/v) Formulation 3 D-Antigen in 2 mM PB + Trehalose 5%(w/v) Formulation 4 D-Antigen in 2 mM PB + Maltose 5%(w/v) Formulation 5 Base 1 + D-Antigen in 2 mM PB + Sucrose 5%(w/v) Formulation 6 Base 1 + D-Antigen in 2 mM PB + Trehalose 5%(w/v) Formulation 7 Base 1 + D-Antigen in 2 mM PB + Maltose 5%(w/v) Formulation 8 D-Antigen in 2 mM PB + Sucrose 5%(w/v) + Trehalose 5%(w/v) Formulation 9 D-Antigen in 2 mM PB + Sucrose (5%(w/v) + Maltose 5%(w/v) Formulation 10 D-Antigen in 2 mM PB + Trehalose 5%(w/v) + Maltose 5%(w/v) Formulation 11 Base 1 + D-Antigen in 2mM PB + Sucrose 5%(w/v) + Trehalose 5%(w/v) Formulation 12 Base 1 + D-Antigen in 2mM PB + Sucrose 5%(w/v) + Maltose 5%(w/v) Formulation 13 Base 1 + D-Antigen in 2 mM PB + Sucrose 5%(w/v) + Trehalose 5%(w/v) + Maltose 5%(w/v)

[0168] The above mentioned various formulations were used for studying the stability of the inactivated Poliovirus type-1 D-Antigen on various days till one month. These formulations comprise of inactivated polioviruses along with said buffers and excipients. This formulation step is elucidating the addition of excipients after inactivation of poliovirus was done. Following data shows the D-antigen content in DU/mL units:

TABLE-US-00007 TABLE 7 D-Antigen (DU/mL) at 37 C. D-Antigen at D-Antigen at D-Antigen at D-Antigen at 0 Day 7.sup.th Day 14.sup.th Day 30.sup.th Day (DU/mL) (DU/mL) (DU/mL) (DU/mL) Control 288.4 84.49 58.51 99.86 Formulation 1 113.61 103.66 111.14 Formulation 2 145.09 113.31 111.22 Formulation 3 148.41 120.38 126.12 Formulation 4 152.79 84.64 86.1 Formulation 5 143.66 133.2 118.93 Formulation 6 194.48 202.4 232.8 Formulation 7 256.1 196.35 140.94 Formulation 8 247.9 132.27 115.74 Formulation 9 159.03 123.1 84.84 Formulation 10 151.33 140.64 94.81 Formulation 11 167.08 149.37 114.19 Formulation 12 182.6 172.16 117.68 Formulation 13 158.77 164.51 99.13

[0169] Table 7 above depicts the D-Antigen stability after inactivation with various combinations of excipients from Day 0 to Day 30. It is claimed that the D-Antigen at 37 C. was most stable in Formulation 6. Effects of stabilizers in D-Antigen Recoveries are depicted in FIG. 12.

Example 6

[0170] Excipients such as stabilizers were added to the inactivation buffer, before inactivation of D-Antigen. This reduced one step in the process of vaccine development, wherein the formulation after the inactivation is not required. This also aided in achieving higher yield recovery of the D-antigen as compared to the step in which the inactivation buffer does not contain stabilizers.

[0171] Two different types of Bases were used as mentioned below:

[0172] Base 1: 1 (9.9 g/L) concentrated M-199 with 0.5% glycine and 0.01% tween 80.

[0173] Base 2: 1 (15.1 g/L) concentrated M-199+HEPES with 0.5% glycine and 0.01% tween 80.

[0174] D-antigen was formulated with Base 1 or Base 2, along with inactivation buffers and stabilizers. This was referred to as the Formulated Inactivated Buffers in Table 8 below. These formulations were thereafter subjected to inactivation and checked for virus titres.

[0175] The kinetics of infectivity of a definite virus can be studied by inactivation kinetics. Inactivation is a process of enhancing viral safety in which virus is inactivated with formalin, BPL or any other inactivating agents for particular days to reduce its infectivity. This study can determine the minimal duration of treatment to obtain a given degree of infectivity reduction. The time required for the complete inactivation depends on the concentration of formaldehyde, incubation temperature, pH of the reaction mixture and the initial concentration of the virus. Based on the results obtained from the addition of various excipients for stability, the effect of Formulation 6 was studied during the inactivation with formalin. Following are the four different compositions used for the kinetic study at the pH range of 6.0 to 7.5, in particular at 7.2, depicted in Table 8 below:

TABLE-US-00008 TABLE 8 Compositions of Formulated Inactivation Buffers Composition Formulated Inactivation Buffer-1 (FIB-1) Base 1 + 2 mM PB + Trehalose (5% w/v) Formulated Inactivation Buffer-2 (FIB-2) Base 2 + 2 mM PB + Trehalose(5% w/v) Formulated Inactivation Buffer-3 (FIB-3) Base 1 + 2 mM PB Formulated Inactivation Buffer-4 (FIB-4) Base 2 + 2 mM PB

[0176] Inactivation Kinetics of Poliovirus Type-1

[0177] Inactivation kinetics study for Poliovirus type-1 was studied by taking the samples every 24 hours from the time of inactivation till 13 days. The samples were tested for virus titration, D-Antigen and virus inactivation tests.

TABLE-US-00009 TABLE 9 Concentration of D-Antigen after Inactivation Day 0 Day 0 (BI) (AI) Day 3 Day 6 Day 9 Day 13 % D-Ag/ml D-Ag/ml D-Ag/ml D-Ag/ml D-Ag/ml D-Ag/ml recovery FIB-1 1454.6 1459.5 1345.9 1423.4 1321 1388.6 95.4 FIB-2 1522.8 1356 1464 1482.3 1184.8 1432.8 94 FIB-3 1445.5 1403.7 1102 1289.2 1278 1331.4 92.1 FIB-4 1513.7 1627.5 1283.8 1283.6 1356.9 1314.8 86.8

[0178] Table 9 above shows the concentration of D-Antigen in the inactivated bulks up to 13.sup.th day, where the recoveries in all the inactivated buffers are more than 85%. The antigen recovery was higher with the inactivation buffers used along with the stabilizers as compared with the inactivation without stabilizers.

[0179] Inactivation Kinetics Results for Type-I:

TABLE-US-00010 TABLE 10 Type-1 Virus titers of Inactivated Samples FIB-1 FIB-2 FIB-3 FIB-4 Day 0 (Before 10.sup.9.2/ml 10.sup.9.09/ml 10.sup.9.37/ml 10.sup.9.6/ml Inactivation) Day 0 (After 10.sup.7.58/ml 10.sup.7.89/ml 10.sup.7.57/ml 10.sup.7.68/ml Inactivation) Day 1 10.sup.5.4/ml 10.sup.5.64/ml 10.sup.5.22/ml 10.sup.5.48/ml Day 2 10.sup.3.59/ml 10.sup.4.09/ml 10.sup.2.66/ml 10.sup.2.66/ml Day 3 0 0 3 wells 4 wells infected in10.sup.1 infected in 10.sup.1 dilution dilution Day 4 0 0 1 well 0 infected in 10.sup.1 dilution Day 5 0 0 0 0 Day 6 0 0 0 0 Day 7 0 0 0 0 Day 8 0 0 0 0 Day 9 0 0 0 0 Day 10 0 0 0 0 Day 11 0 0 0 0 Day 12 0 0 0 0 Day 13 0 0 0 0

[0180] Table 10 above shows the titers of different component inactivated buffers after addition of formalin from 0.sup.th day to 13.sup.th day. The virus titers decreased till 4th day and there were no titers observed after day 5 indicating that the poliovirus was getting completely inactivated within 5 days. Viral amplification tests were also negative for poliovirus type-1 from Day 6 to Day 13.

Example 7

[0181] Inactivation Kinetics of Poliovirus Type-3

[0182] Inactivation kinetics study for Poliovirus type-3 was studied by taking the samples every 24 hours from the time of inactivation till 13 days. The samples were tested for virus titration, D-Antigen and virus inactivation tests.

TABLE-US-00011 TABLE 11 Concentration of D-Antigen after Inactivation Day 0 Day 0 (BI) (AI) Day 3 Day 6 Day 13 % D-Ag/ml D-Ag/ml D-Ag/ml D-Ag/ml D-Ag/ml recovery FIB-1 678.8 679.5 607.3 545.7 634.5 93.4 FIB-2 750.7 669.1 661.7 670.5 667.4 88.9 FIB-3 781.2 767.9 752.8 747.9 664.2 85.02 FIB-4 722.6 677.8 642 672.3 662.7 91.7

[0183] Table 11 above shows the concentration of D-Antigen in the inactivated bulks upto 13.sup.th day, where the recoveries in all the inactivated buffers were more than 85%.

[0184] Inactivation Kinetics Results for Type-III:

TABLE-US-00012 TABLE 12 Type-3 Virus titers of Inactivated Samples FIB-1 FIB-2 FIB-3 FIB-4 Day 0 (Before 10.sup.8.68/ml 10.sup.8.67/ml 10.sup.8.7/ml 10.sup.8.12/ml Inactivation) Day 0 (After 10.sup.7.89/ml 10.sup.7.89/ml 10.sup.7.95/ml 10.sup.7.4/ml Inactivation) Day 1 10.sup.5.89/ml 10.sup.5.86/ml 10.sup.5.74/ml 10.sup.4.78/ml Day 2 10.sup.3.44/ml 10.sup.3.77/ml 10.sup.3.12/ml 10.sup.3.21/ml Day 3 10.sup.2.89/ml 10.sup.2.8/ml 10.sup.2.79/ml 10.sup.2.87/ml Day 4 2 wells 3 wells 3 wells 4 wells infected infected infected infected i in 10.sup.1 in 10.sup.1 in 10.sup.1 n 10.sup.1 dilution dilution dilution dilution Day 5 0 0 0 0 Day 6 0 0 0 0 Day 7 0 0 0 0 Day 8 0 0 0 0 Day 9 0 0 0 0 Day 10 0 0 0 0 Day 11 0 0 0 0 Day 12 0 0 0 0 Day 13 0 0 0 0

[0185] Table 12 above shows the titers of different component inactivated buffers after addition of formalin from 0.sup.th day to 13.sup.th day. The virus titers decreased till 4th day and there were no titers observed after day 5 indicating that the poliovirus is getting completely inactivated within 5 days. Viral amplification tests were also negative for poliovirus type-3 from Day 6 to Day 13.

Example 8

[0186] D-antigen content determination by ELISA.

[0187] The D-antigen content determination was out through ELISA following the below mentioned steps:

[0188] Plate coating: The 96 well plate was coated with specific anti-polio sera in bicarbonate buffer and incubated overnight at 2-8 C.

[0189] Blocking: The plates were washed (Washing/dilution buffer 0.05% Tween 20 in 1 PBS) once and incubated at 371 C. for 60 minutes with block buffer (1% BSA in PBS).

[0190] Sample addition: After washing the plate with 0.05% PB ST, Serial four-fold diluted samples were added to the 96 well plate and then incubated at 37 C. for 2 hr.

[0191] Monoclonal antibody addition: The plate was washed thrice with 0.05% PBST and 1:1000 type specific monoclonal antibodies were added. The plates were sealed and incubated for 1 hour at 37 C.

[0192] Conjugate: Diluted conjugate of Anti-mouse IgG raised in goat with horse radish peroxidase at 1:2500 was added and incubated for 1 hour at 37 C.

[0193] Substrate addition: OPD substrate was added to all wells and mixture was incubated at room temperature for 20 minutes. Reaction was stopped by addition of 2M H2504. Plate was read at 490 nm. D-antigen concentration was calculated by using My assays software.

[0194] Although the invention has been illustrated and described in greater detail with reference to the preferred exemplary embodiments, the invention is not limited to the examples disclosed, and further variations can be inferred by a person skilled in the art, without departing from the scope of protection of the invention.

[0195] For the sake of clarity, it is to be understood that the use of a or an throughout this application does not exclude a plurality, and comprising does not exclude other steps or elements.