Methods for enterovirus inactivation, adjuvant adsorption and dose reduced vaccine compositions obtained thereof

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

• (i) an inactivated polio virus antigen selected from the group consisting of IPV Type 1 at a dose less than 15 D-antigen units (DU), IPV Type 2 at a dose less than 18 D-antigen unit (DU), and IPV Type 3 at a dose less than 17 D-antigen unit (DU), per 0.5 ml;
• (ii) a diphtheria toxoid (D) antigen in an amount of 1 to 50 Lf per 0.5 ml;
• (iii) a tetanus toxoid (T) antigen in an amount of 1 to 30 Lf per 0.5 ml;
• (iv) a whole cell pertussis (wP) antigen in an amount of 1 to 50 IOU per 0.5 ml or acellular pertussis (aP) comprising one or more of modified adenylate cyclase, Pertussis toxoid (PT) 1-50 μg, Filamentous hemagglutinin (FHA) 1-50 μg, Penactin (P69 or PRN) 1-20 μg or Fimbrial proteins (FIM 1, 2 and 3) 2-25 μg;
• per 0.5 ml;
• (v) a hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml;
• and
• (vi) a Haemophilus influenzae type b antigen (Hib) in an amount of 1 to 20 μg per 0.5 ml;
• wherein the IPV Type 1, IPV Type 2 or IPV Type 3 antigen is produced by formaldehyde inactivation in a non-phosphate buffer selected from the group consisting of TRIS, TBS, MOPS, HEPES and bicarbonate;
• the process comprising the steps of:
• a) adsorbing the IPV (Sabin/Salk strain) bulk individually on aluminium hydroxide, followed by pH adjustment to 6.2-6.6;
• b) adsorbing the D on aluminium phosphate, followed by pH adjustment to 5.5-6.5;
• c) adsorbing the T on aluminium phosphate, followed by pH adjustment to 5.5-6.5;
• (d) adsorbing the HBsAg on aluminium phosphate, followed by pH adjustment to 6.0-6.5;
• (e) blending a mixture obtained from steps b), c), and d) by agitation at room temperature for 18-24 hours;
• (f) blending a content of step a) with the mixture obtained in step e), followed by pH adjustment to 6.4-6.6 and agitation at room temperature for 60 minutes;
• (g) adding an inactivated wP antigen/acellular pertussis antigen and a stabilizer (Histidine amino acid buffer solution 100-300 mM) to the mixture in step f), followed by agitation for 60 minutes and left in static condition for overnight at 2-8° C.;
• (h) adding the Hib antigen to the mixture obtained in step g) at 2-8° C., followed by pH adjustment to 6.4-6.6;
• and
• (i) adjusting the pH to 6.0 to 7.0 with sodium hydroxide/sodium carbonate and adding normal saline (0.9% NaCl) or Water For Injection (WFI) (q.s.) to make up a volume of the mixture obtained in step h), followed by agitation for 2 hours.

A method for producing a composition comprising Enteroviral poliovirus particles, wherein the method comprises the steps of:

• a) purifying the Enteroviral poliovirus particles in a medium comprising a phosphate buffer;
• b) exchanging the phosphate buffer of the purified Enteroviral poliovirus particles of step a) for a non-phosphate buffer selected from the group consisting of TRIS, TBS, MOPS, HEPES or bicarbonate buffer and a combination thereof;
• c) stabilizing the purified and buffer-exchanged Enteroviral poliovirus particles of step b);
• d) inactivating the Enteroviral poliovirus particles of step c) by:
• (i) adding formalin to the purified Enteroviral poliovirus particles in the non-phosphate buffer of step b) and stirring for 7 days;
• (ii) filtering the product of step (i);
• (iii) adding formalin to the product of step (ii) and stirring for 6 days;
• and
• (iv) filtering the product of step (iii),
• wherein the non-phosphate buffer is present at a concentration of 30-70 mM, aggregation of the Enteroviral poliovirus particles is prevented or reduced and the D-antigen losses are reduced post inactivation by 8 to 10 fold as compared to inactivation in phosphate buffer.

A combination vaccine comprising a vaccine portion and a buffer portion, wherein the vaccine portion consists essentially of:

• diphtheria toxoid (D);
• tetanus toxoid (T);
• whole cell pertussis (wP);
• hepatitis B virus surface antigen (HBsAg);
• Haemophilus influenzae b PRP-Carrier protein conjugate (Hib);
• and
• an inactivated polio virus antigen selected from the group consisting of IPV Type 1 at a dose between 5 D-antigen units and 10 D-antigen units (DU), IPV Type 2 at a dose between 1 D-antigen units and 16 D-antigen units (DU), and IPV Type 3 at a dose between 1 D-antigen units and 16 D-antigen units (DU)
• wherein the IPV Type 1, IPV Type 2 or IPV Type 3 antigen is produced by formaldehyde inactivation in a non-phosphate buffer selected from the group consisting of TRIS, TBS, MOPS, HEPES and bicarbonate.