Methods for enterovirus inactivation, adjuvant adsorption and dose reduced vaccine compositions obtained thereof
AssigneeSerum Institute of India Pvt Ltd
InventorsRajeev Mhalasakant Dhere, Sambhaji Shankar Pisal, Jagdish Kamalaji Zade, Rajendra Narayan Sabale
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Abstract
The present invention is directed to improved methods of Enterovirus inactivation by formaldehyde in presence of tromethamine buffer resulting in maximum recovery of D-antigen. Subsequent adsorption of said sIPV on aluminium hydroxide provides significantly dose reduced sIPV compositions.
Claims21
A method for producing a composition comprising Enteroviral poliovirus particles, wherein the method comprises the steps of:
- a) purifying Enteroviral particles in a medium comprising a phosphate buffer;
- b) exchanging the phosphate buffer of the purified Enteroviral particles of a) for a TRIS buffer;
- c) stabilizing the purified and buffer-exchanged Enteroviral particles of b);
- d) inactivating the Enteroviral particles of c) by:
- (i) adding formalin to the purified Enteroviral particles 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 TRIS buffer is present at a concentration of 30-70 mM;
- aggregation of the Enteroviral particles is prevented or reduced;
- and D-antigen losses are reduced post inactivation by 8 to 10 fold as compared to inactivation in phosphate buffer;
- and
- e) adsorbing the inactivated Enteroviral particles on an aluminum salt adjuvant, whereby the percentage adsorption on the aluminum salt adjuvant is at least 95%.
A method for preparing a dose reduced inactivated Polio vaccine containing Salk or Sabin poliovirus, comprising:
- a) purifying the poliovirus from a medium comprising a poliovirus and a phosphate buffer;
- b) exchanging the phosphate buffer of the purified Enteroviral particles of a) for a TRIS buffer;
- c) stabilizing the purified and buffer-exchanged poliovirus of b) by adding a M-199 medium containing glycine;
- d) inactivating the poliovirus of c) using formaldehyde 0.025% at 37° C. for 5 to 13 days in presence of TRIS buffer at a concentration between 30 mM to 60 mM to prevent or reduce aggregation of the poliovirus particles thereby reducing the D-antigen losses post inactivation by 8 to 10 fold as compared to phosphate buffer;
- and
- e) adsorbing the inactivated poliovirus on an Alum hydroxide adjuvant having concentration between 2 to 2.5 mg/dose, whereby percentage adsorption on Alum hydroxide is greater than 95% for Type 1, Type 2 and Type 3.
A method for producing a composition comprising Enteroviral poliovirus particles, wherein the method comprises the steps of:
- a) purifying Enteroviral particles comprising:
- (i) concentrating a solution comprising the Enteroviral particles using tangential flow filtration;
- (ii) diafiltering the product of step a-i with a medium comprising a phosphate buffer;
- and
- (iii) concentrating the product of step a-ii using column chromatography and a phosphate buffer;
- b) exchanging the phosphate buffer of the product of step a-iii for a TRIS buffer having a concentration of 30 to 50 mM of TRIS and a pH of 6.8 to 7.2;
- c) stabilizing the purified and buffer-exchanged Enteroviral particles by adding a medium comprising glycine to the product of step b;
- d) inactivating the Enteroviral particles of c) by:
- (i) adding formalin to the purified Enteroviral particles 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:
- aggregation of the Enteroviral particles is prevented or reduced;
- and D-antigen losses are reduced post inactivation by 8 to 10 fold as compared to phosphate buffer;
- and
- e) adsorbing Enteroviral particles on an Al(OH)3 adjuvant, whereby the percentage adsorption on the adjuvant is at least 95%.
Dependencies are inferred from the claim text.