Methods for enterovirus inactivation, adjuvant adsorption and dose reduced vaccine compositions obtained thereof
Inventors
DHERE, Rajeev Mhalasakant • Pisal, Sambhaji Shankar • ZADE, Jagdish Kamalaji • SABALE, Rajendra Narayan
Assignees
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Publication Number
US-10485862-B2
Publication Date
2019-11-26
Expiration Date
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.
Core Innovation
The invention relates to methods for producing a composition comprising Enteroviral poliovirus particles by purifying the particles in a phosphate buffer, exchanging the phosphate buffer for a TRIS buffer, and stabilizing the buffer-exchanged particles. The approach inactivates the Enteroviral particles with formalin while maintaining a TRIS buffer present at a concentration of 30–70 mM, where aggregation of the Enteroviral particles is prevented or reduced and post inactivation D-antigen losses are reduced by 8 to 10 fold as compared to inactivation in phosphate buffer.
After formalin inactivation under the specified TRIS conditions, the inactivated Enteroviral particles are adsorbed on an aluminum salt adjuvant such that the percentage adsorption on the aluminum salt adjuvant is at least 95%. In embodiments, the TRIS buffer is specified with a pH of about 6.8 to 7.2 and the aluminum salt adjuvant is selected from aluminum hydroxide, aluminum phosphate, or a mixture of both.
Claims Coverage
The consolidated content includes three independent claims. The main inventive features are directed to TRIS buffer exchange prior to formalin inactivation, formalin inactivation under TRIS conditions to reduce aggregation and D-antigen losses, and adsorption of the inactivated poliovirus onto an aluminum salt adjuvant with at least 95% adsorption, including dose-reduction aspects for an inactivated polio vaccine.
Producing an Enteroviral poliovirus composition using TRIS-buffered formalin inactivation with reduced D-antigen losses
Purifying Enteroviral particles in a phosphate buffer; exchanging the phosphate buffer for a TRIS buffer; stabilizing the purified and buffer-exchanged Enteroviral particles; and inactivating the Enteroviral particles by adding formalin with a TRIS buffer present at 30–70 mM, where aggregation is prevented or reduced and D-antigen losses are reduced post inactivation by 8 to 10 fold as compared to inactivation in phosphate buffer.
Adsorption onto an aluminum salt adjuvant with at least 95% adsorption after TRIS-buffered inactivation
Adsorbing the inactivated Enteroviral particles on an aluminum salt adjuvant such that the percentage adsorption on the aluminum salt adjuvant is at least 95%.
Preparing a dose reduced inactivated Polio vaccine using TRIS-buffered formaldehyde inactivation and alum hydroxide adsorption
Purifying the poliovirus from a medium comprising a poliovirus and a phosphate buffer; exchanging the phosphate buffer for a TRIS buffer; stabilizing by adding a M-199 medium containing glycine; inactivating using formaldehyde 0.025% in presence of TRIS buffer at 30–60 mM to prevent or reduce aggregation and thereby reducing the D-antigen losses post inactivation by 8 to 10 fold as compared to phosphate buffer; and 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.
Producing an Enteroviral poliovirus composition using tangential flow filtration and TRIS-buffered formalin inactivation followed by Al(OH)3 adsorption
Purifying Enteroviral particles comprising concentrating using tangential flow filtration, diafiltering with a medium comprising a phosphate buffer, and concentrating using column chromatography with a phosphate buffer; exchanging the phosphate buffer for a TRIS buffer having a concentration of 30 to 50 mM TRIS and a pH of 6.8 to 7.2; stabilizing by adding a medium comprising glycine; inactivating by adding formalin in staged stirring steps with filtering between additions, where aggregation is prevented or reduced and D-antigen losses are reduced post inactivation by 8 to 10 fold as compared to phosphate buffer; and adsorbing Enteroviral particles on an Al(OH)3 adjuvant such that percentage adsorption is at least 95%.
Across the independent claims, the core coverage centers on using TRIS buffer during formalin/formaldehyde inactivation to prevent or reduce poliovirus aggregation and to reduce post-inactivation D-antigen losses by 8 to 10 fold versus phosphate buffer, then adsorbing the inactivated particles onto aluminum salt adjuvants to achieve at least 95% adsorption. The dose-reduced polio vaccine claim further specifies M-199 with glycine stabilization and formaldehyde 0.025% in presence of TRIS at 30–60 mM.
Stated Advantages
Aggregation of the Enteroviral/poliovirus particles is prevented or reduced during inactivation.
D-antigen losses are reduced post inactivation by 8 to 10 fold as compared to inactivation in phosphate buffer.
High adsorption onto aluminum salt adjuvant is achieved, with percentage adsorption at least 95% (and, for alum hydroxide in the dose reduced vaccine claim, greater than 95% for Type 1, Type 2 and Type 3).
Documented Applications
Production of a composition comprising Enteroviral poliovirus particles by purifying, TRIS buffer exchanging, formalin inactivating, and adsorbing on an aluminum salt adjuvant.
Preparation of a dose reduced inactivated polio vaccine containing Salk or Sabin poliovirus with reduced D-antigen losses and alum hydroxide adsorption greater than 95%.
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