Method for inactivating zika virus and related methods

Abstract

The present disclosure relates to methods for inactivating a Zika virus which can be used in vaccines and immunogenic compositions. The present disclosure also relates to a method for determining the completeness of inactivation of an arbovirus preparation and to a method for determining the residual formaldehyde content in a pharmaceutical composition comprising an inactivated virus.

1 .- 57 . (canceled) 
     
     
         58 . A method for determining the residual formaldehyde content in a pharmaceutical composition comprising an inactivated virus, comprising the steps of:
 (a) providing a composition comprising a virus which has been treated with formaldehyde;   (b) mixing the composition of (a) with phosphoric acid and 2,4-dinitrophenyl¬hydrazine (DNPH), thereby providing a mixture;   (c) incubating the mixture of (b) under suitable conditions; and   (d) analyzing the mixture for the presence of residual formaldehyde.   
     
     
         59 . The method of  claim 58 , wherein the composition of (a) contains an adjuvant. 
     
     
         60 . The method of  claim 59 , wherein the adjuvant is aluminum hydroxide. 
     
     
         61 . The method of  claim 58 , wherein the composition of (a) contains 0.1 mg/ml to 1.0 mg/ml aluminum hydroxide as adjuvant. 
     
     
         62 . The method of  claim 58 , wherein step (b) comprises mixing 50 parts of the composition of (a) with 1 part of 15 to 25% (v/v) phosphoric acid and 2.5 parts of 0.9 to 1.1 mg/ml DNPH. 
     
     
         63 . The method of  claim 58 , wherein the mixture of the composition of (a) with phosphoric acid and 2,4-dinitrophenylhydrazine (DNPH) is incubated at room temperature. 
     
     
         64 . The method of  claim 58 , wherein the mixture of the composition of (a) with phosphoric acid and 2,4-dinitrophenylhydrazine (DNPH) is incubated for 10 to 30 minutes. 
     
     
         65 . The method of  claim 58 , wherein the mixture of the composition of (a) with phosphoric acid and 2,4-dinitrophenylhydrazine (DNPH) is analyzed by HPLC. 
     
     
         66 . The method of  claim 65 , wherein the HPLC is reversed-phase HPLC. 
     
     
         67 . The method of  claim 66 , wherein a mixture of water and acetonitrile (1:1, v/v) is used as a mobile phase in HPLC. 
     
     
         68 . The method of  claim 58 , wherein the virus is an inactivated Zika virus. 
     
     
         69 . The method of  claim 68 , wherein the inactivated Zika virus has been treated with 0.01% (w/v) formaldehyde for 10 days at 22° C. 
     
     
         70 . The method of  claim 68 , wherein the Zika virus has been inactivated by the following steps:
 (a) isolating a Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation, wherein isolating the Zika virus preparation comprises one or more steps selected from:
 (i) depth filtration, 
 (ii) buffer exchange and/or dilution; 
 (iii) ion exchange chromatography; and 
   (b) treating the Zika virus preparation with formaldehyde, wherein the numerical result of the multiplication of the formaldehyde concentration as measured in % (w/v) with the period of incubation with formaldehyde as measured in days is 0.025 to 0.5.   
     
     
         71 . The method of  claim 70 , wherein the cells are non-human cells. 
     
     
         72 . The method of  claim 70 , wherein the cells are Vero cells. 
     
     
         73 . The method of  claim 70 , further comprising a step (c) comprising neutralizing the formaldehyde-treated Zika virus preparation with sodium metabisulfite. 
     
     
         74 . The method of  claim 73 , wherein the formaldehyde-treated Zika virus preparation is neutralized at least five, at least seven, at least nine, at least 11, or at least 14 days after formaldehyde treatment. 
     
     
         75 . A pharmaceutical composition comprising an inactivated Zika virus having a residual formaldehyde content of less than 30 μg/ml. 
     
     
         76 . A pharmaceutical composition comprising an inactivated Zika virus having a residual formaldehyde content of less than 50 μg/ml, wherein the residual formaldehyde is determined by the method of claim  1 .