Purification of hbv antigens for use in vaccines

Abstract

The present invention relates to a method for the production of a hepatitis B antigen suitable for use in a vaccine, the method comprising purification of the antigen in the presence of cysteine, to vaccines comprising such antigens.

1  A hepatitis B vaccine comprising a purified hepatitis B antigen, the antigen comprising less than 0.025 μg mercuy per 20 μg protein, the antigen being obtainable by purification in the presence of a reducing agent having a free —SH group.  
     
     
         2  A vaccine according to  claim 1  which is without preservative.  
     
     
         3  A vaccine according to  claim 2  wherein the preservative is thiomersal.  
     
     
         4  A vaccine according to any of claims  1 - 3  wherein the hepatitis antigen is adsorbed onto aluminium hydroxide.  
     
     
         5  A vaccine according to any preceding claim wherein the hepatitis B antigen is obtainable by subjecting a crude hepatitis B preparation to the following steps: 
 (a) gel permeation chromatography;  
 (b) ion-exchange chromatography; and  
 (c) mixed with a reducing agent having a free —SH group  
 
     
     
         6  A vaccine according to any preceding claim wherein the reducing agent is cysteine, glutathione,dithiothreitol or β-mercaptoethanol.  
     
     
         7  A vaccine according to  claim 6 , wherein the reducing agent is cysteine.  
     
     
         8  A vaccine according to  claim 7  wherein the cysteine is added to a final centration of between 1-10 mM.  
     
     
         9  A vaccine according to  claim 8  wherein the cysteine is added to a final centration of about 2 mM.  
     
     
         10  A vaccine according to any preceding claim wherein the antigen is purified in the presence of thiomersal before treatment with the reducing agent.  
     
     
         11  A vaccine according to any of claims  1 - 9  wherein the purified antigen is completely free of thiomersal.  
     
     
         12  A vaccine according to  claim 11  which is a thiomersal free vaccine.  
     
     
         13  A vaccine composition according to any preceding claim in conjunction with an adjuvant.  
     
     
         14  A vaccine composition according to  claim 13  wherein the adjuvant is an aluminium salt.  
     
     
         15  A vaccine composition according to  claim 14  wherein the aluminium salt is aluminium hydroxide  
     
     
         16  A vaccine composition as claimed in claim  13 - 15  which comprises a TH-1 inducing adjuvant.  
     
     
         17  A vaccine composition according to  claim 16  wherein the TH1-inducing adjuvant is selected from the group comprising: 3-DMPL, QS21, 3-DMPL and QS21, and aCpG oligonucleotide.  
     
     
         18  A vaccine composition as claimed in any preceding claim which additionally comprises one or more of the antigens selected from the group consisting of: 
 diptheria toxoid (D), tetanus toxoid (T) acellular pertussis antigens (Pa), inactivated polio virus (IPV), haemophilus influenzae antigen (Hib), hepatitis A antigen, herpes simplex virus (HSV), chlamydia, GSB, HPV, streptococcus pneumoniae and neisseria antigens.  
 
     
     
         19  Use of a hepatitis antigen comprising less than 0.025 μg mercury per 20 μg protein in the preparation of a preservative free hepatitis vaccine, the antigen being obtainable by purification in the presence of a reducing agent having a free —SH group.  
     
     
         20  A method for producing a hepatitis B antigen suitable for use in a vaccine, the method comprising purification of the antigen in the presence of a reducing agent having a free —SH group, wherein the antigen is purified in the presence of thiomersal before treatment with the reducing agent.  
     
     
         21  A method for producing a hepatitis B antigen according to  claim 20 , wherein the purified antigen product comprises less than 0.025 μg mercury per 20 μg protein  
     
     
         22  A method of producing a hepatitis B antigen according to  claim 20  or  21  wherein a crude hepatitis B antigen preparation is: 
 (a) subjected to gel permeation chromatography;  
 (b) subjected to ion-exchange chromatography; and  
 (c) mixed with a reducing agent having a free —SH group  
 
     
     
         23  A method of producing a hepatitis B antigen according to claim  20 - 22  wherein the reducing agent is cysteine, glutathione, dithiothreitol or β-mercaptoethanol.  
     
     
         24  An immnunogenic hepatitis B antigen, the antigen comprising less than 0.025 μg mercury per 20 μg protein, the antigen being obtainable by purification in the presence of a reducing agent having a free —SH group and wherein the antigen is purified in the presence of thiomersal before treatment with the reducing agent.  
     
     
         25  A vaccine comprising a hepatitis B antigen as claimed in  claim 24   
     
     
         26  A method, antigen or vaccine composition according to any preceding claim wherein the hepatitis B antigen is a surface antigen.  
     
     
         27  A method of treatment and/or prophylaxis of hepatitis B virus infections, the method comprising administering to a human or animal subject suffering from or susceptible to hepatitis B infection a safe and effective amount of a vaccine according to any of claims  1 - 18  and  25 .