Method for the purification of alphavirus replicon particles

Abstract

Methods of production and purification for viruses and virus-derived vectors, including those related to alphaviruses, are disclosed. in one aspect, methods of purification that subject alphavirus replicon particle preparations to one or more steps of chromatographic purification, such as using an ion exchange resin, are provided. Also disclosed are methods of characterizing alphavirus replicon particles and utilizing these materials for vaccines and gene-based therapeutics.

1 . A method for purifying alphavirus replicon particles comprising:
 (a) contacting a preparation containing alphavirus replicon particles with a tentacle ion exchange resin, under conditions and for a time sufficient to bind to said resin;   (b) removing the portion of the preparation which is not bound to said ion exchange resin from said ion exchange resin;   (c) eluting the bound alphavirus replicon particles from said ion exchange resin; and   (d) recovering said replicon particles.   
   
   
       2 . The method according to  claim 1  wherein said tentacle ion exchange resin is a cationic exchange resin. 
   
   
       3 . The method according to  claim 1  wherein said tentacle ion exchange resin is an anionic exchange resin. 
   
   
       4 . A method for purifying alphavirus replicon particles comprising:
 (a) contacting a preparation containing alphavirus replicon particles with a first chromatography resin selected from the group consisting of a tentacle ion exchange chromatography resin, a size exclusion chromatography resin, a hydrophobic interaction chromatography, and an affinity chromatography resin;   (b) recovering a first portion of the preparation from the first resin, the first portion comprising the alphavirus replicon particles;   (c) contacting the first portion comprising the alphavirus replicon particles with a second chromatography resin selected from the group consisting of a tentacle ion exchange chromatography resin, a size exclusion chromatography resin, a hydrophobic interaction chromatography, and an affinity chromatography resin; and   (d) recovering a second portion from the second resin, the second portion comprising the alphavirus replicon particles,   
     thereby purifying alphavirus replicon particles. 
   
   
       5 . The method according to  claim 4  wherein the first or second resin comprises a tentacle ion exchange chromatography resin. 
   
   
       6 . The method according to  claim 4  wherein a first resin is a tentacle ion exchange chromatography resin and a second resin is a size exclusion chromatography resin. 
   
   
       7 . A method for purifying alphavirus replicon particles comprising
 (a) contacting a preparation containing alphavirus replicon particles with a first chromatography resin selected from the group consisting of an ion exchange chromatography resin, a size exclusion chromatography resin, a hydrophobic interaction chromatography, and an affinity chromatography resin;   (b) recovering a first portion of the preparation from the first resin, the first portion comprising the alphavirus replicon particles;   (c) contacting the first portion comprising the alphavirus replicon particles with a second chromatography resin selected from the group consisting of an ion exchange chromatography resin, a hydrophobic interaction chromatography, and an affinity chromatography resin; and   (d) recovering a second portion from the second resin, the second portion comprising the alphavirus replicon particles,   
     thereby purifying alphavirus replicon particles. 
   
   
       8 . A method for purifying alphavirus replicon particles comprising:
 (a) contacting a preparation containing alphavirus replicon particles with a first chromatography resin selected from the group consisting of a tentacle ion exchange chromatography resin, a size exclusion chromatography resin, a hydrophobic interaction chromatography, and an affinity chromatography resin;   (b) recovering a first portion of the preparation from the first resin, the first portion comprising the alphavirus replicon particles;   (c) contacting the first portion comprising the alphavirus replicon particles with a second chromatography resin selected from the group consisting of an ion exchange chromatography resin, a size exclusion chromatography resin, a hydrophobic interaction chromatography, and an affinity chromatography resin; and   (d) recovering a second portion from the second resin, the second portion comprising the alphavirus replicon particles,   
     thereby purifying alphavirus replicon particles. 
   
   
       9 . The method of  claim 7  wherein the alphavirus replicon particles are capable of expressing an antigen derived from a pathogenic agent. 
   
   
       10 . The method of  claim 9  wherein said pathogenic agent is selected from the group consisting of viruses, bacteria, fungi, parasites, and cancerous cells. 
   
   
       11 . The method of  claim 7  wherein the alphavirus replicon particle preparation comprises a therapeutic. 
   
   
       13 . The method of  claim 7  wherein said alphavirus replicon particle preparation expresses a lymphokine, cytokine, or chemokine. 
   
   
       14 . The method of  claim 13  wherein said lymphokine, cytokine or chemokine is selected from the group consisting of IL-2, IL-10, IL-12, gamma interferon, GM-CSF, macrophage inflammatory protein (MIP)3α, MIP3β, and secondary lymphoid tissue chemokine (SLC). 
   
   
       15 . A method for stimulating. an immune response within a warm-blooded animal, comprising administering to a warm-blooded animal a preparation of alphavirus replicon particles prepared according to  claim 7 . 
   
   
       16 . The method according to  claim 15  wherein said alphavirus replicon particle preparation expresses a lymphokine, cytokine, or chemokine. 
   
   
       17 . The method according to  claim 16  wherein said lymphokine, cytokine or chemokine is selected from the group consisting of IL-2, IL-10-, IL-12, gamma interferon, GMCSF, macrophage inflammatory protein (MIP)3α, MIP3β, and secondary lymphoid tissue chemokine (SLC). 
   
   
       18 . A method of producing alphavirus replicon particles comprising:
 (a) infecting alphavirus packaging cells with a seed stock of alphavirus replicon particles;   (b) incubating the infected packaging cells in a bioreactor, under conditions and for a time sufficient to permit the production of alphavirus replicon particles; and   (c) harvesting culture supernatants containing said replicon particles.   
   
   
       19 . The method of  claim 18  wherein said bioreactor is selected from the group consisting of an external component bioreactor, a suspension culture bioreactor, and a hollow fiber bioreactor.