Human papillomavirus vaccine with disassembled and reassembled virus-like particles

Abstract

Human Papillomavirus vaccine formulations which contain virus-like particles (VLPs) can be made more stable and have an enhanced shelf-life, by treating the VLPs to a disassembly and reassembly process. Also provided are formulation buffers to long term stable storage of VLPs.

What is claimed is:  
     
         1 . A process for making a stable human papillomavirus (HPV) vaccine comprising HPV virus-like particles (VLPs), the process comprising the steps of: 
 (a) incubating VLPs in a dissociation mixture comprising a relatively low concentration of a reducing agent, a salt present in a range of approximately physiological ionic strength up to 1.25 M, a non-ionic surfactant, a metal chelating agent and a buffer until disassembled VLPs are produced;    (b) removing the reducing agent from the dissociation mixture; and    (c) reassembling the disassembled VLPs.    
     
     
         2 . A process according to    claim 1    wherein the reducing agent in the dissociation mixture is about 2-20 mM dithiothreitol (DTT).  
     
     
         3 . A process according to    claim 1    wherein the salt in the dissociation mixture is selected from the group consisting of: NaCl, KCl, Na 2 SO 4 , (NH 4 ) 2 SO 4  sodium phosphate, sodium citrate and mixtures thereof.  
     
     
         4 . A process according to    claim 1    wherein the non-ionic surfactant in the dissociation mixture is selected from the group consisting of: Polysorbate 80, Polysorbate 20, polyoxyethylene alkyl ethers, Triton X-100®, Triton X-114®, NP-40®, Span 85 and a Pluronic non-ionic surfactant.  
     
     
         5 . A process according to    claim 4    wherein the non-ionic surfactant is 0.01-0.5% Polysorbate 80.  
     
     
         6 . A process according to    claim 1    wherein the buffer in the dissociation mixture is TRIS or phosphate buffer, at pH 7-10.  
     
     
         7 . A process according to    claim 1    wherein the metal chelating agent in the dissociation mixture is EDTA at 0.5-5 mM.  
     
     
         8 . A process according to    claim 1    wherein step (a) takes place for less than about one hour at room temperature.  
     
     
         9 . A process according to    claim 8    wherein step (a) takes place for 30-40 minutes at room temperature.  
     
     
         10 . A process according to    claim 3    wherein the salt is NaCl.  
     
     
         11 . A process according to    claim 10    wherein the salt in the dissociation mixture is 0.10M to −0.2M NaCl.  
     
     
         12 . A process according to    claim 10    wherein the salt in the dissociation mixture is 0.50-1.25M NaCl.  
     
     
         13 . A process according to    claim 1    wherein step (b) comprises a dialysis or diafiltration/ultrafiltration step.  
     
     
         14 . A process according to    claim 13    wherein the dialysis step comprises a dialysis or diafiltration/ultraflitration against a solution of physiological salt or a higher salt concentration, non-ionic surfactant, and buffer at a lower pH than is present in the dissociation mixture.  
     
     
         15 . A process according to    claim 13    wherein the dialysis step comprises a dialysis or diafiltration/ultrafiltration in a reassembly buffer, said reassembly buffer comprising: ionic strength salt in the range of 0.5-1.35M NaCl, a metal ion source, and a buffer at pH 6.0-6.5.  
     
     
         16 . A process according to    claim 1    wherein step (c) comprises a dialysis or diafiltration/ultrafiltration step with a reassembly buffer, the reassembly buffer comprising: ionic strength salt in the range of 0.5-1.35M NaCl a metal ion source, a buffer at pH 6.0-6.5.  
     
     
         17 . A process according to    claim 16    wherein the metal ion source is a Ca +2  or a Mg +2  source.  
     
     
         18 . A process according to    claim 17    wherein the metal ion source is CaCl 2  or MgCl 2  at 0.5-5.0 mM.  
     
     
         19 . A process according to    claim 16    wherein the buffer is selected from the group consisting of: a) glycine and phosphate, b) citrate and phosphate; and c) citrate.  
     
     
         20 . A process according to    claim 16    wherein the reassembly buffer comprises 1 M NaCl, 2 mM Ca °2 , 50 mM citrate, pH 6.2 and 0.03% polysorbate 80.  
     
     
         21 . A process according to    claim 1    further comprising: 
 (d) further purifying with a dialysis step comprising a final buffer comprising: 0.25-1.25 M NaCl, a nonionic detergent and optionally, a histidine buffer at pH 6.0-6.5.  
 
     
     
         22 . A process according to    claim 21    wherein the nonionic detergent is polysorbate 80 or polysorbate 20.  
     
     
         23 . A process according to    claim 1   , further comprising adsorbing the dis/reassembled VLPs onto aluminum adjuvant.  
     
     
         24 . A process according to    claim 1    wherein the VLPs are selected from the group consisting of HPV 6a, HPV 6b, HPV 11, HPV 16, HPV 18, and combinations thereof.  
     
     
         25 . A process for making a storage stable human papillomavirus (HPV) vaccine comprising HPV virus-like particles (VLPs), the process comprising the steps of: 
 (a) incubating VLPs in a low salt dissociation mixture for 30-40 minutes, the dissociation mixture comprising: 0.16-0.18 NaCl, 2-20 mM DTT, 0.01-0.03% Polysorbate 80, 0.5-5 mM EDTA and 5-15 mM TRIS buffer, at pH 8.2 to produce dissembled VLPs;    (b) optionally removing the DTT from the dissociation mixture using a dialysis against a buffer comprising 0.16-0.18 M NaCl, 0.01-0.03% polysorbate 80, and 10 mM phosphate at pH 7.0;    (c) reassembling the disassembled VLPs using dialysis against a reassembly buffer, the reassembly buffer comprising 1.0 M NaCl, 2 mM CaCl 2 ; and a pH 6.2 buffer selected from the group consisting of: 50 mM sodium citrate; 50 mM glycine and phosphate; and 50 mM citrate; and    (d) further purifying the reassembled VLPs using dialysis against a final buffer, the final buffer comprising 0.5M NaCl, and 10 mM histidine, pH 6.2.    
     
     
         26 . A process according to    claim 25   , further comprising 
 (e) adsorbing the reassembled VLPs from step (d) onto aluminum adjuvant.    
     
     
         27 . A process for making a storage stable human papillomavirus (HPV) vaccine comprising HPV virus-like particles (VLPs), the process comprising the steps of: 
 (a) incubating VLPs in a high salt dissociation mixture for 30-40 minutes, the dissociation mixture comprising: 0.5-1.25 M NaCl, 2-20 mM DTT, 0.01-0.03% polysorbate 80, 0.5-5 mM EDTA and 5-100 TRIS buffer, and 0-50 mM phosphate at pH 8.2 to produce disassembled VLPs;    (b) optionally removing the DTT from the dissociation mixture using a dialysis against a buffer comprising at least 1 M NaCl, 0.01-0.03% polysorbate 80, and 10 mM phosphate at pH 7.0;    (c) reassembling the disassembled VLPs using dialysis against a reassembly buffer, the reassembly buffer comprising 1.0-1.35 M NaCl, 2 mM CaCl 2 ; and a pH 6.2 buffer selected from the group consisting of: 50 mM sodium citrate; 50 mM glycine and phosphate; and 50 mM citrate; and    (d) further purifying the reassembled VLPs using dialysis against a final buffer, the final buffer comprising 0.5M NaCl, and 10 mM histidine, pH 6.2.    
     
     
         28 . A process according to    claim 27   , further comprising 
 (e) adsorbing the reassembled VLPs from step (d) onto aluminum adjuvant.    
     
     
         29 . A vaccine made by the process of    claim 1   .  
     
     
         30 . A vaccine made by the process of    claim 23   .  
     
     
         31 . A vaccine made by the process of    claim 25   .  
     
     
         32 . A vaccine made by the process of    claim 28   .  
     
     
         33 . A vaccine formulation comprising VLPs and a formulation buffer, said formulation buffer comprising: 0.15-0.32M NaCl, 5-10 mM histidine, pH 6.2, and 0.005-0.015% polysorbate 80.  
     
     
         34 . A vaccine formulation comprising aluminum adsorbed VLPs and a formulation buffer, said formulation buffer comprising: 0.15-0.32M NaCl, 5-10 mM histidine, pH 6.2, and 0.005-0.015% polysorbate 80.