High pressure refolding of monoclonal antibody aggregates

Abstract

Methods for refolding antibodies, particularly monoclonal antibodies, from aggregated and/or denatured preparations by subjecting the antibody preparation to high hydrostatic pressure are provided. Refolded preparations of antibodies produced by the methods described herein are also provided.

1 . A method for refolding a sample of an antibody, comprising the steps of:
 obtaining an antibody sample comprising a solution of antibody exposed to low pH;   adjusting the pH of the solution to above pH 5.0 if necessary;   exposing the antibody sample to high hydrostatic pressure for a period of time; and   reducing the hydrostatic pressure to atmospheric pressure;   wherein the antibody sample after pressure exposure has a higher content of monomeric antibody, a higher content of properly folded antibody, or a lower content of aggregated antibody than the antibody sample prior to the pressure exposure.   
   
   
       2 . The method of  claim 1 , wherein the antibody is monoclonal. 
   
   
       3 . The method of  claim 2 , wherein the content of monomeric or properly folded antibody is measured by a method selected from a binding assay specific for the antibody, analytical ultracentrifugation, size exclusion chromatography, field flow fractionation, light scattering analysis, light obscuration analysis, fluorescence spectroscopy, gel electrophoresis, GEMMA, nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy, or reverse-phase chromatography. 
   
   
       4 . The method of  claim 2 , wherein the high hydrostatic pressure is between about 1500 bar and a value about 50 bar below the denaturation pressure of the native antibody. 
   
   
       5 . The method of  claim 4 , wherein the high hydrostatic pressure is between about 1500 bar and about 3000 bar. 
   
   
       6 . The method of  claim 2 , wherein the ionic strength of the antibody sample comprising an antibody solution is less than a value about 25 mM below the denaturation point of the native antibody. 
   
   
       7 . The method of  claim 6 , wherein the ionic strength of the antibody sample comprising an antibody solution is less than about 200 mM. 
   
   
       8 . The method of  claim 2 , wherein the antibody is abatacept. 
   
   
       9 . The method of  claim 8 , wherein the pH of the abatacept antibody solution is at or above about 7. 
   
   
       10 . The method of  claim 9 , wherein sucrose is present in the antibody solution at a concentration of about 5% to about 15%. 
   
   
       11 . The method of  claim 10 , wherein sucrose is present in the antibody solution at a concentration of about 10%. 
   
   
       12 . The method of  claim 7 , wherein the ionic strength of the antibody solution is less than about 100 mM. 
   
   
       13 . The method of  claim 8 , wherein the amount of aggregated antibody present in the antibody sample after pressure exposure is decreased by at least about 20% as compared to the amount of aggregated antibody present in the antibody sample before pressure exposure. 
   
   
       14 . The method of  claim 8 , wherein the amount of aggregated antibody present in the antibody sample after pressure exposure is less than about 1%. 
   
   
       15 . A preparation of abatacept antibody comprising less than about 1.0% aggregated antibody. 
   
   
       16 . A preparation of abatacept antibody comprising about 0.8% or less aggregated antibody.