Methods and Reagents for Purification of Proteins

Abstract

The present disclosure provides compositions and methods for purifying a protein such as DNase using a chromatographic process. The methods include a single chromatographic step and the use of high concentration salt buffers. The present disclosure provides methods for purification of a protein from a sample comprising loading the sample onto a chromatography column and washing the column with at least one buffer having very high conductivity. In some embodiments, the wash buffer has a conductivity of about 50 mS/cm, about 60 mS/cm, about 70 mS/cm, about 80 mS/cm, or higher. In further embodiments, the protein is eluted from the column with an elution buffer following the wash with the very high conductivity buffer.

1 . A method for purification of a protein from a sample comprising: loading the sample onto a chromatography column,
 washing the column with a wash buffer, wherein the wash buffer has a conductivity of 80 mS/cm or higher, and   eluting the protein from the column with an elution buffer.   
     
     
         2 . The method of  claim 1 , wherein the wash buffer comprises a salt selected from the group consisting of (NH 4 ) 2 SO 4 , Na 2 SO 4 , and CH 3 COONa. 
     
     
         3 . The method of  claim 2 , wherein the wash buffer comprises a concentration of about 0.8 M (NH 4 ) 2 SO 4 . 
     
     
         4 . The method of  claim 1 , wherein the elution buffer has a conductivity of 80 mS/cm or higher. 
     
     
         5 . The method of  claim 1 , wherein the elution buffer comprises a salt selected from the group consisting of NaCl, KCl, and NaOAc. 
     
     
         6 . The method of  claim 5 , wherein the elution buffer comprises a concentration of about 1 M NaCl. 
     
     
         7 . The method of  claim 1 , wherein the method comprises at least two washing steps. 
     
     
         8 . The method of  claim 7 , wherein a first washing step comprises washing the column with a buffer having a conductivity of 2 mS/cm or less. 
     
     
         9 . The method of  claim 8 , further comprising a third washing step, wherein the third washing step comprises washing the column with a buffer having a conductivity of 2 mS/cm or less. 
     
     
         10 . The method of  claim 1 , wherein the protein is an enzyme. 
     
     
         11 . The method of  claim 10 , wherein the enzyme is DNase or RNase. 
     
     
         12 . The method of  claim 11 , wherein the DNase is a recombinant DNase. 
     
     
         13 . The method of  claim 10 , wherein the enzyme is DNase, and wherein the buffers used in the method comprise CaCl 2 . 
     
     
         14 . The method of  claim 13 , wherein the buffers used in the method comprise 1 mM CaCl 2 . 
     
     
         15 . The method of  claim 1 , wherein the chromatography column is an ion exchange or a mixed mode chromatography column. 
     
     
         16 . The method of  claim 15 , wherein the mixed mode column is a multimodal anion exchanger. 
     
     
         17 . The method of  claim 16 , wherein the mixed mode column is a Capto Adhere column. 
     
     
         18 . The method of  claim 1 , wherein the sample is a cell culture sample. 
     
     
         19 . The method of  claim 1 , wherein the pH of the loaded sample is between 5.0 and 9.0. 
     
     
         20 . The method of  claim 19 , wherein the pH of the loaded sample is between 6.0 and 8.0. 
     
     
         21 . The method of  claim 20 , wherein the pH of the loaded sample is about 7.0. 
     
     
         22 . The method of  claim 1 , wherein the pH of the loaded sample is the same or higher than the pI of the protein. 
     
     
         23 . The method of  claim 1 , wherein the pH of the wash buffer is between about 5.0 and about 9.0. 
     
     
         24 . The method of  claim 23 , wherein the pH of the wash buffer is about 7.0. 
     
     
         25 . The method of  claim 1 , wherein the pH of the elution buffer is between about 5.0 and about 9.0. 
     
     
         26 . The method of  claim 25 , wherein the pH of the elution buffer is about 7.0. 
     
     
         27 . The method of  claim 1 , wherein the method further comprises adding a detergent to the sample prior to loading the sample onto the chromatography column. 
     
     
         28 . The method of  claim 27 , wherein the detergent is Triton X-100. 
     
     
         29 . The method of  claim 28 , wherein the detergent is added to the sample at a final concentration of 0.5%. 
     
     
         30 . A method for purification of DNase from a cell culture sample comprising: adding a detergent to the cell culture sample;
 directly loading the sample to a mixed mode chromatography column; washing the column with a first wash buffer;   washing the column with a second wash buffer; and eluting DNase from the column with an elution buffer.   
     
     
         31 . The method of  claim 30 , wherein the second wash buffer and the elution buffer each have a conductivity of 80 mS/cm or more. 
     
     
         32 . The method of  claim 30 , wherein the second wash buffer comprises 0.8M (NH 4 ) 2 SO 4 . 
     
     
         33 . The method of  claim 30 , wherein the elution buffer comprises 1 M NaCl. 
     
     
         34 . A kit comprising a wash buffer having a conductivity of about 80 mS/cm or more and an elution buffer having a conductivity of about 80 mS/cm or more. 
     
     
         35 . The kit of  claim 34 , wherein the wash buffer comprises about 0.8 M (NH 4 ) 2 SO 4 , and the elution buffer comprises about 1 M NaCl.