Protein purification using bis-tris buffer

Abstract

The invention provides a two-step chromatography process for small and large-scale purification of proteins, specifically monoclonal antibodies, using only four buffer solutions made from a mother solution.

1 . A method for purifying a protein from solution comprising:
 (a) a first chromatography step comprising:
 passing said solution over a first chromatography column; 
 eluting a crude protein eluent from the first chromatography column using a first elution buffer; and 
   (b) a second chromatography step comprising:
 passing the crude protein eluent obtained at the end of step (a) over a second chromatography column; 
 recovering purified protein from the second chromatography column using a second elution buffer 
   wherein each of the buffers comprises Bis Tris.   
     
     
         2 . The method of  claim 1 , wherein each one of the two chromatography steps comprises:
 passing equilibration buffer over the chromatography column;   passing the solution or the crude protein eluent over the chromatography column;   passing equilibration buffer over the chromatography column;   optionally passing wash and sanitation buffer over the chromatography column;   optionally passing equilibration buffer over the chromatography column;   eluting the crude protein eluent or recovering purified protein from the chromatography column using an elution buffer,   
       wherein each of the buffers comprises Bis Tris. 
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein each of the buffers consists of Bis Tris, acetic acid, NaCl, and water. 
     
     
         5 . The method of  claim 1 , wherein the method for purifying a protein from solution only comprises two chromatographic steps. 
     
     
         6 . The method of  claim 1 , wherein one of the chromatography columns is an affinity chromatography column. 
     
     
         7 . The method of  claim 1 , wherein said affinity chromatography column is a Protein A column. 
     
     
         8 . The method of  claim 1 , wherein one of the chromatography columns is a multi-modal resin chromatography column. 
     
     
         9 - 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein said method comprises the steps of:
 (a) a first chromatography step comprising:
 (i) passing equilibration buffer over a first chromatography column; 
 (ii) passing the solution over the first chromatography column; 
 (iii) passing equilibration buffer over the first chromatography column; 
 (iv) passing wash and sanitation buffer over the first chromatography column; 
 (v) passing equilibration buffer over the first chromatography column; 
 (vi) eluting a crude protein eluent from the first chromatography column using a first elution buffer; and 
 (vii) optionally adjusting the pH of the crude protein eluent using a Bis Tris solution; and 
   (b) a second chromatography step comprising:
 (i) passing equilibration buffer over a second chromatography column; 
 (ii) passing the crude protein eluent from step (a) over the second chromatography column; 
 (iii) passing equilibration buffer over the second chromatography column; and 
 (iv) recovering purified protein from the second chromatography column using a second elution buffer. 
   
     
     
         13 - 15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein said method comprises the steps of:
 (a) a first chromatography step comprising:
 (i) passing equilibration buffer over a first chromatography column; 
 (ii) passing the solution over the first chromatography column; 
 (iii) passing equilibration buffer over the first chromatography column; 
 (iv) eluting a crude protein eluent from the first chromatography column using a first elution buffer; and 
 (v) optionally adjusting the pH of the crude protein eluent using a Bis Tris solution; and 
   (b) a second chromatography step comprising:
 (i) passing equilibration buffer over a second chromatography column; 
 (ii) passing the crude protein eluent from step (a) over the second chromatography column; 
 (iii) passing equilibration buffer over the second chromatography column; 
 (iv) passing wash and sanitation buffer over the second chromatography column; 
 (v) passing equilibration buffer over the second chromatography column; and 
 (vi) recovering purified protein from the second chromatography column using a second elution buffer. 
   
     
     
         17 - 20 . (canceled) 
     
     
         21 . The method of  claim 16 , wherein the protein is a monoclonal antibody. 
     
     
         22 . (canceled) 
     
     
         23 . The method of  claim 16 , further comprising, after step (b), a step (c) of passing the crude protein eluent over a membrane adsorber. 
     
     
         24 . The method of  claim 23 , wherein said membrane adsorber is a salt tolerant interaction chromatography membrane adsorber. 
     
     
         25 . The method of  claim 16 , further comprising a nanofiltration step after step (b) or (c). 
     
     
         26 . The method of  claim 25 , further comprising an ultrafiltration and diafiltration step after the nanofiltration step. 
     
     
         27 . The method of  claim 16 , wherein the first elution buffer comprises 15 to 25 mM Bis Tris, and 15 to 25 mM NaCl, adjusted to a pH comprised between 3 and 4 with acetic acid. 
     
     
         28 - 34 . (canceled) 
     
     
         35 . The method of  claim 1 , wherein the purified protein is recovered with a yield of at least 85%. 
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 1 , further comprising the step of formulating the recovered purified protein into a pharmaceutical composition. 
     
     
         38 - 43 . (canceled)