Human Antibodies that Bind Human TNF-Alpha and Methods of Preparing the Same

Abstract

Methylglyoxal (MGO)-modified recombinant TNF-alpha antibodies (e.g., Adalimumab) are identified. MGO modification decreases binding between Adalimumab and TNF-alpha. Methods are disclosed for reducing the presence of MGO-modified antibodies in the production of Adalimumab TNF-alpha antibodies.

We claim: 
     
         1 . A composition comprising a binding protein capable of binding TNF-alpha, wherein said binding protein comprises at least one methylglyoxal (MGO)-susceptible amino acid, and wherein at least a portion of said binding protein comprises one or more MGO-modified amino acids. 
     
     
         2 . The composition of  claim 1 , wherein the portion of the binding protein that comprises at least one MGO-modified amino acid is less than 12%. 
     
     
         3 . The composition of  claim 1 , wherein the portion of the binding protein that comprises at least one MGO-modified amino acid is less than 6%. 
     
     
         4 . The composition of  claim 1 , wherein the MGO-susceptible amino acid is an arginine. 
     
     
         5 . The composition of  claim 1 , wherein the binding protein is a human antibody or an antigen-binding portion thereof, wherein the binding protein dissociates from human TNF-alpha with a K d  of 1×10 −8  M or less and a K off  rate constant of 1×10 −3  s −1  or less, both as determined by surface plasmon resonance, and wherein the binding protein neutralizes human TNF-alpha cytotoxicity in a standard in vitro L929 assay with an IC 50  of 1×10 −7  M or less. 
     
     
         6 . A composition comprising a binding protein capable of binding TNF-alpha, said binding protein comprising a methylglyoxal (MGO)-susceptible amino acid, wherein said composition is prepared by substantially removing molecules of said binding protein that comprise at least one MGO-modified amino acid. 
     
     
         7 . The composition of  claim 6 , wherein more than 70% of said molecules that comprise at least one MGO-modified amino acid is removed. 
     
     
         8 . The composition of  claim 6 , wherein more than 90% of said molecules that comprise at least one MGO-modified amino acid is removed. 
     
     
         9 . The composition of  claim 6 , wherein the MGO-susceptible amino acid is an arginine. 
     
     
         10 . The composition of  claim 6 , wherein the binding protein is a human antibody or an antigen-binding portion thereof, wherein the binding protein dissociates from human TNF-alpha with a K d  of 1×10 −8  M or less and a K off  rate constant of 1×10 −3  s −1  or less, both determined by surface plasmon resonance, and wherein the binding protein neutralizes human TNF-alpha cytotoxicity in a standard in vitro L929 assay with an IC 50  of 1×10 −7  M or less. 
     
     
         11 . A method for purifying a composition comprising a target protein, said method comprising:
 (a) loading the composition to a cation exchange adsorbent using a loading buffer, wherein the pH of the loading buffer is lower than the pI of the target protein;   (b) washing the cation exchange adsorbent with a washing buffer, wherein the pH of the washing buffer is lower than the pI of the target protein;   (c) eluting the cation exchange adsorbent with an elution buffer, said elution buffer being capable of reducing the binding between the target protein and the cation exchange adsorbent; and   (d) collecting the eluate, wherein the percentage of the target protein is higher in the eluate than the percentage of the target protein in the composition.   
     
     
         12 . The method of  claim 11 , wherein the conductivity of the elution buffer is higher than the conductivity of the washer buffer. 
     
     
         13 . The method of  claim 12 , wherein the conductivity of the elution buffer is raised by increasing the salt concentration of the elution buffer. 
     
     
         14 . The method of  claim 12 , wherein the pH of the elution buffer is between 5.5 and 9.0. 
     
     
         15 . The method of  claim 13 , wherein the salt concentration of the elution buffer is between 20 mM NaCl and 200 mM NaCl. 
     
     
         16 . The method of  claim 11 , wherein the target protein is a human antibody or an antigen-binding portion thereof, wherein the target protein dissociates from human TNF-alpha with a K d  of 1×10 −8  M or less and a K off  rate constant of 1×10 −3  s −1  or less, both determined by surface plasmon resonance, and wherein the target protein neutralizes human TNF-alpha cytotoxicity in a standard in vitro L929 assay with an IC 50  of 1×10 −7  M or less. 
     
     
         17 . A method for purifying a composition comprising a target protein, said method comprising:
 (a) loading the composition to an anion exchange adsorbent using a loading buffer, wherein the pH of the loading buffer is lower than the isoelectric point (pI) of the target protein;   (b) allowing the majority of the target protein to pass through without binding to the anion exchange adsorbent;   (c) collecting the pass-through loading buffer containing said unbound target protein;   (d) washing the anion exchange adsorbent with a washing buffer;   (e) allowing the target protein bound to the anion exchange adsorbent to disassociate from the anion exchange adsorbent; and   (f) collecting the washing buffer containing said disassociated target protein.   
     
     
         18 . The method of  claim 17 , wherein the loading buffer comprises an anionic agent and a cationic agent, wherein the conductivity and pH of the loading buffer is adjusted by increasing or decreasing the concentration of a cationic agent and maintaining a constant concentration of an anionic agent in the loading buffer. 
     
     
         19 . The method of  claim 18 , wherein the anionic agent is selected from the group consisting of acetate, citrate, chloride anion, sulphate, phosphate and combinations thereof. 
     
     
         20 . The method of  claim 18 , wherein the cationic agent is selected from the group consisting of sodium, Tris, tromethalmine, ammonium cation, arginine, and combinations thereof. 
     
     
         21 . The method of  claim 17 , wherein the target protein is a human antibody or an antigen-binding portion thereof, wherein the target protein dissociates from human TNF-alpha with a K d  of 1×10 −8  M or less and a K off  rate constant of 1×10 −3  s −1  or less, both determined by surface plasmon resonance, and wherein the target protein neutralizes human TNF-alpha cytotoxicity in a standard in vitro L929 assay with an IC 50  of 1×10 −7  M or less.