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