Purification method for proteins, in particular antibodies, utilizing a wash solution comprising arginine at high pH for the affinity chromatography step
The invention provides a washing method for affinity chromatography in which a wash solution comprising arginine, or an arginine derivative, at pH greater than 8.0, is effective in removing impurities without the presence of a nonbuffering salt, while simultaneously increasing product concentration in the eluate and maintaining a high percent yield of recovered product.
1. A method of producing a purified protein of interest using an affinity chromatography (AC) matrix to which the protein of interest is bound, the method comprising washing the AC matrix with a wash solution comprising arginine, or an arginine derivative, at a pH of at least 8.5, and thereafter eluting the protein of interest from the AC matrix, wherein the wash is performed without the presence of a nonbuffering salt.
2. The method of claim 1 , wherein the method comprises:
a) loading a mixture comprising the protein of interest onto the AC matrix;
b) washing the AC matrix with a wash solution comprising arginine, or an arginine derivative, at a pH at least 8.5; and
c) eluting the protein of interest from the AC matrix,
wherein the wash is performed without the presence of a nonbuffering salt.
3. The method of claim 1 , wherein the protein of interest is an antibody, antibody fragment, or Fc fusion protein.
4. The method of claim 3 , wherein the AC matrix is a Protein A column.
5. The method of claim 1 , wherein the pH is 8.5-9.5.
6. The method of claim 1 , wherein the pH is about 8.9-9.0.
7. The method of claim 1 , wherein the pH is 9.0 or about 9.0.
8. The method of claim 1 , wherein the arginine derivative is selected from the group consisting of acetyl arginine, agmatine, arginic acid, N-alpha-butyroyl-L-argimine and N-alpha-pyvaloyl argimine.
9. The method of claim 1 , wherein arginine is at a concentration in a range of 0.1-0.5 M.
10. The method of claim 9 , wherein arginine is at a concentration of or about 0.25 M.
11. The method of claim 1 , wherein the wash solution comprises arginine-HCl.
12. A method of producing a purified antibody, antibody fragment, or Fc fusion protein using a Protein A column, the method comprising
a. loading a mixture comprising the antibody, antibody fragment, or Fc fusion protein onto the Protein A column;
b. washing the Protein A column with a wash solution comprising arginine, or an arginine derivative, at a pH at least 8.5; and
c. eluting the antibody, antibody fragment, or Fc fusion protein from the Protein A column,
wherein the wash is performed without the presence of a nonbuffering salt.
13. The method of claim 12 , further comprising equilibrating the Protein A column with an equilibration buffer prior to loading and/or eluting the antibody, antibody fragment, or Fc fusion protein from the Protein A column.