Affinity chromatography matrices and methods of making and using the same

1. A method or purifying a target molecule from a mixture comprising:

(1) contacting the mixture comprising the target molecule with an affinity chromatography matrix under a set of pH and/or salt conditions, wherein the affinity chromatography matrix comprises: a) a solid support; b) a protein ligand bound to the solid support, wherein the protein ligand binds to the target molecule under the set of pH and/or salt conditions; c) an activating group bound to the solid support which interacts covalently with the protein ligand: and d) an associative group bound to the solid support, where the associative group interacts non-covalently with the protein ligand and wherein the activating group and the associative group are both linked separately to the solid support: and

(2) altering the set of pH and/or salt conditions of (1) such that the target molecule no longer binds to the protein ligand of the matrix, thereby purifying the substance of interest.

2. The method of claim 1 , wherein the target molecule is an immunoglobulin.

3. The method of claim 1 , wherein the target molecule is a fusion protein.

4. The method of claim 3 , wherein the fusion protein is an Fc fusion protein.

5. The method of claim 2 , wherein the immunoglobulin is a monoclonal antibody.

6. The method of claim 1 , further comprising an intermediate washing step to remove contaminants comprising, contacting the solid support with a wash buffer after step (1) and before step (2).

7. The method of claim 6 , wherein the contaminants comprise a small molecule, a protein, a nucleic acid or any combination thereof.

8. The method of claim 6 , wherein the wash buffer comprises an acidic pH and a salt or both.

9. The method of claim 8 , wherein the pH ranges from 5-6.99.

10. The method of claim 6 , wherein the wash buffer has a pH ranging from 5.0-8.0.

11. The method of claim 6 , wherein the wash buffer has a basic pH and comprises more than 0.0001M NaCl, but less than 0.8M NaCl.

12. The method of claim 11 , wherein the pH is 7.4.

13. The method of claim 6 , wherein the wash buffer comprises an amino acid or an alkylated amino acid.

14. The method of claim 13 , wherein the amino acid or alkylated amino acid is selected from the group consisting of from L-alanine, betaine, glycine valine, glutamic acid, and lysine.

15. The method of claim 14 , wherein the amino acid is a charged amino acid.

16. The method of claim 1 , wherein the solid support is an agarose bead.

17. The agarose bead of claim 16 , wherein the bead comprises at least 10g/L of protein A bound to the surface of the agarose bead.