Anionic exchange-hydrophobic mixed mode
Solid supports and ligands are provided for purification of biomolecules by mixed-mode anion exchange-hydrophobic chromatography. Compositions can have the formula Support-(X)—N(R 1 , R 2 )—R 3 -L-Ar, or a salt thereof, wherein: Support is a chromatographic solid support; X is a spacer or absent; R 1 and R 2 are each selected from hydrogen and an alkyl comprising 1-6 carbons; R 3 is an alkyl comprising 1-6 carbons or a cyclo alkyl comprising 1-6 carbons; L is NR 4 , O, or S; wherein R 4 is hydrogen or an alkyl comprising 1-6 carbons; and Ar is an aryl. Methods are also provided for using solid supports and ligands to purify biomolecules such as monomeric antibodies.
1. A method of purifying a biomolecule from a sample, the method comprising
contacting the sample to a chromatographic solid support linked to a ligand according to formula (I):
Support-(X)—N(R 1 ,R 2 )—R 3 -L-Ar, or a salt thereof (I),
wherein
X is a spacer or absent;
R 1 and R 2 are each selected from hydrogen and an alkyl comprising 1-6 carbons;
R 3 is an alkyl comprising 1-6 carbons or a cyclo alkyl comprising 1-6 carbons;
L is NR 4 , O, or S; wherein R 4 is hydrogen or an alkyl comprising 1-6 carbons;
Ar is an aryl; and
the nitrogen in the N(R 1 , R 2 ) is positively charged during the contacting; and
collecting a purified biomolecule.
2. The method of claim 1 , wherein the purified biomolecule is a protein.
3. The method of claim 2 , wherein the protein is an antibody.
4. The method of claim 1 , wherein the sample comprises a monomeric antibody and antibody aggregates, the purified biomolecule comprises the monomeric antibody, and the method comprises separating the monomeric antibody from the antibody aggregates.
5. The method of claim 1 , wherein the purified biomolecule is a monomeric antibody.
6. The method of claim 5 , wherein the monomeric antibody is immobilized on the chromatographic solid support and subsequently eluted.
7. The method of claim 5 , wherein the purification operates in flow-through mode, and the monomeric antibody flows past, without binding to, the chromatographic solid support, and is collected.
8. The method of claim 5 , wherein R 1 and R 2 are hydrogen.
9. The method of claim 5 , wherein R 3 is an alkyl comprising 1-4 carbons.
10. The method of claim 5 , wherein R 3 is an alkyl comprising 2 carbons.
11. The method of claim 5 , wherein the aryl is a phenyl.
12. The method of claim 5 , wherein the aryl is a substituted phenyl.
13. The method of claim 12 , wherein the substituted phenyl is an alkyl-substituted phenyl.
14. The method of claim 5 , wherein R 1 and/or R 2 are an alkyl comprising 1-6 carbons and Ar is an alkyl-substituted phenyl.
15. The method of claim 5 , wherein the ligand is N-phenylethylenediamine or a salt thereof.
16. The method of claim 5 , wherein the ligand is 2-phenoxyethylamine or a salt thereof.
17. The method of claim 5 , wherein X is absent.
18. The method of claim 5 , wherein X is a spacer.
19. The method of claim 18 , wherein the spacer is up to 30 carbon atoms in length.
20. The method of claim 5 , wherein the purification operates in bind-elute mode.
21. The method of claim 20 , wherein the biomolecule is eluted by a step comprising reduction of pH of solution in contact with the ligand.