Buffer system for protein purification
A method for purifying a protein using a simplified, sodium chloride-free buffer system that consists of two components (acid and base pairs) for appropriate solution pH control; and a third component for ionic strength control, where the third component is the sodium salt conjugate base.
1. A method for purifying a protein from a contaminated solution thereof comprising the steps of:
(1) passing the protein through a series of chromatography steps operated in a mode chosen from: bind-elute mode and flow-through mode, using a sodium chloride-free multi-component buffer system,
wherein the chromatography steps comprise affinity chromatography and at least one additional chromatography step chosen from: anion exchange chromatography, cation exchange chromatography, and mixed-mode chromatography, and
wherein the sodium chloride-free multi-component buffer system comprises:
(a) an organic acid,
(b) at least one component chosen from an alkaline metal and an ammonium salt of the conjugate base of the organic acid of (a), and
(c) an organic base; and
wherein the sodium chloride-free multi-component buffer system is used throughout the series of chromatography steps; and
(2) recovering the purified protein.
2. The method as claimed in claim 1 , wherein each chromatography step comprises:
(1) an equilibration step using an equilibration buffer solution,
(2) a wash step using a wash buffer solution and, optionally,
(3) an elution step using an elution buffer solution,
wherein the equilibration buffer, wash buffer and elution buffer solutions are all made using the sodium chloride-free multi-component buffer system.
3. The method as claimed in claim 1 , wherein the organic acid comprises acetic acid.
4. The method as claimed in claim 1 , wherein the organic base is tris base.
5. The method as claimed in claim 1 , wherein the alkaline metal or ammonium salt of the conjugate base of the organic acid is chosen from the sodium, potassium, and ammonium salt of the conjugate base of the organic acid.
6. The method as claimed in claim 1 , wherein the organic acid is acetic acid, and the conjugate base of acetic acid is the sodium salt.
7. The method as claimed in claim 1 , wherein the protein is an antigen binding protein.
8. The method as claimed in claim 7 , wherein the antigen binding protein is an antibody of the IgG class.
9. The method as claimed in claim 7 , wherein the antigen binding protein is an immunoglobulin single variable domain.
10. The method as claimed in claim 1 , wherein the series of chromatography steps comprises affinity chromatography and anion exchange chromatography, optionally followed by cation exchange chromatography.
11. The method as claimed in claim 1 , wherein the affinity chromatography comprises Protein A chromatography.
12. The method as claimed in claim 11 , wherein the affinity chromatography comprises Protein A chromatography performed in the presence of tris base and acetic acid, at about pH 7.5.
13. The method as claimed in claim 1 , wherein the series of chromatography steps comprises anion exchange chromatography performed in the presence of tris base and acetic acid, at about pH 7.5.
14. The method as claimed in claim 1 , wherein the series of chromatography steps comprises cation exchange chromatography performed in the presence of sodium acetate and acetic acid, at about pH 5.0.
15. The method as claimed in claim 1 , wherein the affinity chromatography steps comprise:
(a) equilibrating a Protein A immobilized on a solid phase with a Protein A equilibration buffer solution;
(b) adsorbing the protein from the contaminated solution to the Protein A immobilized on the solid phase;
(c) removing contaminants by washing the solid phase with a Protein A wash buffer solution; and
(d) recovering the protein from the solid phase with a Protein A elution buffer solution,
wherein all of the buffer solutions are made using the sodium chloride-free multi-component buffer system.
16. The method as claimed in claim 1 , wherein the affinity chromatography is followed by anion exchange chromatography comprising the steps of:
(a) equilibrating an anion exchange matrix with an anion exchange equilibration buffer solution;
(b) applying the contaminated solution to the anion exchange matrix and collecting the first flowthrough; and
(c) applying an anion exchange wash buffer solution to the anion exchange matrix and collecting the second flowthrough,
wherein all of the buffer solutions are made using the sodium chloride-free multi-component buffer system.
17. The method as claimed in claim 16 , wherein the first flowthrough and the second flowthrough are combined into a single flowthrough solution.
18. The method as claimed in claim 1 , wherein the affinity chromatography is followed by cation exchange chromatography comprising the steps of:
(a) equilibrating a cation exchange matrix with a cation exchange equilibration buffer solution;
(b) adsorbing the protein from the contaminated solution to the cation exchange matrix;
(c) removing contaminants by washing the solid phase with a first cation exchange wash buffer solution; and
(d) recovering the protein from the solid phase with a cation exchange elution buffer solution,
wherein all the buffer solutions are made using the sodium chloride-free multi-component buffer system.