of 25 cm, according to manufacturer’s recommendation, in 1.1 cm diameter Vantage columns obtained from Millipore Corporation (Bedford, Mass., USA). The IgG monoclonal antibodies used for this work are recombinantly expressed using mammalian cell culture at the GlaxoSmithKline Upper Merion site (King of Prussia, Pa., USA). All chemicals are obtained from JT Baker (Phillipsburg, N.J., USA) or Sigma Aldrich (St Louis, Mo., USA) and are of USP grade. Protein A Chromatography 

Purification of monoclonal antibodies by Protein A affin- ity chromatography utilizing MabSelect SuRe are carried out according to Table 2. First, the column is equilibrated with 55 mM Tris Base, 45 mMAcetic acid, pH 7.5. Clarified mammalian cell culture broth is then applied to the colunm until sufficient load mass had been applied to the colunm. The colunm is then washed with 55 mM Tris Base, 45 mM Acetic acid, 300 mM Sodium Acetate, pH 7.5. Prior to elution, the column is re-equilibrated with 55 mM Tris Base, 45 mM Acetic Acid, pH 7 .5. The column is then eluted with 1.8 mM Sodium Acetate, 28.2 mM Acetic Acid, pH 3.6. Low pH Treatment for Virus Inactivation 

Protein A Eluate from the previous step is adjusted to pH 3.5 with 30 mM Acetic Acid, 100 mM HCI. The low pH adjusted material is held for 30 to 60 minutes then neutral› ized to pH 7 .5 with 1 M Tris Base. The neutralized pool is then filtered in preparation for subsequent purification steps. Anion-Exchange Flow-Through Chromatography 

Further purification is accomplished by equilibrating the CaptoQ colunm with 55 mM Tris Base, 45 mM Acetic Acid, pH 7.5 following a pre-equilibration rinse with WFI (water for injection). Note that the same equilibration buffer used for the protein A equilibration is re-used in this step, offering the advantage of minimizing buffer solutions that need to be prepared. The neutralized pool is then applied to the column where the protein of interest flowed through and is collected while the contaminants remained bound to the colunm. Following the application of protein, the column is then washed with adequate equilibration buffer such that the remaining protein is washed from the colunm and able to be collected. 

Cation-Exchange Chromatography 

Protein collected from the anion-exchange flow-through step is titrated to pH 5 .0 with 30 mM Acetic Acid, 100 mM HCI. Again, note the re-use of the low pH treatment solution for pH adjustment in this step. The GigaCapS 650M column is equilibrated with 25 mM Sodium Acetate, 12.1 mM Acetic Acid, pH 5.0. The titrated load is then applied to the colunm until the desired load mass is achieved. The column is then re-equilibrated with 25 mM Sodium Acetate, 12.1 mM Acetic Acid, pH 5.0. Next the colunm is step eluted by applying to the colunm 175 mM Sodium Acetate, 75 mM Acetic Acid, pH 5.0. The colunm effluent is collected and retained for further processing. 

1. Ghose, S.; McNemey, T. M. Hubbard, B. pH transitions in ion-exchange systems: Role in the development of a cation exchange process for a recombinant protein. Bio› technol. Prag. 2002, 18, 530-537. 

2. Soto Perez, J. and Frey, D. D. Behavior of the Inadvertent pH Transient Formed by a Salt Gradient in the Ion› Exchange Chromatography of Proteins, Biotechnol. Prag. 2005, 21, 902-910 

3. Pabst, T. M., Carta, G. pH transitions in cation exchange chromatographic columns containing weak acid groups. (2007) Journal of Chromatography A, 1142, pp. 19-31. 

## REFERENCES 

4. Zhou, J. X., et al. pH-conductivity hybrid gradient cation› exchange chromatography for process-scale monoclonal antibody purification. Journal of Chromatography A, 1175 (2007) 69-80 

5. Butler, J. N., Ionic Equilibrium: Solubility and pH Cal› culations. John Wiley and Sons (1998). 

What is claimed: 

1. A method for purifying a protein from a contaminated solution thereof comprising the steps of: 

(1) passing the protein through a series of chromatogra› phy 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 chroma› tography step chosen from: anion exchange chroma› tography, 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 chro› matography 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, option› ally, 

(3) an elution step using an elution buffer solution, 

wherein the equilibration buffer, wash buffer and elu› tion 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 chromatogra› phy performed in the presence of tris base and acetic acid, at about pH 7.5. 

