US 9,868,761  B2  16  15  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:  (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  wherein the sodium chloride-free multi-component  buffer system is used throughout the series of chro› matography steps; and  (a) an organic acid,  ( c) an organic base; 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. 