The "loading buffer" is that which is used to load the  mixture of the protein and contaminant(s) onto the chroma› tography matrix. The equilibration and loading buffers can 15  be the same.  is generally one which comprises a glass, silica, agarose or  polystyrene surface. The solid phase may be a purification  colunm or a discontinuous phase of discrete particles. In  preferred embodiments, the solid phase is a controlled pore  glass colunm or a silicic acid colunm. In certain embodi- ments, the solid phase is coated with a reagent (such as  glycerol), which is intended to prevent nonspecific adher› ence of contaminants to the solid phase.  A "buffer" is a buffered solution that resists changes in pH  by the action of its acid-base conjugate components.  An "equilibration buffer" herein is that used to prepare the  solid phase for chromatography.  The "elution buffer" is used to elute proteins from the  chromatography matrix.  A "salt" is a compound formed by the interaction of an  acid and a base.  In one embodiment, the organic acid includes, but is not  limited to, formic acid, acetic acid, lactic acid, citric acid,  malic acid, maleic acid, glycine, phosphoric acid, glycyl› clycine,  succ1mc  acid,  TES  (2-{[tris(hydroxymethyl)  methyl]amino }ethanesulfonic  acid),  MOPS  (3-(N-mor› pholino )propanesulfonic acid), PIPES (piperazine-N,N’-bis  (2-ethanesulfonic  acid)),  and MES  (2-(N-morpholino)  ethanesulfonic acid).  In one embodiment, the organic base includes, but is not  limited to, the group consisting of tris base, arginine, Bis› Tris, Bis-Tris-Propane, Bicine (N,N-bis(2-hydroxyethyl)  glycine), HEPES ( 4-2-hydroxyethyl-1-piperazineethanesul- fonic  acid),  TAPS  (3-{[tris(hydroxymethyl)methyl]  amino }propanesulfonic  acid),  and  Tricine  (N-tris  (hydroxymethyl)methylglycine ).  In one embodiment, the conjugate base of the organic acid  is the sodium, potassium, or ammonium salt of the conjugate  base of the organic acid. In one embodiment, the organic  acid is acetic acid and the conjugate base of acetic acid is the  sodium salt.  In one embodiment, the protein is an antigen binding  protein. In one embodiment, the antigen binding protein is  an antibody. In one embodiment the antibody is of the IgG  class. In one embodiment, the antigen binding protein is a  immunoglobulin single variable domain.  In one embodiment, the sequence of chromatographic  steps comprises protein A chromatography and flowthrough  anion exchange chromatography. In one embodiment, the  sequence of chromatographic steps comprises protein A  chromatography, flowthrough anion exchange chromatogra- phy, and cation exchange chromatography.  In one embodiment, the sequence of chromatographic  steps comprises protein A chromatography performed in the  presence of about 55 mM tris base, about 45 mM acetic acid,  at about pH 7.5.  In one embodiment, the sequence of chromatographic  steps comprises flowthrough anion exchange chromatogra› phy performed in the presence of about 55 mM tris base,  about 45 mM acetic acid, at about pH 7.5.  In one embodiment, the sequence of chromatographic  steps comprises cation exchange chromatography performed  in the presence of about 25 mM sodium acetate, about 12.1  mM acetic acid, at about pH 5.0.  In one aspect, the present invention is directed to a  method for purifying a protein from a contaminated solution 65  thereof by Protein A chromatography comprising: (a) equili› brating a Protein A immobilized on a solid phase with a  Protein A equilibration buffer comprising about 55 mM tris  base, about 45 mM acetic acid, at about pH 7 .5; (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 first Protein  A wash buffer comprising about 55 mM Tris Base, about 45  mM Acetic acid, 300 mM Sodium Acetate, at about pH 7 .5;  and ( d) recovering the protein from the solid phase with a  Protein A elution buffer comprising about 1.8 mM Sodium  Acetate, about 28.2 mM Acetic acid, at about pH 3.6,  wherein all the buffers are made without the addition of  NaCl.  In one embodiment, the method further comprises the  following step after step (c) and before step (d): removing  contaminants by washing the solid phase with a second  Protein A wash buffer comprising about 55 mM Tris Base,  about 45 mM Acetic acid, at about pH 7.5, wherein the  second Protein A wash buffer is made without the addition  of NaCl.  In one embodiment, the method further comprises the  following steps after step ( d): ( e) titrating the solution  containing the recovered protein to about pH 3.0 with about  30 mM acetic acid, about 100 mM HCl; (f) allowing the  solution of step (e) to remain at about pH 3.0 for about 30  to about 60 minutes; and (g) adjusting the pH of the solution  of step (f) to about pH 7.5 with about 1 M Tris.  In one embodiment, the method further comprises filter› ing the solution produced by step (g) above.  In one aspect the present invention is directed to a method  for purifying a protein from a contaminated solution thereof  by flowthrough anion exchange chromatography compris› ing: (a) equilibrating an anion exchange matrix with an  anion equilibration buffer comprising about 55 mM tris base,  about 45 mM acetic acid, at about pH 7 .5; (b) applying the  contaminated solution to the anion exchange matrix and  collecting the first flowthrough; and ( c) applying an anion  wash buffer comprising 55 mM Tris Base, about 45 mM  Acetic acid, about 300 mM Sodium Acetate, at about pH 7 .5  to the anion exchange matrix and collecting the second  flowthrough, wherein all the buffers are made without the  addition of NaCl.  In one embodiment, the contaminated solution is the  solution produced by step (g) of a method for purifying a  protein from a contaminated solution thereof by Protein A  chromatography comprising: (a) equilibrating a Protein A  immobilized on a solid phase with a Protein A equilibration  buffer comprising 55 mM tris base, 45 mM acetic acid, at  about pH 7.5; (b) adsorbing the protein from the contami› nated solution to the Protein A immobilized on the solid  phase; ( c) removing contaminants by washing the solid  phase with a first Protein A wash buffer comprising 55 mM  Tris Base, 45 mM Acetic acid, 300 mM Sodium Acetate, at  about pH 7.5; and (d) recovering the protein from the solid  phase with a Protein A elution buffer comprising 1.8 mM  Sodium Acetate, 28.2 mM Acetic acid, at about pH 3.6,  wherein all the buffers are made without the addition of  NaCl. or the filtered solution of a method of purifying a  protein from a contaminated solution thereof by Protein A  chromatography comprising: (a) equilibrating a Protein A  immobilized on a solid phase with a Protein A equilibration  buffer comprising 55 mM tris base, 45 mM acetic acid, at  about pH 7.5; (b) adsorbing the protein from the contami› nated solution to the Protein A immobilized on the solid  phase; ( c) removing contaminants by washing the solid  phase with a first Protein A wash buffer comprising 55 mM  Tris Base, 45 mM Acetic acid, 300 mM Sodium Acetate, at  about pH 7.5; and (d) recovering the protein from the solid  US 9,868,761 B2  10  9 