Method for purifying recombinant protein

A method for purifying a recombinant protein from a mixture comprising the recombinant protein and its related proteins, which comprises the following steps performed sequentially:

• A. binding the recombinant protein to an ion exchange medium using a first equilibration buffer, wherein the first equilibration buffer is at a first conductivity and pH;
• the ion exchange medium is a cation exchange medium;
• B. equilibrating the protein-bound ion exchange medium continually using a second equilibration buffer, wherein the second equilibration buffer is at a second conductivity and pH;
• the conductivity of the second equilibration buffer is less than that of the first equilibration buffer, but the pH of both are the same;
• C. washing the ion exchange medium using a wash buffer which has different pH, so as to elute a first class of the related proteins from the ion exchange medium, wherein the wash buffer is at a third conductivity and a gradually increased pH;
• the conductivity of the wash buffer is less than that of the first equilibration buffer, but the pH of the wash buffer is greater than that of the first and/or second equilibration buffer;
• D. washing the ion exchange medium using a first elution buffer, so as to elute the target recombinant protein from the ion exchange medium, wherein the first elution buffer is at a fourth conductivity and pH;
• the pH of the first elution buffer is greater than that of the wash buffer, but the conductivity of both are essentially the same;
• and
• E. then washing the ion exchange medium using a second elution buffer, so as to elute a second class of related proteins from the ion exchange medium, wherein the second elution buffer is at a fifth conductivity and pH;
• the pH and/or conductivity of the second elution buffer are/is greater than that of the first elution buffer.