Methods for the purification of refolded Fc-peptide fusion protein

A method for producing of a refolded Fc-peptide fusion protein, the method comprising the following steps in the following order:

• 1) Expression of an Fc-peptide fusion protein in microorganisms wherein the Fc-peptide fusion protein is present in inclusion bodies;
• 2) Lysis of the microorganisms;
• 3) Sedimentation of the inclusion bodies;
• 4) Solubilization of the inclusion bodies;
• 5) Refolding of the Fc-peptide fusion protein present in the inclusion bodies;
• 6) Adaption of the refold solution containing the refolded Fc-peptide fusion protein;
• 7) Purification of the Fc-peptide fusion protein comprising the following steps in the following order:
• a) performing an affinity capture chromatography in bind-elute mode;
• b) performing a mixed-mode chromatography;
• c) performing a cation exchange chromatography;
• and
• d) performing an ultrafiltration/diafiltration;
• wherein the chromatographies a), b), and c) are performed in bind-elute mode,
• and wherein step b) is performed with a positively charged mixed-mode chromatography medium and wherein the mixed-mode chromatography of step b) is preceded by a conditioning step, wherein the conditioning step comprises dilution, pH adjustment, incubation, and filtration of the eluate of the affinity capture chromatography of step a).