Separation of pre-peak and post-peak in fusion protein sample by using size exclusion high performance liquid chromatography

An improved method for the separation of CTLA4-IgG1 fusion protein mixture comprising CTLA4-IgG1 fusion protein, pre-peak impurity thereof and post-peak impurity thereof, the process comprising:

• a) loading the CTLA4-IgG1 fusion protein mixture onto Size Exclusion High Performance Liquid Chromatography (SE-HPLC) column;
• b) separating the CTLA4-IgG1 fusion protein, the pre peak impurity and the post peak impurity present in the CTLA4-IgG1 fusion protein mixture through SEC-HPLC column with a mobile phase comprising a combination of salts, at a suitable pH, wherein the suitable pH is more than pI of the CTLA4-IgG1 fusion protein, and wherein the mobile phase maintains a flow rate of about 0.4 ml/min;
• and
• c) separating the pre-peak impurity and the post-peak impurity from the CTLA4-IgG1 fusion protein;
• wherein the pre-peak impurity resolution is more than 1.3, wherein the area of the pre-peak impurity and the post-peak impurity is not merged or interfered with the area of the CTLA4-IgG1 fusion protein,
• wherein the CTLA4-IgG1 fusion protein is abatacept;
• and
• wherein the mobile phase has a pH of about 6.5 to about 6.7.

An improved method for the separation of CTLA4-IgG1 fusion protein mixture comprising CTLA4-IgG1 fusion protein, pre-peak impurity thereof and post-peak impurity thereof, the process comprising:

• a) loading the CTLA4-IgG1 fusion protein mixture onto Size Exclusion High Performance Liquid Chromatography (SE-HPLC) column;
• b) separating the CTLA4-IgG1 fusion protein, the pre peak impurity and the post peak impurity present in the CTLA4-IgG1 fusion protein mixture through SEC-HPLC column with a mobile phase comprising a combination of salts, at a suitable pH, wherein the suitable pH is more than pI of the CTLA4-IgG1 fusion protein, and wherein the mobile phase maintains a flow rate of about 0.6 ml/min;
• and
• c) separating the pre-peak impurity and the post-peak impurity from the CTLA4-IgG1 fusion protein;
• wherein the pre-peak impurity resolution is more than 1.3, wherein the area of the pre-peak impurity and the post-peak impurity is not merged or interfered with the area of the CTLA4-IgG1 fusion protein,
• wherein the CTLA4-IgG1 fusion protein is abatacept;
• and
• wherein the mobile phase has a pH of about 6.5 to about 6.7.