Method for using light scattering in real time to directly monitor and control impurity removal in purification processes

A method for determining the purity of a recombinant protein in the eluate pool from a cation exchange resin comprising:

• loading a mixture containing a recombinant protein and biological impurities on to a cation exchange chromatography resin;
• eluting by a salt gradient the recombinant protein from the cation exchange chromatography resin;
• measuring the intensity of light scattered by the eluate with a laser light scattering detector to obtain a light scattering (LS) signal;
• measuring the absorbance of the eluate with a UV absorbance detector to obtain an absorbance signal;
• calculating a ratio of the LS signal to the absorbance signal for each elution fraction to obtain a LS/UV fraction ratio;
• monitoring in real time during the elution of the recombinant protein the LS/UV fraction ratio over time for each elution fraction;
• and
• detecting a change in the LS/UV fraction ratio over time, wherein an increase in the LS/UV fraction ratio over time indicates an increase of biological impurities in the eluate pool.

A method for controlling the separation of an antibody from high molecular weight species of the antibody comprising:

• loading a mixture containing the antibody and high molecular weight species of the antibody on to a cation exchange chromatography resin;
• eluting by a salt gradient the antibody from the cation exchange chromatography resin;
• measuring the intensity of light scattered by the eluate with a laser light scattering detector to obtain a light scattering (LS) signal;
• measuring the absorbance of the eluate with an ultraviolet (UV) absorbance detector to obtain an absorbance signal;
• calculating a ratio of the LS signal to the absorbance signal for each elution fraction to obtain a LS/UV fraction ratio;
• monitoring in real time during the elution of the antibody the LS/UV fraction ratio over time for each elution fraction, wherein an increase in the LS/UV fraction ratio over time indicates an increase in high molecular weight species of the antibody in the eluate;
• and
• terminating the elution when an increase in the LS/UV fraction ratio over time is detected.