Abstract: The objective of this work is to establish a two-step series chromatography method for purification of anti-TNF-α monoclonal antibodies(mAbs)from recombinant CHO cell culture medium. Anti-TNF-α mAbs from harvested cell culture fluid(HCCF)after 2 times centrifugation and 1 filtration were captured using filler of protein A, from which the eluant was then further finely purified using the positive ion filler Source30 resin. During fine purification, the effects of pH and salt concentration of the Source30 on the purification was investigated using the DoE method and CCF(Central Composite Face)to achieve the optimal mAbs recovery and purity as well as the lowest aggregate content. The concentration and aggregation ratio of the purified mAbs were determined by HPLC and the purity by CE-SDS. Results were as the followings. The optimal elution pH for protein A affinity chromatography was 4.0. After DoE optimization, in order to reach the quality target(> 90% recovery, > 97% purity, and < 0.3% aggregate), the optimal elution range was 0.05-0.13 mol/L NaCl at pH 5.7-6.0. The elution buffer containing 0.10 mol/L NaCl at pH 6.0 was chosen as the optimal condition of Source elution with 94.3% product recovery, 97.3% purity, and 0.3% aggregate, i. e. , very close to those in the reference standard. In conclusion, we have successfully developed and optimized a two-step series chromatography method at the laboratory scale for the purification of anti-TNF-α mAbs with high recovery, high purity, and low aggregate content.

Key words: two-step chromatography antibody purification platform, anti-TNF-α monoclonal antibody, affinity chromatography, cation exchange chromatography