Optimization of immobilized metal affinity chromatography for a recombinant protein expressed in CHO cells

Immobilized metal affinity chromatography (IMAC) is widely used in research laboratories to purify His-tagged proteins expressed in Escherichia coli (E. coli) by enabling direct capture and moderate removal of process-related impurities, such as host cell proteins (HCPs) and DNA. However, its application for purifying recombinant proteins secreted by mammalian cells is limited due to incompatibility issues between cell culture media and IMAC resins. In this study, we purified a CHO-expressed and secreted recombinant protein with His-tag using a Ni Sepharose excel resin, resistant to EDTA and reducing agents, and optimized loading, washing, and elution conditions to maximize protein recovery and HCP clearance. The resin demonstrated a maximum load capacity of 10 mg/mL. Results show that incorporating Triton X-100, PS80 & TnBP, or 2-propanol in washing buffers significantly improves HCP removal while maintaining low salt concentrations in the elution buffer enhances both yield and product quality. Resin lifetime studies conducted under optimal conditions showed acceptable yields, stable product quality attributes (PQAs), and effective cleaning over 54 purification cycles. This protocol provides a robust IMAC purification strategy, potentially broadening its industrial applications.