Systematic optimization of histidine-tagged enhanced green fluorescent protein (His-EGFP) purification using packed bed immobilized metal affinity chromatography: A multistage design of experiments approach.

A systematic purification process for His-tagged enhanced green fluorescent protein (His-EGFP) from recombinant E. coli was developed using immobilized metal affinity chromatography (IMAC) in a packed bed format. Large-scale fermentation was conducted in a 5 L bioreactor, followed by cell harvesting and sonication-based disruption at 20 kHz and 4 °C. The highest EGFP release from 25 % (w/v) disrupted cells reached 8.39 × 10 AU. Ni-charged STREAMLINE Chelating adsorbent served as the IMAC adsorbent. Dynamic binding capacity (DBC) at 5 % breakthrough was evaluated through one-factor-at-a-time (OFAT) screening, then optimized via a sequential design of experiments (DOE) approach, including two-level factorial design, steepest ascent, and central composite design (CCD). Optimal adsorption conditions were identified as pH 8.08, flow rate 6.15 mL/min, cell concentration 64.7 % (w/v), and adsorbent bed height 15.37 cm, achieving a predicted DBC of 2.15 × 10 AU/mL. Elution optimization revealed the most effective 300 mM imidazole in 500 mM NaCl (3 column volumes, 300 cm/h). Under these optimized conditions, adsorption efficiencies reached 86.68 % and 97.94 % for 30 mL and 10 mL feed volumes, respectively. Final His-EGFP yields were 85.45 % (purification factor 3.09) and 97.21 % (purification factor 3.20), confirming the robustness and scalability of the developed purification strategy.