Non-functionalized silica for low-cost and sustainable affinity purification in comparison to IMAC

Immobilized metal affinity chromatography (IMAC) is widely employed for affinity-tagged protein capture but presents challenges, including high resin cost, toxic metal waste, and reduced performance in crude feeds. These limitations were observed during the capture of the engineered IgG-binding ligand B8-(RH) 4 (rSpA), motivating an extensive screening of IMAC resins across ligand chemistries and metal ions. As an alternative, non-functionalized silica was evaluated as a low-cost and sustainable capture resin. Frontal analysis was performed on silica and Ni 2+ -IMAC under single- and multi-component conditions across a tenfold titer range. At comparable equilibrium capacities, dynamic behavior differed markedly due to distinct mass transfer regimes. Silica binding is presumably predominantly localized at the external particle surface, leading to steeper breakthrough profiles and superior DBC 10% Silica 2.7–9.0 mg mL −1 vs. IMAC 1.7–4.5 mg mL −1 , enabling rapid uptake. In contrast, IMAC benefits from combined film and pore diffusion within its macroporous agarose matrix, yielding higher capacities at near saturation (DBC 70% (IMAC 23.0–34.4 mg mL −1 vs. silica 7.0–29.5 mg mL −1 ) but broader breakthrough profiles. At elevated titers, deviations from monolayer adsorption on silica suggest multilayer-like adsorption and/or surface-associated protein-protein interactions, supporting higher-than-expected capacities. Preparative-scale tests confirmed silica's robustness and delivered a threefold higher concentration factor compared to IMAC, albeit at lower purity (67% vs. >95%), consistent with a non-optimized capture step. Economic analysis highlights a >75-fold resin cost advantage for silica while eliminating metal recharging and toxic waste, positioning it as a viable alternative prioritizing productivity and sustainability.