Volumetric Interpretation of Protein Adsorption: Ion-Exchange Adsorbent Capacity Protein pI and Interaction Energetics

摘要

Adsorption of lysozyme (Lys), human serum albumin (HSA), and immunoglobulin G (IgG) to anion- and cation-exchange resins is dominated by electrostatic interactions between protein and adsorbent. The solution-depletion method of measuring adsorption shows, however, that these proteins do not irreversibly adsorb to ion-exchange surfaces, even when the charge disparity between adsorbent and protein inferred from protein pI is large. Net-positively-charged Lys (pI = 11) and net-negatively-charged HSA (pI = 5.5) adsorb so strongly to sulfopropyl sepharose (SP; a negatively-charged, strong cation exchange resin, −0.22 mmol/mL exchange capacity) that both resist displacement by net-neutral IgG (pI = 7.0) in simultaneous adsorption-competition experiments. By contrast, IgG readily displaces both Lys and HSA adsorbed either to quarternary-ammonium sepharose (Q; a positively-charged, strong anion exchanger, + 0.22 mmol/mL exchange capacity) or octadecyl sepharose (ODS, a neutral hydrophobic resin, 0 mmol/mL exchange capacity). Thus it is concluded that adsorption results do not sensibly correlate with protein pI and that pI is actually a rather poor predictor of affinity for ion-exchange surfaces. Adsorption of Lys, HSA, and IgG to ion-exchange resins from stagnant solution leads to adsorbed multi-layers, into-or-onto which IgG adsorbs in adsorption-competition experiments. Comparison of adsorption to ion-exchange resins and neutral ODS leads to the conclusion that the apparent standard free-energy-of-adsorption ΔGadso of Lys, HSA, and IgG is not large in comparison to thermal energy due to energy-compensating interactions between water, protein, and ion-exchange surfaces that leaves a small net ΔGadso. Thus water is found to control protein adsorption to a full range of substratum types spanning hydrophobic (poorly water wettable) surfaces, hydrophilic surfaces bearing relatively-weak Lewis acid/base functionalities that wet with (hydrogen bond to) water but do not exhibit ion-exchange properties, and surfaces with strong Lewis acid/base functional groups that exhibit ion-exchange properties in the conventional-chemistry sense of ion exchange.>Impact Statement: This work yields detailed insights into the physical chemistry of protein adsorption by elucidating relationships among adsorbent surface charge, capacity to adsorb proteins with different net charge inferred from pI, and the energy required to displace water at the solution-material interface.