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Driven by the prevalent use of ion exchange chromatography (IEC) for polishing therapeutic proteins, many rules have been formulated to summarize the different dependencies between chromatographic data and various operational parameters of interest based on statically determined interactions. However, the effects of the unfolding of protein structures and conformational stability are not as well understood. This study focuses on how the flexibility of proteins perturbs retention behavior at the molecular scale using microscopic characterization approaches, including hydrogen–deuterium (H/D) exchange detected by NMR and a quartz crystal microbalance (QCM). The results showed that a series of chromatographic retention parameters depended significantly on the adiabatic compressibility and structural flexibility of the protein. That is, softer proteins with higher flexibility tended to have longer retention times and stronger affinities on SP Sepharose adsorbents. Tracing the underlying molecular mechanism using NMR and QCM indicated that an easily unfolded flexible protein with a more compact adsorption layer might contribute to the longer retention time on adsorbents. The use of NMR and QCM provided a previously unreported approach for elucidating the effect of protein structural flexibility on binding in IEC systems. [ABSTRACT FROM AUTHOR]

Highlights: [•] Gigaporous microspheres were hydrophilic modified by poly(ethylenimine) (PEI). [•] PEI in the microspheres was in situ branched to resist non-specific adsorption for proteins. [•] The modified microspheres gave more than 90% proteins recovery. [•] The branched PEI in the microspheres could change its construction in different environment of solutions. [•] Dynamic binding capacity of proteins on the modified media didn’t show obviously decrease at the high flow rates. [Copyright &y& Elsevier]

Highlights: [•] Pore size effects of media on adsorption capacity, kinetics and activity of VLPs were investigated. [•] Gigaporous media show higher capacity and faster kinetics for VLPs than agarose media. [•] Gigaporous structure also improved the structural stability and activity of VLPs. [•] Mechanism of pore size effects on the aggregation and/or disassociation of VLPs was discussed. [•] Gigaporous media exhibit the best performance in many aspects compared to agarose media. [ABSTRACT FROM AUTHOR]

Abstract: Ion-exchange chromatography (IEC) using commercial ionic absorbents is a widely used technique for protein purification. Protein adsorption onto ion-exchange adsorbents often involves a multipoint adsorption. In IEC of multimeric proteins or “soft” proteins, the intense multipoint binding would make the further desorption difficult, even lead to the destruction of protein structure and the loss of its biological activity. In this paper, DEAE Sepharose FF adsorbents with controllable ligand densities from 0.020 to 0.183mmol/ml were synthesized, and then the effect of ligand density on the static ion-exchange adsorption of bovine serum albumin (BSA) onto DEAE Sepharose FF was studied by batch adsorption technique. Steric mass-action (SMA) model was employed to analyze the static adsorption behavior. The results showed that the SMA model parameters, equilibrium constant (K a), characteristic number of binding sites (υ) and steric factor (σ), increased gradually with ligand density. Thus, it was feasible to regulate BSA multipoint adsorption by modulating the ligand density of ion-exchange adsorbent. Furthermore, IEC of hepatitis B surface antigen (HBsAg) using DEAE Sepharose FF adsorbents with different ligand densities was carried out, and the activity recovery of HBsAg was improved from 42% to 67% when the ligand density was decreased from 0.183 to 0.020mmol/ml. Taking the activity recovery of HBsAg, the purification factor and the binding capacity into account, DEAE Sepharose FF with a ligand density of 0.041mmol/ml was most effective for the purification of HBsAg. Such a strategy may also be beneficial for the purification of macromolecules and multimeric proteins. [ABSTRACT FROM AUTHOR]