Effect of dextran layer on protein uptake to dextran‐grafted adsorbents for ion‐exchange and mixed‐mode chromatography

Abstract

In the current research, a series of dextran-grafted adsorbents were prepared using sulfopropyl and 4-(1H-imidazol-1-yl) aniline as chromatographic ligands for ion-exchange (IEC) and mixed-mode chromatography (MMC) to respectively investigate the influence of dextran layer on adsorption of γ-globulin. Experimental evidences of static adsorption on dextran-grafted IEC adsorbents showed that adsorption capacity of γ-globulin increased with dextran content. It could be attributed to the multilayer adsorption of charged protein in dextran layer and thus further induced a significant electrical potential gradient at the boundary of adsorbed area and its proximity, improving mass transfer in combination with concentration gradient. In contrast to IEC adsorbents, adsorption capacity and effective diffusivity of dextran-grafted MMC adsorbents did not change obviously with dextran grafting. It was considered that hydrophobic ligands immobilized onto dextran-grafted MMC adsorbents were stuck together at pH 8.0, resulting in the collapse of dextran layer. In concert with measured effective porosity for γ-globulin at pH 4.0, it was confirmed that dextran layer in MMC adsorbent was more complicated and influenced significantly by buffer pH. It was also manifested by protein adsorption at different pHs. Thus, it revealed the complexity in intraparticle mass transfer of the protein in dextran-grafted MMC adsorbent.

Protein adsorption and chromatography on novel mixed-mode resins fabricated from butyl-modified poly(ethylenimine)-grafted Sepharose

“…of uptake rate is mainly attributed to the decreased "adsorptioncaused hindrance" from the adsorbed BSA molecule to the free BSA molecule, as the adsorption capacity decreased with increasing butyl density. Additionally, that the severer collapse of the PEI layer with increasing hydrophobic groups (Yu et al, 2011) enlarged the pore size and then decreased the diffusion hindrance (Liu et al, 2014a;) also contributed.…”

“…Static adsorption experiments of BSA and γ-globulin were carried out at 0 or 1.0 mol/L NaCl in 20 mmol/L Tris-HCl (pH 8.0) with the method reported previously (Yu et al, 2011). The Langmuir model was used to describe the adsorption isotherms,…”

“…In addition, as a substitute of protein adsorption capacity, the adsorption density (q c ) of BSA and γ-globulin in a wide salt concentration range (0-1.5 mol/L NaCl), were measured at an equilibrium liquid-phase concentration of 1 mg/mL according to the method described by Liu et al (2014a), and the average value with its standard deviation is reported. Protein uptake kinetics were conducted in 20 mmol/L Tris-HCl (pH 8.0) plus 0 or 1.0 NaCl concentrations (Yu et al, 2011). After equilibration, a known amount of drained resin was added into 100 mL of BSA or γ-globulin solution.…”

“…The flow rate was 20 mL/min and a UV-900 detector (ÄKTA Explorer 100, GE Healthcare, and Uppsala, Sweden) was used to monitor the real-time protein concentration in the suspension. The effective pore diffusivity (D e ) was fitted by pore diffusion model (Yu et al, 2011) using the Matlab 7.5.0 (Mathworks, USA). Herein, D e cannot represent the real kinetic mechanism, which is a lumped kinetic parameter describing the overall uptake rate of protein adsorption.…”

Fabrication of high-capacity protein ion-exchangers with polymeric ion-exchange groups grafted onto micron-sized beads by atom transfer radical polymerization

“…Further evidence of protein adsorption in poly(ethylenimine) (PEI)-grafted Sepharose FF reported by Yu et al demonstrated that there was a critical ionic capacity or PEI chain density above which the protein adsorption capacity and effective diffusivity increased sharply [14]. However, the intrinsically heterogeneous architecture of these ion-exchange beads caused by the distribution of the added polymer and ligand leads to a tendency to complicate and intensify mechanistic and synthetic issues of chromatography, which must be addressed [11,15].…”