A comparative investigation of random and oriented immobilization of protein A ligands on the binding of immunoglobulin G

Increasing immunoglobulin G adsorption in dextran‐grafted protein A gels

“…Figure 3 shows IgG adsorption on Z 1 ‐CMD10‐6FF‐IC250 and Z 1 ‐CMD40‐6FF‐IC260 as well as non‐grafted Z 1 ‐CM Sepharose FF (Z 1 ‐CMSep) at different salt concentrations and neutral pH (pH 7.4). With the help of zeta potential measurement at various buffer pH, zero point of zeta potential for IgG was determined to be pH 5.8, far from experimental pHs in this work [18]. Therefore, IgG had the charge with the same sign as carboxyl group on the surface of the gels at pH 7.4 and above, and contribution of electrostatic attraction to IgG binding could be ignored.…”

“…At the similar ligand density, q m values increased by over 220% as IC increased from 200 mmol/L to 300–350 mmol/L. In this case, IgG binding was dominant by affinity interaction with protein A ligand because electrostatic attraction between IgG and negative‐charged matrix surface could completely be ignored at buffer pH much higher than p I of IgG [18]. A further increase in IC led to a little decrease of q m to 60.3 mg/g gel on Z 1 ‐CMD10‐4FF‐IC400 gel.…”

“…A decreased ligand availability was also found previously by Yang et al . on non‐grafted protein A gels [18]. At low ligand density, q m and ligand availability of Z 1 ‐CMD10‐4FF‐IC300 were higher than that of Z 1 ‐CM Sepharose, which can be considered that the three‐dimensional binding space provided by the dextran graft layer did have a positive effect on reducing steric hindrance.…”

“…Protein A gel was prepared by coupling protein A ligands onto CMD‐grafted Sepharose gel as described by Yang et al . with a modification [18]. As a reference, non‐grafted protein A gel was prepared on CM Sepharose gel.…”

“…So far, engineered Z domain and its derivatives is the most frequently used ligands in commercial protein A chromatography. In protein A chromatography, binding capacity for antibody was influenced by many factors including ligand types [12][13][14] and density [15][16][17][18], immobilization techniques [13,17,18], matrix properties [15,19,20], operation conditions [21] and molecular orientation of adsorbed antibody [22]. Adsorption capacity for antibody is largely dependent on ligand density of protein A adsorbents [13,16,17,23].…”