Increasing immunoglobulin G adsorption in dextran-grafted protein A gels

Abstract

The formation of a stable spatial arrangement of protein A ligands is a great challenge for the development of high-capacity polymer-grafted protein A adsorbents due to the complexity in interplay between coupled ligands and polymer chain. In this work, carboxymethyl dextrans (CMDs) with different molecular weight were introduced to provide stable spatial ligand arrangement in CMD-grafted protein A gels to improve IgG adsorption. The result showed that coupling of protein A ligand in CMD-grafted layer had no marked influence on pore size and dextran layers coupled with the ligands were stable in experimental range of salt concentrations. The result of IgG adsorption revealed that carboxymethyl dextran T10, a short CMD, was more suitable as a scaffold for the synthesis of high-capacity protein A gels. Moreover, the maximal adsorption capacity for IgG was obtained to be 96.4 mg/g gel at ionic capacities of 300-350 mmol/L and a ligand density of 15.2 mg/g gel. Dynamic binding capacity for IgG exhibited a higher capacity utilization in CMD-grafted protein A gels than non-grafted protein A gel. The research presented a tactics to establish a stable dextran layer coupled with protein A ligands and demonstrated its importance to improve binding capacity for IgG.

Keywords: IgG adsorption; carboxymethyl dextran; grafting density; ligand density; protein A chromatography.

FIGURE 1

Synthesis of CMD‐grafted Sepharose gel and protein A gels

FIGURE 2

Dextran calibration curves for Sepharose 6FF and CMD‐grafted protein A gels using Sepharose 6FF matrix

FIGURE 3

IgG adsorption on CMD‐grafted protein A gels and non‐grafted protein A gel at different salt concentrations. (A) Z₁‐CMSep gel, (B) Z₁‐CMD10‐6FF‐IC250 gel, and (C) Z₁‐CMD40‐6FF‐IC260 gel

FIGURE 4

IgG adsorption on CMD‐grafted protein A gels and non‐grafted protein A gel at different buffer pHs. The experiment was conducted in 20 mmol/L phosphate buffer containing 100 mmol/L NaCl at pH 7.4 and 10.0. Protein A gels used in this work were (A) Z₁‐CMSep gel, (B) Z₁‐CMD10‐6FF‐IC250 gel, and (C) Z₁‐CMD40‐6FF‐IC260 gel

FIGURE 5

IgG adsorption on CMD‐grafted protein A gels with different molecular weight and grafted amount of CMD. (A) Sepharose 6FF based CMD‐grafted protein A gels, (B) Sepharose 4FF based CMD‐grafted protein A gels

FIGURE 6

IgG adsorption on CMD‐grafted protein A gels with different grafted amount of CMD10 based on Sepharose 4FF

FIGURE 7

Schematic of IgG adsorption in non‐grafted and CMD‐grafted protein A gels

FIGURE 8

IgG adsorption on CMD‐grafted protein A gels coupling respectively with Z₁, Z₂, and Z₄ ligands

FIGURE 9

IgG adsorption on CMD‐grafted protein A gels at different ligand densities. (A) adsorption isotherms, (B) adsorption capacity for IgG and ligand availability CMD‐grafted protein A gels

FIGURE 10

Breakthrough curves of non‐grafted Z₁‐CMSep gel and Z₁‐CMD10‐4FF‐IC300 gel at different ligand densities. The ligand densities (LDs) were (A) 10 mg/g gel and (B) 20 mg/g gel