Oriented covalent immobilization of recombinant protein A on the glutaraldehyde activated agarose support.

High IgG-binding capacity of protein A affinity chromatography is crucial to its application in the antibody purification and autoantibody-associated disease treatment. An oriented immobilization strategy was used to covalently conjugate the recombinant protein A (rSpA) on the glutaraldehyde activated agarose. By controlling the glutaraldehyde concentration, pH and reactivity time, one or two molecules of glutaraldehyde per primary amino group were anchored on agarose supports. The structure differences of activated supports were evaluated. Moreover, the 3D surface structure of B domain was modeled to explore the distribution of reactive and adsorptive groups. Compared with the monomeric glutaraldehyde agarose (Aga@MG), the dimeric glutaraldehyde agarose (Aga@DG) seems to be involved with more amino acid groups of rSpA during the immobilization. The leaked rSpA of 0.24 ng/mg IgG from Aga@DG@rSpA was slightly lower than that of 0.36 ng/mg IgG from Aga@MG@rSpA. However, Aga@MG is more suitable for oriented immobilization of rSpA which endows the prepared adsorbents to higher IgG-binding capacity. When rSpA was immobilized on Aga@MG at the low and high ionic strength, the maximum capacities from Langmuir model were 56.2 and 59.2 mg/g, respectively. The Aga@MG provided shorter spacer arm compared with the Aga@DG, which contributed to the oriented immobilization of rSpA.