Highly efficient antibody purification with controlled orientation of protein A on magnetic nanoparticles

Abstract

In this study, we prepared protein A grafted magnetic nanoparticles for the industrial large-scale purification of antibodies with enhancement of binding capacity and immobilization by controlled orientation with chlorophenylsilane (CPTMS) on the surface. For site-specific immobilization of protein A, genetically modified protein A with a cysteine residue was expressed in E. coli and purified by affinity chromatography. To improve the surface area to volume ratio and increase the immobilization amount of protein A, chlorophenylsilane functionalized magnetic nanoparticles (CPTMS@MNPs) were prepared, which are smaller nanoparticles with an average diameter of 20 nm compared to commercial magnetic microparticles (Dynabeads) with an average size of 2.8 μm. The CPTMS@MNPs showed the enhancement of protein A immobilization and binding capacity to antibodies, being 11.5-fold and 7-fold higher than those of commercial Dynabeads, respectively. In addition, the CPTMS@MNPs retained about 80% of the initial protein binding capacity until the third stage of recycling. Therefore, protein A grafted CPTMS@MNPs may be useful for the industrial large-scale purification of antibodies.

Fig. 1. Schematic description of (a) un-oriented immobilization of protein A on commercial magnetic microparticles (Dynabead@protein A) and (b) oriented immobilization on chlorophenylsilane-functionalized magnetic nanoparticles (CPTMS@MNPs@protein A).

Fig. 2. The preparation of CPTMS@MNPs and genetically modified protein A containing a cysteine residue (protein A-cys). (a) TEM image of CPTMS@MNPs; (b) the expression and purification of protein A-cys (N: non-induction, I: induction, FT: flow-through, W: waste, E: elution).

Fig. 3. The immobilization condition test of protein A-cys on magnetic nanoparticles using (a) reaction concentrations from 25 to 300 μg of protein A in 20 mM PB buffer (pH 7) and (b) different pH values between pH 5 and pH 9 in 200 μg ml^–1 of protein A-cys.

Fig. 4. The antibody binding capacity and re-usability. (a) The measurement of protein A amount in Dynabead@protein A and CPTMS@MNPs@protein A. (b) The antibody binding capacity of Dynabead@protein A and CPTMS@MNPs@protein A. (c) SDS page electrophoresis of eluted antibodies in Dynabead@protein A (lanes 3 and 4) and CPTMS@MNPs@protein A (lanes 2 and 3). (d) The re-use of CPTMS@MNPs@protein A.