Magnetic Separation of Antibodies with High Binding Capacity by Site-Directed Immobilization of Protein A-Domains to Bare Iron Oxide Nanoparticles

Abstract

The demand for purified antibodies is ever-rising. This study presents a nanoparticle-based material for efficient magnetic separation of immunoglobulin G (IgG) with high binding capacity. The characteristics include: (i) Cost-effective bare iron oxide nanoparticles are used as the solid phase on which optimized protein A-based ligands are directly immobilized. An additional chemical modification or activation is not needed. (ii) Oriented immobilization of the ligands is promoted using a C-terminal peptide tag, containing amino acids with an affinity for iron oxide. (iii) The immobilized ligand consists of eight polymerized B-domains of Protein A. This affinity adsorbent for antibody capture allows a recovery of up to 418 mg IgG per gram of particle, which exceeds the state of the art of magnetic nanoparticles as well as microparticles. Particles with a lower ligand density show a higher percentage recovery of IgG, which allows for a cost-effective design of the adsorbent. Furthermore, the selectivity of the immobilized ligand is shown by means of purification of rabbit polyclonal IgG using rabbit serum.

Competition at the Bio-nano Interface: A Protein, a Polysaccharide, and a Fatty Acid Adsorb onto Magnetic Nanoparticles

“…As can be seen in Figure , the highest adsorption capacities for BSA, SO, and dextran were 0.28 g g –1 , 0.40 g g –1 , and 0.18 g g –1 , respectively. In general, adsorption loadings of proteins onto iron oxide nanoparticles are between 0.1 and 0.3 g g –1 , ,, except for antibodies, which are very big proteins that additionally agglomerate, leading to very large protein quantities onto the surface; adsorption loadings of sodium oleate of 0.35 g g –1 have been reported, and our values are within the same range . The quantities of dextran adsorbed are significantly lower than the ones of SO and BSA; however, these quantities are also common values for sugars adsorbed onto iron oxide materials. , …”

Direct capture and selective elution of a secreted polyglutamate‐tagged nanobody using bare magnetic nanoparticles

“…[17,18] In contrast, the use of magnetic nanoparticles (MNPs) is suggested to be a costefficient and promising alternative as it combines magnetic properties with a high specific surface area, a fast and simple handling, and easy upscale possibilities. [17,[19][20][21] MNPs are applied in many nano-bio applications with a functionalized surface, however, functionalization is costly. [21,22] Therefore, the use of bare, non-functionalized MNPs saves costs and appears to be worthwhile.…”

Matrices and Affinity Ligands for Antibody Purification and Corresponding Applications in Radiotherapy

“…Magnetic beads of different sizes are fabricated via entrapping magnetite within agarose, cellulose, polystyrene, or other polymeric materials, onto which ligands are fixed. Protein affinity separation using the magnetic beads offers the advantages of a low cost, robustness, rapid separation, few handling steps, and reduced system costs [ 4 , 18 ]. For example, an affinity sorbent was prepared by coupling protein A to magnetic monodisperse-porous SiO 2 microspheres and employed to isolate immunoglobulin G (IgG) from rabbit serum in shorter isolation periods [ 19 ].…”