Immobilizing calcium-dependent affinity ligand onto iron oxide nanoparticles for mild magnetic mAb separation

Abstract

Current downstream processing of monoclonal antibodies (mAbs) is limited in throughput and requires harsh pH conditions for mAb elution from Protein A affinity ligands. The use of an engineered calcium-dependent ligand (Z_Ca) in magnetic separation applications promises improvements due to mild elution conditions, fast processability, and process integration prospects. In this work, we synthesized and evaluated three magnetic nanoparticle types immobilized with the cysteine-tagged ligand Z_Ca-cys. Ligand homodimers were physically immobilized onto bare iron oxide nanoparticles (MNP) and MNP coated with tetraethyl orthosilicate (MNP@TEOS). In contrast, Z_Ca-cys was covalently and more site-directedly immobilized onto MNP coated with (3-glycidyloxypropyl)trimethoxysilane (MNP@GPTMS) via a preferential cysteine-mediated epoxy ring opening reaction. Both coated MNP showed suitable characteristics, with MNP@TEOS@Z_Ca-cys demonstrating larger immunoglobulin G (IgG) capacity (196 mg g ^-1) and the GPTMS-coated particles showing faster magnetic attraction and higher IgG recovery (88 %). The particles pave the way for the development of calcium-dependent magnetic separation processes.

Keywords: Anything but conventional chromatography (abc); Downstream processing; Epoxy; Physical and covalent immobilization; Silica.

Graphical abstract

Fig. 1

Schematic illustration of the three particle types investigated for immobilizing the cysteine-tagged, calcium-dependent Protein A ligand (Z_Ca). MNP: magnetic iron oxide nanoparticle, TEOS: tetraethyl orthosilicate, GPTMS: (3-glycidyloxypropyl)trimethoxysilane. Note that the hydrolyzed silane coupling agents (TEOS and GPTMS) react with hydroxyl groups (-OH) of bare MNP by condensation. However, a high degree of crosslinking between the multiple silanol groups also occurs (forming Si-O-Si bonds), which is, for simplicity, not shown here.

Fig. 2

(A) TEM images and specific surface areas of the three particle types determined by BET nitrogen sorption isotherms. (B) FT-IR spectra of the particles. For better comparability, the spectra were normalized to their respective absorbance at ∼ 580 cm⁻¹, representing Fe-O vibrations of iron oxide [39]. MNP: magnetic nanoparticle, TEOS: tetraethyl orthosilicate, GPTMS: (3-glycidyloxypropyl)trimethoxysilane.

Fig. 3

(A) Particle magnetizations determined by SQUID. (B) Hydrodynamic diameters of the particles as z-average values and zeta potential measurements in the exemplary process buffers TBSC (50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, pH 7.5) and EDTA (100 mM, pH 5.5). MNP: magnetic nanoparticle, TEOS: tetraethyl orthosilicate, GPTMS: (3-glycidyloxypropyl)trimethoxysilane.

Fig. 4

FT-IR spectra for the investigation of EDTA binding to (A) MNP, (B) MNP@TEOS and (C) MNP@GPTMS. Spectra were normalized to the absorbance at around 580 cm⁻¹, assigned to Fe-O in the iron oxides [39]. Particles incubated in water serve as a reference (grey/orange/green lines). Particles incubated in 100 mM EDTA (pH 5.5, 1 h) were rebuffered into water by exchanging 95 vol% three times before the measurements (blue line). Pure EDTA (not normalized) is illustrated as a reference (blue dashed). MNP: magnetic nanoparticle, TEOS: tetraethyl orthosilicate, GPTMS: (3-glycidyloxypropyl)trimethoxysilane.

Fig. 5

Investigation of ligand immobilization onto the three magnetic nanoparticle types. (A) Z_Ca-cys ligand immobilization isotherms. (B) Ratio of rSpA-cys to untagged rSpA ligand immobilization. (C) Non-reducing SDS-PAGE analytics of particles with immobilized (1) Z_Ca-cys, (2) rSpA and (3) rSpA-cys ligands (from different experiments). (D) FT-IR spectra of MNP@GPTMS, MNP@GPTMS@Z_Ca-cys and pure Z_Ca-cys in water. The spectra of particle samples were normalized to the absorbance at around 580 cm⁻¹, assigned to Fe-O [39]. MNP: magnetic nanoparticle, TEOS: tetraethyl orthosilicate, GPTMS: (3-glycidyloxypropyl)trimethoxysilane.

Fig. 6

IgG adsorption and desorption onto/from magnetic nanoparticles@Z_Ca. (A) Adsorption isotherms onto particles in TBSC buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, pH 7.5). (B) IgG recoveries after incubation of 110 mg IgG per g particles, washing, and elution in 100 mM EDTA (pH 5.5). The bound IgG was calculated from the initially loaded and unbound IgG. (C) Reduced SDS-PAGE of loaded IgG (L) and elution samples taken in three subsequent particle reuse cycles (E 1–3) of the three functionalized particles. The heavy (H; ∼ 50 kDa) and light (L; ∼ 25 kDa) chains of the IgG can be seen. MNP: magnetic nanoparticle, TEOS: tetraethyl orthosilicate, GPTMS: (3-glycidyloxypropyl)trimethoxysilane.