Adsorption behavior of a human monoclonal antibody at hydrophilic and hydrophobic surfaces

Abstract

One aspiration for the formulation of human monoclonal antibodies (mAb) is to reach high solution concentrations without compromising stability. Protein surface activity leading to instability is well known, but our understanding of mAb adsorption to the solid-liquid interface in relevant pH and surfactant conditions is incomplete. To investigate these conditions, we used total internal reflection fluorescence (TIRF) and neutron reflectometry (NR). The mAb tested (“mAb-1”) showed highest surface loading to silica at pH 7.4 (~12 mg/m²), with lower surface loading at pH 5.5 (~5.5 mg/m², further from its pI of 8.99) and to hydrophobized silica (~2 mg/m²). The extent of desorption of mAb-1 from silica or hydrophobized silica was related to the relative affinity of polysorbate 20 or 80 for the same surface. mAb-1 adsorbed to silica on co-injection with polysorbate (above its critical micelle concentration) and also to silica pre-coated with polysorbate. A bilayer model was developed from NR data for mAb-1 at concentrations of 50–5000 mg/L, pH 5.5, and 50–2000 mg/L, pH 7.4. The inner mAb-1 layer was adsorbed to the SiO₂ surface at near saturation with an end-on” orientation, while the outer mAb-1 layer was sparse and molecules had a “side-on” orientation. A non-uniform triple layer was observed at 5000 mg/L, pH 7.4, suggesting mAb-1 adsorbed to the SiO₂ surface as oligomers at this concentration and pH. mAb-1 adsorbed as a sparse monolayer to hydrophobized silica, with a layer thickness increasing with bulk concentration - suggesting a near end-on orientation without observable relaxation-unfolding.

Keywords: :

• mAb
• interface
• desorption
• polysorbate
• neutron reflectometry
• self assembled monolayer

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

R.G.C. is the recipient of a doctoral training scholarship from MedImmune Ltd. The NR beam-time at ISIS was funded by the Science and Technology Facilities Council, experiment number RB1120015.