Antibody adsorption on the surface of water studied by neutron reflection

Abstract

Surface and interfacial adsorption of antibody molecules could cause structural unfolding and desorbed molecules could trigger solution aggregation, resulting in the compromise of physical stability. Although antibody adsorption is important and its relevance to many mechanistic processes has been proposed, few techniques can offer direct structural information about antibody adsorption under different conditions. The main aim of this study was to demonstrate the power of neutron reflection to unravel the amount and structural conformation of the adsorbed antibody layers at the air/water interface with and without surfactant, using a monoclonal antibody 'COE-3 0 as the model. By selecting isotopic contrasts from different ratios of H 2 O and D 2 O, the adsorbed amount, thickness and extent of the immersion of the antibody layer could be determined unambiguously. Upon mixing with the commonly-used non-ionic surfactant Polysorbate 80 (Tween 80), the surfactant in the mixed layer could be distinguished from antibody by using both hydrogenated and deuterated surfactants. Neutron reflection measurements from the co-adsorbed layers in null reflecting water revealed that, although the surfactant started to remove antibody from the surface at 1/100 critical micelle concentration (CMC) of the surfactant, complete removal was not achieved until above 1/10 CMC. The neutron study also revealed that antibody molecules retained their globular structure when either adsorbed by themselves or co-adsorbed with the surfactant under the conditions studied.

Interfacial Adsorption of Monoclonal Antibody COE-3 at the Solid/Water Interface

“…This is consistent with previous observations for other proteins. [18][19][20][21][22][23] These results were also obtained in the simulations showing that the dominant influence to equilibrated values is the effective area of adsorbed COE-3, with adjustments caused by the pH induced changes to electrostatic interactions involving adsorbing COE-3 molecules as indicated by changes in equilibrated values in Figures 4(a) and 4(b).…”

“…Adsorption of COE-3 on the surface of water has also been undertaken recently under similar solution conditions using a combined study of NR and surface tension. 22,23 The most noticeable feature was that COE-3 surface adsorption caused only a small decrease in surface tension at bulk concentrations up to 1 mg/ml. This effect could be linked to a high globular stability which is also revealed in the data acquired here and reflected in…”

“…[17][18][19][20][21] A key value of NR lies in its high depth resolution and therefore capability to determine the thickness and composition of the adsorbed layer(s) from which the structural conformation of the adsorbed molecules can be inferred. 18,22,23 NR data together with the measured globular structures (e.g from X-ray crystal diffraction) of proteins enables us to determine the probable conformational orientations of the adsorbed protein molecules at a given interface, therefore also implying the extent of structural deformation and unfolding. 18 However, each NR experiment generally requires a minimum of 30 min to acquire sufficient data for statistical analysis, making NR an impractical technique to follow a fast, dynamic adsorption process.…”

Neutron Reflection Study of Surface Adsorption of Fc, Fab, and the Whole mAb

“…Prior work by our group in this area set out to study mAb adsorption at the air/liquid interface in the presence of polysorbate 80, which is widely added to mAb drug substances. 17 Comparing Fc and Fab adsorption under similar conditions will lead to an understanding of how these fragments affect the behavior of the whole mAb. As a first step towards this goal, Fc and Fab domains were obtained by digestion and re-purification of an IgG1 termed 'COE-3'.…”

Coadsorption of a Monoclonal Antibody and Nonionic Surfactant at the SiO2/Water Interface

“…In contrast, neutron reflection (NR) together with deuterium labelling can reveal the thickness and composition of adsorbed protein with and without surfactant. 5,14,[16][17][18][19][20] NR data together with the known globular structures of proteins allow us to determine how interfacial adsorption and surfactant binding affect the conformational orientation of the adsorbed protein molecules, from which the extent of structural rearrangement can be inferred.…”