Neutron reflectivity measurement of protein A-antibody complex at the solid-liquid interface

Abstract

Chromatography is a ubiquitous unit operation in the purification of biopharmaceuticals yet few studies have addressed the biophysical characterisation of proteins at the solution-resin interface. Chromatography and other adsorption and desorption processes have been shown to induce protein aggregation which is undesirable in biopharmaceutical products. In order to advance understanding of how adsorption processes might impact protein stability, neutron reflectivity was used to characterise the structure of adsorbed immunoglobulin G (IgG) on model surfaces. In the first model system, IgG was adsorbed directly to silica and demonstrated a side-on orientation with high surface contact. A maximum dimension of 60Å in the surface normal direction and high density surface coverage were observed under pH 4.1 conditions. In chromatography buffers, pH was found to influence IgG packing density and orientation at the solid-liquid interface. In the second model system, which was designed to mimic an affinity chromatography surface, protein A was attached to a silica surface to produce a configuration representative of a porous glass chromatography resin. Interfacial structure was probed during sequential stages from ligand attachment, through to IgG binding and elution. Adsorbed IgG structures extended up to 250Å away from the surface and showed dependence on surface blocking strategies. The data was suggestive of two IgG molecules bound to protein A with a somewhat skewed orientation and close proximity to the silica surface. The findings provide insight into the orientation of adsorbed antibody structures under conditions encountered during chromatographic separations.

Keywords: Adsorption; Antibody; Chromatography; Interface; Neutron reflectivity; Protein A.

Fig. 1

Schematic representation of the flow cell used for reflectivity experiments. The flow-through cavity has a volume of approximately 1.5 mL and a height of 0.1 mm. During experiments all space inside the cell is filled with aqueous solution at all times. For a bare silicon surface the incident angle of the collimated neutron beam is equal to the reflected angle. Neutrons reflect at interfaces present inside the reflectivity cell, such as the SiO₂-protein interface and the protein-water interface. The reflectivity profile generated by adsorbed material depends on five major factors: its chemical composition, the number of layers present across the z axis, their respective depths and volume fractions, and the resolution or smoothness of transition between layers (roughness).

Fig. 2

Left-hand plots (A) and (B): reflectivity (relative intensity) against momentum transfer vector, Q (Å⁻¹), for sequential IgG adsorption and wash steps carried out in D₂O based (A) and H₂O based (B) sodium citrate buffer. In all cases H₂O buffer was at pH 4.00 and D₂O buffer was at an apparent pH of 4.10, which approximates to pD 4.5. Data sets are offset down the y-axis for clarity; in D₂O the critical edge always occurs when reflectivity is equal to unity. Different colours represent different experimental conditions/stages. From top to bottom: black is the bare silicon wafer; red is IgG adsorption at 5.6 mg/mL; blue is adsorbed IgG after a 6.0 mL rinse. Solid lines show the fitted models. Plot (C) shows the corresponding profiles of scattering length density, SLD (Å⁻²), against distance from the silicon surface, each generated by simultaneous fitting of D₂O and H₂O reflectivity data sets. Curves that terminate at the upper and lower ends of the SLD scale represent D₂O and H₂O data, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Reflectivity data for sequential IgG adsorption and wash steps carried out in D₂O based (A) and H₂O based (B) sodium citrate buffer. From top to bottom: black is the bare silicon wafer; red is 5.6 mg/mL IgG adsorption at pH 6.2; blue is adsorbed IgG after a 6.0 mL rinse with pH 6.2 buffer; green is after a subsequent 6.0 mL rinse with pH 3.7 buffer. Solid lines show the fitted models. Plot C shows the corresponding profiles of scattering length density, SLD (Å⁻²), against distance from the silicon surface. Layer thicknesses in each SLD-distance profile are shown by double-ended arrows. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

FTIR spectra for sequential stages of silica surface modification, IgG adsorption and elution. Within each plot, the event sequence is analogous to the following colour sequence: black (initial) – red – blue (end). The instrument was blanked and SPDP-modified rSPA was introduced into the cell; reaction with cross-linkers on the silica surface resulted in cross-linked rSPA (A); BSA was then used to block un-reacted or “sticky” sites (B). The cell was equilibrated in adsorption buffer, the instrument was blanked and IgG was introduced (C). Adsorbed IgG was eluted first at pH 4.1 and then at pH 3.7 (D). Cross-linked rSPA was cleaved by DTT reduction and reactive cross-linkers were blocked with denatured BSA; IgG was then introduced to check for non-specific binding (E). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Reflectivity data for attachment of rSPA to an amino-silylated silica surface, and addition of the blocking agent BSA. Left-hand plots A and B: reflectivity (intensity) against Q (Å⁻¹) in D₂O-based (A) and H₂O-based (B) buffer at various stages of surface modification. From top to bottom: black is the amino-silylated wafer; red is with the cross-linker attached, blue is after rSPA cross-linking and green is after blocking with BSA. Solid lines show the fitted models. Plot C shows the corresponding profiles of scattering length density, SLD (Å⁻²), against distance from the silicon surface. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Reflectivity (intensity) against Q at three solution phase contrasts for IgG4 adsorbed at pH 6.7 to rSPA-modified silica with BSA blocking. Black, red and blue data points represent D₂O, H₂O and silicon matched water (SMW, 38% D₂O) solution phases, respectively. H₂O and SMW data sets are offset down the y-axis to prevent overlap. Solid lines represent fitted models. The pink dotted line represents the fitted model for the amino-silylated surface in D₂O (Fig. 5) and is shown for comparison. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

(A) Profile of scattering length density against distance for IgG4 adsorbed at pH 6.7 to protein A-modified silica with BSA blocking. Data was fitted simultaneously for three solution phase contrasts: D₂O (black), H₂O (red) and silicon matched water (SMW) (blue). The pink dotted line represents the bare amino-silylated silicon wafer in D₂O. Shading is to aid layer visualisation. Layer identities are suggested in black text. (B) Schematic of one suggested configuration for IgG4 adsorbed to protein A on a silicon substrate; BSA has been omitted for simplicity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Reflectivity data for attachment of rSPA to an amino-silylated silica surface, and addition of the blocking agent PEG₆₀₀₀. Left-hand plots A (D₂O contrast) and B (H₂O contrast): black is the amino-silylated wafer with cross-linker attached; red is after rSPA cross-linking and blue is after blocking with PEG₆₀₀₀. Solid lines show the fitted models. Plot C shows the corresponding profiles of scattering length density, SLD (Å⁻²), against distance from the silicon surface. Where data was collected at both solution phase contrasts, the profile was generated by simultaneous fitting of D₂O and H₂O reflectivity data sets to a single layer depth profile. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 9

Reflectivity data for IgG4 adsorbed at pH 6.7 to rSPA-modified silica with PEG₆₀₀₀ blocking. Left-hand plot (A): reflectivity (intensity) against momentum transfer, Q (Å⁻¹), at three solution phase contrasts: D₂O (black), H₂O (red) and silicon-matched water (blue). H₂O and SMW data sets are offset down the y-axis to prevent overlap. The fitted curve for the amino-silylated surface with cross-linker only is shown for comparison (green dotted line). Plot B shows the corresponding profiles of scattering length density (Å⁻²) against distance from the silicon surface. Black, red and blue curves represent D₂O, H₂O and SMW data, respectively. SLD-distance profiles for the surface with cross-linked rSPA (dotted lines) and with cross-linker only (green dashed line) are shown for comparison. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 10

SLD-Distance profiles for IgG adsorbed to protein A with BSA surface blocking (black line) and PEG₆₀₀₀ surface blocking in Cell B (red line) and Cell C (blue line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)