In situ neutron scattering of antibody adsorption during protein A chromatography

Highlights

• Antibody structure is measured during purification protein A chromatography.

• 1mL flow cell enabled in-situ  measurement using Small Angle Neutron Scattering.

• Protein A chromatography resin and bound antibody studied at nano-mesoscales.

• Buffer conditions alter the antibody structure in its adsorbed state.

Abstract

A deeper understanding of the nanoscale and mesoscale structure of chromatographic adsorbents and the distribution of proteins within the media, is critical to a mechanistic understanding of separation processes using these materials. Characterisation of the media's architecture at this scale and protein adsorption within, is challenging using conventional techniques. In this study, we propose a novel resin characterisation technique that enables in-situ measurement of the structure of the adsorbed protein layer within the resin, under typical chromatographic conditions. A quartz flow-through cell was designed and fabricated for use with Small Angle Neutron Scattering (SANS), in order to measure the nanoscale to mesoscale structures of a silica based protein A chromatography resin during the monoclonal antibody sorption process. We were able to examine the pore-to-pore (˜133 nm) and pore size (˜63 nm) correlations of the resin and the in-plane adsorbed antibody molecules (˜ 4.2 nm) correlation at different protein loadings and washing buffers, in real time using a contrast matching approach. When 0.03 M sodium phosphate with 1 M urea and 10 % isopropanol buffer, pH 8, was introduced into the system as a wash buffer, it disrupted the system's order by causing partial unfolding of the adsorbed antibody, as evidenced by a loss of the in-plane protein correlation. This method offers new ways to investigate the nanoscale structure and ligand immobilisation within chromatography resins; and perhaps most importantly understand the in-situ behaviour of adsorbed proteins within the media under different mobile phase conditions within a sample environment replicating that of a chromatography column.

Introduction

In commercial separations for therapeutic proteins, binding capacity is a critical factor. An example of such a process is protein A-based affinity chromatography, which is the crucial purification stage for monoclonal antibodies. Maximised binding capacity leads to improved productivity, which in turn leads to better process intensification (PI). Although PI is ultimately the goal for several scale-up procedures, product stability and quality must always be maintained. It is widely accepted that low pH in such processes leads to increased aggregation propensity [1], [2], [3]. This increase in aggregation is likely to contribute to the enhanced interaction with Fcg Receptors observed by Lopez et al. [4]. They also highlight that IgG purification methods can alter the Fcg Receptors binding behaviour and biological activity significantly, and that the purification approach selected might be a contributing factor to the pure reproducibility across current assays employed to evaluate Fc-mediated antibody effector functions [4]. However, pH is not the only parameter that can affect aggregation levels. Structural stability of the eluted IgG can be jeopardized by protein A, making at least a subpopulation of eluted Immunoglobulin G (IgG) more prone to aggregation. This can be attributed to the protein A destabilising effect (conformational relaxation) on the upper portion of IgG's second constant domain [5,6], in addition to the denaturing effects of low pH. Gagnon et al. have demonstrated that during the protein A chromatographic elution step. Size and conformation of the IgG1 (150 kDa) undergo significant changes due to protein A mediated (42 kDa) denaturation, pH, ionic strength and high IgG concentrations [7,8]. It is suggested that the decrease in hydrodynamic radius of the IgG1 molecules in solution arises predominantly from the propensity of IgG to adopt smaller conformations at higher concentrations, low pH and ionic strength. However, when binding to protein A, an increase in IgG size accompanied the loss of secondary structure. This change was attributed to the excess α-helices, extending the hydrodynamic axis of the protein. The high degree of changes in the secondary structure appeared to result from the dual-site interaction between both IgG heavy chains and distinct protein A molecules (one IgG is bound to two protein A molecules) [8]. Other studies from Shukla et al. and Mazzer et al. also support that adsorption / binding to the protein A resin has a destabilizing effect on the antibody molecules and promotes the formation of aggregation-prone species. They quantified the increase in aggregation rate following low pH elution from a protein A column, which was significantly higher when compared to the aggregation rates arising from low pH alone [9,10]. Kulsing also experimented using lysozyme adsorbed to a chromatography column at different temperatures. Conformational changes upon elution where measured ex-situ using Small Angle X-ray Scattering (SAXS) which was connected to the column's outlet and showed that the protein was larger at higher concentrations. These conformational changes were also attributed to the ‘on-column residency effects rather than just detecting a temperature-induced shift’ [11].

The architecture of the media impacts desorption, adsorption, stability, retention and transport rates of the protein during the chromatographic process [12]. It is thus vital to not only characterise the architecture of the media, but also to understand its effect on protein behaviour within the material itself. However, this has proven to be challenging to characterise following traditional approaches. Imaging techniques, such as optical microscopy that can be used to visualise the macro and microstructure of these materials, lack the resolution required to reveal structural information. Methods with the required nanoscale resolution, such as atomic force microscopy and electron microscopy, often demand tailored sample preparation, for instance drying, and thus the output may not reflect the in situ conditions. Techniques like inverse size-exclusion chromatography (ISEC) that can measure surface area and pore distribution at these length scales do not provide information on the material's architecture [12]. Heigl et al. used MIR/NIR imaging to monitor the stationary phase compositions down to the low micrometer range of a polymer material. By using only one measurement, was able to determine the physicochemical characteristics about the chemical composition and the pore volume/area distributions [13]. However, there is still an opportunity for new characterisation techniques that not only measure the intrinsic structure of the media, but also the nanoscale distribution of the protein under the same conditions found during the chromatographic separation process. Therefore, novel techniques that can reveal the media's nanoscale structure, and the spatial distribution of the protein within the media, are needed, which will eventually benefit the design of new media as well as improve the modelling of existing ones [12]. The work presented here aims to help address these issues.

Neutron and x-ray scattering techniques are powerful, non-destructive probes for studying the structure and dynamics of materials on the nano to micron scale and from picoseconds upwards. Neutrons are particularly useful for hydrogenous/soft materials under processing conditions due to their high sensitivity to light elements, and hydrogen in particular, and to their high penetrating power allowing them to probe samples contained within a complex experimental apparatus, such as pressure cells and temperature regulators [14]. For example Mazzer et al. recently used Neutron Reflectivity to characterise the structure and orientation of adsorbed IgG on a model surface designed to mimic the affinity chromatography surface [10,12]. Moreover, neutron scattering power differs significantly between isotopes, allowing the judicial use of hydrogen/deuterium isotopic substitution to highlight one component in the presence of several others which are rendered invisible [15].

In the present work, we used Small Angle Neutron Scattering (SANS) to measure protein adsorption and the resulting resin bound protein structuring on the chromatographic media in a flow-through cell. Even though this technique is widely used by the scientific community, to the best of our knowledge, there are no previous studies on protein adsorption in chromatographic media using a flow through cell with SANS to study the chromatographic process. Pozzo used SANS to probe the conformation of SDS-BSA protein surfactant complexes during electrophoresis in cross-linked polyacrylamide gels [16]. This work demonstrated that SANS has the unique potential to probe nanoscale structures on complex systems that contain multiple components and that this technique is useful in understanding complex phenomena, such as polyelectrolyte electrophoretic migration in hydrogels[16]. In another study by Plewka et al., a flow cell was used during SAXS experiments when studying IgG adsorption on MabSelect Sure resin. The authors state that ‘It was, therefore, possible for the first time to directly correlate the nanostructure changes inside the column, which is otherwise a black box, with the adsorption and elution process' [17]. As described by Koshari et al., SANS is very well suited for this application as it can resolve spatial features from the micrometre to the nanometre length scale. It can also characterise the static, as well as the dynamic aspects of the structure, without being disruptive to the sample [12,18].

Here, we aim to establish SANS as a feasible characterisation technique to study protein adsorption in chromatographic media at the nanoscale to mesoscale. We also wish for the first time to perform these experiments under real-time affinity chromatography conditions using SANS. For this purpose, a custom-made quartz flow-through cell was built mimicking the affinity chromatography columns, and the IgG1 was adsorbed onto Prosep Ultra Plus resin (Fig. 1).