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

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.

Specific-ion effects on the aggregation mechanisms and protein-protein interactions for anti-streptavidin immunoglobulin gamma-1.

Non-native protein aggregation is common in the biopharmaceutical industry and potentially jeopardizes product shelf life, therapeutic efficacy, and patient safety. The present article focuses on the relationship(s) among protein-protein interactions, aggregate growth mechanisms, aggregate morphologies, and specific-ion effects for an anti-streptavidin (AS) immunoglobulin gamma 1 (IgG1). Aggregation mechanisms of AS-IgG1 were determined as a function of pH and NaCl concentration with sodium acetate buffer and compared to previous work with sodium citrate. Aggregate size and shape were determined using a combination of laser light scattering and small-angle neutron or X-ray scattering. Protein-protein interactions were quantified in terms of the protein-protein Kirkwood-Buff integral (G22) determined from static light scattering and in terms of the protein effective charge (Zeff) measured using electrophoretic light scattering. Changing from citrate to acetate resulted in significantly different protein-protein interactions as a function of pH for low NaCl concentrations when the protein displayed positive Zeff. Overall, the results suggest that electrostatic repulsions between proteins were lessened because of preferential accumulation of citrate anions, compared to acetate anions, at the protein surface. The predominant aggregation mechanisms correlated well with G22, indicating that ion-specific effects beyond traditional mean-field descriptions of electrostatic protein-protein interactions are important for predicting qualitative shifts in protein aggregation state diagrams. Interestingly, while solution conditions dictated which mechanisms predominated, aggregate average molecular weight and size displayed a common scaling behavior across both citrate- and acetate-based systems.

Protein adsorption, desorption, and aggregation mediated by solid-liquid interfaces.

Adsorption of proteins to solid-fluid interfaces is often empirically found to promote formation of soluble aggregates and larger, subvisible, and visible particles, but key stages in this process are often difficult to probe directly. Aggregation mediated by adsorption to water-silicon oxide (SiOx) interfaces, akin to hydrated glass surfaces, was characterized as a function of pH and ionic strength for alpha-chymotrypsinogen (aCgn) and for a monoclonal antibody (IgG1). A flow cell permitted neutron reflectivity for protein layers adsorbed to clean SiOx surfaces, as well as after successive "rinse" steps. Aggregates recovered in solution after gently "rinsing" the surface were characterized by neutron scattering, microscopy, and fluorescence spectroscopy. IgG1 molecules oriented primarily "flat" against the SiOx surface, with the primary protein layer desorbed to a minimal extent, whereas a diffuse overlayer was easily rinsed off. aCgn molecules were resistant to desorption when they appeared to be unfolded at the interface, but were otherwise easily removed. For cases where strong binding occurred, protein that did desorb was a mixture of monomer and small amounts of HMW aggregates (for aCgn) or subvisible particles (for IgG1). Changes in adsorption and/or unfolding with pH indicated that electrostatic interactions were important in all cases.

Investigating the structural impact of the glutamine repeat in huntingtin assembly.

Acquiring detailed structural information about the various aggregation states of the huntingtin-exon1 protein (Htt-exon1) is crucial not only for identifying the true nature of the neurotoxic species responsible for Huntington's disease (HD) but also for designing effective therapeutics. Using time-resolved small-angle neutron scattering (TR-SANS), we followed the conformational changes that occurred during fibrillization of the pathologic form of Htt-exon1 (NtQ42P10) and compared the results with those obtained for the wild-type (NtQ22P10). Our results show that the aggregation pathway of NtQ22P10 is very different from that of NtQ42P10, as the initial steps require a monomer to 7-mer transition stage. In contrast, the earliest species identified for NtQ42P10 are monomer and dimer. The divergent pathways ultimately result in NtQ22P10 fibrils that possess a packing arrangement consistent with the common amyloid sterical zipper model, whereas NtQ42P10 fibrils present a better fit to the Perutz ß-helix structural model. The structural details obtained by TR-SANS should help to delineate the key mechanisms that underpin Htt-exon1 aggregation leading to HD.

Protein-protein interactions in dilute to concentrated solutions: α-chymotrypsinogen in acidic conditions.

Protein-protein interactions were investigated for α-chymotrypsinogen by static and dynamic light scattering (SLS and DLS, respectively), as well as small-angle neutron scattering (SANS), as a function of protein and salt concentration at acidic conditions. Net protein-protein interactions were probed via the Kirkwood-Buff integral G22 and the static structure factor S(q) from SLS and SANS data. G22 was obtained by regressing the Rayleigh ratio versus protein concentration with a local Taylor series approach, which does not require one to assume the underlying form or nature of intermolecular interactions. In addition, G22 and S(q) were further analyzed by traditional methods involving fits to effective interaction potentials. Although the fitted model parameters were not always physically realistic, the numerical values for G22 and S(q → 0) were in good agreement from SLS and SANS as a function of protein concentration. In the dilute regime, fitted G22 values agreed with those obtained via the osmotic second virial coefficient B22 and showed that electrostatic interactions are the dominant contribution for colloidal interactions in α-chymotrypsinogen solutions. However, as protein concentration increases, the strength of protein-protein interactions decreases, with a more pronounced decrease at low salt concentrations. The results are consistent with an effective "crowding" or excluded volume contribution to G22 due to the long-ranged electrostatic repulsions that are prominent even at the moderate range of protein concentrations used here (<40 g/L). These apparent crowding effects were confirmed and quantified by assessing the hydrodynamic factor H(q → 0), which is obtained by combining measurements of the collective diffusion coefficient from DLS data with measurements of S(q → 0). H(q → 0) was significantly less than that for a corresponding hard-sphere system and showed that hydrodynamic nonidealities can lead to qualitatively incorrect conclusions regarding B22, G22, and static protein-protein interactions if one uses only DLS to assess protein interactions.

Observation of small cluster formation in concentrated monoclonal antibody solutions and its implications to solution viscosity.

Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals. It is hypothesized that some concentrated mAb solutions exhibit formation of a solution phase consisting of reversibly self-associated aggregates (or reversible clusters), which is speculated to be responsible for their distinct solution properties. Here, we report direct observation of reversible clusters in concentrated solutions of mAbs using neutron spin echo. Specifically, a stable mAb solution is studied across a transition from dispersed monomers in dilute solution to clustered states at more concentrated conditions, where clusters of a preferred size are observed. Once mAb clusters have formed, their size, in contrast to that observed in typical globular protein solutions, is observed to remain nearly constant over a wide range of concentrations. Our results not only conclusively establish a clear relationship between the undesirable high viscosity of some mAb solutions and the formation of reversible clusters with extended open structures, but also directly observe self-assembled mAb protein clusters of preferred small finite size similar to that in micelle formation that dominate the properties of concentrated mAb solutions.

Structural formation of huntingtin exon 1 aggregates probed by small-angle neutron scattering.

In several neurodegenerative disorders, including Huntington's disease, aspects concerning the earliest of protein structures that form along the aggregation pathway have increasingly gained attention because these particular species are likely to be neurotoxic. We used time-resolved small-angle neutron scattering to probe in solution these transient structures formed by peptides having the N-terminal sequence context of mutant huntingtin exon 1. We obtained snapshots of the formed aggregates as the kinetic reaction ensued to yield quantitative information on their size and mass. At the early stage, small precursor species with an initial radius of gyration of 16.1 ± 5.9 Å and average mass of a dimer to trimer were monitored. Structural growth was treated as two modes with a transition from three-dimensional early aggregate formation to two-dimensional fibril growth and association. Our small-angle neutron scattering results on the internal structure of the mature fibrils demonstrate loose packing with ~1 peptide per 4.75 Åß-sheet repeat distance, which is shown to be quantitatively consistent with a ß-helix model. This research provides what we believe to be new insights into the structures forming along the pathway of huntingtin exon 1 aggregation and should assist in determining the role that precursors play in neuronal toxicity.