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.

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.

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.

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.

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.

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.

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.