Structure of Human Serum Albumin at a Foam Surface.

Proteins can be adsorbed on the air-water interface (AWI), and the structural changes in proteins at the AWI are closely related to the foaming properties of foods and beverages. However, how these structural changes in proteins at the AWI occur is not well understood. We developed a method for the structural assessment of proteins in the foam state using hydrogen/deuterium exchange mass spectrometry. Adsorption sites and structural changes in human serum albumin (HSA) were identified in situ at the peptide-level resolution. The N-terminus and the loop (E492-T506), which contains hydrophobic amino acids, were identified as adsorption sites. Both the structural flexibility and hydrophobicity were considered to be critical factors for the adsorption of HSA at the AWI. Structural changes in HSA were observed after more than one minute of foaming and were spread widely throughout the structure. These structural changes at the foam AWI were reversible.

A Multi-Method Approach to Assess the Self-Interaction Behavior of Infliximab.

Attractive self-interaction processes in antibody formulations increase the risk of aggregation and extraordinarily elevated viscosity at high protein concentrations. These challenges affect manufacturing and application. This study aimed to understand the self-interaction process of Infliximab as a model system with pronounced attractive self-interaction. The association mechanism was studied by a multi-method approach comprising analytical ultracentrifugation, dynamic light scattering, small angle X-ray scattering, self-interaction bio-layer interferometry and hydrogen-deuterium exchange mass spectrometry. Based on our results, both Fab and Fc regions of Infliximab are involved in self-interaction. We hypothesize a mechanism based on electrostatic interactions of polar and charged residues within the identified areas of the heavy and the light chain of the mAb. The combination of fast and reliable screening methods and low throughput but high resolution methods can contribute to detailed characterization and deeper understanding of specific self-interaction processes.

Physicochemical Characterization of Sabin Inactivated Poliovirus Vaccine for Process Development.

Upscaling the production capacity of inactivated poliovirus vaccines (IPV) is urgently needed to eradicate polio worldwide. For the development of a robust manufacturing process for IPV, the impact of stresses on the properties of the poliovirus during manufacturing needs to be carefully evaluated. In this study, the physicochemical properties of Sabin poliovirus after low pH exposure were analyzed by asymmetrical flow field-flow fractionation coupled to multi-angle laser light scattering (AF4-MALS), sedimentation velocity analytical ultracentrifugation (SV-AUC), transmission electron microscopy (TEM), dynamic light scattering (DLS) and surface plasmon resonance (SPR). Low pH stress caused structural changes and aggregation of inactivated poliovirus virions, whereas degraded virion particles would not revert to native virions even after neutralization. Importantly, a complete loss of the D-antigenicity of IPV by low pH stress, followed by neutralization, was observed in SPR. These results suggest that the exposure of poliovirus particle to low pH stress would induce irreversible denaturation and aggregation of virus particles and lead to the loss of D-antigenicity; thus, low pH stress during the manufacturing of poliovirus vaccine should be minimized. The analytical methods above can be efficiently utilized in the development of high-integrity manufacturing processes and high-quality vaccines.