RESUMEN
When highly concentrated, an antibody solution can exhibit unusual behaviors, which can lead to unwanted properties, such as increased levels of protein aggregation and unusually high viscosity. Molecular modeling, along with many indirect biophysical measurements, has suggested that the cause for these phenomena can be due to short range electrostatic and/or hydrophobic protein-protein interactions. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for investigating protein conformation, dynamics, and interactions. However, "traditional" continuous dilution labeling HDX-MS experiments have limited utility for the direct analysis of solutions with high concentrations of protein. Here, we present a dialysis-based HDX-MS (di-HDX-MS) method as an alternative HDX-MS labeling format, which takes advantage of passive dialysis rather than the classic dilution workflow. We applied this approach to a highly concentrated antibody solution without dilution or significant sample manipulation, prior to analysis. Such a method could pave the way for a deeper understanding of the unusual behavior of proteins at high concentrations, which is highly relevant for development of biopharmaceuticals in industry. Graphical Abstract á .
Asunto(s)
Anticuerpos Monoclonales/química , Medición de Intercambio de Deuterio/instrumentación , Diálisis/instrumentación , Espectrometría de Masas/instrumentación , Animales , Células CHO , Cricetulus , Diseño de Equipo , Modelos Moleculares , Conformación Proteica , SolucionesRESUMEN
Nanoparticle metal oxides offer a wide variety of potential applications in medicine due to the unprecedented advances in nanobiotechnology research. In this work, the effect of zinc oxide (ZnO) nanoparticles prepared by mechano-chemical method on the antibacterial activity of different antibiotics was evaluated using disk diffusion method against Staphylococcus aureus and Escherichia coli. The average size of ZnO nanoparticles was between 20 nm and 45 nm. Although ZnO nanoparticles (500 microg/disk) decreased the antibacterial activity of amoxicillin, penicillin G, and nitrofurantoin in S. aureus, the antibacterial activity of ciprofloxacin increased in the presence of ZnO nanoparticles in both test strains. A total of 27% and 22% increase in inhibition zone areas was observed for ciprofloxacin in the presence of ZnO nanoparticles in S. aureus and E. coli, respectively. The enhancing effect of this nanomaterial on the antibacterial activity of ciprofloxacin was further investigated at three different contents (500, 1000, and 2000 microg/disk) against various clinical isolates of S. aureus and E. coli The enhancing effect of ZnO nanoparticles on the antibacterial activity of ciprofloxacin was concentration-dependent against all test strains. The most enhancing activities were observed in the contents of the 2000 microg/disk.