Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A ) from 15 laboratories. Laboratories reported ∼89 800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (∼78 900 centroids), giving ∼100% coverage, and ∼10 900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87%) exhibited centroid mass laboratory repeatability precisions of ⟨ sLab⟩ ≤ (0.15 ± 0.01) Da (1σx̅). All laboratories achieved ⟨sLab⟩ ≤ 0.4 Da. For immersions of protein at THDX = (3.6 to 25) °C and for D2O exchange times of tHDX = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σreproducibility15 Laboratories( tHDX) = (9.0 ± 0.9) % (1σ). A nine laboratory cohort that immersed samples at THDX = 25 °C exhibited reproducibility of σreproducibility25C cohort( tHDX) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements.

Progress towards blue emitting MgO-ZnO-Ga2O3 nanocomposites synthesized by bio mediated route.

MgO-ZnO-Ga2O3 nanocomposites are synthesized by solution combustion method using Aloe Vera gel as a reducing agent to increase the efficiency of blue emission. The appearance of Bragg reflections corresponding to MgO, ZnO and Ga2O3 clearly indicates the formation of nanocomposites. The surface morphology consists irregular shape and sized NPs. The Energy dispersive X-ray analysis confirms the purity of the sample. The band energy gap was tuned to 3.1 eV. The Photoluminescence excitation and emission spectra was discussed and compared it with emission spectra of individual oxides as well as with other reported blue emitted nanophosphors. Further, the chromaticity coordinates and Color correlated temperature coordinates clearly confirms their warm blue emission. Further, the powder dusting method was employed to collect the latent fingerprints on the pores and non-pores surfaces. The synthesized MgO-ZnO-Ga2O3 nanocomposites exhibits well-resolved ridge patterns that can be used to identify latent finger prints with clarity. From all these results, the present synthesized MgO-ZnO-Ga2O3 nanocomposite might find an application in display technology as a blue nanophosphor material and for latent finger print detection in crime investigation.

Application of Dual Protease Column for HDX-MS Analysis of Monoclonal Antibodies.

A co-immobilized, dual protease column was developed and implemented to more efficiently digest IgG molecules for hydrogen/deuterium exchange mass spectrometry (HDX-MS). The low-pH proteolytic enzymes pepsin and type XIII protease from Aspergillus were packed into a single column to most effectively combine the complementary specificities. The method was optimized using an IgG2 monoclonal antibody as a substrate because they are known to be more difficult to efficiently digest. The general applicability of the method was then demonstrated using IgG1 and IgG4 mAbs. The dual protease column and optimized method yielded improved digestion efficiency, as measured by the increased number of smaller, overlapping peptides in comparison with pepsin or type XIII alone, making HDX-MS more suitable for measuring deuterium uptake with higher resolution. The enhanced digestion efficiency and increased sequence coverage enables the routine application of HDX-MS to all therapeutic IgG molecules for investigations of higher order structure, especially when posttranslational and storage-induced modifications are detected, providing further product understanding for structure-function relationships and ultimately ensuring clinical safety and efficacy.