Integration of NMR Spectroscopy in an Analytical Workflow to Evaluate the Effects of Oxidative Stress on Abituzumab: Beyond the Fingerprint of mAbs.

The assessment of the higher-order structure (HOS) by NMR is a powerful methodology to characterize the structural features of biologics. Forced oxidative stress studies are used to investigate the stability profile, to develop pharmaceutical formulations and analytical methods. Here, the effects of forced oxidative stress by H2O2 on the monoclonal antibody Abituzumab have been characterized by a multianalytical approach combining NMR spectroscopy, mass spectrometry, differential scanning calorimetry, surface plasmon resonance, computational tools, and bioassays. This integrated strategy has provided qualitative and semiquantitative characterization of the samples and information at residue level of the effects that oxidation has on the HOS of Abituzumab, correlating them to the loss of the biological activity.

Expression of genes involved in the T cell signalling pathway in circulating immune cells of cattle 24 months following oral challenge with Bovine Amyloidotic Spongiform Encephalopathy (BASE).

BACKGROUND: Bovine Amyloidotic Spongiform Encephalopathy (BASE) is a variant of classical BSE that affects cows and can be transmitted to primates and mice. BASE is biochemically different from BSE and shares some molecular and histo-pathological features with the MV2 sub-type of human sporadic Creutzfeld Jakob Disease (sCJD). RESULTS: The present work examined the effects of BASE on gene expression in circulating immune cells. Ontology analysis of genes differentially expressed between cattle orally challenged with brain homogenate from cattle following intracranial inoculation with BASE and control cattle identified three main pathways which were affected. Within the immune function pathway, the most affected genes were related to the T cell receptor-mediated T cell activation pathways. The differential expression of these genes in BASE challenged animals at 10,12 and 24 months following challenge, vs unchallenged controls, was investigated by real time PCR. CONCLUSIONS: The results of this study show that the effects of prion diseases are not limited to the CNS, but involve the immune system and particularly T cell signalling during the early stage following challenge, before the appearance of clinical signs.

CyTOF Mass Cytometry for Click Proliferation Assays.

Novel cell analyzers, including polychromatic flow cytometers and isotopical cytometry by time of flight (CyTOF) mass cytometers, enable simultaneous measurement of virtually bondless characteristics at the single-cell level. BrdU assays for quantifying cellular proliferation are common but have several limitations, including the need for a DNA denaturation step and inability to simultaneously resolve multiple parameters and phenotypic complexity. Click chemistry reactions have become popular in the past decade, as they can resolve these issues. This protocol introduces a novel assay able to bridge flow cytometry and CyTOF analysis for active S-phase determination in cell cycle applications, combining well-established click chemistry with a novel iodo-deoxyuridine (IdU) azide derivative and a cross-reactive anti-IdU antibody for detecting incorporated EdU during DNA synthesis. This method is preferred over traditional BrdU-based assays for complex and multiparametric experiments. It provides a feasible cost-effective approach for detecting ethynyl-labeled nucleotides, with the advantage of combining flow and mass cytometry analyses. © 2017 by John Wiley & Sons, Inc.