Stability indicating ion-pair reversed-phase liquid chromatography method for modified mRNA.

Modified messenger RNA (mRNA) represents a rapidly emerging class of therapeutic drug product. Development of robust stability indicating methods for control of product quality are therefore critical to support successful pharmaceutical development. This paper presents an ion-pair reversed-phase liquid chromatography (IP-RPLC) method to characterise modified mRNA exposed to a wide set of stress-inducing conditions, relevant for pharmaceutical development of an mRNA drug product.

Investigation of factors influencing the separation of diastereomers of phosphorothioated oligonucleotides.

This study presents a systematic investigation of factors influencing the chromatographic separation of diastereomers of phosphorothioated pentameric oligonucleotides as model solutes. Separation was carried out under ion-pairing conditions using an XBridge C column. For oligonucleotides with a single sulfur substitution, the diastereomer selectivity was found to increase with decreasing carbon chain length of the tertiary alkylamine used as an ion-pair reagent.

HPLC-UV-MS Analysis: A Source for Severe Oxidation Artifacts.

HPLC coupled to both UV and MS is an established setup for purity assessments in many areas. With evolving technology, instrument sensitivity increases, calling for lower sample concentration, while light flux in a commercial UV detector cell is considerably higher than earlier. This evolution has now reached the point where radicals formed by UV light are abundant enough, compared to the analyte levels, to generate unwanted artifact signals in the MS spectrum.

Neprilysin Inhibits Coagulation through Proteolytic Inactivation of Fibrinogen.

Neprilysin (NEP) is an endogenous protease that degrades a wide range of peptides including amyloid beta (Aβ), the main pathological component of Alzheimer's disease (AD). We have engineered NEP as a potential therapeutic for AD but found in pre-clinical safety testing that this variant increased prothrombin time (PT) and activated partial thromboplastin time (APTT). The objective of the current study was to investigate the effect of wild type NEP and the engineered variant on coagulation and define the mechanism by which this effect is mediated.

Matrix effect explained by unexpected formation of peptide in acidified plasma.

Aim: Peak distortion and strong signal enhancement was observed when applying a bioanalytical method based on mixed-mode SPE, hydrophilic interaction chromatography and ESI-MS to acidified rabbit plasma samples.

Results: High-resolution ESI-MS and N-terminal peptide sequencing revealed a peptide NFQNAL, which was confirmed by H/D exchange ESI-MS.

Conclusion: The peptide causing the observed matrix effect was formed by enzymatic degradation of serum albumin at pH 3.

X-ray structural analysis of tau-tubulin kinase 1 and its interactions with small molecular inhibitors.

Tau-tubulin kinase 1 (TTBK1) is a serine/threonine/tyrosine kinase that putatively phosphorylates residues including S422 in tau protein. Hyperphosphorylation of tau protein is the primary cause of tau pathology and neuronal death associated with Alzheimer's disease. A library of 12 truncation variants comprising the TTBK1 kinase domain was screened for expression in Escherichia coli and insect cells.

FAM3B PANDER and FAM3C ILEI represent a distinct class of signaling molecules with a non-cytokine-like fold.

The FAM3 superfamily is predicted to contain classical four-helix bundle cytokines, featuring a typical up-up-down-down fold. Two members of FAM3 have been extensively studied. FAM3B PANDER has been shown to regulate glucose homeostasis and β cell function, whereas the homologous FAM3C ILEI has been shown to be involved in epithelial-mesenchymal transition and cancer.

Current methods for phosphoprotein isolation and enrichment.

The phosphorylation of proteins is a central paradigm of signal transduction. The substitution of neutral hydroxyl groups of serine, threonine and tyrosine with a negatively charged phosphate group alters the physicochemical and immunogenic properties of the protein, which then can be used to isolate these isoforms. In the last decades several different techniques were applied, attempting to selectively enrich protein populations with this post-translational modification.