Improved Mass Accuracy and Precision for Multi-Attribute Methods Using a New Internally Calibrated High Resolution Orbitrap Mass Detector.

In the development of biotherapeutics, a thorough understanding of a molecule's product quality attributes (PQAs) and their effect on structure-function relationships and long-term stability is essential for ensuring the safety and efficacy of the product. First published in 2015, the multi-attribute method (MAM), based on LC-MS peptide mapping and automation principles, can be used to support biotherapeutic process and product development. The MAM provides simultaneous site-specific detection, identification, quantitation, and quality control monitoring of selected PQAs. In this article, a low-maintenance MAM-ready mass detector with a small footprint was evaluated for its ability to monitor PQAs on proteolytically digested proteins with high mass accuracy and precision. Optimized source parameters enable robust relative quantitation of attributes with high sensitivity and minimal in-source fragmentation. A combination of a built-in one-point mass calibration procedure prior to data acquisition and Scan-to-Scan on-the-fly mass correction allows monitoring of most peptides for at least 54 days with sub-1 ppm mass accuracies at high-resolution (180,000 at m/z 200). This enables the use of <3 ppm mass tolerances for peptide monitoring, supporting high method specificity and robustness. LC-MS based MAM data from this instrument compares well to data collected by earlier MAM systems and conventional HPLC profile-based drug substance release assays.

Oligonucleotide mapping via mass spectrometry to enable comprehensive primary structure characterization of an mRNA vaccine against SARS-CoV-2.

Oligonucleotide mapping via liquid chromatography with UV detection coupled to tandem mass spectrometry (LC-UV-MS/MS) was recently developed to support development of Comirnaty, the world's first commercial mRNA vaccine which immunizes against the SARS-CoV-2 virus. Analogous to peptide mapping of therapeutic protein modalities, oligonucleotide mapping described here provides direct primary structure characterization of mRNA, through enzymatic digestion, accurate mass determinations, and optimized collisionally-induced fragmentation. Sample preparation for oligonucleotide mapping is a rapid, one-pot, one-enzyme digestion. The digest is analyzed via LC-MS/MS with an extended gradient and resulting data analysis employs semi-automated software. In a single method, oligonucleotide mapping readouts include a highly reproducible and completely annotated UV chromatogram with 100% maximum sequence coverage, and a microheterogeneity assessment of 5' terminus capping and 3' terminus poly(A)-tail length. Oligonucleotide mapping was pivotal to ensure the quality, safety, and efficacy of mRNA vaccines by providing: confirmation of construct identity and primary structure and assessment of product comparability following manufacturing process changes. More broadly, this technique may be used to directly interrogate the primary structure of RNA molecules in general.

O-Linked ß-N-acetylglucosamine (O-GlcNAc) regulates emerin binding to barrier to autointegration factor (BAF) in a chromatin- and lamin B-enriched "niche".

Emerin, a membrane component of nuclear "lamina" networks with lamins and barrier to autointegration factor (BAF), is highly O-GlcNAc-modified ("O-GlcNAcylated") in mammalian cells. Mass spectrometry analysis revealed eight sites of O-GlcNAcylation, including Ser-53, Ser-54, Ser-87, Ser-171, and Ser-173. Emerin O-GlcNAcylation was reduced ~50% by S53A or S54A mutation in vitro and in vivo. O-GlcNAcylation was reduced ~66% by the triple S52A/S53A/S54A mutant, and S173A reduced O-GlcNAcylation of the S52A/S53A/S54A mutant by ~30%, in vivo. We separated two populations of emerin, A-type lamins and BAF; one population solubilized easily, and the other required sonication and included histones and B-type lamins. Emerin and BAF associated only in histone- and lamin-B-containing fractions. The S173D mutation specifically and selectively reduced GFP-emerin association with BAF by 58% and also increased GFP-emerin hyper-phosphorylation. We conclude that ß-N-acetylglucosaminyltransferase, an essential enzyme, controls two regions in emerin. The first region, defined by residues Ser-53 and Ser-54, flanks the LEM domain. O-GlcNAc modification at Ser-173, in the second region, is proposed to promote emerin association with BAF in the chromatin/lamin B "niche." These results reveal direct control of a conserved LEM domain nuclear lamina component by ß-N-acetylglucosaminyltransferase, a nutrient sensor that regulates cell stress responses, mitosis, and epigenetics.