DOI: 10.5061/dryad.xpnvx0kk9
title: Data for: Oligonucleotide mapping via mass spectrometry to enable comprehensive primary structure characterization of an mRNA vaccine against SARS-CoV-2

description: Oligonucleotide mapping via liquid chromatography mass spectrometry mass
 spectrometry (LC-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
 &gt;98% 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.
description: Oligonucleotide mapping was developed with a representative batch
 of Comirnaty® BNT162b2 Original DS (i.e., the original Pfizer-BioNTech
 COVID-19 vaccine that encodes for the spike glycoprotein (S) of the
 SARS-CoV-2 virus, the Wuhan-Hu-1 isolate: GenBank: QHD43416.1), and it has
 been applied to subsequent Comirnaty® BNT162b2 constructs (BNT162b2s04
 [Delta] and BNT162b2s05 [Omicron]) and other portfolio mRNA molecules.
 Fifty micrograms of mRNA DS was digested with 2500 U of RNase
 T<sub>1</sub> in a 50 mM Tris(hydroxymethyl)aminomethane
 (Tris) pH 7.5 buffer with 20 mM Ethylenediaminetetraacetic acid (EDTA) 1 h
 at 37°C. The resulting enzymatic fragment solution was spiked with 10×
 triethylamine (TEA) and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) emulsion
 to give a final v/v concentration of 0.1% TEA 1% HFIP. A 4 µg load was
 injected and fragments were separated by ion-pair reversed-phase ultrahigh
 performance liquid chromatography (IP RP-UHPLC) with UV detection at 260
 nm using a 1290 Infinity II Bio LC System (Agilent) paired with an ACQUITY
 Premier Oligonucleotide C18 column: 130Å, 1.7 µm, 2.1 x 150 mm (Waters).
 Each mobile phase contained 0.1% TEA and 1% HFIP. The TEA functions as the
 ion-pairing agent, and the HFIP provides MS-compatible buffering as a
 volatile weak acid. The gradient progressed from 1% to 17% mobile phase B
 (50% methanol) in 195 min, then 17% to 35% B in 60 min, followed by wash
 and equilibration segments. The flow rate was 0.2 mL/min with a
 post-column split: 50 µL/min to the UV diode array detector, and 150
 µL/min to an Orbitrap Eclipse Tribrid Mass Spectrometer (Thermo Fisher
 Scientific). The on-line electrospray ionization (ESI) MS acquisition was
 done in negative ion mode with a spray voltage of 2700 V. MS scans were
 from 400 to 2000 m/z at 120000 resolving power (RP) at 400 m/z. Tandem
 mass spectrometry (MS/MS) was accomplished at 30000 RP by a 17, 21, 25
 stepped higher-energy collisional dissociation (HCD) of multiply charged
 precursor candidates selected by the data-dependent acquisition (DDA)
 algorithm. Biopharma Finder version 5.0 software
 (Thermo Fisher Scientific) was used to identify oligonucleotides based on
 both MS and MS/MS matches to theoretical RNase T<sub>1</sub>
 digest products. An MS match required the observed oligonucleotide neutral
 mass to be within 5 ppm of the theoretical mass. An MS/MS match required
 that all major fragments were identified and that the complete sequence
 could be inferred from fragment ions containing the 5′ or 3′ ends (not
 internal fragments). To ensure that the automated software employed
 stringent MS/MS matching, the software also searched against theoretical
 RNase T<sub>1</sub> digests of decoy constructs having random
 arrangements of the same composition of nucleotides as the mRNA molecule.
 To augment the list of automated software identifications, Excel Visual
 Basic for Applications (VBA) scripts were employed to examine unidentified
 LC/UV features and underlying mass spectra one-by-one. In the case of the
 73-mer R1062 and its related poly(A)-tail species, identifications were
 made using a deconvolved, zero-charge mass spectrum, without
 MS/MS.
description: The Thermo mass spectrometer .raw files can be opened by Xcalibur
 Qual Browser (Thermo Fisher Scientific), which is (proprietary) commercial
 software. An open-source alternative is ThermoRawFileParser (https://doi.org/10.1021%2Facs.jproteome.9b00328). Automated identification of oligonucleotides was accomplished using BioPharma Finder v5.0 (Thermo Fisher Scientific), which is (propriety) commercial software. An open-source alternative is attached in this dataset: three Excel VBA spreadsheets that may be used to in the identification of any MS feature. As Excel VBA spreadsheets, they are fully compiled and do not require any library code extensions or external links. The "Oligonucleotide MS Peak ID Given Sequence v8.xlsm" spreadsheet may be used to generate a list of theoretical RNaseT1 digest oligonucleotides and match observed precursor masses to candidate theoretical RNaseT1 digest oligonucleotides. Inputs are a single or list of observed masses and a target mRNA construct sequence. The "Oligonucleotide MS2 Spectrum Matcher v11.xlsm" spreadsheet may be used to check a candidate oligonucleotide sequence with a single MS/MS spectrum. Inputs are the candidate sequence and the m/z vs intensity "XY" coordinate list. The "Oligonucleotide Composition from Mass Calculator v2.xlsm" may be used to generate a list of possible nucleotide compositions given an input mass. A 4th Excel VBA spreadsheet is also provided, "Oligonucleotide Mapping UV Annotation Tool v11.xlsm", to facilitate the annotation of the LC-UV chromatogram with LC-MS/MS-identified oligonucleotides in a PowerPoint file in which the chromatogram is plotted with annotations placed as separate, overlayed text objects in the .pptx. Inputs to this spreadsheet include a Qual Browser LC/UV peak list, LC/UV chromatogram time vs intensity (XY) coordinate list, the mRNA construct sequence, and the BioPharma Finder-exported component table. The use of these spreadsheets and details of the entire method are provided in Oligonucleotide Mapping and Data Analysis Protocol.pdf