mRNA Mapping with IP-RPLC-MS on a Low-Adsorption Flow Path

Importance of the topic

Messenger RNA (mRNA) therapeutics have revolutionized vaccine and biopharmaceutical development, but their inherent fragility and complexity require precise characterization of sequence, 5’ cap structure, 3’ poly(A) tail length, and chemical modifications. Ion-pair reversed-phase liquid chromatography coupled to mass spectrometry (IP-RPLC-MS) offers high sensitivity for oligonucleotide mapping but is hampered by adsorption on stainless steel surfaces, leading to reduced recovery and peak tailing.

Objectives and study overview

This work demonstrates a robust IP-RPLC-MS workflow for comprehensive mRNA mapping using a low-adsorption flow path. Key goals included:

• Assessing metal adsorption effects on oligonucleotide analysis.
• Performing parallel RNase T1 and RNase 4 digestions of in vitro transcribed Firefly luciferase (Fluc) mRNA.
• Evaluating sequence coverage, cap efficiency, and poly(A) tail length distribution in a single platform.

Methodology and instrumentation

• Sample preparation: Fluc mRNA was digested by RNase T1 (cleavage 3’ of G) or RNase 4 (cleavage at UG/UA) under optimized buffer and temperature conditions, then concentrated to 3 µg/µL.
• Chromatography: Agilent 1290 Infinity II Bio LC system with deactivated stainless steel C18 column; mobile phases contained triethylamine (TEA) and hexafluoroisopropanol (HFIP); gradients at 0.2–0.35 mL/min, column at 65 °C.
• Mass spectrometry: Agilent 6530 Q-TOF in negative electrospray mode; full MS (m/z 500–3200) and Auto MS/MS for sequence confirmation.
• Data processing: Agilent OpenLab CDS and MassHunter for maximum entropy deconvolution; R scripts matched observed monoisotopic/average masses to in silico RNase digests; MS/MS fragmentation (a, c, w, y ions) resolved isomeric subsequences.

Main results and discussion

• Conventional stainless steel columns exhibited progressive signal increase across multiple RNA standard injections, indicating surface adsorption. A deactivated column stabilized recovery within a single injection and limited non-eluted oligonucleotides to ~4%.
• RNase T1 digestion yielded predominantly short fragments (2–5 nt), achieving 49.4% coverage of the Fluc ORF. RNase 4 produced longer oligonucleotides, achieving 88.6% coverage.
• Combining RNase T1 and RNase 4 datasets improved single-site coverage to 94.7% and overall coverage (including multi-site fragments) to 96.9%.
• Simultaneous analysis of the 3’ poly(A) tail revealed a narrow, symmetric distribution (118–133 A units; mode 124). MS/MS-based extracted ion chromatograms of capped (m7G–2’-O-MeA G…) versus noncapped termini yielded a capping efficiency of 99.2%.

Benefits and practical applications

This integrated workflow offers high sequence coverage, reliable cap and tail analysis, and excellent chromatographic performance. It supports quality control of mRNA therapeutics, accelerates process development, and meets stringent regulatory requirements.

Future trends and opportunities

• Adoption of additional RNases or partial digestion protocols to fill sequence gaps.
• Automation and high-throughput LC-MS platforms for routine mRNA QC.
• AI-driven data analysis for rapid identification of modifications and variants.
• Extension of low-adsorption methods to other oligonucleotide modalities (siRNA, antisense, saRNA).

Conclusion

The use of a deactivated stainless steel column and low-adsorption flow path on the Agilent 1290 Infinity II Bio LC system enables robust IP-RPLC-MS mapping of mRNA digests. Parallel RNase T1 and RNase 4 digestions maximize sequence coverage, while simultaneous cap and poly(A) tail analysis ensures comprehensive primary structure characterization for mRNA-based therapeutics.

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