Introduction

 The recent development and approval of the two COVID mRNA-based vaccines has brought RNA-based therapeutics to the forefront of the biopharma industry. As such, development of analytical methods for monitoring the CQAs of RNA-based therapeutics has become a high priority for ensuring proper control of manufacturing processes. CQAs for nucleic acid therapeutics can include sequence confirmation, 5’ capping efficiency and structure (mRNA), 3’ Poly(A) tail heterogeneity (mRNA) analysis, localization of post transcriptional modifications (PTMs), and purity assessment of the active RNA product. This study describes two UPLC-MS assays coupled with customized software for streamlining the CQA characterization of RNA molecules.

 Methods

Samples used for the analytical method development included: (1) 100-mer sgRNA, used to optimize the RNase T1 digestion and software testing; (2) synthetic 25-mer nucleotide with Cap-1 structure and intermediate byproducts, used to establish Cap-1 identification and quantitation; (3) 120-mer long synthetic polyadenosine mRNA oligonucleotide, used to optimize the LC-MS conditions for Poly(A) Tail analysis; (4) Firefly luciferase mRNA (Fluc-beta mRNA from AmpTec, 1971 nt), used to illustrate the sequence mapping of mRNA. All data were collected using a benchtop UPLC-TOF MS system. Oligonucleotide mixtures were separated by IP-RP chromatography on a 2.1 x 150 mm UPLC column (1.7 µm C18 particles) containing a modified internal surface [1]. Mobile phase A contained 8 mM DIPEA (diisopropylethylamine) and 40 mM HFIP (hexafluoroisopropanol), while mobile phase B had 4 mM DIPEA, 4 mM HFIP in 75% ethanol. Automated data processing was performed using the INTACT Mass software.

Preliminary Data

A long (120 minutes) UPLC gradient was used to perform the sequence mapping and 5’-capping assays, while a short (10 min) gradient was used for the Poly(A) Tail LC-MS assay. A longer gradient is required to resolve the complex RNase T1 digestion mixture. The short gradient is beneficial for focusing the heterogenous Poly(A) Tail components to produce a high-quality ESI-MS spectrum.

Three separate workflows for each of the CQAs for RNA molecules were demonstrated: (1) 5’-Cap structure and capping efficiency were established for a 25-mer synthetic oligo construct; (2) 3’-Poly(A) Tail heterogeneity was investigated for Fluc mRNA (AmpTec firefly luciferase mRNA, 126.5-mer average Poly(A) measurement); (3) sequence mapping coverage for a 100-mer sgRNA (91% coverage) and for the 1971 nt Fluc mRNA (75% coverage). Further sequence elucidation in the case of ambiguous RNase T1 digestion fragments, can be done using the CONFIRM Sequence software, provided that data-independent fragmentation spectra (MS^E) are collected for the Fluc mRNA digest.

Both INTACT Mass and CONFIRM Sequence software applications are fully integrated with the waters_connect software which operates the BioAccord UPLC-MS system.

References:

1. Anal Chem, 2021, 93, p5773.

Take-home messages:

 1) The INTACT Mass software supports three independent workflows for three different CQAs commonly used for characterization of RNA molecules

2) The three CQAs workflows demonstrated here are for 5’-Cap and 3’-Poly(A) Tail analysis, as well as for RNA sequence confirmation