Characterisation and analysis of mRNA critical quality attributes using liquid chromatography based methods

Figures

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  Fig. 1. Schematic illustration of the structure of synthetic mRNA. The key features of mRNA therapeutics/vaccines are shown with their functional roles highlighted. The 5′ cap reduces RNA degradation and facilitates ribosome initiation during translation. The 5′ UTR influences translation efficiency and stability. Coding sequence is essential for encoding the protein of the gene of interest. The coding sequence can be chemically modified using modified nucleoside triphosphates (NTPs) in the enzymatic reaction, which can reduce immunogenicity and enhance stability. The 3′ UTR regulates translation efficiency. Poly(A) tail enhances mRNA stability and also facilitates ribosomal initiation and translation efficiency. Created in BioRender. Dickman, M. (2024) https://BioRender.com/c49v683 .

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  Fig. 2. Synthesis of mRNA using in vitro transcription. Synthetic mRNA is synthesized from NTPs using T7 RNA polymerase incubated with a linearised plasmid DNA template. Created in BioRender. Dickman, M. (2024) https://BioRender.com/f29f724.

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  Fig. 3. IP-RP HPLC analysis of mRNA. A) IP-RP HPLC chromatogram of a typical mRNA. The chromatogram show the presence of peaks before and after the main peak. The corresponding peaks before the main full length mRNA product are typically generated from mRNA species with different poly(A) tail lengths and partial hydrolysis of the full length mRNA resulting in reduced hydrophobicity and earlier retention times. The corresponding peaks after the main peak are typically associated with dsRNA by-products resulting from 3′ loop back formation. B) IP-RP HPLC stability indicating analysis. eGFP mRNA was heat treated prior to IP-RP HPLC analysis. The chromatogram in black shows an early time point and blue a later time point. The increase in abundance of peaks prior to the main full-length mRNA peak demonstrate the increased abundance of shorter length hydrolysis products resulting in lower mRNA integrity. IP-RP HPLC analysis was performed using mobile phase A) 0.1 M TEAA; mobile phase B) 0.1 M TEAA, 25% ACN. Gradient elution starting at 35% B to 55% B over 15 mins on a DNAPac RP column (2.1 × 100 mm) with UV detection at 260 nm.

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  Fig. 4. mRNA cap structures. Cap 0 is a N7-methyl guanosine connected to the 5′ nucleotide through a 5′ to 5′ triphosphate linkage. The presence of 2′-O-methyl groups at either R1 only or both R1 and R2 determine whether a cap structure is Cap 1 or Cap 2 respectively. Created in BioRender. Dickman, M. (2025) https://BioRender.com/a44e767.

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  Fig. 5. RNase T1 based LC-MS workflow for parallel analysis of 5′ cap and 3′ poly(A) tail. There are four key steps; 1) Complete digestion of mRNA with RNase T1 yields 5′ capped and uncapped oligonucleotides in addition to fragments containing the poly(A) tail. 2) IP-RP HPLC separations are interfaced with LC-MS and LC-MS/MS. 3) 5′ capping efficiency is determined based on the abundance of the 5′ capped and uncapped fragments 4) 3′ poly(A) tail analysis is performed using intact mass deconvolution. An example is shown from a typical poly(A) tail, where each of the individual masses identified correspond to an additional A residue. Created in BioRender. Dickman, M. (2024) https://BioRender.com/f93c835.

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  Fig. 6. Schematic representation of direct mRNA sequencing using LC MS/MS workflows. RNase(s) are used to digest the mRNA into smaller oligoribonucleotide fragments. These fragments are separated using IP-RP HPLC interfaced with high resolution tandem mass spectrometry (LC-MS/MS). Data analysis and visualisation tools are then employed to match experimentally identified oligoribonucleotides with the theoretical mRNA sequence. Created in BioRender. Dickman, M. (2024) https://BioRender.com/c73t543.