Current Analytical Strategies for mRNA-Based Therapeutics

Abstract

Recent advancements in mRNA technology, utilized in vaccines, immunotherapies, protein replacement therapies, and genome editing, have emerged as promising and increasingly viable treatments. The rapid, potent, and transient properties of mRNA-encoded proteins make them attractive tools for the effective treatment of a variety of conditions, ranging from infectious diseases to cancer and single-gene disorders. The capability for rapid and large-scale production of mRNA therapeutics fueled the global response to the COVID-19 pandemic. For effective clinical implementation, it is crucial to deeply characterize and control important mRNA attributes such as purity/integrity, identity, structural quality features, and functionality. This implies the use of powerful and advanced analytical techniques for quality control and characterization of mRNA. Improvements in analytical techniques such as electrophoresis, chromatography, mass spectrometry, sequencing, and functionality assessments have significantly enhanced the quality and detail of information available for product and process characterization, as well as for routine stability and release testing. Here, we review the latest advancements in analytical techniques for the characterization of mRNA-based therapeutics, typically employed by the biopharmaceutical industry for eventual market release.

Keywords: chromatography; electrophoresis; functionality; mass spectrometry; messenger RNA; quality attributes; sequencing.

Figure 1

Conventional analytical methods for characterizing the quality attributes of IVT mRNA.

Figure 2

CGE-UV electropherograms of isolated poly(A) tails obtained from different mRNA constructs with poly(A)120 spikes. The most abundant peak is labeled with asterisk (*). Modified with permission from [29].

Figure 3

AEX separations obtained for the analysis of EPO (859 nts) and Cas9 mRNAs (4500 nts) using a classical NaCl gradient (left panel) versus those with an ion-pairing TMAC gradient (IPAX, right panel). Modified with permission from [46].

Figure 4

(A) SEC-UV profiles obtained for EGFP mRNA using an ultrawide pore column. The profiles were obtained before heating the samples (blue traces) and after heating (red traces). (B) Mass photometry profiles obtained for eGFP, Fluc, and β-Gal mRNAs. The number of nucleotides and the percentages of monomer, dimer, and trimer forms are indicated. Figures have been adapted with permission from [30,47].

Figure 5

Proof of concept for the application of the SC retention mode for the baseline separation of the main 4.0 knt mRNA product (see blue ssRNA ladder) and 4.0 kbp dsRNA impurities (see black dsDNA ladder), one important byproduct of IVT processes to be quantified. Adapted with permission from [63].

Figure 6

IP-RP chromatograms showing the response of LNP loaded with FLuc mRNA and sgRNA or formulated without any payload (empty—black trace). Overlay of IP-RP chromatograms for a fresh LNP sample (black) and a LNP sample stored at room temperature for 1 month (red) showing separation of lipid-adducted mRNA and loss of intact mRNA peak. Modified with permission from [68].

Figure 7

Schematic representation of different approaches for the MS analysis of mRNA variants and the attributes that can be resolved.

Figure 8

Oligonucleotide mapping via MS to enable comprehensive primary structure characterization of an mRNA vaccine against SARS-CoV-2. (A) IP-RP LC-MS RNase T1 oligonucleotide map of BNT162b2 mRNA. “*” denotes a sequence-repeat oligonucleotide, where the single peak assignment represents all identical oligonucleotides in the sequence. Each color distinguishes the number of nucleotides per digestion product: blue, 4, 10, and 16; green, 5 and 11; gold, 6 and 12; red, 7 and 13; purple, 8 and 14; black, 9 and 15: magenta, >16. (B) (1) Extracted ion chromatograms of uncapped (5′ppp-AG) and capped (5′ cap-AG) versions of the 5′ terminal oligonucleotide for BNT162b2 variant constructs Original, Delta, and Omicron. (2, 3) Deconvoluted mass spectra of uncapped (5′ppp-AG) and capped (5′ cap-AG) versions, respectively, of the 5′ terminal oligonucleotide for BNT162b2 variant constructs Original, Delta, and Omicron. (C) UV chromatograms of the poly(A)tail region for BNT162b2 variant constructs Original, Delta, and Omicron. The blue box highlights the chromatographic distribution of L70 poly(A) oligonucleotides (unseparated), which are further described in panels (5) and (6). (5) Deconvoluted mass spectra of the L70 poly(A) oligonucleotide distribution. (6) Extracted deconvolved chromatograms of BNT162b2 Original L70 poly(A) oligonucleotide distribution highlighted in panel (5). Modified with permission from [79].

Figure 9

Representative intact mass analysis of mRNA variants by native MS. Superposition of the deconvoluted spectra of mRNA (A) with and (B) without poly(A) synthesized by five different T7 polymerases. WT, wild-type T7; E1–E4, four engineered T7 polymerases. n + 1/n + 2 transcripts are represented in blue, and the presence of 3′-loopback dsRNA is represented in orange. Adapted with permission from [51].

Figure 10

Overview of Illumina-based methods for sequencing RNA 3′ ends. Solid red blocks represent barcode sequences, which may include or exclude an oligo(dT) sequence. Purple regions indicate 5′ barcodes, while the 3/4 purple semicircles represent biotin modifications attached to the 3′ adapter. Poly(A) tail measurement techniques have evolved significantly since the introduction of PAL-Seq and TAIL-Seq (left panel). Despite advancements, challenges remain, particularly in balancing read length, adapter compatibility, and computational processing. Newer methods, including poly(A)-Seq and TED-Seq (right panels), improve upon earlier techniques but require careful consideration of sequencing depth, sample recovery, and bioinformatic tools. Each method presents trade-offs in resolution, efficiency, and applicability, underscoring the need for continued development in mRNA tail analysis.

Figure 11

Overview of long-read RNA sequencing methods for capturing full-length RNA, including poly(A) sequences. Panels (A,B) depict methods utilizing the PacBio sequencer with green SMRTbell adapters. Panels (C,D) illustrate methods employing the Oxford Nanopore sequencer. In panel (A), the orange region represents the unique molecular identifier (UMI).