With USP’s guidelines emphasising quality standards of mRNA analytical methods for characterisation and release testing, Eurofins BPT delivers solutions

Jeremy Johnston, Scientific Advisor, Drug Product Operations, Eurofins BioPharma Product Testing, jeremy.johnston@bpt.eurofinsus.com; Joe Page, President, Eurofins Advantar Laboratories, joe.page@bpt.
eurofinsus.com

The global mRNA therapeutics market is expected to expand tremendously in the next 10 years due to these modalities being more effective, faster to design and produce, as well as more flexible compared to traditional approaches. In order to emphasise the quality and consistency of analytical methods used for
mRNA-based vaccines and therapies, the United States Pharmacopeia (USP) has recently published guidelines on analytical methods covering the characterisation and release testing for mRNA drug substances and mRNA drug products. In addition, the European Pharmacopoeia (Ph. Eur.) Commission released three guidelines in 2023 with the goal of setting common quality standards across Europe related to the production and control of mRNA-LNP medicinal products and their components. While there are several common methods required to ensure safety, quality, and purity let’s focus on a few of the more challenging assays that pharmaceutical organisations often lack expertise and instrumentation availability.

The 5’ cap structure and 3’ poly-adenosine tail are crucial modifications that are introduced either co-transcriptionally or in a post-transcriptional reaction on the mRNA construct. The percentage of capped
and polyadenylated mRNA, as well as the poly(A) tail length and distribution, will have a direct
impact on the translational efficiency. Assessing these structural features requires complex methodology and instrumentation and would typically involve digesting the mRNA with enzymes that allow isolation
of the relevant 3’ or 5’ end and analysis, using oligo specific synthesised probes. Both the efficiency of 5’ capping and poly A tail length can be characterised by LC/MS/MS with a high-resolution mass spectrometer (Q-TOF or Orbitrap) with IP-RP-HPLC.

An appropriate potency assay is a key CQA for any mRNA product. Eurofins BioPharma Product Testing has experts in the development and validation of potency assays for both mRNA therapeutics and vaccines. These methods are usually based on the transfection of appropriate cell lines with the mRNA drug substance or product, followed by monitoring of an induced effect, such as protein expression or other quantitative effect in the cell. While the endpoint readout may be an ELISA, often Flow Cytometry can be utilised for specific mRNA products with complex mechanisms of action.

LNPs (Lipid nanoparticles) are currently the foremost non-viral delivery vector employed to transport mRNA into cells. LNPs are typically composed of four components: an ionisable cationic lipid, a helper phospholipid, cholesterol, and a PEGylated lipid. These lipids encapsulate the mRNA payload and protect the nucleic acid core from degradation. Characterisation and monitoring of the LNP physicochemical properties such as particle size, lipid content and composition, and percent mRNA encapsulation are required to
support regulatory submissions. These CQAs can be monitored by DLS, zeta potential, HPLC-CAD, and RiboGreen assays.

Eurofins BioPharma Product Testing offers full mass spectrometry, cell-based potency, and flow cytometry GMP capabilities from U.S. coast to coast in Lancaster, PA; Columbia, MO; as well as San Diego, CA, locations.

In this quickly developing area, we are constantly re-evaluating the best approach and improving methodology as well as introducing new technology with the latest being the introduction in early 2025 of GMP next-generation sequencing (NGS) services in Lancaster to confirm sequence as well and to identify any sequence variants. For more information, visit: www.eurofinsus.com/bpt