With USP’s recent guidelines emphasizing quality standards of mRNA analytical methods for characterization and release testing, Eurofins BPT delivers solutions
Jeremy Johnston, Scientific Advisor, Drug Product Operations, Eurofins BioPharma Product Testing; Joe Page, President, Eurofins Advantar Laboratories
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 emphasize 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 characterization 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 organizations 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 posttranscriptional 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 synthesized probes. Both the efficiency of 5’ capping and poly A tail length can be characterized 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 BPT 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 utilized 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 ionizable cationic lipid, a helper phospholipid, cholesterol, and a PEGylated lipid. These lipids encapsulate the mRNA payload and protect the nucleic acid core from degradation. Characterization 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.
EBPT offers full mass spectrometry, cell-based potency, and flow cytometry GMP capabilities from 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, 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.