Guest Column | May 1, 2024

Forum Notebook: Quick Takes From USP's mRNA Virtual Summit

By Sarita K. Acharya, United States Pharmacopeia, and Khaled Yamout, consultant


Nucleic acids, particularly mRNA, have found applications in various therapeutic areas such as vaccines, infectious diseases, and cancer treatments, among others. As the adoption of this therapeutic modality has accelerated, there is a need to apply and expand existing as well as novel analytical methodologies to comprehensively characterize mRNA both as drug substance (DS) and within drug products (DP). This expansion is crucial to support drug development, meet regulatory requirements, and identify critical quality attributes (CQAs).

In February, the U.S. Pharmacopeia (USP) held an open forum called “Collaborating to Pave the Way for mRNA,” a virtual event to convene stakeholders from industry and academia to exchange perspectives on the advancement of current and emerging analytical methodologies for characterization and release testing. The discussions focused on how these methodologies can be used for more complete analytical characterization of mRNA and their significance in monitoring identified CQAs to help ensure products are safe, effective, and of high quality before they are introduced to the market. By engaging in this dialogue, stakeholders aimed to foster a deeper understanding of the evolving landscape of mRNA-based therapeutics and the analytical tools necessary to support their development and regulatory approval processes.

Consensus: Complementary Analytical Methods Needed

The presenters offered a diverse range of methodologies and concepts aimed at addressing the ongoing need for improvements in characterization and analysis of mRNA DS and DP. Following an introductory overview of analytical methodologies and mRNA CQAs, the speakers delved into their specific topics. One focal point was the importance of employing orthogonal methods for various purposes such as identification, purity assessment, particle size measurement, and assessment of poly(A) size and distribution.

Presenters emphasized the importance of these complementary approaches in ensuring robust characterization of mRNA. Moreover, speakers discussed the adaptation of established methods from other therapeutic modalities. For instance, Fourier-transform infrared spectroscopy (FT-IR) was highlighted as a valuable tool for characterizing mRNA formulations and assessing compatibility with excipients. Additionally, it was discussed how FT-IR can be utilized to study phosphodiester bond cleavage as an indicator of mRNA degradation.

Other innovative techniques presented included the use of microarray platforms for identifying multivalent drug products, nano-pore sequencing for characterizing plasmid and mRNA sequences, and field flow fractionation coupled with multi-angle light scattering (FFF-MALS) for comprehensive characterization of both mRNA DS and DP. Novel approaches to testing potency and the use of cryo-transmission electron microscopy (Cryo-TEM) to study the morphology and payload of mRNA-lipid nanoparticle (LNP) drug products were discussed. The presentations also covered several methodologies that have been deemed fit for use and qualified to support IND submissions. These methods represent established tools with demonstrated reliability and applicability in regulatory contexts.

The Absence Of Reference Standards Remains An Analytical Hurdle

In recent years, significant progress has been made in developing analytical methods to characterize and monitor CQAs for mRNA testing. However, despite these advancements, there remain several gaps and challenges that need to be addressed within this field. One of the primary challenges is the availability of assay standards for method development and system suitability. Having standardized reference materials is crucial for ensuring the accuracy and reliability of analytical methods used in mRNA characterization and release testing.

Another challenge relates to development and selection of assay standards that are representative of the mRNA being tested. It is essential that the standards used accurately reflect the properties and characteristics of the specific mRNA molecules under examination to ensure the validity of the analytical results such as the length of mRNA, type of cap structure, and quality of raw materials, such as the modified NTPs and enzymes. Additionally, there is a need to build consensus around reporting poly(A) results as a release method.

Determining the appropriate approach for assessing polyadenylation levels and reporting these results in a consistent and standardized manner is vital for regulatory compliance and comparability across different studies. Furthermore, there is a recognized necessity for more robust potency and double-stranded RNA (dsRNA) detection methods. Potency assays are critical for determining the biological activity of mRNA-based therapeutics, while dsRNA detection methods are essential for assessing potential impurities and ensuring product safety.

Other Highlights From The Forum

The forum facilitated in-depth discussions on analytical methodologies, advancements, and emerging trends. The presentations featured substantial scientific content, fostering a thought leadership environment that encouraged creative thinking and exploration of innovative solutions. Participants engaged in discussions around optimizing methods, defining CQAs, and identifying practical applications for mRNA DS and DP characterization. Overall, the forum provided a platform for collaboration and knowledge exchange, driving progress in the field of mRNA analytics.

Next Steps

During the discussion, participants explored potential solutions to address the analytical challenges related to analysis of mRNA-based therapies. They prioritized ideas for establishing standards based on their perceived impact/need and feasibility.  The proposed standards included:

  • Poly(A) standards of different length
    Recognizing the importance of poly(A) tail length in mRNA constructs, it was suggested to establish standards for poly(A) of varying lengths to provide reference materials for accurately characterizing mRNA molecules with different poly(A) tail lengths.
  • Well-characterized guide RNA used in the CRISPR/Cas system for gene editing
    Participants emphasized the necessity for well-characterized guide RNA that falls under mRNA therapeutics, to assess the suitability of analytical methods for evaluating mRNA purity, impurities, structural integrity, and other critical parameters. These standardized guide RNAs would serve as benchmarks for method validation and quality control.
  • DP/LNP related standards
    Addressing the complexities associated with delivery systems like LNPs and mRNA DP, it was proposed that DP/LNP-related standards will be useful to establish criteria and benchmarks for assessing the quality, safety, and efficacy of mRNA DP and the delivery systems. In addition, these standards can be used to evaluate encapsulation efficiency and size distribution.

Additionally, participants expressed a desire for more events on mRNA-based therapies, focusing on areas such as potency assessment. This reflects the need for continued education and collaboration within the scientific community to address the evolving challenges in mRNA-based therapy development and evaluation.

USP asks that interested individuals please contact Sarita K. Acharya at if you have questions or clarifications for the above summary or additional thoughts on the topics that were discussed.

About The Authors:

Sarita K. Acharya, M.S., is a principal scientist in the global biologics department at USP. Prior to joining USP, she worked for several biotech companies, academia, contract research organizations, and pharmaceutical companies, including GlaxoSmithKline (GSK) and Janssen Pharmaceuticals. She has held several lab-based roles in method development and validation, CMC, and quality control, and she has worked on monoclonal antibodies and protein- and mRNA-based vaccines. At USP, Acharya identifies and advances development of standards to support vaccine manufacturing, analytical testing of monoclonal antibodies, oligonucleotides, genomics, and personalized medicine.

Khaled Yamout is an independent consultant and industry expert in analytical sciences, quality, and manufacturing. Previously, he held a position as a senior director at TriLink Biotechnologies where he oversaw the Analytical Sciences Center of Excellence and all analytical aspects of method development and validation to product release and stability to support regulatory filings for both small and large molecules. Prior to TriLink, Khaled held various positions in quality control, research and development, and manufacturing at large and small companies.