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Characterization is key: how to evaluate mRNA therapeutic safety and efficacy

The rapid development of mRNA-based therapeutics such as vaccines, monoclonal antibodies, and protein replacement therapies has been an exciting advancement in the prevention and treatment of diseases. Although we have already seen great successes with the SARS-CoV-2 vaccines and clinical trials for other therapies, several challenges remain for the development of an mRNA-based medicine. Included in these challenges are effective and thorough product characterization to ensure that the expected therapeutic is being produced at high purity while remaining effective and safe. In this eBlog we review some of the principal areas of mRNA drug substance and product characterization.

Identity: is the drug substance the expected sequence?

One of the strengths of mRNA-based therapeutics is that they use nucleotide sequences and cellular machinery to produce the desired protein within the patient, enabling rapid changes to be implemented such as with infectious disease where variants can reduce the efficacy of the original design. As such, the correct nucleotide sequence must be manufactured each time to ensure the correct therapeutic protein or antigen is produced1.

To confirm the sequence of the mRNA, several approaches have been developed ranging from gel-based methods to next-generation sequencing. For the SARS-CoV-2 vaccines developed by Moderna and Pfizer sequence identity was confirmed with Sanger sequencing, which uses fluorescent dyes and chain termination to determine the sequence of an mRNA2. Although Sanger sequencing is reliable, the throughput is low and interest in next-generation sequencing approaches has increased as an alternative. For example, the USP’s draft guidelines for assessing mRNA vaccine quality describe assessing identity using a mRNA-Seq approach1.  Other alternatives include RT-PCR (limited to examining short stretches of the mRNA rather than the whole sequence) and nanopore sequencing (which can have a lower throughput compared to next-generation sequencing)1,2,3.

Integrity: is the drug substance intact?

To produce the correct protein, the mRNA needs to be full-length but during the manufacturing process truncated versions of the mRNA can be produced. Typically, integrity is examined using agarose gel or capillary electrophoresis2,5. Both approaches examine the distribution of RNA fragments to determine if unexpected mRNAs are present. A limitation of these approaches is that they are relatively low throughput and can only provide information on the size of the fragment and a separate paired assay is needed to look at identity. As discussed at this year’s mRNA Analytical Development Summit6, there is a need for next-generation sequencing approaches to support higher throughput and multimodal studies of the drug substance and product to ensure both integrity and identity.

Also included under integrity, is the presence of the 5’ cap and 3’ tail on the mRNA product (although depending on the standard-setting agency this can also be considered a purity or quality metric instead)1,3,5. The cap and tail are required for recognition by the cellular machinery for translation and maintaining stability. The cap can be added during transcription using a cap analog or added enzymatically after, while the tail can be directly encoded in the DNA template (although this limits the maximum length) or added enzymatically (leading to variable tail lengths)5.  Current analytical methods typically use HPLC and mass spectrometry to determine the percentage of cap and tail present1. These approaches can be time-consuming and require extensive expertise.

Purity: are process-related impurities retained in the drug product?

To be effective and reduce side effects, the manufacturing process needs to limit impurities as much as possible. For example, dsRNA generated by IVT run-off during mRNA synthesis can activate the innate immune system leading to decreased protein production and inflammation. dsRNA is typically measured through a dot blot or an ELISA using an antibody that recognizes dsRNA such as J2 or K11. Two limitations of J2-based approaches are that they cannot detect shorter regions of dsRNA that can be immune-activating, and they only indicate the presence of dsRNA not the source making it a challenge to optimize the manufacturing process8.  As such, there is a need for a robust and reproducible method for quantifying dsRNA less than 40 nucleotides long with positional information to address these challenges.

In addition to measuring dsRNA, other impurities need to be detected and controlled including the presence of the DNA template and enzymes used during the manufacturing process3. DNA templates can be detected using qPCR2, although the use of primers means that unexpected sequences are not captured. Alternatively, next-generation sequencing approaches can be used to detect DNA templates in addition to detecting dsRNA. This makes next-generation sequencing a powerful tool for obtaining an unbiased, comprehensive view of impurities in mRNA-based therapeutics.

Eclipsebio provides integrated support for mRNA therapeutic characterization

In this eBlog we have reviewed some of the many recommended characterization assays to ensure an effective and safe therapeutic, in addition to these assays there are orthogonal analyses that can help to understand the behavior of an mRNA-based drug including measurements of RNA structure, LNP effectiveness, and host changes in response to drug product delivery. Examining all of these features together provides a deeper understanding of mRNA behavior, critical for achieving regulatory requirements and making sure that only the most efficacious and safe therapeutic proceeds to clinical trials.

At Eclipsebio, our focus is on deep understanding of RNA from the latest mRNA-based therapeutics to novel discoveries in RNA biology. Through our experience supporting our partners as they design and manufacture cutting edge mRNA therapeutics, we have developed a suite of next-generation sequencing based approaches for the comprehensive characterization of drug substances and products. Contact us today to discover our solutions for measuring identity, integrity, safety, and more.

References
  1. 1. US Pharmacopeia
  2. 2. Guimaraes et al.
  3. 3. Pharmeuropa
  4. 4. Tinari
  5. 5. World Health Organization
  6. 6. mRNA Analytical Development Summit
  7. 7. Verbeke et al.
  8. 8. Bonin et al.

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