Analyze This

February 26, 2025 by Nate Spangler, Ph.D.

Messenger RNA, the revolution in targeted therapies

Messenger RNA (mRNA) technology has rapidly evolved from a laboratory curiosity to a leading force in targeted drug therapies. The remarkable success of mRNA-based COVID-19 vaccines illuminated the immense potential of mRNA-based medicines, ushering in a new era of medical innovation.

This burgeoning field encompasses a diverse array of approaches, including formulations with multiple mRNA active pharmaceutical ingredients (APIs) or combinations of different RNA types, such as mRNA with guide RNAs (gRNA).

The Growing Importance of mRNA Quality
As mRNA-based therapies and vaccines gain prominence, there is a growing demand for increasingly sensitive analytical methods. The efficacy and safety of these medicines hinge on the quality of the synthesized mRNA, with the 5'-end cap modification being a critical feature. In the in vitro transcription (IVT) synthesis of mRNA, cap addition is commonly achieved through two methods:

1. Post-transcriptional Capping
   A cap structure is added to the 5' end of the RNA post hoc using capping enzymes. While highly efficient, this method can generate cap-related byproducts due to the inherent complexity of the enzymatic process.
2. Co-transcriptional Capping
   A di- or tri-nucleotide cap analog is incorporated at the 5' end of the mRNA during transcription. However, the efficiency of this method can vary depending on the specific analog and RNA polymerase used, often resulting in a mixture of capped and uncapped RNA.

Traditional techniques like High-Performance Liquid Chromatography (HPLC) and capillary electrophoresis can be used to distinguish between capped and uncapped mRNA. However, these methods lack the necessary resolution to identify specific cap intermediates and byproducts, which is crucial for a comprehensive understanding of mRNA quality.

LC-MS: A Powerful Tool for mRNA Characterization
Liquid chromatography-mass spectrometry (LC-MS) offers a robust solution for the detailed characterization of IVT-synthesized mRNA. Established as a key technique for the analysis of traditional biologics, LC-MS provides identification and quantification of post-translational modifications.

For mRNA, this technique enables the quantitative assessment of capping efficiency and the identification of potential unwanted modifications, as well as information about other structural features that may compromise safety.

Aldevron, a pioneer in nucleic acid production, is at the forefront of integrating LC-MS into analytical workflows. The company utilizes LC-MS as an orthogonal supplement to traditional quality control (QC) release testing, providing a deeper level of product characterization. This includes the detailed analysis of the 5' end structure, as well as the 3' poly(A) tail, another critical quality attribute of mRNA.

Due to the large size and complex structure of mRNA molecules, direct analysis of the 5' cap and 3' tail is challenging. To overcome this limitation, Aldevron has developed enzymatic cleavage strategies to generate smaller, analyzable fragments. These fragments are then quantified and characterized using Ion-Pair Reverse-Phase (IP-RP), HPLC and RP-LC-MS techniques, providing robust measurements and detailed insights into the 5' and 3' end structures.

The Future of mRNA Quality Control
The integration of MS into QC processes marks a significant advancement in the field of mRNA-based medicines. By combining the high-resolution, highly detailed characterization capabilities of MS with traditional QC methods, Aldevron is setting new standards for the evaluation of nucleic acid therapeutics. This approach not only enhances the quality and safety of these products but also ensures compliance with stringent regulatory standards, ultimately benefiting patient outcomes.

As the field of nucleic acid therapeutics continues to rapidly evolve, so must the analytical techniques used to ensure their quality, safety, and efficacy. Mass spectrometry, with its unparalleled sensitivity and specificity, is poised to play a pivotal role in shaping the future of mRNA-based medicines.

By embracing innovative analytical approaches like LC-MS, companies like Aldevron are paving the way for a new era of precision medicine. So, as we continue to explore the full potential of mRNA, the advancements in quality control and analytical methods will play a crucial role in delivering the next generation of life-saving medicines to patients worldwide.

View the poster, Building Mass Spectrometry Based Analytics in Support of Oligonucleotide Drug Substance and Drug Product Characterization

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