Messenger-RNA (mRNA) drug therapeutics have recently emerged as promising and increasingly viable treatment modalities for cancer, cardiovascular diseases, rare genetic disorders and autoimmune diseases. Furthermore, the potential of using mRNA technology during mass vaccination campaigns was successfully demonstrated during the COVID-19 pandemic.

With an increased number of RNA therapies currently in various stages of clinical trial around the world, the requirements for reliable analytical methods to ensure the quality and safety of this new class of therapeutics are high.

Here, we present an overview of the latest advancements in analytical platforms for the characterisation and batch release testing of mRNA-based therapeutics, focusing on those typically available in the biopharmaceutical industry.

Following the success of mRNA vaccines in response to the COVID-19 pandemic, mRNA-based therapeutics have demonstrated significant potential in treating various diseases,
including cancer, cardiovascular diseases, infectious diseases, rare genetic disorders and autoimmune conditions. Currently, over 3000 mRNA therapeutics are in various stages of clinical trials worldwide, substantially increasing the demand for robust analytical platforms to monitor drug quality, safety and efficacy throughout development and for batch release testing.

Linear mRNA is the conventional form used in many mRNA-based therapies. It consists of: 1) a 5′ cap (typically a modified guanine nucleotide) necessary for ribosome binding and translational initiation, to increase stability and reduce immune-response; 2) a single, linear strand of nucleotides with a sugar-phosphate backbone encoding the desired protein; and
3) a 3′ poly(A) tail to improve mRNA stability.

Despite the incorporation of chemically modified nucleosides to enhance mRNA stability, a key challenge in producing reproducible, high-quality mRNA therapeutics is their inherent instability. This, coupled with the high heterogeneity, large size and negative charge of mRNA molecules, makes developing analytical workflows for mRNA characterisation and batch release testing to meet regulatory requirements a complex task.