mRNA Purity and Integrity Assessment
Determining mRNA purity and integrity is essential for mRNA-based drug R&D, enabling the improvement of cGMP compliance, cost reduction of mRNA-based drugs, and the availability of these drugs. Creative Biogene can help our customers speed up their mRNA-based drugs' production capacity and market availability.
The necessity of testing mRNA purity and integrity
To date, based on phage RNA polymerase, a large quantity of in vitro-transcribed (IVT) mRNA can be yielded. The in vitro synthesis process of mRNA molecules is relatively complex. After in vitro transcription, the reaction solution contains mRNAs and multiple other contaminants, including remaining reagents and side products. For mRNA biopharmaceuticals, the purification of IVT mRNA as well as the purity testing are crucial steps- both directly affecting the potency of mRNA-based drugs. Besides, RNAs, including mRNAs, are sensitive and unstable molecules, and can be easily fragmented by the external environment, such as the existence of ubiquitous nucleases, heat and UV. Therefore, assessment of the purity and integrity of mRNA is very essential to subsequent mRNA-based drugs' development to ensure the safety profile and availability of the products.
Methods for testing RNA purity and integrity
The most straightforward and convenient method to assess RNA purity is ultraviolet (UV) spectroscopy. The method is based on measuring the optical density (OD) and calculating the ratio of the absorbance contributed by the nucleic acid and the contaminants. However, the method lacks sensitivity and specificity, and cannot distinguish the multiple forms of nucleic acid.
Due to the ubiquitous nucleases, the integrity of RNA can easily be compromised, especially in the in vitro synthesis process. Therefore, the assessment of RNA integrity is necessary. A few methods have been proposed to measure RNA integrity so far. Among them, agarose gel electrophoresis with ethidium bromide staining has been the most popular method, allowing one to measure RNA size and integrity. But the disadvantage of the method is less sensitive, labor-intensive and time-consuming. Moreover, RNA integrity can be determined by reverse transcriptase qPCR (RT-qPCR), which is fast, high-throughput, and extremely sensitive, but is costly. Over the last decade, microfluidic capillary electrophoresis has emerged as the preferred technology for RNA integrity testing. And based on the method, several instruments have become the standard for RNA quality control.
Our strategy for mRNA purity and integrity assessment
To provide the reliable and reproducible assessment of mRNA, we employ the current state-of-the-art RNA quality assessment method, combining UV/Vis spectrophotometry and microfluidic capillary electrophoresis. We guarantee our data's accuracy and validity with our advanced testing platform. In addition, we also provide high-pressure liquid chromatography (HPLC) and fast protein liquid chromatography (FPLC) to quantify or characterize the mRNA. A major contaminant in IVT mRNA synthetic reaction system is double-stranded RNA (dsRNA), characterized by strong immunogenicity. The methods mentioned above can also be employed to remove dsRNA from mRNA. A dot blot can then be used to detect the purified mRNA.
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- Poveda, C., et al. (2019). "Establishing preferred product characterization for the evaluation of RNA vaccine antigens." Vaccines, 7(4), 131.