Comparison of capillary electrophoresis-based methods for the analytical characterization of purity and stability of in vitro transcribed mRNA.

Messenger RNA (mRNA) is rapidly growing as a therapeutic modality for vaccination and the treatment of a wide range of diseases. As a result, there is an increased demand for mRNA-based analytical methods capable of assessing purity and stability, which are considered critical quality attributes (CQAs). In recent decades capillary electrophoresis (CE) has emerged alongside liquid chromatography (LC) as an important tool for the assessment of purity and stability of mRNA therapeutics. CE offers a variety of advantages over conventional LC or gel-based analytical methods, including reduced injection volume, increased resolution, and increased separation efficiency. In this study we compared CE-based analytical methods: the Agilent RNA 6000 Nano Kit, the Revvity RNA Reagent Kit, the Sciex RNA 9000 Purity and Integrity Kit, and the Agilent HS RNA Kit. These methods were evaluated on their vendor-recommended instruments: the Bioanalyzer, LabChip GXII, PA800 Plus, and Fragment Analyzer, respectively. We assessed the ability of these methods to measure mRNA integrity, purity, and stability. Furthermore, several parameters for each method were also assessed: selectivity, precision, resolution, analysis time, and ease of use. Based on our results, all four methods are suitable for use in the characterization of in vitro transcribed (IVT) mRNA, depending on the intended application. The Sciex RNA 9000 Purity and Integrity kit method achieved the highest selectivity and resolving power compared with the other methods, making it the most suitable for high-resolution, in-depth sample characterization. In comparison, the Agilent RNA 6000 Nano Kit, Revvity RNA Reagent Kit, and Agilent HS RNA Kit achieved lower selectivity and resolution, but their faster analysis times make them more suitable for high-throughput and screening applications.

Comprehensive chromatographic assessment of forced degraded in vitro transcribed mRNA.

Heightened interest in messenger RNA (mRNA) therapeutics has accelerated the need for analytical methodologies that facilitate the production of supplies for clinical trials. Forced degradation studies are routinely conducted to provide an understanding of potential weak spots in the molecule that are exploited by stresses encountered during bulk purification, production, shipment, and storage. Consequently, temperature fluctuations and excursions are often experienced during these unit operations and may accelerate mRNA degradation. Here, we present a concise panel of chromatography-based stability-indicating assays for evaluating thermally stressed in vitro transcribed (IVT) mRNA as part of a forced degradation study. We found that addition of EDTA to the mRNAs prior to heat exposure reduced the extent of degradation, suggesting that transcripts may be fragmenting via a divalent metal-ion mediated pathway. Trace divalent metal contamination that can accelerate RNA instability is likely carried over from upstream steps. We demonstrate the application of these methods to evaluate the critical quality attributes (CQAs) of mRNAs as well as to detect intrinsic process- and product-related impurities.

A single-nucleotide resolution capillary gel electrophoresis workflow for poly(A) tail characterization in the development of mRNA therapeutics and vaccines.

The 3' poly(A) tail is an important component of messenger RNA (mRNA). The length of the poly(A) tail has direct impact on the stability and translation efficiency of the mRNA molecule and is therefore considered to be a critical quality attribute (CQA) of mRNA-based therapeutics and vaccines. Various analytical methods have been developed to monitor this CQA. Methods like ion-pair reversed-phase liquid chromatography (IPRP-LC) can be used to quantify the percentage of mRNA with poly(A) tail but fail to provide further information on the actual length of poly(A). High-resolution methods such as liquid chromatography coupled with mass spectrometry (LC-MS) or next generation sequencing (NGS) can separate poly(A) tail length by one nucleotide (n/n + 1 resolution) but are complicated to implement for release testing of manufactured mRNA. In this study, a workflow utilizing capillary gel electrophoresis (CGE) for characterizing the poly(A) tail length of mRNA was developed. The CGE method demonstrated resolution comparable with the LC-MS method. With UV detection and the addition of poly(A) length markers, this method can provide poly(A) tail length information and can also provide quantitation of each poly(A) length, making it a suitable release method to monitor the CQA of poly(A) tail length.

Permeability for intestinal absorption: Caco-2 assay and related issues.

In vitro permeability assays remain a valuable tool of screening scientists for lead compound optimization. As a majority of discovery projects are focused on the development of orally bioavailable drugs, the need for predictability and correlation of in vitro permeability data to in vivo absorption results has never been greater. For more than a decade, the Caco-2 screening assay has remained a popular, in vitro system to test compounds for intestinal permeability and efflux liability. Despite advances in artificial membrane technology and in silico modeling systems, drug compounds still benefit from testing in cell-based epithelial monolayer assays for lead optimization and SAR. This review discusses the strengths and limitations of the Caco-2 permeability assay, and puts into context the power of combining multiple assays and approaches to improve predictability and rank-ordering for lead compound optimization. Technical information for dealing with some of the most pressing issues with in vitro permeability assays (i.e. low aqueous solubility and low post-assay recovery) is also discussed. Insights are offered to help researchers avoid common pitfalls in the interpretation of in vitro permeability data, which can often lead to the perception of misleading results for correlation to in vivo data. In addition, the advantages of addressing the issue of efflux liability early in the drug development process is discussed, detailing the usefulness of Caco-2 cells for this type of screening paradigm.