In vitro transcription (IVT) of mRNA is a versatile platform for a broad range of biotechnological applications. Its rapid, scalable, and cost-effective production makes it a compelling choice for the development of mRNA-based cancer therapies and vaccines against infectious diseases. The impurities generated during mRNA production can potentially impact the safety and efficacy of mRNA therapeutics, but their structural complexity has not been investigated in detail yet. This study pioneers a comprehensive profiling of IVT mRNA impurities, integrating current technologies with innovative analytical tools. We have developed highly reproducible, efficient, and stability-indicating ion-pair reversed-phase liquid chromatography and capillary gel electrophoresis methods to determine the purity of mRNA from different suppliers. Furthermore, we introduced the applicability of microcapillary electrophoresis for high-throughput (<1.5 min analysis time per sample) mRNA impurity profiling. Our findings revealed that impurities are mainly attributed to mRNA variants with different poly(A) tail lengths due to aborted additions or partial hydrolysis and the presence of double-stranded mRNA (dsRNA) byproducts, particularly the dsRNA 3'-loop back form. We also implemented mass photometry and native mass spectrometry for the characterization of mRNA and its related product impurities. Mass photometry enabled the determination of the number of nucleotides of different mRNAs with high accuracy as well as the detection of their size variants [i.e., aggregates and partial and/or total absence of the poly(A) tail], thus providing valuable information on mRNA identity and integrity. In addition, native mass spectrometry provided insights into mRNA intact mass, heterogeneity, and important sequence features such as poly(A) tail length and distribution. This study highlights the existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, thus contributing to the refinement and streamlining of mRNA production, paving the way for continued advancements in biotechnological applications.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10918618 | PMC |
| http://dx.doi.org/10.1021/acs.analchem.3c05539 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Distinguishing abiotic corrosion from two types of microbiologically influenced corrosion (MIC) using a new electrochemical biofilm/MIC test kit.
J Environ Manage
January 2025
Department of Chemical & Biomolecular Engineering, Institute for Corrosion and Multiphase Technology, Ohio University, Athens, 45701, USA; Department of Biological Sciences, Molecular & Cellular Biology Program, Ohio University, Athens, OH, 45701, USA. Electronic address:
Biofilms can cause biofouling, water quality deterioration, and transmission of infectious diseases. They are also responsible for microbiologically influenced corrosion (MIC) which can cause leaks, resulting in environmental disasters. A new disposable biofilm/MIC test kit was demonstrated to distinguish abiotic corrosion of carbon steel from MIC.
View Article and Find Full Text PDFThe CeHRes Roadmap 2.0: Update of a Holistic Framework for Development, Implementation, and Evaluation of eHealth Technologies.
J Med Internet Res
January 2025
Section of Psychology, Health & Technology, Centre for eHealth and Wellbeing, University of Twente, Enschede, Netherlands.
To ensure that an eHealth technology fits with its intended users, other stakeholders, and the context within which it will be used, thorough development, implementation, and evaluation processes are necessary. The CeHRes (Centre for eHealth and Wellbeing Research) Roadmap is a framework that can help shape these processes. While it has been successfully used in research and practice, new developments and insights have arisen since the Roadmap's first publication in 2011, not only within the domain of eHealth but also within the different disciplines in which the Roadmap is grounded.
View Article and Find Full Text PDFEnhancing HIV Cognitive Abilities and Self-Management Through Information Technology-Assisted Interventions: Scoping Review.
J Med Internet Res
January 2025
First Hospital of China Medical University, Shenyang, China.
Background: HIV/AIDS remains a significant global challenge, and with the rapid advancement of technology, there has been an increasing number of interventions aimed at improving HIV/AIDS cognition and self-management behaviors among patients. However, there is still a lack of detailed literature integrating relevant evidence.
Objective: This study aims to comprehensively review existing research on interventions using modern information methods to improve HIV/AIDS cognition and enhance self-management behaviors among patients.
Trends, challenges, and research pathways in emerging contaminants: a comprehensive bibliometric analysis.
Integr Environ Assess Manag
January 2025
Department of Civil and Environmental Engineering, Florida State University, Tallahassee, FL, United States.
The growing concern over environmental pollution has spurred extensive research into various contaminants impacting ecosystems and human health. Emerging contaminants (ECs), including pharmaceuticals, personal care products, endocrine-disrupting chemicals, nanomaterials, and microplastics, have garnered significant attention due to their persistence, bioaccumulation, and toxicity. This study presents a comprehensive bibliometric analysis of EC research, aiming to detail the research landscape, highlight significant contributions, and identify influential researchers and pivotal studies.
View Article and Find Full Text PDFExploring the Frontiers of Nanopore Sequencing in Food Safety and Food Microbiology.
Annu Rev Food Sci Technol
January 2025
1Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida, USA; email:
Foodborne illnesses are a significant global public health challenge, with an estimated 600 million cases annually. Conventional food microbiology methods tend to be laborious and time consuming, pose difficulties in real-time utilization, and can display subpar accuracy or typing capabilities. With the recent advancements in third-generation sequencing and microbial omics, nanopore sequencing technology and its long-read sequencing capabilities have emerged as a promising platform.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!