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Capillary electrophoresis methods for determining the IVT mRNA critical quality attributes of size and purity
Denise A Warzak1, Whitney A Pike1, Kyle D Luttgeharm1
1Agilent Technologies, Ankeny, IA 50023, USA.
Abstract:
One result of the Covid-19 pandemic has been an increased awareness of IVT mRNA vaccines and the speed at which they can be produced for disease outbreaks. Currently the only approved IVT mRNA therapeutics are the Covid-19 vaccines, however IVT mRNA is being investigated for other non-Covid prophylactic vaccines, therapeutics, and therapeutic vaccines. IVT mRNAs can range from less than 100 nt in length to longer than 9,000 nt. When producing any IVT mRNA, quality control of the IVT mRNA is essential to ensure that the product is the correct length and does not contain truncated or degraded mRNA. Capillary gel electrophoresis provides high resolution separations of the IVT mRNA of interest from the degraded or truncated impurities allowing for the accurate purity assessment of IVT mRNA. Specialized capillary electrophoresis gels can also be used to provide analysis of purified poly(A) tails enabling characterization of multiple Critical Quality Attributes on a single platform. Here we describe methods for the purity assessment of IVT mRNAs through either 6,000 or 9,000 nt and determination of poly(A) tail length using different capillary gel electrophoresis methods.
Insights
Quality control for in vitro transcribed messenger RNA (IVT mRNA) is crucial. Capillary gel electrophoresis effectively assesses IVT mRNA purity and poly(A) tail length for therapeutic applications.
Area of Science:
- Molecular Biology
- Biotechnology
- Analytical Chemistry
Background:
- The COVID-19 pandemic highlighted the importance and rapid production capabilities of in vitro transcribed messenger RNA (IVT mRNA) vaccines.
- While currently limited to COVID-19 vaccines, IVT mRNA holds potential for various prophylactic vaccines, therapeutics, and therapeutic vaccines.
- IVT mRNA molecules vary significantly in length, from under 100 nucleotides (nt) to over 9,000 nt.
Purpose of the Study:
- To establish and describe methods for assessing the purity of IVT mRNA, specifically for lengths of 6,000 or 9,000 nt.
- To detail techniques for determining the length of the poly(A) tail of IVT mRNA.
- To demonstrate the utility of capillary gel electrophoresis (CGE) for characterizing critical quality attributes of IVT mRNA.
Main Methods:
- Utilized capillary gel electrophoresis (CGE) for high-resolution separation of IVT mRNA from impurities like truncated or degraded fragments.
- Employed specialized CGE gels to analyze purified poly(A) tails, enabling comprehensive characterization.
- Developed and applied distinct CGE methods tailored for purity assessment and poly(A) tail length determination.
Main Results:
- Demonstrated that CGE provides accurate purity assessment by effectively separating full-length IVT mRNA from degradation products.
- Showcased the capability of specialized CGE gels to analyze poly(A) tail lengths, a critical quality attribute.
- Validated CGE as a versatile platform for characterizing multiple critical quality attributes of IVT mRNA on a single system.
Conclusions:
- Capillary gel electrophoresis is an essential tool for ensuring the quality and purity of IVT mRNA.
- CGE methods described allow for accurate assessment of IVT mRNA length and poly(A) tail integrity.
- This analytical approach supports the development and quality control of diverse IVT mRNA-based therapeutics and vaccines.
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