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

Autor: Di Grandi D; Protein Biochemistry, Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA., Dayeh DM; Protein Biochemistry, Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA., Kaur K; Regeneron Genetic Medicines, Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA., Chen Y; Regeneron Genetic Medicines, Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA., Henderson S; Protein Biochemistry, Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA., Moon Y; Protein Biochemistry, Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA., Bhowmick A; Regeneron Genetic Medicines, Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA., Ihnat PM; Protein Biochemistry, Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA., Fu Y; Protein Biochemistry, Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA. Electronic address: yue.fu@regeneron.com., Muthusamy K; Protein Biochemistry, Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA. Electronic address: kathir.muthusamy@regeneron.com., Palackal N; Protein Biochemistry, Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA., Pyles EA; Protein Biochemistry, Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591, USA.
Jazyk: angličtina
Zdroj: Journal of pharmaceutical and biomedical analysis [J Pharm Biomed Anal] 2023 Nov 30; Vol. 236, pp. 115692. Date of Electronic Publication: 2023 Sep 02.
DOI: 10.1016/j.jpba.2023.115692
Abstrakt: 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.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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