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Chromatography in mRNA Production From Analytics to Purification

Speaker: Rok Sekirnik, Sartorius BIA Separations Title of the lecture: Chromatography in mRNA Production: From Analytics to Purification Date & Time: June 20, 2023 The talk will present case studies of leveraging the power of at-line HPLC monitoring for process development to increase purity and yield of mRNA production. Approaches to optimize plasmid purification and linearization, IVT reaction and mRNA purification will be described, including purification methods to achieve more than 95% supercoiled plasmid purity, optimize linearization reaction, and increase of IVT reaction yield from 3-5 mg/mL to 10-12 mg/mL. Further, use of at-line analytics to monitor automated, continuous production in single use bioreactors to produce multi-gram amounts of mRNA will be demonstrated. Oligo dT-based affinity purification will be described with approaches to optimize dynamic binding capacity to reach up to 6 g/L. Multimodal chromatography and reverse-phase chromatography cases will be discussed for capture of non-polyadenylated RNA, and removal of dsRNA. OUR mRNA LIBRARY: https://www.biaseparations.com/en/sea... GET in TOUCH: Webpage: https://www.biaseparations.com/ E-mail: [email protected] Tech support: [email protected] 0:00 Introduction 4:08 Structure and Properties of mRNA 10:04 mRNA process starts with pDNA 11:40 Quantitative Isoform Analysis of pDNA with AEX HPLC 14:41 Pre-Chromatography: Lysis Step is Critical in pDNA Processing 17:49 Plasmid Capture on CIMmultus DEAE 19:36 Plasmid Polishing in Bind-Elute Mode 20:52 Tunable HIC for high purity of supercoiled DNA plasmids 23:56 Linearisation of pDNA as IVT Raw Material 26:14 Linear pDNA Purification Process Overview 27:34 In Vitro Transcription Reaction - Very Fast Production of mRNA from DNA Template 29:00 mRNA analytics: Monitoring IVT - paradigms for rapid at-line analytics 34:21 Optimisation of IVT reaction with monitoring 35:52 CIMac PrimaS Provides Tight Control Over IVT Reaction 37:42 Optimize fed-batch protocol by monitoring NTP depletion 38:53 A Step Towards Continuous Manufacturing: Fed-Batch 41:08 Toolbox for Purification of mRNA or RNA 43:42 mRNA Binding Kinetics Affect Column Binding Capacity 47:17 CIM Oligo dT18 Purification Increases mRNA Stability Post-Capture 53:30 Take Home Message 54:34 mRNA Applications in Discovery & Development