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.

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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

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