Messenger RNA (mRNA) vaccines and therapeutics have emerged as a transformative platform technology due to their rapid development timelines, manufacturing flexibility, and broad therapeutic applicability. However, downstream purification remains a critical bottleneck in mRNA manufacturing because purification processes must simultaneously achieve high product recovery, high molecular integrity, impurity removal, scalability, and cost-effectiveness. This thesis investigates oligo-deoxythymidine (oligo-dT) affinity chromatography as a platform purification strategy for polyadenylated mRNA produced by in vitro transcription (IVT), with emphasis on process intensification, matrix optimisation, and upstream–downstream integration.
First, a quality-by-design (QbD)-guided continuous oligo-dT chromatography process was developed and optimised by systematically linking critical process parameters (CPPs), critical material attributes (CMAs), critical quality attributes (CQAs), and key performance indicators (KPIs). Optimisation of loading salt conditions, flow rate, and mRNA loading concentration enabled a four-column continuous process achieving >90% mRNA recovery, >95% integrity, and >99% purity. Compared with equivalent batch purification, the continuous process increased estimated 24-hour productivity by approximately 5.75-fold while reducing operating cost by ~15%.
Second, membrane-, monolith-, and bead-based oligo-dT matrices were comparatively evaluated using multifactorial optimisation approaches. Membrane matrices demonstrated the highest mRNA binding capacity, recovery, and productivity, whereas bead-based matrices offered the lowest overall process cost. Monolith matrices provided balanced performance across productivity and economics. These findings establish practical matrix-selection criteria for platform mRNA purification.
Third, an integrated IVT–oligo-dT workflow was developed to directly connect mRNA synthesis with affinity capture and enable recycling of high-value IVT reagents, including cap analogue and plasmid DNA. Across five sequential recycling cycles, the integrated process maintained high mRNA integrity (~98%), low double-stranded RNA content, preserved protein expression, and reduced reagent cost per equivalent mRNA output to approximately 43% of a conventional non-recycling process.
Collectively, this work establishes a robust, scalable, and techno-economically balanced oligo-dT purification platform that advances continuous and integrated manufacturing of mRNA vaccines and therapeutics.
