Integrated in vitro transcription and oligo-dT affinity chromatography enable multi-cycle reagent recycling for mRNA...

A platform process underpins the manufacturing of RNA-based vaccines and therapeutics. However, it remains constrained by high operational expenditures from costly reagents, inefficient raw-material utilization, and extensive purification. We report an integrated sequential-batch in vitro transcription (IVT)-oligo-dT chromatography process that links RNA synthesis and purification via a shared buffer, enabling reagent recycling. Across five cycles, 5' cap analog utilization improved 3.72× and calculated raw-material cost efficiency improved 2.21× for the NaCl workflow at the demonstrated 8 mL scale, while stable RNA production was maintained across recycling cycles. Product quality was assessed across seven critical quality attributes (CQAs), including RNA integrity, 5' capping, poly(A)-tail heterogeneity, sequence identity, residual nucleotides, dsRNA content, and cell-based functional activity, measured by protein expression and cytokine responses. Purified recycling-process RNA contained ≈70%-77% less double-stranded RNA (dsRNA) than purified standard-process RNA, purified RNA integrity exceeded 90%, and five-cycle mean 5' capping exceeded 80% with NaCl; 3' polyadenylate (poly(A)) tail length and heterogeneity remained stable across recycling cycles. THP-1 cell assays showed no progressive cycle-dependent loss of protein expression or increase in cytokine secretion across recycling cycles. This integrated framework advances cost- and resource-efficient mRNA production while identifying candidate control points for extended reagent recycling.