Increasing Dynamic Binding Capacity of Oligo(dT) for mRNA Purification: Experimental Results Using CIM 96-Well Plates

Nina Mencin, Andreja Krušic, Jure Ličen, Sebastijan Peljhan, Jana Vidič, Urh Černigoj, Tomas Kostelec, Aleš Štrancar and Rok Sekirnik

BioProcess International’s Special Report, June 2022

Messenger RNA (mRNA) emerged as a powerful therapeutic tool for treatments in gene therapy, oncology, and infectious diseases, as recently demonstrated by vaccines against Covid-19. mRNA is produced by an enzymatic reaction that can be rapidly designed and scaled-up, and the platform is highly adaptable to different targets. One of the greatest challenges in mRNA production is the removal of process-related impurities stemming from in vitro transcription (IVT) reaction, such as residual nucleotide triphosphates, DNA template, enzymes, abortive transcripts.

Affinity-based chromatographic isolation of mRNA is robust and simple, lending itself as a useful industrial platform. mRNA constructs typically contain a 3’ polyA tail to increase stability in vivo, thereby enabling affinity purification using oligo-deoxythymidinic acid (Oligo dT) probes covalently coupled to a solid support. Macro-porous polymethacrylate monoliths offer high binding capacity and resolution for mRNA due to the convective nature of interconnected flow-through channels (>1.5 μm) modified with ligands that are easily accessible for mRNA. Typical binding capacity for CIMmultus™ Oligo dT for mRNA is 2-4 mg/mL, depending on construct length and loading concentration of NaCl.

Due to an increasing productivity of IVT reaction protocols, which routinely reach 5-10 mg/mL, elucidation of conditions that increase binding capacity of Oligo dT has been an intense focus of development. CIM® Oligo dT 0.05 mL Monolithic 96-well Plates were used for multi-parallel screening of binding conditions. Binding capacity could be significantly increased if NaCl is replaced with Gu-HCl, with DBC values of >6 mg/mL demonstrated, and scalability of binding capacity shown on CIMmultusTM Oligo dT preparative scale, which spans bed volume range 1 mL – 40 L, thereby theoretically supporting the purification of >200 g mRNA in a single run.

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