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

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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