Purification of linearized DNA for use as an in vitro transcription template mRNA therapeutics, such as those created for the COVID-19 pandemic, are manufactured using an enzymatic reaction referred to as an in vitro transcription (IVT) reaction. The desired sequence is expressed as a plasmid from E. coli. Super coiled and open circular DNA are digested enzymatically to give a linear template. This IVT reaction uses the linearized plasmid to create multiple copies of the mRNA sequence required. Purity of the linearized template is critical, since any remaining host cell DNA impurities or other nucleic acid contaminants will give rise to off-target mRNA products, which consume costly reagents and are difficult to remove. We propose a process whereby the primary capture of pDNA is carried out on an ion exchange resin. In this case, DEAE PuraBead® HF is loaded with the clarified lysate conditioned to a high enough conductivity so that only the DNA species is bound, and the RNA is removed in the flow-through. The elution pool from the AEX step, can then be desalted, linearized enzymatically, and loaded onto an orthogonal purification step using a hydrophobic adsorbent, the pDNAHERO® device. The linear DNA is eluted from the column using a TRIS EDTA buffer.
AEX capture 1. Alkaline lysis process 2. Calcium chloride precipitation 3. Diafiltration into 50 mM TRIS, 10 mM EDTA, pH 7.5, 300 mM NaCl 4. Adjust to 35–37 mS/cm 5. Load onto DEAE PuraBead® HF Conditions for AEX capture Equilibration buffer A1: 50 mM TRIS, 10 mM EDTA, pH 7.5 Elution buffer B1: 50 mM TRIS, 10 mM EDTA, 1 M NaCl, pH 7.5 CIP A2: 50 mM TRIS, 10 mM EDTA, 0.5 M NaOH
Issue date: 1 July 2025
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