Selective removal of immunogenic double-stranded RNA from mRNA feeds using affinity chromatography

Double-stranded RNA (dsRNA) is a byproduct of in vitro transcription (IVT) that can trigger innate immune responses in mRNA therapeutics. Existing purification methods can remove dsRNA, but they often scale poorly due to solvent requirements and limited binding capacities. This article describes the development and validation of a dsRNA-selective affinity resin, summarizes the size distribution of dsRNA byproducts measured by immuno-northern blotting, and outlines a high-throughput filter-plate workflow to optimize binding conditions. Finally, it shows that pairing upstream IVT optimization with dsRNA affinity chromatography can reduce dsRNA to sub-immunogenic levels, including elimination of the interferon response in A549 cells for mRNA produced with standard nucleotides.

dsRNA is an immunogenic IVT byproduct that existing purification methods struggle to remove at scale. This article describes a selective dsRNA affinity resin that reduces dsRNA to sub-immunogenic levels – eliminating the interferon response in A549 cells.

What you will learn

01

Why dsRNA is difficult to remove and why existing methods scale poorly

02

How a dsRNA-selective affinity resin captures byproducts while mRNA flows through

03

How combining IVT optimization with affinity purification eliminates the interferon response

The purification workflow

1

IVT reaction & dsRNA characterisation

2

Filter-plate condition screening

3

Affinity column purification

4

Immunogenicity assessment

Key findings

dsRNA byproducts are larger than the primary mRNA transcript, informing selection of a macroporous, convective base bead to enable efficient binding

Column purification achieved an 80-fold dsRNA reduction (1,100 → 14 ppm) with ~83% mRNA recovery across four cycles

Pairing IVT optimisation with affinity purification reduced dsRNA to 0.73 ppm and fully eliminated the interferon response — without modified nucleotides

Key interests

mRNA Manufacturing

Analytical Development & QC

mRNA Stability

Purification

Immunogenicity