(302e) Engineered Peptide Modified Affinity Membranes for the Removal of Immunogenic dsRNA Species

The messenger-RNA (mRNA) biologic toolbox is rapidly expanding beyond vaccines to encompass a broad range of therapeutic applications. However, realizing the full potential of mRNA therapeutics requires scalable purification technologies that preserve mRNA integrity while effectively removing immunogenic impurities. This work proposes a paradigm shift from traditional diffusion-limited resin technologies to convective membrane separators, enhancing both efficiency and process economics¹. Double stranded RNA (dsRNA), a key immunogenic process impurity², presents a particular challenge as it exists as a heterogenous population of varying lengths and degrees of hybridization. Additionally, secondary structures in single stranded mRNA (ss-mRNA) can exhibit partial double stranded characteristics, further complicating purification due to their similar physiochemical properties³. These complexities necessitate a selective and cost-effective affinity-based approach. Peptides, with their biological origin, structural flexibility, sequence diversity, and facile synthesis, hold immense potential as affinity ligands for dsRNA removal⁴. Their ability to mimic endogenous interactions allows for highly specific binding, making them an ideal candidate for targeting immunogenic dsRNA contaminants in in vitro transcription (IVT) mRNA synthesis.

In this presentation we explore the development, characterization, and optimization of peptide-functionalized cellulose filter paper membranes as a scalable and cost-effective platform for mRNA purification. Here, we investigate (1) membrane surface modification via solid-phase peptide synthesis, integrating peptide synthesis and membrane functionalization, (2) validation studies evaluating dsRNA removal efficiency and mRNA recovery, and (3) robustness assessments, including reusability, flow rate dependence and performance under IVT-relevant conditions, with the goal of scaling up for broad application in mRNA-based biologics.

The results demonstrate that the affinity peptide-modified membranes provide a highly selective, scalable, and cost-effective solution for dsRNA removal, offering an alternative to current industrial purification technologies. By harnessing the convective nature of microporous membranes, the system achieves high mRNA recovery and effective dsRNA removal, presenting a promising strategy for reducing the immunogenicity of mRNA-based therapeutics while ultimately enhancing process economics.

References

1 Banik, R. et al. Convection rather than diffusion for fast efficient mRNA vaccine purification. Separation and Purification Technology 354 (2025). https://doi.org/10.1016/j.seppur.2024.129310

2 Lenk, R. et al. Understanding the impact of in vitro transcription byproducts and contaminants. Front Mol Biosci 11, 1426129 (2024). https://doi.org/10.3389/fmolb.2024.1426129

3 Martínez, J., Lampaya, V., Larraga, A., Magallón, H. & Casabona, D. Purification of linearized template plasmid DNA decreases double-stranded RNA formation during IVT reaction. Frontiers in Molecular Biosciences 10 (2023). https://doi.org/10.3389/fmolb.2023.1248511

4 Mullerpatan, A. et al. Single-step purification of a small non-mAb biologic by peptide-ELP-based affinity precipitation. Biotechnol Bioeng 117, 3775-3784 (2020). https://doi.org/10.1002/bit.27539