(93c) Molecular Analysis of the Effect of Ligand Grafting Density on Membrane-Based mRNA Purification

The success of mRNA-based vaccines against SARS CoV-2 has underscored the necessity for scalable manufacturing processes and robust analytical methods to ensure safety, potency, and purity. Membrane-based purification operations offer an appealing convective-transport based alternative to porous bead-based chromatography due to their relatively large available pore surface areas and short residence times, preserving mRNA length and secondary structure integrity. Oligo dT affinity ligands selectively capture full-length mRNA transcripts by specifically targeting their poly(A) tails through A-T base pairing. This method efficiently eliminates various contaminants, including DNA template, nucleotide substrate, enzymes, buffer components and mRNA lacking poly(A) tails. Oligo dT affinity systems reported in literature have ~3.2% ligand utilization for mRNA capture, assuming 1:1 binding between dT-dA1.This work aims to incorporate the effects of grafting density and membrane pore size on the adsorptive capacity of membranes. We also emphasize the fact that expensive oligo-dT affinity ligands immobilized on the membrane surface are mostly unutilized during adsorption. Efforts will be made to bridge the gap between over-grafting and under-grafting to identify the sweet spot for optimal grafting density that maximizes capture and elution performance while minimizing separation media costs.

The distance between ligands at the molecular scale influences the hybridization valency between the poly-A tail and oligo-dT ligands. Atomic Force microscopy will be employed to give further insight into the binding energies between different lengths of mRNA poly-A tail. And oligo-dT grafted ligand. Different lengths of oligo-dT nucleotides will be immobilized on AFM probe tips which can then be utilized to generate a pulling force on an mRNA immobilized on a glass slide. The effect of the chaotropicity of different salts in the binding buffer such as NaCl, Gu-HCl, arginine and urea will be investigated at a pH range of 7-7.5 to identify the solution conditions for optimal binding. Combining these different strategies, the global objective of the study is to discover the optimal ligand utilization for maximum adsorptive capacity of pure mRNA and hence reduced cost of the process.

* Part of this work has been submitted as a manuscript Banik R, Neumann T, Al Sharabati M, Hao Z, et al., Convection Rather than Diffusion for Fast Efficient mRNA Vaccine Purification, 2024 (Submitted)

1. Mencin, N.; Štepec, D.; Margon, A.; Vidič, J.; Dolenc, D.; Simčič, T.; Rotar, S.; Sekirnik, R.; Štrancar, A.; Černigoj, U., Development and scale-up of oligo-dT monolithic chromatographic column for mRNA capture through understanding of base-pairing interactions. Separation and Purification Technology 2023, 304, 122320.