Tuning hydrogen bonds and electrostatics with convection for purifying mRNA: A paradigm shift

Area of Science:

• Biotechnology
• Materials Science
• RNA Therapeutics

Background:

• Clinical trials for single-stranded mRNA (ss-mRNA) therapeutics are increasing, necessitating efficient, scalable, and economical purification methods.
• Current purification techniques are limited by diffusion, low productivity, and reliance on expensive oligo(dT) ligands for poly(A) tail hybridization.
• Immunogenic double-stranded RNA (dsRNA) is a critical impurity that must be removed from therapeutic ss-mRNA.

Purpose of the Study:

• To develop a ligand-less, cost-effective purification strategy for ss-mRNA therapeutics.
• To separate ss-mRNA from dsRNA impurities using interfacial molecular forces and modified membranes.
• To enhance purification efficiency through multimodal interactions and selective charge neutralization.

Main Methods:

• Utilized a positively charged synthetic microporous membrane to capture ss-mRNA via charge and hydrogen bonds.
• Investigated optimized surface densities (4000 to 10,000 nmol/m²) and pH conditions (~9.0) for membrane binding.
• Employed the polyamine spermine to selectively neutralize dsRNA charge, enhancing separation at specific amine-to-phosphate ratios (>450).

Main Results:

• Achieved high binding capacities (1.28 mg/m²) and up to 100% ss-mRNA recovery.
• Demonstrated rapid purification flow rates (1.5 ml/min, 1000 MV/min) with membrane reusability (>10 cycles).
• Observed negligible ligand leaching, confirming the ligand-less nature of the approach.

Conclusions:

• The multimodal, ligand-less membrane purification strategy offers an efficient, scalable, and economical alternative to current methods.
• Selective charge neutralization of dsRNA using spermine is key to achieving high purity ss-mRNA.
• This approach significantly advances the production of safe and effective ss-mRNA therapeutics.