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Structure-performance analysis of oligo(dT) resin affinity chromatography for RNA separations.

Julian Grinsted1, Emir Bouleghlimat2, Sara S Rosa1

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Journal of Chromatography. A
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Oligo deoxythymidine (oligo(dT)) resins are crucial for messenger RNA (mRNA) purification but have low capacity. Structural analysis revealed pore size impacts RNA binding, informing future resin designs for improved mRNA purification efficiency.

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Area of Science:

  • Biotechnology
  • Chemical Engineering
  • Materials Science

Background:

  • Messenger RNA (mRNA) is a rapidly developing therapeutic and prophylactic drug modality.
  • Oligo deoxythymidine (oligo(dT)) ligand chemistries are vital for mRNA chromatographic purification.
  • Beaded oligo(dT) resins exhibit limitations in dynamic binding capacity (DBC).

Purpose of the Study:

  • To establish a structure-performance relationship for beaded oligo(dT) resins.
  • To understand the impact of resin structure on messenger RNA (mRNA) binding.
  • To inform the design of next-generation resins for mRNA purification.

Main Methods:

  • Surface area analysis using Brunauer-Emmett-Teller (BET) method.
  • Pore size distribution and morphology characterization via mercury intrusion porosimetry and scanning electron microscopy (SEM).
  • Poly-adenine RNA binding and breakthrough experiments to assess dynamic binding capacity (DBC).

Main Results:

  • Resin surface areas ranged from 13-18 m²g⁻¹, with modal pore diameters between 298-526 nm.
  • RNA binding occurred primarily on the bead exterior, with gradual penetration into the pore network.
  • A 257% increase in DBC₁₀ was observed with increasing residence time, emphasizing diffusive transport.

Conclusions:

  • Resin structural properties significantly influence messenger RNA (mRNA) binding and purification performance.
  • Diffusive transport limitations impact the dynamic binding capacity (DBC) of beaded oligo(dT) resins.
  • Understanding structure-performance relationships is key to optimizing resin design for efficient mRNA purification.