Affinity 2025

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Speaker: Urh Černigoj, Head of R&D, Sartorius BIA Separations
Time and Date: Wednesday, June 3 | 11:15 -11:30 AM
Title: Optimizing Monolith-Based Oligo-dT Chromatography Through Understanding the Hybridization Kinetics

Abstract:

Oligo-deoxythymidilic acid (oligo-dT) probes, conjugated to solid-phase supports, exhibit a high affinity for poly-adenylated (poly-A) targets due to strong base-pairing interactions. This is basis of hybridization-affinity chromatography, where oligo-dT ligand captures messenger RNA (mRNA) containing a poly-A tail in a high ionic strength mobile phase, while IVT reaction components and mRNA fragments without a poly-A tail flow-through. One of the scalable stationary phases with bound oligo-dT ligand are CIM® chromatographic monoliths. Examples of mRNA purification via oligo-dT modified CIM columns reported DBCs ranging from 2 up to 6 mg/mL, depending on buffer composition.

Due to their specific architecture, where mass transport is primarily governed by convective flow, CIM chromatographic monoliths have consistently demonstrated that their capacity for large biomolecules is relatively independent of flow rate. However, contrary to expectations, testing the dynamic binding capacity of mRNA to oligo-dT monoliths as a function of residence time revealed that the mass of bound mRNA increased by approximately 50% when the flow rate was reduced from 9 to 0.33 CV/min. This unexpected result prompted us to investigate whether the underlying cause is linked to the hybridization kinetics between the surface-bound oligo-dT ligand and the sterically hindered poly-A tail of mRNA. Chromatographic experiments were combined with dynamic modeling to gain a deeper understanding of this complex system.

Location: Raleigh, USA

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