Dynamic Binding Capacity of Oligonucleotides on PLRP-S Columns and Stationary Phases

Significance of the Topic

The dynamic binding capacity (DBC) of oligonucleotides on reversed-phase stationary phases is a critical metric for preparative and analytical workflows in molecular biology and biopharmaceutical research. By quantifying the saturation point of a chromatographic column under continuous loading, DBC measurements guide the selection of appropriate pore sizes and column dimensions to maximize yield, resolution, and throughput in oligonucleotide purification.

Objectives and Overview of the Study

This study aimed to evaluate the DBC of custom-synthesized oligonucleotides of four different lengths (25, 50, 75, and 100 bases) on Agilent PLRP-S polystyrene/divinylbenzene stationary phases with pore sizes of 100, 300, 1 000, and 4 000 Å. The goal was to determine how pore accessibility and internal surface area influence loading capacity, mass transfer behavior, and peak sharpness, thereby providing practical guidelines for method development and scale-up.

Methodology and Instrumentation

All experiments were performed on an Agilent 1260 Infinity II quaternary LC system equipped with a high-performance autosampler, thermostatted column compartment, and diode array detector (262 nm).

• Mobile phases: 0.1 M triethylamine acetate (TEAA) in water (A) and in 90% acetonitrile (B), with oligonucleotide solutions prepared at 1 mg/mL in phase A as eluent C.
• Columns: PLRP-S, 2.1×50 mm, 5 μm particles with pores of 100, 300, 1 000, and 4 000 Å.
• Flow rate: 0.21 mL/min; temperature: 25 °C.

Before each DBC run, columns underwent a 60 min cleanup gradient to remove residual contaminants. System void volume was determined using a barrel connector and the delay in oligonucleotide detection at 262 nm.

Main Results and Discussion

DBC measurements revealed that the smallest pore size (100 Å) provided the highest capacity for the 25 mer oligonucleotide but showed reduced loading for 50 and 75 mers due to restricted pore accessibility. The 300 Å phase offered a balanced performance for mid-sized oligos, with sharper breakthrough curves and improved mass transfer compared to the 100 Å material. The largest pores (4 000 Å) exhibited the lowest capacity across all oligo sizes but delivered the most efficient mass transfer and narrowest analytical peaks.

Analytical separations of crude oligonucleotides confirmed that pore size critically affects peak width at half height and impurity resolution. Close-up comparisons showed approximately 12% narrower peak widths on the 300 Å column versus the 100 Å phase for the 50 mer target.

Benefits and Practical Applications of the Method

This approach enables rapid screening of stationary phases to identify the optimal pore size for specific oligonucleotide lengths, streamlining method development for preparative purification. By using 5% of the measured DBC as a starting load, practitioners can estimate injection quantities for columns of various internal diameters and bed depths, facilitating scale-up from analytical to preparative formats.

Used Instrumentation

• Agilent 1260 Infinity II quaternary pump (G1311B)
• Agilent 1260 Infinity II autosampler with cooler (G1367E)
• Agilent 1260 thermostatted column compartment (G1316C)
• Agilent 1290 Infinity II diode array detector (G4212B)

Future Trends and Possibilities

Continued development of polymeric stationary phases with tailored pore structures and surface chemistries will expand the applicability of DBC measurements to emerging oligonucleotide therapeutics such as LNA, 2′-O-methyl, and conjugate-modified sequences. Integration with high-throughput screening platforms and predictive modeling could further accelerate optimization and reduce experimental burden.

Conclusion

DBC profiling on PLRP-S phases provides a robust and practical framework for selecting column pore size and estimating load capacities for oligonucleotide purification. The balance between surface area and pore accessibility determines binding capacity, while larger pores enhance mass transfer and resolution. Employing these insights can significantly improve both analytical and preparative workflows for diverse oligonucleotide applications.

References

1. Lloyd, L. L.; et al. Journal of Chromatography A 2003, 1009, 223–230.