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Small-Footprint Capillary UHPLC/MS Technology Significantly Reducing Consumption of PFAS Containing Modifiers for Fast and High-Resolution Separation of Synthetic Oligonucleotides

Xiaoli Dong1, Cary Simpson2, Sam Foster2, Greg Ward2, Patrick Batoon1
1Agilent Technologies Inc, Santa Clara, CA, 95051
2Axcend, Lehi, UT, 84043

Poster Reprint

ASMS 2025
Poster number  MP 230

 

Introduction

Synthetic oligonucleotides are important biotherapeutic drugs because of their broad applications in genetic research, drug development, diagnostics, and personalized medicine and has gained popularity as a therapeutic modality in the past few years.

HFIP is a perfluoroalkyl (PFAS) substance

Hexafluoroisopropanol (HFIP) is often used as a mobile phase modifier in cation exchange (ion pairing) LC/MS at high pH to determine the presence of the active pharmaceutical ingredient (API) and various other impurities.1 HFIP is a perfluoroalkyl substance (PFAS) aka “Forever Chemical”, contributing to PFAS contamination of the lab environment due to the aerosolization of droplets in the electrospray process.

Therefore, the reduction of HFIP consumption in routine LC analysis of oligonucleotide drugs is of major interest in the long term.

TEA to adjust pH and augment ion pairing

To adjust pH, Triethylamine (TEA) has been identified as the ideal reagent since it augments the ion-pairing chromatography process. However, in the context of LC/MS operation, ion pairing reagents exist in high concentration, which introduces persistent contamination risk of the overall system over time.


In this work, we present a robust method for oligonucleotide analysis which dramatically reduces the consumption of HFIP and TEA, while maintaining chromatographic and mass spectral performance of typical “standard flow” rates of analytical systems.

Focus LC Pro IQAxcend Focus LC® coupled with the Agilent LC/MSD Pro iQ Plus


Experimental

Instrumentation

Axcend Focus LC with Autosampler
Agilent Pro iQ Plus (G6170A) with ESI source
OpenLab CDS Acquisition and Data Analysis 2.8


LC Method Parameters

LC Parameter

Value

Column Acquity M-Class HSS T-3 100 mm x 0.15 mm with 1.8 um fully porous particle
Mobile Phase A 100 mM HFIP and 15 mM TEA in water
Mobile Phase B

Methanol

Flow Rate 2 μL/min
Injection Volume 250nL
Gradient Program Time (min) %B
  0.0 20
  10 27
  11 95
  12 95
  12.1 20


MS Method Parameters

MS Parameter

Value

Ion Source ESI
Polarity Negative
Drying Gas Temp

300 °C

Drying Gas Flow 6 L/min
Nebulizer 15 psi
Capillary Voltage 4000 V


Data Processing

Sample data was processed directly in OpenLab CDS Data Acquisition. Each analyte yielded mass spectra with charge state distributions. Molecular Weight (Measured Mass) was determined using the built-in Deconvolution algorithm


Samples Analyzed

Agilent DNA ladder standard
(15, 20, 25, 30, 35, and 40-mer; Part No. 5190-9029 )
Custom 103-mer oligonucleotide
Givosiran Standard

 

Results and Discussion

DNA ladder standard, 1 mL, with 15, 20, 25, 30, 35, and 40-mer oligos

Agilent Image 1Asset 2

DNA Ladder Expected Mass Measured Mass Delta Mass (Da)
15mer 4501.0 4500.5 0.5
20mer 6022.0 6021.5 0.5
25mer 7543.0 7542.5 0.5
30mer 9063.9 9063.1 0.8
35mer 10584.9 10584.5 0.4
40mer 12105.9 12105.4 0.5

 

Agilent Image 2aAsset 3

Agilent Image 2bAsset 4


100-mer Oligonucleotide Standard

A custom 103-mer oligonucleotide crude sample of unknown sequence (mass known) was analyzed, demonstrating excellent chromatographic peak shape and generation of mass spectrum.

Agilent Image 3

  Expected Mass Measured Mass Delta Mass (Da)
103-mer 32394.3 32394.5 0.2


Agilent Image 4Asset 1


Givosiran (Givlaari) a siRNA therapeutic oligonucleotide

Givosiran is an N-acetylgalactosamine (GalNAc)-conjugated siRNA targeting aminolevulinate synthase 1 (ALAS1). This drug contains two “tagged” oligonucleotide strands, sense (SS) and antisense (AS).

Strand Sequence Expected Mass Measured Mass Delta Mass (Da)
22SS mC*mA*mGmAmAmAfGmAfGmUfGmUfCmUfCmAmUmCmUmUmA/L96/ 7563.84 7562.97 0.87
23AS mU*mG*mGfUmCfUmUfUfCmUfCfAmCfAmGfAmGfUmAmGfA*fA*mU 8736.50 8735.97 0.53

 

Agilent Image 5Asset 1


Agilent Image 6Asset 1


Solvent Consumption Comparisons

A major advantage to microflow based chromatography is the significant reduction in consumption of organic solvents, HFIP, and TEA. Based on the comparison of methods below.

This method results in 220x less (>99.5% reduction) of Methanol, HFIP, and TEA consumption compared to a conventional method.

LC Param. Microflow Method Standard Flow Method 2
Mobile Phase A 100 mM HFIP and 15 mM TEA in water 100 mM HFIP and 15 mM TEA in water
Mobile Phase B Methanol Methanol
Flow Rate 2 μL/min 500 μL/min
Injection Volume 250 nL 2 μL
Gradient Program Time (min) %B Time (min) %B
  0.0 20 0.0 20
  10.0 27 10.0 27
  11.0 95 11.0 95
  12.0 95    
  12.1 20    
Runtime 12-17 minutes 11-15 minutes
Calculation Equation 2Asset 4 Equation 2Asset 1
Solvent Use 24-34 μL per run 5500-7500 μL per run

 

Conclusions

  • A standard flow oligonucleotide analysis method containing HFIP and TEA was transferred to microflow chromatography using the Axcend Focus LC.
  • This method resulted in a 220x-less consumption (>99.5% reduction) of Methanol, HFIP, and TEA consumption compared to a conventional method.
  • This method was able to chromatographically resolve the DNA Ladder Standard (15-40mer oligonucleotides) and custom 103-mer oligonucleotide standard, producing a clean mass spectrum for each analyte.
  • This analysis was applied to real sample, Givosiran. Both Sense (SS) and Antisense (AS) components of the drug provided correct MW assignment
  • Mass spectral deconvolution resulted in correct MW assignments. ~Δ0.5 Da from theoretical values.

 

References

1Advancements in the characterisation of oligonucleotides by high performance liquid chromatography-mass spectrometry in 2021: A short review (10.1002/ansa.202100066)

2Molecular Weight Confirmation of Oligonucleotides Using Agilent LC/MSD XT and OpenLab CDS (5994-7083EN)

 

 

 

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This information is subject to change without notice.
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© Agilent Technologies, Inc. 2025
Published in USA, May 15, 2025