The impact of low adsorption surfaces for the analysis of DNA and RNA oligonucleotides

Honorine Lardeux; Alexandre Goyon; Kelly Zhang; Jennifer M Nguyen; Matthew A Lauber; Davy Guillarme; Valentina D'Atri

doi:10.1016/j.chroma.2022.463324

The impact of low adsorption surfaces for the analysis of DNA and RNA oligonucleotides

J Chromatogr A. 2022 Aug 16:1677:463324. doi: 10.1016/j.chroma.2022.463324. Epub 2022 Jul 9.

Authors

Honorine Lardeux¹, Alexandre Goyon², Kelly Zhang², Jennifer M Nguyen³, Matthew A Lauber³, Davy Guillarme¹, Valentina D'Atri⁴

Affiliations

¹ Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, Geneva 4 1211, Switzerland; School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, Geneva 4 1211, Switzerland.
² Small Molecule Pharmaceutical Sciences, Genentech Inc., DNA Way, South San Francisco, CA 94080, USA.
³ Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.
⁴ Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, Geneva 4 1211, Switzerland; School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, Geneva 4 1211, Switzerland. Electronic address: valentina.datri@unige.ch.

PMID: 35858489
DOI: 10.1016/j.chroma.2022.463324

Abstract

As interest in oligonucleotide (ON) therapeutics is increasing, there is a need to develop suitable analytical methods able to properly analyze those molecules. However, an issue exists in the adsorption of ONs on different parts of the instrumentation during their analysis. The goal of the present paper was to comprehensively evaluate various types of bioinert materials used in ion-pairing reversed-phase (IP-RPLC) and hydrophilic interaction chromatography (HILIC) to mitigate this issue for 15- to 100-mer DNA and RNA oligonucleotides. The whole sample flow path was considered under both conditions, including chromatographic columns, ultra-high-performance liquid chromatography (UHPLC) system, and ultraviolet (UV) flow cell. It was found that a negligible amount of non-specific adsorption might be attributable to the chromatographic instrumentation. However, the flow cell of a detector should be carefully subjected to sample-based conditioning, as the material used in the UV flow cell was found to significantly impact the peak shapes of the largest ONs (60- to 100-mer). Most importantly, we found that the choice of column hardware had the most significant impact on the extent of non-specific adsorption. Depending on the material used for the column walls and frits, adsorption can be more or less pronounced. It was proved that any type of bioinert RPLC/HILIC column hardware offered some clear benefits in terms of adsorption in comparison to their stainless-steel counterparts. Finally, the evaluation of a large set of ONs was performed, including a DNA duplex and DNA or RNA ONs having different base composition, furanose sugar, and modifications occurring at the phosphate linkage or at the sugar moiety. This work represents an important advance in understanding the overall ON adsorption, and it helps to define the best combination of materials when analyzing a wide range of unmodified and modified 20-mer DNA and RNA ONs.

Keywords: Bioinert surfaces; Hydrophilic interaction chromatography (HILIC); Ion-pairing reversed-phase chromatography (IP-RPLC); Low adsorption surfaces; Oligonucleotides.

MeSH terms

Adsorption
Chromatography, Reverse-Phase / methods
DNA
Hydrophobic and Hydrophilic Interactions
Oligonucleotides* / analysis
RNA*
Sugars

Substances

Oligonucleotides
Sugars
RNA
DNA