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The impact of low adsorption surfaces for the analysis of DNA and RNA oligonucleotides
Honorine Lardeux1, Alexandre Goyon2, Kelly Zhang2
1Institute 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.
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.
Insights
Adsorption of oligonucleotides (ONs) during analysis can be reduced by using bioinert materials in chromatography. Column hardware choice significantly impacts adsorption, with bioinert options outperforming stainless steel for DNA and RNA analysis.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Chromatography
Background:
- Oligonucleotide (ON) therapeutics are gaining prominence, necessitating robust analytical methods.
- Non-specific adsorption of ONs to analytical instrumentation poses a significant challenge.
- Developing effective strategies to mitigate ON adsorption is crucial for accurate analysis.
Purpose of the Study:
- To comprehensively evaluate bioinert materials for mitigating oligonucleotide adsorption in ion-pairing reversed-phase (IP-RPLC) and hydrophilic interaction chromatography (HILIC).
- To assess the impact of different materials across the entire sample flow path, including columns, UHPLC systems, and UV flow cells.
- To identify optimal material combinations for analyzing a wide range of DNA and RNA oligonucleotides.
Main Methods:
- Evaluation of various bioinert materials in IP-RPLC and HILIC systems.
- Analysis of 15- to 100-mer DNA and RNA oligonucleotides.
- Assessment of adsorption across chromatographic columns, UHPLC systems, and UV flow cells.
- Testing of diverse ONs including duplexes, varying base compositions, sugar types, and modifications.
Main Results:
- Non-specific adsorption to chromatographic instrumentation was generally negligible.
- UV flow cell material significantly affected peak shapes for larger ONs (60- to 100-mer), necessitating sample-based conditioning.
- Bioinert column hardware (walls and frits) demonstrated significant benefits in reducing adsorption compared to stainless steel.
- Any type of bioinert RPLC/HILIC column hardware provided clear advantages over stainless-steel counterparts.
Conclusions:
- The choice of column hardware is the most critical factor in minimizing oligonucleotide adsorption during chromatographic analysis.
- While UV flow cell material requires careful conditioning, column hardware material is paramount for overall adsorption reduction.
- Bioinert materials offer a substantial improvement over stainless steel for analyzing diverse oligonucleotides, aiding in method development.
