Hydrophilic Interaction Chromatography for the Analysis of Therapeutic Oligonucleotides: An Interview with Davy Guillarme

Davy Guillarme, Institute of Pharmaceutical Sciences of Western Switzerland and the School of Pharmaceutical Sciences at the University of Geneva (Switzerland)

Davy Guillarme of the Institute of Pharmaceutical Sciences of Western Switzerland and the School of Pharmaceutical Sciences at the University of Geneva (Switzerland) states in a recent paper that the U.S. Food and Drug Administration’s approval of 14 therapeutic oligonucleotides (ON) drugs since 2018 clearly shows that these drugs are on the radar for the pharmaceutical manufacturing industry (1). These ONs are complicated to analyze; however, the unprecedented need for powerful analytical methods to serve this scope makes the challenge worthwhile. While ion-pairing reversed-phase liquid chromatography (IP-RPLC) has been determined to be the benchmark for ON characterization, Guillarme said that hydrophilic interaction chromatography (HILIC) has clearly emerged as an alternative option. The paper critically examines recent advances in HILIC-mass spectrometry (MS) analytical developments for the analysis of therapeutic ONs and their impurities, as well as reporting on several applications of HILIC-MS in the context of pharmaceutical analysis and bioanalysis. Guillarme recently spoke with LCGC International about his work analyzing therapeutic oligonucleotides.

What are oligonucleotides? How do they differ from nucleotides? What is their benefit?
Oligonucleotides are polymeric molecules made up of 20 to 100 nucleotide units. Each nucleotide consists of a nitrogenous base (either purine or pyrimidine), a sugar unit, and a phosphate group. As a new generation of drugs, oligonucleotides are gaining increasing popularity. Currently, 20 oligonucleotide-based products have been approved by the Food and Drug Administration (FDA) in the United States, and many more are in development pipelines at various pharmaceutical companies.

Why is the accurate characterization analysis of oligonucleotides important?
Oligonucleotides are produced through solid-phase chemical synthesis, a multi-step process where nucleotides are added sequentially. This method can generate various impurities that may significantly affect the drug's efficacy and toxicity. Therefore, it is crucial to develop analytical methods to assess the purity of oligonucleotide products.

In your paper (1), you discuss current challenges in hydrophilic-interaction chromatography (HILIC) for the analysis of therapeutic oligonucleotides. Can you briefly explain these challenges?
There are several challenges associated with using HILIC for oligonucleotides. Firstly, the selectivity of closely related peaks in HILIC is often more limited compared to ion-pairing reversed-phase liquid chromatography (IP-RPLC), necessitating the exploration of new stationary and mobile phase conditions. Additionally, oligonucleotides have a significant number of free negative charges, leading to more pronounced adsorption on the metallic parts of the system in HILIC. In IP-RPLC, these charges are partially masked by ion pairing agents, reducing such issues. This can cause recovery and peak tailing problems in HILIC. Finally, although HILIC can be easily coupled with MS without ion pairing agents, it has limited sensitivity due to the presence of alkali ion adducts.

You state in your paper that IP-RPLC is the “gold standard” for oligonucleotide characterization. What benefits does this technique offer?
IP-RPLC is an effective strategy for analyzing polar charged compounds like oligonucleotides. These molecules are not retained by conventional RPLC using a C18 column, but the formation of an ion pair between the negatively charged phosphate group and a positively charged alkylamine makes the oligonucleotide more hydrophobic, allowing for compatibility with C18 column analysis. In IP-RPLC, oligonucleotides are primarily eluted based on their lengths, and it is possible to separate modified oligonucleotides (such as phosphorothioate oligonucleotides, locked nucleic acids, methylated cytosine). Additionally, adding HFIP to control the mobile phase pH enhances MS sensitivity compared to using acetate in IP-RPLC. This method, utilizing alkylamine and HFIP, has been in use since 1997 and is currently recognized by regulatory authorities.

You also state that HILIC is emerging as an attractive alternative option. Why is this? What benefits does it offer that surpass IP-RPLC?
HILIC is emerging as an alternative to IP-RPLC. While it may offer lower selectivity and higher susceptibility to adsorption, its key advantage lies in its inherent compatibility with mass spectrometry (MS). Unlike IP-RPLC, HILIC does not require the use of alkylamines, which can contaminate the MS instrument. Instead, the mobile phase in HILIC typically consists only of ammonium acetate, which poses no issues for MS.

Can you briefly describe your “hands-on” experience with these techniques for oligonucleotide analysis?
HILIC is a relatively straightforward approach for analyzing oligonucleotides, unlike ion pairing reversed-phase chromatography, which can be more challenging experimentally. However, a critical issue with HILIC is the adsorption of oligonucleotides on the metallic parts of the column, due to the absence of ion pairing reagents and the potential for numerous ionic interactions. Therefore, using a bioinert column is necessary and it is now available from several suppliers. Even with a bioinert HILIC column, it is strongly recommended to perform a few sacrificial injections of oligonucleotides on a new column to condition it properly. Additionally, plastic glassware should be used in HILIC-MS experiments to minimize alkali ion adducts.

Was there anything you observed that was unexpected in terms of analysis results and that stands out from your perspective?
Chromatographic selectivity of oligonucleotides in HILIC is often limited and generally lower than in ion pairing reversed-phase chromatography. There is a clear need to further optimize mobile phase conditions and develop more suitable stationary phase chemistries than those currently available. Additionally, MS sensitivity under HILIC conditions has been disappointing and lower than in ion pairing reversed-phase, mainly due to the presence of cation adducts from the glassware. Solutions must be found to reduce these adducts and improve HILIC-MS sensitivity.

Can you please summarize the feedback that you have received from others regarding this review paper?
When I recently attended the International Symposium on Hyphenated Techniques in Chromatography and Separation Science (HTC) conference in Leuven, Belgium, several colleagues mentioned that they greatly enjoyed reading this recent review paper on the use of HILIC for oligonucleotides. Additionally, colleagues have advertised this review paper on LinkedIn, highlighting it as an important resource for those interested in starting with HILIC for oligonucleotides. The paper can significantly aid in understanding the process and best practices.

What can you share about your next research steps, or new review articles you have planned?
Our next objective is to make some experiments with alternative mobile phase conditions to enhance the potential of HILIC further. Currently, most studies utilize acetonitrile (ACN) as the organic modifier, ammonium acetate as the additive, and a mobile phase temperature of 40°C. We believe better conditions can likely be identified to improve the selectivity of oligonucleotide impurities. Additionally, finding a solution to limit the formation of alkali ion adducts is another key objective of our research.

Reference
(1) Lardeux, H.; D’Atri, V.; Guillarme, D. Recent Advances and Current Challenges in Hydrophilic Interaction Chromatography for the Analysis of Therapeutic Olgionucleotides. TrAC, Trends Anal. Chem. 2024, 176, 117758 DOI: 10.1016/j.trac.2024.117758