Development of Enhanced Separation Techniques for Oligonucleotides Utilizing Mixed-Mode Chromatography and 2D-LC/UV/MS Analysis

Abstract

Oligonucleotides are traditionally analyzed using ion-pair reversed-phase liquid chromatography. In this project, we explored an alternative approach utilizing a reversed phase-weak anion exchange mixed-mode column, which combines separation based on both charge and hydrophobicity. Our findings indicate that the mixed-mode column provided stronger retention and superior separation compared to the C18 column, eliminating the need for ion-pairing reagents. Additionally, selectivity can be adjusted using gradient pH and buffer concentration as well as acetonitrile content. However, the use of a phosphate buffer was necessary to ensure adequate retention and separation, despite its incompatibility with MS detection. To address this issue, a 2DLC-UV-qTOF setup with multiple loops was established, where the second-dimension LC, connected to the MS detector, required only an MS-compatible mobile phase. Using a HILIC-type LUNA Omega Sugar column in the second-dimension LC, the MS intensity of the heart-cut from the first dimension was increased 100-fold, allowing for the characterization of the main peak.

Keywords: 2D liquid chromatography; mass spectrometry; mixed‐mode chromatography; oligonucleotide; reversed‐phase/weak anion‐exchange.

FIGURE 1

Illustration of 2DLC setup, with focus on the different valve's positions. (A) ¹D‐separation of oligonucleotide sample, (B) heart cut of ¹D eluent and collection in loop, (C) injection of stored loop volume onto the ²D‐system. Finally, (D) shows a schematic of the multi‐loop setup.

FIGURE 2

Analysis of degraded oligonucleotide sample on the Acclaim Mixed‐Mode RP/WAX‐1 column with an ammonium phosphate mobile phase buffer and 30% ACN with a pH and buffer concentration linear gradient from pH 7 (100 mM) to pH 8 (200 mM).

FIGURE 3

Analysis of oligonucleotide sample with impurities on the Acclaim Mixed‐Mode RP/WAX‐1 column with an ammonium phosphate mobile phase buffer and 30% ACN and pH and buffer concentration gradient from pH 7 (100 mM) to pH 8 (200 mM). Chromatograms illustrate different gradient settings with (A) 40%−100% B; (B) 80%−100 % B; and (C) 90%−100% B.

FIGURE 4

Analysis of oligonucleotide sample with impurities on the Acclaim Mixed‐Mode RP/WAX‐1 column with an ammonium phosphate mobile phase buffer and 15% (A, bottom) to 35% (E, top) ACN in 5% increments; pH and buffer concentration gradient from pH 7 (100 mM) to pH 8 (200 mM).

FIGURE 5

Changes in retention time for main oligonucleotide and selected impurities as a function of ACN content in the mobile phase.

FIGURE 6

Analysis of oligonucleotide sample with impurities on the Acclaim Mixed‐Mode RP/WAX‐1 column at different temperatures from 30°C (A) to 50°C (E) in 5°C increments. The mobile phase comprised an ammonium phosphate mobile phase buffer and 20% ACN with a pH and buffer concentration linear gradient from pH 7 (100 mM) to pH 8 (200 mM).

FIGURE 7

Overlay of UV‐traces from the (red) ¹D method using Acclaim Mixed‐Mode RP/WAX‐1 column with ammonium phosphate buffer and 30% ACN, with 30 min pH  (7−8) and concentration (100−200 mM) gradient; and the (black) ²D method using a 250 µL loop and 1 min heart cut of the main oligonucleotide peak at 24 min injected on LUNA Omega SUGAR stationary phase with a 66.5%–19 % ACN gradient over 15 min in water. The cut‐out shows the zoomed‐in area around heart‐cut region. Peak labelling according to Table 1: (b) n‐2 short‐mer, (d) n‐1 short‐mer, (j) main 16‐mer oligonucleotide.

FIGURE 8

MS spectrum from heart cut main oligonucleotide peak at 28 min in previous Figure 7: ¹D method using Acclaim Mixed‐Mode RP/WAX‐1 column with ammonium phosphate buffer and 30% ACN, with 30 min pH  (7−8) and concentration (100−200 mM) gradient; ²D method using a 250 µL loop and 1 min heart cut of the main oligonucleotide peak at 24 min injected on LUNA Omega SUGAR stationary phase with a 66.5%– 9% ACN gradient over 15 min. Letters in zoomed in section corresponds to (a) Na‐adduct, (b) K‐adduct, (c) Fe‐adduct, (d) ACN‐adduct, (e) P‐adduct, (f) P+Na‐adduct, (g) P+K‐adduct.