Detecting the Major Degradation Products of Phosphorothioate Oligonucleotides by Chemical Derivatization and Weak Anion Exchange Chromatography

Abstract

Novel polar cysteine analogues have been synthesized for the derivatization of oligonucleotide depurination impurities that may be formed under acidic conditions. Depurination impurities belong to a group that includes deamination and phosphate diester impurities, which are similar in chemical structure to each other and the parent oligonucleotide, and thus coelute by most chromatographic separation methods. The polar cysteine analogues react with depurination impurities and enable their complete separation from the parent oligonucleotide by weak anion exchange (WAX) chromatography. Optimized conditions for the derivatization reaction and the WAX analysis are presented. The ability of the WAX method to chromatographically separate deamination and phosphate diester impurities is also demonstrated, and therefore, the combination of chemical derivatization and WAX chromatography permits detection and quantification of the three major degradation products of phosphorothioate (PS) oligonucleotides.

Figure 1

Example detection of the main oligonucleotide degradation products of PO, depurination, and deamination by the TBuAA IP-RP method. (A) Total ion current (TIC), and extracted ion chromatogram (EIC) traces of a mixture of three model impurity compounds Ab(A), PO, DA, and the parent oligonucleotide n, in the ratios: Ab(A):PO:n:Da 1:1:3:1. (B) Average mass spectrum (z = −4) obtained across the composite chromatographic peak of (A). Analysis done using a Waters XBridge C18 column (2.1 × 150 mm, 3.5 μm), 60–90% B in 30 min with 10 min post run time, 0.25 mL/min, 50 °C, 3 μL of 0.1 mg/mL injected.

Figure 2

Reaction of aldehyde with l-cysteine.

Figure 3

Effect of temperature on reaction. (A) Ab(A)+n (1:1) in H₂O, ambient temperature. (B) Ab(A)+n (1:1) in 25 mM l-cysteine, ambient temperature. (C) Ab(A)+n (1:1) in 25 mM l-cysteine, 50 °C, 12 min. Waters Gen-Pak column (4.6 × 100 mm, 2.5 μm), MPA: H₂O, MPB: 30% WAX Stock (20 mM Na₃PO₄, 1 M NaBr, 1 M guanidinium chloride (GnDCl), 50% MeOH, 20% H₂O), 50–95% B in 20 min, 5 min post run, 0.4 mL/min, 30 °C, 2 μL of 0.1 mg/mL injected.

Figure 4

Effect of l-cysteine ester length. (A) Ab(A)+n (1:1) in 30 mM l-cysteine methyl ester, 55 °C, 40 min; chromatography parameters: 60–90% B in 20 min, 5 min post run, 0.6 mL/min. (B) Ab(A)+n (1:1) in 30 mM l-cysteine ethyl ester, 55 °C, 40 min; chromatography parameters: 60–90% B in 30 min, 5 min post run, 0.6 mL/min. (C) Ab(A)+n (1:1) in 30 mM l-cysteine butyl ester, 55 °C, 45 min; chromatography parameters: 50–95% B in 20 min, 5 min post run, 0.4 mL/min. Column and mobile phases as in Figure 3. Other parameters: 20 °C, 2 μL of 0.1 mg/mL injected.

Figure 5

Effect of reaction pH. Ab(A)+n (1:1) in 30 mM l-cysteine ethyl ester, 50 °C, 20 min. (A) pH 4.28. (B) pH 8.95 (+30 mM triethylamine (TEA)). (C) pH 10.71 (+60 mM TEA). Column and mobile phases as in Figure 3. Other parameters: 60–95% B in 20 min, 5 min post run, 0.4 mL/min, 30 °C, 2 μL of 0.1 mg/mL injected.

Figure 6

Effect of column temperature. Ab(A)+n (1:1) in 30 mM l-cysteine ethyl ester, 50 °C, 20 min, pH 4.28. (A) 20 °C column temperature. (B) 60 °C column temperature. Other chromatographic conditions as in Figure 5.

Scheme 1

Reagents and conditions: (a) 1.5 eq. R-NH₂, 2 eq. Ni-Pr₂Et, CH₂Cl₂ (2 mL/mmol), 20 °C, 2 h, (b) TFA (5 mL/mmol), 40 eq. H₂O, 4.1 eq. Et₃SiH, 20 °C, 1 h.

Figure 7

Derivatization of Ab(A) with different cysteine polar analogue reagents. Ab(A)+n in 30 mM of each Cys-1–4 reagent, (pH 5.1, 4.83, 4.91, 5.03, respectively), 52 °C, 45 min. (A) Cys-1, MW 208. (B) Cys-2, MW 222. (C) Cys-3, MW 266. (D) Cys-4, MW 252. (E) l-cysteine methyl ester. Column and mobile phases as in Figure 3. Other parameters: 50–95% B in 20 min, 5 min post run, 0.4 mL/min, 20 °C, 2 μL of 0.1 mg/mL injected.

Figure 8

Separation and quantification of spiked Ab(A) impurity in 30 mM reagent Cys-3, 52 °C, 45 min. (A) UV chromatograms (260 nm); (B) UV area vs % amount spiked. Two μL of 0.1 mg/mL injected. Column and mobile phases as in Figure 3. Other parameters: 60–90% B in 20 min, 5 min post run, 0.3 mL/min, 20 °C, 2 μL of 0.1 mg/mL injected.

Figure 9

Effect of % MeOH and amount of sample injected on n-DA resolution. UV chromatograms (260 nm) of model oligonucleotide mixtures by WAX. MPA: H₂O. Stock solution for MPB: 20 mM Na₃PO₄, 1 M NaBr, 1 M GnDCl. (A) 30% stock solution, 20% H₂O, 50% MeOH, 10 μL of 0.1 mg/mL injected, 20 min, 5 min post run, 0.3 mL/min. (B) 25% stock solution, 5% H₂O, 70% MeOH, 10 μL of 0.1 mg/mL injected, 30 min, 5 min post run, 0.4 mL/min. (C) 25% stock solution, 5% H₂O, 70% MeOH, 2 μL of 0.1 mg/mL injected, 30 min, 5 min post run 0.4 mL/min. Other parameters: 20 °C, 60–90%B.

Figure 10

pH application study. UV chromatograms (260 nm) of 0.1 mg/mL oligonucleotide n samples at pH 7, and pH 2.2, and oligonucleotide n spiked with Ab(A) impurity, in 30 mM reagent Cys-3, 52 °C, 45 min. Conditions as in Figure 8.

Figure 11

Nature of peaks A, B, and C produced at pH 2.2 in Figure 10, by WAX x IP 2D-LC HRMS. One mg/mL oligonucleotide n at pH 2.2, in 20 mM reagent Cys-3, at 52 °C for 45 min. Average mass spectrum of: (A) Peak A in Figure 10. (B) Peak B in Figure 10. (C) Peak C in Figure 10.