High-Resolution HPLC for Separating Peptide-Oligonucleotide Conjugates

Abstract

Peptide-oligonucleotide conjugates (POCs) are chimeric molecules that combine the specificity of oligonucleotides with the functionality of peptides, improving the delivery and therapeutic potential of nucleic acid-based drugs. However, the analysis of POCs, particularly those containing arginine-rich sequences, poses major challenges because of aggregation caused by electrostatic interactions. In this study, we developed an optimized high-performance liquid chromatography (HPLC) method for analyzing POCs. Using a conjugate of DNA and nona-arginine as a model compound, we systematically investigated the effects of various analytical parameters, including column type, column temperature, mobile-phase composition, and pH. A column packed with C18 resin with wide pores combined with butylammonium acetate as the ion-pairing reagent and an optimal column temperature of 80 °C provided superior peak resolution and sensitivity. The optimized conditions gave clear separation of POCs from unlinked oligonucleotides and enabled the detection of nucleic acid fragments lacking an alkyne moiety as a linkage part, which is critical for quality control. Our HPLC method is robust and reproducible and substantially reduces the complexity, time, and cost associated with the POC analysis. The method may improve the efficiency of quality control in the production of POCs, thereby supporting their development as promising therapeutic agents for clinical applications.

1

Compounds used for the LC analysis in this study.

2

Chromatograms of the crude conjugated product (pink lines) and blank (black lines) obtained with different columns. (a) Accura Triart C18 (pore size: 12 nm), (b) Accura Triart Bio C18 (pore size: 30 nm), and (c) Accura Triart Bio C4 (pore size: 30 nm).

3

Chromatograms of the crude conjugated product obtained with different mobile-phase solutions: (a) 15 mM TEA, 400 mM HFIP/MeOH, (b) 100 mM DEAA (pH 7.0)/ACN, (c) 100 mM TEAA (pH 7.0)/ACN, and (d) 100 mM BAA (pH 7.0)/ACN.

4

Chromatograms of the crude conjugated product obtained with different gradient elution conditions where the percentage of mobile-phase solution B (ACN) was gradually increased: (a) 7–17% B (0–10 min), 100% B (10–15 min); (b) 7–17% B (0–5 min), 100% B (5–10 min); (c) 5–25% B (0–5 min), 100% B (5–10 min); (d) 7–27% B (0–5 min), 100% B (5–10 min); (e) 10–30% B (0–5 min), 100% B (5–10 min); and (f) 10–40% B (0–5 min), 100% B (5–10 min). Black arrows indicate R9-ER­(dec)-R peaks. Red triangles indicate shoulder peaks partially separated from the R9-ER­(dec)-R peaks.

5

Chromatograms of the crude conjugated product (pink lines) and blank (black lines) obtained with BAA buffer at pH (a) 6.0, (b) 7.0, and (c) 8.0. Red triangles indicate shoulder peaks partially separated from the R9-ER­(dec)-R peaks.

6

Chromatograms of the crude conjugated product (pink lines) and blank (black lines) obtained at column temperatures of (a) 40, (b) 60, (c) 70, and (d) 80 °C. Red triangles indicate shoulder peaks partially separated from the R9-ER­(dec)-R peaks. At 40 °C, no elution of R9-ER­(dec)-R was observed, although the UV absorption signal showed a slight increase from 7 to 9 min (green triangle).

7

Chromatograms of the crude conjugated product (R9-ER­(dec)-R, pink line), unmodified oligonucleotide (ER­(dec)-R, brown line), and modified oligonucleotide (5′-Hexynyl-ER­(dec)-R, orange line).