Trichloroacetaldehyde modified oligonucleotides

A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

“…Characterization and determination of the source origin of the impurities is the first step in the development of mitigation measures for prevention and elimination of their formation. Phosphorothioate oligonucleotide impurities can be classified in several categories depending on the type of reaction leading to their formation: (1) failure sequences due to coupling inefficiency, incomplete capping, or detritylation (shortmers) [20,21]; (2) molecules larger that the full length product (longmers) [22,23]; and (3) other impurity products formed by depurination, deamination, sulfur loss, thermal stress, adduct formation, and other reactions [24][25][26][27][28]. It is possible for some of these impurities to be formed by mixed consecutive or parallel secondary reactions initiated by solvent and raw materials initial impurities, process variables, or the chemical nature of the target oligonucleotide structure.…”

Chromatographic approaches for the characterization and quality control of therapeutic oligonucleotide impurities

“…Reversed‐phase liquid chromatography, especially using ion pairing agents in the mobile phase and coupling with mass spectrometric detection, has been the chief technique for the analysis of oligonucleotides impurities and degradation products (Figure ; Gilar et al, ; Nikcevic et al, ). IP‐RP‐HPLC has been conducted using several alkylammonium salts as ion pairing agents, including tripentylammonium acetate (Capaldi et al, ; Gaus et al, ), tributylamine (Roussis, ), triethylammonium formate (Gaus et al, ), triethylammonium acetate (Fearon et al, ; Fountain et al, ; Gilar, ; Gilar et al, , ; Rentel et al, ), tetrabutylammonium phosphate (Metelev & Agrawal, ), tributylammonium acetate (Kurata et al, ; Rentel et al, ) and hexylammonium acetate (Cramer et al, ; Smith & Beck, ). Moreover using a buffering system containing the ion pairing agents and hexafluoroisopropanol and triethylamine (Farand & Beverly, ; Fountain et al, ; Gilar, ; Gilar et al, , ; Li et al, ; Liu et al, ; Nikcevic et al, ) and diisopropylethylamine and hexafluoroisopropanol (Chen & Bartlett, ; McGinnis et al, ) has provided significant chromatographic separation and higher signal intensity, as well as higher selectivity and chromatographic resolution (Apffel et al, ; Basiri & Bartlett, ; McGinnis et al, ).…”

“…Generally separations have been performed using C 18 columns (Gaus et al, ; Kurata et al, ; Rentel et al, ) such as YMC (An et al, ; Capaldi et al, ), Acquity BEH (Chen & Bartlett, ; McGinnis et al, ; Smith & Beck, ), Acquity UPLC OST (Roussis, ), Xbridge OST (Roussis, ), XTerra® MS (Farand & Beverly, ; Fountain et al, ; Gilar, ; Gilar et al, , ; Li et al, ), NovaPak (Metelev & Agrawal, ), Inertsil ODS‐2 (Suzuki et al, ) and Zorbax Extend‐C 18 columns (Culf et al, ). XBridge BEH columns have shown higher‐quality separations while retaining long‐term stability and reproducibility when compared with other columns such as Xterra MS, Xbridge OST and Clarity Oligo‐RP columns (Nikcevic et al, ).…”

“…Ion‐exchange chromatography with UV detection is also an outstanding method for the separation of impurities owing to the highly charged nature of oligonucleotides (Figure ; Srivatsa et al, ). Typical mobile phases include NaCl in sodium phosphate buffers (Gaus et al, ), Tris/1 m m EDTA or Tris/1 m m EDTA/NH 4 Cl (Cohen et al, ; Li et al, ; Liu et al, ), KH 2 PO 4 and (NH 4 ) 2 SO 4 (Cohen et al, ; Metelev & Agrawal, ), Tris/NaCl (McGinnis et al, ), Tris/NaCl, Tris/NaBr and sodium phosphate dibasic/NaCl (Srivatsa et al, ), NaOH/NaCl (Fearon et al, ), ammonium phosphate, KBr and NaSCN (Bergot & Egan, ). Tris–HCl/1 m m EDTA has been used with NaCl, NaBr, NaI and NaSCN where NaSCN showed the most efficient separation (Yang et al, ).…”

“…Further investigations were done after digestion of the oligonucleotide sample with snake venom phosphodiesterase and alkaline phosphatase where HPLC‐UV analysis showed a number of small later‐eluting peaks in addition to the four natural nucleosides. Analysis of these peaks indicated a series of dinucleotides modified by the addition of two carbon, one hydrogen, one oxygen and three chlorine atoms which were added to an oligonucleotide in the form of chloral that is present as an impurity in the DCA (Gaus et al, ). Also chloral adducts in oligonucleotide samples have been detected using IP‐RP‐HPLC coupled with high resolution Fourier transform mass spectrometry (Nikcevic, Wyrzykiewicz, & Limbach, ).…”