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

Abstract

Abstract.A new method is presented for determining relationships between components in complex analytical systems. The method uses the mass differences between peaks in high resolution electrospray ionization (ESI) mass spectra. It relates peaks that share common mass differences. The method is based on the fundamental assumption that peaks in the spectra having the same exact mass difference are related by the same chemical moiety/substructure. Moreover, the presence (or absence/loss) of the same chemical moiety from a series of molecules may reflect similarities in the mechanisms of formation of each molecule. The determined mass differences in the spectra are used to automatically differentiate the types of components in the samples. Contour plots and summary plots of the summed total ion signal as a function of the mass difference are generated, which form powerful tools for the rapid and automated determination of the components in the samples and for comparisons with other samples. For the first time, in this work a unique profile contour plot has been developed that permits the interactive interrogation of the mass range by mass difference data matrix to obtain valuable information about components that share a common mechanism of formation, and all possible mechanisms of formation linked to a selected precursor molecule. The method can be used as an additional and complementary method to the existing analytical methods to determine relationships between components in complex chemical systems.

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

“…5′ ‐O‐ DMTr adducts have been detected in an oligonucleotide sample using IP‐RP‐HPLC coupled with high‐resolution FT‐MS (Nikcevic et al, ). Also LC–MS has been used for the analysis of purified oligonucleotides with the DMTr group either on or off (Roussis, ).…”

“…Under the strong basic conditions used in oligonucleotide deprotection, cyanoethyl adducts can react with the hetereocyclic bases, mostly thymine, to give a full‐length oligonucleotide in which thymine is replaced by an N 3 ‐cyanoethylthymine residue (Figure d). LC–MS data analysis reveals the presence of an impurity with a characteristic mass 53 Da greater than the full‐length oligonucleotide corresponding to the N 3 ‐cyanoethylthymine (Capaldi et al, ; Roussis, ). Further investigations were done after digestion with snake venom phosphodiesterase and alkaline phosphatase where RP‐HPLC analysis of the products indicated the presence of a later‐eluting product in addition to thymidine and LC–MS confirmed its identity as N 3 ‐cyanoethylthymidine.…”

“…Similar separations have been accomplished using IP‐RP‐HPLC, resulting in improved selectivity and reproducibility when compared with capillary gel electrophoresis and ion‐exchange HPLC for the same set of compounds (Li et al, ). The PO analogs have been identified using both ESI‐MS (Gilar et al, ; Roussis, ; Smith & Beck, ) and MALDI‐TOF‐MS (Li et al, ; Liu et al, ; Yang et al, ). In addition, 31 P–NMR spectroscopy has been used for further examination of PO analogs (Bergot & Egan, ; Smith & Beck, ; Srivatsa et al, ; Yang et al, ).…”

“…The 3′‐TPT monoester has been analyzed using capillary gel electrophoresis, strong anion‐exchange HPLC and 31 P NMR spectroscopy (Ravikumar et al, ). LC–MS has also been used successfully to characterize and identify the monoester (Ravikumar et al, ; Roussis, ). This class of process‐related impurity can be reduced by a simple reverse of the way in which the first deoxynucleoside is attached to the solid support.…”

“…Increasing the contact time between phosphoramidites and the solid support and increasing the acidity of the activator used in the coupling step will both contribute to higher levels of longmer impurities. Analysis of these products has been achieved using capillary gel electrophoresis (Krotz et al, ) and LC–MS (Gilar et al, ; Roussis, ). Longmers may also be produced as full‐length oligonucleotides containing branched impurities consisting of an additional oligonucleotide chain attached via a P(O)SH group either to the exocyclic amino group where the benzoyl group that is normally used for the protection of this position is absent (Figure i) or on the 3′‐terminal hydroxyl group (Figure j).…”