Liquid chromatography
Therapeutic oligonucleotides place high demands on analytical method development, selectivity and robustness. Variations in sequence length, modifications and impurity profiles require precisely tailored chromatographic strategies. Reproducible and high-performance separation methods are becoming increasingly important, particularly in development, quality control and process monitoring.
Two new Expert Tips are aimed at scientists working in pharmaceutical research, bioanalytics, quality control and analytical development. The content supports the efficient selection of suitable chromatographic conditions for the analysis of therapeutic oligonucleotides. The focus is on providing structured guidance for method development using IP-RP and AEX chromatography.
The documents combine the latest scientific findings with practical recommendations. Tables, selectivity overviews and methodological guidance facilitate the selection of suitable ion-pair systems, buffer systems, temperatures and stationary phases. At the same time, the content supports the faster development of robust, reproducible and MS-compatible methods.
Drawing on many years of expertise in chromatographic materials, bioinert technologies and oligonucleotide separations, YMC continues to support laboratories developing next-generation analytical methods for therapeutic oligonucleotides, mRNA analysis and process control.
The Expert Tip describes the methodological selection of suitable IP-RP conditions for the analysis of oligonucleotides of varying lengths. IP-RP chromatography is widely regarded as the gold standard for the separation and characterisation of therapeutic oligonucleotides. The focus is on the targeted combination of ion-pair system, temperature, organic modifier and stationary phase.
The overview structures the method selection according to oligonucleotide length. Short oligonucleotides require more hydrophobic ion-pair reagents to ensure sufficient retention. Medium sequence lengths provide high robustness, precise peak shapes and strong LC-MS compatibility. Long oligonucleotides benefit from elevated temperatures and optimised ion-pair concentrations to reduce secondary structures and peak broadening.
The influence of pore size, temperature and stationary phase on selectivity and elution behaviour is also explained. Particular attention is paid to LC-MS-compatible TEA/HFIP systems as well as alternative approaches without fluorinated alcohols.
Application examples from the YMC Application Database complement the theoretical principles. The content supports the precise and efficient development of robust methods for ASOs, siRNA, sgRNA and other therapeutic oligonucleotides.
The second Expert Tip describes the methodological selection of suitable conditions for anion exchange chromatography of oligonucleotides of varying lengths. The focus is on the targeted optimisation of the buffer system, salt gradient, temperature and denaturing conditions.
The overview structures the choice of method according to oligonucleotide length. Short sequences exhibit low retention and often benefit more from RP or IP-RP methods. Medium-length oligonucleotides represent the optimal working range for AEX. The method provides high length selectivity, robust separations and precise resolution of n-1 and n+1 impurities.
Long oligonucleotides require elevated temperatures and denaturing conditions to reduce secondary structures and peak broadening. Very long sequences exhibit reduced chromatographic resolution due to high structural complexity and small relative charge differences.
The document also explains the influence of organic modifiers, salt gradients and denaturants on retention and selectivity. Particular attention is given to ammonium-based buffer systems for MS-compatible workflows, as well as optimised conditions for phosphorothioate-modified oligonucleotides.
YMC application examples complement the methodological recommendations and support the efficient development of high-performance AEX methods for therapeutic oligonucleotides, mRNA analysis and process control.
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