In-line Phosphoramidite Identification by FTIR to Support Real-Time Oligonucleotide Sequence Confirmation

A classification method was developed for the identification of antisense oligonucleotide phosphoramidite starting materials to support sequence confirmation in real-time during solid-phase oligonucleotide synthesis. Calibration data were collected using spectral data from a Fourier transform infrared analyzer and an attenuated total reflectance (ATR) probe. Eight different phosphoramidites were prepared in acetonitrile and sampled in-line using the ATR probe under various concentrations, flow conditions, and instrument configurations to reproduce anticipated process variability. Additional test sets were constructed from spectra that represented specific types of variability to challenge the model and set forth boundaries to define a prediction space. The identification model was confirmed suitable for checking the oligonucleotide sequence as an in-process and real-time release test.