Real-Time Process Monitoring Ammonolysis of Oligonucleotides with Online FTIR Spectroscopy

In recent years, oligonucleotide drugs, as a novel class of drug molecules, have provided new treatment modalities for patients with chronic diseases. Although the traditional synthesis process system for oligonucleotide drugs is continuously improving and maturing, the ammonolysis process still commonly uses offline methods. Especially during industrial scale-up, limitations such as difficulties in material transfer, nonuniform sampling, and monitoring of high-concentration supersaturation have highlighted the lack of an efficient, real-time, and accurate online control system for monitoring the oligonucleotide ammonolysis process. In this study, a novel online ammonolysis process system for oligonucleotides was constructed for the first time. This system is equipped with online Fourier transform infrared spectroscopy (FTIR), enabling real-time monitoring and endpoint control of the oligonucleotide ammonolysis process. The experimental results showed that under conditions of different temperatures and various types of solid supports, this method can stably and accurately reflect the oligonucleotide ammonolysis process. The detection results at multiple wavelengths (IR 1232 cm –1, 1457 cm –1, 1504 cm –1, 1635 cm –1 ) are comparable to the traditional offline ultraviolet (UV) detection method (UV 260 nm) (Pearson correlation coefficient r > 0.9). To the best of our knowledge, this study is the first to achieve real-time online infrared monitoring and control of the ammonolysis process for oligonucleotides within the public literature. This achievement lays a solid foundation for deploying IR process analytical technology on an industrial scale and realizing online quality control and process optimization for the production of oligonucleotide drugs.