Background Antisense oligonucleotide (ASO) synthesis is conducted on a solid-phase support by flowing phosphoramidites (ASO building blocks) one unit at a time to build the chain of nucleotides which make up the ASO. ASO sequences in Biogen’s pipeline contain up to eight different phosphoramidites. The crude intermediate (after synthesis) is sampled and tested off-line by high resolution mass spectrometry to confirm that the correct sequence was made. Goal/Opportunity: Develop a robust in-line Process Analytical Technology (PAT) method to identify incoming phosphoramidites during oligonucleotide synthesis in order to obtain real-time ASO sequencing. This would eliminate the need for a laborious sequencing release test and can be used to identify synthesis errors in real time thus reducing downstream efforts and the associated costs. Methods: A principal component analysis (PCA) model based on different types of phosphoramidites from different lots and vendors was established via off-line mid-infrared measurements. A high-pressure attenuated total reflection (ATR) flow cell was connected to the ASO synthesizer to collect real-time spectra as a proof-of-concept study. However, this flow cell created extra volume that may impact the process. Therefore, a customized transmission flow cell combined with fiber optics has also been evaluated. Results: The off-line PCA model was optimized via a series of data processing and key parameter optimizations. All eight phosphoramidites were uniquely identified by this model. Both high-pressure ATR and transmission flow cells were shown to be amenable for in-line implementation with the synthesizer. The phosphoramidites spectra were collected in real-time and unknown amidites were successfully identified. Conclusions: Real-time phosphoramidites identification and ASO sequence confirmation was achieved via an in-line mid-IR PAT method. These results provide evidence that PAT can be used in ASO manufacturing. Furthermore, implementation of this method removes the need for the more complex sequencing release test which provides data after the downstream process has already been completed.

Process analytical technology for flow synthesis of antisense oligonucleotides: Real-time starting material assurance and ASO sequence confirmation

255th National Meeting (2018)

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