Fig. 4: Optimized flow synthesis produces PMOs with similar purity to manual batch protocols.

a Synthesis variables were changed in sequence, and relative crude purities were tracked by comparison of the crude LC–MS traces. Crude purity and deletion sequences quantified, as described in Supplementary Section 6. *The Python script was edited to slowly deliver the monomers, reducing flow inaccuracies due to the viscous solution of the G monomer. N.d. = levels were below the sensitivity of detection with the described quantification method—not detected. Column colors indicate which category the variables correspond to pink—detritylation, green—neutralization, blue—coupling, and orange—instrument. Red lettering in-line 11 denotes the best observed conditions. b The total ion current chromatogram (TICC) of a batch-synthesized sample of the 18-mer PMO IVS2-654 is shown along with the mass spectrum and associated deconvoluted mass spectrum. c The TICC of the batch-synthesized sample after purification by cation exchange chromatography is shown along with the mass spectrum and associated deconvoluted mass spectrum. d The TICC, mass spectrum and deconvoluted mass spectrum are shown for the crude product from a flow synthesis of the same 18-mer sequence. e The TICC of the flow-synthesized sample after purification along with the mass spectrum and deconvoluted mass spectrum. Note: traces c and e were acquired using different LC–MS methods.