Speciality Chemicals Magazine SEP / OCT 2026 | Page 24

PHARMACEUTICALS

DCA consumption in mL

4800 4600 4400 4200 4000 3800 3600 3400

4224

DeTrityl Ultrapure

Figure 3- Average required amount of detritylation-solution for a complete synthesis

class impurity levels for impurities from n-1 to n-15, as well as for depurination( Figure 2).

The UV-monitored detritylation process showed significant differences in reagent consumption. DeTrityl Ultrapure showed an up to 8 % reduction in DCA consumption by volume compared to the Asian DCA grades( Figure 3). This led to reduced contact time of the DCA with the support and the growing oligonucleotide chain.

Discussion

The present study demonstrates that the quality of the detritylation reagent can influence oligonucleotide synthesis beyond its primary function of removing the 5’-dimethoxytrityl( DMTr) protecting group. Although all the grades investigated yielded crude oligonucleotides of acceptable quantity, significant differences were observed in the formation of critical process-related impurities.

Because these related substances can adversely affect downstream purification, overall yield, and final API quality, minimising their formation during synthesis is of considerable importance. DeTrityl Ultrapure resulted in significantly lower levels of key related substances, including N-1, N-2 and depurination products, as well as a reduced impurity burden across the broader N-1 to N-15 range.

4649 4556

A 1 Asian DCA 2

The study further showed that approximately 8 % less DCA was required to achieve complete detritylation with DeTrityl Ultrapure than with the Asian DCA grades. In addition to reducing reagent consumption, the lower DCA volume required shortens the exposure of the growing oligonucleotide chain to the highly acidic conditions of the detritylation step.

Acid-mediated side reactions, particularly depurination, are known to depend strongly on acid concentration and contact time, making detritylation conditions a critical process parameter. 4 Paul et al. demonstrated that localised acid accumulation during detritylation can affect reaction kinetics and impurity formation. 5

Consistent with these observations, lower levels of critical related substances, including depurination products and N-1, N-2 and N-3 species, were detected in the study groups with lower DCA consumption. Although the present data establish a correlation rather than a direct causal relationship, they suggest that reducing acid exposure may contribute to improved crudeproduct quality.

These findings are particularly relevant to the synthesis of longer oligonucleotides, including guide RNAs, for which cumulative

side reactions and increasingly complex impurity profiles make downstream purification substantially more challenging.

Preventing the formation of related substances during synthesis may therefore be more effective than relying solely on downstream purification to achieve the required product quality. This conclusion is consistent with a recent review identifying impurity control as a key consideration in the scalable manufacture of therapeutic oligonucleotides. 6

Because the present study evaluated only a single phosphorothioate antisense oligonucleotide sequence, further studies are required to determine whether the observed effects are transferable to other DNA and RNA sequences and to oligonucleotides with different modification patterns. Future work will also examine the impact of the improved crude-product impurity profile on downstream processing, final API purity, process yield and large-scale manufacturing performance. ●

Prof. Dr. Tobias Pöhlmann

CEO

24 SPECIALITY CHEMICALS MAGAZINE ESTABLISHED 1981