Scale Purification of Therapeutic Oligonucleotides

After purification the DMT group from the oligonucleotide is removed by acid treatment, such as, acetic acid or some other milder acid in aqueous solution. After final detritylation, the salts if excessive are removed via precipitation, and then the product is lyophilized. Several process improvements have been tried and implemented to enable processing at the kilogram scale in our GMP facilities.1,47 

The final aqueous detritylation is a complicated step that requires careful process optimization, such as control of pH, oligo and salt concentrations etc. 49 After detritylation, the oligonucleotide is precipitated quantitatively from the acidic DMT cation containing solution under optimized conditions. This step can be labor intensive at the large scale, and may be inconvenient for the high-throughput small-scale synthesis. One way to circumvent the problem is to use on column detritylation, where the RP and detritylation steps are combined in one chromatographic operation. Since the acid can leach the silica based columns, this is more useful on polymeric supports. 

FIGURE 8 AX Purification of DMT-off oligonucleotide crude. A 20 mer phosphorothioate, DMT-off crude was purified on a AX column (POROS HQ/50, PE BioSystems). A 200 mL column (50 X 200 mm length) was used and approximately 10 g of sample was loaded on the column ( 50 mg / mL column volume). Buffer A 20 mM NaOH Buffer B 20 mM NaOH + 2.5 M NaCI. Optimized gradient was used, with a flow rate of 35 mL/min (107 cm/hr). The experiment was performed on the BioCad 60 workstation and monitored at A = 260 and 290 nm. 

Additional results from AX purification of DMT-off synthetic oligonucleotides are discussed in the following section, based on the principles of displacement and sample self-displacement chromatography. 

Displacement Chromatography and Sample Self-Displacement Chromatography 

---