Last week, at the TIDES conference in Boston, Martin Deetz, PhD, research laureate at DuPont, and his colleagues presented a poster on using reverse-phase high-performance chromatography (RP-HPLC) to purify DNA and RNA oligos. These oligos are “used in a wide variety of applications from research to diagnostics to therapeutics,” Deetz says. “Today’s oligo-based therapeutics mostly focus on gene silencing, but there are many other gene-modification technologies being explored.” Purification makes up a key step in oligo bioprocessing.
After synthesis, an oligo solution is only 40–80% pure. “There are a lot of salts and small molecules and failure sequences,” Deetz explains. “There are many ways to accomplish the purification, but most of them really rely on some form of chromatography—most commonly reverse phase chromatography or ion exchange.”
The diversity of potential oligos makes purification challenging. As an example, Deetz points out that a DNA oligo “of just 20 nucleotide bases has over a trillion possible sequences.”
To create a process that produces the desired oligos, many factors must be considered. “You really have to find the right option that matches your target molecule,” Deetz explains. To do that, a bioprocessor must consider the impurities to be removed, as well as the production platform and facility. Plus, the composition and manufacturing of the chromatography resin plays a key role.
Addressing all of those variables depends on experience. “At the end of the day, the process developers really need to be familiar with multiple chromatography techniques,” according to Deetz.
In thinking about taking on the purification of an oligo, Deetz notes that he would start by “thinking about the specific oligo chemistry that I have and pair it with the right mode of chromatography.” For longer sequences, he turns to RP-HPLC. Then, as Deetz and his colleagues showed at TIDES, finding the most efficient resin takes testing. Only then can a bioprocessor find the best path to purifying a specific oligo.