HPLC, particularly preparative HPLC, has become increasingly important in the isolation and purification of products used in biological research, diagnostic assays, and drug development. With that trend, a focus on improving efficiency by increasing yields and/or shortening purification times has become more important as laboratories try to do more with less. In addition to improving efficiencies, laboratories need better insight into their results, so they can identify issues earlier in the process and make the necessary adjustments sooner.
In this article, Joshua Pletzke, a chemist at Luminex, will help provide a better understanding of some of the challenges chemists face in large-scale purification of oligonucleotides and also explain how the chemists at Luminex are tackling those issues.
Q: Can you briefly discuss the work you do at Luminex, particularly in relation to oligonucleotide synthesis?
A: I spend a significant amount of time synthesizing our novel phosphoramidites and nucleotide triphosphates using the necessary bench chemistry techniques. I’ve also been trained to synthesize R&D oligonucleotides on two different synthesizers and carry them throughout the synthetic process, all the way through final testing for purity, quantification, and identification. I also spend a significant amount of time performing analytical method development for oligonucleotides using the HPLC and capillary electrophoresis (CE) equipment at Luminex Madison. I carry out the method development through its conception, feasibility, and all the way through the necessary validations.
Q: What challenges were you experiencing when conducting oligonucleotide synthesis?
A: There were two main challenges that Luminex Madison experienced prior to the installation of our GX-271 system. One major issue was always scale. The high-throughput preparatory design has allowed us to purify larger amounts of oligonucleotide per purification run. We also actively use our Gilson systems for purification of some of our other materials, like our phosphoramidites and triphosphates.
Another challenge was the fact that all of our other preparatory systems were only capable of monitoring single UV wavelengths during purification. The GX-271’s ability to monitor up to four separate UV wavelengths has allowed us to dissect our purifications, and instantly know more about the peaks contained within the chromatogram. This actively helps us makes quick determinations on the performance of our method designs, as well as helps us understand more about the composition of our crude mixtures.
Q: What improvements have you been able to make to your workflows?
A: Obviously, by being able to purify larger amounts of oligonucleotides and other synthesized materials at Luminex Madison, we’re able to shorten our lead times for synthetic goods. In some instances, our purification injection volumes were actually doubled. Consequently, this allowed us to cut down the time needed for our purification in half.
From a research perspective, our ability to create methods quickly and dissect purification chromatograms down using multiple wavelengths allows us to see how our purifications actively work without having to separately run additional multiple wavelength analytical methods. This has helped us better understand our synthetic performance, and helps suggest when we may need to make synthetic adjustments prior to purification. It also allows our chemistry group to remain flexible and adaptive in a dynamic work environment.
Q: In what ways are you now able to achieve your goals and overcome previous barriers?
A: Our chemistry group goes through our work responsibilities at a breakneck pace. Any ability to shorten our lead times and carry out more necessary repetitions in a shorter amount of time is always welcome. Our new system also allows us to investigate new purification methods easily and refine the methods we currently use. Although, it’s the hope that every purification would go perfectly, that is not the case in reality, and our new GX-271 system allows us to troubleshoot and investigate aberrant results by making use of multiple wavelengths, easily altering our method parameters, cutting smaller or larger fractions, etc. Admittedly, we still need to do some more work in order to maximize our productivity using the analytical HPLC capabilities of the GX-271 system.
Q: What are the greatest benefits of the new system in the lab?
A: For me, personally, the large-scale throughput and built-in investigational ability compared to anything else we use on site saves me countless hours a year. I wish I had more time to investigate and experiment with the system because I bet I could find more capabilities available to me to make me even more efficient.
Q: Does the system improve the results of your oligonucleotide synthesis?
A: Yes. It allows us increased throughput and increased investigational ability. This shortens our lead times, and allows us to quickly optimize our purification methods in order to produce better quality chemical compounds and oligonucleotides.
The Gilson GX-271 Oligo Purification System provides automated preparative and analytical HPLC capabilities that can be used to isolate, analyze, and desalt oligonucleotides in one continuous run. The large injection volumes for preparative work speeds up the process, while the ability to monitor up to four wavelengths simultaneously improves analysis, allowing for quicker decisions on method design performance. These features and others of GX-271 system tackle many of the challenges in oligonucleotide purification, improving lab efficiency.