The Pentelute Lab at MIT uses Biotage® Selekt flash chromatography to rapidly purify synthetic peptides, accelerate hit discovery and move research forward with less time spent on purification.
Dr. Bradley Pentelute is a Professor in Chemistry at the Massachusetts Institute of Technology (MIT), where his team works on projects that include the development of new chemistries for site-selective modification of natural proteins, designing peptides and proteins for intracellular cargo delivery, and discovery of peptide binders for various protein targets.
We spoke with Wayne Vuong, PhD, and Alexia Telios, two members of Prof. Pentelute’s team, to learn how they are using flash chromatography to purify synthetic peptides and other molecules of interest to move their research forward.
Dr. Vuong: That's a great question. The use of the peptides spans a pretty broad range, with our overall theme being peptide applications across different areas. This includes pharmaceuticals, of course, but also extends to biological tools, discovery, development, and diagnostics.
We also work in the functional peptide space, like cell penetrating peptides. The projects I've personally worked on involve pharmaceutically relevant compounds as well as tool discovery and just developing research tools to probe biological systems, using peptides as targeting entities to do that.
Dr. Vuong: Our lab uses custom automated synthesizers to do flow chemistry, which allows us to rapidly generate sequences of interest. I'll phrase my answer in the context of hit discovery from library screening, typically from a one-bead-one-compound peptide library, though we have now other versions as well.
We undergo affinity selection mass spectrometry campaigns, and from these we discover putative hits and sequences of interest. Each of these is then rapidly synthesized using our automated synthesizers. For example, a 10-mer peptide can be synthesized in about half an hour. After cleavage using standard protocols, one of the nice things about the Biotage® Selekt system is that we can take our cleaved material after it's precipitated out and head directly into purification.
In the hit discovery workflow, we very rapidly want to have the answers for the functionality of sequences that we've discovered, and the Biotage® Selekt allows us to quickly purify these sequences up to around 80% purity. At that point, we can attach them to probes and move directly to assays like BioLayer Interferometry (BLI), experiments to look at cursory kinetic binding data.
Dr. Vuong: Yes, there was one time that I ended up having a list of about 30 sequences, and we were able to synthesize, cleave, and purify all of them within a week. It just wouldn't be possible with an HPLC system, purely due to the speed of runs with the Biotage® Selekt. Especially because we have two of them, we can run both at the same time. A single purification might take around 20 minutes, and you can very rapidly get usable, even excessive quantities, of material, purified to a point that’s fully usable in your subsequent assays.
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30 sequences Synthesized, cleaved and purified within a week |
~20 min Per purification run |
Dr. Vuong: I can't speak for everybody in our lab, but from my own personal experience, I have a handful of different gradients that I choose from based on what I believe the peptide of interest will elute in. Generally speaking, because the system is so fast, we don't really need to do much method optimization. We just have a gradient that spans a good range of %B solvent, and that's able to just elute off.
Because we get a very nice and easy readout, we can quickly identify the relevant fractions, check them on an LCMS, and get our answer fast. So overall, it's very easy to pick up, and there's very little optimization needed. Of course, you can fine-tune it a lot, but for my purposes, it's been a very reliable general instrument.
One thing I like about it is that, unlike with HPLC, it’s less sensitive to clogs because we're working with such varied sequence compositions, solubility can fluctuate a lot from one sequence to another. Having the ability to purify something that you cannot immediately get fully into solution is a big advantage and one that I think is often underappreciated for the type of work that we do.
Dr. Vuong: Generally, it's fine. We're able to get enough material out of our synthesizers that we can just inject a good amount into the Biotage® Selekt and still get sufficient, usable quantities at the end. You have two advantages there: as you ramp up the % B, you'll probably wash everything out anyway, and things will dissolve. Secondly, if the column really does clog, it's not super expensive to replace.
Alexia Telios: As someone who started to do full time peptide synthesis recently and has been learning all these methods, I’d say the Biotage® Selekt system was one of the easiest purification methods to use and the least intimidating for a beginner. It's easy to replace these columns that are cheaper, and the system’s user interface is very straightforward, making it easy to use.
Dr. Vuong: Actually, no, I have not seen the system become over-pressurized. There have been times when I couldn’t physically inject something, but the pump still works. If I'm able to get the tubing screwed on, it'll still go, which is nice. The Biotage® Selekt can go up to a gram of material, and in peptide chemistry, it's rare to go past 100 mg or so. A column for that is around USD 100, whereas an HPLC column would cost a couple of thousand dollars, at least.
Overall, it's really easy to use, everything's visual. Everything's pretty self-explanatory in terms of the user interface perspective, making it easier to train new users quickly.
Dr. Vuong: Well, without it, you’d be looking at maybe an hour and a half per purification, compared to just 20 minutes. That's a, what, three, four, maybe five times increase in your purification time.
It's also difficult to get simultaneous access to HPLC systems, whereas the Biotage® Selekt, we have two systems we can use at the same time. And since purification times are shorter, there’s less competition for system time, so access is easier. Without the Biotage® Selekt, the process would be an order of magnitude slower, five times slower, maybe even ten.
Another benefit of the Biotage® Selekt is that the material doesn't elute over such a long gradient, so the volumes that you need to reduce at the end, through lyophilization, are smaller. So to answer your question, without it, purification might take one to two weeks, instead of just a couple days.
Alexia Telios: We had a few peptides we needed to finish quickly, including some cyclized peptides that were slightly challenging to purify, just based on the LCMS data. But we decided to give the Biotage® Selekt a try, and it gave us some pretty pure fractions.
In terms of timing, it's been really great. For many cases, I've been using mass-directed purification (MDP) to get the mass data. However, when I know there are certain retention time differences, and where my product is expected to be at a certain retention time, Biotage® Selekt system has been great. It’s easier than MDP because with some prior knowledge about where to look for the molecule, transitioning directly from analytical LCMS to the Biotage® Selekt system is straightforward.
Literature number: CC05