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HTS becoming game of the fast and the curious
Bigger may be better when it comes to licensing fees, royalties and sales for new drugs, but smaller is where you want to go with screening technologies. So, with many thousands or even millions of candidates to go through, it's a good thing high-throughput screening is finding ways to get smaller and use less material. More and more, companies are embracing the 1536-well plate formats and using increasingly sophisticated robotics and microfluidics technologies to make their libraries more fruitful.
But as 1536 potentially edges toward being par for the course, some instrument providers are already looking toward 3456-well plate formats and more pharma and biotech companies are pushing the envelope by trying to get high-throughput performance out of cell-based assays.
A lot of this is possible and even productive now, note some experts in the field. But the question is: "Is it worth it?" And in a few cases, they note, that answer may be "no."
Making 1536 the norm
Dr. Richard Eglen, from his perspective of president of PerkinElmer's Bio-discovery business unit, says most of the major HTS groups that serve as PerkinElmer's customers are now using 1536-well formats.
"You have 384 wells in almost every screening laboratory right now. That's pretty much a given," Eglen says. "But many of those who are doing larger volumes are almost standardized now on 1536. That is being done for a number of reasons, one of which is simple economies of scale, once you've gotten past the initial capital outlay for the equipment. The cost per well is cheaper when you're on 1536 and many of the liquid handling solutions, reader systems and reagent systems are very well optimized now to the 1536 base."
Because screening can go so much faster in 1536, that format is also allowing many companies to get around the need for multiplexing in many cases, Eglen notes.
Companies are also getting more mileage out of their compound libraries, he notes, and reducing the amount of materials needed for each well, while getting more results out of each run.
So, with the great success of 1536, is it inevitable that we'll end up with even more wells on a plate with even smaller microfluidics handling? The consensus seems to be that 1536 won't be the end of the story. But at the same time, most think higher-numbered formats will serve, at best, a highly niche role for some time.
"Some people are pushing the envelope to the 3456-well format already," observes Graham Threadgill, director of Life Science Automation at Beckman Coulter. "So we're not done with this yet, and 1536 certainly won't be the end of the line."
What will likely be one of the bigger hurdles for 3456-well technology is running up against detection issues, Threadgill notes, saying, "Can you get enough signal or a good enough signal-to-noise out of such a small amount of material in a well?"
There is a 3456-well format already available from Aurora Biotechnologies, for example, points out Eglen, but he says many groups just don't see the need to go that far.
"Once you start getting into fluid volumes below the 8 microliter range—and with 1536 you're already in 12 microliter territory—you start getting into some really high-precision fluid dispensing, for one thing, and that means buying yet more new stuff to handle that," Eglen notes. "Also, the sheer volume of data that you are going to end up with all at once from a run with a 3456-well format could be overwhelming, so the question is are you getting much gain from the technology for the investment you have to put into it?"
At the vast majority of screening groups, that answer is, "no" he says, and the groups that are using 1536 are more than happy with their output and results. Too happy to be chasing after 3456 any time in the near future, anyway.
Many companies now aren't always screening their whole libraries, points out Dr. Rob Kilkuskie, executive director of the Michigan High Throughput Screening Center. With more focused library work or only a portion of their libraries being screened now, it's just not worth the time for most companies to consider 3456—or sometimes even bother with 1536 right now.
"The fact that a company has two million compounds doesn't mean it plans to run them all," Kilkuskie notes. "And for what they are doing, there really isn't a need always to even use 1536, much less go beyond it. The need just isn't there yet. People are already going into that realm, and I'm sure someone will make it work well, but I'm not sure how much of a market there will be for it, and I certainly don't see 3456-well formats being used on a routine basis. For one thing, you'd be pulling down more data than you really want to handle in many cases."
Also, let's not sound the death knell of 96-well and 384-well formats, even with the success of the 1536-well format. As Eglen notes, the level of high-throughput you need drops once you move past initial screening and go into optimizing or profiling for secondary activity, and 96-well and 384-well formats often work quite well even for the big players in drug development.
"Once you get into secondary screening, you're dealing with compounds in the thousands instead of potentially in the millions, so your output needs go down correspondingly," Eglen says.
Kilkuskie, who doesn't even have a 1536-well setup for his center's contract research work, is a case in point.
"We use the 384-well format here," he says. "The compound library we have is 100,000 compounds, so for our purposes, 384 wells is really appropriate, and that applies equally for our cell-based assay work as it does for the enzymatic assay work. We just don't need 1536 wells for the kind of experiments we do; that's for companies doing work with bigger libraries."
Cell-based assays speeding up fast
And since Kilkuskie brought it up, what about those cell-based assays? Are they ready for the world of HTS? The opinions there remain mixed, but even those who don't think cell-based assays have truly broken the high-throughput barrier acknowledge that point is just around the corner.
"The industry is definitely approaching or reaching the high-throughput realm with cell-based assays," Kilkuskie asserts. "There is much more a feeling out there that the engineering problems of treating cells more like reagents and the quality control issues with cells have largely been solved. And these factors make using cell-based assays more routinely a greater reality for a greater number of researchers."
Eglen says that for the most part, cell-based assays are already firmly in the high-throughput realm, noting that the main classes people are looking at with cell-based assays are G protein-coupled receptors (GPCRs), kinases, nuclear hormone receptors and ion channels.
"Of those, all of them with the possible exception of ion channels can be handled by cell-based assays that in turn can be handled with high-throughput screening technologies," he maintains. "Cell dispensing and cell-based assays appear to be highly amenable to 1536-well screening, though ion channels have a ways to go, I'll admit. Especially if you are doing patch clamping, you really just can't get up to 384 wells very easily, much less more than 384."
Beckman Coulter's BioRAPTR screening technology, for example, has vertical lines and low heat, both of which make cell-based assays at the 384-well level—and even the 1536-well level—feasible without harming the samples, notes Alisa Jackson, product manager in the Strategic Planning Business Center for Life Sciences Automation at Beckman.
Threadgill isn't quite as enthusiastic as Eglen in saying that cell-based assays have arrived at the 1536-well level, noting it is still a challenge to get cells to work properly at such small volumes. But clearly, he says, that is becoming more practical, and routine use of 1536-well formats for cell-based assays is simply a matter of time.
"There's an increasing trend toward doing cell-based assays in high-throughput," Threadgill says, all the way up to the 1536 level in some companies and maybe beyond that as well. "A lot of people said we'd never get out of 96-well formats with cell-based assays, and I admit that I was one of the skeptics who figured we'd never reach 384 wells with cells. Now that I've seen that barrier broken, I'm never saying 'never' again with the potential for high-throughput cell-based assays." DDN