Advances in flash purification

For decades, liquid chromatography has been the primary tool used to purify synthesised compounds in drug discovery. A target compound’s successful progression from hit to lead status depends not only on its structure-based biological activity, but also on compound purity.

The medicinal chemist who created and purified the target compound relies on biologists to verify and test for biological activity. All too often, invisible contaminants and inadequate purification of the target compound have led to finger-pointing and tension between chemists and biologists.

Historically, the purity and stability of entire compound collections have worried the pharmaceutical industry and has been the focus of discussion and debate among analytical chemists. Since the early 1990s, pharma has been plagued with compound purity concerns following the industry’s failed attempt to turbo-boost their pipelines with high-throughput synthesis efforts. This lead generation strategy, originally known as ‘combinatorial’ chemistry, was criticised for producing highly impure targets and false hits.

Despite the power of combinatorial chemistry to produce enormous libraries of compounds, the lack of consensus on either the definition or the impact of purity made it nearly impossible to make sense of the bioactivity associated with library compounds.

Better detection, better results

What has led to this debate over purity? Why do the medicinal chemist and the bioassay chemist get two different results for the same compound? The answer lies in detection during purification.

Flash purification today relies almost exclusively on ultraviolet (UV) detection. However, UV can only see compounds with chromophores, and the response relates to the strength of the chromophore (the extinction coefficient) rather than the quantity of the compound. Non-chromophoric impurities are not detected, which can increase the apparent quantity collected and give misleading purity values. Purity analysis using high-performance liquid chromatography (HPLC) systems with multiple detection capabilities later reveals the true purity of the target compound. Additionally, ‘hits’ in bioassays can be a result of the impurity, rather than the target. Detecting only by UV can also lead to productivity concerns. To avoid missing something, medicinal chemists collect everything, peak and off peak, and check and recheck their work by thin-layer chromatography (TLC).

Exploiting the benefits of different detection options for early stage purifications could result in better quantified and higher purity compounds entering the screening pipeline. For the pharmaceutical company, this means less time wasted on false hits and rework, and more time spent on truly promising targets.

Assessing purity and removing contaminants sounds easy enough, but many chemists agree it is completely dependent on the ability to correctly detect target compounds as well as all contaminants present in a sample at submission.

Optimised Analytical Solutions (OpAns) is a contract analytical services company serving the preclinical therapeutic drug discovery market. In a recent interview, its co-founder and CEO, Dr Ken Lewis, discussed the need for post-flash analysis to confirm purity.

“For general collections as a whole, the focus is on determining whether samples are of reasonable purity. Purity is the primary driver, and identity is secondary. Our standard analytical platform is a ‘hyphenated’ system consisting of multiple detection capability Â… We use four online detectors. The foundation of the system is good chromatography. Without it we cannot perform accurate purity or quantity assessments. The mass spectrometer is used for molecular weight confirmation. The CLND and ELSD are used for quantitative assessment.”

Figure 1: One HPLC analysis with four compounds in equivalent amounts shows the peaks that can be missed by UV or MS alone.

Applying a multi-detector approach earlier in the process - at the flash purification stage - could significantly improve the quality and quantity of promising targets submitted by medicinal chemists for screening.

Not as easy as it sounds …

Solving the known deficiencies of relying solely on UV for flash purifications is not as simple as adding a stand-alone detector, such as an ELSD, to an existing flash system. ELSDs and flash instruments were developed for two very different purposes - low-concentration, small sample size analytical work versus high-concentration, large sample size preparative purifications. Bringing these two diverse technologies together and expecting them to be compatible is more complicated than one might think. Along with the significant increase in bench space required, there are a number of drawbacks both at the front end (fluidics) and the back end (collection and analysis of data) of the process.

… Until now

Grace Davison Discovery Sciences has addressed these challenges with the invention of the Reveleris flash purification system with RevealX detection. This proprietary technology identifies and collects a broad range of compounds, including UV-invisible compounds, while providing a real-time indication of the mixture’s relative mass balance. By combining the strengths of ultraviolet and light-scattering technologies into a single detection mechanism, and processing these multiple signals to precisely control fraction collection, this technology can provide optimal peak volumes, high sample purity and recovery, solvent flexibility and waste reduction, and overall faster purifications.

The Reveleris flash purification system with RevealX detection technology lets medicinal chemists gather more information about their sample than is possible by UV alone, and lets them strategically collect fractions to reduce post-run analysis.

Figure 2: Multiple detection allows non-chromophoric compounds to be easily identified and isolated. Note the first peak, which would not have been visible by UV alone.

Technology-driven benefits

There are three novel technologies behind these benefits. First is the ability to see compounds undetectable by UV. Second, the accurate mass representation provided by light scattering detection. Third, the ability to synchronise multiple detection signals to collect a single set of fractions of accurate identity and quantity.

Non-chromophoric compounds

Because UV detectors only see compounds with chromophores, they will miss non-chromophoric compounds when purifying mixtures. If the chemists do not know they have non-chromophoric compounds in their mixtures, they may be passing along compounds with ‘invisible’ contaminants. This can result in inaccurate assessments of purity and misleading results in bioassay screening. If the chemists do know they have non-chromophoric compounds, they will collect all possible fractions and do extensive post-flash analysis to sort them out. This can include days of work by HPLC, LC/MS and TLC, or even handing the mixture off to another laboratory which can create custody concerns and possible delays.

By incorporating both UV and ELS detection technologies into one detector, the Reveleris system reveals both chromophoric and non-chromophoric compounds, reducing uncertainty and the need for extensive post-flash analysis. Chemists are able to spend less time sorting and reconstituting fractions, evaporating solvents and re-purifying failed samples - common bottlenecks in existing flash processes.

Mass balance

During synthetic transformations, chemists continuously monitor their reactions using TLC spotting and manual UV detection as indicators of reaction completion. With its ability to represent mass balances better than UV alone, RevealX technology can give chemists more confidence about the relative amounts of the compounds being purified as well as the corresponding fractions being collected.

Chemists will better understand their true reaction yields because they can determine how efficiently a transformation progressed and the relative amounts to expect in return after fractionation. Having this knowledge helps them make critical decisions about reaction optimisations and future syntheses, and potentially recover important (or costly) starting materials. The Reveleris system allows chemists to maximise collection of the target compound and minimise the amount left behind in the system or column.

Fraction Collection

Complementing UV detection with light scattering detection reveals important information about a sample mixture. But, without the ability to collect fractions based on all detection signals, the medicinal chemist would still have to collect everything and spend significant time on post-flash fraction work-up.

The RevealX detection technology is more than just multiple detectors - it also uses advanced signal processing and software algorithms to synchronise the signals so the fraction collector knows which peaks are which, and collects accordingly.

Proper flow splitting is key to this technology. Grace’s splitting design eliminates the variability surrounding flow rates, sample volumes and solvent conditions. It synchronises delay times so the fraction collector can trigger from three sets of peaks (UV1, UV2, ELS) in perfect alignment. It also simplifies solvent choices - the chemist can choose the best solvents for their flash purification needs rather than their detector’s needs.

The chemist can modify triggering settings, including threshold and slope, to collect as much or as little of the sample as needed. For example, the chemist can optimise collection of all peaks while maximising purity, since even slight changes in the peak slope will trigger the change to a new collection tube. This ability to finetune fraction collection results in putting the right fraction into the right tube at the right time.

Don’t Forget the Chromatography

To get the most out of a flash purification, one must not forget that fundamental chromatography principles apply. To use a computer analogy, detectors are ‘garbage in, garbage out.’ The most advanced detector in the world will only take chemists so far if their separation is poor.

To capitalise on the advanced detection capabilities now available for flash purifications, chemists also need to use a flash cartridge specifically designed for good chromatography. The specifications on the silica, the pressure limits and overall design of the cartridge hardware, and the quality of the packing procedure all combine to impact issues such as voids and channelling, contaminants, peak shouldering or tailing, and in general whether compounds are or are not separated sufficiently to collect pure fractions. Solvent choices, flow rates and pH are also important factors.

Detect more, collect smarter

When purity analysis is completed with only UV detection, any contaminants that remain hidden can undermine the entire discovery process by giving falsely positive results in bioassays. New detection advancements allow familiar techniques like flash to benefit from multiple detectors. The critical issues associated with producing target compounds with compromised purities are addressed early in the process - at the point of synthesis - instead of after screening resources have potentially been wasted.

Until now, the medicinal chemist had to either rely on the incomplete information provided by UV detection, or separately repeat the experiment by ELSD and manually attempt to correlate the two sets of data. With the Reveleris system with RevealX technology, the medicinal chemist can now gather more information about their sample in a single experiment and pass more promising drug candidates to the downstream laboratories in less time. The end result is an overall acceleration of the drug discovery and development pipeline, allowing pharmaceutical companies to bring important new drugs to market more quickly.