As the current COVID-19 crisis has revealed, uncovering the deep secrets of clinically significant molecules is absolutely critical for developing effective treatments and therapies. We identify the usefulness of testing and laboratory instrumentation by the depth of the resolution these instruments can achieve. Throughput, ease of use and speed are also important, but resolution – being able to see deeply into the unseen – of whatever clinically significant molecule is important to that researcher, is paramount.
If we could dramatically improve the resolution of such instrumentation, imagine how profoundly that could affect the pharmaceutical industry, clinical research and patient care.
Now, imagine that the same technology that allows for such an industry step change could reduce reliance on typical labor-intensive traditional methods and works instead of on computer printed circuit boards. After all, why hasn’t more laboratory instrumentation moved into the Digital Age?
That’s just a small part of how High-Resolution Ion Mobility (HR-IM) will help bring dramatic improvements in medicine. Currently uncovering the deepest secrets of the COVID-19 molecule, prestigious research organizations including the Complex Carbohydrate Research Center of the University of Georgia have been putting this technology to work.
Ion mobility separation has been around for almost 20 years, but to be more broadly practical, the path for those ions had to be made longer and more compact to achieve breakthrough performance. The key development was the ability to move ions through long separation path lengths, on printed circuit boards, and around corners, making90° turns without loss of ions. This opened the door to a compact serpentine arrangement providing ultra-high resolution.
So far, the technology has led to improvements in biotherapeutics characterization as well as accelerating biomarker discovery to facilitate early disease detection. HRIM technology enables fast, efficient, high-resolution lipid analysis, glycan analysis, peptide mapping, and intact and subunit protein analysis – all areas in which current technology is deficient or just plain slow.
One company has taken the lead in the development of this technology, and their SLIMTM technology (Structures for Lossless Ion Manipulation), being used in conjunction with Agilent’s high-resolution mass spectrometry platform, has researchers realizing there is a future state of resolution that will make today’s LC-MS resolution appear as quaint as Kodak film appears to current cell phone photographers.
The Challenges of Conventional Separation Technology
In general, liquid chromatography and mass spectrometry (LC-MS) has some significant challenges when compared to HRIM or SLIM technology. For example, it can be difficult and time-consuming to achieve molecular and structural characterization of complex molecules. Classic techniques are simply too slow, too complicated, and not powerful enough for the increasingly complex demands of biologic drug development, early disease detection and clinical diagnostics. After all biologics, with their hundreds of thousands of sugars, proteins or complex combinations of these substances, have presented quite a challenge when researchers probe to “see the unseen.”
Scientists and researchers often are faced with making trade-offs when using traditional instruments. For example, they may need enhanced resolution to identify molecules, but to get a better resolution they may need to compromise throughput. With HRIM, resolution, speed and efficient operation are all considered. SLIM technology also has greater instrument uptime as compared to LC-MS.
SLIM technology also has the benefit of being able to use much smaller sample sizes. Researchers in proteomics can obtain measurements even in extreme cases – for example, instances in which there might be insufficient tumor tissue. Previous technology was impractical for that purpose.
SLIM technology also offers a distinct speed advantage, providing unprecedented resolution in two-minute analysis times. In practical terms, this means for example that for a biomarker discovery study or for clinical trial sample analysis, SLIM can analyze 1000 samples in a week, which might require up to one year with LC – in other words, 98 percent faster than when using conventional technology.
The Difference Between LC and SLIM
In its simplest terms, liquid chromatography separates based on chemical interactions. Because of the columns, pumps, solvents, buffers, tubing and plumbing involved, the workflow requires time – both time for scientists to prepare the buffer solutions and the time required for an analyte to travel through the LC system following liquid-phase kinetics. Changeout between samples also takes considerable time when using LC, as the operator sets up the column and method again and again, for each different sample class.
Ion mobility separations differ fundamentally from liquid chromatography in that the work occurs in the gas phase. In this case, ionized molecules separate based on their size, charge and shape and this happens much faster in a gas phase. Consequently, analytes with the same molecular mass and chemical formula can be separated by their size, shape and structure, rather than their chemical affinity with said buffer/solvent.
Ultimately, SLIM is a more “analyte agnostic” approach. Separations are conducted on ionized molecules in the gas phase through an inherent physical property of the analyte, and hence separations are more reproducible. It is possible to achieve separations of isomeric molecules (that is, molecules of the same mass and same chemical formula). Back-to-back samples can be run without component change out, method development is faster, and instrument uptime is greater because of SLIM’s relative ubiquity across analyte classes.
The separation of isomers described above is directly related to the higher resolution possible with SLIM. In general, with ion mobility approaches, the longer the paths, the greater the resolution of the results. Unfortunately, incumbent ion mobility instruments are limited in size because of the space available in a typical laboratory. In other words, LC might enable similar resolution but one would need many columns to achieve it.
By contrast, SLIM allows ions to move around corners and follow serpentine paths. This results in very long paths in a comparatively small footprint and provides very high-resolution separations. A pair of 30 cm x 30 cm SLIM boards create a 13 meter (40 foot) path in a device the size of a personal computer. SLIM can also accumulate ions for ease of analysis and selectively switch them as a group to other locations, all without any losses.
SLIM has a superior capability to separate and identify the most challenging clinically significant molecules – even many that escape LC-MS detection. With software-driven methods, better instrument uptime, and reduced operator cost, SLIM can perform analysis significantly faster.
Typical analysis times tend to be around two minutes. Comparable LC separations run by a highly qualified expert can take from as little as 15 minutes to as much as two or three hours and are less reproducible.
Not surprisingly, these results have led to a veritable avalanche of scientific papers – more than 35 have been published about the science behind SLIM since 2014. In 2019, one such paper discussed SLIM’s ion mobility technology as useful in pharmaceutical and clinical fields because of its capabilities in glycosylation monitoring of biological drugs as well as in Vitamin D analysis.
Practical Applications: Biologics, Diagnostics and COVID Analysis
The advantages of such high resolution in a laboratory instrument is applicable to a range of areas of study:
Biotherapeutic Drug Development. The dramatic improvement in resolution when using SLIM technology translates immediately into better characterization so drugs are safer, more effective and get to market faster. For characterization of protein-based biologic therapeutics, SLIM provides run times of two to five minutes for glycan analysis and peptide mapping; LC-MS requires up to 90 minutes to complete the same two functions with significant component change out and instrument downtime.
BiomarkerDiscovery and Validation. In clinical research settings, SLIM’s improved resolution and speed mean accelerated biomarker discovery and validation. With SLIM it is not only possible but much easier to run population-scale studies. The sheer number of samples required to validate biomarkers requires large-scale studies that previously took many months or even years before test results were completed.
Early Disease Detection: The fast, high-resolution analysis of key disease biomarkers allows SLIM to detect critical molecules that LC-MS cannot. These include biomarkers that can predict heart attacks before they happen, identify the location of cancer from a blood test, and diagnose Alzheimer’s Disease before symptoms appear.
COVID-19. The nodule in images of the COVID-19 viral capsule that has been seen across all media is known as “spike glycoprotein.” The CCRC project, using SLIM technology, is working to detail the glycosylation microheterogeneity of this glycoprotein. By understanding its heterogeneity, researchers will gain a greater understanding of how the virus binds to its target; that, in turn, is essential information when developing effective treatment.
Conclusion
Does all of this mean that traditional means of separation and extraction have had their day? Absolutely not.
There always will be applications in which one technology or another may be preferable or circumstances in which one technology may be used together with the other.
In general, however, particularly driven by the trends discussed here, the ability to reveal the unseen will become more and more of a must-have, not simply a “nice-to-have.” The benefits of High-Resolution Ion Mobility (HRIM) allow a level of resolution previously unachievable, which in turn deepens understanding.
For most pursuits in the biopharmaceutical and clinical research arenas, the capability to efficiently and quickly reveal the unseen will inevitably lead to faster discoveries in disease prediction, diagnosis and treatment.
Melissa Sherman is CEO of MOBILion Systems Inc.