Lending Ion Chromatography a ‘Hand’
A decades-old theory and a new super-localization microscopy method have teamed to advance the efficiency of ion chromatography in drug discovery applications.
A marriage between theory and practice in ion chromatography could lead to more efficient protein capture during drug manufacturing. Researchers at Rice Univ., Houston, are using an in-house method to provide pinpoint locations for single proteins in combination with a well-established theory that describes those proteins’ interactions with other molecules in work in an effort to make protein isolation five times more efficient.
The work is being conducted by Rice physical chemist Christy Landes and her research group.
A critical step in drug manufacturing is the separation of proteins of interest—the active elements in drugs—from other material. The method of choice, ion chromatography, has been around for some time, but according to Landes, there is ample room for improvement.
“As a physical chemist, I’ve somewhat ignored this technique because of the nagging sensation that the process was too empirical,” says Landes. “But after seeing the nice statistical theory developed by Giddings and Eyring, [which] has never been applied because access to the molecular-scale data was previously impossible, it became clear that it is possible to take this field in a more quantitative direction.”
In ion chromatography, a separation column removes proteins from water and other cellular material. The raw material containing the proteins is either pulled or pushed through a column. Along the way, the liquid encounters a stationary phase that incorporates ligands—binding ions or molecules.
“Our fundamental understanding of this process at the level where proteins bind to ligands, which basically drives several different industries, is ridiculously small,” she says. “We should take care to understand everything about separation, because up to half the cost of bringing a drug to market is separation and purification.”
Old theory, new application
Remarkably, the stochastic theory of chromatography that describes single molecule interactions has been around for decades but nobody had a tool to validate it through experimentation. The Rice team used a super resolution technique called mbPAINT (motion blur point accumulation for imaging in nanoscale topography), which they originally developed to identify individual sequences along strands of DNA, and found it could work in other processes involving the capture and release of single molecules.
“The trick here is to work in exactly the opposite regime that you’d desire for a true separation,” says Landes. “For bench- or industrial-scale separations, you’d work in a large 3-D column and you’d load up with as high a ligand density as possible in order to separate the most analyte you could at a time. But to actually understand what is happening at the moment of protein capture, we want to decrease the dimension of the column geometry to two dimensions so that we can truly focus on the
mobile phase-stationary phase interface. We also decrease the loading density so that we can observe, in real-time, the capture of a single protein by a single ligand.”
In earlier work with mbPAINT, the Rice researchers resolved structures as small as 30 nanometers, at least 10 times smaller than the wavelength of light, by building up pictures over time of a probe molecule that would fluoresce when temporarily captured by the immobilized DNA. The ability to map the location of proteins as they attach to ligands gives a much more precise look at the mechanism that makes column chromatography possible, says Landes.
The results showed that at the molecular level, ligands embedded in an agarose-based stationary film would only capture proteins—in this case, a synthesized peptide—when at least three ligands were clustered together. This was corroborated by Richard Willson at Univ. of Houston, who performed the initial binding assays and determined that clusters of ligands improve protein capture.
“Industry uses charged ligands as a hand to grab a protein flowing by,” says Jixin Chen, a member of the Landes group. “Once the protein encounters a ligand, their charges attract each other and they stick together. But now we can see that one single, charged ligand isn’t enough to grab it. It only really happens when multiple ligands are clustered in a small area and work together to grab a protein.”
“We show that there’s a spatial arrangement to the ligands that’s also important,” says Landes. “We’ve learned that although the accepted way to improve ion-exchange is to increase the number of fingers grabbing each protein, those fingers have to be ideally organized as a hand.”
She added that the team is planning a round of experiments to examine the spacing effects.
The combination of mbPAINT and stochastic theory could work equally well in other areas, she says, like the optimization of diagnostic tests that depend on the capture of analytes in a flowing fluid, in water purification columns and in catalysis for oil and gas refineries.
More about mbPAINT
• Motion blur point accumulation for imaging in nanoscale topography, or mbPAINT, relies on high-tech optics and super-localization microscopy to pinpoint the location of specific sequences along single strands of DNA.
• Proof-of-concept experiments identified DNA sequences as short as 50 nucleotides at room temperature, which is not possible with standard or electron microscopes.
• The technique can resolve structures as small as 30 nanometers by rapidly imaging DNA probes flowing over a known target sequence along an immobilized single strand of DNA.
• The probes are labeled with a fluorescent dye that lights up only when attached to the target DNA.
• In experiments, a few probes would do just that for a few milliseconds, just long enough to be captured by the camera.
• Processing images of these brief events amidst the background blur allows the researchers to image objects smaller than the natural diffraction limits of light-based imaging, which do not allow for the resolution of targets smaller than the wavelength of light used to illuminate them.
• The images taken of the fluorescing probes are just a pixelated blur at first. But it’s a blur with a bright spot, and careful analysis of multiple images allows researchers to pinpoint a specific spot along a strand.