Calcium-assisted ion-assisted chromatography unlocks scalable purification of high polarity drug candidates

A serendipitous water-system failure at Southwest Research Institute has led to ion-assisted chromatography, a calcium-enabled approach that allows rapid, scalable purification of highly polar active pharmaceutical ingredients and peptides that have traditionally defeated silica gel columns

Silica gel columns remain the workhorse for purification, although they perform poorly with highly polar organic compounds, which include many active pharmaceutical ingredients. The result has often been time-consuming workarounds, expensive specialist materials and scale-up bottlenecks that can delay – or derail drug – candidates as they move from discovery to development.

Researchers at Southwest Research Institute (SwRI) in San Antonio, Texas, have now reported a calcium-enabled approach that they believe could remove many of these obstacles. The team has developed ion-assisted chromatography, in which calcium salts in the mobile phase alter the retention and elution of very polar molecules on conventional silica gel. In trials, the method allowed efficient separations of charged species, including amines, ammonium salts and even peptides, while the use of inexpensive reagents and standard equipment preserved scalability.

The concept arose from a chance observation when SwRI’s deionised water system failed during work on a demanding separation for an upcoming preclinical study. The team needed around 20g of an ammonium salt at a purity of at least 96 %, but conventional chromatographic approaches on silica gel could not deliver the required specification. The timelines for the study ruled out systematic crystallisation development, which would usually require extended optimisation and often specialist modified silica to handle highly polar drugs.

In search of inspiration, the scientists reviewed the literature on thin-layer chromatography systems for amino acids and related polar compounds. They identified conditions that used aqueous solvent blends on normal-phase silica and decided to adapt these for column use. Because the deionised water system was offline, they switched temporarily to tap water, a practice that chemists normally avoid because dissolved ions can interfere with reactions and separations.

“Chromatography is a necessary evil,” said Darrel Johnston of Southwest Research Institute.

“I have yet to meet a chemist who enjoys column chromatography, but for fast and general purification it is still the most effective method.

“In this case we faced a tight deadline, highly polar material and no obvious route to crystallisation, so we needed a solution that delivered results quickly,” he added.

Despite the use of untreated tap water, the team recorded unexpectedly strong purification performance with the adapted aqueous solvent system. When the deionised water supply returned and they repeated the experiments, the performance deteriorated. This contrast immediately suggested that the tap water had introduced a key component into the system.

Most freshwater in South Texas flows through limestone aquifers rich in calcium carbonate. Calcium carbonate is almost insoluble in water, apart from trace amounts, but those traces are enough to leave mineral deposits on domestic and industrial surfaces.

For most chemists, dissolved limestone is an impurity to remove. SwRI therefore invested in systems that remove dissolved minerals and salts from process water. In this case, however, a malfunction had ensured that tap water reached the laboratory at the precise moment the team worked on a particularly intractable chromatographic problem.

As the scientists buffered the tap water with hydrochloric acid (HCl), the acid reacted with calcium carbonate to yield dissolved calcium chloride. A chemist on the team recognised that the combination of tap water and HCl could have produced calcium chloride in situ and proposed that calcium ions might explain the superior chromatographic performance.

“Once we suspected calcium, we realised we could recreate the conditions very easily,” said Shawn Blumberg at SwRI.

“Calcium chloride is inexpensive and readily available. When we added it deliberately, we recovered the same favourable behaviour on silica columns, which confirmed that calcium ions sat at the heart of the effect.”

With that insight, ion-assisted chromatography at SwRI began to take shape. The institute’s internal research and development funding programme – which supports exploratory work by staff scientists – provided resources and protected time to expand the initial observation into a general method. The availability of internal funding allowed the team to investigate solvent systems and salt combinations in a more systematic way than routine project budgets might have allowed.

Because traditional column screening can consume large volumes of solvent and material, the group turned to high-performance liquid chromatography (HPLC) to explore conditions with smaller quantities of sample. Automated HPLC systems allowed rapid testing of mobile-phase compositions and salt types, which proved pivotal in identifying the operating space for the calcium-enabled process. Using this platform, the researchers evaluated more than 20 salts of calcium, sodium, potassium, magnesium and zinc.

The data indicated that no salts performed as well as calcium-based systems in the separation of very polar, charged compounds. Even within the calcium set, however, the team observed substantial variation in performance between individual salts. At the same time, they found that some organic solvents paired poorly with aqueous calcium solutions. After further optimisation, acetonitrile emerged as a particularly effective organic component that supported high-resolution separations when combined with calcium salts.

Ultimately, calcium trifluoroacetate proved to be the best-performing salt, with calcium chloride as a close second. The team has reported that calcium-ion-assisted chromatography allowed rapid and inexpensive purification of highly polar compounds on silica at scales relevant to development, without the need to resort to crystallisation or costly modified stationary phases.

Although the method has shown clear practical value, the underlying mechanism is not yet fully understood. The scientists have suggested that calcium ions may play a dual role. They appear to enable elution of very polar molecules along the silica column, yet also exert a retentive effect that slows and separates analytes more effectively than conventional systems.

“As of now, we are still working to understand exactly how calcium ions operate in this context,” said Travis Menard at Southwest Research Institute.

“Macro-scale chromatographic systems are very difficult to study and almost impossible to model in full detail. We see that calcium helps with elution, but we also see enhanced retention and separation. We hope that as more groups adopt the method, additional data will clarify the mechanism,” he said.

The SwRI team has identified two main research priorities. The first is a more detailed mechanistic investigation of calcium–silica–analyte interactions, although this lies largely outside the group’s immediate remit. The second is a broader exploration of peptide purification, where the method shows early promise. Peptides have long posed challenges for normal-phase silica chromatography, with analysts often forced towards reverse-phase systems or bespoke stationary phases.

Initial experiments with selected peptides have produced encouraging results, with improved separations of target sequences from truncated oligopeptides. Nevertheless, the scientists have stressed that much more work must take place to establish the scope of the method across peptide classes. Progress in this direction will require a reliable supply of impure oligopeptides that can serve as test systems and benchmarks.

The group is currently preparing a peer-reviewed publication that will describe the calcium-assisted approach in detail and present case studies that illustrate its generality for polar small molecules and peptide-like compounds. They intend to show that the method can address a broad range of chemical purification problems rather than a single idiosyncratic separation.

Beyond the initial discovery programme, ion-assisted chromatography has already found a place in SwRI’s applied work on medical countermeasures. The institute maintains an active portfolio in the development of agents to counteract organophosphate poisoning. Oximes, which are one of the principal scaffolds used as reactivators of organophosphate-inhibited enzymes, are permanently charged ammonium salts and therefore difficult to purify on conventional silica.

The calcium-enabled method has allowed the team to purify oxime candidates to more than 98 per cent purity at meaningful scales, which supports ongoing toxicology and efficacy studies. According to the researchers, this capability has removed a significant bottleneck in the progression of organophosphate countermeasures towards preclinical evaluation.

The story of the discovery has also highlighted the importance of place in scientific work. SwRI occupies around 1,500 acres of land that previously formed part of a South Texas ranch. The institute’s founder, Thomas Baker Slick Jr., envisaged the site as a centre for scientific and technological research. In the case of ion-assisted chromatography, the local limestone aquifers and their trace calcium content have literally fed into the laboratory, where they interacted with a minor equipment failure and an urgent drug project to reveal a novel technique.

The researchers have emphasised, however, that institutional culture was as important as geology in the outcome. SwRI’s internal research model and its encouragement of multidisciplinary approaches allowed chemists who work in drug discovery, purification, formulation and process development to pursue an unconventional lead based on tap water.

Clients now approach the institute for solutions that draw on this breadth of expertise in research and development. The serendipitous origin of calcium-assisted chromatography has therefore reinforced a broader theme in science: where rigorous methods, institutional support and an open mindset meet, even a water-system failure can become the starting point for a useful advance.