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Purifying the First Hundred Grams

18 October 2011

Preparative FLASH chromatography expedites the production of clinical trial compounds.

Tobi Williams is an application chemist specializing in pilot and production purification at Biotage Inc., Charlottesville, Va.


Thin-layer chromatography correlates closely with the order of and spacing between the preparative FLASH fractions.

Pilot-scale manufacturers are experiencing a significant increase in the number of projects they must cope with, and they must begin pilot studies for a growing number of lead compounds. Facilities are asked to complete more projects in less time with limited resources. Consequently, producing pure active pharmaceutical ingredients for clinical trials in a timely manner requires a new development strategy.

Purifying pilot quantities is often achieved by crystallizing the active pharmaceutical ingredients out of solution. A good crystallization procedure will yield a high-purity product; however, development may require several weeks. Preparative FLASH chromatography eliminates the rate-limiting step of crystallization process development, freeing more development time for other projects.

Preparative FLASH chromatography is used late in the synthesis process where purity and yield are critical. Often, a crude product matrix is too complex for successful crystallization. FLASH chromatography gives consistent, high yields of pure product, which is easily crystallized into a pure powder.

FLASH chromatography permits pilot or substantial volumes of compounds to be purified quickly. These columns use 40- to 60-µm dia silica particles to permit high flow rates and effective separations with modest system pressure--40 to 100 psi. Large silica particles and radial compression technology create stable, effective separation beds at a fraction of the cost of HPLC systems. Prepacked FLASH columns are easily scaled from re-search quantities to kilogram lab and pilot facilities. The technology per mits significant savings in reduced development time and improved product recovery.

Preparative FLASH chromatography methods are developed in as little as three days. With the proper equipment and a structured approach, small-scale synthesis batches can be scouted with thin-layer chromatography (TLC). TLC separations are correlated to the results of the separation on a silica FLASH column. To optimize separation conditions, dozens of TLC evaluations can be simultaneously run in 100-mL beakers, with each beaker containing a different solvent system. Each TLC system requires 10 min or less to run.

Transferring methods

The TLC retention factor (Rf) values directly correlate to the separation on a silica column. TLC Rfs are inversely proportional to FLASH cartridge retention, which is measured in column volumes. Biotage Inc., Charlottesville, Va., provides silica packed in cartridges and TLC plates so that results are consistently converted to preparative separations. The company also provides cartridges in sizes ranging from 4 g to 40 kg for scaling processes.

The TLC separation can be transferred directly to a FLASH chromatography system. Small cartridges with 4 to 8 g of silica can purify up to 100 to 500 mg of product and are ideal for confirming a TLC separation. When the process is confirmed, scaling is simple and predictable. With prepacked cartridges, silica beds are consistently well-packed, and the purification process can be scaled in a linear manner.

The purified product has the same yield and purity as with the small column. Production runs require approximately the same amount of process time as lab-scale runs. Developing a pilot-scale purification process requires two to five hrs of TLC work and two to four hrs of lab work to confirm a small-scale cartridge. Chromatographic method development thus requires only a day, as opposed to several weeks for crystallization. A typical production-scale process can be run in four hrs or less, including setup and cleaning.

Prepacked cartridges give a high degree of confidence in the consistency and scalability of preparative chromatography processes. Ready-to-use cartridges permit a pilot facility to concentrate on synthesis rather than packing and testing silica beds. They also save substantial production time, because installing a new cartridge requires less than 20 min. Cleaning procedures are simple and only require removing the cartridges. All of the silica and trace impurities remain inside the cartridges, increasing safety by reducing operator exposure.