Chiral Technology Forum

Chiral separations are important in several application areas, most notably in pharmaceutical analysis. Participants in this Chiral Technology Forum include Chris Hamman and Mengling Wong of Genentech, Marc Jacob of Phenomenex, and Zachary S. Breitbach of the University of Texas at Arlington.

What impact has the recent introduction of so many new chiral selective phases had on chiral method development?

Hamman and Wong: For starters, it has made our screening process a little more involved! Unfortunately, since there is no column that resolves all racemates, it is great to have a few more tools in the shed. We're fortunate in our group that we have the resources to purchase many of the new phases to see their utility. Some of the new phases are giving us great results. In particular, the 4-chloro-3-methylphenyl carbarmate cellulose polysaccharide phase has provided excellent results for us. We utilize sub-5-µm particle size packing with high flow rates on 50-mm columns in effort to shorten our run times. The shorter run times allow us to efficiently screen many more phases. The increase in the number of vendors offering similar phases has helped drive the costs down (a nice perk for us). Additionally, the increased competition of the vendors seems to drive the research of creating even more phases.

Jacob: The introduction of so many new chiral phases has dramatically increased the chance of enantiomeric separation success to virtually 100%. With the improvement of analytical instrumentation, chemists can now screen up to 10 chiral columns in a short period of time — about 30 min under supercritical fluid chromatography (SFC) conditions. At the same time, the introduction of so many phases has compounded the screening process, as more phases mean more choices. The challenge is to decide which phase to use in one’s primary screening protocol. The relatively recent commercialization of chlorinated polysaccharide-based chiral stationary phases has been well received by the pharmaceutical industry as more companies are achieving success on these phases and thus placing them in their primary chiral screening protocol.

Breitbach: The large number of broadly selective chiral phases available has stimulated advancements in HPLC instrumentation. For example, there are now high-throughput, multicolumn (that is, 10 columns running simultaneously) screening systems, which allow researchers a brute-force technique for screening the most successful chiral phases in the most common operating conditions to quickly determine an acceptable set of separation conditions.

Alternatively, some chiral phases have given separation scientists some rational choice in their column selection, such that a certain class of compounds should always be screened on a particular phase (for example, amino acids on teicoplanin or primary amine containing racemates on aliphatic derivatized cyclofructans).

With no universal chiral selector likely to be developed in the near future, it is necessary for column manufacturers to continue to develop improved broadly selective chiral phases or niche chiral phases as indicated above.

What are some of the factors influencing the decision between performing chiral purification versus chiral-specific synthesis?

Hamman and Wong: Speed, scale and cost are the three biggest factors that determine our decision to do a chiral purification versus chiral-specific synthesis. Early in the discovery process, we do not require a much of material to get preliminary data. It is less expensive and faster to obtain racemic material and do a purification on either the racemic starting material or the racemic analogs. As the project and chemistry matures, the scale increases to accommodate advanced studies. Additionally, the chemistry becomes more focused yielding one or two cores. At this point in the project, if one of the cores has a chiral center, it is usually cost effective to find a chiral-specific synthetic route to the make the core. We currently have the capacity to purify a few hundred grams of racemic material, but generally once things get to the kilogram scale it is more cost and time effective to go the chiral-specific synthesis route.

Jacob: Looking at the pharmaceutical industry in particular, the stage of a drug in the development pipeline is the major factor influencing the decision to choose chiral purification over chiral synthesis. In drug discovery and early phase development, purification is used to quickly obtain pure enantiomers, but as the project scales up or moves toward the final phase of the drug approval process, chemists tend to use the asymmetric synthesis pathway rather than purification. Moreover, the use of SFC as well as prepacked columns makes the purification process very efficient and may push chemists to pursue that technique. Other factors, such as equipment availability and cost, may also influence the decision to perform a purification process at large scale over synthesis.

Breitbach: The key factors that drive the decision to either choose prep high performance liquid chromatography (HPLC) or asymmetric synthesis to obtain enantiomerically pure products are how much product is needed and how quickly is it needed.

Typically, when going from milligram to gram scale it is quickest and most cost effective to perform semi-prep HPLC to obtain pure enantiomers. With the large number of chiral semi-prep and SFC compatible phases available, it has become very common to take an analytical separation to the semi-prep level, and quickly and effectively be able to produce grams of purified products. At the gram to kilogram scale, it is too costly to work out an asymmetric synthesis, which is inherently more difficult and time consuming than chromatographic purification. However, when going to the kilogram or process scale, the operating cost of prep HPLC will often outweigh the cost in time and money of developing a successful chiral synthetic route. Simply put, if you need a little, HPLC or SFC is the way to go. If you need a lot, synthesis wins out.

It should be noted that developments are being made in both the fields of preparative separations and asymmetric synthesis, which could shift the paradigm either way. For example, cheaper packing materials (such as resin-based chiral stationary phases) and the savings in operating costs when using SFC could make chromatographic resolution most attractive across the board. On the other hand, improved screening methods (for example, high-throughput, 96-well plate, synthetic screens) for developing ideal synthetic approaches may make synthesis more amenable even at the sub-kilogram scale.

Is the resurgent interest in SFC for chiral analysis enough to support the instrument companies that are offering the technology? Would it just be better to include the ability to handle carbon dioxide as a fluid in LC systems?

Hamman and Wong: Phew, that is a tough question. While SFC has found a home as an excellent means to separate chiral samples, it is certainly not limited to only chiral samples. We don't work for the instrument companies, but we're sure they are expecting SFC to replace LC in many other domains beyond just chiral analysis. The new instruments are very impressive and have excellent sensitivities that enable them to penetrate the GMP world. If LC systems can be built to handle carbon dioxide as a fluid and still give excellent results, then there would be no need to have a dedicated system. If such an LC system can be built, hopefully it will be less expensive and allow SFC to expand into other labs with smaller budgets.

Jacob: SFC is a proven technology for chiral analysis, and instrumentation is justified whether the systems are purely dedicated to SFC or have the ability to handle carbon dioxide as a fluid. There are instrument companies on the market currently offering the option to add carbon dioxide as a mobile phase to an existing HPLC instrument, and others that allow the analyst to switch between SFC and HPLC modes on a single instrument platform. These options are great as they allow the chiral analyst to screen more mobile phases and separation modes such as reversed phase, normal phase, and SFC using the same instrument. Presently, however, these combination instruments are limited to the analytical scale only.

Breitbach: Enantiomeric separations by SFC of course have the advantage of speed and low operating costs, and they are considered “greener” than HPLC. For this reason, SFC is an important tool for separations scientists, especially at the semi-prep to prep levels. However, supercritical mobile phases are essentially additional normal-phase conditions, and considering that method development occurs at the analytical level first, instruments that perform both SFC and HPLC (or ultrahigh-pressure liquid chromatography [UHPLC]) are of great importance. These systems have currently become available and will prove to be essential in the chiral analysis field.