Professor Debby Mangelings from the Vrije Universiteit, Belgium, estimates that 60% of newly commercialized drugs possess chiral properties. In this interview with LCGC, Mangelings discusses the importance of chiral chromatography in pharmaceutical analysis, the challenges of current methods, and where the field is headed next.
Q. What are your main research interests and how did you become interested in your field?
A: My main research interests are separations with chromatographic separation techniques and the use of miniaturized separation techniques, with a special focus on chiral separations. We have several research lines within chiral separations, such as the definition of generic separation strategies for different high performance liquid chromatography (HPLC) modes, capillary electrophoresis (CE), and supercritical fluid chromatography (SFC); the implementation of capillary electrochromatography (CEC) as a chiral separation technique; and the use of monolithic chiral stationary phases in CEC. In addition, our department is also involved in the development of fingerprint chromatograms of herbal extracts and the use of chemometrics on these fingerprints for identification, classification, and calibration purposes.
The interest of my main research field originates from my studies as a pharmacist: As a first year student, I became fascinated by the fact that mirror images of molecules exist, and that they display different activities in the human body. Later, I discovered that chirality had an enormous impact on the pharmaceutical industry, and I was lucky that the department where I did my Master and PhD thesis was doing research on chiral separations.
Q. What are you focusing on at the moment?
A: For chiral separations, we have two research projects: The first focuses on SFC used as a separation technique and in the second we are investigating new types of chiral stationary phases in CEC mode. In all research we try to implement the use of experimental designs, for example in method optimization, to reduce the number of experiments.
Besides these projects, we are working on precision improvement and method transferability of CE methods between different instruments and laboratories. Also here, we are using chiral separations as test cases.
In SFC, we also initiated a project concerning the development of drug impurity profiles.
A final topic is the development of fingerprint chromatograms by means of liquid chromatography–mass spectrometry (LC–MS) and LC with diode array detection (LC–DAD), and subsequent chemometric analysis. The idea is to compare both fingerprint types and see how much information is kept, gathered, or lost for data analysis when switching from LC–DAD to LC–MS fingerprints.
Q. Why is there so much emphasis on chiral analysis and the detection of enantiomers in pharmaceutical products?
A: Chiral analysis is a very important topic in the pharmaceutical industry because enantiomers of a given compound exhibit different pharmacodynamic and pharmacokinetic profiles in living systems. This originates from the fact that receptors, enzymes, and ion channels in the human body are composed of chiral building blocks, such as amino acids and carbohydrates, and therefore are also chiral.
In this chiral environment, different interactions are seen for individual enantiomers. For chiral medicines, it is often seen that only one enantiomer (eutomer) displays the therapeutic activity, while the other (distomer[s]) may exert no effect, another effect, an antagonist effect, may cause side effects, or can even be toxic. When a chiral molecule is commercialized in a pharmaceutical formulation, the presence of distomeric forms may be inconvenient. Therefore, this should be investigated properly in advance. Softenon is an often-handled example when discussing the risks of introducing chiral drug molecules. Here, racemic thalidomide (equimolar mixture of enantiomers) was incorporated as active pharmaceutical ingredient (API). One enantiomer (probably S-thalidomide) had teratogenic properties and administration of Softenon to pregnant women led to thousands of newborns with serious fetal malformations.
Q. Why is the single enantiomer important and how is this regulated?
A: When commercializing a chiral pharmaceutical product, the use of a single enantiomer drug is preferred because it presents many advantages, such as dose reduction, reduced pharmacokinetic and pharmacodynamic variability among patients, simpler dose-response relationships, and the reduction of side effects and toxicity. The commercialization of a racemate (a mixture of enantiomers) is still allowed, but only when the eutomer is converted into the other enantiomers by metabolization processes in the human body (in vivo racemization). This occurs, for example, with thalidomide. Therefore, even when this product would have been commercialized as pure enantiomer, the severe outcomes could not have been avoided because R-thalidomide is converted fast into S-thalidomide during metabolization.
The effects of the enantiomers in a chiral drug molecule must be carefully investigated for the registration file. The pharmacological and toxicological profile of each enantiomer must therefore be well documented in regulatory files, because when a single enantiomer drug is commercialized, other enantiomers are considered as an impurity of the API. Identification tests should be able to distinguish the enantiomers of the drug molecule. In addition, the enantiomers must be separated and quantified during production, fabrication, and quality control processes.
Knowing that at least 60% of newly commercialized drugs have chiral properties, there is no need to state that the development of chiral separation methods is very important in the pharmaceutical industry.
Q. What methods are used for chiral separation in industry at the present time?
A: There are two ways to achieve a chiral separation: The indirect methods use derivatization reactions to convert enantiomers in diastereomers, which can be separated with conventional achiral methods because these have different physico-chemical properties. However, this method is less used because derivatizations are generally avoided in the pharmaceutical industry. Therefore, direct methods are mostly preferred.
Direct methods use a chiral selector that forms transient diastereomeric complexes with the enantiomers, enabling their separation. This selector can be added to the mobile phase but this approach is mostly used in miniaturized techniques such as CE and CEC because of the high selector consumption with techniques such as HPLC. The selector may also be coated or immobilized on a chromatographic matrix, creating a chiral stationary phase (CSP). The use of a CSP is by far the most applied approach in the pharmaceutical industry to separate enantiomers, with the most popular CSP those containing polysaccharide-, macrocyclic antibiotic-, or cyclodextrin-based selectors. CSPs are used in combination with chromatographic techniques, such as HPLC, SFC, and gas chromatography (GC), of which HPLC is definitely the most popular one. Some companies also use SFC and CE, but the application of those techniques is far below that of HPLC, but there has been a kind of a revival in SFC recently.
Q. What are the limitations of these methods?
A: A major problem with chiral separations is that enantioselectivity cannot be predicted. Several research groups have tried, but with limited success. This also implies that the development of a chiral separation method is often a trial-and-error approach. Therefore, our research focused on the development of generic chiral separation strategies, which help the analysts in chiral method development. In principle, these strategies should be applicable on any compound, independent of their structure. Our separation strategies consist of a screening step, where a limited number of experiments are performed on a CSP with a broad and complementary enantioselectivity. The second step of such a strategy is to apply an optimization step when a (partial) separation is obtained and a second attempt with new conditions when it was not the case. Screenings are often also defined “in-house” in pharmaceutical companies.
Chiral HPLC consumes a relatively high amount of organic solvent for the analysis. Particularly for less environmentally friendly modes of HPLC such as normal-phase- and polar organic solvent chromatography, this can represent a major hurdle on the level of waste disposal. The analysis times can also be quite long, but this is really case-dependent.
Q. Are there other methods in development or emerging to replace such established methods?
A: SFC is definitely a candidate technique to complement HPLC as a separation technique. I have recently seen what SFC can do, and I am quite impressed about the capability of the technique for chiral separations. What you can do in one hour in HPLC, you can easily achieve in less than half an hour in SFC. In addition, the waste problems of HPLC are not applicable for SFC, because the largest fraction of the mobile phase is CO2 and this simply evaporates after the analysis. There are still some hesitations in the scientific world about the potential of SFC because the instruments of former generations were not what they should have been in terms of repeatability and baseline noise, but instrument manufacturers have invested much in this technology recently, and I am convinced that the new generation analytical instruments are significantly better.
I also still believe in the potential of CEC because it uses a combined separation principle (chromatographic partition and electrophoretic mobility) that is quite unique. However, for this technique, there are not only instrumental issues, development of proper CEC stationary phases is also needed.
With regards to stationary phase technology, polysaccharide-based selectors are the most popular. Recently, several companies introduced chlorinated polysaccharide-based selectors and some even have a broader enantioselectivity than non-chlorinated. Polysaccharide-based selectors were also immobilized to cope with the restriction of using special solvents, like acetone, tetrahydrofuran (THF), and chloroform, on coated polysaccharide CSP. Therefore, these selectors will probably remain the most frequently used. What may be an interesting development is the use of new chromatographic matrices, for example, the use of smaller particle sizes and fused-core materials. However, at the moment these are still at the research level.
Q. What will it take for these methods to become industry standards?
A: SFC has the most potential to become an industry standard in the short-term. In fact, it is already used as a technique in several pharmaceutical companies, and increasingly at the level of preparative separations. Exact control of instrumental parameters and much more fundamental research is still required to get SFC to the level where HPLC is at the moment, but with the introduction of the new generation instruments, there are new ways to get there.
Q. Where do you see your research taking you in the future?
A: In SFC, we want to investigate its potential for non-chiral separations and see in what way it can be related to HPLC. Is it a replacement technique for HPLC or is it complementary? The coupling of our SFC instrument to mass spectrometry is also a future research topic.
In CEC, our research will remain in the chiral separation field, where the investigation of polysaccharide CSPs with smaller particles and fused-core particles will be future topics.
More fundamental research in chiral separations is a necessity that remains, so comparative studies will be introduced in our future research. We will also focus on including more chemometrics in this field of our research.
Debby Mangelings is a professor at the Vrije Universiteit Brussel, Belgium. She graduated as a pharmacist in 2001, obtained a PhD in Pharmaceutical Sciences in 2006, and became a full professor in 2010. She is a member of the Department of Analytical Chemistry and Pharmaceutical Technology, headed by Yvan Vander Heyden. Together, they currently supervise the work of seven PhD students. Her main research interests are chiral separations and miniaturized separation techniques. Debby is author or co-author of eight book chapters and 59 manuscripts, 57 of which are published in peer-reviewed journals.