Capillary Electrophoresis Evolving In Niche Pharm, Bio Applications
After a decade of living in the shadow of high performance liquid chromatography (HPLC), capillary electrophoresis (CE) has settled into niche industrial and analytical applications that exploit its strengths: analyzing charged or highly polar molecules. Unlike gel electrophoresis, a slow method primarily used by biologists, CE is rapid and its results may be quantified. These benefits make it suitable for non-biotech pharmaceutical QC/QA, especially for chiral drugs whose importance will continue to increase. Most experts today recognize CE and HPLC as "orthogonal" analytical methods that complement, but in general do not substitute, for each other.
Mass Spectrometry: Tandem Technique Makes CE More Useful
Where CE Fits In
Summary: Still No Replacement for HPLC, But…
According to Ray Lombardi, president of Scientific Resources, Inc. (SRI; Eatontown, NJ), after the initial excitement over CE users became turned off by the technique's analytical variability, low sample loading requirements and dependence on ultraviolet detection. "Researchers tried to apply the principles and philosophy of HPLC to CE," Lombardi said. "Since the two techniques are so different, disappointment was inevitable. Today, I'm happy to say there are more seasoned CE users than ever getting the analytical results they need."
Electroosmotic force (EOF) variability is inevitable with unmodified capillaries. "You can buy a capillary from a top supplier and get one result. The next time you use the same capillary, or a different one from the same batch, you get a different result." The solution? SRI's Celixir, a proprietary coating buffer that makes capillary inner walls uniform, resulting in even EOFs across very wide pH ranges. Celixir uses a two-buffer system—initiator plus stabilizer—to eliminate absorption and interference problems. Lombardi claims that Celixir reduces EOF variability to less than 0.1%. "Celixir is a fully dynamic coating that can be removed after a run. It even works with capillaries from different manufacturers. Most CE suppliers suggest working at low pH to reduce the incidence of wildly erratic EOFs in uncoated capillaries, but we'd like to think Celixir has tamed EOF variability."
Mass Spectrometry: Tandem Technique Makes CE More Useful
Inexpensive desktop mass spectrometers (MSs) have boosted the utility and attractiveness of CE to R&D workers in biotechnology and related laboratories. Modern CE instruments may be interfaced with MS to combine rapid separations and mass analysis. Hewlett Packard's (HP; Wilmington, DE) CE business stresses the CE-MS connection through instruments hooked up with electrospray MS, with a focus on analyzing peptides and proteins through the combined technique. HP's Mass Selective Detector allows switching between HPLC/MS and CE/MS for rapid separations based on either hydrophobicity or charge.
Hewlett-Packard's 3D Capillary Electrophoresis instrument
HP is among instrument companies that have made MS, through its interface with CE, an analytical tool for the masses. "We view easy-to-use benchtop MS as simply one detector for CE instruments," said David Heiger, Americas Business Manager for Liquid Phase Products at HP. "The benefit to biochemists is that they don't need to be MS experts to obtain mass info. Now anyone can perform absolute peak identification."
According to Heiger, getting out the news on CE success stories is key to establishing CE as a routine analytical method, especially in industrial labs. "Many chromatographers who've purchased CE instruments have not been as successful as they or we had hoped. They don't always recognize that CE is governed by the principles of electorphoreses, not of chromatography." For this reason it is probably more reasonable to expect CE to replace slab gel electrophoresis and isoelectric focusing, rather than to cut into HPLC's traditional markets. Slab gels are labor intensive, difficult to quantify, and for the most part not automated. "The advantage of CE is you can achieve a level of automation for electrophoretic separations that customers are used to for GC or LC," Heiger added.
Where CE Fits In
Beckman Coulter, Inc. (Fullerton, CA) is another company that has committed itself to CE's evolution since the early days, when end-users hoped CE's high speed and efficiency might enable it to replace LC. "After ten years with this technology we've discovered it does certain things really well," says Jeff Chapman, Strategic Marketing Manager for Bioseparations at Beckman Coulter. "Yet as with most new technology it has taken time for users to determine where CE best fits into their workflow. CE is by no means a replacement for all HPLC, but rather an additional analytical tool which should be considered first for specific applications."
Chapman views CE as a kind of scientific-intellectual liberator. "CE gives scientists the ability to define their experiments to suit their needs. Rather than working with a predefined separations medium, you start with an empty tube, and fill it with material of your choosing that defines what you can do with it. You have complete control. Clearly, this gives the experimenter the ability to answer critical questions they couldn't answer before." CE's principle drawback, according to Chapman, is that it is strictly an analytical technique and cannot be scaled up.
The rising cost of CE instrumentation (yes, it's simpler and less expensive to run, but not significantly cheaper to acquire than HPLC) has not been a barrier to wider adoption of CE in the pharmaceutical industry. "Companies will invest what they need to invest to solve critical analytical problems, whether it's by GC, LC, or CE," he said. "Once a compound is identified as a viable pharmaceutical candidate, every day in the analytical methods development phase is a day less of potential revenue. With highly charged, polar or chiral compounds, researchers can struggle for quite a long time with the older techniques, while CE methods development for these applications is relatively straightforward and fast. Getting something up and running quickly is a very attractive benefit of CE, and it is these situations where the technique is most widely adopted."
For example the analysis of drug molecules with high pKa is traditionally carried out with GC or LC, but these work best only when the molecular charge is masked or covered up. CE, by contrast, exploits these pesky charges. "Nucleic acids and some proteins give analytical scientists fits, but with CE their analysis is trivial. Don't get me wrong—here are challenges, but they can be more easily worked out. Researchers who begin a method using GC or LC, then switch to CE, often ask why they didn't start with CE in the first place."
Beckman Coulter's P/ACE line of CE instruments, the company's trademark since 1989, consists of instruments tailored for specific applications, i.e. separating glycoproteins or chiral molecules. Each custom-configured P/ACE instrument system features detectors, chemistries, training and documentation suited for the module's intended application. The most recent addition to this line is the P/ACE MDQ (Methods Development Quality Control), designed as single platform systems that may be used in one or more areas of a pharmaceutical or biopharmaceutical company: A single platform can be tailored for research, methods development or quality control.
Specialized hardware, disposables, and software (pictured above) differentiate Beckman's P/ACE MDQ analytical systems, which are suitable for QC/QA as well as research.
Beckman Coulter's CE detection is anchored on laser-induced fluorescence (LIF) detection, around which many novel CE applications have sprung up. Chapman mentioned that nucleic acids and carbohydrates as ideal analytical targets for LIF. Early in 1999 Beckman Coulter expects to extend LIF to its MDQ instruments, which currently use ultraviolet and diode array detectors, plus an external detector adapter for electrochemical, conductivity, and mass spectrum detectors.
Summary: Still No Replacement for HPLC, But…
As you know by now CE doesn't work for every separation. Experts interviewed for this article did, however, agree on applications where CE is a best-bet:
- Highly charged, polar analytes that are difficult to separate using chromatography. Many drugs—both biotech and synthetic—fall into this category. In particular, CE shines for peptide or protein molecular weight determinations, carbohydrate analysis and DNA/RNA. CE's low operating cost and versatility also makes it the clear choice for basic (high pKa) drugs and chiral separations. For chiral molecules, CE capillaries are charged with chiral auxiliary agents costing pennies per run, compared with chiral GC or LC colums costing a thousand dollars or more.
- Because it is not limited to low molecular weight materials, CE/MS is gaining acceptance for protein characterization, including protein derivatives formed by deamidation and loss of amino acids. In genetic analysis, CE is currently used for structural studies on synthetic oligonucleotides (purity and size determinations) and gene sequencing.
- Even CE's staunchest proponents don't believe it will replace HPLC, but for some applications the advantages of CE are difficult to resist. CE instruments use no pistons, seals, or moving parts, so the technique is considered simpler and faster, with less troublesome instrumentation, than HPLC. CE is not less costly to acquire, but it is cheaper to run since capillaries run a tiny fraction of the cost of HPLC columns.
- Pharmaceutical QC/QA engineers can try CE in cases where analysis of polar components of a reaction do not separate easily by HPLC or gas chromatography.
- Individuals in biology or biotechnology laboratories who think of CE as a replacement for gel electrophoresis won't be disappointed. Sensitivity is about equal to that of Coomassie blue staining, but not as sensitive as silver staining unless a laser-induced fluorescence (LIF) detector is used. Keep in mind that LIF requires derivatization.
For more information:
Ray Lombardi, president, Scientific Resources, Inc., 1 Industrial Way West, Eatontown, NJ 07724. Tel: 732-542-5000. Fax: 732-542-0089.
David Heiger, PhD, America's business manager, Liquid Phase Products, Hewlett Packard Corp., 2850 Centerville Rd., Wilmington, DE 19808. Tel: 302-633-8000.
Jeff Chapman, strategic marketing manager for Bioseparations, Beckman Coulter, Inc., 4300 N. Harbor Boulevard, PO Box 3100 Fullerton CA 92834-3100. Tel: 714-871-4848.