Putting Capillary Electrophoresis to Work
Jeff Chapman and John Hobbs, Beckman Coulter Inc., Fullerton, CA
Q: Which applications are ideally suited to CE?
Q: Is HPCE robust enough for routine QC?
Q: How easy is it to develop a method from scratch?
Q: Can HPCE be used in a "preparative mode"?
Q: How Sensitive is HPCE?
Q: Is capillary temperature control really necessary?
Q: Does HPCE require extensive sample preparation?
It's been over 10 years since the first commercial instrument for capillary electrophoresis (CE) was introduced with a fanfare and a list of promises to revolutionize the world of separation science. Although the company responsible (Microphoretic Systems) vanished shortly thereafter, CE is still very much with us, and in numerous incarnations. While many of you have read countless reviews on ways that CE has been applied, we will focus this article on areas where CE is being put to work. To allow you to best assess this approach, we will answer a series of real world questions from the perspective of using CE in routine assay environments.
To provide perspective, let's first take a brief look at what has transpired during CE's formative years. Early research instruments, while demonstrating exceptional resolving power and phenomenal efficiencies, left a lot to be desired when it came to assay ruggedness and reproducibility. Over time, commercial systems continually improved as refinements were made to both injection design and the efficiency of controlling capillary temperature. As improved commercial instruments came to market, more companies got involved, more detectors became available, more chemistries expanded the range of application and as with most new technologies it was tried for almost everything!
This led to some success but also to disillusionment when struggling with an inappropriate application. In due time, as the technique became better understood (especially capillary surfaces) the logical areas of application emerged and CE began to find it's place as a robust analytical tool. Many, who looked at the technique in its early years and concluded that it wasn't for them, could well find it worthwhile to check it out today. A lot has happened in the last ten years.
The following collection of frequently asked questions with real world answers highlight those applications where CE has become a real analytical workhorse not simply a treasure of technology.
Q: Which applications are ideally suited to CE? (back to top)
Clearly, those which have been difficult by other analytical systems. However, this is not a technology of last resort but rather the first that should be considered when dealing with highly polar, charged analytes. CE has excelled in the analysis of ions where rapid results are desired, and has become most predominant for the impurity analysis of both basic and chiral pharmaceuticals. This technology is making its mark in biotechnology replacing traditional electrophoresis for the characterization and analysis of macromolecules such as proteins, carbohydrates and nucleic acids. Indeed, CE is bringing a whole new meaning to the phrase "current good manufacturing processes" as they apply to biotechnology products. The "in solution" approach which is the trademark of this technique, has also been ideal to create environments in which molecular interactions may be detected and studied.
Ion Analysis
By nature, ions are highly charged polar species which lend themselves nicely to the capillary electrophoresis format. Typically, most of the routine CE work that has been done uses bare-fused silica capillaries, with simple buffer systems carrying a cationic surfactant to reverse and modulate electroosmotic flow. The primary mode of detection has been indirect UV absorption, requiring an appropriate choice of background electrolyte. Dabek et.al., have demonstrated that by changing the background electrolyte from phthalate to 2,6-napthalene-dicarboxylic, they could improve the sensitivity of their organic anion assay greater than 5 fold. This same group also illustrated the successful separation of both anions and cations from environmental aresols using standard indirect methodology.
Basic Pharmaceutical Analysis
The highly polar nature of basic pharmaceuticals makes for fundamentally complex chromatography. Ion pairing reagents and stringent column regeneration is often required to reduce non-specific ionic interactions. With CE, these highly functional groups are favored and can be exploited to effectively provide the separation.
When analyzing basic compounds two approaches have primarily been adopted. 1) Utilization of an amine capillary surface to repel cationic interactions with the capillary wall, allowing a wide range of buffer pH to be used. 2) Analysis at low pH where the capillary surface is essentially neutral and the amine functional groups on the solutes are maximally ionized.
The generation of amine-coated surfaces have been accomplished in a variety of ways. These include the creation of novel capillary surfaces bearing quaternary ammonium ions or a dynamic coating procedure with proprietary commercial reagents like Microcoat from Applied Biosystems or the eCAP amine regenerator from Beckman Coulter. An example of this approach is illustrated in Figure 1.0 with the analysis of seven tricyclic antidepressants. These highly charged pharmaceuticals are notoriously difficult to separate using chromatographic approaches yet lend themselves well to the electrophoretic format.
The second approach, which is much more straight forward, is the use of bare-fused silica at a well defined acidic pH. Figure 2.0 illustrates an example of this approach with the analysis of 20 basic drugs. The robust nature of this tool and the ruggedness of this single method approach lends itself well to screening protocols used in drug discovery and forensic toxicology. Indeed, Hudson et al., (1998) have reported the routine use of this approach to screen complex samples from a library of greater than 500 basic pharmaceuticals of toxicological interest.
Chiral Pharmaceutical Analysis
Pharmaceuticals with asymmetric carbons that exist as enantiomers pose a difficult challenge for the analytical chemist in designing rugged assay methodology. As these stereoisomers are physically and chemically identical, one has to focus on the construction of chiral environments to facilitate separation.
Of all of the chiral additives employed, we have seen the best success obtained when using a family of sulfated cyclodextrins. Of course not all sulfated cyclodextrins are alike as both the degree and site of sulfation appear to significantly impact resolution. With this approach, separation of racemates yielding resolution values of 5 or greater are common, while the best separations produce values greater than 20. The direct consequence of this, is the ability to rapidly detect enantiomeric impurities at levels as low as 0.1%. Although this approach is not highly visible in the recent press, it is in routine use as an effective enantiomeric purity assay in many pharmaceutical companies. With such high resolving power, it is usually inconsequential whether the enantiomeric impurity resides on the front or the tail of the peak. An example of this high resolving power is illustrated in figure 3.0 with the separation of amphetamine enantiomers.
A paradox of the successful implementation of tools within industry is that you usually only hear about them when a product has been rejected as a drug candidate and is deemed publishable or once a product has finally reached market. Be assured that CE is being utilized a lot more in industry than most people realize. Surely, the simplicity of this approach will ensure that CE becomes the primary analytical methodology utilized for the assessment of enantiomeric impurities.
Glycoprotein Analysis
Biotechnology products, especially glycoprotein-based pharmaceuticals must be analyzed and characterized as rigorously and completely as current technology allows. As an important goal of analytical biotechnology is to have adequate characterization, specification and control assays for the chemistry manufacturing and control sections of a regulatory filing, technology advancement is always at issue. CE has been applied effectively to many of these functions, improving processes, which assist in bringing macromolecular drugs to market faster.
Peptide mapping is extensively used for the quality control and characterization of recombinant products. Although reversed-phase chromatography continues to be the main tool used here, CE is being rapidly adopted. The CE separation is based on differences in a peptide's electrophoretic mobility at a defined pH, giving additional information orthogonal to that of reversed phase chromatography. This ultimately increases one's confidence when assessing a product's purity and identity.
An increasing number of glycoproteins are being developed as potential biopharmaceuticals, owing much to their activity in biological systems. These proteins require special consideration as their physiological function; i.e. activity and specificity may often be dependent on the carbohydrate moieties. Differences in how a protein is glycosylated may affect its biological activity and as such the heterogeneity generated in its production must be evaluated and controlled. CE in solution provides one of the simplest approaches to assessing the distribution of glycovariants. In such a system one can easily create an environment to discriminate between differences in glycosylation. An example of this is shown in figure 4.0 with the analysis of ribonuclease B. In this example, borate added to the buffer system complexes differentially with the ribonuclease B glycans. This differential glycovariant complexation allows an enhancement of resolution through a shift in mobility induced by the additive charges of the borate ions. The inset table compares the values of the glycoform distribution obtained by borate complexation with the data received from the size exclusion chromatography of the released glycans. The high correlation between the two results highlights the power of CE in being able to quantitatively assess heterogeneity of glycoproteins directly from the protein.
Enhancements to the capillary surface have also proven beneficial in the analysis of glycovariants. Polymeric amine surfaces have been used to generate repellent forces to cationic functional groups on proteins. An example of the utility of these polyamine surfaces is illustrated by Girard et. al. Here the authors demonstrate the separation of recombinant human erythropoietin glycoforms using a polymeric amine capillary and a buffer near the iso-electric point of the protein. In this paper they highlight distribution differences of the rHuEPO variants in the final formulations from two manufacturers.
Carbohydrate Analysis. Carbohydrate analysis is essential to fully characterize a glycoprotein-based biopharmaceutical and continues to be a challenging and formidable task. At first glance CE does not appear to be ideal in that most carbohydrates lack readily ionizable functional groups and the ability to either absorb or fluoresce. This situation is easily remedied with a simple reductive amination reaction using 1-amino-pyrene-3,6,8 trisulfonate. Figure 5.0. Illustrates this reaction which provides specificity, ample charge for mobility and strong fluorescence when excited with an argon ion laser at 488 nm.
Monosaccharide Composition.
Essentially there are eight sugars commonly found in eucaryotic glycoproteins that once labeled, can be easily resolved and quantified by CE-LIF. This assay is rapid and the labeling efficiency is similar for all monosaccharides, allowing relative quantities to be determined directly without the need of standards.
A clear advantage of this solution-based approach to monosaccharide composition analysis is in the specificity of the assay. The reductive amination and the open tube format allows this analysis to be done directly, without the need for post hydrolysis clean up. Figure 6.0 illustrates the separation of the monosaccharides hydrolysed from bovine fetuin. The electropherogram in panel A illustrates the separation and quantitation of the sialic acid released using a weak acid hydrolysis. Panel B represents the remaining sugars released upon a strong acid hydrolysis, while panel C represents the separation of the monosaccharide standards. The data generated showed good correlation with values reported using other analytical tools.
Oligosaccharide Profiling. The distribution of glycans released from a glycoprotein yields a fingerprint, which may be, used it its identification. This same fingerprint is often used to monitor process to insure that no change has occurred in the production of the protein. Of particular note is the significant resolving power, which is sensitive to even positional isomers of the glycans. Figure 7.0 illustrates the analysis of glycans released from Ribonuclease B. The mannose-7 glycan is represented here with a distribution of three peaks representing a possible 2-2, 2-3 and 2-6 mannose linkage.
Iso-Electric Focusing. Identity can be partially established with the determination of a protein's isoelectric point. cIEF offers advantages over slab gel analyses in speed, ease of automation and most importantly direct quantification. Essentially, cIEF provides information in three areas critical for discovery research and for characterization and control of biotechnology products. 1) Evidence of identity can be obtained from the determination of pI. 2) Quantity can be assessed from peak area. 3) Heterogeneity can be determined from the relative area percent of the various species resolved. Although several vendors offer pre-packaged iso-electric focusing kits, most routine laboratories are opting to develop their own methodology, owing to the unique nature of individual proteins. Vendors such as Biorad offer ampholytes which are purified to allow more suitable detection at 280 nm.
Separation by Molecular Weight. Traditionally, one of the most common methods used to assess the molecular weights and purities of proteins has been SDS-PAGE. An adaptation of this technique to the capillary format has had significant benefits including more accurate MW assignment, a greater degree of automation and direct quantitation. Glycoproteins, which tend to bind SDS in an irregular manner, can more effectively be evaluated using the capillary format. Using a free solution approach, the gel dilution format of the Ferguson method can be easily automated, helping to correct for molecular weight discrepancies. Vendors such as Applied Biosystems, Beckman Coulter, and Biorad all sell commercial chemistries to achieve this task.
Identity by Immunoassay. Immunoassays are commonly used in biotechnology for the detection and quantification of both small and macro-molecules in biological fluids and for detecting host cell contaminants. The free solution approach by CE-LIF has brought an exciting alternative to solid-phase immunoassay. The CE-LIF format eliminates antigen immobilization and avoids many solid phase associated problems. This methodology makes use of either a purified antigen labeled with a stable fluorescent dye (competitive assay) or an affinity probe labeled with the dye (direct assay). An example of the use of a direct immunoassay was recently published by M.J. Schmerr et al., where they developed a CE-LIF immunoassay to detect prions associated with transmissible spongiform encephaopathies like that of Scrapie and "Mad Cow" disease.
Genetic Analysis
Since the first publication of DNA's double helical structure in 1953 (Watson and Crick), electrophoresis has been a standard among analytical tools used in modern biochemistry. Forty-five years later we find electrophoresis still going strong as the methodology of choice for the analysis of nucleic acids. However, with the automation and quantitation provided for by CE it is a natural progression that this technology replaces the slab gel format for genetic analysis.
Specific applications where CGE is actively being used include oligonucleotide quality control, quantitative viral load determination, gene expression studies, analysis of DNA- protein interactions, genotyping and most recently automated DNA sequencing.
Oligonucleotide Quality Control
It is well acknowledged that there is a lot of work that goes on downstream of a produced primer or probe and that when something goes wrong in a hybridization reaction, the synthesized oligo is usually the first to be targeted. By providing good quality assurance (QA) the quality of the oligonucleotide is confirmed and attention is appropriately focused on the downstream process. Of course with the development of nucleic acid based pharmaceuticals a rigorous characterization of the product is demanded.
Denaturing capillary gel electrophoresis has worked well for this process. Typically, linear polyacrylamide is used to create a physical gel. Urea (7M) is commonly used as a denaturant to minimize secondary structure forming within complimentary sequences of the oligonucleotide such that it can be separated by size as a linear single stranded fragment. Figure 8.0 illustrates the analysis of a 45-mer oligonucleotide providing an assessment of its purity level. In this example there appears to be the presence of a significant quantity of the n-1 product, with the low-level failure sequences clearly visible along the run. Most work along these lines have utilized commercial oligonucleotide analysis chemistry available from vendors such as Beckman Coulter Inc., Biorad, Hewlett Packard and J&W.
Quantitation of Gene Expression
Accurate measurement of the expression of a gene has long been difficult to assess, yet it is critical to better understanding cellular processes. Although competitive PCR has advanced the ability to measure changes in mRNA expression, inaccuracies still remain in the quantitation of the final amplified product.
CGE with LIF detection not only provides a separation of the target and competitor product, but also accurately quantitates them at a sensitivity equivalent to autoradiography. The quantitation is not an estimation of band density but rather, a count of all the material passing though the detection cell. As the separated sequences are based on size, confirmation of fragment identity is achieved, ensuring the measurement of the correct sequence.
Viral Load Quantitation
Quantitation of viral load in patient specimens is important to assess the stage of disease progression and to monitor the effectiveness of drug therapy. A good case in point can be found with acquired immunodeficiency syndrome (AIDS), where the level of human immunodeficiency virus (HIV) has been linked to the progression of the disease. Competitive PCR methods have allowed quantitation of proviral DNA or plasma viral RNA in patients with HIV-1 infection and this methodology has been adapted to the capillary format. However, with HIV-1, novel advanced anti-retroviral therapies have been reported to reduce virus titres below the detectable limit.
To address both the amplification based-variability and inadequate detection limits, Kolesar et al., explored the use of CGE with direct probe hybridization. In this example the "open tube" approach of a physical gel separation allows a probe-hybridized viral RNA to be readily detected and resolved from the unhybridized probe. The probe complex is stabilized with the use of an intercalator and probe label. Stringency is imparted by the choice of the medium and the separation process. In this study, 21 copies of HIV were detected from a single ml of plasma (without amplification), representing a significant increase in sensitivity and reduction in assay variability.
DNA-Protein Interactions
DNA-protein interactions are involved in the control of replication, recombination, modification, repair and transcription processes. Analysis in solution is an ideal way to study the processes of interacting species, and is clearly one of the benefits that CE brings. As the capillary acts as simply a vessel, the separation environment can be defined to examine these interactions. Essentially, the assay looks at a shift in mobility which occurs while one species is interacting with another. A good example of this is shown by Xian et al., with the analysis of a sea urchin transcription factor important in embryo development. In this method they are able to rapidly determine dissociation constants at levels 100 times more sensitive than conventional gel mobility-shift assays.
Genotyping
CGE-LIF has also been successfully applied to the analysis of genetic markers used for human identification. Restriction fragment length polymorphisms, short tandem repeats and variable number tandem repeat (VNTR) analysis have all been successfully accomplished with CGE methodology. Figure 9.0 illustrates an electropherogram of the separation of the D1S80 ladder (300 to 700 bp) a common allelic ladder having repeat units of 16 bp. In this example the physical gel utilized is polyethylene oxide based and thiazole orange is used as an intercalator to provide fluorescence for detection.
Automated DNA Sequencing
Many of the inherent capabilities of CGE lend itself well to DNA sequencing. The automation of capillary filling and purging negates the need of a technician to learn gel pouring and lifting. The ability to control and maintain even temperature of a capillary, produces sequencing bands that are both better resolved and more reproducible. Automated fluorescence signal detection allows for faster turnaround and an improved determination of band migration order.
Q: Is HPCE robust enough for routine QC? (back to top)
Yes, with the development of more simplified robust instrumentation geared for the industrial laboratory, CE is being adopted for many routine assays. Simple components precisely controlled provide the reproducibility and robustness necessary for validating methods. CZE in its purest form is a very simple technology, essentially just an open tube bridged by a power supply with a detection mechanism at one end. By understanding the surface of the capillary and its potential interactions, you can alleviate the largest source of error, interactions with the capillary surface. CZE is being used as a routine quality control assay in many corporations for applications such as oligonucleotide purity determination, enantiomeric excess determination, glycoprotein analysis and even screening for basic drugs.
In forensic toxicology, the robustness and reproducibility of the assay is critical, as there is no more difficult task than screening for the "general unknown". To evaluate the robustness of this methodology Hudson et al., (1998), performed an extensive four month study on the reproducibility of migration behavior (for purposes of identification). In this study they evaluated a 20 component test mixture run twice daily, every day for four months. Figure 10.0 illustrates the results of this study, highlighting mobility R.S.Ds less than 0.3% over this lengthy study.
Q: How easy is it to develop a method from scratch? (back to top)
As with any analytical tool, the greater the flexibility in methods design the more potentially complex the methods development can appear. Since in CE, one has the flexibility to define the separation "environment" the task of developing methodology can appear quite daunting. On the surface one must evaluate capillary temperature, buffer pH, buffer salt composition, buffer concentration, capillary surface, applied field strength, capillary length, and sample injection volume. Of course all of this must take into consideration the solubility characteristics of the analyte and the composition of the matrix with which it can be found.
As with HPLC and GC, experience simplifies the degree of methods development. Ones accumulated experience often narrows a methods development strategy to a very small subset of conditions. In reality, most successfully implemented CE analyses require very little "methods development", as the core methodology for that application needs only to be "tweaked". A good example of this can be found with our chiral methods development strategy using the highly sulfated cyclodextrins (figure 11.0). Here we essentially use one method to screen three different cyclodextrins for resolution of the enantiomers of interest. So far, we have achieved significant resolution of the enantiomers in 131 of 134 compounds that we have analyzed.
Q: Can HPCE be used in a "preparative mode"? (back to top)
The answer to this question is usually qualified by the questioner's definition of "preparative". CE as a technology is by its very definition an analytical technique. The use of the capillary format results in reduced joule heating and diffusion making analyses in solution using high field strength's possible. The apposing side to this is that very small volumes and mass loads are introduced; limiting CE to an analytical tool. When pushed this technique has been described as "micro-preparative", where with the use of larger column dimensions (200 um) enough mass can be collected for further analysis. This process has been routinely automated with fraction collection algorithms which allow collection by time or detection gates.
Q: How Sensitive is HPCE? (back to top)
Although the use of nanolitre injection volumes is listed by many as an advantage, this same attribute becomes a liability with regards to assay sensitivity. By limiting the volume of sample introduced you also limit the mass load required for detection. For this reason assay sensitivity becomes a function of maximizing mass load and concentrating the separation zone. An assays sensitivity can be impacted by more than 100 fold by simply loading larger sample volumes and concentrating the zones during a pre-separation phase using isotachophoresis.11 Additional strategies used to improve detection sensitivity have included the manipulation of capillary path-length using bubble cells and Z-Cell arrangements.
For many routine protein applications, CE is typically operated at in the ug - mg/ml concentration range. Basic pharmaceutical analysis is being accomplished at the 10 ng/ml range when incorporating extraction protocols and using selective electrokinetic injection techniques. For DNA analysis, pg-ng/ml concentrations are typically seen when using intercalator dyes and laser-induced fluorescence detection.
Q: Is capillary temperature control really necessary? (back to top)
The answer here is an emphatic yes! The first step to successful CE is in the definition of the actual set temperature as this will influence viscosity impacting sample load and electroosmotic flow. The heat in the system will also impact the kinetics of any potential interaction that you are evaluating. The second step is in controlling the capillary at that defined temperature to maintain the reproducibility of migration behavior and injection volume.
Q: Does HPCE require extensive sample preparation? (back to top)
This is another one of these very subjective questions. As a general rule of thumb, sample preparation should be approached in the same manner as HPLC. This may involve solid phase extraction protocols to remove protein or excipients, dialysis to remove interfering salts or buffer exchange for compatibility and solubility purposes.
Although CE appears to use much smaller tubes than traditional HPLC, the actual "pore" is much larger. In combination with the use of selective injection techniques (electrokinetic) one can quite often "get away" with more complex sample matrices than traditional chromatographic approaches. However, in the interest of assay robustness and operating in a routine environment, the cleaner the sample matrix the simpler the methods required.
In summary, high performance capillary electrophoresis is a valuable tool which is now finding its place as a workhorse in the analytical laboratory. So if you are looking for an analytical separation technique to resolve highly charged polar analytes or resolve enantiomers of compounds with chiral centers then you would do well to look to CE first.
For more information: Jeff Chapman, Beckman Coulter, 4300 N. Harbor Blvd., PO Box 3100, Fullerton, CA 92834-3100. Tel: 714-871-4848. Fax: 714-443-8283.