June 1, 2011 (Vol. 31, No. 11)

Automation and Spatial Resolution Improvements Drive Broader Usage

When coupled with mass spectrometry, liquid chromatography forms a powerful technique with high sensitivity and selectivity. The technology is especially relevant to the specific detection and identification of chemicals in complex mixtures, which will be confirmed by the studies reviewed in this article.

“Lectin-affinity chromatography is a popular medium for the purification of oligosaccharides, glycopeptides, and glycoproteins,” noted Srinivasa Rao, Ph.D., technical manager at Dionex. Concanavalin A (Con A), a lectin derived from the jack bean, Canavalia ensiformis, is the lectin of choice for many purification protocols. At neutral and alkaline pH, it exists as a tetramer of four identical subunits weighing in at approximately 104 kDa.

Con A is noted as one of the best characterized and widely used lectins, given that it binds to α-mannose and to α-glucose, although with weaker affinity to the latter. “However, its utility is limited by the fact that it is ordinarily incorporated into manually operated agarose bead-based spin columns.”

As Dr. Rao explained, this limits the number of purification cycles for which the column can be used, with the result that costs are driven up and time is lost. With the growing interest in investigations in the realm of glycoproteomic studies, such as biomarker identification, there is an increasing need for a robust HPLC lectin column.

For this reason, Dr. Rao and his coworkers have focused on the development and function of new monolithic affinity columns. Their recently designed column is known as the ProSwift ConA-1S, a polymeric monolith prepared by in situ polymerization followed by functionalization with Con A.

The monolith is a cylindrical polymer rod containing uninterrupted, interconnected flow-through pores, with surface area greater than nonporous bead-based columns. The structure consists of small pores that contribute surface area and larger openings that allow reduced back pressure at elevated flow rates. This approach results in short mass-transfer distances that produce improved efficiency, even at elevated flow rates, Dr. Rao explained. The HPLC compatibility of this column allows automatic sample injection, high throughput, and excellent reproducibility, he remarked.

To give an example of successful application of their column technology, Dr. Rao described the purification of horseradish peroxidase, a glycoprotein rich in high-mannose type glycans. Bound to the Con A column, it was eluted with α-methyl-mannopyranoside. This high-capacity Con A column retained the high specificity toward alpha mannose residues as shown by binding and elution investigations of several standards.

Dionex’ new HPLC monolithic Con A affinity column, ProSwift™ ConA-1S, allows automatic injection and on-line elution monitoring, resulting in sharper peak shape and requiring smaller elution volume of the more enriched fractions, he explained. The column can also be used for glycan and glycoprotein analysis and purification. The ProSwift ConA-1S column can be regenerated easily by washing with conditioning buffer after sample binding and elution, said Dr. Rao. “Our durable column chemistry allows hundreds of run cycles with minimal capacity loss.”

Porous Graphitic Carbon

Luisa Pereira, Ph.D., principal scientist in chromatography consumables and speciality products at Thermo Fisher Scientific, is developing analytical tools based on porous graphitic carbon columns to advance protein phosphorylation studies.

“Unfortunately proteins tend to be too large to analyze quantitatively in their native states, and so a formal process of denaturation and digestion of the protein had to be undertaken,” said Dr. Pereira.

This strategy generates a series of peptides among which the extremely polar class of glycopeptides and phosphopeptides are of particular interest. However, these compounds present a challenge due to their poor retention with standard reverse-phase liquid chromatography packing media. For this reason Thermo Fisher Scientific began using porous graphitic carbon, noted for its strong retention of polar compounds.

To assess the utility of this approach, Dr. Pereira and her colleagues evaluated the separation of a collection of polar and nonpolar peptides. The retention of polar peptides GHK and TSK is greatly affected by the pH of the mobile phase. Both these peptides have a basic terminal lysinyl residue and feature no acidic residues other than the carboxy terminus. When the pH of the mobile phase is increased, reaching near-pI values, the retention of the peptides is greatly increased. The overall increase in charged states at higher pH may result in decreased hydrophobic interactions, explaining the drop in retention. For polar fragments, the increase in temperature was found to improve peak shape.

“Furthermore, we found that capillary columns allowed for increased sensitivity, at the same time allowing rapid elution methods. The fact that we are able to analyze complex mixtures of polar peptides establishes that porous graphitic carbon columns are an effective complement to reverse-phase platforms.”

Automating Separations

Thomas E. Wheat, Ph.D., research scientist at Waters, is working with automated systems for controlling pH in the chromatographic separation of biological macromolecules. “The basis for separations is employing a medium that confronts the net surface charge on the target proteins with oppositely charged groups on the ion-exchanger.” Because the protein surface is covered with both weakly acidic and weakly basic functionalities, both the net charge and the 3-D charge distribution can be controlled with the buffer pH.

There are two purification strategies; the bound proteins can be eluted from the column using a gradient of increasing salt concentration, the conventional approach, or a gradient of changing pH, which is a more demanding and therefore less common method. However, pH optimization is the most effective means of achieving selectivity. The classic analytical approach to constructing a pH gradient is the use of solutions of disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4), an acid/base conjugate pair.

Dr. Wheat and his colleagues have developed a software system to automatically calculate the proportions of the buffers for a given pH. The simple implementation of this function is based on calculations from the pKa. This software algorithm simplifies the development and routine execution of bioseparation methods.

“The platform, known as Auto Blend Plus Technology, provides accurate, automated pH control for chromatographic mobile phases. The algorithms are designed to compensate for ionic strength effects on pH.”

These functions, although relatively straightforward, are extremely powerful. Dr. Wheat’s team has been able to separate mixtures of monoclonal antibodies, which differ in relatively subtle properties, including their amino acid sequences and secondary modifications.

“We are able to identify a specific monoclonal antibody from a panel and say which of these 10 antibodies in a mixture we have isolated, based on retention time,” Dr. Wheat said. “While I can’t separate every antibody mixture, by manipulating the pH I can get a separation of just about all of them if I pick the right 10.”

The Auto Blend Plus Technology is not a clinical tool at this point, but it has numerous applications in biopharmaceutical quality control and will no doubt be of interest to R&D pharma groups. “We have been focusing on this technology for a long time and I am happy to see it progressing to this level.”

Detection of Organic Contamination

Water supplies throughout the U.S. are showing unprecedented levels of contamination from drugs, antibiotics, hormones given to farm animals, and personal-care products such as lotions, beauty products, soaps, and hair dyes.

According to Maricar Tarun, Ph.D., an application scientist at EMD Millipore, at least 46 million Americans are supplied with drinking water that tested positive for at least one of these substances. Contaminants run the gauntlet, including acetaminophen, caffeine, codeine, nicotine, and sulfamethoxazole.

With water supplies deeply compromised, the question arises as to what extent these molecules wend their way into the high-purity water so essential to the operation of scientific laboratories.

Using reversed-phase HPLC with a quadrupole ion trap mass spectrometry system, Dr. Tarun and her co-workers examined water that had been subjected to a number of different purification regimes. They detected six pharmaceutical molecules in tap water, between 2 and 76 ng/L.

After reverse osmosis and deionization, Dr. Tarun was able to detect low nanogram levels of all the molecules except acetaminophen. In the HPLC-grade water tested, most of the molecules that were examined yielded levels in the range of 200 pg/L, yet one molecule, caffeine, was much higher, giving values of about 11 ng/L. In LC/MS grade bottled water, half of the antibiotics and drugs were undetectable, although caffeine still weighed in at ca. 10 ng/L. And this was even after an elaborate series of purification steps such as reverse osmosis, ion-exchange resins, UV photo-oxidation, and treatment with activated carbon.

Dr. Tarun noted that high-purity water plays a critical role in reversed-phase HPLC separations. Contaminants in the water used to prepare the aqueous mobile phase accumulate in the column and could be responsible for high background noise and other interference, rendering meaningless studies on such pressing questions as environmental contamination. Without a legitimate baseline, it will be difficult to rely on values obtained from samples.

Dr. Tarun’s work points out the difficulties that environmental scientists are faced with in an era when extremely powerful technology is available that can detect contaminants as rare as one in a billion of even one in a trillion. The amount of caffeine in one cup of coffee is 400 million times higher than what she detected in her water samples. Assigning health risks to such miniscule levels of contaminants would be virtually impossible. So while the health risks may be negligible or undetectable, clearly it is essential to monitor the water used in scientific investigations.

According to EMD Millipore, high-purity water plays a critical role in reversed-phase HPLC separations. Contaminants in the water could be responsible for high background noise and other interferences.

Superficially Porous Columns

“Higher productivity is the new catch phrase in the pharmaceutical industry,” says William J. Long, Ph.D., research scientist at Agilent Technologies. In order to move productivity forward, companies are examining their options, including investment in ultra high pressure liquid chromatography instruments as well as alternative column technologies with existing equipment. Dr. Long notes that there is now more flexibility in FDA/USP regulations.

The United States Pharmacopeial Convention (USP) is a volunteer-driven, not-for-profit, scientific organization that sets standards that are enforced by the FDA. In recent years new flexibility has been introduced so that today new methods validation is not always necessary, and many productivity enhancements can be made with simple adjustments to the methods. Additionally, there is more focus on cost-per-analysis, so that a high-use method can be modified to achieve large savings, taking into account physical time, reagents, and reduced instrument usage.

To meet these challenges, Dr. Long has been evaluating superficially porous HPLC columns for use in pharmaceutical quality control applications. Superficially porous column technology is based on particles with a solid core and a superficially porous shell. These particles consist of a 1.7 μm solid core with a 0.5 μm porous silica shell. In total, the particle size is about 2.7 μm. They provide 40–50% lower back pressure and 80–90% of the efficiency of a sub-2 μm totally porous particle or approximately twice the efficiency of a 3.5 micron particle.

Dr. Long reviewed the adaptation of the Poroshell 120 columns to the analysis of naproxen, ibuprofen, and cefipime in accordance with the requirements for method adjustments under USP Chapter 621. Long indicated that these adjusted methods can meet the requirements using lower amounts of solvent and substantially less time. Since these columns are constructed with 2 μm frits, no changes to sample preparation are needed. Lifetime of these columns and the adjusted methods was demonstrated using over 5,000 injections using a prepared ibuprofen sample, Dr. Long explained.

“We have demonstrated that the use of the Poroshell 120 column meets or exceeds the performance requirements for Compendial methods (those that are reviewed in the USP compendium) while staying within adjustment guidelines. In addition, the potential for even higher productivity is demonstrated within guidelines of proposed changes. Substantial improvements in throughput and reduction of analysis time can be accomplished using instrumentation already in the laboratory with even higher productivity by using newer equipment.”

Researchers from Agilent Technologies are evaluating a range of HPLC methodologies in order to facilitate productivity enhancements in pharmaceutical quality control applications.

Chiral Compounds

Chiral chemistry is based on the phenomenon of isomerism, in which two compounds may have the same composition, but the arrangement of the atoms is different, resulting in molecular structures which are the mirror image of one another. Because living systems use one form exclusively, the same enzymatic machinery cannot process both forms, or enantiomers. Ettigounder Ponnusamy, Ph.D., and Mark Nowlan, research scientists at Sigma-Aldrich, have taken this issue into account in developing an HPLC-based method for D-luciferin, the biologically active form of this commercially important compound.

D-luciferin is found in many insects, where it acts as a sexual attractant. It is the natural substrate for luciferase, catalyzing the production of the typical yellow-green light of fireflies. The bioluminescence of this substance is widely applied as an important marker throughout the biological sciences for such purposes as in vivo luminescence monitoring, characterizing gene expression, and detection of the level of cellular ATP in cell viability assays.

Dr. Ponnusamy and Nowlan designed a chemical synthesis of D-luciferin by reacting 2-Cyano-6-hydroxybenzothiazole and D-cysteine in aqueous methanol, ending with a high-quality product generated in large amounts. A chiral HLPC method suitable for luciferin was developed. The optical purity of the D-luciferin was determined by chiral HPLC.

The reversed-phase chiral HPLC method took advantage of a Chrom Tech AGP (α1-acid glycoprotein) column, preferred because of in-house availability and smaller particle size, Dr. Ponnusamy explained. The α1-acid glycoprotein is immobilized onto 5 µm spherical silica beads capable of withstanding organic solvents and higher temperatures within a pH stability range of 4–7. It provides both chiral and general reversed-phase separation power, which can be modulated through shifts in buffer ionic strength and pH, as well as type and level of organic modifier.

The HPLC chiral methodology offers a versatile approach in terms of alternative UV wavelengths, detection modes (ultraviolet and fluorescence), chiral phases (AGP and Dacel Chiralpak WH), and adjustable conditions for enantiomeric purity screening of synthetic formulations. “While we are pleased with the performance of this method,” said Dr. Ponnusamy, “there are a number of areas that we are following including impurity characterization, stability studies, in-process monitoring, and faster, smaller-particle methods.”

Scientists at Sigma-Aldrich have developed an HPLC-based method for purifying D-luciferin that utilizes a Chrom Tech AGP column.

An Advancing Frontier

As improvements in HPLC technology move forward, the precision of spatial resolution and automation of methods allows broader application and more rapid processing of samples and increases in the number of compounds detectable. Widely adopted analytical techniques that are often paired with liquid chromatography include capillary electrophoresis, enzyme assays, sensors, and mass spectrometry. The advances profiled here are only a part of a constantly changing industry.

K. John Morrow Jr., Ph.D. ([email protected]), is president of Newport Biotech and a contributing editor for GEN.

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