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

Versatility of HPLC Sets Technology Apart

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.”

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