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Feature Articles : Sep 15, 2009 (Vol. 29, No. 16)

HPLC Advances for Biomolecular Analysis

Enabling Tools and Technologies Make Cutting-Edge Research Possible
  • Susan Aldridge, Ph.D.

HPLC is at the heart of both downstream processing and research applications such as biomarker discovery. New columns and systems for bioseparations and applications, including proteomics, metabolomics, and environmental and food analysis, were on display at “HPLC” held in Dresden recently. Vendors were ready to show how they were keeping up with customers’ increasing bioseparations needs, while academic groups had a wealth of new discoveries to show off.

Waters is fine-tuning its focus on the biopharmaceutical market, addressing some of the difficult challenges involving protein separation and characterization. “By 2014, seven out of the top ten drugs are likely to be either monoclonal antibodies or recombinant proteins,” noted Dorothy J. Phillips, Ph.D., director strategic marketing chemistry operations.

Waters’ Acquity UltraPerformance LC, or UPLC®, technology is based on sub-2 micron particle-size chromatography and offers speed, high resolution, high sensitivity, and solvent savings of up to 95% over traditional HPLC, according to Dr. Phillips. The company is now developing new column chemistries for this system designed specifically for bioseparations. These new products are designed for both recombinant proteins and antibodies, and peptide digests of these products. Dr. Phillips added that Waters will continue to develop applications for synthetic oligonucleotides because of the many RNAi drugs in the pipeline.

Waters’ protein-separation technology is based on patented Bridged Ethyl Hybrid (BEH) particles that work at high pH and high temperature—conditions often used in protein separation. Another type of BEH column has been developed for the separation and accurate quantization of fluorescently labeled glycans using HILIC (hydrophilic interaction liquid chromatography).

Having technology like this that can analyze glycoprotein-derived glycans is becoming increasingly important for researchers and biopharmaceutical manufacturers. In biosimilars, for instance, regulatory authorities will want convincing protein-characterization data to help show equivalence between an original protein-based drug and its generic version.

Mannose-containing glycans, for example, can be challenging to analyze, but Waters’ Acquity BEH-Glycan columns are able to separate critical mannose-containing pairs, reported Dr. Phillips. “We also look at what is needed to get the sample onto the column, and we offer reagents such as RapiGest ™ SF Surfactant, which increases the quality of enzymatic protein digests and is also mass spec friendly.” 

Glycopeptides can also be analyzed, with determination of the glycosylation site, if this is required. Additional UPLC-based technology solutions from Waters include high-throughput and amino acid analyses from cell culture, as well as technology useful for the analysis of host-cell contaminants in downstream processing.

Meanwhile, Sergio Guazzotti, Ph.D., global product marketing manager liquid chromatography at Thermo Fisher Scientific discussed how the company’s latest U-HPLC systems can boost productivity and reduce costs.

It is now widely accepted that U-HPLC shortens run times, allows faster results and method development, has improved resolution and peak capacity, and significantly reduces solvent costs. “There is a huge trend worldwide toward U-HPLC,” Dr. Guazzotti said. “Growth in HPLC sales worldwide has been around 4 percent but in U-HPLC it has been around 15–20 percent.”

Yet, despite the obvious advantages, some users may not be ready for a shift from HPLC to U-HPLC. The Thermo Scientific Accela Systems offer HPLC and U-HPLC in the same instrument, allowing for stepwise transfer of methods from one to the other. “We believe we offer optimum performance in both, and we have the data to prove this,” Dr. Guazzotti added.

Accela high-speed pumps offer a quaternary mixing capability, which is useful when four solvents are needed for particularly difficult separations. The mixing is done in a single pump, allowing for more rapid method development. “We are the only vendor that can provide quaternary U-HPLC at 15,000 psi,” reported Dr. Guazzotti.

Another important pump feature, introduced at “HPLC” is force-feedback control, a mechanism that allows the pump to guarantee performance by delivering accurate and precise flow and gradients over the whole range of conditions, resulting in exceptional reproducibility.

“It assesses the compressibility of the mobile phase in every stroke. Other pumps merely give an average. This is a unique way of compensating for the variation in the mobile phase and guaranteeing the performance of the pump,” Dr. Guazzotti said. Put simply, force feedback control involves continuous adjustment of valve timing and pumping efficiency based upon the measured compressibility of the solvents.
One of the advantages of U-HPLC is that it reduces solvent usage, which has obvious benefits as the acetonitrile shortage continues. Switching to Thermo U-HPLC is just one option for those concerned about the acetonitrile shortage. Recycling and swapping acetonitrile are also possible, although the latter will produce changes in viscosity and, therefore, pressure of operation. The new Thermo Scientific SRS Pro Solvent System makes the former option easier by redirecting untainted mobile phase to the solvent reservoir during isocratic HPLC. It is powered directly from a USB connection, allowing plug-and-play operation in labs wanting to conserve solvent.

Thermo Fisher Scientific also offers its Transcend™ TLX systems with Aria™ software, which allows multiplexing of two or four channels of HPLC-MS and U-HPLC-MS. A mass spectrometer is a significant investment, yet for much of the time it lies idle—because it only detects the compounds of interest during a fraction of the complete LC run. If a lab has four HPLCs and one MS, higher productivity is possible by having the HPLC systems multiplexed, so that the MS can be dedicated to the HPLC system that is eluting the compounds of interest.

“The concept is easy,” said Dr. Guazzotti. “But the execution is difficult. We have developed a solution within Thermo Fisher Scientific that saves buying additional mass spectrometers by enabling users to increase the throughput of their existing MS.”

A further Thermo Fisher Scientific development on the Transcend TLX system is TurboFlow™ technology, which allows for online sample preparation. Complex biological samples often need to be preprepared—for example, protein might otherwise be precipitated and phospholipids can induce ion suppression which hinders detection in the MS. There are various well-accepted ways of cleaning up the sample prior to HPLC, but they are all labor intensive and may be expensive. It would be better if the sample could just be directly injected and cleaned up automatically before entering the analytical column.

With TurboFlow technology, the sample is introduced into a TurboFlow column and subjected to fast flow conditions that do not allow large molecules, like proteins, time to diffuse. The smaller target molecules with an affinity to the stationary phase, bind to the column. Then the flow is reversed, and the bound molecules elute and go to the analytical column, while unwanted molecules are washed away. “You see an improvement in data quality and a significant decrease in sample preparation time when you use TurboFlow technology compared to standard preparation,” explained Dr. Guazzotti.

HPLC is also being applied in plasma proteomics. There is growing awareness that glycoproteins may exhibit different glycan patterns in cancer and in healthy tissue. As part of the NCI’s new glycobiology initiative—the Alliance of Glycobiologists—William Hancock, Ph.D., of Northeastern University, described the contribution of multiple lectin affinity chromatography (MLAC-MS) to the comparison of the glycan plasma proteome  between women with early breast cancer and healthy controls. 

Andrew Alpert, Ph.D., president of PolyLC Inc. in Columbia, MD, presented the application of ERLIC (Electrostatic Repulsion HILIC) to the separation of phosphopeptides and sialylated glycopeptides, which would be difficult to separate with HILIC alone. ERLIC is proving particularly useful in investigating post-translational modifications of peptides in proteomic studies, Dr. Alpert said.

Meanwhile, Albert Sickmann, Ph.D., Institute for Analytical Sciences, Dortmund, Germany, explained that a knowledge of platelet proteomics is important to better understand the role of platelet activation in hemostasis and thrombosis. But studying platelets is challenging—there are more than 100,000 analytes in a platelet sample and many protein isoforms.

Moreover, the time course of platelet activation is hard to follow, and there are issues around reproducibility and sensitive quantitation of analyses. Dr. Sickmann and colleagues, however, have made progress on a comprehensive study of the platelet proteome, with the identification of 2,500 proteins, more than 1,000 phosphorylation sites, and more than 350 glycosylation sites. They are also using quantitative MS to identify changes in post-translational modification during platelet activation.

Finally, Gabriella Massolini, Ph.D., of the University of Pavia, presented work on the phosphoproteome of amniotic fluid using HPLC-MS. “Amniotic fluid is a dynamic and complex sample that reflects the state of the fetus,” she said. Many biomarkers have been reported to be involved in fetal growth; IGFBP-1 (insulin-like growth factor binding protein) is one example. Phosphoproteins are a new type of biomarker; aberrant phosphorylation has been shown to be important in disease.

However, there are various challenges in phosphoproteins analysis, arising from their heterogeneity, low stoichiometry, low abundance in the cell, and limitations in the dynamic range of analytical methods.
Dr. Massolini’s team uses hybrid instruments, like LTQ-Orbitrap coupled with nano-LC, to pursue the analysis of phosphoproteins. They are looking at a new analytical platform with online digestions, online selective enrichment, and online LC-MS.

The team has examined the phosphorylation state of IGFBP-1 which is only present at 0.2 mg/L. Although this represents 0.01% of the whole proteome, it is still one of the most abundant proteins. They carried out the analysis in miniature, using amniotic fluid from a single patient, with a view to using this approach for prenatal diagnosis. This setup seems to work well for acquiring the complete proteome and phosphoproteome of amniotic fluid. As with other proteomics studies, the challenge now is analysis and interpretation.