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Sep 15, 2009 (Vol. 29, No. 16)

HPLC Advances for Biomolecular Analysis

Enabling Tools and Technologies Make Cutting-Edge Research Possible

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    According to Waters, the Acquity UPLC® System allows chromatographers to work at higher efficiencies with a much wider range of linear velocities, flow rates, and backpressures.

    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.

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