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June 15, 2016 (Vol. 36, No. 12)

Chromatography Reaches New Peaks

Before Any Biopharma Claims Can Be Staked, Tools and Techniques Need To Be Optimized For Biotherapeutic Recovery

  • In the early 1900s, a Russian-Italian botanist named Mikhail Semyonovich Tswett was investigating plant pigments when he hit upon a curiously effective separation technique.

    At the time, just two plant pigments were known, but several more came to light after Tswett’s ministrations. He used a mixture of ether and ethanol to extract pigments from ground-up plants, and he decanted the resulting tinctures to a vertical glass tube that contained powdered calcium carbonate.

    As the pigment-laden mixture, the mobile phase, was washed downward through the calcium carbonate, the immobile phase, as many as 10 bands of color appeared. Tswett surmised that each band corresponded to a different pigment, and that each pigment passed through the immobile phase at a different rate, presumably because the immobile phase was more or less adsorbent with respect to the different pigments.

    When Tswett discovered this phenomenon—separation by means of differential adsorption—he called it “chromatography,” after the Greek words meaning color and writing. Chromatography has become more sophisticated since then. For example, demanding chromatography applications have become commonplace in the biotechnology and pharmaceutical industries.

    Although it is a mainstay of many laboratories, chromatography continues to be refined. It was an important part of the program at the recent Pittcon Conference and Expo, which took place in Atlanta. Pittcon’s chromatography coverage included the selection of chromatographic media, the fine-tuning purity analysis, and the rigorous identification of harmful genotoxins. Chromatography, Pittcon’s presenters made clear, can still become much more efficient and economical.

  • Genotoxic Impurities

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    A Waters team integrated UV and mass detection using an Acquity QDa detector to enable correct peak identification. The team reported that this approach also minimizes the need for multiple standard runs to confirm the identity of peaks by retention times.

    Genotoxic impurities (GTIs) can develop during many stages of drug development, for example, during synthesis, formulation, or storage. They may represent byproducts or result from degradation. According to Margaret Maziarz, a senior scientist at Waters, GTIs are considered unusually toxic: “They have potential to react with DNA and induce genetic mutation, which may consequently lead to tumor development. Therefore, GTIs must be controlled at low levels to ensure quality of the pharmaceutical product and patient safety.”

    Rigorous identification and quantification of these impurities early in the drug development process requires reliable and highly sensitive methods for accurate determination of both drug substance and drug products. “This is one area in particular where leveraging optical detection (UV), mass spectrometry (MS), and ultra-high-performance liquid chromatography (UHPLC)-based technologies can have a big impact,” Maziarz stated. “It’s something Waters and many analytical vendors are working hard to improve.” For their part, Maziarz and colleagues set out to develop a sensitive and robust UHPLC method with dual detection for routine monitoring of genotoxic impurities in pharmaceuticals.

    “By closely integrating UV and mass detection using a Waters Acquity QDa detector, we enabled correct peak identification while minimizing the need for multiple standard runs to confirm the identity of peaks by retention times,” Maziarz explained. “Using both UV and MS spectral data, we were able to quickly confirm spectral homogeneity and method specificity of each analyte in the active pharmaceutical ingredient matrix using Empower 3 Software.”

    Maziarz asserted that employing mass detection provides greater sensitivity for the identification of low-level GTIs in pharmaceutical samples. Further, she emphasized that high-performance technologies are more accessible than many people realize.

    “UHPLC, column chemistries, MS, and informatics have evolved to a point where they are becoming mainstream,” Maziarz said. “In addition, we see an increasing need to transfer methods and technology from older high-performance liquid chromatography (HPLC), MS, and UV instruments to platforms that are capable of analyzing increasingly potent (and low dosage) medicines, combination therapeutics, novel formulations, and biotherapeutics that present an entirely unique set of challenges often while continuing to address the challenges of managing legacy methods.

    “These technologies need to be implemented across the product development pipeline. Regulatory agencies expect adoption of new tools to address challenges, such as GTIs, faced in the pharmaceutical industry today and in the future.”

  • Methods Transfer

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    When the Agilent 1290 Infinity II LC is augmented with Intelligent System Emulation Technology (ISET), it can emulate other systems and achieve a transfer of methods between LCs, regardless of brand.

    The transfer of methods from conventional HPLC to UHPLC can present significant challenges. At Agilent Technologies, Gregory Hunlen, an applications engineer, and Michael Woodman, an application scientist, reported that they are tackling this problem head on. “We are observing an acceleration of interest in this transition,” Hunlen said. “We wanted to demonstrate how emulation technology may be used for bridging the gap between performances in the multivendor LC environment of the laboratory.”

    The notion of having one powerful UHPLC system that can perform like the varieties of other systems in our laboratories is a unique concept, suggested Hunlen. “Agilent introduced Intelligent System Emulation Technology (ISET) in 2011,” he continued. “This allows the 1290 Infinity LC to be a truly universal system capable of duplicating the chromatographic results of Agilent and non-Agilent LC systems without having to make method or instrument changes.

    “The important point to make is that not only delay volumes are being emulated but the complete mixing behavior of the targeted system.”

    According to Hunlen, the Agilent system was challenged to perform a gradient method transfer from HPLC to UHPLC where different column geometries were explored and modifications of method parameters were necessary. “The investigation was performed on the Agilent 1290 Infinity II LC system in emulation mode to duplicate results from the targeted system, an Agilent 1200 Series RRLC,” he summarized. “The basic chromatographic calculations for injection volumes, flow rates, and gradient slope were simplified by using the Agilent Method Translator.

    “The comparative analysis, using the same chromatographic conditions on the two systems, resulted in excellent agreement in chromatography and compound retention times. This means all operations took place on just one system—running the legacy method, optimizing the new method, and finally, running the new UHPLC method.  Intelligent System Emulation Technology allows system adaptability for nearly universal instrument to instrument transfer methods.”

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