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May 1, 2012 (Vol. 32, No. 9)

Leading-Edge Separation Techniques

  • Crystallization

    According to Jörg Peters, Ph.D., of Bayer Pharma global drug discovery, Bayer is the biopharmaceutical company with the most experience in protein crystallization, and its work seems about to bear fruit. The difficulties in crystallizing therapeutic proteins aside, the technique has lagged behind chromatography because the unit operations—borrowed from chemicals and small molecule drugs—are foreign to bioprocessors.

    Bayer’s process is described as an “integrated, disposable, and closely operated” piece of equipment that integrates harvest, washing, drying, sampling, and storage of product. Because it is contained in a disposable system, cleanroom use and cross-contamination are minimized.

    Dr. Peters explained that, in principle, protein crystallization is applicable to any kind of protein, peptide, or crystallizable virus. “However in certain cases finding the correct conditions in multi-dimensional space is difficult.”

    But by no means is this a platform technology. The additives and physico-chemical conditions employed in protein crystallization are in the public domain, but the precise conditions for development-stage projects remain trade secrets. The “filtration-drying-storage” device was developed at Bayer Healthcare Pharmaceuticals in cooperation with sister unit Bayer Technology Services.

    Interestingly the technique is not suitable for the equivalent of a capture step since those involve large volumes and relatively low titers. No chance, then, that this can replace expensive affinity media. “It’s a post-capture step and has been shown by Bayer and other companies to fit into the purification sequence,” noted Dr. Peters, who added that Bayer plans to make the technology available for licensing and perhaps for “further co-development.”

  • Applying a Toolbox to a Platform

    Platform approaches to production and purification have been popularized by the emergence of mAb therapeutics. Matteo Costioli, Ph.D., associate manager for downstream processing at Merck Serono, described what he calls a “toolbox approach” for mAb purification platforms.

    The toolbox idea can be thought of as adding versatility to manufacturing platforms, and thereby reducing costs. An example is the three-column platform for mAbs. Rather than stick with expensive unit operations like protein A capture, Merck Serono process engineers evaluate older, trusted technologies or new ones with the idea of plugging-and-playing them when the need arises.

    “The main goals of our approach are to reduce costs, shorten timelines, and increase process knowledge,” said Dr. Costioli.

    Regardless, this is a major undertaking involving significant behind-the-scenes development and probably some in-process tweaking as well. “Toolboxing” requires first identifying different purification technologies, matching them to processes on molecules, and testing with different mAbs under different conditions.

    When a similar situation arises down the road—a highly hydrophobic protein or one that tends to aggregate—Merck Serono reaches into its toolbox, pulls out the right operation, and applies it. The company is accumulating a database of techniques and conditions, which it hopes to apply to its future bioseparations projects.

    “Everything will be tested, known to work, and suitable for including in the separation platform,” pointed out Dr. Costioli.

    His Prague presentation specifically focused on a process that involves precipitating impurities in-line with the bioreactor, then capturing on a cation exchange column, or capturing and clarifying in one step using a novel expanded bed adsorption technology.

    Originally developed by Upfront Chromatography and now exclusively owned by DSM Biologics, the so-called second generation expanded bed adsorber has applications in food and feed industries as well as pharmaceuticals. Its main claim to fame is reducing two unit operations (harvest, capture) to one at high yield and throughput.

    So why haven’t biotech companies been knocking down Dr. Costioli’s door to get a piece of these cost-saving technologies? “There’s always a balance between the effort you have to put in at the beginning, to change a technology, and the benefit at the end,” he said.

  • Mixed-Mode Ligands

    Click Image To Enlarge +
    The simplified structure of Pall Life Sciences’ synthetic mixed-mode ligands: They operate by a combination of hydrophobic and ionic interactions, and protein desorption is prompted by electrostatic charge repulsion. 4-MEP: 4 mercapto-ethyl-pyridine; HEA : hexylamine; PPA: Phenyl propyl amine. These ligands are bound to a robust industry-scalable matrix (MEP, HEA, and PPA HyperCel™ sorbents), used as alternatives/complement to affinity (protein A), ion exchange or hydrophobic interaction chromatography for the separation of monoclonal antibodies (mAbs) and various recombinant proteins.

    Sylvio Bengio, Ph.D., scientific communications manager for bioproduction at Pall Life Sciences, rounded out the talks on bioprocess separations with a case study on mixed-mode (or multimode) chromatography for mAb purification.

    We tend to think of mixed-mode resins as novel but they are as old as column chromatography itself. In 1903 Mikhail Tsvet, then a newly minted professor at Warsaw University, discovered that hydroxyapatite could separate plant pigments. Tsvet did not realize it at the time, but the mineral’s ability to separate chlorophyll from carotenoids was based on a mixed-mode effect.

    Tsvet’s last name, coincidentally, means “color” in Russian, the same word from which “chromatography” was derived. Tsvet’s discovery lay dormant for several decades, and he died without official recognition of his invention.

    Pall introduced the first mixed-mode chromatography resin in 2000, and today numerous vendors sell mixed-mode resins combining nearly every combination of adsorption mode. Hydrophobic interaction plus some type of ion exchange predominate.

    In a bioprocess setting, mixed-mode replaces mostly hydrophobic interaction chromatography. They provide the benefit of not requiring binding-promoting salts, at concentrations of up to 3 M, required to run hydrophobic interaction columns. “Salts are a problem at such high concentrations, and recycling them is a problem,” explained Dr. Bengio.

    In addition to intermediate-step chromatography, mixed-mode ligands are useful in nonantibody purification as a low- or no-salt alternative to hydrophobic interaction chromatography. Once optimized, mixed mode’s resolving power is quite high. “We’ve published on a separation of two proteins differeing only by a methionine group,” noted Dr. Bengio.

    The method has gained steam with the advent of high-throughput screening methods and design of experiment-based screening for both chromatography media.


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