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Feb 1, 2011 (Vol. 31, No. 3)

Making Strides in Protein Characterization

Researchers Increasingly Rely on Enabling Tools and Strategies to Boost Operations

  • Click Image To Enlarge +
    Researchers at Novo Nordisk use Waters’ Synapt G2 ion mobility mass spectrometer to produce a detailed picture of complex molecules.

    The bioprocessing industry continues to transform and reinvent itself as a range of new technologies is introduced and adopted. The sequencing of the human genome has made available a wealth of targets for drug development, translating into a demand for appropriate biotherapeutics. Moreover, the expiration of patent protection for many biologics is fueling increasing interest in biosimilars.

    Improvements at the upstream end, including vast increases in production titers of cell cultured proteins, has made feasible products that previously would have been ignored because of the initial difficulty and astronomical cost of preparing adequate amounts for pilot studies. Finally, the demand for higher performance in purification processes has motivated biotech companies to drive their technologies to the edge.

    Better characterization, cleaning up contaminating material, and overcoming molecular heterogeneity were among the issues that researchers were talking about at “BioProduction” held recently in Barcelona.

    Ion mobility mass spectrometry (IM-MS), a recently developed technology for molecular characterization, is a major interest of Rune Salbo, a Ph.D. student at Novo Nordisk.  “We had substantial experience with mass spectrometry, but neither myself nor anyone else in Denmark had worked with ion mobility mass spec, so I invested a lot of time and effort optimizing the parameters of this instrument.”

    The fundamental basis of ion mobility is straightforward; it measures how fast a given ion moves against an electrical field through an atmosphere composed of neutral drift molecules. The ions under investigation are introduced into the drift chamber and subjected to a homogeneous electric field where they interact with the neutral drift molecules contained within the system.

    Novo Nordisk uses the traveling wave IM-MS developed by Waters in which a sequence of symmetric potential waves continually propagating through a tube propels the ions, and different species transit the tube in unequal times. These modifications allow more accurate determination of fundamental properties of the molecule under investigation, including its overall shape, subunit packing, and topology.

    The Waters system is reportedly the first commercially available instrument able to study intact proteins and protein complexes. Prior to this, the only available instruments were hand-designed, a challenge which many investigators chose not to pursue. “My goal in these studies was to employ the new Waters instrument to measure collision cross sections, but the calibration of the instrument failed,” Salbo said. “In a collaboration with Matthew F. Bush and Carol V. Robinson of the department of chemistry at Oxford University, we have now solved this problem.”

    Salbo reported on studies using insulin as a model system, with the goal of gaining accurate measurements of the collisional cross-sectional dimensions of the molecule. To obtain an accurate picture, he stressed the use of native-like calibrant molecules that span the molecular weight and CCS values for the insulin molecule. Salbo invested considerable effort in measuring at a range of wave velocities in order to develop consistent results.

    Salbo strongly endorses the use of IM-MS for producing a detailed picture of complex molecules. “X-ray crystallography is looked upon as the gold standard for a detailed description of a molecule’s structural properties, but x-ray crystallography and most other methods in structural biology give an average picture of all species in the solution. This means that homogeneity and high purity is essential.

    “With ion mobility, you look at each species individually and the presence of other species does not interfere with the measurement. For instance, in a mixture of insulin monomers, dimers, and hexamers, we observe each individual state and not an average of the three combined.”

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