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Apr 1, 2008 (Vol. 28, No. 7)

Regulations Still Driving Stability Testing

Scope of Characterization and Validation Level Should Match the Phase of Development

  • Wolf uses LC/MS, gel electrophoresis, isoelectric focusing, light scattering, peptide mapping, and activity assays to test for protein stability. The latter are becoming increasingly important as analytical tests may show nothing out of the ordinary. “But if a molecule does not pass the activity assay, you know something is wrong,” comments Dr. Augustine.

    Conversely, a physical or chemical test may unveil problems that are not apparent in activity assays conducted in vitro but which may indicate inferior performance when drugs are injected or infused. Activity assays are only one piece of the larger picture. “They’re important for obtaining a panoramic view of a molecule’s stability that includes data from electrophoresis, light scattering, and LC/MS,” Dr. Augustine says.

    Among the techniques used for stability testing, microcalorimetry has been seriously underutilized considering its broad applicability. Differential scanning calorimetry (DSC), for example, detects the most subtle changes in a protein while consuming only a few micrograms of sample. Because it works on such small quantities, DSC is ideal for measuring stability, particularly folding and unfolding, at every stage of biopharmaceutical development, from discovery through fill/finish and post-release quality testing. DSC is particularly useful for determining optimal process and storage conditions, for example how pH, excipients, and buffers affect folding.

    All folded biomolecules such as proteins and antibodies have a characteristic thermal midpoint corresponding to the 50% folded/unfolded state. Developers can use that information to screen for stability during formulation, purification, and even fermentation.

    “Developers have to worry about stability from early on,” explains Eric Reese, Ph.D., director of business development for biotherapeutics products at Microcal. “Microcalorimetry allows them to do that with minimal method development.”

    Microcalorimetry detects protein folding and unfolding by measuring the minute quantities of heat absorbed or released during these events. As a protein solution is heated its temperature rises. During the transition from folded to unfolded configuration, an endothermic event, the molecule absorbs the applied heat and the temperature curve levels off temporarily, similar to how cooling stops near the point where water freezes. The solution resumes heating up when the protein is denatured.

    Transitions are normally sharp for highly homogeneous proteins, less so for molecules produced in several isoforms or with varying degrees of post-translational modification. “Peak broadening implies there is more than one degradation product,” says Dr. Reese.

    Similarly, isothermal titrating calorimetry (ITC), which is carried out at constant temperature, can serve as an activity assay for molecular affinity. Through this method, an antibody solution is titrated with substrate at constant temperature. The heat of binding measured over time is directly proportional to the amount of binding. After the antibody is saturated only the heat of dilution is observed. Analogous with DSC, antibody-antigen binding through ITC is characteristic of the binding pair.

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