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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

  • A common protocol for biopharmaceuticals is the freeze-thaw study that looks for degradation or denaturation around a solution’s freezing temperature. Proteins studied for freeze-thaw degradation undergo reverse-phase HPLC, polyacrylamide gel electrophoresis, and isoelectric focusing.

    Most of PPD’s biotech customers approach the company with early-stage molecules. Another common entry point for stability testing is during formulation development to determine whether the proposed coingredients enhance or diminish stability. As products move forward, investigators add more characterization testing while increasing the level of analytical method validation. Stability studies build on each other, eventually yielding a GMP-worthy stability profile and expiration dates under various conditions.

    Critical to the process is understanding stability-indicating analytical methods, which are identified through forced-degradation studies. As the name implies, forced degradation involves conditions—heat, acid/base, light, oxidation, humidity, freeze-thaw cycling, and deamidation in TRIS buffer to name a few—that are certain to generate degradation products.

    For deamidation, PPD uses HPLC with a high-resolution quadrupole time-of-flight (QTOF) mass detector. QTOF is capable of picking up small differences in large molecules. Because of the method’s high cost, PPD will sometimes employ HPLC-UV after the peaks have been identified by MS.

    Aggregation, which occurs readily once proteins begin degrading, is associated with immunogenicity. PPD therefore characterizes aggregates with size-exclusion HPLC, light scattering, and analytical ultracentrifugation. The company has a differential scanning microcalorimeter but has not worked it into its protocols yet.

    Developing a robust, forced-degradation package becomes complicated as the conditions and variables begin to add up. “You may have eight or nine assays plus the variable of time, which expands and complicates the analysis matrix,” Dr. Lively reports. “These studies require tremendous planning to organize many analysts to perform a battery of tests simultaneously when the specific degradation time point is reached for each condition.”

    To prepare for these studies, PPD performs an initial range-finding study to select the correct conditions for arriving at the desired degradation level. This study consists of varying the time and level of the condition such as different temperatures and/or acidity conditions as functions of time.

    Activity’s the Thing

    Identifying molecule-specific instabilities (oxidation, deamidation, or aggregation) is crucial for developing a formulation to prevent those instabilities. “By the preformulation stage, it may already be too late to set up a stability study,” observes John Augustine, Ph.D., principal scientist at Wolfe Laboratories. “Lacking that information, developers will have no idea of what to look for during later development in the clinic or even in manufacturing.” Wolfe offers a gamut of stability testing for small molecule and protein-based therapeutics.

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