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Feature Articles : Apr 1, 2008 ( )
Regulations Still Driving Stability Testing
Scope of Characterization and Validation Level Should Match the Phase of Development!--h2>
While stability testing for small molecule drugs is well established, its counterpart for biologicals is still evolving. Dozens of techniques have emerged for characterizing proteins, ranging from the standard and reliable (electrophoresis and chromatography), to the cutting edge (calorimetry), to the downright mysterious (atomic force microscopy).
Stability testing is broadly structured around ICH guidances Q1A through Q10, which cover pharmaceutical quality. ICH Q1A is devoted to small molecule drugs while Q5C deals with biologicals.
“Stability testing for biomolecules is still quite a new field in many ways,” notes Emma Waite, Ph.D., laboratory manager for biopharmaceutical services at Tepnel, which provides a range of molecular analytical services. Perhaps due to the novelty and greater inherent uncertainty in the production of biotherapeutics, regulators increasingly expect developers to choose multifaceted, orthogonal analysis techniques that clearly demonstrate stability and activity.
Part of a Package
Companies providing stability testing services generally offer a range of related services. NewLab Bioquality’s pharmaceutical-development assistance includes cell line characterization, molecular biology, protein chemistry, and viral process validation in addition to stability testing of intermediates, actives, and formulated products.
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.
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.
MicroCal offers four instruments: VP-ITC, VP-DSC, VP-Capillary DSC (ten times the throughput of the VP-DSC), and the AutoITC, which runs 100 samples per week unattended.
How Much Testing?
The ability to characterize biomolecules has exploded in the last 10–15 years, which creates a tempting situation where developers of biotherapeutics can overtest. “This conservative thinking can sometimes cause people to hold a molecule back,” observes Dr. Lively of PPD. Developers need to be aware of their stage of development, he says, and test appropriately and in line with what regulators expect. “Investigators need to match the scope of characterization and the level of validation to the phase of development that they are in and they need to discuss this scope with the agency.”
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