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

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

As a product matures through its life cycle, so does the sophistication of stability testing. Bioprocessors enjoy significant leeway early in development to apply tests they think make sense, particularly those that provide feedback on process optimization. Testing becomes more formalized as a product enters human testing. “ICH guidelines call for stability studies, at least of the accelerated variety, before Phase I,” says Guy Berg, Ph.D., svp at NewLab. “There are check-off lists of tests to conduct depending on the clinical phase and molecule.”

Erythropoietin, for example, is tested for appearance, pH, peptide sequence, types and extent of post-translational modifications, aggregation, and potency. Many more assays are possible and would surely yield more information, “but at some point, one must limit the number of tests for no reason other than cost,” observes Dr. Berg.

Similarly, Pharmaceutical Product Development (PPD) performs stability testing, methods development, validation, and QC release in more than 60,000 cubic feet of controlled-environment space. Large, walk-in units maintain standard ICH temperature and humidity conditions, while reach-in units can duplicate any reasonable condition that might affect stability like photodegradation at variable temperature. A central computer system controls condition monitoring for all stability chambers, which are protected from power grid failures by backup electrical generators. Larger walk-in chambers employ redundant monitoring.

Early in a drug’s development, samples will be subjected to their most likely storage conditions, for example, frozen, refrigerated, or ambient temperature, notes Chris Lively, Ph.D., director of biopharmaceutical services. “We will also likely add challenge conditions such as higher temperature or humidity, which will indicate the conditions under which products are likely to degrade.”

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.

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.

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.”

How much testing is enough becomes a perennial question in an era where analytics improve constantly. Capillary-zone electrophoresis was once considered unreliable for stability testing but now has become more robust. The technique’s high sensitivity and resolving power are compelling, but that is a good news, bad news proposition because the more one sees, the more questions one (or one’s regulatory reviewer) will ask.

Developers of biotherapeutics should, therefore, be prepared for long discussions with regulators, especially for new molecules, warns Dr. Waite of Tepnel. “But in the end, each sponsor must be prepared to make their own case for stability.”

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