Targeting Key Parameters
BASi is a contract research services provider that offers preclinical and bioanalytical services, large molecule bioanalysis, method development, and pharmaceutical analysis. The company’s pharmaceutical analysis group primarily fields requests to analyze small molecule drug compounds and also works with proteins and other large molecules.
For stability testing the main focus is on potency and purity analysis, as well as physical characterization (such as moisture content and tablet hardness or dissolution), according to Josef Ludwig, director of pharmaceutical analysis. Studies to assess a compound’s stability typically begin early in development. “They may start even before toxicology studies,” Ludwig said.
Stability testing on an API can give companies an idea of how long they can work with a batch of material before they need to reproduce it. For smaller biotech companies looking to license a compound, information on its stability adds to the database they can provide to potential development partners.
Ludwig described increasing demand for analysis of experimental samples given to animals during toxicology testing. These samples have often been frozen for several months, he noted, and after tox studies are complete, BASi may be asked to assess the stability of simulated samples to show that material maintained at a defined concentration in a given matrix and stored under specified conditions would not have degraded, lost potency, or undergone changes in its physical or chemical properties.
Potency and purity are the biggest concerns, Clark agreed. Appearance is also important—does the product change color over time, for example, even if its potency does not change? The presence of moisture is a key issue as well. If a compound is hydrophilic it is important to know how it will react in the presence of water. Clark emphasized the value of integrated data evaluation and trending analysis, putting together the various components of stability analysis to see the big picture.
Ludwig advised companies to establish robust analytical methodologies early on. In his view, the biggest mistake companies make is to rely on quick-and-dirty methods that may not capture an accurate and precise potency or complete degradation profile.
Too often young companies “do not have an overall stability platform plan” detailing what they will do at each stage, what they are looking for, and how much material they will need, said Barry Rosenblatt, director of technology development for the biopharmaceutical services North America division of Charles River Laboratories.
HPLC is the standard analytical tool used to validate the identity and quantity of a compound. Ultra-high pressure liquid chromatography (UPLC) is gaining momentum as a higher-throughput alternative that can accelerate testing and enable higher-resolution analysis. Binding assays are used to assess potency, and functional assays to evaluate activity. Nuclear magnetic resonance imaging may be used to evaluate the identity and proper folding and tertiary structure of proteins and other complex compounds.
Analyzing larger, more complex molecules such as proteins for stability may require additional testing beyond determination of the amount of a chemical entity or the amount of degradants in a sample. “You also have to confirm activity,” said Ludwig. A compound may be present, but it may have reduced or no activity due to changes in its tertiary structure.
Selecting analytical tools for stability studies, especially for larger biopharmaceuticals, is a continuing challenge, particularly because of the variability inherent in available cell-based potency assays, according to Rosenblatt. “The holy grail is to find an in vitro, clinically relevant potency assay,” he said, one that can be validated and yields a reliable stability indication.
Some molecules will form aggregates during stability testing and may exhibit super-activity, explained Rosenblatt. In some cases, aggregates can have greater potency than the monomeric form of the molecule. Aggregation has been associated with increased risk for immunogenicity as well as drug resistance.
“People are concentrating on particulates, and especially ‘sub-visible’ particulates”—a new buzzword being used to describe small, aggregated products that are “invisible in terms of the nascent activity of the molecules and are more of an issue from a safety standpoint” due to their propensity to cause adverse reactions, Rosenblatt explained.
Light scattering is one technique being used to detect and quantify these sub-visible particles. Rosenblatt expects a variety of laser-based imaging tools to become available that will enable visualization of particulates. To support ongoing PAT initiatives, these will likely be microfluidics-based instruments that will allow for real-time analysis of samples as they come off a HPLC instrument.
One of the challenges these new technologies will introduce relates to interpretation of the findings, he said. For example, is the researcher seeing something truly new, or something that has been there all along but can now be detected?
In the future, Clark envisions a growing emphasis on detecting impurities in final formulations (and determining their toxicologic profile) as new and more complex compounds move through development. Analytical methods will rely on a broader menu of technologies, including, for example, mass spectrometry. “UPLC will be used almost exclusively,” replacing HPLC, Clark predicted, especially for highly potent compounds dosed at very low concentrations (sub-milligram to 1 mg).