In terms of our CreDA™ platform, Creative Biolabs supplies a comprehensive assessment of the physical stability of our clients’ drug candidates, addressing the influence of pH, temperature, mechanical stress, and diverse formulation buffers on the protein aggregation and biological activity. These studies can be informative for downstream processing and help customers select candidates that perform well in the preferred formulation platforms.
Fig.1 Differential scanning calorimetry for thermal stability determination.
Stability at low pH
The stability of biotherapeutics at low pH is particularly important, especially for antibody drugs, which will be exposed to low pH during the purification process with protein A affinity chromatography, and during the viral inactivation step. Exposure to low pH is known to destabilize the antibody, causing aggregation and misfolding. Stability at low pH is studied by incubating the candidates at the desired pH for a few hours, followed by a series of analyses which include size exclusion chromatography (SEC), polyacrylamide gel electrophoresis (PAGE), and activity assays to detect possible increase in aggregation and retention of protein activity.
Freezing and thawing studies
Stability studies are performed to assess the resistance of drug candidates to storing and processing such as freezing and thawing or cryogenic lyophilization. These experiments typically consist of subjecting the samples to several cycles of freezing and thawing or lyophilization and resolubilization, and analyzing the samples for aggregate content and biological activity.
Based on the assumption that proteins exhibiting transitions at higher temperatures will be more stable at physiological temperature and storage conditions, the structural stability of biotherapeutics is estimated by measuring the temperature-dependent transition points (Tm) in different formulation conditions. These assays are usually performed with differential scanning calorimetry (DSC) or differential scanning fluorimetry (DSF). For antibody drugs, they often display two characteristic thermal transitions around ~70°C and ~80°C corresponding to the CH2 and CH3 domains, respectively. And the Fab domain often appears as a third band with a Tm that varies for different antibodies. In our developability assessment, a minimum Tm threshold of 60°C is typically set as the pass criterion for the lowest thermal transition.
Sensitivity to mechanical stress
Our CreDA™ developability assessment campaign also involves test of the drug candidates for their tolerance to mechanical stress. Proteins tend to aggregate at the air-water interface where foaming may occur under agitation. These studies are thus performed by mechanically rotating or shaking the candidates and measuring the formation of aggregates at different time intervals.
Long-term stability is the ability of biotherapeutics to remain in solution under different storage conditions for a relatively long time. Variants of the drug candidates will be tested at different temperatures, typically 2°C-8°C, 25°C, and 37°C for several weeks. Samples are collected at different time points and analyzed for the formation of aggregates using analytical SEC and SDS-PAGE. Functional assays will also be performed to determine retention of the biological activity.
Equipped with the most advanced analytical instrument, Creative Biolabs has the ability to thoroughly assess the physical stability of your drug candidates, aiming to help your selection of the most robust molecule for further development. Please contact us for more information and a detailed quote.