A comprehensive approach to predict the long-term stability of monoclonal antibodies
Monoclonal antibodies and their derivatives, such as antibody-drug conjugates and bispecific antibodies, are essential in modern medicine. However, these complex molecules can undergo changes during manufacturing and storage. To ensure their effectiveness and safety, stability studies are critical early in the drug development process.
“Traditional forced degradation studies, which involve exposing monoclonal antibodies to harsh conditions, provide valuable insights but fall short in predicting long-term shelf-life stability,” says Geert Van Raemdonck, senior scientist mass spectrometry at AnaBioTec. “To address this gap, modeling programs such as ASAP - which stands for Accelerated Stability Assessment Program - were developed.”
Comprehensive analysis with high-resolution mass spec
At AnaBioTec, we apply our analytical expertise and instruments, including liquid chromatography (Vanquish Flex UHPLC) coupled with high-resolution mass spec (Orbitrap Exploris 240), to compare different approaches to evaluate the stability of monoclonal antibodies.
Van Raemdonck: “We used a commercially available monoclonal antibody and subjected it to stability testing. We focused on temperature and oxidation as the main stressors in forced degradation studies. For ASAP modeling, the antibody solutions were stored in temperature-controlled chambers at various temperatures over different durations.”
We analyzed the antibodies at intact, subunit and peptide levels, using reversed-phase C4 and reversed-phase C18 separation after tryptic digestion. Additionally, we evaluated structural changes using mass spectrometry.
Modeling degradation processes over six months
“While the forced degradation study revealed important information about monoclonal antibody aggregation, oxidation and fragmentation, the ASAP modeling software enabled us to model Asn residue deamidation and antibody fragmentation over time,” he says. “Using LC-MS/MS, we identified several post-translational modification hotspots.”
The team also pinpointed four methionine residues prone to oxidation, and peptide mapping showed increased levels of deamidated peptides under elevated temperatures. Heating the monoclonal antibodies at temperatures above 45°C led to precipitation and non-soluble particles. ASAP software effectively modeled these degradation processes over several months.
Combining forced degradation studies with ASAP modeling software provides a comprehensive approach to predict long-term shelf-life stability of therapeutic proteins including monoclonal antibodies. This innovative method enhances our ability to ensure the safety and efficacy of monoclonal-antibody-based therapies.
Geert Van Raemdonck
Senior Scientist Mass Spectrometry at Anabiotec
