Ligand binding assays (LBAs) are the gold-standard tool for the bioanalysis of therapeutic proteins owing to their high sensitivity, robustness and capability to meet the high-throughput demands of routine sample analyses. In case of complex sample composition, e.g. two administered therapeutic proteins, the LBA should be capable of analyzing each co-administered protein independently. When proteins bind to the same soluble circulating target, information regarding size of the formed complexes of both therapeutic proteins with the target can’t be collected from LBA. A combination of size exclusion chromatography (SEC), a separation technique based on the difference in the stokes radius and geometry of molecules/complexes, with LBA would provide data regarding the molecular size of the complexes.
Molecular weight standards are commonly used for constructing a calibration curve for SEC. For proteins or protein-complexes of >1 MDa, proper standard proteins are missing. For non-globular proteins, an extrapolation of the calibration curve to >1MDa is misleading due to their different morphology. Complex matrices like serum/plasma can’t be analyzed by well established technologies like light scattering detectors.
This presentation will cover an alternative calibration function for SEC based on the morphology of the protein complexes confirmed by atomic force microscopy. The protein complexes were formed during a transitions phase of protein A to protein B. The alternative calibration supports the setup of a correct “Mathematical mechanistic modeling of drug-target-drug complex” and enables a deeper understanding of the size of the formed complexes and could thus support the understanding of the transition phase of the patients. Additionally the presented solution enables an investigation regarding immunogenicity related questions with the focus on hypothetical presentation of neo-epitopes of the in vivo formed complexes for which no specific assays are available.
...reflect on the limitations of current bioanalytical approaches to analyze large protein complexes but will be able to develop appropriate methods for accurate successful bioanalytical support of a clinical trial.
...appreciate how important a combination of knowledge and expertise from different scientific areas is to cope with complex bioanalytical challenges.
...to work on solutions to cover immunogenicity related questions for which no defined approach is available in the scientific community.