An Optimized Analytical Methodology for Monoclonal Antibodies (mAb) Testing
The efficient characterization of antibody drugs is important to both regulatory agencies and pharmaceutical companies to ensure the safety and efficacy of biopharmaceutical products. Proteins with different sizes may show similar hydrophobicity and are therefore difficult to separate by traditional reversed-phase chromatography (RP).
Optimized Size Exclusion Chromatography (SEC) coupled with Electrospray Ionization with Mass Spectrometry (ESI-MS) methodologies are ideal for routine characterization of reduced humanized Monoclonal antibodies (mAb).
Size Exclusion Chromatography (SEC) is widely used in the biopharmaceutical industry to detect antibody aggregates and fragments. However, traditional SEC mobile phases are incompatible with Electrospray Ionization Mass Spectrometry (ESI-MS). Therefore, optical detection methods with lower specificity historically have been used. The significant improvement in size-based separation techniques linked to mass spectrometric detection offers a powerful, routine and generic solution for antibody characterization.
Moreover, this optimized SEC method and newly developed UPLC BEH125 SEC columns allows accurate determination of aggregate levels in less time for proteins ranging from 1,000 to 80,000 daltons, ensuring batch-to-batch consistency, specificity and increased confidence in validated methods.
Ideally, SEC separations are based on the size of the proteins in a solution. For this reason, the Size Exclusion Chromatography of biomolecules is performed under aqueous, native conditions. However, the presence of mixed mode interactions can complicate size measurement. More specifically, the charged sites on the packing material can interact with the proteins, resulting in an ion-exchange effect. To determine the influence of these effects, the mobile-phase conditions of the separation need to be evaluated. The conditions of the chromatographic separation can alter the protein structure and state. The concentration and identity of the salt and pH can affect the 3D-structure and protein-protein interactions.
Therefore, a suggested set of experiments for SEC separation should evaluate a number of conditions
mobile phase (pH and ionic strength)
Use 0f UPLC-SEC allows method optimization to be predicted in less time with a high level of efficiency and a higher degree of confidence that includes improvement in resolution and sensitivity. If factors affecting the protein's biological activity are limited, PBS is the recommended starting mobile phase.
UPLC-SEC-UV/MS can be successfully used for quantitative analysis of protein aggregates (SEC-UV) as well as to identify individual components in the aggregate peak by SEC-MS.
To find out more about how these techniques can be used, explore the case studies in: