Impact of Monoclonal Antibody Aggregates on Effector Function Characterization

Area of Science:

• Biopharmaceutical development
• Immunology
• Protein aggregation

Background:

• Therapeutic monoclonal antibodies, often IgG1, utilize Fc gamma receptor (FcγR) interactions for effector functions.
• Controlling antibody aggregates is crucial for therapeutic efficacy and safety.
• Previous studies on antibody aggregate impact on FcγR binding primarily used surface plasmon resonance (SPR) in avidity-based formats.

Purpose of the Study:

• To investigate which Fc gamma receptor (FcγR) is most affected by monoclonal antibody (mAb) aggregation.
• To determine how mAb aggregates influence FcγR binding in solution-based and cell-based assays.
• To compare FcγR binding to mAb aggregates across different assay formats.

Main Methods:

• Forcible degradation and size exclusion chromatography of an IgG1 monoclonal antibody (mAb1) to enrich aggregates.
• Evaluation of FcγR binding to mAb1 aggregates using surface plasmon resonance (SPR) in various formats and solution-based assays.
• Assessment of FcγR binding using FcγR reporter cell-based assays.

Main Results:

• All tested Fcγ receptors showed increased binding to mAb1 aggregates in avidity-based SPR and solution assays, with FcγRIIa being most affected.
• FcγRIIa binding to mAb1 aggregates did not increase in an antibody-down SPR format, which is less susceptible to avidity effects.
• mAb1 aggregate activity showed a slight increase in an FcγRIIa cell-based assay but decreased in an FcγRIIIa assay, potentially due to fucosylation differences.

Conclusions:

• Monoclonal antibody aggregation significantly influences FcγR binding, especially in avidity-based formats.
• Elevated FcγRII binding is observed even with low levels of antibody aggregation.
• Understanding these interactions is critical for the development and quality control of therapeutic antibodies.