Impact of mAb Aggregation on Its Biological Activity: Rituximab as a Case Study

Monoclonal antibody (mAb) products are presently the dominant class of therapeutic proteins. When stressed, they are known to be prone to molecular instabilities like aggregation, fragmentation, oxidation and reduction, of which aggregation is typically the most significant concern. These stresses may be experienced during manufacturing, storage, filling, formulation development and shipping. This paper investigates how mAb aggregates generated by a variety of mechanical, thermal and chemical stresses impact the biological activity of a biotherapeutic. Increased aggregation resulted in a decrease in biological activity, as confirmed by cell based assays such as antibody dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) and ligand binding assays such as surface plasmon resonance (SPR). It was observed that aggregates formed due to extreme pH (pH 3.5 and pH 11.0), stirring (1 d stir and 3 d stir), thermal stress, and oxidation via CuSO₄ have the most impact on potency of the therapeutic. In contrast, aggregates formed due to stresses from pipetting, milder pH (4.3 and 8.5), oxidation via H₂O₂, freeze-thaw (Ft-slow and Ft-fast) have relatively less impact on the potency of the mAb biotherapeutic. The results affirm that understanding of the mechanism of aggregation is critical for achieving consistent product quality and the resulting efficacy.