(1182-C) Binding and Functional Characterization of Anti-HER2 Antibodies Using Advanced Flow Cytometry and BLI Label-Free Detection
Tuesday, February 6, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: Monoclonal antibody (mAb) therapeutics comprise a large segment of the biopharmaceutical market and have been developed across a huge range of disease areas, from cancers to infectious diseases. To facilitate the discovery of novel mAbs to feed this growing market, we need robust and high-throughput methods for screening and characterizing mAb drug candidates. Here, we present a streamlined approach for high-throughput profiling of mAbs, using both iQue® Advanced Flow Cytometry and Octet® Bio-Layer Interferometry (BLI) Label-Free Detection. We have created a multiphase workflow, which utilizes the two instruments in parallel, to comprehensively characterize therapeutic antibodies. The iQue® assays employ live cell-based models to profile binding, competition, and Fc function of test antibodies. To complement this, the Octet® can provide antibody characterization through the determination of antigen binding kinetics, relative antibody glycosylation content and Fc-gamma receptor (FcγR) binding. During the study, three anti-HER2-hIgG1 antibodies were characterized: a trastuzumab biosimilar; Kadcyla® (trastuzumab emtansine; a therapeutic antibody-drug conjugate) and a pertuzumab biosimilar (Proteogenix). For the iQue® live-cell binding assay, the anti-HER2 antibodies were either analyzed in their native state, or after heating in a water bath at 69°C for 45 or 90 minutes. The Trastuzumab biosimilar had both the highest level of binding to HER2-expressing AU565 cells and the greatest resistance to heat denaturation, with the EC50 remaining constant across the native, 45- and 90-minute heated antibodies (EC50 160 to 180 ng/mL). EC50 values for binding of Kadcyla were highest and it was sensitive to heat denaturation, with a two-fold increase in EC50 from the native state (530 ng/mL) compared to after 90 minutes of heating (1250 ng/mL). This suggests that the modification of the Fc portion to include the ADC payload, compared to the native Trastuzumab, has resulted in a loss in both thermal and cell binding stability. The Pertuzumab biosimilar in its native state had similar binding to Trastuzumab, with an EC50 of 230 ng/mL, but it was also sensitive to heating and saw a two-fold loss in binding activity after 90 minutes of heating (EC50 530 ng/mL). Together, the power of these instruments can be leveraged to provide comprehensive assessment of novel mAbs, to improve the identification and qualification of hits during screening and to advance drug discovery workflows.