Figure 9.

Antibody reconstitution in 384-well format fully automated by BiomekFX HTS platform. Antibody reconstitution was conducted in 386 wells by mixing anti-CD40 Fab Int^N with huFc Int^C in a 2:1 molar ratio, resulting in a final volume of 40 µL with a total protein amount of 14 µg, followed by addition of 0.5 mM TCEP for PTS activation. Non-reconstituted antibody fragments were removed by magnetic Ni²⁺ bead addition and reconstituted anti-CD40 antibodies were supplemented with a 10-fold molar excess over TCEP with DHAA for reoxidation. (a) ELISA readout of reconstituted anti-CD40 antibodies (e-v) derived from 384-well plate generated by fully automated BiomekFX HT platform. Each column from E to V represents a cluster of 16 wells including the mean with standard deviation for each cluster. The heat map shows signal detection at 450 nm after ELISA was performed for each well in the 384-well plate. An antibody concentration of 0.1 µg mL⁻¹ was used for ELISA analysis. (b) CD40 activation assay was performed to evaluate cellular functionality of reconstituted anti-CD40 antibodies derived from high throughput reconstitution. Human CD40-expressing HEK293 cells were treated with 4 nM reconstituted antibodies (E-V) and 200 nM APX005M (positive control) for 6 h, activating an engineered NF-κB pathway for luciferase transcription. Luminescence signal was recorded and relative luminescence units (RLU) is representing normalized luminescence to untreated cells. 200 nM trastuzumab was used as a negative control, showing no CD40 activation. The heat map shows CD40 activation for each single well on the plate. Error bar: Mean with standard deviation (SD).