Amicon ultra 0.5 ml centrifugal filters

10 μM of h38C2 IgG1 or h38C2_Lys99Ala IgG1 was incubated with 100 μM (5 eq per each of the two Lys99 residues) of compound 1 (MS-PODA-fluorescein) or compound 2 (β-lactam-hapten-TAMRA) in PBS for 3 h at RT. Of the reduced and nonreduced conjugation mixture, 2.5 vg was loaded onto a 10-well NuPAGE 4-12% Bis-Tris Protein Gel (Thermo Fisher Scientific). Fluorescent bands were visualized by blue light on an E-gel Imager and the gel was subsequently stained by PageBlue Protein Staining Solution (all form Thermo Fisher Scientific). Chemically programmed h38C2 IgG1_3, _4, _5, and _6 and the two ADCs (anti-HER2 DVD-IgG1_7 and _8) were assembled analogously, purified with illustra NAP-5 Columns (GE Healthcare), and concentrated with Amicon Ultra 0.5-mL Centrifugal Filters with 30-kDa MWCO.

To generate ADCs in DVD-IgG1 format, 10 μM homodimeric h38C2_Arg DVD-IgG1 or heterodimeric h38C2_Arg/h38C2_Lys DVD-IgG1 was incubated with 50 μM phenylglyoxal-MMAF (compound 4) in PBS (pH 6.6) for 3 h at 37°C. In parallel, 10 μM homodimeric h38C2_Lys DVD-IgG1 or heterodimeric h38C2_Arg/h38C2_Lys DVD-IgG1was incubated with 50 μM β-lactam-hapten-MMAF (compound 6) at RT for 4 h. Following incubation, illustra NAP-5 Columns (GE Healthcare) were used to remove free compounds and the ADCs were concentrated with Amicon Ultra 0.5-mL Centrifugal Filters to 2 mg/mL in PBS (pH 7.4).

10 μM h38C2_Arg and h38C2_Lys DVD-Fab were incubated with three different concentrations (10 μM, 50 μM, 100 μM) of phenylglyoxal-TAMRA (compound 1) in 50 mM HEPES, 50 mM NaHCO₃ (pH 6.0) for 3 h at 37°C. As a positive control, 10 μM of h38C2_Lys DVD-Fab was incubated with β-lactam-hapten-TAMRA (compound 3) in PBS (pH 7.4) for 3 h at RT in parallel. 7.5 μg of each conjugation mixture was loaded onto a 10-well NuPAGE 4-12% Bis-Tris Protein Gel. Fluorescent bands were visualized by blue light on an E-gel Imager (Thermo Fisher) and the gel was subsequently stained by PageBlue Protein Staining Solution. To test whether DVD-Fab conjugation to phenylglyoxal can be blocked with β-lactam-hapten derivatives, 10 μM h38C2_Arg and h38C2_Lys DVD-Fab were pre-incubated with 100 μM β-lactam-hapten-azide (compound 2) for 3 h at RT and then processed and analyzed as described above. To generate ADCs in DVD-Fab format, 10 μM h38C2_Arg DVD-Fab was incubated with 50 μM phenylglyoxal-MMAF (compound 4) in 50 mM HEPES, 50 mM NaHCO₃ (pH 6.0) for 3 h at 37°C. In parallel, 10 μM h38C2_Lys DVD-Fab was incubated with 50 μM β-lactam-hapten-MMAF (compound 6) at RT for 4 h. Following incubation, illustra NAP-5 Columns (GE Healthcare) were used to remove free compounds and the ADCs were concentrated with Amicon Ultra 0.5-mL Centrifugal Filters to 1 mg/mL in PBS (pH 7.4).