Ultra performance liquid chromatography (uplc)

A standard reaction mixture was prepared: 100 μM Fab, 500 µM linker 1–4, 10 µM eTl, 100 μM ZnCl₂ in HEPES buffer (100 mM HEPES/NaOH, pH 7.8, 100 mM NaCl, 10 mM CaCl₂). When reaching the product maximum, the mixture was diluted in phosphate buffer (50 mM NaH₂PO₄; pH 7.0) (1:5). Anti-ErbB2- and anti-ErbB3-Fab mixtures were loaded onto a Protein G and A Spin Column, respectively (NAb Protein G or A Spin Columns, ThermoFisher Scientific (Waltham, MA, USA)). Purification was performed by 5 washing steps with phosphate buffer (50 mM NaH₂PO₄; pH 7.0) and 4 elution steps (100 mM glycine/HCl; pH 2.7). The elution fractions were pooled, and the buffer was exchanged by cross filtration to phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄; pH 7.4). Click anchor modified Fabs were mixed together in a ratio of 1:2 according to the manufacturer’s instructions (Jena Bioscience (Jena, Germany)) for the respective click reaction type and monitored by 10% SDS-PAGE, UPLC (waters system (Milford, MA, USA), Aeris^TM 3.6 µm WIDEPORE XB-C18 column, Phenomenex (Torrance, CA, USA)) and LC-MS. Purification of the bsFabs was done by SEC (S200 pg, 16/600, Cytiva (Chalfont St Giles, UK)) in PBS.

High-resolution mass spectra were recorded on a Synapt G2-S HDMS by Waters Co. (Milford, UK) equipped with an Acquity UPLC and a Phenomenex Jupiter C-18 column (3 × 250 mm, 5 μm, 300 Å). The ionization and transfer parameters of the MS system have been optimized for a standard sample (insulin) and were applied without modification. Deconvolution of measurement results was performed by the MaxEnt1 method of the MassLynx software by Waters Co. The most important source parameters are: capillary voltage: 3.3 kV, sampling cone: 40 V, source offset: 60 V, source temperature: 100 °C, desolvation temperature: 250 °C, desolvation gas flow: 600 l h⁻¹. Chromatographic conditions: start 95% solvent A, within 35 min linear gradient to 1% solvent A, 5 min 1% solvent A, within 4 min back to 95% solvent A. Solvent composition: A—water, 0.1% formic acid; B—acetonitrile, 0.1% formic acid. Flow rate: 0.5 ml min⁻¹. For mass spectra of protease–inhibitor complexes in this article, see Supplementary Figs 30–35.