Skip to main content
. 2016 Aug 25;2(9):637–646. doi: 10.1021/acscentsci.6b00180

Figure 4.

Figure 4

Ammonium sulfate accelerated site-specific modification of antibody. (a) Scheme for the site-specific biotin/drug conjugation to π-clamp trastuzumab 5. Val-Cit-PABC is the valine-citrulline p-aminobenzyl carbamate linker. (b) The deconvoluted mass spectra for antibody before conjugation (left), after conjugation in the presence of ammonium sulfate (middle), and after conjugation without ammonium sulfate (right). Reaction conditions: 40 μM π-clamp trastuzumab 5, 500 μM probe 6, 100 mM phosphate, 10 mM TCEP, ± 1.25 M (NH4)2SO4, 37 °C, 3 h. (c) Biotinylated trastuzumab (5-biotin) binds to recombinant HER2 in Octet BioLayer Interferometry assay (KD = 122 ± 1.5 pM). 5-biotin was immobilized on the streptavidin biosensors and sampled with serially diluted concentrations of recombinant HER2; see Figure S24 for fitting and data analysis. (d) The deconvoluted mass spectra for 5-MMAF and 5-MMAE synthesis. Reaction conditions for 5-MMAF: 40 μM π-clamp trastuzumab 5, 500 μM probe 6B, 100 mM phosphate, 10 mM TCEP, ± 1.25 M (NH4)2SO4, 37 °C, 210 min. Reaction condition for 5-MMAE: 40 μM π-clamp trastuzumab 5, 500 μM probe 6C, 100 mM phosphate, 10 mM TCEP, ± 1.25 M (NH4)2SO4, 37 °C, 16 h. (e) 5-MMAF and 5-MMAE prepared with ammonium sulfate were functional, selectively killing HER2 positive BT474 cells, while they were significantly less toxic for HER2 negative HEK 293T cells. Experiments were performed in triplicate. Error bars indicate the standard deviation from the average of three experiments.