Optimisation of the dibromomaleimide ( DBM ) platform for native antibody conjugation by accelerated post-conjugation hydrolysis †

Disulfide bridging offers a convenient approach to generate site-selective antibody conjugates from native antibodies. To optimise the reagents available to achieve this strategy, we describe here the use of dibromomaleimides designed to undergo accelerated post-conjugation hydrolysis. Conjugation and hydrolysis, which serve to ‘lock’ the conjugates as robustly stable maleamic acids, is achieved in just over 1 h. This dramatic acceleration is also shown to infer significant improvements in homogeneity, as demonstrated by mass spectrometry analysis.

Absorbance spectra were obtained in 10% DMF in acetate buffer pH 4.5.

Synthesis and Characterization of Compounds
Scheme S1 -Synthesis of DBMs 1-6

General procedure to prepare 3,4-Dibromo-maleimide acids
This procedure was adapted from one already reported. 3 3,4-dibromomaleic acid (250 mg, 913 μmol) in AcOH (5 mL) was refluxed for 1 h. Then, the amino acid (1.1 eq.) was added and the mixture was stirred under reflux for another 3 h. The mixture was allowed to cool to 20 °C. AcOH was removed by concentrating under vacuum and traces of AcOH were removed by adding toluene (10 mL) and concentrating once more.

Conjugation of DBM reagents 4 -6 to trastuzumab at pH 8.0
This conjugation protocol was performed in BBS (pH 8.0) following a previous method 3 described in the literature with some modifications. Briefly, to trastuzumab (22.9 μM, 100 μL, 2.3 nmol, 1 eq.) was added TCEP (10 mM, 1.8 μL, 18.4 nmol, 8 eq.) and the reaction was incubated at 37 °C for 2 h under mild agitation. Next, the DBM reagent in dry DMF (10 mM, 2.3 μL, 23 nmol, 10 eq.) was added to the reduced trastuzumab. The concentration of DMF was corrected to 10% (v/v) and the reaction was incubated at 20 °C for 30 min.. Afterwards, excess reagents were removed by ultrafiltration (10 kDa MWCO) with BBS to afford the modified trastuzumab.
Hydrolysis was achieved by incubating the conjugates for 48 h in conjugation buffer, at 37 ºC. The final trastuzumab conjugates were deglycosylated and characterized by LC-MS. All conjugation reactions were carried out in triplicate.
The final trastuzumab conjugates were deglycosylated and characterized by LC-MS.

One-pot conjugation and hydrolysis protocol
Conjugation protocol was performed as described above in BBS (pH 8.5) using 5 and 6 with one modification; excess reagent was only removed by ultrafiltration after hydrolysis step. LC-MS data matched that obtained above.

Determination of fluorophore to antibody ratio (FAR) and drug to antibody ratio (DAR)
UV/Vis spectra were recorded on a Varian Cary 100 Bio UV/Vis spectrophotometer, operating at 20 °C. Sample buffer was used as blank for baseline correction. Calculation of fluorophore-antibody ratio (FAR) and drug-antibody ratio (DAR) follows the formula below using ɛ280 = 215380 M -1 cm -1 for trastuzumab mAb, ɛ495 = 71000 M -1 cm -1 for Alexa Fluor 488® (0.11 as a correction factor (Cf) of the dye Excitation at 280 nm) and ɛ495 = 8030 M -1 cm -1 for doxorubicin (0.724 as a correction factor of the drug absorbance at 280 nm), respectively.

Analysis of antibodies under denaturing LC-MS conditions
An aliquot (20 μL) of trastuzumab or trastuzumab conjugates in ammonium acetate buffer (50 mM, pH 6.9) is diluted to concentration of 0.7 μM (0.1 mg·ml -1 ) and deglycosylated as follows: PNGase F (1 μL, 15,000 units·mL -1 , in 20 mM Tris-HCl, 50mM NaCl, 5 mM EDTA, pH 7.5) (purchased from New England BioLabs Inc.) was added to trastuzumab or conjugates and the resultant solution was incubated at 37 °C overnight. After this time the solution was analysed on an Agilent 6510 QTOF LC-MS system (Agilent, UK). Ten μL of each sample was injected onto a PLRP-S, 1000 Å, 8 μm, 150 mm x 2.1 mm column, which was maintained at 60 °C. Chromatrographic separation was optimised for the separation of a light and heavy chains and PNGase F using mobile phase A (5% MeCN in aqueous 0.1% formic acid) and B (95% MeCN, 5% water, 0.1% formic acid) using a gradient elution. The flow rate was 300 µL/min and the gradient as follows: 15% B for the first 2 min followed by increase to 32% B over 1 min, remained at 32% B for 1 min. After 4 min, mobile phase B was increased to 50% over 10 min, with further increase of B to 95% over 4 min and maintained at 95% B for 2 min.. At 22 min., the mobile phase B was changed to 15% B for the next 3 min to initial conditions The column effluent was continuously electrosprayed into capillary ESI source of the Agilent 6510 QTOF mass spectrometer and ESI mass spectra were acquired in positive electrospray ionization (ESI) mode using the m/z range 800−5,000 in profile mode.
The acquisition rate was 1 spectra/sec and acquisition time 1000 msec/spectrum corresponding to 9,652 transients/spectrum. The raw data was converted to zero charge mass spectra using maximum entropy deconvolution algorithm over the region between the vertical lines in the total ion chromatogram (TIC) with MassHunter software (version B.07.00).

Alexa Fluor 488 ® conjugate linker pH stability
To

Hydrolysis Study
Hydrolysis kinetics study preand postconjugation of NGMs was performed at an initial concentration of approximately 230 μM.
Hydrolysis data were further analysed in units of molar concentration to determine kinetic constants. Pseudo first-order rate constants were determined from the slopes of curves generated from plotting ln [NGM] versus time and linear regression analysis.
Hydrolysis reaction half-lives were calculated from the pseudo first order rate constants using equation below:
Hydrolysis kinetics of DBMs and DTPMs reagents was monitored by spectrophotometry, using absorbance at 325 nm and 425 nm, respectively.
Samples (200 μL) were prepared as described above (conjugation section). Extinction coefficient of dicysteine maleimide-alkyne (12, 13 and 14) prepared above was used to estimate the concentration of conjugated reagent.   pH stability study of NGM linker in antibody conjugate