The purified Ig(B12) was conjugated with MMAE using a protease-cleavable valine-citruline linker with a carbonyl acrylic acid head-group. In brief, inter-chain disulfide bonds of a full-length IgG(B12) were reduced using excess amounts of DTT and reacted with 60 equivalents of carbonylacrylic acid-valine citruline-MMAE molecule(25 (link)). The reaction was stirred at 37° C for 8-10hrs. The antibody-drug conjugate was then purified using a desalting column and concentrated using Vivaspin devices (GE Healthcare). The average drug-to-antibody ratio was 4:1 as determined by native mass-spectrometry.
Protocol detail
Antibody-Drug Conjugation Protocol
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Acid
Acrylic acid
Antibody
Antibody drug conjugate
Citruline
Devices
Disulfide
Drug
Head
Mass spectrometry
Protease
Valine
Corresponding Organization : University of Cambridge
Other organizations : University of Lisbon, Center for Cancer Research, National Cancer Institute, Universidad de La Rioja, Philochem (Switzerland), ETH Zurich
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Variable analysis
independent variables
- Reduction of inter-chain disulfide bonds of a full-length IgG(B12) using excess amounts of DTT
- Reaction of reduced IgG(B12) with 60 equivalents of carbonylacrylic acid-valine citruline-MMAE molecule
- Average drug-to-antibody ratio of the antibody-drug conjugate
- Reaction temperature of 37°C
- Reaction time of 8-10 hours
- No positive or negative controls were explicitly mentioned in the provided information.
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