Maleimide conjugation

Based on

Equilibrate all moisture-sensitive reagents to room temperature before opening to avoid condensation.

Column

Reagents

  • mTz-PEG4-maleimide
  • Tris-EDTA 100 mM Tris-HCl pH 7.2 2.5 mM EDTA
    • Need high buffering capacity for the reduction reaction. EDTA chelates divalent cations to reduce oxidation.
  • TBS: 20 mM Tris-HCl pH 7.0 150 mM NaCl
    • Maleimide reactions prefer sulfhydryls over amines at lower pH (pH 6.5-7.5).
  • High-salt PBS: PBS + 1.0M NaCl

Stock concentration

  • 20 mM mTz-PEG4-maleimide. Dissolve 10 mg in 970 μl anhydrous DMSO. Maleimide hydrolyzes in alkaline solution.
    • Dilute 5-fold in DMSO to 4 mM for working concentration.

For 100 μg = 0.67 nmol IgG.

  1. Partially reduce antibody

    • Prepare fresh 4 mM TCEP solution from 0.5 M stock. (2 μl of 0.5 M to 8 μl Tris-EDTA, then 4 μl + 96 μl Tris-EDTA)
    • Buffer exchange into Buffer R with a 50 kDa filtration unit. Centrifuge at 14,000g for 4 minutes (can be more). Volume should not exceed 20 μl after spin.
    • Add 100 μl of 4 mM TCEP to the filtered antibody inside the column and mix by pipetting.
    • Incubate at 37°C in a water bath for exactly 30 min.
    • Wash with TBS 3 times (400 μl). TCEP can reduce conjugation yield but this is not as bad as DTT.

  2. Conjugate with mTz-PEG4-maleimide

    • Use 10-fold molar excess of mTz-PEG4-maleimide. Final concentration is 66.7 μM. Use 1.675 μl from 4 mM working concentration and 78.325 μl TBS and add directly to antibody in the column.
    • Incubate at 37°C in a water bath for 90 min.
    • Wash in TBS and wash 3 times in BBS pH 8.5. (Maleimide-thiol conjugates can undergo a reverse reaction. Hydrolysis of maleimide in an alkaline solution prevents this. Mild Method for Succinimide Hydrolysis on ADCs: Impact on ADC Potency, Stability, Exposure, and Efficacy Michael addition of the thiol to the maleimide results in the maleimide-thiol conjugate, the thiosuccinimide. The reaction is fast, but the conjugate is relatively unstable and can undergo further reaction via one of two pathways: (i) it can undergo irreversible ring-opening hydrolysis to yield a stable hydrolyzate (succinamic acid thioether), which prevents elimination of the maleimide-thiol conjugate; (ii) it can undergo a retro-Michael conversion back to the starting thiol and maleimide.
    • The concentrated antibody should have a slight pinkish hue from the methyltetrazine.

  3. Conjugate with TCO-labelled oligo

    • Mix with 3 nmol of oligo for a conjugation ratio of 5:1. Incubate at room temperature O/N. Probably overkill but at least this opens more of our maleimide ring.
    • Wash with High-salt PBS 4 times (tested oligo retention in high-salt PBS in a 50 kDa unit, undetectable after 3 washes).
    • Wash last time with PBS.
    • Flip the centrifugation unit into a new tube and centrifuge at 1,000g for 2 min to elute.
    • Quantify with BCA.