Butyl-Dependant Hydrophobic Interaction Liquid Chromatography: A Technique to Characterize Antibody-Drug Conjugates Based on Hydrophobic Interactions

1. Produce and Characterize the Antibody

1. Express the antibody
   1. Thaw a vial of HEK suspension cells in a 250 mL flask containing 50 mL of expression medium supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 250 ng/mL amphotericin B. Keep the cells at 37 °C with 8% CO₂ in humidified incubators equipped with a shaker at 125 rpm. Split cells to 0.3-0.5 x 10⁶ cells/mL (every 2-3 days) at least 2 times before transfecting.
   2. When a density of 2.5 x 10⁶ cells/mL is reached (2-3 days after splitting), prepare a fresh solution of 100 mM CypK. For this purpose, weigh 64 mg of CypK, add 2.5 mL of 0.1 sodium hydroxide, vortex, spin down to recover all undissolved particles and sonicate.
   3. Add 2.5 mL of CypK (100 mM in 0.1 M NaOH) to 42.5 mL of expression medium supplemented with antibiotics. Mix well, add 250 µL of 0.1 M HCl, and sterilize using a 0.22 µm filter.
   4. Dilute 50 µg of HC and LC pKym1 plasmids to 2.5 mL with reduced serum medium. In a separate tube, dilute 135 µL of transfection reagent to 2.5 mL with reduced serum medium.
   5. Five minutes after preparing the solutions, mix the plasmids and the transfection reagent solution and incubate for 20 min to allow the formation of complexes between the DNA and the transfection reagent.
   6. In the meantime, centrifuge 125 million cells at the target density for 5 min at 500 x g, resuspend with the expression medium containing CypK and add the DNA–transfection reagent mixture. CypK can be purchased or synthesized as reported previously.
   7. After incubating cells for 20 h, add 250 µL of transfection reagent enhancers included in the kit.
   8. Harvest antibodies from the supernatant 6-7 days after addition of CypK (no change of medium is required during expression).
      *Alternatively, to obtain higher and more consistent yields, a stable cell-line can be generated as described in Oller-Salvia et al. 2018. In this case, trastuzumab(CypK)₂ is expressed simply by addition of CypK 5 mM in the expression medium.
2. Purify the antibody
   1. Centrifuge the cells for 15 min at 3000 x g.
   2. Filter the supernatant with a 0.45 or a 0.22 µm filter. If the filter becomes occluded, replace it for a new one and continue filtering. If this happens after only a few milliliters, centrifuge the supernatant for an additional 15 min at 7000 x g.
   3. Add protein A resin (2 mL/100 mL of supernatant) in an empty polypropylene chromatography column and equilibrate the resin with at least 5 volumes of bead wash buffer (0.1 M sodium phosphate, 150 mM NaCl).
   4. Split the supernatant into two 50 mL conical tubes and add 5x bead wash buffer (0.5 M sodium phosphate, 150 mM NaCl) followed by the pre-equilibrated protein A resin. Place the conical tube on a roller for 3 h at room temperature to pull down the antibody in the supernatant.
   5. Alternatively: Add the supernatant on the column with at least double the recommended volume of resin and allow to flow through. Make sure there is not a significant amount of antibody left in the supernatant by SDS-PAGE. If there is, elute the supernatant through the resin once again.
   6. Transfer protein A resin with the supernatant into a column and allow the liquid to flow through.
   7. Add 25 mL of wash buffer (or at least 10 resin volumes) and allow to flow through.
   8. Elute the antibody with 4 mL of 0.1 M sodium citrate, pH 3, on 1 mL of 1 M phosphate buffer, 150 mM NaCl.
   9. Dilute the antibody with 10 mL of PBS, concentrate to 0.5-1 mL by centrifugal filtration, and exchange buffer three times with PBS.
   10. Purify the samples by FPLC using a butyl HIC column with a flow of 0.5 mL/min and a 0-100% gradient in 30 min of low-salt buffer (50 mM sodium phosphate pH 7.0, 20% isopropanol) in high-salt buffer (1.5 M (NH4)₂SO₄, 50 mM sodium phosphate pH 7.0, 5% isopropanol). Collect all fractions and monitor the elution at 280 nm. Small amounts (< 1 mg) can be purified with HPLC-HIC under the conditions described in 2.4 to maximize yield and purity.
   11. Pool the fractions that contain antibody, dilute them to at least 4 mL with PBS, concentrate them by centrifugal filtration and exchange buffer three times with PBS.
3. Quantify the antibody
   1. Obtain an approximate concentration by measuring absorbance at 280 nm using a micro-volume spectrophotometer.
   2. If an accurate measurement is required, use an ELISA kit to measure human IgGs. For a rough estimate, use SDS-PAGE with a Coomassie-based stain as follows:
      1. Prepare standards by diluting six times two-fold a trastuzumab standard quantified by ELISA at 1 mg/mL.
      2. Boil the standards and the samples at approximately 0.25 mg/mL in reducing loading buffer.
      3. Run them in a 4-12% Bis-Tris SDS-PAGE using MES-SDS buffer and stain with a Coomassie-based dye. Finally measure color density of the band corresponding to the light or the heavy chain using ImageJ and interpolate the signal from the samples in the standard curve.
4. Store the antibody at 4 °C.

2. Conjugate the Antibody and Characterize the ADC

1. Conjugate the antibody with the tetrazine-bearing molecule
   1. Dilute 10 molar equivalents of tetrazine-vcMMAE (4 µL, 3.4 mM in dimethyl sulfoxide (DMSO)) with 20 µL of acetonitrile and 76 µL of PBS in a small conical tube (e.g.,PCR tube).
   2. Add 1 molar equivalent of trastuzumab(CypK)₂ (100 µL, 2 mg/mL in PBS), mix thoroughly, and allow to react for 3 h at room temperature (25 °C) to form trastuzumab(MMAE)₂.
      NOTE: Other molecules such as tetrazine-5-TAMRA can be linked to the antibody instead of tetrazine-vcMMAE using this protocol.
2. Purify the ADC
   1. Pre-equilibrate a size exclusion spin column with PBS following the manufacturer's instructions.
   2. Add the whole volume of the reaction on the spin column and centrifuge at 1500 x g for 1 min.
3. Quantify the ADC and analyze the conjugate by SDS-PAGE
   1. Quantify the ADC as described in 1.3.2. by measuring the color density of the light chain on an SDS-PAGE using the non-modified antibody for the standard curve.
   2. The heavy chain should have slightly shifted showing an increase in molecular weight upon the conjugation.
      Note: If the antibody is modified with a fluorophore such as in trastuzumab(TAMRA)₂, only the band corresponding to the heavy chain should show in-gel fluorescence before staining.
4. Analyze the conjugate by HPLC-HIC
   1. Equilibrate the HPLC-HIC column with 100% buffer A (1.5 M (NH₄)₂SO₄, 50 mM sodium phosphate pH 7.0, 5% isopropanol) for 5 min.
   2. Mix 15 µL (67 pmol) of a 1 mg/mL solution of trastuzumab(CypK)2 with 15 µL of 2x buffer A in a vial. Then program a 10 µL injection in the HPLC.
   3. Elute in an isocratic 1 mL/min flow with 100% buffer A for 1 min followed by a 15 min gradient from 100 to 0% of buffer A in B (50 mM sodium phosphate pH 7.0, 20% isopropanol). Monitor the elution at 280 nm.
   4. Calculate the DAR by integrating the peak corresponding to each species and using the resulting areas in the following equation:
      DAR = (1x trastuzumab(MMAE) + 2x trastuzumab(MMAE)₂)
      /(trastuzumab(MMAE)₀ + trastuzumab(MMAE)₁ + trastuzumab(MMAE)₂)
      Expected retention time for trastuzumab(CypK)₂ is 7.5-8.0 min, for trastuzumab(CypK, MMAE) is 9.1-9.6 min, and for trastuzumab(MMAE)₂ is 10.5-11.0 min.