Preparation of Dual-Labeled and Site-Specific Antibody and Fab Conjugates by Solid-Phase

During the early stages of ADC drug discovery, various variables such as linker-drugs, site of conjugation, and optimal clone of target antibodies will impact the effectiveness of the drug. The combination of these variables generates a significant number of ADC molecules that need to be screened. As a result, researchers are eagerly seeking a high-throughput generation and purification method that can significantly enhance the efficiency of ADC synthesis. Based on the affinity between antibodies or Fab fragments to the solid-phase reactants, a high-throughput, solid-phase mediated antibody conjugation method has been discovered to significantly facilitate early-stage research efforts.

This article introduces some solid-phase conjugation methods for the preparation of dual-labeled antibodies and Fab conjugates using antibodies and engineered cysteines. This protocol aims to assist our customers in gaining a better understanding of how to perform conjugation through solid-phase coupling reactions.

Disclaimer

This procedure is a guideline only. Please note that Creative Biolabs is unable to guarantee experimental results if it is conjugated by the customer.

• High-Throughput, Solid-Phase Site-Specific Conjugation of Engineered Cysteines on Antibodies

Material:
1. 1 mL Protein A agarose beads
2. Dulbecco's phosphate buffered saline (DPBS, 1x, 9.5 mM PO4 without calcium or magnesium), pH 7.4
3. Human IgG1 with engineered cysteines (1–10 mg/mL)
4. 0.5 M tris (2-carboxyethyl) phosphine hydrochloride (TCEP) solution, pH 7.0
5. 2,20',200'',2'''-(ethane-1,2-diyldinitrilo) tetraacetic acid (EDTA) solution, pH 7.5
6. Fresh reducing buffer: add TCEP and EDTA to DPBS to make 2.22 mM TCEP (100 equivalents), 5 mM EDTA, DPBS
7. Fresh reoxidation buffer: add DHA solution in DPBS to obtain a final concentration of 0.85 mM DHA (40 equivalents), DPBS
8. Antibody elution buffer: 0.1 M Glycine.HCl, pH 3.0 or Pierce elution buffer #21004
9. Neutralization buffer: 100 mM Tris buffer, pH 8.0
10. 50 mM (L)-Dehydroascorbic acid (DHA) (in 1:4:5, DMSO: DPBS: Ethanol)
11. 5 mM maleimide payload: Mix 10 μL of 5 mM maleimide linker-drug (6 equivalents) solution to 290 μL of DPBS, 5 mM EDTA, pH 7.4
12. Dimethyl sulfoxide (DMSO)
13. 96-well filter plates
14. 96-well deep-well collection plates
15. MultiScreen®HTS vacuum manifold

Procedure:
1. Add 100 μL of Protein A bead suspension into each well of a 96-well filter plate (See Note 1 and 2).
2. Place a 96-well collection plate in the MultiScreen® HTS vacuum manifold and apply a vacuum of -20 kPa to -50 kPa to filter the solution from the Protein A bead suspension, then discard the waste.
3. Wash Protein A beads: Add 300 μL of DPBS to rinse the beads and use the vacuum manifold to remove the flow-through from the 96-well filter plate, then discard the waste.
4. Antibody binding step: Switch off the vacuum and add 1 mg of human IgG1 antibody engineered with extra-engineered cysteines to the Protein A beads while suspending the beads. Place the filter plate on top of the collection plate and incubate on a shaker at room temperature for 15 minutes at a speed of 50–150 rpm. After the antibody has bound to the Protein A beads, wash the beads with 300 μL of fresh DPBS, following the same steps as in step 3. Repeat the washing process two more times.
5. Antibody reduction step: Add 300 μL of fresh reduction buffer to the antibody bound to the beads and incubate on a shaker at room temperature for 1 hour at a speed of 50–150 rpm. Then, use the vacuum manifold to remove the reduction buffer and discard the waste. Rinse the beads with 300 μL DPBS and 5 mM EDTA, repeating the washing process three times.
6. Antibody reoxidation step: Add fresh reoxidation buffer to the antibody bound to the beads and incubate on a shaker at room temperature for 1 hour at a speed of 50–150 rpm. Then, use the vacuum manifold to remove the re-oxidation buffer and discard the waste. Rinse the beads with 300 μL DPBS and 5 mM EDTA, repeating the washing process three times.
7. Site-specific cysteine conjugation step: Add 5 mM maleimide linker-drug solution (See Note 3) to the reoxidized antibody bound to the agarose and incubate on a shaker at room temperature for 2 hours at a speed of 50–150 rpm.
8. Linker-drug washing steps: After the conjugation step, use the vacuum manifold to remove the linker-drug. Wash the bead-bound ADCs seven times with 500 μL 10% DMSO in DPBS, ensuring the removal of all conjugation drugs. Perform three additional washes using 300 μL of DPBS and discard the waste.
9. Elution of ADC: Add 75 μL of neutralization buffer to a 96-well collection plate. Add 250 μL of elution buffer to each well containing ADC bound to agarose beads. Using the vacuum manifold, collect the eluate containing the ADC into the 96-well collection plate with neutralization buffer. Repeat the elution step twice.
10. Mix the neutralized ADC solution using a multi-channel pipette and measure the absorbance at 280 nm to determine the concentration (See Note 4).

Note
1. Equilibrate all buffers and agarose beads to room temperature.
2. Seal unused wells with tape to ensure an effective vacuum in the manifold.
3. For higher reaction efficiency, use haloacetamide linker-drug instead of maleimide at 100 mM. Tris pH 8.0, extend the reaction time to 6 hours or overnight, and incubate the reaction mixture at 37°C.
4. Calculate drug-to-antibody ratio (DAR) using an appropriate liquid chromatography-mass spectrometry (LC-MS) method.

• Solid-Phase Dual-Labeled Site-Specific ADC

Material:
1. 1 mL Protein A agarose beads
2. Dulbecco's Phosphate Buffered Saline (DPBS, 1x, 9.5 mM PO4 without calcium or magnesium), pH 7.4
3. Human IgG1 with engineered cysteines (2–10 mg/mL)
4. 0.5 M tris (2-carboxyethyl) phosphine hydrochloride (TCEP) solution, pH 7.0
5. 2,20',200'',2'''-(ethane-1,2-diyldinitrilo) tetraacetic acid (EDTA) solution pH 7.5
6. Fresh reducing buffer: add TCEP and EDTA to DPBS to make 2.22 mM TCEP (100 equivalents), 5 mM EDTA, DPBS
7. Fresh reoxidation buffer: add DHA solution to DPBS to obtain a final concentration of 0.85 mM DHA (40 equivalents), DPBS
8. Antibody elution buffer: 0.1 M Glycine.HCl, pH 3.0 or Pierce elution buffer #21004
9. Neutralization buffer: 100 mM Tris buffer, pH 8.0
10. 50 mM (L)-Dehydroascorbic acid (DHA) (in 1:4:5, DMSO: DPBS: Ethanol)
11. 5 mM maleimide payload: Mix 10 μL of 5 mM maleimide linker-drug (6 equivalents) solution to 290 μL of DPBS, 5 mM EDTA, pH 7.4
12. Dimethyl sulfoxide (DMSO)
13. 96-well filter plates
14. 96-well deep-well collection plates
15. MultiScreen®HTS vacuum manifold
16. PNGase F enzyme for deglycosylation
17. Bacterial transglutaminase enzyme
18. TG (transglutaminase) reaction mix: mix 20 mg of transglutaminase/maltodextrin powder to 0.555 mM BCN-amine (5.55 μL of 30 mM BCN amine in 300 μL PBS).
19. Azido-linker-drug: mix 0.133 mM azido-linker-drug (6 equivalents) with up to 10% DMSO in DPBS,

Procedure:
1. Add 0.2 μL of PNGase F to 1 mg of human IgG1 antibody engineered with selective extra cysteines and incubate overnight at 37°C.
2. Incubate the reaction mixture from step 1 with Protein A agarose beads. Follow steps 1–4 of the first coupling method ("High-Throughput, Solid-Phase Site-Specific Conjugation of Engineered Cysteines on Antibodies").
3. Add 300 μL of TG (thioglycolamide) reaction mix to the antibody-bound beads, and incubate on a shaker at 37°C for 24–48 hours at a speed of 50–150 rpm.
4. After enzymatic conjugation of the antibody, use the vacuum manifold to remove the TG reaction mixture and discard the waste. Rinse the beads with 300 μL of DPBS and 5 mM EDTA, and discard the waste. Repeat the washing process 3 times.
5. Conjugate the cysteine-engineered antibody with the second linker-drug following steps 5-8 of the first coupling method.
6. Couple the strained alkyne on BCN to the azide carrying linker-drug using copper-free click chemistry.
7. Add the azide linker-drug solution to the agarose-bound antibody and incubate at room temperature on a shaker for 4 hours at a speed of 50–150 rpm.
8. Follow linker-drug washing and antibody elution steps following steps 8–10 of the first coupling method.

• Dual-Labeled Fab Conjugation on Solid-Phase

Material:
1. 1 mg cysteine-engineered human Fab fragment
2. Pierce protein-L plus agarose beads.
3. Dulbecco's phosphate buffered saline (DPBS, 1x, 9.5 mM PO4 without calcium or magnesium), pH 7.4
4. 0.5 M tris (2-carboxyethyl) phosphine hydrochloride (TCEP) solution, pH 7.0
5. 2,20',200'',2'''-(ethane-1,2-diyldinitrilo) tetraacetic acid (EDTA) solution, pH 7.5
6. Fresh reducing buffer: add TCEP and EDTA to DPBS to make 0.444 mM TCEP (20 equivalents), 5 mM EDTA, DPBS
7. Fresh reoxidation buffer: add DHA solution in DPBS to obtain a final concentration of 0.85 mM DHA (40 equivalents), DPBS
8. Antibody elution buffer: 0.1 M Glycine.HCl, pH 3.0 or Pierce elution buffer #21004
9. Neutralization buffer: 100 mM Tris buffer, pH 8.0
10. 50 mM (L)-dehydroascorbic acid (DHA) (in 1:4:5, DMSO: DPBS: Ethanol)
11. 5 mM maleimide payload: mix 10 μL of 5 mM maleimide linker-drug (6 equivalents) solution to 290 μL of DPBS, 5 mM EDTA, pH 7.4
12. Dimethyl sulfoxide (DMSO)
13. 96-well filter plates
14. 96-well deep-well collection plates
15. MultiScreen®HTS vacuum manifold

Procedure:
1. Bind 1 mg of engineered human Fab fragment with cysteine to 200 μL of Pierce Protein L Plus agarose beads. Follow steps 1–3 of the first coupling method for antibody binding and washing.
2. Follow steps 4–8 of the first coupling method to selectively conjugate the first linker-drug to the engineered cysteine.
3. Add 300 μL of fresh reduction buffer to the antibody-bound beads and incubate on a shaker at room temperature for 1 hour at a speed of 50–150 rpm.
4. After reducing the antibody with excess TCEP, use the vacuum manifold to remove the reduction buffer and discard the waste. Wash the beads with a solution containing 300 μL of DPBS and 5 mM EDTA, and discard the waste. Repeat the washing process 3 times.
5. Follow steps 7–10 of the first coupling method to conjugate the second linker-drug to the reduced endogenous cysteine and elute the Fab double-drug conjugate.

We provide a comprehensive solution for Antibody Fragment-Drug Conjugates, Drug-Linker Synthesis services and DAR and Payload Distribution Analysis services, making your bioconjugation experiments simple and efficient.

For Research Use Only. NOT FOR CLINICAL USE.