The Cysteine Rebridging Approach for ADC Conjugation

The native lysine and cysteine residue linkage strategy is the primary chemical conjugation method used in the first generation of ADCs. The lysine conjugation method is relatively straightforward but can lead to highly heterogeneous mixtures in many ADCs due to the high abundance of lysine residues available for conjugation in a random distribution. Cysteine conjugation can also result in heterogeneous mixtures of ADC species and may increase the physical-chemical instability under stress conditions, affecting pharmacokinetic (PK) properties. Improved disulfide rebridging strategies can address issues of site selectivity and uniformity, resulting in a more homogeneous and more stable ADC.

There are various reagents used for antibody disulfide rebridging conjugation. This article introduces a method based on bis-thiol alkylating reagents to help more researchers understand the application of rebridging strategies in ADC conjugation.

Disclaimer

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

• 3.1 Preparation of the hIgG1 Antibody

Material:
hIgG1 Antibody (Concentration > 8 mg/mL)
Reaction Buffer: 20 mM sodium phosphate buffer, pH 7.5, containing 150 mM NaCl and 20 mM EDTA
Eppendorf Thermoblock
PD-10 Column

Procedure:
1. Equilibrate a PD-10 column with 25 mL of reaction buffer. Add 2.5 mL of antibody to the column, allowing it to pass into the bed. Add 3.5 mL of reaction buffer to elute the antibody from the column, and collect the eluate in a clean tube. Mix the solution, and then centrifuge it.
2. Using a nanodrop spectrophotometer, measure the antibody concentration at UV A280 and dilute the antibody solution to 5.2 mg/mL using reaction buffer.
3. Transfer 981 µL of the antibody (5.1 mg) into a clean tube and incubate the solution at 40°C for 15 minutes.

• 3.2 Interchain Disulfide Reduction

Material:
5 mM tris(carboxyethyl)phosphine (TCEP) solution
Iodoacetamide
Eppendorf thermoblock

Procedure:
1. Add 40.8 μL of 5 mM TCEP to the antibody solution and stir the solution using a vortex mixer. Incubate the mixture at 40°C for 1 hour.
2. After the incubation, cool the antibody solution for 15 minutes to 22°C. Briefly centrifuge the tube, and then open it. Transfer 10 microliters of the antibody solution to a clean tube and freeze it at -80°C. Take another 10 microliters of the antibody solution and use it for iodacetamide alkylation of samples, followed by SDS-PAGE analysis.

• 3.3 Cysteine Rebridging Conjugation

Material:
Acetonitrile
Cysteine rebridging reagent

Procedure:
1. Take 0.256 μmol of cysteine rebridging reagent and add it to 85.7 μL of acetonitrile to prepare a 3.0 mM reagent solution. Mix the solution until it is completely dissolved, and then centrifuge the reagent solution.
2. Take 5 mg of the reduced antibody solution from step 3.2, add 188 μL of reaction buffer, and then add 62.5 μL of the reagent solution. Use a vortex mixer to thoroughly mix the solution, ensuring complete conjugation, and then incubate it overnight at 22°C.
3. After 16 hours, take a sample and analyze the progress of the reaction through analytical HIC (hydrophobic interaction chromatography).

• 3.4 Preparative HIC Purification

Material:
ÄKTA prime plus
ToyoPearl Phenyl-650S column
0.2 M NaOH
Endotoxin-free deionized water
HIC buffer B: 50 mM sodium phosphate buffer, pH 7.0, containing 20% v/v 2-propanol
HIC buffer A: 50 mM sodium phosphate buffer, pH 7.0, containing 2 M NaCl
HIC loading buffer: 50 mM sodium phosphate buffer, pH 7.0, containing 4 M NaCl
15 mL Falcon tubes<
Syringe

Procedure:
1. Mount the ToyoPearl Phenyl-650S column onto the ÄKTA prime, and wash the system with 0.2 M NaOH followed by endotoxin-free deionized water. Ensure that both buffer lines, the injection loop, and the outlet tube to the fraction collector are all washed.
2. Prior to starting purification, wash the system with HIC buffer B, followed by washing with HIC buffer A.
3. Load 15 mL Falcon tubes onto the fraction collector turntable.
4. Mix the ADC sample from step 3.3 in 1:1 v/v ratio with HIC load buffer. Centrifuge the vial, and then use a syringe to load the sample into the injection loop.
5. After loading the sample, initiate elution of the ADC with a linear gradient from 0–100% B at a flow rate of 1 mL/min. Collect appropriate fractions.
6. Wash the ÄKTA system with 100% B until the conductivity and UV measurements reach a stable state, and then stop the run.
7. Analyze the eluted fractions by analytical HIC to determine the fractions containing DAR 4 ADC.

• 3.5 Sample Processing

Material:
Endotoxin-free, deionized water
70% ethanol
Dulbecco's phosphate buffered saline (DPBS)
Vivaspin 20 centrifugal concentrator
PD-10 desalting column
Syringe
0.22 μm PVDF sterile filter

Procedure:
1. Wash the Vivaspin 20 centrifugal concentrator with endotoxin-free deionized water, 70% ethanol, and DPBS. Combine the fractions containing DAR 4 ADC and concentrate the sample to approximately 2 mL using a centrifuge (3000 x g).
2. Use a pipette to remove the ADC from the centrifugal concentrator. Rinse the centrifugal concentrator with 0.5 mL of DPBS and add the rinse solution to the ADC, bringing the final solution volume to 2.5 mL.
3. Equilibrate a PD-10 column with 25 mL of DPBS. Add the 2.5 mL of ADC to the column and place it in the bed. Add 3.5 mL of DPBS to elute the antibodies into a clean tube. Mix the solution, and then centrifuge it. Finally, filter the ADC through a 0.22-micron PVDF syringe filter.

For Research Use Only. NOT FOR CLINICAL USE.