Site-Specific Conjugation Strategy Mediated by Transglutaminase

Microbial transglutaminase possesses the capability to catalyze the crosslinking of proteins and certain non-protein substances, making it suitable for site-specific conjugation reactions between antibodies and small molecule drugs. The conjugation mediated by transglutaminase not only yields homogeneous antibody-drug conjugates (ADCs) but also allows for flexible adjustments of the drug-to-antibody ratio (DAR) and antibody binding regions through methods such as amino acid mutations and the use of branched linkers.

This article takes the human IgG1 antibody as an example to introduce a protocol for site-specific conjugation based on transglutaminase, aiming to assist a broader audience of researchers in understanding strategies related to antibody chemical conjugation.

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.

• Deglycosylation

Material:
2 mg/mL human IgG1
500 units/μL PNGase F
100 mM dithiothreitol (DTT)
50 kDa centrifugal filter units
Reverse-phase HPLC column

Procedure:
1. Thaw the frozen antibodies rapidly in a water bath at 37°C and add PBS to adjust the antibody concentration to 2 mg/mL.
2. Then add 2 μL of PNGase F to the sample.
3. Incubate the mixture at 37°C for 16–20 hours.
4. Take 5 μL of the reaction mixture for monitoring purposes. Dilute the sample with 5 μL of PBS, add 1 μL of 100 mM DTT, and incubate at 37°C for 5–20 minutes. Finally, inject 10 μL of the diluted sample into the LC/MS system for heavy and light chain analysis (see Note 1).
5. Load the mixture into a 50 kDa centrifugal filter units, and centrifuge at 4°C, 4,000 g for 20 minutes. Wash the sample with PBS three times or more (see Note 2).

Note:
1. If antibody deglycosylation is incomplete, add PNGase F (2 μL), and allow the reaction mixture to incubate for an additional 16–20 hours at 37°C until maximum conversion is achieved.
2. Centrifugation conditions can be adjusted based on the number of collected samples.

• MTGase-Mediated Antibody-Linker Conjugation

Material:
5–15 mg/mL deglycosylated human IgG1
Branched linker (40 equivalents per reaction site)
MTGase: 40% (w/v) Activa TI®
Column for size-exclusion chromatography (SEC)
0.22 μm centrifuge tube filters
PBS eluent

Procedure:
1. Centrifuge the antibodies collected in the previous step to concentrate them to 10 mg/mL (see Note 3).
2. Mix the collected monoclonal antibody solution (final concentration > 5 mg/mL), branched linker, and Activa TI® together, and incubate the mixture at room temperature overnight without shaking or inverting (see Note 4).
3. Monitor the reaction using an LC/MS system.
4. Purify the conjugated compound with SEC chromatography (see Note 5), and concentrate the solution to 4 mg/mL or higher. This solution can be stored at 4°C.

Note:
3. High concentrations of mAb are crucial for achieving antibody-linker conjugation, so please ensure that the mAb concentration is within the range of 5–20 mg/mL.
4. The coupling conditions need to be optimized for MTGase concentration, pH, temperature, and incubation time parameters to achieve complete conversion.
5. The product purity should exceed 95%.

• Strain-Promoted Azide—Alkyne Cycloaddition for Payload Installation

Material:
DBCO-spacer-MMAF (4 mM in DMSO, spacer: PEG3-glutamic acid-valine-citrulline-p-aminobenzyloxycarbo-nyl)
Column for SEC
PBS eluent

Procedure:
1. Mix DBCO-spacer-MMAF with mAb-conjugated linker in PBS (final concentration: 4 mg/mL, with DMSO level at 10% or lower) and incubate at room temperature for 1 hour.
2. Monitor the reaction using LC/MS (see Note 6).
3. Purify the sample using SEC (flow rate: 0.6 mL/minute) to obtain the ADC and concentrate it to 1 mg/mL. Store the solution at 4°C for later use.
4. Analyze the purified ADC using LC/MS and determine the average DAR value based on UV peak areas (see Note 7).

Note:
6. If the reaction is not complete, add the payload module and incubate the reaction mixture for an additional hour at room temperature.
7. HIC analysis is an alternative method for determining ADC hydrophobicity and average DAR.

We offer customers a variety of different conjugation strategies, including but not limited to Lysine-Based Conjugation, Cysteine-Based Conjugation, Tyrosine-Based Conjugation, EnCys-mAb-Based Conjugation, Unnatural Amino Acid (UAA) Based Conjugation, and Carbohydrate-Based Conjugation. Feel free to reach out, and we can customize the most suitable bioconjugation solution for your research needs.

For Research Use Only. NOT FOR CLINICAL USE.