Conjugation Strategy of Endogenous Cysteine Residues

There are several ways to covalently couple small molecule drugs to monoclonal antibodies, such as coupling the drug to a lysine or endogenous cysteine residue on the monoclonal antibody via a linker and using site-specific coupling of the drug to an engineered functional gene on the antibody. One of the most common methods is the former one.

This essay shares a detailed protocol for traditional cysteine coupling, taking the preparation of anti-Notch3-targeted ADCs as an example.

Reagents Required

Dimethyl sulfoxide (DMSO), Solution of 10 N sodium hydroxide, Tris(2-carboxyethyl)-phosphine hydrochloride TCEP HCl (0.5 M solution), Sucrose (Low Endotoxin), _L-Histidine, _L-Histidine monohydrochloride monohydrate, Polysorbate-80, Disodium EDTA dihydrate, 20 mM sodium phosphate buffer (pH 7.2), 400 mM sodium chloride.

Mobile phase A: Ammonium sulfate (1.5 M) solution, 50 mM potassium phosphate dibasic, pH 7

Mobile phase B: 10% isopropanol, 50 mM potassium phosphate dibasic, pH 7

PF-06424469 Linker-Payload. Anti-notch 3 IgG1 monoclonal antibody stock solution

Materials Required

Tubing (Platinum cured silicon tubing (50 A), L/S^® 24), Tubing (Platinum cured silicon tubing (50 A), L/S^® 35), Serological pipettes (50 mL, 20 mL, 10 mL), Pellicon 3 UF/DF Ultracel Membrane (30 kD MWCO) (0.11 cm2) (Millipore), Sterile filter (0.45 + 0.22 μm)

Equipment Required

4 L & 2 L Glass reactor with an impeller, pH meter, Temperature probe, UF/DF system, Glove box balance for weighing PF-06424469 (see Tips 1), Axichrom™ 70/300 purification column, YMC-Pack Diol-200 (300 * 8 mm ID), Thermo Propac HIC10 column (4.6 mm * 10 cm, 5 μm)

Operating Procedure

Conjugation of Anti-notch 3 mAb to PF-06424469 Linker-Payload

• The solution of anti-Notch 3 antibody (1 equivalent) is placed in a jacketed reactor equipped with a magnetic stir bar or overhead agitator. The pH of the anti-Notch 3 antibody solution is adjusted to 6.5-7 (see Tips 2). The antibody solution is heated to 37°C. An aqueous solution of TCEP hydrochloride (2.2 equivalents) is added to the antibody solution (see Tips 3). The obtained solution is stirred at 37°C for 90 minutes.
• The solution is cooled to 25 °C. A solution of PF-06424469 (6 equiv.) in DMSO (7.7 mg/mL, see Tips 4) is slowly added to the antibody solution over 10-15 min. Stir the reaction mixture for 1 h. Add L-cysteine (4 equiv.) (see Tips 5) to quench the mixture and stir for 30 min at 25 °C. The reaction mixture is filtered through a 0.2 μm filter.
• The resulting unpurified product included an ADC (hydrophobic interaction chromatography (HIC) to assess the average DAR, which should be ~4%, and size exclusion chromatography to assess the aggregate level, which should be ~5%).

Purification of the Reaction Mixture

• The ADC mixture is purified by column chromatography over ceramic hydroxyapatite resin (type 1, 40 μm), with a column volume of 7.5 mL (see Tips 6).
• The following purification parameters are used for the separation of aggregates from monomeric species.
• Mobile phase A: 5 mM sodium phosphate, pH 7;
• Mobile phase B: 200 mM sodium phosphate, pH 7;
• Buffer gradient is 10-70% mobile phase B over 20 column volumes. The protein loading on the column is 15 mg/mL, with a flow rate of 1 mL/min and a column bed height of 22 cm.
• The first fraction is collected from the main peak continuously to the next 6.9 column volume, and then collecting next three fractions at every 1/3 column volume. The fractions are analyzed for DAR values by in-process UV assay and for aggregates by SEC analytical methods.
• Most of the monomer elutes in fraction 1 of the collected fractions to provide the ADC product in 85-88% yield (based on protein yield). The aggregates are in the last peak as shown in the chromatogram. (The average DAR of the isolated ADC material should be 3.6 (HIC assay) and the aggregate content should be about 1% (SEC assay))

Concentration

• The ADC material obtained after column chromatography is purified and the buffer is exchanged via ultrafiltration/diafiltration process (UF-DF) with Ultracel membrane cartridges (30 kD) and 20 mM histidine buffer as diafiltration buffer (pH 5.8). The retentate is collected and filtered through a 0.2 μm filter.

ADC Formulation & Storage

• Purified ADC material is prepared with standard formulation excipients, such as sucrose, EDTA, and PS 80. The prepared ADC solution is stored frozen for long-term storage.

Determination of Average Drug Loading and Drug Distribution of the ADC

• The drug load and drug load distribution of ADCs are determined by analytical HIC methods as follows.
• Reference material (25 μL) and test samples are diluted to 2 mg/mL with diluent (half strength of mobile phase A) and then injected into a Thermo Propac HIC10 column (4.6 mm 10 cm, 5 μm).
• The mobile phase A is 1.5 M ammonium sulfate, 50 mM potassium phosphate dibasic, pH 7.
• The mobile phase B is 10% isopropanol, 50 mM potassium phosphate dibasic, pH 7.
• Sample elution flow rate: 0.8 mL/min
• Sample elution column temperature: 30 °C
• Species with different DAR are separated with a gradient of salt and detected by UV absorption at 280 nm. The average DAR and distribution are determined by the percentage of peak area of each species.

Determination of Aggregate Content by Analytical SEC

• The SEC method is adopted to determine the purity of the product and aggregate content. The test samples are diluted with mobile phase (20 mM sodium phosphate, 400 mM sodium chloride, pH 7.2) at a concentration of 2 mg/mL and injected into a YMC-Pack Diol-200 (300 8mm ID) column at 0.75 mL/min for 20 min. Aggregate and monomer species in the crude ADC preparation are reported as a percentage of the total area of all protein-related peaks.

Determination of Average Drug Loading (DAR) by Reduced Reverse Phase HPLC

• Samples are denatured in 4 M guanidinium-HCL, 50 mM Tris, pH 7.8, and 20 mM DTT at 37 °C for about 30 min. The reduced samples (10 μL) are separated and monitored at 214 nm with Agilent HPLC using an Agilent Zorbax 300SB-CN column (150*4.6 mm, 3.5 μm) at a column temperature of 75 C. Samples are eluted using mobile phase A (0.1% TFA, water) and mobile phase B (80% acetonitrile, 20% isopropanol, and 0.1% TFA) at a flow rate of 0.75 mL/min.

Tips:

1. Linker-payload PF-06424469 is highly toxic. Care must be taken to protect during operation.
2. The anti-Notch 3 antibody is prepared in 20 mM histidine buffer and the pH of the antibody solution should be adjusted to 7.8 with 200 mM histidine buffer. The final concentration of histidine in the final reaction mixture of the reaction should be 100 mM histidine.
3. The rate of addition of TCEP should be strictly controlled to obtain a homogeneous antibody solution. Before the large-scale experiment begins, a small-scale titration experiment is conducted to determine the correct charge of TCEP. The thiol content of the mAb formed after reduction can be determined by DTNB. Alternatively, the reduced McAb sample can be combined with excessive joint payload and the DAR of the sample can be determined by analytical HIC or the reductive reversed-phase high-performance liquid chromatography.
4. Control the addition rate of the DMSO solution and do not make the temperature of the reaction mixture too high.
5. _L-cysteine is used as a quenching reagent to react with the unconjugated linker-loadings remaining in the reaction mixture. When the histidine buffer is used for the conjugation reaction, the linker-loaded maleimide group slowly reacts with the histidine, resulting in the formation of a linker-loaded histidine adduct.
6. Optimal purification of the ADC can be obtained by using ceramic hydroxyapatite resin and the sodium phosphate gradient reported in this procedure.

For Research Use Only. NOT FOR CLINICAL USE.