Purification of ADCs by HIC

Antibody-drug conjugate (ADC) in vitro potency is dependent on drug load, with a higher drug load providing lower IC50 values. However, in vivo potency is affected by intrinsic biological effects as well, such as plasma clearance and dose-limiting toxicity. Hydrophobic interaction chromatography (HIC) can take advantage of the differences in hydrophobicity of different constituents present in complex protein matrices to purify ADCs.

This article takes the ADC with a specific drug load, two monomethyl auristatin E (MMAE) payloads (E2) as an example to detailly describe the method for purification of ADCs by hydrophobic interaction chromatography, aiming to provide the researcher with an in-depth understanding about the purification of ADCs.

Disclaimer

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

• Load Solubility Screening and Preparation Methods

Materials:
HIC Load Diluent: 25 mM Sodium Phosphate, 3.0 M Ammonium Sulfate, pH 7.0.
Mobile Phase A: 25 mM Sodium Phosphate, 1.0 M Ammonium Sulfate, pH 7.0.
Mobile Phase B: 25 mM Sodium Phosphate, pH 7.0.

Procedure:
A Direct Titration Method
1. Transfer 500 μL of sample to a clear 1.5 mL Eppendorf tube.
2. Add 100 μL of HIC Load Diluent to the tube to adjust to 0.5 M (NH₄)₂SO₄, and then thoroughly mix by tube inversion or vortexing.
3. Centrifuge at ≥3000g for ≥ 2 min.
4. Inspect the base of the tube for the pellet.
5. If there is a pellet, dilute the solution by 10% using Mobile Phase B and vortex thoroughly until the pellet is fully suspended, or dissolved. Centrifuge again. Repeat this step until the pellet is dissolved.
6. If there is no pellet, repeat steps 2-5 adding HIC Load Diluent in sequence 25 μL (0.60 M), 27 μL (0.70 M), 30 μL (0.80 M), 33 μL (0.90 M), 35 μL (1.00 M), 40 μL (1.10 M), 43 μL (1.20 M), 50 μL (1.30 M), 55 μL (1.40 M), 62 μL (1.50 M).

B. Screening Panel Method
1. Label 11 clear 1.5 mL Eppendorf tubes from 0.5 to 1.5 in increments of 0.1.
2. Transfer 500 μL of sample to each tube. Add sequentially 0.100, 0.125, 0.152, 0.182, 0.215, 0.250, 0.290, 0.333, 0.383, 0.438, 0.500 μL HIC Load Diluent to each tube, and then mix thoroughly by inverting or vortexing the tube.
3. Centrifuge tubes at ≥3000g for ≥ 2 min.
4. Inspect the base of the tube for the pellet. Alternatively, measure the supernatant for absorbance at 280 nm and compare it to the expected concentration to determine when the precipitation has occurred.

• HIC Analysis

Materials:
Mobile Phase A: 25 mM Sodium Phosphate, 1.0 M Ammonium Sulfate, pH 7.0.
Mobile Phase B: 25 mM Sodium Phosphate, pH 7.0.
Sanitization Buffer: 0.5 N Sodium Hydroxide.
Storage Buffer: 20% Ethanol.

Procedure:
1. Temper Mobile Phase A and B to RT.
2. Adjust the ADC load solution to 0.5 M ammonium sulfate.
3. Perform the linear gradient as follows:
Equilibrate 5 column volumes (CV) with 33.3% mobile phase B.
Wash 5 CV with 33.3% mobile phase B.
Elute 30 CV with 33.3% to 100% mobile phase B.
Post-elution wash 3 CV with 100% mobile phase B
CIP/sanitization 3 CV with 0.5 N NaOH.
Storage with 4 CV 20% EtOH.
4. Following the linear gradient elution, determine the mixture points from the gradient where individual drug-load species begin to elute.
5. Analyze the product pool for purity and recovery.

Our preload MMAE-based ADC product catalog

For Research Use Only. NOT FOR CLINICAL USE.