Analysis of Small Molecule and Endotoxin Contaminants in ADC Preparations

Incomplete removal of free (unconjugated) drugs or drug-linker species used to prepare ADCs results in contaminated ADC samples, which may pose a risk for toxicity. To prepare a safe and effective final ADC drug product, it is often necessary for the detection, quantification, and removal of residual free drug and endotoxin present in ADC samples.

This article describes a general method for the detection and removal of small molecules and endotoxins, aiming to provide a guide for researchers working in the fields of analytical chemistry and biochemistry.

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.

• Free Drug Detection

Materials:
UPLC with a PDA detector and a high-sensitivity flow cell
Column, 4.6 × 150 mm, 3.5 μm
10 mM Linker-payload (LP) stock solution
2 mg/mL unconjugated antibody stock solution
2-5 mg/mL ADC samples
Buffer A: 97.9% water +2% acetonitrile +0.1% formic acid
Buffer B: 99.9% acetonitrile + 0.1% formic acid

Procedure:
1. Dilute the initial LP stock solutions (10 mM) to 100 μM in DMSO.
2. Add 1 μL of the LP stock (100 μM, DMSO) to prepare a 1 μM LP standard.
3. Prepare a 5 μM LP standard by spiking 5 μL of the LP stock into 95 μL of an antibody solution (2 mg/mL in PBS).
4. Set up the UPLC method as shown below:
Column temperature: 80°C.
Gradient: 5% B to 95% B over 8 min, hold at 95% B for 1 min, then 2 min re-equilibration at 5% B. The total run time is 11 min.
Flow rate: 1 mL/min.
Injection: 25 μL.
PDA parameters: 3D data collection enabled (210-350 nm), 80 pts/s sample rate, 4.8 nm resolution, fast filter time constant.
5. Inject samples in the following order, 25 μL per injection:
Blank PBS - single injection.
ADC injection 1 - record AUC (see Note 1).
ADC injection 2 - record AUC.
Blank PBS - single injection (see Note 2).
2 mg/mL mAb + 1 μM LP spike - record AUC.
2 mg/mL mAb + 1 μM LP spike - record AUC.
2 mg/mL mAb + 5 μM LP spike - record AUC.
2 mg/mL mAb + 5 μM LP spike - record AUC.
6. Estimate the free drug concentration by comparing the average AUC for the test sample to the AUC for the 1 μM and 5 μM spikes.

Note:
1. Using the HPLC method outlined, the ADC typically elutes at ~2 min, while the small molecule contaminants elute between 2.5 and 3.5 min, depending on their polarity.
2. Inject a PBS blank between ADC sample injections to prevent cross-contamination due to potential ADC carryover.

• Free Drug Removal

Materials:
Non-polar polystyrene adsorbent, 20-50 mesh
Reagent grade methanol
Cellulose-acetate centrifugal filter

Procedure:
1. Add 2 mL methanol to the non-polar polystyrene adsorbent and gently shake at room temperature for a few minutes, then remove the solvent by pipette.
2. Resuspend the beads in 2 mL of fresh methanol and gently shake at 37°C overnight. Remove the MeOH and rinse the beads for ~30 min at room temperature successively in 1 × 1 ml distilled water and 2 × 2 mL PBS.
3. Filter the final PBS suspension to afford dry beads, and then use immediately or freeze for a long time.
4. The ADC sample (~5-10 mg/mL) is treated with ~50 mg of pre-washed non-polar polystyrene adsorbent per mL of sample. The sample is gently agitated at 37°C for ~3 h.
5. Take an aliquot, filter through a 0.2 μm cellulose acetate filter, and analyze by LC-MS to confirm the removal of the free drug (see Note 5).
6. Once removal of the free drug is confirmed, the beads are removed using a centrifugal sterile filter unit with a 0.2 μm membrane by spinning for ~30 s at 300 rpm to remove the beads.

Note:
It is typically recommended that aliquots be removed and monitored by HPLC in "real time" to determine when complete removal of drug-linker contaminants has been achieved. The process typically takes 2-24 hours of agitation.

• Endotoxin Removal

Materials:
Endotoxin removing columns
DPBS, 1× 9.5 mM PO₄ without calcium or magnesium
1% solution of sodium deoxycholate in water
Endosafe assay instrumen
Endosafe assay kit

Procedure:
1. 4.2 mL of an ADC in DPBS buffer was tested via an Endosafe assay instrument to contain endotoxin at >25 EU/mL (see Note 4).
2. Two separate 1 mL prepacked columns were washed with 5 resin bed volumes each of 1% sodium deoxycholate solution to regenerate the active resin.
3. Equilibrate the columns of activated columns with 6 resin bed volumes each of DPBS.
4. Load 2.1 mL ADC solution onto each column and gravity elute.
5. Column flow-through was recycled through each column 3× to increase the exposure time of conjugate to resin (see Note 5).
6. Wash columns with an additional 2 mL of DPBS each.
7. The diluted ADC sample was re-concentrated via a 50KD ultrafiltration device to afford 2.5 mL final ADC.
8. The purified ADC sample was tested to contain endotoxin at 2.13 EU/mL (see Note 6).

Note:
1. 1 mL of resin removes >9995 EU from a 5 mL sample challenge containing 10,000 EU. In the procedure outlined, the resin was conservatively loaded with the contaminated sample, as the exact total amount of endotoxin present in the sample was not known.
2. It is also possible to stop elution once the sample has been applied to the column and incubate the sample on the resin for up to 1 h before ultimately collecting the sample as an alternative.
3. The entire process could be repeated as necessary to further decrease the endotoxin load.

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