The Strategy for Characterizing ADCs by Capillary Electrophoresis

Capillary electrophoresis (CE), a highly efficient separation technique that resolves ions based on their electrophoretic mobility in the presence of an applied voltage, has been broadly applied for characterizing biotherapeutics, including ADCs.

This article briefly describes how to characterize ADCs using CE, including reduced and non-reduced capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and imaged capillary isoelectric focusing (iCIEF), aiming to help researchers understand the latest advancements in ADC characterization techniques.

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• ADC Sample Preparation for CE-SDS Purity Analysis

Materials:
2-Mercaptoethanol.
SDS MW analysis kit.
0.5 M iodoacetamide solution.
Microcentrifuge.

Procedure:
A. Reduced ADC Sample Preparation (see Note 1)
1. Dilute the ADC sample to 2 mg/mL with water, then take 100 μL of the diluted ADC sample into a 0.5 mL microcentrifuge tube.
2. Add 100 μL of SDS-MW sample buffer and 10 μL of 2-mercaptoethanol into the above microcentrifuge tube.
3. Vortex the microcentrifuge tube for 10 s, heat the mixture at 70 °C for 10 min, and cool the mixture to room temperature. The final protein concentration is about 1 mg/mL (see Note 2).
B. Non-reduced ADC Sample Preparation (see Note 1)
1. Dilute the ADC sample to 2 mg/mL with water, then pipette 100 μL of the diluted ADC sample into a 0.5 mL microcentrifuge tube.
2. Add 100 μL of SDS-MW sample buffer and 10 μL of 0.5 M iodoacetamide into the above microcentrifuge tube (see Note 3).
3. Vortex the microcentrifuge tube for 10 s, heat the mixture at 55 °C for 10 min, and cool the solution to room temperature. The final protein concentration is about 1 mg/mL (see Note 2).

Note:
1. The ADC reference materials can be prepared following the sample preparation procedures.
2. It is recommended that protein concentration be 1 mg/mL for best results as high protein concentration can result in broad peaks and poor resolution, while low protein concentration can result in low signal. However, this could be varied and optimized depending on the properties of ADC samples.
3. For non-reducing conditions, iodoacetamide was added to the sample buffer as an alkylating agent.

• ADC Purity Analysis by CE-SDS

Materials:
CE instrument.
Detector, Ultraviolet or Photodiode Array.
Capillary, 50 μm I.D. bare-fused silica.
Capillary cartridge, 100 × 200 μm aperture, approximate length 30.2 cm total.
Microcentrifuge.

Procedure:
1. Follow the manufacturer's procedures to set up the CE instrument.
2. Replace the sample with the formulation buffer. Follow the steps of ADC sample preparation to prepare reduced and non-reduced CE-SDS blank samples, respectively.
3. Centrifuge blank samples and ADC samples (reduced and non-reduced) at 10,000 rpm (9500 × g) for 2 min.
4. Transfer 100 μL of all samples and blanks from the above step into autosampler vials for CE-SDS analysis (see Note 4). The autosampler temperature is typically set at 10 °C.
9. Set up the following separation method:
The UV detection is set at 214 nm.
Capillary temperature is controlled at 25 °C.
The autosampler temperature is set at 10 °C (see Note 5).
Set up the sequence and run (see Note 6).
10. Data analysis (see Note 7).

Note:
1. Make sure there are no bubbles in the sample solution during preparation and transfer. If bubbles exist, centrifuge the microvials and repeat if necessary.
2. The UV detection can be set at other wavelengths depending on the maximum UV absorbance of ADC samples.
3. When both non-reduced and reduced preparations are analyzed in one sequence, begin the sequence with the non-reduced preparations first.
4. For reduced CE-SDS analysis of cysteine-linked ADCs, two major peaks are typically shown: light chain (LC) and heavy chain (HC). The LC or HC with and without drugs is generally not separated or only partially separated. The purity of ADCs produced by reduced CE-SDS was expressed as the sum of the percent areas of light and heavy chains. The percent purity determination of ADC by non-reduced CE-SDS depends on the ADC conjugation sites and its drug-to-antibody ratio. The percent purity will be the sum of the peak area of expected fragment species over the total peak area.

• iCIEF for ADC Charge Heterogeneity Analysis

Materials:
iCE3 analyzer.
cIEF cartridge: 50 mm, 100 μm I.D. fluorocarbon-coated capillary with built-in electrolyte tanks.
Microcentrifuge.
8 M urea after reconstitution with 16 mL of purified water (see Note 8).
Electrolyte kit.
Time transfer measurement (TTM) solution kit.
Freshly prepared pharmalyte mix, consisting of the following one or several kinds:
pH 5–8 pharmalytes.
pH 8–10.5 pharmalytes.
pH 3–10 pharmalytes.
1% methyl cellulose.
0.5% methyl cellulose.
pI 7.05 marker and pI 9.77 marker (see Note 9).

Procedure:
Preparation of ADC Sample and Blank
1. Dilute ADC sample to 0.5 mg/mL with water.
2. Add 50 μL of 8 M urea and 100 μL of pharmalyte mix to 100 μL of diluted ADC sample and mix well (see Note 10). The resulting sample is in the final composition of 0.35% methyl cellulose, 2% pH 5–8 , 2% pH 8–10.5 Pharmalytes, 0.4% pI 7.05 marker, 0.4% pI 9.77 marker, 1.6 M urea, and 0.2 mg/mL ADC (see Note 11).
3. Mix 100 μL of formulation buffer, 50 μL of 8 M urea, and 100 μL of pharmalyte mix to prepare a blank.
4. Centrifuge the sample and blank solutions at approximately 10,000 rpm (9500 × g) for at least 5 min, and transfer approximately 200 μL of the supernatant to an autosampler vial for analysis (see Note 12).

iCIEF Analysis
1. Set up iCE3 analysis and all the solutions following the software and instrument manual.
2. Set up a run sequence for blank and sample in the iCE software.
3. Isoelectric focusing method parameters were set as pre-focusing at 1500 V for 1 min, and focusing at 3000 V for 10 min, sample transfer time as 60 seconds, and the autosampler tray temperature is typically set to 10 °C (see Note 13).
4. Run the iCE3 analyzer.
5. Process the images in the iCE software by identifying the lower and upper pI markers and calibrating the pI scale in all electropherograms following the user’s manual.

Note:
1. The urea solution needs to be prepared freshly and kept away from heat to avoid thermal degradation.
2. pI markers should be selected based on the pI of ADCs. All peaks of interest from ADC samples should be bracketed by the pI markers. Two pI markers are needed for calibration of the pI scale of the electropherogram.
3. Urea is used as an additive to prevent precipitation and aggregation of ADCs during focusing.
4. It is recommended that the protein concentration be 0.2 mg/mL, as a high protein concentration can overload the capillary and cause poor resolution, while a low protein concentration can result in a low signal.
5. Try to avoid bubble formation during sample preparation and transfer, as the bubbles could interfere with focusing and separation.
6. It is recommended to evaluate and optimize the focusing time based on the properties of ADC samples.

For Research Use Only. NOT FOR CLINICAL USE.