The Strategy of High-Resolution Characterization of ADCs by Orbitrap LC-MS

ADCs, a type of therapy aimed at targeting tumor cells, hold great promise in the field of medicine. These ADCs have complex molecular structures as they are not only composed of both small and large molecules, but they often undergo biotransformation over time in circulation, which further increases their structure complexity. For the ADC undergoing biotransformations that result in small mass changes, the traditional time-of-flight mass spectrometry (TOF-MS) lacks the necessary resolution to fully characterize the ADC. However, the use of orbitrap technology presents an exciting new opportunity for characterizing ADCs with greater resolution.

A high-resolution Orbitrap MS approach for the characterization of both intact and reduced ADCs was described in this article, particularly ADC biotransformations with small mass changes, aiming to help researchers understand the latest advancements in ADC characterization techniques.

Disclaimer

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

• Affinity Capture

Material:
96 magnetic particle processor
Streptavidin
HBS-EP buffer: 10 mM Hepes (pH 7.4), 150 mM NaCl, 3.4 mM ethylenediaminetetraacetic acid (EDTA), 0.005% surfactant P20
N-Glycanase
Elution buffer: 30% acetonitrile, 1% formic acid
Centrifuge

Procedure:
You can use the 96 magnetic particle processor to conduct affinity capture in the following steps, thus selectively extracting ADCs from plasma samples. (see Note 1)
1. In a 96-well plate, immobilize the extracellular domain (ECD) (see Note 2) of biotinylated antigen onto beads by incubating them with streptavidin for 1 hour.
2. Wash the beads with HBS-EP buffer twice, and proceed to incubate the ECD-coupled beads with plasma samples containing ADCs at room temperature for 2 hours to capture the ADCs.
3. Wash the beads twice with HBS-EP buffer, and then digest the captured ADCs on the beads using N-glycanase (10 mU per sample) at 37 °C overnight with moderate shaking (200–400 rpm) (see Note 3).
4. Wash the beads extensively with HBS-EP buffer (twice), water (twice), and 10% acetonitrile (see Note 4), and then elute ADCs using 50 μL of elution buffer (see Note 5).
5. Centrifuge the above 96-well plate at 3700 × g for 10 minutes at room temperature.
6. Put the above 96-well plate onto a magnetic platform and carefully move 60 μL of supernatant to a fresh 96-well plate (see Note 6).
7. Spin the new 96-well plate in a centrifuge at a speed of 3700 × g for a duration of 10 minutes at room temperature, then the supernatant was taken and injected onto the LC-MS for analysis.

Note:
1. It is highly recommended to use a 96-bit magnetic particle processor for affinity capture, as it allows for higher throughput and greater consistency, although it can also be done manually.
2. For nonclinical samples, biotinylated anti-human IgG can be used if ECD is not available.
3. To ensure efficient deglycosylation, N-glycanase treatment should be conducted with moderate shaking.
4. This step is very important to remove most of the nonspecific binding molecules and salts before the elution step.
5. This method is not suitable for analyzing intact ADCs where the linker-drug is conjugated to the antibody via reduced inter-chain disulfide bonds because the elution buffer, which contains 30% acetonitrile and 1% formic acid, will denature ADCs.
6. This step will help remove most of the residual beads from each sample, preventing them from clogging the LC system.

• Separation of ADCs by Reverse Phase Liquid Chromatography

Material:
Ultra-performance liquid chromatography (UPLC)
PS-DVB monolithic column (500 μm i.d. × 5 cm)
PS-DVB monolithic column (200 μm i.d. × 25 cm)
Mobile phase A: 0.1% formic acid
Mobile phase B: acetonitrile, 0.1% formic acid
Tris(2-carboxyethyl)phosphine (TCEP)

Procedure:
A. Separation of Intact ADCs
1. The intact ADC sample (5 μL) was injected and loaded onto a PS-DVB monolithic column (500 μm i.d. × 5 cm) with mobile phase A.
2. Perform chromatographic separation at 60 °C with a gradient elution (15 μL/min ) (see Note 7). The gradient is as follows:

• 0% B (0–4 min)
• 0–40% B (4–8 min)
• 40% B (8–11 min) (see Note 8)
• 40–100% B (11–12.5 min)
• 100% B (12.5–13.5 min)
• 100–0% B (13.5–14.2 min)
• 0% B (14.2–15 min)

B. Separation of Reduced ADCs
1. Following affinity capture, add 10 mM tris (2-carboxyethyl)phosphine (TCEP) to the elution buffer to reduce ADCs, and incubate the mixture at 37 °C for 40 minutes.
2. The reduced ADC sample (5 μL) was injected and loaded onto a PS-DVB monolithic column (500 μm i.d. × 25 cm) with mobile phase A.
3. Perform chromatographic separation of the ADC light chain and heavy chain at 65 °C with a gradient of elution (3 μL/min) (see Note 9). The gradient is as follows (see Note 10):

• 0% B (0–4min)
• 0–25% B (4–4.1min)
• 25% B (4.1–7min)
• 25–35% B (7–29min)
• 35–60% B (29–32min)
• 60–100% B (32–32.1min)
• 100% B (32.1–34min)
• 100–0% B (34–34.1min)
• 0% B (34.1–40min).

Note:
1. Microflow LC is strongly recommended as it enables more sensitive detection of ADCs in comparison to analytical flow LC.
2. Under these conditions, intact ADCs generally elute in approximately 9–10 minutes.
3. To achieve effective separation of light and heavy chain species, a longer column and a lower flow rate are employed.
4. The reduction of ADCs may generate various light-chain or heavy-chain species carrying different drug quantities, depending on the ADC's structure. Consequently, these reduced species usually exhibit different retention times under the specified separation conditions.

• ADC Analysis by High-Resolution Orbitrap MS

Material:
Orbitrap MS
Protein Deconvolution Software

Procedure:
1. Obtain mass spectra by employing a full MS method at a resolution setting of 17,500 (see Note 11).
2. Adjust the following key parameters:

• Set the spray voltage at 3.2 kV.
• Maintain a sheath gas flow rate of 8.
• Keep the S-lens RF level at 100.
• Utilize in-source CID at a value of 100 ev (see Note 12).
• Set the AGC target at 3 x 106.
• Set the maximum injection time at 150 ms.
• Perform 10 microscans.

3. Obtain full MS spectra from the total ion chromatograms (TICs) and conduct deconvolution using the ReSpect™ algorithm integrated into Protein Deconvolution software. Set the mass tolerance to 25 ppm.
4. Characterize ADC biotransformation and drug-to-antibody ratio (DAR) distribution (see Note 13).

Note:
1.Typically, a resolution setting of 17,500 is utilized for the analysis of complete ADCs and heavy chain species. It is recommended to set a higher resolution setting (70,000 or 140,000) when specifically analyzing light chain species.
2. Due to the fact that in-source CID might result in the fragmentation of certain linkers or drugs, it is recommended to carefully monitor any potential fragmentation of linkers or drugs that may occur when using high in-source CID energy in this process.
3. ADC biotransformations are typically characterized according to the mass differences between the newly formed and the original DAR species. The average DAR of the ADC is calculated according to the relative abundance of different DAR species.

For Research Use Only. NOT FOR CLINICAL USE.