Characterization of the Primary Structure of Cysteine Linked ADCs by CE-ESI-MS

Capillary electrophoresis-mass spectrometry (CE-MS) enables the characterization of the primary structure of ADCs in one injection. An analytical method based on a bottom-up proteomic workflow allows in-depth insights into the amino acid sequence, glycosylation profile, and drug conjugation site on the peptide backbone.

This article takes brentuximab vedotin as an example to detail the protocol for the characterization of cysteine-linked ADC using a sheathless CE-ESI-MS instrument, aiming to provide researchers with a comprehensive guide for the analysis 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.

• ADC Sample Preparation

Material:
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
Sanitization Buffer: 0.5 N Sodium Hydroxide
Storage Buffer: 20% Ethanol

Procedure (see Note 1):
A IdeS Digestion
1. Mix 30 μL of 5 μg/μL ADC with 117.25 μL of 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.4, in a 500 μL microtube.
2. Add 2.25 μL of IdeS (see Note 2) to the above 500 μL microtube and heat at 37°C for 30 min.
3. After digestion completion, exchange sample buffer with 50 mM ammonium bicarbonate buffer, pH 7.0, using centrifugal filters 10 kDa at 10°C and 14,000g for 20 min.
4. Evaporate to dryness and reconstitute in a volume of 10 μL to obtain a final ADC concentration of 12.5 μg/μL (see Note 3).

B Trypsin Digestion
1. Mix 10 μL ADC at 12.5 μg/μL and 10 μL of 0.1% RapidGest surfactant in a 500 mL microtube, and heat the sample at 40°C for 10 min.
2. Add 1 μL DTT solution (500 mM), and then heat the sample at 80°C for 10 min.
3. Once cooled down to room temperature, add 2 μL of ACN (10% (v/v)) to conserve hydrophobic drug-loaded peptides in
solution (see Note 4) .
4. Two 2 μL portions of trypsin (0.5 μg/μL) are added sequentially, and the samples are left at room temperature for 3 h in the first portion while heated at 37°C overnight in the second portion.
5. Add 1 μL DTT solution (1 M) to a final concentration of around 35 mM and heat the sample at 56°C for 45 min.
6. Once cooled down to room temperature, add 11.5 μL of IPA (40% (v/v)) (see Note 4).
7. Add 0.5 μL formic acid solution (1% (v/v)) and leave the sample at room temperature for 2 h (see Note 5) .
8. Before CE-MS analysis, mix 1 μL of ADC and 9 μL ammonium acetate 200 mM to obtain a sample with a final concentration in protein of 2.1 μM (see Note 6).

Note:
1. The procedure is dedicated to the primary structure characterization of ADCs conjugated to hinge cysteine residues. Concerning lysine-linked ADC, IdeS digestion could be removed, and other enzymes such as chymotrypsin could be evaluated.
2. IdeS digestion before trypsin digestion enables the formation of a THTCPPCPAPELLG peptide potentially loaded with 0 to 2 drugs and then facilitates the characterization of the drug-loaded peptides.
3. To achieve an ADC concentration of 12.5 μg/μL and, at the end of the digestion protocol, a final concentration of 21 μM for analysis by CE-ESI-MS, the sample volume must be evaporated to dryness and reconstituted to a volume of 10 μL.
4. Organic solvents, especially acetonitrile and 2-propanol, have been added during reduction and digestion steps for the conventional enzymatic digestion of mAbs, which can avoid hydrophobicity of drug molecules-induced precipitation of drug-loaded peptides in aqueous buffers such as ammonium bicarbonate and didn't affect the characterization of peptides and glycopeptides.
5. Rapigest is an acid-cleavable anionic detergent used to enhance the enzymatic digestion of proteins. The addition of an acid is mandatory to cleave the surfactant, promoting MS detection of peptides.
6. Dilution of the ADC sample in ammonium acetate at 200 mM, pH 4.0, modifies the sample buffer and allows the sample to be in a favorable condition to perform online pre-concentration by transient isotachophoresis during the CE-ESI-MS analysis.

• CE-ESI-MS Analysis

Material:
Sheathless CE-ESI-MS instrument
The time-of-flight (TOF) mass spectrometer is equipped with a hybrid analyzer composed of a quadrupole followed by a TOF analyzer.
Calibration solution: 8 μM digest of β-galactosidase, 200 mM ammonium acetate at a pH 4.0.

Procedure:
1. Add 10 μL of ADC sample (2.1 μM) in a microvial for analysis.
2. Apply 10 psi for 1 min to perform a hydrodynamic injection of 90 nL.
3. Separations by a voltage of +20 kV for 50 min.
4. Set ESI source parameters as follows:
ESI voltage IS 1.45 kV while Gas supplies (GS1 and GS2) are deactivated.
The source heating temperature is 150°C, and the curtain gas value is 4 (see Note 7).
The mass/charge (m/z) range is 100-2000 in MS and 50-2000 in MS/MS.
5. Calibrate all spectra using a digest of β-galactosidase.

Note:
Due to the ultra-low flow rate obtained with the sheathless CE-ESI-MS interface, curtain gas must be less than 10 to get astable spray.

• MS and MS/MS Data Analysis

Procedure:
1. Use analyst software to perform the identification of the primary structure of the ADC. The mass tolerance for search algorithm identification was set to 5 ppm for precursor ions and 0.05 Da for fragmentation ions.
2. For the missing peptides not found by the search algorithm, calculate the theoretical monoisotopic masses using a fragment ion calculator. Extract the masses found in the MS-raw data using Peakview software and validate the presence of missing peptides using MS/MS data.
3. For glycopeptide and drug-loaded peptide characterization, enter the theoretical modification in the search algorithm and perform the identification.
4. For the missing glycopeptides or drug-loaded peptides, follow the same protocol described above in step 2 with the masses of modified peptides.

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