Drug Loading and Distribution of ADCs by Mass Spectrometry

Enhanced analytical and bioanalytical characterization of antibody-drug conjugates (ADCs) through the assessment of critical quality attributes (CQAs) is critical for their development and optimization. High-resolution native mass spectrometry (MS) is a widely used analytical tool that not only provides accurate mass measurements of intact ADCs (within 30 ppm) but can also be used in parallel with hydrophobic interaction chromatography (HIC) to yield drug loading distributions (DLD) and the average drug-to-antibody ratio (DAR). For unconjugated mAbs and smaller mAb fragments, ADC profiles can be simplified by reduction or IdeS digestion for the more straightforward RP-HPLC-MS analysis of their subunits.

This article takes Brentuximab vedotin as an example to describe an orthogonal method in which LC-MS following ADC reduction or IdeS (fabricator) digestion and reduction was used to measure drug load distribution (DLD) and average drug-to-antibody ratio (DAR), aiming to provide researchers with a faster and more efficient method to develop and optimize ADCs.

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.

• ADC Sample Preparation

Material:
Brentuximab vedotin
Dithiothreitol (DTT)
Ethylenediamine tetraacetic acid (EDTA)
Tris–HCl (Trizma Base)
Acetic Acid 90%.
6 M reduction buffer at pH 8 with Tris-HCl, EDTA, and guanidine HCl
DTT Reducing Reagent

Procedure:
You can choose either of the following two methods:

A. Reduction
1. Place 25 μg of the ADC sample into an Eppendorf tube.
2. Use a reduction buffer to dilute the sample to a volume of 23.5 μL.
3. Add 1.5 μL of 500 mM DTT. The ADC concentration should be 1 mg/mL, and the DTT concentration should be 30 mM.
4. Place the Eppendorf tube in the thermomixer and incubate for 45 minutes at 56 °C with agitation at 750 rpm.
5. Stop the reaction by adding 1 μL of acetic acid.

B. IdeS Digestion and Reduction
1. Place 25 μg of the ADC sample into an Eppendorf tube.
2. Add 1.25 μL of fabricator (IdeS, see Note 1) and complete to 10 μL with MilliQ Water.
3. Incubate for 30 minutes at 37 °C with 750 tr/min.
4. Use a reduction buffer to dilute the sample to a volume of 23.5 μL.
5. Add 1.5 μL of 500 mM DTT. The ADC concentration should be 1 mg/mL, and the DTT concentration should be 30 mM.
6. Incubate for 45 minutes at 56 °C with 750 tr/min.
7. Stop the reaction by adding 1 μL of acetic acid.

Note:
1. FabRICATOR (IdeS) is a cysteine protease that digests antibodies at a specific site below the hinge, generating a homogenous pool of F(ab’)2 and Fc/2 fragments for human IgG1-4, IgG from monkey, rat, rabbit, and sheep.

• LC-MS Analysis

Material:
Acetonitrile.
Trifluoroacetic Acid (TFA)
Ultra-Performance Liquid Chromatography (UPLC)
UPLC Column
Mobile Phases
Eluting Solution A: MilliQ Water +0.05% TFA
Eluting Solution B: Acetonitrile +0.05% TFA

Procedure:
1. Let 70% solvent A run through it for 10 minutes at a flow rate of 0.5 mL/min to equilibrate the column.
2. Set up the mass spectrometer parameters:

• The voltage applied to the capillary cap is about 3000 V.
• Ions Scan range: 1000–5000 m/z
• Source and desolvation temperatures: 150 and 300 °C
• Cone and aperture voltages: 120 and 40 V
• Nitrogen gas flow rates for the cone: 50 L/h
• Nitrogen gas flow rates for desolvation: 800 L/h

3. The ADC sample preparation (8 μL) from the above two procedures was injected into the column for analysis.
4. Linear gradient elution (0.5 mL/min):

• Elute from 30% B to 45% B in 13 minutes.
• Change solvent B from 45% B to 95% B within 2 minutes.
• Switch from 95% B to 30% B within 2 minutes, followed by a 10-minutes equilibration period at 30% B.

5. Calculate the payload distribution and average DAR from the LC-UV chromatogram (see Notes 2 and 3).

• DAR(L) = Σ[nALn/ΣAL] (in principle, from n = 0 to n = 1).
• DAR(H) = Σ[nAHn/ΣAH] (in principle, from n = 0 to n = 3).
• Av.DAR = 2 × [DAR (L) + DAR (H)].

Note:
2. The payload distribution (i.e., % of each fragment) calculated from the LC-UV chromatogram should be within ±5 around the following values: L0 = 45%; L1 = 55%; H0 = 18%; H1 = 32%; H2 = 30%; H3 = 20%.

• L0, L1, H0, H1, H2, and H3 (reduced ADC).
• L0, L1, Fd0, Fd1, Fd2, and Fd3 (IdeS digested and reduced ADC).

3. The calculated average DAR of the control ADC, brentuximab vedotin, should be 4 ± 0.5.

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For Research Use Only. NOT FOR CLINICAL USE.