Biophysical Methods for Characterization of ADCs

ADCs are made up of three components, which are mAb, drug molecule, and linker. The addition of the linker and drug moieties during processing, as well as the hydrophobicity of the drug itself, can lead to structural changes that may affect the stability and functional profile of the conjugated molecule. Therefore, during the ADC development process, they must be fully characterized to ensure safety and effectiveness.

This article details several characterization methods, such as circular dichroism, intrinsic fluorescence, ANS fluorescence, differential scanning calorimetry, and dynamic scanning fluorimetry, to determine the biophysical characteristics of the ADC, aiming to help researchers better understand and optimize the properties of these important therapeutic molecules.

See our ADC biochemical analysis capabilities:

• ADC structural analysis
• ADC stability analysis
• Drug to antibody ratio (DAR) evaluation
• Drug load distribution assessment
• Linker and conjugation site analysis

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.

• Circular Dichroism Analysis

Material:
Circular dichroism instrument
Cuvettes

Procedure:
1. Prepare protein samples of a certain concentration according to experimental requirements (see Note 1 and Note 2).
2. Set the instrument parameter.

• For the routine CD spectra, set spectra with a bandwidth that is less than or equal to 1 nm. For the resolution of CD spectra related to phenylalanine, set spectra with a bandwidth of 0.1 nm.
• Set a scan rate of 0.2-0.5/min.

3. Data analysis was performed based on the following characteristic CD bands associated with different secondary and tertiary structures of the protein.

• α-Helixes exhibit three major absorbance bands, with two negative minima at approximately 208 and 222 nm and a positive maxima between 190 and 195 nm.
• β-Sheets are recognized by two signature peaks with negative minima between 217 and 218 nm and positive maxima between 195 and 197 nm.
• The random coil is typically represented by negative minima around 200 nm.
• Peaks in the region of 250-270 nm are attributable to phenylalanine residues.
• Peaks from 270 to 290 nm are attributable to tyrosine residues.
• Peaks from 280 to 300 nm are attributable to tryptophan residues.
• Disulfide bonds have weak, broad peaks throughout the near UV region (260-300 nm).

Note:
1. It is preferred that the total absorbance of the cuvette, buffer sample does not exceed 0.8.
2. In cases where the concentration of the protein is high or the buffer absorbs significantly in the given region of interest, it is recommended to use cuvettes of a small path length (e.g., 0.01 cm).

• Intrinsic Fluorescence Analysis

Material:
Spectrophotometer
Cuvettes

Procedure:
1. Prepare protein samples at a certain concentration according to experimental requirements (see Note 3).
2. Set the instrument parameter

• Set excitation for Trp is at 295 nm (> 95% Trp emission), with emission recorded from 300 to 400 nm.
• Set excitation for Tyr is at 280 nm, with emission recorded from 290 to 390 nm.
• Set excitation and emission slit widths are 5 nm.

3. Measure emission peak maxima as a function of temperature, pH, concentration, or ionic strength to determine the thermodynamics of denaturation.
4. Analyze the raw data using certain plotting software and compare the fluorescence spectra before and after conjugation to determine the microenvironmental changes of the tryptophan residues in the mAb.

Note:
1. Cuvettes should be cleaned thoroughly to get rid of any protein residues by thoroughly rinsing with water and soaking in a detergent, strong acid, base, or protease-based solution.

• 1-Anilinonaphthalene-6-Sulfonic Acid (ANS) Fluorescence Analysis

Material:
Spectrophotometer
Cuvettes

Procedure:
1. Prepare protein samples of a certain concentration according to experimental requirements (see Note 4).
2. Set the instrument parameter.

• Set excitation wavelengths at 280 or 372 nm to utilize for ANS-bound ADC samples.
• Set excitation and emission slit widths are 5 nm.

3. Measure emission peak maxima as a function of temperature, pH, concentration, or ionic strength to determine the thermodynamics of denaturation.
4. Analyze raw data by using graphing software and comparing the fluorescence spectra pre- and post-conjugation to determine the change in the microenvironment of the ANS bound to protein.

Note:
1. 80 μM of ANS and 0.4 mg/mL mAb are mixed thoroughly and equilibrated for half an hour before use. ANS can be added in excess to bind all the proteins present in the solution.

• Differential Scanning Calorimetry Analysis

Material:
1 M HCl, or 1 M NaOH concentrated detergent
High-throughput DSC with an autosampler
Buffer: Glycine, phosphate, acetate, sodium citrate, and sodium cacodylate buffers

Procedure:
1. Prepare protein samples at a certain concentration according to experimental requirements.
2. The protein sample is dialyzed with the corresponding buffer.
3. After dialysis, the sample is filtered through a 0.22-0.45 μm filter or centrifuged for ~ 15 min at ~ 8,000 × g at the temperature where protein is most stable (typically at 2-8 °C).
4. Set the instrument parameter.

• Set the final set point temperature to 90 to 100 °C.
• Set the scan rate to 1 °C/min.

5. Fill the sample cell to measure the exact concentration of protein.
6. Extract the raw data from either single or multiple samples and subtract the reference thermogram from the sample thermogram. Use a nonlinear square fit applying either a two-state or non-two-state model to perform peak integration.

• A two-state model is used for thermograms with cooperative transition observed represented by a single peak.
• A non-two-state model is used for thermograms, with multiple peaks or for unknown samples.

7. The denaturation onset point (T_onset), melting temperature Tm, and denaturation enthalpy (ΔH) results were obtained according to the above thermogram.

• Differential Scanning Fluorimetry Analysis

Material:
Sypro Orange stock solution
4-(dicyanovinyl)julolidine (DCVJ) stock solution
Thioflavin T stock solution
PCR thermal cycler

Procedure:
1. Prepare protein samples at a certain concentration according to experimental requirements.
2. Set the instrument parameter.

• Sypro Orange concentrate solution is diluted 2000-fold into the assay samples. After excitation at 450-490 nm, the fluorescence emissions are collected using a FAM filter (515-530 nm).
• Both DCVJ and Thioflavin T concentrated solutions are diluted 200-fold to 20 μM working concentration in the assay samples (see Note 5). Excitation set at 560-590 nm, the fluorescence emission is collected using a ROX filter (600-630 nm).

3. Add 25 μL of sample solution into each cell in triplicate on a PCR plate.
4. Use a PCR thermal cycler to monitor the fluorescence.
5. Set the sample plates subject to a temperature ramp from 5 to 95 °C at an increment of 1 °C.
6. The fluorescence data and the second derivatives obtained from the built-in software are exported.
7. Use the corresponding software to analyze the above data.

• The midpoint of the fluorescence curve of Sypro Orange is defined as the temperature of hydrophobic exposure (T_h).
• The midpoint of the DCVJ fluorescence curve is defined as the temperature of aggregation (T_agg).
• The midpoint of the Thioflavin T fluorescence curve is defined as the temperature of formation of amyloid-like fibrils.

Note:
1. Dye should be diluted in the assay samples immediately before the thermal cycle study starts to avoid a quench of fluorescence due to environmental factors.