Physical Stability Studies of ADCs

Identification of key product quality attributes is an integral step in therapeutic antibody development. Heterogeneous ADCs with different physical stabilities are generated when cytotoxic drugs are combined with antibodies. Furthermore, the conjugation of the cytotoxic small molecule drugs to antibodies results in a species that has an overall higher hydrophobic character as compared to the unconjugated parent antibodies. These characteristics determine that ADC is a physically unstable protein. In order to develop effective and safe ADCs, appropriate forced degradation studies must be performed to understand their manufacturability, and formulation development, assess comparability between batches, develop analytical methods, and understand degradation pathways.

This article details a series of indicative analytical methods and tools for determining the thermodynamic stability of ADCs, aiming to provide researchers with comprehensive guidance for ADC stability analysis.

See our ADC analytics services:

ADC In Vivo Analysis

• ADC Pharmacokinetics Characterization
• ADC Safety Assessment
• ADC In Vivo Efficacy Evaluation
• ADC Immunogenicity Analysis

ADC In Vitro Analysis

• Biochemical Analysis
• In Vitro Efficacy Evaluation
• 3D In Vitro

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.

• Elevated Temperature Stress Analysis

Material:
Protein samples (antibody or ADC)

Procedure:
1. Formulating ADC samples in a high ionic strength buffer.
2. Aliquot-formulated antibody sample into glass vials.
3. Store the above glass vials at 30 or 40 °C for up to 8 weeks with appropriate light protection, while the unstressed control (T = 0) sample is at -70 °C for future analysis.
4. Pull out samples from stress temperature storage based on the desired time points and test immediately or freeze at -70 °C until analysis.

• Protein Concentration Measurement

Material:
Protein samples (antibody or ADC)

UV-Vis spectrophotometer

Procedure:
1. Dilute the protein sample with formulation buffer or water so that the absorbance maximum around 280 nm is between 0.5 and 1.0 absorbance units. (see Note 1).
2. Use a cuvette to load samples with an appropriate sample volume, and make sure to fill the cuvette above the light path window with the sample.
3. Use a spectrophotometer with a diode array instrument to measure protein concentration.
4. Calculate protein concentration using the equation below (Eq. 1).

Note:
1. If the protein sample's UV absorbance is between 0.5 and 1 absorbance unit, dilution is not necessary.

• Differential Scanning Calorimetry (DSC) Analysis

Material:
Differential thermal analyzer

Procedure:
1. Prepare a minimum of 500 μL of protein sample at 1 mg/mL protein concentration diluted in formulation buffer.
2. Prepare a reference solution and make sure it exactly matches the protein sample dilution buffer.
3. Load samples on a 96-well plate, and use one protein sample, one reference solution, and two sets of reference solutions to subtract background noise from the instrument.
4. Scan each sample-buffer pair over the temperature range 15-95 °C at a rate of 1 °C per minute.
5. Analyze the data and determine Tm onset and other thermal transition temperatures using Origin software.

• Size Exclusion Chromatography High-Performance Liquid Chromatography (SE-HPLC) Analysis

Material:
SE-HPLC stationary phase (column): 7.8 mm × 30 cm, 5 μm particle size column.
SE-HPLC mobile phase: 0.2 M potassium phosphate and 0.25 M potassium chloride, pH 6.95.
HPLC with a multiple wavelength detector.

Procedure:
1. Equilibrate the stationary phase (column) with the mobile phase, and maintain ambient column temperature and 2-8 °C autosampler temperature.
2. Load samples into SE-HPLC vials and inject samples using the autosampler. Employ isocratic elution at a flow rate of 0.5 mL/min for a minimum run time of 30 min.
3. Analyze the data using a suitable electronic integrator or computer system.
4. Integrate protein-related peaks and classify them as HMWS, monomer, and LMWS, and report the percentage of peak areas for the HMWS, main peak, and LMWS.
5. Use the following equation to calculate the percent peak area (Eq. 2):

• Hydrophobic Interaction Chromatography (HIC) Analysis

Material:
HIC mobile phase A: 1.5 M ammonium sulfate, 25 mM sodium phosphate, pH 6.95.
HIC mobile phase B: 25 mM sodium phosphate, pH 6.95, with 25% (v/v) IPA.
HIC stationary phase (column): butyl-NPR 4.6 mm ID × 3.5 cm, 2.5 μm or equivalent.
HPLC system with diode array detector.

Procedure:
1. Equilibrate the stationary phase with buffer A, and set the column temperature at 25 °C during equilibration and throughout the chromatography run.
2. Inject a maximum of 5-10 μL (50-100 μg) of neat protein sample onto the column using the autosampler.
3. Run a linear gradient from 100% mobile phase A to 100% mobile phase B over 18 minutes with a flow rate of 0.8 mL/min.
4. Analyze the data by integrating each DAR species peak to determine the percent peak area of peaks of interest.
5. Calculate the percentage peak area of each DAR species using the equation below (Eq. 3):