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AAPS Interlaboratory Study

Tier 2 Method: Differential Scanning Calorimetry

Introduction

DSC is a thermoanalytical method used to measure thermal unfolding and conformational 

stability of a protein. It measures the difference in heat energy uptake by a sample relative to a 

reference during a controlled temperature change. 

This protocol describes measurement of thermal unfolding of proteins by differential scanning 

calorimetry (DSC). The result of the measurements are reported as onset temperature (Tonset) and 

melting temperature (Tm). Only the unstressed mAbs will be analyzed on this method. 

Important Notes: This protocol is not a Standard Operating Protocol and assumes the required 

instrument is in good working order and the analysis is performed by experienced user(s). 

Required calibration or check standards should be run according to the manufacturer 

instructions. This protocol does not contain all the details of the analysis. The analyst should 

rely on their best judgment, routine practices, and knowledge of the technique to conduct the 

study; this protocol should be used as a guideline for the analysis. Samples should be analyzed 

immediately after thawing and preparation.

Equipment and Materials List 

• Microcal DSC instrument appropriate for small-volume sampling (< 2 mL).

• Standard recommended by the manufacturer or Lysozyme standard (1 mg/mL).

• Protein samples (at 1 mg/mL) and formulation buffers supplied by sample originators 

o Buffers and aliquots of unstressed samples ONLY from Shipping #1 

• Appropriate dilution buffer for the mAbs

• Pipets and pipette tips as recommended by manufacturer.

• Cleaning solutions or detergents to run between samples, as recommended by the 

manufacturer 

• Particle free water/deionized ultra-filtered water
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Reagents and Solutions 

Table 1: The following samples and their buffers will be provided by sample originators. Buffers 

and aliquots of unstressed samples from Shipping #1 should be used for this analysis. They are 

nominally at 1 mg/mL. 

Proteins Formulations Samples

Amgen IgG2 10 mM sodium acetate, pH 

5.0 Unstressed, 1 mg/mL

NISTmAb IgG1

12.5 mM L-histidine, 12.5 

mMol L-histidine HCl, pH 

6.0

Unstressed, 1 mg/mL

Procedures

Instrument Set up and Sample Preparation 

1. Thaw the samples from Shipping #1 and aliquot the Tier 2 vials (for the unstressed mAb 

samples only) as described in the instructions into smaller aliquots for the various methods

and freeze until ready to use. Be sure to follow the procedure for vigorously mixing the 

sample prior to aliquoting. 

2. For this analysis, thaw the aliquots designated for DSC measurements by bringing them to 

room temperature for no more than 15 minutes. This method should only be used on the 

unstressed samples. 

3. Turn on the instrument and supply N2 to the system according to the manufacturer’s 

recommendations.

4. Ensure the cleaning solvents are filled to the required volume in their designated reservoirs. 

5. Set the temperature of the sample holding compartment to 5 ̊C to maintain the integrity of 

the sample prior to the experiment.

6. For concentration measurements, follow the recommended protocol described in the UV-Vis 

protocol. Actual concentrations are needed to normalize the data. The samples are at 

nominally 1 mg/mL so no dilution is required. A minimum sample volume of ~400uL will 

be required for each sample well. 

7. Convert the concentration of protein (mg/mL) into mM by dividing by the Molecular weight 

in kDa - i.e. Concentration in mM = (Concentration in mg/mL) / (Molecular Weight in 

kDa).

8. Degas the samples under vacuum for 30 mins to get rid of microbubbles that can cause 

volume inaccuracy. This step can be skipped for newer calorimeter models. 
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Setting up a 96-well plate for Analysis 

1. In a laminar flow biocontainment cabinet, load the samples (unstressed only) and their 

respective buffers in pairs into 96 well plates. The analysis of each sample is performed by 

comparing the thermal profile of that sample against its reference (buffer). The source well 

for each of these solutions has to be specified for each sample to be analyzed. This is the 

general sequence of runs: 1) water, 2) standard, 3) samples, and 4) a final clean. There is an 

intermediate cleaning step between each of these injections. 

2. The following runs are recommended:

a. Lysozyme at 1 mg/mL (in water) can be used as a standard for system suitability and run 

before and after every run. You may also use vendor recommended standard(s) for 

system suitability. 

b. A contrad-contrad cleaning sample set is run after every three samples, followed by two 

water sample sets to ensure all the detergent is out of the system 

c. Buffer-buffer scans verify the suitability of the instrument prior to sample measurement 

(i.e. assessment of instrumentation error) as well as establish a baseline; while water 

scans are run to clean the cells. 

3. Cover the well plate before taking the plate out of the Biosafety Cabinet to avoid sample 

contamination.

4. Place the plate in the sample holding compartment in the proper orientation.

Sample Measurements

1. Review the DSC Data template for the requested information before beginning the 

measurements and input the set-up information. 

2. Depending on the instrumentation, samples can be loaded into the cell manually or 

automatically. The autosampler is preferred. 

3. In the acquisition software, enter the sample information in the order the plate was loaded. 

Enter sample concentration values into the software. 

4. Ensure that an automated cleaning cycle is set to run between samples so that cells are 

cleaned with detergents before every sample scan, which should be followed by multiple 

water rinses steps to ensure no detergent residue is left in the cells. 

5. Ensure that the instrument parameters are set to the following as shown in Table 2 below. It 

is important to keep the scan rate at 120°C/h. Scan rates should be kept the same to obtain 

comparable data between samples. Variation in scan rate can lead to variation in the Tm and 

melting profile of the molecule of interest. 

6. Specify 25°C for Auto-Fill Cell temperature. This value should be higher than the starting 

temperature and ensures that sample analysis begins after the temperature ramp has been 

initiated. 

7. Prior to initiating analysis, ensure all solvent reservoirs are full and periodically monitor 

solvent volume during the run to ensure that there is enough volume for all of the rinse/wash 

cycles throughout the run. 

8. Initiate the run sequence. 
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Table 2: Recommended Scan Parameters

Starting Temperature (°C) 20

Final Temperature (°C) 110

Scan rate (°C/h) 120

PreScan Thermostat (min.) 5

Post Scan Thermostat (min.) 2

# of rescans 0 (for samples); 2 or more (for 

buffers being scanned for the 

first time)

Filtering period (sec.) 10

Feedback Mode/Gain Low

Post cycle Thermostat (°C) 20

Data reporting

1. Data analysis should be performed using the software provided with the instrument. 

2. Subtract baseline and buffer scans from the sample scans and normalize to the concentration 

in the units kcal/mole/oC.

3. Using the software, pick the apparent Tms of the sample. Note the Tm onset, apparent ∆H 

(enthalpy) and apparent Tms. 

4. If samples show peaks with more than one transition Non 2 state curve fitting can be used to 

model the peaks.

5. Report the curve fit Tm’s and curve fit enthalpy in the DSC Data reporting template. 

Note: Post peak aggregation can be misinterpreted as domain unfolding. In these cases 

samples can be run at low concentration (0.2 mg/ml) and compare it with the thermogram of 

high concentration (1 mg/ml). Decrease in the signal intensity of the aggregation peak 

confirms its identity.

References

1. Durowoju, I. B., Bhandal, K. S., Hu, J., Carpick, B., Kirkitadze, M. Differential Scanning 

Calorimetry-A Method for Assessing the Thermal Stability and Conformation of Protein 

Antigen. JOVE, 2017, 1-8. 

2. Malvern Instruments. Characterization of Bioparmaceutical Stability with Differential Scanning 

Calorimetry: Candidate Selection for Developability. 2017. 

Further Information

For any specific questions regarding this method, please contact Prakash Manikwar at 

prakash.manikwar@astrazeneca.com. Please copy aapsinterlab@nist.gov on your emails. 
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