Differential Scanning Calorimetry — A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

The overall goal of this procedure is to assess the thermal stability and structural conformation of proteins in an industrial setting using differential scanning calorimetry. Differential scanning calorimetry measures the molar heat capacity of samples as a function of temperature and has been successfully used to assess the thermal stability and structural conformation of proteins. This relatively simple procedure does not depend on structural helicity or intrinsic fluorophores as it is the case for other biophysical methods.

Another advantage of this technique is that it directly measures the thermal transition temperature and the energy required to disrupt the interaction stabilizing the tertiary structure of proteins. When used in conjunction with the enthralpy of unfolding, the thermal transition temperature can serve as a useful parameter to monitor lot-to-lot consistency of manufacturing processes for biologics. Visual demonstration of this method can serve as an interactive medium to effectively assist the new users with critical steps.

To begin, switch on the differential scanning calorimeter. Then supply nitrogen into the system. This will increase the pressure in the cells to suppress boiling of the samples as well as prevent the formation of bubbles at elevated temperatures.

Depending on the constituting material of the cell, adjust the pressure of the nitrogen gas supply according to the manufacturer's recommended pressure to avoid damaging the cell. Ensure that all the cleaning agent reservoirs are filled to the required volume. Required cleaning agents include detergent to wash the cell and water to clean the cell after each sample run.

Set the temperature of the sample holding compartment to a suitable value preferably five degrees Celsius to maintain the integrity of the sample prior to the experiment. It is important to equilibrate the sample against the buffer to ensure that the only difference between the solutions is the protein. Therefore, the observed difference in heat capacity can be correctly attributed to the protein.

Determine the concentration of the protein sample using a suitable protein concentration determination method such as the Lowry method. For the instrument used in this protocol, the preferable working range is 0.5 to one milligram of protein per milliliter. Dialyze the sample against the buffer that will be used as the reference for the experiment.

Degas the sample and reference buffer in a vacuum to get rid of micro bubbles that can cause volume inaccuracy. Working in a laminar flow biocontainment cabinet, use a micropipette and sterile tips to load the samples in their respective buffer in pairs into 96 well plates. Fill the first two pairs of wells with buffer to perform buffer-buffer scans to verify the suitability of the instrument prior to sample measurement.

Fill the last two pairs of wells with water for the water scan to clean the cells. Then cover the 96 well plate with sealing film. Ensure that the wells are properly sealed prior to taking the plate out of the biosafety cabinet to avoid sample contamination.

Finally, place the plate in the sample holding compartment in the proper orientation. Using the acquisition software, enter the sample information in the order the plate was loaded. Enter protein concentrations if available.

Otherwise, enter the concentration into the analysis software prior to data analysis. Select the option that ensures cleaning of the cell with detergent before every sample scan. The cleaning should be followed by multiple water rinse steps to ensure no detergent residue is left in the cells.

Set the starting temperature of the experiment to 20 degrees Celsius which can vary depending on the sample. For known proteins, a predetermined starting temperature can be used while a lower starting temperature can be applied for unknown samples. Then set the final temperature of the experiment.

The final temperature may also vary depending on prior knowledge of the sample. Next, set the scan rate of the experiment. It is advisable to scan unknown samples at different scan rates to assess the kinetics of unfolding.

Set up the acquisition software to rescan the samples to examine the reversibility of the thermal transition. The unfolding of a protein is considered reversible if the enthalpy obtained for the second scan is at least 80%of the enthalpy value from the first scan. Set the post experiment thermostat to 10 degrees Celsius to preserve the integrity of the calorimeter cell.

Verify that the experiment setup parameters are correct before executing the experiment. If everything is in place, start the experiment. Following the experiment, perform data analysis as described in the text protocol.

For the analysis, baseline subtraction is performed manually. Consistency at this step is crucial to obtain comparable results. Shown here a representative raw data of an experimental run including the buffer and water scans.

The samples being analyzed are toxins in its native and detoxified states. The sample scans are processed separately to derive the melting temperature and enthalpy values for each sample. For the native state, the melting temperature is 55.55 degrees Celsius and the enthalpy of the toxin is 3.157 times 10 to the fifth calories per mole.

Conversely, the melting temperature of the toxin in its detoxified state is 81.21 degrees Celsius and the enthalpy is 3.656 times 10 to the fifth calories per mole. The overlay of the processed data of the toxin in its native and detoxified states demonstrates that the detoxified sample is more thermally stable that its native state according to the melting temperature values. It also indicates that the detoxification process introduces structural changes to the tertiary structure of the toxin.

While attempting this procedure, it is important to remember to provide an adequate supply of detergent and water to prevent cross contamination of the cell and the syringe as their clearness is crucial for accurate results. After watching this video, you shall have a good understanding of the differential scanning calorimetry as a method for assessing the thermal stability and conformation of proteins. You shall also be able to make meaningful deductions from resulting thermal runs.

Following this procedure, other methods like circular dichroism can be performed in order to answer additional questions regarding the changes to the secondary structures during unfolding. Data collected for an array of lots of the same product have been used to create empirical baselines to examine the impact of process changes, formulation and storage conditions on the structure conformation of protein antigens for vaccine production.