DSC as a multipurpose biophysical technique for stability assay and higher-order structure investigation in biopharmaceutical development
Author: Shailesh Dugam (Ph.D Scholar, Institute of Chemical Technology)
The thermal stability of therapeutic proteins is a prime concern in the course of each stage of biopharmaceutical development. Changes introduced in process development and formulation can have a major impact on the stability, efficacy, and safety of protein molecules. Thus, the thermal stability of therapeutic proteins is considered an imperative factor that needs to be addressed during production, manufacturing, long term storage which endorses the safety, potency, and efficacy of the protein milieu. Higher the stability of the protein, the fewer the issues during the development process. Stabilizing conditions render a greater probability of the protein to remain in its functional state without any propensity to undergo chemical modifications and aggregation. The thermal stability assessment of protein molecules includes monitoring the unfolding of protein structure as a function of temperature. There are several techniques that have been adopted to characterize the thermal stability of proteins which include differential scanning fluorimetry, circular dichroism, etc. However, “differential scanning calorimetry (DSC) remains an unparalleled technique to assess the thermodynamic stability of proteins” [1].
DSC is the measure of the heat capacity as a function of temperature, as the protein is exposed to an increase in temperature. Protein starts to denature leading to the unfolding of protein witnessed by a change in heat capacity. At lower temperatures, when protein is predominantly in its native folded state, DSC tests are started. When temperatures rise above a certain threshold, proteins begin to unfold (T[onset]). The temperature at which 50% of protein remains in the folded and 50% in the unfolded state, is called thermal midpoint/melting temperature (Tm), where, heat capacity reaches a maximum value. Above the Tm value, proteins remain primarily in their unfolded state. The Tm value obtained from DSC studies is considered as an indicator of protein stability. However, in the real scenario, thermodynamic stability is difficult to measure in pharmaceutically relevant, multidomain proteins like monoclonal antibodies as their variable domains vary widely in the intrinsic thermodynamic stability, also the Fab fragment is most sensitive to heat treatment [2]. In such cases, the domain-specific melting temperatures and the change from dual to single domain transition are considered to analyze the stability of the protein. DSC is thus, able to characterize and quantitate the different domains, and determine the individual Tm for each transition (depicted in Figure 1). The Tm for a given fold of a protein domain and for a given set of solution conditions can be expected to remain constant. In addition to that, the various parameters (depicted in Figure 1) obtained from DSC investigation such as a change in unfolding enthalpy (ΔH, which is measured by the area under the curve), heat capacity (ΔCp, heat capacity change of unfolding), T[onset] (the temperature at which protein starts to unfold) are also used to measure the rank-ordering stability, validation of DSC data, quantitative analysis of protein unfolding, and higher order structure ‘fingerprinting’. The measured values are also used to calculate entropy (ΔS a measure of molecular disorder) and free energy (ΔG). Free energy describes the overall stability of the system, and Positive ΔG indicates, the folded protein is more stable than the unfolded protein. The free energy can be calculated using the following formula;
ΔG = ΔH –TΔS
(Wherein, ΔG = Gibbs free energy, ΔH = change in enthalpy, T = temperature, ΔS=change in entropy).
Figure 1. Schematic representation of DSC thermogram of Rituximab (RmAb)
We at NRG, ICT Mumbai use this technique for the investigation of forced degradation conditions viz., oxidation, deamidation, and glycation, in a model therapeutic trastuzumab biosimilar. Here, the results obtained from DSC data confirmed that, in the case of oxidation and glycation, the Tm1 values decreased with increasing stress, indicating that these stress conditions significantly affect the CH2 domain of the mAb and thus, did not affect the Fab fragment. While in the case of deamidation, the increase in Tm value was observed. This could be attributed to the protein–protein interactions that lead to the formation of aggregates that ultimately results in increased Tm values [3]. Additionally, we used DSC an as orthogonal technique to investigate the role of 2-methyl imidazolium dihydrogen phosphate in preventing aggregation of Bevacizumab at different pH stress conditions.
Alternate techniques such as Circular Dichroism (CD) and Differential Scanning Fluorimetry (DSF) have been used for the investigation of thermal stability. However, during investigations, these techniques are able to detect only the most dominant Tm in the case of multidomain proteins. The archival of more than one Tm from multidomain proteins using these techniques require complex data fitting, leading to irregular reproducibility of data. Also, some proteins and buffer conditions are not compatible with CD and fluorescence, which can make the calculation of Tm differences very complicated [4]. Thus, DSC serves as a multipurpose biophysical technique for stability assay and higher-order structure investigation in biopharmaceutical developments.
To learn and understand more about biophysical characterization techniques used in industry, please visit our website (https://biosimilarworkshop.com/) and participate in our Biosimilar Workshop 2023 event in Goa. This workshop will provide a great opportunity to learn about ongoing biosimilar research and interact with distinguished scientists to unravel the mystery of science. You can also get a chance to present a poster of your research work by submitting your abstract and can win awards from different sponsors of #Biosimilarworkshop2023.
References:
1. Gokarn, Y., et al., Biophysical techniques for characterizing the higher order structure and interactions of monoclonal antibodies, in State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 2. Biopharmaceutical Characterization: The NISTmAb Case Study. 2015, ACS Publications. p. 285–327.
2. Vermeer, A.W. and W. Norde, The thermal stability of immunoglobulin: unfolding and aggregation of a multi-domain protein. Biophysical journal, 2000. 78(1): p. 394–404.
3. Kamble, R., et al., Characterization of outcomes of amino acid modifications using a combinatorial approach to reveal physical and structural perturbations: A case study using trastuzumab biosimilar. Journal of Chromatography B, 2022. 1209: p. 123430.
4. Yatin Gokarn, 1 Sanjeev Agarwal,1 Kelly Arthur,2, et al., Biophysical Techniques for Characterizing the Higher Order Structure and Interactions of Monoclonal Antibodies American Chemical Society, 2016. 2: p. 286–327.
