Journal of Physical Chemistry & Biophysics
Open Access

Biophysical Approaches: A Revolutionary in Protein-Based Biomedical Device Identification

John Luke^{* *}Correspondence: John Luke, Department of Biophysics, K.N. Toosi University of Technology, Tehran, Iran, Email:

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Description

The sustainable community for a biopharmaceutical drug substance is essential for ensuring a long shelf life for the drug. Optimized formulation screening is the first step in the development process. Among the most critical factors for sustaining potency and activity involve protein conformation (tertiary or three-dimensional structure), folding (secondary structure), and proper subunit interaction (quaternary structure). Higher-Order Structure (HOS) is the collective term for these structures, which can be heavily modified by the formulation environment of a protein medicinal product. However, due to time restrictions, the complexity of most conventional approaches, and the inherent lack of sensitivity of some biophysical methods to small structural changes, analytical monitoring of protein conformation is typically excluded from preformulation. When standard biophysical tools are combined with more sensitive orthogonal methodologies, a full understanding of HOS in biopharmaceutical formulation can be obtained.

Normalization and conceptualization of therapeutic proteins against physical instability, both structural alterations as well as aggregation, seems to be particularly difficult due to each protein's unique physicochemical characteristics, as well as their susceptibility to the surrounding milieu (pH, ionic strength, excipients, etc.) and various environmental stress (temperature, agitation, lyophilization, etc.). By allowing a more quick review of a huge matrix of possible combinations, high-throughput approaches can considerably aid in the evaluation of stabilizing solution conditions. The utility of several high-throughput biophysical approaches to aid in protein formulation development and decrease the occurrence of important physical degradation pathways found for protein-based therapeutics in this discussion. Therapeutic protein candidates of various sizes, shapes, and physicochemical qualities were used to investigate various analytical problems in protein physical instability monitoring. The following a case study, these include an IgG2 monoclonal antibody, an albumin-fusion protein, a recombinant pentameric plasma glycoprotein, and an antibody fragment.

For instance, the physical stability of the pentameric rhPTX-2 protein was studied. To explore coagulation under different pH and temperature conditions, a complete biophysical evaluation of the rhPTX-2 was performed using Circular Dichroism (CD), intrinsic tryptophan fluorescence, extrinsic fluorescence, and optical density measurement at 350 nm. The results of the various procedures were combined into an Empirical Phase Diagram (EPD), which made it easier to see how the structure of the protein changed as a function of pH and temperature. Thermal stability of the protein was discovered. Partially unfolded and aggregated states were only detected at temperatures over 75 C-80 °C. Notwithstanding the protein's remarkable structural stability, soluble aggregates were discovered when it was stored under accelerated conditions at 65°C for several days.

Several researchers investigated the impact of ionic strength on the physical and chemical stability of an immunoglobulin Fragment Antigen Binding (FAB). First, the conformational stability of the Fab was investigated using several biophysical approaches as a function of temperature at various salt concentrations (0, 0.02, 0.2, 0.5, 1, and 2 M). The overall structural stability of the Fab was assessed using Differential Scanning Calorimetry (DSC) at various salt concentrations. At lower NaCl concentrations, the Fab exhibits two thermal transitions. Differential Scanning Calorimetry (DSC) revealed a single significant peak as the salt concentration was raised. Despite increased salt concentrations, the transition temperature climbed from 72°C to 80°C, and the overall area of the DSC thermograms increased significantly, demonstrating that the conformational stability of the Fab increased. The thermally induced aggregation behaviour of the protein was then assessed by measuring the change in optical density at 350 nm at varied salt concentrations. With increasing salt content from 0 to 2 M NaCl, the onset time was shown to decrease and then increase. However, as the NaCl content was increased, the extent of turbidity development reduced. These findings revealed that as salt concentrations increased after thermal stress, the degree of Fab aggregation decreased.

Conclusion

Physical techniques are used to explore and characterise biological systems. Such methods might be regarded as embracing a variety of disciplines. Circular Dichroism (CD), Fourier-Transform Infrared (FTIR) spectroscopy, Differential Scanning Calorimetry (DSC), intrinsic and extrinsic fluorescence, Dynamic Light Scattering (DLS), and Sedimentation Velocity Analytical Ultra Centrifugation (SVAUC) are some of the spectroscopic, thermodynamic, and hydrodynamic techniques used to elucidate three-dimensional protein structure. Such investigations can provide important details regarding protein molecules' secondary, tertiary, and quaternary structures, as well as evidence of their conformational stability (e.g., monoclonal antibodies).