Protein structure studies for biological products are critical for understanding their biological activity, efficacy, stability, and safety. The structure of mAbs is highly complex, involving primary, secondary, tertiary, and quaternary structures that determine their function. Detailed structural characterization ensures that the monoclonal antibodies retain their intended properties throughout development, production, and clinical use, and it is essential for ensuring product quality and regulatory compliance.
Types of structural studies used to characterize monoclonal antibody (mAbs).
| S. No | Type of the Structure & Purpose | Techniques to Determine | Importance of the study |
|---|---|---|---|
| 1 | Primary Structure (Amino acid sequence confirmation) | Peptide Mapping by LC/LC-MS | Ensures that the produced product is identical to the intended design. |
| 2 | Secondary Structure (α-helix or β-sheet, random coil & amide bands) | Far UV by Circular Dichroism (CD) Spectroscopy | Proper secondary structure is essential for maintaining the functional activity of the antibody. Misfolded secondary structures can lead to aggregation and loss of bioactivity. |
| 3 | Fourier Transform Infrared (FTIR) Spectroscopy | ||
| 4 | Nuclear Magnetic Resonance (NMR) Spectroscopy | ||
| 5 | Near UV by Circular Dichroism (CD) | Near UV by Circular Dichroism (CD) | The variable regions of the antibody must adopt a specific 3D fold to ensure the proper formation of the antigen-binding site. |
| 6 | Tertiary Structure (Covalent bond, Hydrophobic interactions & 3D Folding) | Intrinsic Fluorescence (FLR) | The Fc region must also fold correctly to enable interactions with Fc receptors. |
| 7 | Disulfide linkage confirmation by LC-MS | ||
| 8 | Nuclear magnetic resonance (NMR) spectroscopy | ||
| 9 | Hydrogen-deuterium exchange (HDX)-MS | ||
| 11 | Quaternary Structure (3D Folding & Assembly of Subunits) | Nuclear magnetic resonance (NMR) spectroscopy | Ensures that the antibody is correctly assembled into a functional tetramer (two light chains and two heavy chains). Misassembly or fragmentation could lead to loss of function or immunogenicity. |
| 12 | Hydrogen-deuterium exchange (HDX)-MS |
SEC-MALS and SV-AUC are powerful techniques for determining the absolute molar mass of monoclonal antibodies (mAbs) and other biological molecules. SEC-MALS is particularly useful for analyzing size distributions and detecting aggregation in a high-throughput manner, while AUC offers deep insights into molecular weight, heterogeneity, and shape. Together, these techniques provide a comprehensive approach to understanding the structure, stability, and quality of monoclonal antibodies.