Post-Translational Modification in Biopharmaceuticals
In protein-based biopharmaceuticals, common post-translational modifications (PTMs) include glycosylation, phosphorylation, acetylation, methylation, ubiquitination, PEGylation, lipidation, and hydroxylation. These modifications are essential mechanisms by which proteins acquire functional diversity after synthesis. PTMs can significantly influence the stability, conformation, solubility, and half-life of protein drugs, as well as regulate their interactions with receptors or ligands, thereby determining therapeutic activity and efficacy. For example, glycosylation directly impacts the immune effector functions of monoclonal antibodies, PEGylation can extend the circulation time of recombinant protein drugs in vivo, and phosphorylation plays a critical role in signal transduction pathways. Thus, PTMs are not only key determinants of the quality and safety of biopharmaceuticals but also central to ensuring product consistency, functional optimization, and clinical value.
Table 1. Common PTMs in Protein Biopharmaceuticals and Their Roles
| Protein Biopharmaceuticals | Common PTMs | Roles |
| Monoclonal antibodies (mAbs) | Glycosylation | Determines ADCC/CDC effects, impacts immune activity and therapeutic efficacy |
| Recombinant protein drugs | PEGylation, lipidation | Improves pharmacokinetics, reduces dosing frequency |
| Vaccine antigens | Phosphorylation, glycosylation | Enhances immune recognition, increases immunogenicity |
| Fusion proteins | Acetylation, glycosylation | Maintains biological activity, improves adaptability |
| Therapeutic enzymes | Glycosylation, hydroxylation | Prolongs in vivo activity, enhances enzymatic performance |
| Hormones and cytokines | Phosphorylation, glycosylation | Regulates receptor binding, optimizes pharmacological effects |
PTM Characterization Methods in Biopharmaceuticals
1. Mass spectrometry (MS)
MS is at the forefront of PTM characterization, offering unmatched precision in defining modification types and sites. Tandem MS techniques such as electrospray ionization (ESI-MS) and matrix-assisted laser desorption/ionization (MALDI-MS) are widely applied for PTM identification and quantification. High-resolution MS instruments (e.g., Orbitrap, FT-ICR) enable site-specific mapping, providing deep insights into the molecular complexity of protein therapeutics.
2. Glycosylation analysis
As a critical PTM affecting protein stability and function, glycosylation is typically analyzed using hydrophilic interaction chromatography (HILIC-UHPLC), capillary electrophoresis (CE), and MALDI-TOF-MS. These methods enable glycan separation, structural characterization, and profiling, supporting reliable glycosylation consistency assessments.
3. Immunological methods
Western blotting and ELISA, which utilize modification-specific antibodies, are commonly applied for the validation of PTMs such as phosphorylation, acetylation, and ubiquitination. These approaches are straightforward and complement MS-based findings.
4. Structural characterization
Nuclear magnetic resonance (NMR), X-ray crystallography, and cryo-electron microscopy (Cryo-EM) provide insights into the effects of PTMs on overall protein conformation. These methods are particularly valuable for analyzing protein complexes and aggregates.
5. Biophysical techniques
Surface plasmon resonance (SPR) and bio-layer interferometry (BLI) can be used to study how PTMs regulate protein–ligand or protein–receptor interactions, thereby reflecting their functional impact on biopharmaceutical activity.