Unlocking Protein Stability: Insights into Lyophilized States Using Advanced Spectroscopy

A recent study published in Molecular Pharmaceutics examined the mechanisms that are behind the structural stability of protein drugs in their solid, lyophilized state. By using advanced electron paramagnetic resonance (EPR) spectroscopy, the research team, led by Wolfgang Friess of Ludwig-Maximilians-Universität München, unveiled new insights into protein folding and how it is influenced during the freeze-drying process (1). Understanding this mechanism better is poised to help pharmaceutical companies maintain the integrity of protein-based drugs.

The significance of DNA in the study of proteomics and protein interactions. Generated by AI. | Image Credit: © Masque - stock.adobe.com

A protein-based drug is considered a therapeutic protein (2), which are routinely used in modern medicine. Out of the top selling drugs on the market, protein-based drugs comprise about half of the top ten, showing that these therapeutics are becoming more mainstream in the drug market (3). However, protein-based drugs are susceptible to losing their structural integrity, especially during manufacturing and storage (1). The freeze-drying (lyophilization) process is widely used to improve the shelf life of these drugs; however, the mechanisms by which proteins retain their functional structure in solid form remain poorly understood (1). This issue was what Friess and his team attempted to address in their study, viewing this as a critical knowledge gap in the pharmaceutical industry.

In their study, the researchers utilized two advanced EPR techniques. These techniques, which were double electron–electron resonance (DEER) and electron spin echo envelope modulation (ESEEM), analyzed the behavior of spin-labeled human serum albumin (HSA) (1). Using these two methods allowed the team to probe interprotein distances and molecular environments with great precision.

Both techniques accomplished different objectives. DEER spectroscopy, which is capable of measuring distances up to 200 Å, revealed critical information about protein folding, aggregation, and local concentration in lyophilized states (1). Meanwhile, ESEEM spectroscopy, which examines the environment within 10 Å of the spin label, provided insights into molecular interactions in the immediate vicinity of the proteins (1).

The results highlighted the complex interplay between protein concentration, structural stability, and molecular environment. DEER spectroscopy revealed distinct local concentration patterns corresponding to different protein folding states. At an HSA concentration of 84 g/L, interprotein distances were approximately 2 nm, correlating directly with folding percentages (1).

Even at low HSA concentrations of 2.6 g/L (sucrose-to-HSA molar ratio of 7469), partial structural perturbations were detected in 50% of the molecules (1). This figure rose to 97% at higher HSA concentrations (84 g/L) (1). Despite these perturbations, no signs of irreversible unfolding were observed after reconstitution, indicating that the proteins retained their essential functionality (1).

With the other technique used, ESEEM spectroscopy, the research team demonstrated a pronounced enrichment of sucrose in the immediate vicinity of the protein spin label, particularly at higher sucrose concentrations (1). This phenomenon was likely linked to partial unfolding detected by DEER. This observation is important because it enforces the idea that stabilizing sugars is vital in preserving protein structure during lyophilization (1).

The research team also showed in their study that EPR spectroscopy can be used in conjunction with other analytical techniques. For example, they demonstrated that EPR spectroscopy works well with small-angle neutron scattering (SANS), Fourier-transform infrared spectroscopy (FT-IR), and solid-state nuclear magnetic resonance (ssNMR) (1). By offering unique insights into molecular interactions and protein behavior, EPR expands the toolkit available for studying the solid-state properties of protein drugs.

As the demand for protein-based therapeutics continues to grow, understanding and controlling the stability of these drugs is more critical than ever. Wolfgang Friess and his team provided more information into how scientists can better keep these drugs stable. Because protein-based drugs are becoming more popular, this research has significant implications for future studies in this space, as researchers continue to help improve drug efficacy.

References

1. Isaev, N.; Lo Presti, K.; Frieß, W. Insights into Folding and Molecular Environment of Lyophilized Proteins Using Pulsed Electron Paramagnetic Resonance Spectroscopy. Mol. Pharmaceutics 2025, 22 (1), 424–432. DOI: 10.1021/acs.molpharmaceut.4c01008
2. Amgen, Therapeutic Protein. Amgen. Available at: https://www.amgen.com/stories/2018/08/the-shape-of-drugs-to-come/therapeutic-protein#:~:text=Technically%2C%20any%20protein%2Dbased%20drug,into%20a%20three%2Ddimensional%20shape. (accessed 2025-01-15).
3. Ebrahimi, S. B.; Samanta, D. Engineering Protein-based Therapeutics Through Structural and Chemical Design. Nat. Commun. 2023, 14, 2411. DOI:10.1038/s41467-023-38039-x