An insight into the (un)stable protein formulation

Significance of the Topic

Understanding the stability and structural integrity of therapeutic proteins is critical in pharmaceutical development. Protein formulations must maintain desired activity, avoid aggregation, and assure safety during storage and administration. FTIR spectroscopy provides an early and sensitive means to monitor conformational changes, enabling rapid optimization of buffer composition, pH, temperature, and excipient selection.

Objectives and Study Overview

This application note illustrates how FTIR spectroscopy can detect subtle alterations in protein secondary structure under varying formulation conditions. It focuses on routine screening for stability, identification of unfolding or aggregation events, and quantitative assessment of structural changes during temperature-driven stress tests.

Methodology and Instrumentation

Proteins are prepared in aqueous buffers or as lyophilized powders with sugars, polyalcohols, or amino acids. FTIR spectra are recorded in transmission mode using a CONFOCHECK FTIR system. Second-derivative analysis of the amide I region (1,600–1,700 cm⁻¹) enhances resolution of α-helix, β-sheet, and random coil features. Chemometric methods, such as Partial Least Squares regression, compare unknown spectra to reference datasets for secondary structure quantification.

Main Results and Discussion

• Comparison of myoglobin (∼74% α-helix) and concanavalin A (∼64% β-sheet) demonstrates distinct amide I profiles, confirming the method’s sensitivity to extreme structural compositions.
• Heat-stress experiments on antibody fragments at 70 °C over 3 min versus 60 min reveal increasing β-sheet formation, indicated by a growing peak at 1622 cm⁻¹. Second-derivative spectra and difference plots quantify the denaturation progression.
• FTIR detects conformational shifts prior to visible aggregation, facilitating early decision-making in formulation development.

Benefits and Practical Applications

• Rapid screening of multiple formulation parameters (pH, excipients, storage conditions).
• Early detection of unfolding or aggregation, reducing reliance on chromatography-based assays.
• Quantitative analysis of structural changes supports regulatory documentation and comparability studies.

Future Trends and Potential Applications

Advances in high-throughput FTIR platforms and integration with machine learning could enable automated formulation screening at industrial scale. Emerging attenuated total reflectance (ATR) accessories and microfluidic sampling promise minimal sample use and real-time monitoring. Combined spectroscopic approaches may further elucidate multi-parameter stability landscapes.

Conclusion

FTIR spectroscopy, highlighted through the CONFOCHECK system, offers a powerful, sensitive, and quantitative approach to monitor protein secondary structure in diverse formulation contexts. Implementing FTIR early in development accelerates optimization and increases confidence in product stability.

Instrumentation Used

FTIR system: CONFOCHECK by Bruker Optics, equipped for transmission measurements and second-derivative spectral processing.

Reference

Application Note AN B401: An insight into the (un)stable protein formulation