Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins Using FTIR

Significance of the Topic

Protein aggregation during the manufacturing and storage of therapeutic monoclonal antibodies (mAbs) can compromise efficacy, safety, and immunogenicity. Rapid, non-destructive methods capable of monitoring aggregation at high protein concentrations are essential for process control and quality assurance.

Objectives and Study Overview

This study compared thermal-induced aggregation of innovator and biosimilar rituximab at high concentration using FTIR spectroscopy. Key aims were to determine denaturation temperatures, assess aggregation kinetics, and evaluate the stabilizing effect of formulation buffer.

Methodology and Instrumentation

Samples of innovator and biosimilar rituximab (∼50 mg/mL) were incubated at temperatures from 20 °C to 90 °C for 15 minutes. FTIR spectra were recorded on an Agilent Cary 630 FTIR with single-reflection diamond ATR module. Data acquisition and processing used Agilent MicroLab Expert software. Protein concentrations were confirmed by UV-Vis absorbance (Cary 60). Aggregation kinetics at 60 °C were followed by measuring amide I band absorbance ratios over 0.5 to 4 hours.

Main Results and Discussion

FTIR spectra showed the amide I peak shifting from 1 638 cm⁻¹ to 1 616 cm⁻¹ with increasing temperature, indicating intermolecular β-sheet formation. Melting temperatures (Tm) derived from absorbance ratio crossovers were 70.2 °C for innovator and 71.8 °C for biosimilar, in agreement with DSC literature values. Aggregation kinetics at 60 °C followed first-order behavior. Without buffer, innovator k₁=0.0611 h⁻¹ (t₁/₂=11.3 h) and biosimilar k₁=0.0681 h⁻¹ (t₁/₂=10.2 h). Inclusion of citrate-based formulation buffer reduced k₁ and extended t₁/₂ (innovator t₁/₂=14.3 h; biosimilar t₁/₂=21.5 h), demonstrating enhanced thermal stability.

Benefits and Practical Applications

• Rapid, label-free detection of mAb aggregation without sample dilution
• High sensitivity in concentrated formulations
• Suitable for in-process monitoring and quality control during biosimilar comparability studies
• Facilitates formulation and process optimization by assessing excipient effects

Future Trends and Potential Applications

Advancements may include integration of FTIR into process analytical technology (PAT) for real-time monitoring, application of multivariate data analysis for early aggregation detection, and expanded use in screening novel excipient systems. Coupling FTIR with complementary techniques (e.g., DSC, light scattering) could yield comprehensive stability profiles.

Conclusion

The Agilent Cary 630 FTIR spectrometer provides a robust, user-friendly platform for quantifying mAb aggregation under thermal stress at high concentrations. It delivers reliable denaturation temperatures and kinetic parameters, and effectively evaluates formulation buffers to improve protein stability. This approach supports quality control and process development for both innovator and biosimilar therapeutic proteins.

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