Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins Using FTIR

Importance of the Topic

Protein aggregation during the production and storage of monoclonal antibodies can undermine safety and efficacy of biologic therapeutics. Rapid, nondestructive monitoring of aggregated forms in high concentration formulations is critical for quality control and process development. Fourier transform infrared spectroscopy offers a direct approach to track structural changes associated with aggregation without extensive sample preparation.

Objectives and Study Overview

This application note describes the use of the Agilent Cary 630 FTIR spectrometer to monitor thermal induced aggregation of innovator and biosimilar rituximab at high concentration. Key goals include determining melting temperatures by amide band shifts, comparing aggregation kinetics with and without formulation buffer, and demonstrating the utility of the Cary 630 platform and MicroLab Expert software for biopharmaceutical analysis.

Methodology and Instrumentation

• Instrumentation
  • Agilent Cary 630 FTIR spectrometer with single reflection diamond ATR module
  • Data acquisition via MicroLab Expert software
• Experimental parameters
  • Spectral range 4000 to 650 per cm
  • Resolution 4 per cm with 140 background and sample scans
  • Triangular apodization and ATR sampling
• Sample preparation
  • Innovator and biosimilar rituximab concentrated to 50 mg per mL using centrifugal concentrators
  • Formulation buffer composed of citrate, sodium chloride, and polysorbate 80 at pH 6.5
• Aggregation workflow
  1. Determine protein concentration by UV absorbance using Cary 60 UV Vis
  2. Thermal stress incubation from 20 to 90 degrees C for 15 minutes
  3. ATR FTIR measurement of 10 microL aliquots
  4. Aggregation kinetics at 60 degrees C with timepoints up to 4 hours
  5. Spectral processing blank subtraction, normalization and second derivative analysis

Main Results and Discussion

• Thermal denaturation revealed a shift of the Amide I band from 1638 to 1616 per cm upon aggregation, reflecting conversion from intramolecular to intermolecular beta sheet structures.
• Melting temperatures estimated from the ratio of absorbance at 1616 and 1638 per cm were 70.2 C for the innovator and 71.8 C for the biosimilar, in agreement with literature values.
• Kinetic analysis at 60 C followed first order behavior. Rate constants in formulation buffer were lower and half life values higher than in water, indicating stabilization by excipients.
• Comparative kinetics showed similar destabilization under non-buffer conditions, while the biosimilar exhibited greater stability in the presence of formulation buffer.

Benefits and Practical Applications

• Rapid label free assessment of aggregation in high concentration mAb samples without dilution artifacts.
• Minimal sample preparation and intuitive software guidance reduce operator training and error.
• Orthogonal data to chromatographic techniques, suitable for in process quality control and biosimilarity assessment.
• Flexibility to analyze solids, liquids, and gases for identification and quantitation tasks in pharmaceutical development.

Future Trends and Potential Applications

• Integration of FTIR with advanced chemometric methods for multi attribute monitoring of biotherapeutics.
• Real time aggregation tracking in continuous manufacturing using in line ATR probes.
• Expansion of spectral imaging techniques to map spatial distribution of aggregates in formulations.
• Development of standardized spectral libraries to support rapid identification of protein variants and degradation products.

Conclusion

The Agilent Cary 630 FTIR spectrometer with MicroLab Expert software provides a robust user friendly platform for monitoring monoclonal antibody aggregation at high concentration. Thermal denaturation and kinetic studies demonstrated reliable determination of melting points and buffer dependent stabilization, confirming FTIR as a valuable tool for quality control and process optimization in biopharmaceutical manufacturing.

References

• Carpenter J F et al Overlooking Subvisible Particles in Therapeutic Protein Products J Pharm Sci 98 4 201 205 2009
• Tiernan H Byrne B Kazarian S G ART FTIR Spectroscopy And Spectroscopic Imaging for the Analysis of Biopharmaceuticals Spectrochim Acta A Mol Biomol Spectrosc 241 118636 2020
• Costantino H R et al Fourier Transform Infrared Spectroscopic Analysis of the Secondary Structure of Recombinant Humanized Immunoglobulin G Pharmacy and Pharmacology Comm 3 3 121 128 1997
• Baird G et al FTIR Spectroscopy Detects Intermolecular beta Sheet Formation Protein J 39 4 318 327 2020
• Flores Ortiz L F et al Physicochemical Properties of Rituximab J Liquid Chromatography Related Technologies 37 10 1438 1452 2014
• Byler D M et al Effect of Sucrose on the Thermal Denaturation of a Protein AIP Conference Proceedings 430 1 332 335 1998
• Ma H O Fagain C O Kennedy R Antibody Stability a Key to Performance Analysis Influences and Improvement Biochimie 177 213 225 2020