Causes of Industrial Protein A Column Degradation, Explored Using Raman Spectroscopy.

Area of Science:

• Biopharmaceutical Manufacturing
• Analytical Chemistry
• Spectroscopy

Background:

• Monoclonal antibodies (mAbs) are critical biotherapeutics, but their production is costly.
• Protein A affinity capture is the most expensive step, with resin capacity decreasing over repeated use.
• Previous studies suggested fouling, not ligand degradation, causes this capacity loss.

Purpose of the Study:

• To investigate the cause of protein A resin capacity loss using Raman spectroscopy.
• To characterize the binding behavior and spectral fingerprint of mAbs on protein A resin.
• To identify foulants responsible for reduced binding capacity.

Main Methods:

• In situ ATR-FTIR spectroscopy (previous studies).
• Raman spectroscopy for binding behavior and capacity loss analysis.
• Partial least-squares regression for data analysis.
• Mass spectrometry for foulant identification.

Main Results:

• A distinct Raman spectral fingerprint for mAb bound to protein A ligand was identified.
• Raman spectroscopy coupled with PLS regression can discern reduced static binding capacity (SBC) in used resin.
• Used resin showed significantly lower SBC (35.76 mg mL⁻¹ inlet) compared to unused resin (70.35 mg mL⁻¹).
• Depth profiling revealed inhomogeneous mAb binding in used beads, concentrating on outer regions.
• Irreversibly bound mAb and host cell proteins were identified as major foulants.

Conclusions:

• Resin fouling, specifically by irreversibly bound mAb and host cell proteins, is the primary cause of capacity loss in protein A chromatography.
• Raman spectroscopy is a viable method for assessing resin condition and identifying foulants.
• Understanding fouling mechanisms can optimize biomanufacturing processes and reduce costs.