PEGS: ELSD Performance on Polysorbate Quantification in Infliximab Drug Products

Importance of the topic

Polysorbate 80 (PS80) is a non-UV-active excipient widely used to stabilize therapeutic proteins due to its biocompatibility and low toxicity.
Accurate quantification at trace levels in complex biologics formulations ensures product safety, efficacy, and regulatory compliance.

Study objectives and overview

This study evaluates the performance of evaporative light scattering detection (ELSD) for quantifying intact PS80 in various infliximab drug products.
It compares baseline subtraction workflows against traditional approaches and benchmarks ELSD against charged aerosol detection (CAD).

Methodology and instrumentation

Samples of four infliximab formulations were analyzed using a trap-and-elute configuration on an Oasis Max mixed-mode column (30 µm, 2.1×20 mm) controlled by a Waters Advance Column Manager.
Under acidic conditions (water with 2% formic acid) proteins are diverted to waste, while PS80 is retained and then eluted with isopropanol containing 2% formic acid.
Chromatographic separation employed an ACQUITY UPLC H-Class PLUS Bio System, and detection used a 2424 ELSD (gain 100, power 75%, temperature 80 °C, gas 20 psi).
Data processing and automated baseline subtraction were performed in Empower 3 Software.

Main results and discussion

Applying baseline subtraction extended the dynamic range from one order (0.1–1 mg/mL) to two orders (0.01–1 mg/mL), improving limit of detection and quantitation.
The calibration curve with subtraction achieved R2 = 0.9993 vs. R2 = 0.9722 without.
PS80 levels in Remicade, Avsola, Inflectra, and Renflexis ranged 0.042–0.045 mg/mL, demonstrating RSDs below 3% for ELSD and acceptable comparison to CAD.
Baseline correction eliminated manual peak curation and improved automation reliability.

Benefits and practical applications

• Robust quantification of intact PS80 in biopharmaceutical formulations at formulation-relevant levels.
• Enhanced sensitivity and dynamic range through baseline subtraction.
• Automated data processing reduces manual intervention and potential errors.
• Compliance-ready workflows suitable for quality control laboratories.

Future trends and potential applications

• Adoption of baseline subtraction methods for other non-UV-active excipients.
• Integration with mass spectrometry for structural elucidation of surfactants.
• Implementation of ELSD-based assays in high-throughput pharmaceutical QC.
• Development of advanced aerosol detectors for improved sensitivity.

Conclusion

ELSD coupled with an optimized trap-and-elute UPLC method and baseline subtraction provides a reliable, sensitive, and automated approach for quantifying PS80 in infliximab products, offering performance comparable to CAD while simplifying data processing.

Instrumentation used

• Waters ACQUITY UPLC H-Class PLUS Bio System
• Oasis Max mixed-mode column (30 µm, 2.1×20 mm)
• Waters Advance Column Manager with 2-position 6-port valve
• 2424 Evaporative Light Scattering Detector
• Empower 3 Software

References

1. Han D, Birdsall R, Simeone J et al. Comparing ELSD and CAD Performance on Polysorbate Quantification in Infliximab Drug Products. Waters Application Note; 2022, 720007501EN.
2. McCarthy S. Quantification of Polysorbate Using the ACQUITY UPLC H-Class Bio System with ELS Detection. Waters Application Note; 2015, 720005323EN.