Improved Recovery of Polysorbate 80 by Charged Aerosol Detection: Enhancing Performance with a Double Valve Configuration

Significance of the topic

Accurate quantification of polysorbates (here represented by Polysorbate 80, PS80) in monoclonal antibody (mAb) formulations is essential for monitoring product quality and formulation stability across biopharmaceutical development and manufacturing. Polysorbates are present at low concentrations relative to the protein matrix and lack strong chromophores, which complicates their analysis. Trap-and-elute workflows coupled with charged aerosol detection (CAD) provide a viable approach, but residual protein in the LC fluidics can coelute during high-organic elution and bias CAD response. This study evaluates how valve configuration in a trap-and-elute workflow impacts protein coelution and quantitative recovery of PS80, demonstrating a straightforward hardware modification to improve analytical accuracy without altering chromatographic chemistry.

Objectives and study overview

The primary objectives were to:

• Compare a conventional single-valve trap-and-elute setup to a double-valve configuration that includes an intermediate fluid-sweep step.
• Assess protein coelution using UV monitoring at 280 nm and quantify the effect on CAD-based PS80 recovery across several spike levels and mAb matrix concentrations.
• Demonstrate whether a double-valve fluidic sweep can reduce residual protein carryover and improve accuracy for low-level polysorbate analysis without changing the chromatographic method.

Methodology

Sample preparation and spike levels: A PS80 stock (20 mg/mL) was diluted to calibration standards (0.02–0.50 mg/mL). PS80 spikes at ~0.1, 0.3 and 0.5 mg/mL were prepared in two mAb matrices: NISTmAb RM 8671 (10 mg/mL) and a research-grade mAb at 50 mg/mL to challenge the trap-and-elute system.

Trap-and-elute strategy: Samples were trapped on an Oasis MAX mixed-mode column under aqueous acidic conditions which retain polysorbates while electrostatically repelling protein to waste. After trapping and a wash, elution occurred with 100% isopropanol (IPA) to transfer the retained PS80 to detectors as a single elution peak.

Comparative valve workflows: Two valve configurations were compared while keeping chromatographic conditions constant:

• Single-valve: A diverter valve directs flow to waste during trapping, then switches to route eluate through detectors during IPA elution.
• Double-valve: Two 2-position, 6-port valves (column manager) enable an additional dedicated fluid-sweep step between trapping and elution that flushes downstream fluidic paths before IPA elution.

Used instrumentation

The study used Waters instrumentation and consumables as follows:

• ACQUITY Premier System with Binary Solvent Manager (BSM)
• Charged Aerosol Detector (CAD) configured with sampling rate 5 Hz, normal time constant, ion trap 600 V, evaporator 40 °C
• Tunable UV (TUV) detector monitoring at 280 nm to detect residual protein coelution
• Oasis MAX Column, 30 µm, 2.1 × 20 mm
• ACQUITY Diverter Valve for single-valve configuration and ACQUITY UPLC Column Manager using two 2-position, 6-port valves for double-valve configuration
• Column temperature 30 °C, sample temperature 10 °C, injection volume 30 µL

Main results and discussion

UV monitoring at 280 nm provided orthogonal evidence of protein carryover into the PS80 elution window. The single-valve configuration showed a pronounced UV peak during IPA elution, indicating residual mAb presence in the fluidics that was eluted with PS80. Introducing a double-valve configuration with an intermediate fluid sweep markedly reduced the UV signal in the PS80 elution window, implying more effective removal of residual protein from unswept fluidic volumes.

CAD spike-recovery experiments quantified the practical impact on PS80 measurement. Results summarized from the study include:

• Using NISTmAb (10 mg/mL): both configurations delivered precise results (%RSD < 1%), but the double-valve recoveries were consistently closer to 100% (example: low spike—single 107.3% vs double 101.6%).
• Using concentrated mAb (50 mg/mL): the difference was more pronounced under the harsher matrix. At the lowest PS80 spike, the single-valve recovery was 112.2% while the double-valve recovery improved to 105.9%; intermediate and high spikes followed the same pattern (single: 104.1% and 101.9% vs double: 102.8% and 102.6%).

These data indicate that residual mAb in the fluidic path contributes to CAD signal bias (over-recovery), particularly at low analyte levels and high protein loads. The double-valve fluid sweep reduces unswept-volume protein release during high-organic elution and thereby improves quantitative accuracy without modifying stationary phase chemistry or elution solvent composition.

Benefits and practical applications

Key practical advantages demonstrated:

• Improved quantitative accuracy for low-level polysorbate analysis in protein-rich matrices, reducing positive bias from protein carryover.
• Reduced risk of protein-related interferences in CAD detection by minimizing residual matrix in fluidic paths.
• Implementation requires only hardware reconfiguration (addition of a second valve and programmed sweep step), preserving validated chromatographic methods and simplifying deployment in regulated environments.

These benefits are directly applicable to analytical laboratories performing QC and formulation development where reliable excipient quantitation at low levels is required in the presence of high protein concentrations.

Future trends and opportunities

Potential future directions and applications include:

• Broader application of multi-valve fluidic control to other trap-and-elute analyses (e.g., other surfactants, fatty acids, or low-level impurities) to control unswept volumes and carryover.
• Integration of automated valve sequences and condition checks into chromatography data systems for routine QC workflows and method robustness testing.
• Evaluation of optimized sweep solvents, volumes, and timing to further minimize matrix release while preserving analyte recovery.
• Combining fluidic optimization with advances in detector sensitivity and signal processing to push limits of quantitation for excipients in complex biopharmaceutical matrices.
• Regulatory and method validation considerations as hardware-based improvements are introduced into GMP workflows, including system suitability criteria that account for fluidic sweep performance.

Conclusion

The study demonstrates that valve configuration—and specifically an added fluid-sweep step enabled by a double-valve arrangement—significantly reduces protein coelution in a trap-and-elute CAD assay for PS80. This modification improves recovery accuracy, particularly at low polysorbate concentrations and under high protein loading, without changing chromatographic chemistry. For laboratories seeking to improve excipient quantitation in mAb formulations, adopting a double-valve fluidic strategy offers a practical, low-disruption approach to reduce matrix-related bias and improve analytical reliability.

References

1. Martos A.; et al. Trends on Analytical Characterization of Polysorbates and Their Degradation Products in Biopharmaceutical Formulations. Journal of Pharmaceutical Sciences. 2017;106(7):1722–1735.
2. Hewitt D.; Zhang T.; Kao T. Quantitation of Polysorbate 20 in Protein Solutions Using Mixed-mode Chromatography and Evaporative Light Scattering Detection. Journal of Chromatography A. 2008;1215(12):156–160.
3. Bigos P.; et al. Quantification of Polysorbates Using the Waters Charged Aerosol Detector. Waters Application Note. July 2026.
4. Waters Corporation. ACQUITY QDa and QDa II Detectors - Diverter Valve Configuration. Waters User Manual. 2026.