Performance Evaluation of Flownamics Sampling Devices for the Agilent Online LC

Importance of the Topic

The continuous and aseptic sampling of bioreactors coupled with online chromatographic analysis is essential for real-time monitoring and control of biologic production processes such as monoclonal antibody manufacture. Integrating automated sampling modules with an Online LC platform minimizes manual interventions, ensures consistent sample quality, and supports efficient process optimization.

Objectives and Study Overview

This overview describes the integration of the Agilent 1260 Infinity II Online LC with Flownamics SegFlow S3 and SampleMod modules to enable unattended sampling, dilution, and analysis of reactor contents. The study outlines the plumbing, electronics, and software configurations, and evaluates key performance metrics including dilution precision, linearity, accuracy, and inter-vessel carryover.

Methodology and Instrumentation

Instrumentation:

• Agilent 1290 Infinity II High-Speed Pump
• Agilent 1260 Infinity II Online Sample Manager with external valve and Online LC Monitoring Software
• Agilent 1290 Infinity II Multicolumn Thermostat
• Agilent 1260 Infinity II Diode Array Detector (10 mm cell)
• Flownamics SegFlow S3 automated sampler
• Flownamics SampleMod S3 for pre-dilution
• ZORBAX RRHD Eclipse Plus C18 column (3.0×50 mm, 1.8 µm)
• Agilent OpenLab CDS and FlowWeb control software

Sampling and Flow Path:

• Bioreactor interface via FISP probe to Aseptic Sample Regulator (ASR)
• Peristaltic pump in SegFlow S3 moves sample plugs isolated by air gaps
• SampleMod performs predefined dilutions before transfer to Online LC interface valve

Method Parameters:

• Solvents: water + 0.1% FA (A), acetonitrile + 0.1% FA (B)
• Flow rate: 1.0 mL/min, isocratic 15% B, column at 45 °C
• Injection volume: 1 µL; needle wash: 3 s with water/ACN (1:1)
• Sampling modes: direct injection, dilute to vial, pure to vial

Main Results and Discussion

Precision of peak areas for ten replicates was 0.4–0.8% RSD when using SampleMod dilutions followed by direct injection, and 0.4–0.7% RSD for dilutions performed in the Online LC Sample Manager. Dilution linearity over factors of 1, 2, 5, 10, and 20 showed R² = 0.9992. Default step-motor settings yielded dilution accuracy of 81–91%, adjustable via motor step calibration. Carryover testing between two reactors (15 g/L caffeine vs. water) resulted in 0.44% carryover, well within acceptable limits for online bioprocess analytics.

Benefits and Practical Applications

By automating sampling, dilution, and LC analysis, this configuration reduces manual errors, enhances reproducibility, and supports continuous monitoring of fermentation or cell culture processes. It streamlines QA/QC workflows and accelerates decision-making in process development and manufacturing.

Future Trends and Possibilities

Future developments may include integration of additional inline sensors (e.g., mass spectrometry), advanced software algorithms for adaptive sampling schedules, miniaturized fluidics for reduced reagent consumption, and AI-driven data analytics to predict process deviations in real time.

Conclusion

The combination of the Agilent 1260 Infinity II Online LC with Flownamics SegFlow S3 and SampleMod provides a robust, automated platform for sterile bioreactor sampling and dilution, offering high precision, excellent linearity, and low carryover. This setup enhances bioprocess monitoring and can be tailored for a wide range of online analytical applications.

References

• Herschbach H., Kutscher D., Naegele E., Ortmann T., Wouters S. Reactor Sample Dilution and Mixing Performance of the Agilent 1260 Infinity II Online Sample Manager. Agilent Technologies Technical Overview 5994-3679EN, 2021.
• Herschbach H., Kutscher D., Naegele E., Ortmann T., Spuling I., Wouters S. Performance Characteristics of the Agilent 1260 Infinity II Online Sample Manager. Agilent Technologies Technical Overview 5994-3529EN, 2021.