Automated Bioprocess Monitoring with Agilent 1290 Infinity III Bio Online LC Solutions

Significance of the topic

The ability to monitor bioprocesses in real time is critical for ensuring optimal yields, product quality, and rapid response to deviations. Automated online chromatography can greatly enhance process analytical technology (PAT) by providing continuous, sterile sampling and immediate feedback during microbial fermentations.

Objectives and overview

This study demonstrates an integrated workflow for hourly measurement of L-alanine production in a bioreactor culture of genetically engineered Vibrio natriegens. The aim was to couple sterile sampling with automated derivatization and high-performance liquid chromatography (HPLC) to achieve unattended, near real-time bioprocess monitoring.

Methodology

The fermentation was conducted at 37 °C, pH 7.5 under anaerobic conditions with V. natriegens Δdldh Δlldh Δpfl Δmdh. Hourly samples were withdrawn via a Flownamics Seg-Flow S3 system equipped with a 0.2 µm FISP probe. Samples were automatically derivatized using OPA and FMOC reagents in the autosampler injector program. Calibration standards covering six concentration levels of 20 amino acids plus internal standards were prepared in borate buffer and analyzed without manual intervention.

Used instrumentation

• Agilent 1290 Infinity III Bio Flexible Pump with 0.2 µm inline filter
• Agilent 1290 Infinity III Bio Online Sample Manager with thermostat and reactor valve pod
• Agilent 1290 Infinity III Multicolumn Thermostat with heat exchanger
• Agilent 1290 Infinity III Diode Array Detector (Bio Max-Light cell)
• Agilent 1260 Infinity High-Performance Degasser
• Flownamics Seg-Flow S3 sampling system and FlowWeb software
• Agilent OpenLab CDS and Online LC Monitoring Software

Main results and discussion

Derivatized amino acids were separated in 17 minutes, achieving baseline resolution of 20 analytes and two internal standards. Calibration curves for L-alanine exhibited excellent linearity across the tested range. During the 24-hour fermentation, monitored hourly, L-alanine concentration rose steadily from an initial background level (inherent to the 2× YTN medium) to a maximum at 24 hours. The online LC monitoring software trend plot provided a clear visualization of production kinetics.

Benefits and practical applications

• Fully automated sterile sampling and derivatization reduce manual workload and contamination risk.
• Near real-time data enables rapid process adjustments to maximize yield and quality.
• The approach is adaptable to other small-molecule or peptide targets in complex matrices.

Future trends and potential applications

Advances may include integration with multivariate data analysis, machine-learning–based process control, and extension to multianalyte assays for online metabolic profiling. Miniaturized flow-through reactors and cloud-based dashboards could further enhance scalability and remote monitoring.

Conclusion

The combination of Flownamics sterile sampling and Agilent 1290 Infinity III Bio Online LC enables robust, unattended monitoring of microbial bioprocesses. This platform offers high-throughput sampling and reliable quantitation, supporting improved process control and productivity in biomanufacturing.

References

1. Nikita S., Mishra S., Gupta K., Runkana V., Gomes J., Rathore A. “Advances in Bioreactor Control for Production of Biotherapeutic Products,” Biotechnol. Bioeng. 2023, 120(5), 1189–1214.
2. Hoffart E., Grenz S., Lange J., Nitschel R., Müller F., Schwenter A., Feith A., Lenfers-Lücker M., Takors R., Blombach B. “High Substrate Uptake Rates Empower Vibrio natriegens as Production Host for Industrial Biotechnology,” Appl. Environ. Microbiol. 2017, 83(22).
3. Naegele E., Herschbach H. “Performance Evaluation of Flownamics Sampling Devices for the Agilent Online LC,” Agilent Technologies Application Note 5994-7394EN, 2024.