Online Amino Acid Analysis for Spend Media Control

Significance of the topic

In biopharmaceutical production, maintaining optimal levels of key nutrients such as amino acids is critical to ensure cell viability and high-quality yields of active pharmaceutical ingredients. Real-time monitoring of amino acid concentrations allows timely adjustments in bioreactors, reduces downtime, and minimizes the risk of culture failure.

Study objectives and overview

This application study demonstrates how the Agilent InfinityLab Online LC Solution can continuously monitor amino acid concentrations in spent cell culture media over multiple days. A Dulbecco’s Modified Eagle Medium (DMEM) spiked with glutamine was used to simulate a bioprocess environment, with samples drawn every five hours and processed automatically.

Methodology and instrumentation

Automated sample preparation was performed with precolumn derivatization using o-phthalaldehyde for primary amino acids and FMOC for secondary amino acids. Chromatographic separation employed an Agilent AdvanceBio AAA column under gradient elution, followed by UV–visible diode array detection. Sleep and wake-up sequences were implemented to conserve solvents and maintain instrument readiness. The entire workflow was controlled by Agilent Online LC Monitoring Software integrated with OpenLab CDS.

Used instrumentation

– Agilent 1290 Infinity II High Speed Pump (G7120A)
– Agilent 1260 Infinity II Online Sample Manager Set (G3167A) with external valve (5067-6680) and Valve Drive (G1170A)
– Agilent 1260 Infinity High Performance Degasser (G4225A)
– Agilent 1290 Infinity II Multicolumn Thermostat (G7116B)
– Agilent 1290 Infinity II Diode Array Detector (G7117B) with Max-Light Cartridge Cell (G4212-60008)
– Agilent 1260 Infinity II Isocratic Pump (G7110B)
– Agilent AdvanceBio AAA analytical column (3.0 × 100 mm, 2.7 μm) and guard column (3.0 × 5 mm, 2.7 μm)
– Software: OpenLab CDS v2.6 or later, Online LC Monitoring Software v1.0

Main results and discussion

A 19-compound amino acid calibration covering 90–900 pmol/µL with quality controls of norvaline and sarcosine at 500 pmol/µL showed robust linearity. In the online experiment, glutamine levels averaged 4.635 mM with an RSD of 2.3%, and retention times averaged 3.887 minutes with an RSD of 0.16%. QC samples averaged 510 pmol/µL (RSD 0.56%) with consistent retention at 8.433 minutes (RSD 0.06%). Trending plots enabled near-real-time decision support for nutrient dosing.

Benefits and practical applications of the method

– Unattended operation over extended periods reduces labor and manual sampling errors
– Sleep modes cut solvent consumption and maintenance intervals
– Automated derivatization ensures reproducible sample preparation
– Real-time data trending supports dynamic process control in biomanufacturing

Future trends and potential applications

Integration with advanced process analytical technologies and predictive control models may further enhance bioprocess automation. Expanding the online LC approach to other critical metabolites and process parameters could provide a more comprehensive view of culture health and productivity. Cloud-based data analytics and machine learning are likely to drive next-generation process monitoring solutions.

Conclusion

The Agilent InfinityLab Online LC Solution offers a robust, automated platform for long-term monitoring of amino acids in cell culture media, combining precise chromatographic analysis with flexible software control. This approach streamlines bioprocess monitoring, enabling timely interventions and higher confidence in production outcomes.

References

1. Agilent Technologies. Amino Acid Analysis How-To Guide, publication number 5991-7694EN, 2021.