Analysis of Underivatized Amino Acids by LC/MS for Bioreactor Cell Culture Monitoring

Importance of the Topic

Monitoring the concentration of amino acids and related metabolites in bioreactor cell cultures is fundamental for process control, product consistency, and yield optimization in biopharmaceutical and industrial biotechnology workflows.

Traditional reversed-phase LC often requires derivatization to retain polar compounds, complicating sample preparation. The use of HILIC-MS in negative mode at high pH offers an efficient alternative for direct analysis of underivatized amino acids alongside key nutrients and waste products.

Objectives and Overview of the Study

This study aimed to develop and validate a fast, reproducible LC/MS method for simultaneous quantification of underivatized amino acids, glucose, and lactate in complex fermentation media.

Key goals included:

• Achieving baseline separation of isobaric amino acids (e.g., leucine/isoleucine).
• Demonstrating method robustness in real bioreactor matrices (hollow fiber and roller bottle systems).
• Illustrating real-time monitoring of nutrient consumption and metabolite secretion.

Methodology

Sample Preparation:

• Dilution of cell culture supernatant 1:4 with 50% acetonitrile.
• Centrifugation at 10,000 × g for 10 minutes; direct injection of supernatant.

Chromatographic Conditions:

• Column: Agilent AdvanceBio MS Spent Media HILIC, 2.1 × 150 mm.
• Mobile phase A: 10 mM ammonium acetate in water at pH 9; B: 10 mM ammonium acetate in acetonitrile (90%).
• Gradient: 90% B initial, ramping to 20% B by 13 min, re-equilibration to 90% B by 25 min.
• Flow rate: 0.25 mL/min; column temperature: 30 °C; injection volume: 1 µL.

Used Instrumentation

• Agilent 1290 Infinity II LC System (binary pump, vialsampler, multicolumn thermostat).
• Ultra-low dispersion kit for Agilent 1290 Infinity LC.
• Agilent 6545 Q-TOF mass spectrometer with Jet Stream ESI source.
• MassHunter workstation software B.08.00.

Main Results and Discussion

The method provided baseline resolution of key amino acids, including a resolution of 1.6 for leucine/isoleucine isobars.

• Retention times for 20 monitored analytes ranged from 3.5 to 13.0 minutes (e.g., phenylalanine at 3.55 min, lysine at 13.01 min).
• Reproducibility tests showed consistent peak areas across triplicate injections (<5% RSD).
• Analysis of cell culture samples revealed time-dependent consumption of glucose and amino acids and accumulation of lactate in both hollow fiber (harvest after 2 days) and roller bottle systems (harvest after 2 weeks).

Benefits and Practical Applications of the Method

• Eliminates the need for derivatization, simplifying sample preparation and reducing analysis time.
• Enables simultaneous monitoring of nutrients, amino acids, and waste metabolites in a single run.
• High sensitivity and selectivity in negative ion mode at high pH improve detection of zwitterionic compounds.
• Applicable for routine QA/QC, bioprocess development, and metabolic profiling in research and industrial settings.

Future Trends and Potential Applications

The approach can be extended to online and at-line process analytical technology (PAT) for real-time bioreactor monitoring.

• Integration with automated sampling platforms for continuous monitoring of critical quality attributes.
• Expansion to additional polar metabolites, vitamins, and short peptides.
• Combination with positive-ion mode acquisition for broader metabolome coverage.
• Adaptation to miniaturized flow systems and high-throughput screening in cell-based assays.

Conclusion

The developed HILIC-MS method offers a robust, high-throughput solution for underivatized amino acid analysis in complex bioreactor media. It achieves reliable separation of critical isobaric pairs, delivers excellent reproducibility, and supports simultaneous nutrient and metabolite profiling, making it an invaluable tool for bioprocess monitoring and control.

References

1. Hsiao J., Chu T.-W., Kennedy A., Bivens A., Blackwell A. Analysis of Underivatized Amino Acids by LC/MS for Bioreactor Cell Culture Monitoring. Agilent Technologies Application Note 5991-8816EN, 2018.