Agilent AdvanceBio workflows for spent media analysis

Importance of the Topic

Spent media analysis of bioreactor cell culture plays a critical role in modern bioprocessing by providing insight into nutrient consumption, metabolite accumulation, and cell health. Reliable quantification of amino acids and other small metabolites informs process optimization, ensures product consistency, and supports regulatory compliance in biotech and pharmaceutical industries.

Objectives and Study Overview

This overview describes two AdvanceBio workflows from Agilent Technologies designed for fast, reproducible analysis of spent cell culture media. One workflow employs automated derivatization and LC/UV detection for standard amino acid profiling. The other uses underivatized HILIC separation coupled with MS detection to expand coverage to cell culture metabolites without sample derivatization.

Methodology and Instrumentation

The derivatization workflow uses OPA (o-phthalaldehyde) and FMOC chemistries packaged in a single kit. Automated sample preparation in the autosampler minimizes manual handling and variability. Reverse-phase separation on 2.7 µm Poroshell columns with UV detection resolves amino acids according to European Pharmacopoeia standards.

The underivatized workflow leverages PEEK-lined stainless steel HILIC columns for robust separation of ionic metabolites under high pressure (up to 600 bar). MS-friendly mobile phases deliver high sensitivity and eliminate chromatographic baseline resolution requirements when paired with mass spectrometric detection.

Instrumentation Used

• Agilent 1260 Infinity II LC system with diode array detector (DAD WR)
• Agilent 6230 Time-of-Flight LC/MS and 6545XT Q-TOF systems
• Agilent 1290 Infinity II LC with Ultivo triple quadrupole MS
• Agilent autosampler technologies (vialsampler, multisampler)

Main Results and Discussion

For derivatized amino acid analysis, the AdvanceBio Amino Acid Analysis kit achieved resolution of 4.35 between leucine and isoleucine, well above the Ph. Eur. requirement of 1.5. Automated derivatization in the autosampler ensured reproducible sample processing with minimal hands-on time. Underivatized HILIC LC/MS analysis delivered a resolution of 3.0 for the same isomers in cell culture media and provided sensitive detection of additional metabolites such as glucose, lactate, polyamines, and water-soluble vitamins.

Practical Benefits and Applications

• High-throughput amino acid profiling for quality control of biopharmaceuticals
• Comprehensive metabolic monitoring of cell culture processes without derivatization
• Compatibility with both HPLC and UHPLC systems reduces capital investment and training requirements
• Automated workflows minimize errors and improve laboratory throughput

Future Trends and Potential Applications

Emerging developments may include expansion of metabolite libraries, integration with real-time process analytical technology (PAT), and enhanced automation for continuous monitoring. Coupling advanced separation media with high-resolution MS and data analytics will further accelerate bioprocess optimization and quality assurance.

Conclusion

Agilent AdvanceBio workflows enable rapid, reliable analysis of spent cell culture media. The derivatization LC/UV approach offers industry-standard amino acid quantification, while the underivatized HILIC LC/MS method broadens the metabolite coverage. Both workflows deliver robustness, sensitivity, and automation to support the evolving needs of biotech research, development, and production.

Reference

1. European Pharmacopoeia 9.0 (2.2.56) Amino Acid Analysis
2. Agilent Technologies publication number 5991-7694EN: How-to guide to amino acid analysis
3. Agilent Technologies publication number 5991-8817EN: AdvanceBio workflows for spent media analysis