Monitoring of Mammalian Cell Culture Media with HILIC LC/MS

Importance of the topic

Accurate profiling of metabolites in mammalian cell culture media is vital for optimizing bioprocesses, understanding cell physiology, and ensuring consistent product quality in research and industry settings. Monitoring both nutrient depletion and waste accumulation supports better control of cell growth, metabolic flux, and biomanufacturing efficiency.

Objectives and study overview

This study aimed to establish a single-run HILIC LC/MS workflow capable of quantifying polar and anionic metabolites in high-salt cell culture media. The method was applied to a time-course experiment tracking key nutrients (glucose and amino acids) and waste products (lactate and TCA cycle acids) over six days in K562 leukemia cell cultures.

Methods and instrumentation

Chromatography was performed on an Agilent InfinityLab Poroshell 120 HILIC-Z column with PEEK-lined hardware. A pH 9 buffered ammonium acetate mobile phase with a deactivator additive preserved peak shape and retention time reproducibility under high salt conditions. Gradient elution from high acetonitrile to aqueous composition enabled separation of diverse analytes. Mass spectrometry employed an Agilent 6545 Q-TOF with Agilent Jet Stream source in negative ion mode and continuous reference mass calibration.

• LC system: Agilent 1290 Infinity binary pump, autosampler with thermostat, column compartment
• Column: 2.1 × 50 mm, 2.7 µm HILIC-Z
• Mobile phase: 10 mM ammonium acetate, pH 9, with deactivator additive in water/ACN
• MS parameters: gas temperature 200 °C, drying gas 10 L/min, capillary voltage 3000 V, acquisition m/z 50–1000

Main results and discussion

Salt tolerance experiments demonstrated stable retention times and minimal ion suppression for phosphorylated sugars and nucleotides up to 40 % added urea/NaCl. Time-course analysis revealed progressive glucose depletion at day 6 and amino acid consumption following distinct kinetics. Lactate and organic acids such as citrate, malate, α-ketoglutarate, and glutamate accumulated over time, reflecting metabolic waste production. Quantitative trends highlighted the capacity to monitor key pathways in a single run.

Benefits and practical applications

The described HILIC LC/MS method offers:

• Robust performance in high-salt matrices common to cell culture media
• Simultaneous profiling of polar, chelating, and anionic metabolites
• Reproducible retention and enhanced sensitivity for phosphorylated compounds

Applications include cell line development, metabolic phenotyping, process monitoring in biomanufacturing, and quality control.

Future trends and potential applications

Integration with automated sample preparation and high-throughput workflows could accelerate metabolomics screening. Coupling with stable isotope tracing would expand flux analysis, while miniaturized HILIC columns may reduce solvent use and increase throughput. Data-driven process optimization and real-time monitoring in bioreactors represent promising directions.

Conclusion

An Agilent HILIC-Z column paired with a 6545 Q-TOF in negative mode provides a reliable, single-run solution for monitoring nutrient and waste metabolites in mammalian cell culture media. The approach supports robust retention under high salt conditions and offers broad metabolite coverage for practical bioprocess analytics.

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