Analysis of Underivatized Amino Acids and Metabolites in Cell Culture Media by HILIC-LC/MS

Significance of the topic

Polar metabolites such as amino acids, vitamins, and polyamines are essential indicators of cell health and productivity in bioprocessing. Robust analytical methods that can directly measure these underivatized compounds in high-salt culture media enable real-time monitoring of nutrient availability, waste accumulation, and biopharmaceutical yield.

Objectives and overview

This work established HILIC-LC/MS protocols for rapid profiling of polar metabolites in cell culture supernatants. Two projects were undertaken: evaluation of media formulations for monoclonal antibody (mAb) production, and time-course monitoring of nutrient consumption and waste excretion in different bioreactor systems.

Methodology and instrumentation

Samples of fresh and spent CHO cell media were diluted with acetonitrile or mobile phase and centrifuged to remove debris. Two chromatographic methods were developed:

• Project 1: Low pH, positive ion mode on an AdvanceBio MS Spent Media HILIC column (2.1×100 mm) at 40 °C, using 20 mM ammonium formate buffer, analyzed with an Agilent 1260 Infinity II BioInert LC coupled to a 6230 TOF MS.
• Project 2: High pH, negative ion mode on an AdvanceBio MS Spent Media HILIC column (2.1×150 mm) at 30 °C, using 10 mM ammonium acetate buffer, analyzed with an Agilent 1290 Infinity II LC coupled to a 6545XT QTOF MS.

Main results and discussion

• Baseline separation of isomeric amino acids (leucine/isoleucine) and comprehensive coverage of B vitamins, amino acids, and polyamines were achieved without derivatization.
• Comparative analysis of two commercial media showed that one formulation delivered higher initial amino acid concentrations and produced nearly three times greater mAb titer after two weeks.
• Dynamic monitoring of amino acid depletion, glucose consumption, and lactate accumulation provided insights into cellular metabolism in hollow fiber bioreactors and roller bottle cultures.

Benefits and practical applications

The developed HILIC-MS workflow offers a fast, derivatization-free alternative to traditional LC/UV assays, enabling:

• Rapid screening and optimization of culture media for improved protein yield.
• Real-time tracking of nutrient levels to guide feed strategies.
• Correlation of metabolite profiles with protein titer for comprehensive bioprocess control.

Future trends and potential applications

• Extension of the method to additional polar metabolites, including nucleotides and small peptides.
• Automation of sample preparation workflows for high-throughput process monitoring.
• Integration with process analytical technology (PAT) platforms for real-time feedback control.
• Application of high-resolution MS data for advanced metabolomic modeling and predictive analytics.

Conclusion

The HILIC-LC/MS methods described provide a versatile, sensitive, and derivatization-free approach to profiling underivatized polar metabolites in complex culture media. These protocols support efficient media selection, metabolic monitoring, and enhanced biopharmaceutical production.

Used instrumentation

• Agilent AdvanceBio MS Spent Media HILIC columns (2.1×100 mm and 2.1×150 mm)
• Agilent 1260 Infinity II BioInert LC
• Agilent 6230 TOF MS
• Agilent 1290 Infinity II LC
• Agilent 6545XT QTOF MS

References

• Dumont E; Vandenheede I; Sandra P; Sandra K. mAb Titer Analysis with the Agilent Bio-Monolith Protein A Column. Agilent Technologies Application Note, 2014.