Simultaneous analysis of supernatant components of cell culture using triple quadrupole LC/MS/MS

Significance of the Topic

The precise profiling of culture medium components underpins optimization and quality control in biotechnology, pharmaceuticals, and biofuel production. Tracking variations in nutrients, vitamins, amino acids, and metabolites in real time supports process control, yields improvement, and deeper insight into cellular metabolism.

Objectives and Study Overview

This work presents a streamlined workflow, termed “Method Package for Cell Culture Profiling,” enabling simultaneous quantitation of 95 supernatant analytes in bacterial and Streptomyces fermentations. Key goals included method development for a broad dynamic range—from high‐abundance substrates such as glucose to trace vitamins—and demonstration of time‐resolved metabolic trends across culture periods.

Methodology and Instrumentation

Pretreatment steps:

• Centrifugation of culture aliquots to separate cells.
• Addition of internal standards to supernatant.
• Protein precipitation with acetonitrile, agitation, and re‐centrifugation.
• Dilution of the clarified supernatant with water before analysis.

Chromatography and detection:

• UHPLC conditions: reversed‐phase separation at 40 °C, 0.35 mL/min flow, formic acid–water (0.1%) and formic acid–ACN (0.1%) mobile phases, 17 min cycle including equilibration, 1 µL injection.
• MS/MS detection: Shimadzu Nexera MP UHPLC coupled to LCMS‐8050 triple quadrupole, electrospray ionization in polarity‐switching MRM mode, ultra‐fast polarity switching (5 ms) and high‐speed MRM acquisition (up to 555 transitions/sec).

Main Results and Discussion

Under optimized parameters all 95 target compounds produced robust signals across four orders of magnitude. Representative time‐course observations:

• Bacterial culture supernatant exhibited a steady decline in succinic acid and accumulation of 2-aminoethanol; deoxycytidine peaked at 10 h before decreasing; biotin remained constant.
• Streptomyces cultures showed rapid alanine depletion within 72 h; citrulline dropped initially then rose post–72 h; cytidine peaked at 86 h; gluconic acid accumulated steadily over 120 h.

Mass chromatograms demonstrated clear separation and quantitation of both high‐level substrates and low‐abundance vitamins in a single 17-minute run.

Benefits and Practical Applications

This single‐run, multi‐component profiling approach enables:

• Comprehensive metabolic monitoring without multiple targeted assays.
• Rapid feedback for bioprocess optimization and scale‐up.
• Quality control in biopharmaceutical manufacturing through consistent medium surveillance.

Future Trends and Applications

Advances may include integration with automated sampling and real‐time data analytics, expansion of compound libraries to secondary metabolites, and coupling with high‐resolution mass spectrometry for structure elucidation. Machine learning–driven interpretation of multi‐omics datasets will further enrich process understanding and predictive control.

Conclusion

The developed Method Package for Cell Culture Profiling achieves robust, high‐throughput simultaneous quantitation of 95 supernatant components in under 17 min. Its broad dynamic range and reproducibility make it an invaluable tool for research and industrial bioprocess monitoring.

Instrumentation Used

• UHPLC: Shimadzu Nexera MP system.
• Mass spectrometer: Shimadzu LCMS‐8050 triple quadrupole with electrospray ionization, ultra‐fast polarity switching and MRM capability.