Quantitative and Qualitative Analysis of Cell Culture Medium Using SWATH® Acquisition

Importance of the Topic

Cell culture medium analysis is critical for ensuring consistent performance in biotherapeutic production. Monitoring nutrients, metabolites and impurities supports process optimization, product yield and safety compliance.

Objectives and Study Overview

This study demonstrates a data independent workflow using SWATH acquisition on a SCIEX TripleTOF 6600 system for both quantitative and qualitative profiling of cell culture medium components. The aim is to achieve comprehensive coverage of known and unexpected compounds in complex media matrices.

Methodology and Instrumentation

• Instrumentation: ExionLC system coupled to TripleTOF 6600 mass spectrometer equipped with a Phenomenex Kinetex F5 column.
• Chromatography: 20 minute gradient at 200 µL/min with mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile).
• Mass spectrometry: Variable window SWATH acquisition using 20 windows, MS range 50–700 m/z, MS/MS range 25–700 m/z; accumulation times of 100 ms (MS) and 35 ms (MS/MS); positive and negative ion modes.
• Sample preparation: Standards mixed at 6.67–20 µg/mL; media samples diluted in formic acid–acetonitrile, centrifuged and further diluted prior to analysis.
• Data processing: SCIEX OS 1.5 software for targeted MRM and untargeted DIA workflows, employing high resolution spectral libraries, FormulaFinder, ChemSpider and MarkerView for statistical evaluation.

Key Results and Discussion

• SWATH acquisition enabled quantification and confirmation of all ESI-detectable compounds, including amino acids, vitamins, nucleotides and fatty acids.
• Choline calibration exhibited linear response and specificity via extracted ion chromatograms and MS/MS library matching.
• Quantitative analysis spanned over four orders of magnitude for representative analytes in serial dilutions.
• Untargeted screening identified unexpected metabolites like spermine, confirmed by accurate mass, MS/MS spectral matching and formula prediction.
• Principal components analysis distinguished fresh from spent media, revealing dynamic consumption of L-serine during culture.

Benefits and Practical Applications

• Comprehensive profiling reduces reliance on multiple targeted assays and accelerates method development.
• High resolution MS/MS data minimize false discoveries and enhance compound confirmation.
• Combined targeted and untargeted workflows in a single run support robust QC and process monitoring.

Future Trends and Opportunities

Advancements may include expanded and updated spectral libraries, real time process analytical technology integration, refined variable window algorithms and increased automation for high throughput biomanufacturing analytics.

Conclusion

The SWATH acquisition workflow on the SCIEX TripleTOF 6600 delivers a versatile, high resolution platform for in depth cell culture medium analysis. Its unified approach to targeted and untargeted data acquisition improves throughput, sensitivity and confidence in bioprocess monitoring.

References

• Demianova Z et al. Quantitative LC-MS solution for targeted analysis of cell culture media. SCIEX Technical Note RUO-MKT-02-9746-A.
• SWATH acquisition Variable Window Calculator. SCIEX software tool.
• Improved data quality using variable Q1 window widths in SWATH acquisition. SCIEX Technical Note RUO-MKT-02-2879-B.
• US Patent US20100221823. Method for culturing mammalian cells to improve recombinant protein production.
• Ducker GS, Rabinowitz JD. One-carbon metabolism in health and disease. Cell Metab. 2017;25(1):27–42.