T Cell Media Analysis using Triple Quadrupole Mass Spectrometry

Significance of the topic

This workflow addresses the critical need to comprehensively profile cell culture media used in T cell immunotherapy manufacturing. Reliable analysis of medium composition is essential for process optimization, quality control, and consistent production of therapeutic T cells.

Study Objectives and Overview

The primary goal was to develop and demonstrate a rapid LC-MS method capable of simultaneously quantifying 125 key metabolites in T cell culture media. Two commercial serum-free, xeno-free media were compared using a 17-minute chromatographic run and principal component analysis to reveal formulation similarities and differences.

Methodology

• Sample preparation involved protein precipitation with acetonitrile, centrifugation, and dilution of supernatants prior to injection.
• Chromatographic separation was performed using a 150×2.1 mm cell culture profiling column at 0.35 mL/min and 40 °C with a 0–95 % acetonitrile gradient containing 0.1 % formic acid over 17 minutes.
• Mass spectrometry employed ESI in positive and negative modes with both MRM targeting 125 compounds and full scan acquisition (m/z 200–1000).
• Data processing included MRM quantitation in LabSolutions Insight and multivariate analysis in MS-DIAL.

Used Instrumentation

• Shimadzu LCMS-8050 triple quadrupole mass spectrometer with fast polarity switching.
• Shimadzu LC-40 series UHPLC system and Cell Culture Profiling Column (150×2.1 mm, 3 µm).
• Software packages: LabSolutions for data acquisition, LabSolutions Insight for MRM analysis, and MS-DIAL for PCA.

Results and Discussion

• PCA score plots clearly distinguished T cell media from IVF and DMEM formulations, while the two T cell media clustered closely.
• Common detection of amino acids, nucleotides, sugars, vitamins (including niacinamide, pantothenic acid, folic acid, riboflavin) and other metabolites in both media.
• Key differences identified: biotin was present only in medium A; medium B exhibited higher pipecolic acid levels; medium A contained alanyl-glutamine dipeptide, whereas medium B had elevated free glutamine.

Benefits and Practical Applications

• Enables high-throughput profiling of diverse metabolite classes in a single 17-minute run.
• Supports media formulation assessment, quality control, and research and development workflows.
• Facilitates comparative studies and time-course monitoring during T cell and CAR-T cell expansion processes.

Future Trends and Potential Applications

• Implementation of time-lapse metabolomic monitoring during cell culture to optimize feeding strategies.
• Extension of the method to other cell types, media formulations, and combined targeted/untargeted workflows.
• Integration of automated sample prep and AI-driven data analytics for predictive medium optimization.
• Scaling to higher throughput for routine biomanufacturing quality assurance.

Conclusion

The demonstrated LCMS-8050 workflow provides a robust, rapid, and comprehensive platform for profiling cell culture media, yielding actionable insights into formulation differences and supporting critical applications in T cell therapy development and quality control.

References

1. U.S. FDA Center for Biologics Evaluation and Research. Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products: Draft Guidance for Industry. Silver Spring, MD; 2002.
2. Tsugawa H. MS-DIAL (ver. 4.80) [software]. RIKEN; 2022.
3. Pérez-García F, Brito LF, Wendisch VF. Function of L-pipecolic acid as compatible solute in Corynebacterium glutamicum under hyperosmolar conditions. Front Microbiol. 2019;10:340.
4. Kim DY et al. Fed-batch CHO cell t-PA production and feed glutamine replacement to reduce ammonia production. Biotechnol Prog. 2013;29(1):165-175.
5. Tan B et al. Alanyl-glutamine but not glycyl-glutamine improved proliferation of enterocytes in vitro. Amino Acids. 2017;49(12):2023-2031.