Quantitative LC-MS Solution for Targeted Analysis of Cell Culture Media

Význam tématu

Cell culture media (CCM) analysis is essential in biotherapeutic development and scale-up, enabling understanding of nutrient consumption and metabolite production under quality by design (QbD) principles. Comprehensive profiling informs process optimization and product quality assurance.

Cíle a přehled studie

This study describes the development of a quantitative LC-MS/MS workflow targeting 110 key CCM components—including amino acids, vitamins, carbohydrates, fatty acids, nucleic acids, inorganic and other metabolites—using the SCIEX QTRAP® 6500+ coupled to the ExionLC™ system. The objective is to achieve high sensitivity, selectivity and throughput in a single 20 min run.

Použitá instrumentace

• SCIEX QTRAP® 6500+ and Triple Quad™ 6500+ mass spectrometers
• ExionLC™ UHPLC system
• IonDrive™ Turbo V electrospray source
• Phenomenex Kinetex® F5 column (150 × 2.1 mm, 2.6 µm)
• SCIEX OS-Q 1.5 software with scheduled MRM, MIDAS™, Accurate Mass Metabolite Spectra Library, MQ4 integration algorithm

Použitá metodika

Standards (1 mg/mL) were combined to create a master mix (6.67–20 µg/mL) covering diverse compound classes. Cell culture medium (CD CHO) was diluted, acidified and centrifuged prior to injection. Chromatographic separation used a reversed-phase gradient (0.1% formic acid in water and acetonitrile) at 200 µL/min over 20 min. Scheduled MRM monitored 178 positive and 54 negative transitions, optimizing dwell and cycle times. MIDAS-enabled IDA scans confirmed identities via spectral library matching.

Hlavní výsledky a diskuse

The method achieved baseline separation of polar and nonpolar analytes, including critical isomeric and homologous pairs (e.g., L-leucine vs L-isoleucine, L-cysteine vs L-cystine, L-arginine vs L-ornithine vs L-citrulline) with specific MRMs. Calibration curves exhibited linear dynamic ranges spanning 2.5–6 orders of magnitude and low-ng/mL LLOQs (e.g., L-tryptophan LLOQ 0.025 ng/mL, S/N 46). Concentration metric plots enabled clear visualization of nutrient fluctuations across culture stages.

Přínosy a praktické využití metody

• Comprehensive nutrient profiling in a single 20 min run
• High throughput compatible with process monitoring and QA/QC
• Robust separation across chemically diverse analytes
• Simultaneous acquisition in positive and negative ion modes
• Future heavy internal standard kit for enhanced quantitation accuracy

Budoucí trendy a možnosti využití

Future directions include expanding the targeted analyte library, incorporating isotopic internal standards, automating sample preparation, integrating real-time bioprocess monitoring, and applying machine learning for advanced data interpretation and predictive process control.

Závěr

The presented LC-MS/MS solution on the SCIEX QTRAP® 6500+ with ExionLC™ delivers rapid, sensitive and robust quantitation of 110 CCM components, addressing challenges of chromatographic separation, dynamic range and throughput to support biotherapeutic development and quality by design workflows.

Reference

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