MONITORING NUTRIENT STATUS IN CELL CULTURE MEDIA USING UPLC TECHNOLOGY

Importance of the Topic

Cell culture media composition directly impacts the productivity and quality of biopharmaceutical manufacturing processes.
Monitoring essential nutrients such as amino acids and water‐soluble vitamins in real time enables dynamic control of feeding strategies and supports consistent product yield and purity.

Objectives and Study Overview

This work illustrates the development and application of a unified UPLC®‐based approach to quantify amino acids and vitamins in complex cell culture media.
The primary goals were to achieve high speed, resolution, and sensitivity while demonstrating reliable performance over multiple culture time points.

Methodology and Instrumentation

Amino acids were derivatized using the AccQ·Tag™ Ultra pre‐column reagent and separated by UPLC on a heated (60 °C) HSS T3 column.
Water‐soluble vitamins were analyzed without derivatization using ion‐pair reversed‐phase UPLC with heptafluorobutyric acid (HFBA) in the mobile phase.
Detection was performed with a photodiode array detector at 270 nm for most B vitamins and at 214 nm for those with lower absorbance.

Used Instrumentation

• Waters ACQUITY UPLC system equipped with PDA detector
• ACQUITY UPLC HSS T3 column (1.8 µm, 2.1×50 mm); compared with BEH C18 and HSS C18
• AccQ·Tag™ Ultra amino acid derivatization kit
• Heptafluorobutyric acid and trifluoroacetic acid as ion‐pairing reagents

Main Results and Discussion

The method achieved baseline resolution of 20+ amino acids in under 8 minutes using a 25 nL injection of 10× diluted media, with no interference from matrix components.
Vitamin separations showed optimal peak shape and retention at 0.035–0.04% HFBA, with linear response (R2>0.999) over 2.5–100 ng on column and limits of detection down to 0.1–0.5 ng for most vitamins.
Replicate injections demonstrated %RSD for peak area below 6%, confirming high precision.
Time‐course analysis of cell culture samples at Days 1, 3, 6 (and Day 7 for amino acids) revealed consumption trends for glutamine, arginine, serine, and production or stability of other amino acids; vitamin levels remained reproducible.

Benefits and Practical Applications

The combined UPLC solution offers rapid turnaround, minimal sample preparation, and outstanding sensitivity suitable for high‐throughput labs.
Its ability to track nutrient depletion in real time supports fed‐batch optimization and quality control in biopharmaceutical development and production.

Future Trends and Possibilities

Integration of this UPLC approach with online sampling modules and process analytical technology (PAT) frameworks could enable fully automated nutrient monitoring.
Extension to additional media components, such as trace metals or lipids, and coupling with mass spectrometric detection may further expand capabilities in bioprocess analytics.

Conclusion

The described UPLC methods deliver fast, reliable quantification of amino acids and water‐soluble vitamins in cell culture media.
High resolution, sensitivity, and precision make this approach a valuable tool for controlling nutrient supply and optimizing biomanufacturing processes.

Reference

Jablonski JM, Wheat TE, Hong P, Diehl DM. Monitoring nutrient status in cell culture media using UPLC® technology. Waters Corporation, 2008.