Amino acid analysis in mammalian cell culture media containing serum and high glucose concentrations by anion exchange chromatography and integrated pulsed amperometric detection
Section snippets
Chemicals
Amino acids, glucose, and sodium azide were purchased from Sigma–Aldrich (Munich, Germany). The amino acid standard mix (AA-S-18) contained most “natural” amino acids at a concentration of 2.5 mM; only cysteine was set to a concentration of 1.25 mM. Glutamine, tryptophan, and asparagine together with glucose (250 g/L) were added separately to this standard to result in an aqueous stock solution with all amino acids at a concentration of 0.25 mM (0.125 mM for cysteine) and glucose at 2.5 g/L. All
Separation of medium samples with high-glucose concentrations
A method had to be found for analyzing samples from a process of influenza virus production with MDCK cells. Like many other existing cell culture processes, the samples contained glucose concentrations of 0 to 5.5 g/L, were supplemented with 2 g/L peptone, and during cell growth had an additional 10 vol% FCS, whereas virus production medium did not contain FCS [15]. The existing gradient for amino acid analysis proposed in the literature [5], [7] did not consider such high glucose concentrations
Conclusions
A simple method for the analysis of amino acid concentrations in typical cell culture medium samples by amperometric detection was validated. Neither high glucose concentrations nor the addition of peptone or FCS interfered with the analysis by slightly modifying the gradient recommended by the manufacturer. All samples could be analyzed directly after dilution with water. A working range between 2.5 and 7.5 μM (for cysteine/cystine, between 1.25 and 3.75 μM) that fitted all amino acids was
Acknowledgments
The authors thank R. Neufang from the Dionex application lab (Idstein, Germany) for his advice and discussions regarding our application. The authors also recognize the contributions of A. Bock and B. Kalbfuß, who provided assistance with the calculations involved in assay validation.
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A practical guide for amino acid determinations by high-performance anion-exchange chromatography with integrated pulsed amperometric detection
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