Elsevier

Analytical Biochemistry

Volume 335, Issue 1, 1 December 2004, Pages 119-125
Analytical Biochemistry

Amino acid analysis in mammalian cell culture media containing serum and high glucose concentrations by anion exchange chromatography and integrated pulsed amperometric detection

https://doi.org/10.1016/j.ab.2004.08.020Get rights and content

Abstract

The direct separation detection of amino acids by anion exchange chromatography with integrated pulsed amperometric detection was optimized for the analysis of typical mammalian cell culture broth samples. Existing gradient elution conditions were adapted, considering the additions of peptone (2 g/L) and 10 vol% fetal calf serum to the medium as well as changing concentrations of glucose from 5.5 g/L up to complete consumption. Samples had to be analyzed in two dilutions with water (1:33.3 and 1:200) due to the strongly varying amino acid concentrations in the samples as a result of the medium composition and cell metabolism. The method was validated in a linear working range for the most common amino acids (2.5–7.5 and 1.25–3.75 μM for cystine/cysteine with 15 μl injection volume). The relative standard deviation of the method for all amino acids was less than 5%, with detection limits of less than 0.6 μM and quantitation limits of less than 1.6 μM. As an example, data for the amino acid composition of different media used for the production of inactivated influenza vaccines in cell culture are shown.

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.

References (18)

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