High-performance liquid chromatographic determination of metabolic products for fermentation control of mammalian cell culture: analysis of carbohydrates, organic acids and orthophosphate using refractive index and ultraviolet detectors.

A method for the determination of carbohydrate substrates and excreted metabolic end-products of cell culture supernatants using a strong cation-exchange column in the H+ form has been developed. Organic acids and carbohydrates can be determined in addition to orthophosphoric acid. Pyrrolidone carboxylic acid, resulting from chemical conversion of the amino acid glutamine during the incubation of fresh medium and during the fermentation process, can be determined. The chromatographic method allows the correction of glutamine uptake values for physiological studies. Measured values of pyrrolidone carboxylic acid in supernatants of human hybridoma cell line show that it cannot be consumed by the cells. This technique allows the separation of major metabolites used in process optimization. Peak homogeneity is proved by on-line monitoring of the effluent with an ultraviolet (214 nm) and a refractive index detector connected in series.