Development and Validation of a Liquid Chromatography-Mass Spectrometry Assay for the Quantitation of IPTG in E. Coli Fed-Batch Cultures

Abstract

IPTG (Isopropyl-beta-d-1-thiolgalactopyranoside) is a gratuitous inducer commonly used for the overexpression of heterologous recombinant proteins in Escherichia coli. A reliable method has been developed for the determination of IPTG in E.coli fed-batch fermentation samples with a minimal sample treatment. Analysis was performed in single ion monitoring positive mode using ESI source. The extracted ion was 261 m/z and the retention time of IPTG was 12.4 min with a total run time in samples of 30 min. The flow was directed to mass spectrometer 11 min after the start of the run and diverted from mass spectrometer after 14.5 min in order to avoid interference of salts and other metabolites. The assay was validated for medium and intracellular matrices and linear calibration curves of 3 orders of magnitude were obtained (R(2) >or= 0.99). Quality control samples were analyzed and showed precision and accuracy within the limits according to FDA Guidelines for analytical method validation. Recovery for both matrices was between 95.8 and 113.5%. The limit of detection (LOD) was set at 0.02 microM being the 0.1% of the lowest IPTG concentration used for induction of recombinant protein overexpression. The developed procedure has been applied to determine the IPTG distribution profiles in medium and intracellular samples in high cell density induced cultures for the production of the recombinant protein rhamnulose-1-phosphate aldolase (RhuA).

Quantitative modeling of inducer transport in fed-batch cultures of Escherichia coli

“…1, the model is able to predict the extracellular concentration profile of IPTG for the lacY mutant strain, within a 10% of error. It must be noticed that the error associated to the analytical measurement was already estimated to be in the 10% range [17]. The net transport rate of inducer without the presence of the lactose permeases can be explained as a contribution of both diffusion and non-specific active transport processes.…”

“…A method for IPTG quantification based on high performance liquid chromatography linked to mass spectrometry (HPLC-MS) has been recently developed [17]. This new method allows obtaining direct experimental data of extracellular concentration of IPTG.…”

“…Medium samples were centrifuged and the supernatant was filtered (0.22 m), and diluted if necessary in order to have a concentration within the calibration range. Mass spectrometry and liquid chromatography conditions are described in literature [17]. All the measurements are made by duplicate, with a mean error of 10%, as already reported [17].…”

“…Mass spectrometry and liquid chromatography conditions are described in literature [17]. All the measurements are made by duplicate, with a mean error of 10%, as already reported [17].…”

Determination of IPTG in Recombinant Human Growth Hormone with Ion Chromatography and Pulsed Electrochemical Detection

“…Several different techniques have been used for the analysis of thiocompounds [11], such as liquid chromatography, Capillary electrophoresis and HPLC-MS-based methods [12][13][14][15][16][17], which have attracted much attention in evaluating sulfur-containing compounds because of their good separation and detection ability [12,13]. However, for the absence of strong chromophores or luorophores, the determination of thiocompounds with UV [18,19] or luorescence [20,21] methods should be coupled with derivatization.…”