Phenotyping the quality of complex medium components by simple online-monitored shake flask experiments

Abstract

Background: Media containing yeast extracts and other complex raw materials are widely used for the cultivation of microorganisms. However, variations in the specific nutrient composition can occur, due to differences in the complex raw material ingredients and in the production of these components. These lot-to-lot variations can affect growth rate, product yield and product quality in laboratory investigations and biopharmaceutical production processes. In the FDA's Process Analytical Technology (PAT) initiative, the control and assessment of the quality of critical raw materials is one key aspect to maintain product quality and consistency. In this study, the Respiration Activity Monitoring System (RAMOS) was used to evaluate the impact of different yeast extracts and commercial complex auto-induction medium lots on metabolic activity and product yield of four recombinant Escherichia coli variants encoding different enzymes. Results: Under non-induced conditions, the oxygen transfer rate (OTR) of E. coli was not affected by a variation of the supplemented yeast extract lot. The comparison of E. coli cultivations under induced conditions exhibited tremendous differences in OTR profiles and volumetric activity for all investigated yeast extract lots of different suppliers as well as lots of the same supplier independent of the E. coli variant. Cultivation in the commercial auto-induction medium lots revealed the same reproducible variations. In cultivations with parallel offline analysis, the highest volumetric activity was found at different cultivation times. Only by online monitoring of the cultures, a distinct cultivation phase (e.g. glycerol depletion) could be detected and chosen for comparable and reproducible offline analysis of the yield of functional product. Conclusions: This work proves that cultivations conducted in complex media may be prone to significant variation in final product quality and quantity if the quality of the raw material for medium preparation is not thoroughly checked. In this study, the RAMOS technique enabled a reliable and reproducible screening and phenotyping of complex raw material lots by online measurement of the respiration activity. Consequently, complex raw material lots can efficiently be assessed if the distinct effects on culture behavior and final product quality and quantity are visualized.

Online measurement of the respiratory activity in shake flasks enables the identification of cultivation phases and patterns indicating recombinant protein production in various Escherichia coli host strains

“…Both, soluble enzymes and enzymes in inclusion bodies are industrially relevant. Furthermore, both model enzymes have recently been investigated for recombinant protein production . Besides E. coli BL21(DE3), the standard host strain for recombinant protein production, four other strains of the B cell line, and two host strains derived from the K‐12 cell line are chosen for this comparative study.…”

Prediction of recombinant protein production by Escherichia coli derived online from indicators of metabolic burden

“…At typical cultivation conditions for E. coli (37°C, atmosphere pressure) the maximal oxygen solubility in water is 0.22 mmol/L (and even lower in media including salts and other dissolved ingredients). Depending on the media composition, E. coli reaches OUR values >70 mmol/L/h . Therefore, high k L a values are necessary, to supply the organisms with enough oxygen.…”

“…Depending on the media composition, E. coli reaches OUR values >70 mmol/L/h. 31,48 Therefore, high k L a values are necessary, to supply the organisms with enough oxygen. The relation of OTR and OUR is expressed in Eq.…”

High-level production of (5S)-hydroxyhexane-2-one by two thermostable oxidoreductases in a whole-cell catalytic approach

“…Best conditions could be found in OnEx auto-induction medium at 37 • C and sufficient aeration. Details are published in Diederichs et al [33]. In a second step, co-expression was investigated to combine both enzymes in a classical cascade in one host strain [34].…”