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

Diederichs S, Korona A, Staaden A, Kroutil W, Honda K, Ohtake H, Büchs J - Microb. Cell Fact. (2014)

Bottom Line: In cultivations with parallel offline analysis, the highest volumetric activity was found at different cultivation times.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.

View Article: PubMed Central - PubMed

Affiliation: AVT - Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074, Aachen, Germany. sylvia.diederichs@avt.rwth-aachen.de.

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.

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Related in: MedlinePlus

Overview of conducted experiments. Cultivations were performed with four E. coli BL21 (DE3) variants carrying the genes for 3-hydroxybutyryl-CoA dehydrogenase (E. coli HBD), glucose 1-dehydrogenase (E. coli GDH), 3-hydroxybutyryl-CoA dehydrogenase and glucose 1-dehydrogense (E. coli HBD-GDH), and alcohol dehydrogenase A (E. coli ADH-A) in three different complex media. The non-inducing TB medium and auto-inducing TB+lactose medium were prepared with yeast extracts from five different suppliers and, in total, eight different lots which are listed in Table 1. Additionally, cultivations were performed with two different lots of the commercially available Overnight Express (OnEx) auto-induction medium
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Fig1: Overview of conducted experiments. Cultivations were performed with four E. coli BL21 (DE3) variants carrying the genes for 3-hydroxybutyryl-CoA dehydrogenase (E. coli HBD), glucose 1-dehydrogenase (E. coli GDH), 3-hydroxybutyryl-CoA dehydrogenase and glucose 1-dehydrogense (E. coli HBD-GDH), and alcohol dehydrogenase A (E. coli ADH-A) in three different complex media. The non-inducing TB medium and auto-inducing TB+lactose medium were prepared with yeast extracts from five different suppliers and, in total, eight different lots which are listed in Table 1. Additionally, cultivations were performed with two different lots of the commercially available Overnight Express (OnEx) auto-induction medium

Mentions: Escherichia coli was chosen as model organism because it is the predominant bacterial expression system for the production of heterologous proteins [27-30] and it is frequently cultivated in complex media. Hence, the objective of this study was the employment of the RAMOS device for the evaluation of the impact of in total 8 different yeast extracts (from different suppliers and in different lots) on metabolic activity and recombinant protein yield of different recombinant E. coli variants (Figure 1). In addition, two lots of a commercial complex medium (Overnight Express (OnEx) auto-induction medium, Novagen, Merck, Germany) were analyzed to further prove the impact of lot-to-lot variation on metabolic activity during protein production.Figure 1


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

Diederichs S, Korona A, Staaden A, Kroutil W, Honda K, Ohtake H, Büchs J - Microb. Cell Fact. (2014)

Overview of conducted experiments. Cultivations were performed with four E. coli BL21 (DE3) variants carrying the genes for 3-hydroxybutyryl-CoA dehydrogenase (E. coli HBD), glucose 1-dehydrogenase (E. coli GDH), 3-hydroxybutyryl-CoA dehydrogenase and glucose 1-dehydrogense (E. coli HBD-GDH), and alcohol dehydrogenase A (E. coli ADH-A) in three different complex media. The non-inducing TB medium and auto-inducing TB+lactose medium were prepared with yeast extracts from five different suppliers and, in total, eight different lots which are listed in Table 1. Additionally, cultivations were performed with two different lots of the commercially available Overnight Express (OnEx) auto-induction medium
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230760&req=5

Fig1: Overview of conducted experiments. Cultivations were performed with four E. coli BL21 (DE3) variants carrying the genes for 3-hydroxybutyryl-CoA dehydrogenase (E. coli HBD), glucose 1-dehydrogenase (E. coli GDH), 3-hydroxybutyryl-CoA dehydrogenase and glucose 1-dehydrogense (E. coli HBD-GDH), and alcohol dehydrogenase A (E. coli ADH-A) in three different complex media. The non-inducing TB medium and auto-inducing TB+lactose medium were prepared with yeast extracts from five different suppliers and, in total, eight different lots which are listed in Table 1. Additionally, cultivations were performed with two different lots of the commercially available Overnight Express (OnEx) auto-induction medium
Mentions: Escherichia coli was chosen as model organism because it is the predominant bacterial expression system for the production of heterologous proteins [27-30] and it is frequently cultivated in complex media. Hence, the objective of this study was the employment of the RAMOS device for the evaluation of the impact of in total 8 different yeast extracts (from different suppliers and in different lots) on metabolic activity and recombinant protein yield of different recombinant E. coli variants (Figure 1). In addition, two lots of a commercial complex medium (Overnight Express (OnEx) auto-induction medium, Novagen, Merck, Germany) were analyzed to further prove the impact of lot-to-lot variation on metabolic activity during protein production.Figure 1

Bottom Line: In cultivations with parallel offline analysis, the highest volumetric activity was found at different cultivation times.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.

View Article: PubMed Central - PubMed

Affiliation: AVT - Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074, Aachen, Germany. sylvia.diederichs@avt.rwth-aachen.de.

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.

Show MeSH
Related in: MedlinePlus