Limits...
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.

Show MeSH

Related in: MedlinePlus

Cultivation ofE. coliunder induced conditions using auto-induction media. Oxygen transfer rate during cultivation of two E. coli BL21 (DE3) variants expressing 3-hydroxybutyryl-CoA dehydrogenase (E. coli HBD) and glucose 1-dehydrogenase (E. coli GDH) in (A-B, D-E) self-made auto-inducing TB+lactose medium with various yeast extracts as listed in Table 1 and (G-H) two different lots of commercial Overnight Express (OnEx) auto-induction medium. The respiration behavior is categorized into two patterns. Pattern I (Merck, Roth_2, and OnEx_2) is characterized by an initial increase in OTR to a plateau of 35 to 60 mmol/L/h and a second increase to 70 mmol/L/h, whereas the highest OTR is reached immediately in pattern II (AppliChem, DSM, Difco, Roth_1, Roth_4, and OnEx_1). Conditions: 250-mL flask, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm, and 37°C. (C, F, I) The volumetric enzyme activity of expressed HBD and GDH was measured in triplicates for all cultivations after 14 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Cultivation ofE. coliunder induced conditions using auto-induction media. Oxygen transfer rate during cultivation of two E. coli BL21 (DE3) variants expressing 3-hydroxybutyryl-CoA dehydrogenase (E. coli HBD) and glucose 1-dehydrogenase (E. coli GDH) in (A-B, D-E) self-made auto-inducing TB+lactose medium with various yeast extracts as listed in Table 1 and (G-H) two different lots of commercial Overnight Express (OnEx) auto-induction medium. The respiration behavior is categorized into two patterns. Pattern I (Merck, Roth_2, and OnEx_2) is characterized by an initial increase in OTR to a plateau of 35 to 60 mmol/L/h and a second increase to 70 mmol/L/h, whereas the highest OTR is reached immediately in pattern II (AppliChem, DSM, Difco, Roth_1, Roth_4, and OnEx_1). Conditions: 250-mL flask, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm, and 37°C. (C, F, I) The volumetric enzyme activity of expressed HBD and GDH was measured in triplicates for all cultivations after 14 h.

Mentions: In Figure 4, the OTR during cultivation of E. coli HBD (first column) and E. coli GDH (second column) and the volumetric activities of HBD and GDH at the end of the cultivation (third column) are presented for all tested auto-induction media. In contrast to the very similar respiration activity in non-inducing TB medium, the OTR exhibited tremendous differences in the shape of the curves in auto-inducing TB+lactose medium. The variation in OTR clearly indicated that the auto-inducing media affected the metabolic activity of the host cells depending on yeast extract lots and OnEx medium lots used to prepare the media. Interestingly, the general shapes of the OTR curves as function of time of the two E. coli variants were comparable in the same lots. The different shapes of respiration behavior in auto-induction media compared to cultivation under non-induced conditions (Figure 2) can be assigned to the different availability of carbon sources and to metabolic burden due to overproduction of plasmid-encoded recombinant proteins [34-37]. Different shapes of respiration behavior in the applied auto-induction media could not be found for E. coli BL21 (DE3) without plasmid (Additional file 1). In those cultivations, the shape of the OTR curves in the different media was in principle equivalent, showing a long phase of oxygen limitation interrupted by a diauxic break at about 8 h. Therefore, the impact of metabolic burden could be confirmed for the cultivations in Figure 4.Figure 4


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)

Cultivation ofE. coliunder induced conditions using auto-induction media. Oxygen transfer rate during cultivation of two E. coli BL21 (DE3) variants expressing 3-hydroxybutyryl-CoA dehydrogenase (E. coli HBD) and glucose 1-dehydrogenase (E. coli GDH) in (A-B, D-E) self-made auto-inducing TB+lactose medium with various yeast extracts as listed in Table 1 and (G-H) two different lots of commercial Overnight Express (OnEx) auto-induction medium. The respiration behavior is categorized into two patterns. Pattern I (Merck, Roth_2, and OnEx_2) is characterized by an initial increase in OTR to a plateau of 35 to 60 mmol/L/h and a second increase to 70 mmol/L/h, whereas the highest OTR is reached immediately in pattern II (AppliChem, DSM, Difco, Roth_1, Roth_4, and OnEx_1). Conditions: 250-mL flask, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm, and 37°C. (C, F, I) The volumetric enzyme activity of expressed HBD and GDH was measured in triplicates for all cultivations after 14 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Cultivation ofE. coliunder induced conditions using auto-induction media. Oxygen transfer rate during cultivation of two E. coli BL21 (DE3) variants expressing 3-hydroxybutyryl-CoA dehydrogenase (E. coli HBD) and glucose 1-dehydrogenase (E. coli GDH) in (A-B, D-E) self-made auto-inducing TB+lactose medium with various yeast extracts as listed in Table 1 and (G-H) two different lots of commercial Overnight Express (OnEx) auto-induction medium. The respiration behavior is categorized into two patterns. Pattern I (Merck, Roth_2, and OnEx_2) is characterized by an initial increase in OTR to a plateau of 35 to 60 mmol/L/h and a second increase to 70 mmol/L/h, whereas the highest OTR is reached immediately in pattern II (AppliChem, DSM, Difco, Roth_1, Roth_4, and OnEx_1). Conditions: 250-mL flask, filling volume 10 mL, shaking frequency 350 rpm, shaking diameter 50 mm, and 37°C. (C, F, I) The volumetric enzyme activity of expressed HBD and GDH was measured in triplicates for all cultivations after 14 h.
Mentions: In Figure 4, the OTR during cultivation of E. coli HBD (first column) and E. coli GDH (second column) and the volumetric activities of HBD and GDH at the end of the cultivation (third column) are presented for all tested auto-induction media. In contrast to the very similar respiration activity in non-inducing TB medium, the OTR exhibited tremendous differences in the shape of the curves in auto-inducing TB+lactose medium. The variation in OTR clearly indicated that the auto-inducing media affected the metabolic activity of the host cells depending on yeast extract lots and OnEx medium lots used to prepare the media. Interestingly, the general shapes of the OTR curves as function of time of the two E. coli variants were comparable in the same lots. The different shapes of respiration behavior in auto-induction media compared to cultivation under non-induced conditions (Figure 2) can be assigned to the different availability of carbon sources and to metabolic burden due to overproduction of plasmid-encoded recombinant proteins [34-37]. Different shapes of respiration behavior in the applied auto-induction media could not be found for E. coli BL21 (DE3) without plasmid (Additional file 1). In those cultivations, the shape of the OTR curves in the different media was in principle equivalent, showing a long phase of oxygen limitation interrupted by a diauxic break at about 8 h. Therefore, the impact of metabolic burden could be confirmed for the cultivations in Figure 4.Figure 4

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