appl_03_glucose-lactate_sll1104-e
TRANSCRIPT
Glucose | Lactate Performance and Accuracy
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Application Note
1. Introduction
GlucoseGlucose is by far the most important sub-strate for microorganisms and mammalian cell lines in bioprocesses. In over 90% of all microbial cultivations and in all mammalian cell cultivations it is used as carbon source. Thus, monitoring and control of glucose concentrations is required. A typical example is a fed-batch high cell density cultivation for the pro-duction of recombinant proteins. The concentration must remain below 1 g/L during the production phase to minimize the formation of unwanted byproducts. Total depletion of glucose will immediately lead to starvation and a decrease in cell viability. Thus, the control of the substrate feed requires frequent and reliable mea-surements of the in-situ glucose concen-tration.
LactateLactate is a common metabolic product in microbial and mammalian processes. The monitoring and control of this metabolite is necessary to avoid unwanted effects when lactate concentration increases. The productivity of mammalian cell cul-tures is highly influenced by the lactate concentration in the media. An increase of the lactate concentration over a specific value during the cultivation causes negative cellular affects. Therefore, online monitoring of the metabolite is necessary for an optimal fed-batch process control. In addition, the development of a process where the final lactate concentration is kept to a minimum, results in improved purification procedures.
Analysis methods like HPLC or enzyme kits are capable of measuring Glucose and Lactate concentrations, but are relatively expensive or problematic for online analysis. BioPAT® Trace and BioPAT® Multi Trace allows a rapid and precise determination of Glucose and Lactate concentrations inside the bioreactor within minutes.
3. System PerformanceThese data were compiled in order to give an overview of the system- and sensor- performance in the normal media concen-tration range using the dialysis sampling method.
LinearityBy comparing the actual value with the set value a regression coefficient R2 of not less than 0.9995 will be obtained (Figure 1).
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0 2 4 6 8 10 12 14 16 18 20
Actu
al v
alue
[g/L
]
Set value [g/L]
Linearity Glucose
y = 0,9707x + 0,0709
R² = 0,9998
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y = 1,0165x + 0,0753R² = 0,9996
Actu
al v
alue
[g/L
]
Set value [g/L]
Linearity Glucose
Figure 1: Linearity of Glucose (R2=0.9998) and Lactate (R2=0.9996)
2. Measurement Principle
GlucoseThe enzyme glucose oxidase (GOD) is used for the detection of glucose.
Glucose Gluconolactone
In presence of oxygen, glucose oxidase catalyses the transformation of β-D-Glucose to D-Glucono-δ-lactone and hydrogen peroxide. The Glucose content is measured indirectly via the formed peroxide, which is oxidized to water and oxygen during the amperometric measurement.
LactateThe enzyme lactate oxidase (LOD) is used for the detection of lactate.
L-Lactate Pyruvate
In presence of oxygen, lactate oxidase catalyses the transformation of L-Lactate to Pyruvat and hydrogen peroxide. The Lactate content is measured indirectly via the formed peroxide and the amperometric measurement.
Amperometric measurementThe resulting hydrogen peroxide is oxidizedto water and oxygen generating ameasureable electric current which isdirectly proportional to the parameterconcentration. This signal is based on a calibrated linear range and gives the in-situ concentration.
H2O2 → O2 + 2 H+ + 2 e-
PrecisionThe typical variation about the mean value is below 1.5% (Figure 2), except for the low concentrations (< 2.5%).
0,0%
0,5%
1,0%
1,5%
2,0%
2,5%
3,0%
0,50 1,05 2,06 3,92 10,37 19,90
Varia
tion
[%]
Glucose [g/L]
Precision Glucose
0,0%
0,5%
1,0%
1,5%
2,0%
2,5%
3,0%
0,25 0,53 0,97 1,96 5,03 10,4
Precision Lactate
Lactate [g/L]
Varia
tion
[%]
Figure 2: Precision of Glucose and Lactate
RecoveryThe recovery of the glucose and lactate values is shown in figure 3.
0%
20%
40%
60%
80%
100%
120%
0,50 1,05 2,06 3,92 10,37 19,90
Reco
very
[%]
Glucose [g/L]
Recovery Glucose
0%
20%
40%
60%
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120%
0,25 0,53 0,97 1,96 5,03 10,4
Reco
very
[%]
Lactate [g/L]
Recovery Lactate
Figure 3: Recovery of Glucose and Lactate
Operational stabilityLong term stability for the application Glucose|Lactate has been document tested for 5,000 measurements or 14 days.
Operational stabilityLong term stability for the application Glucose|Lactate is guaranteed for 5,000 measurements or 14 days. Figure 4 shows a typical profile during the load test within the QA procedure over 5.000 assays (every two minutes = 7 days).
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Glu
cose
[%]
Measurement [number]
Long term stability: Glucose
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Lact
ate
[%]
Measurement [number]
Long term stability: Lactate
Figure 4: Load test of Glucose and Lactate sensors
Shelf life Glucose- | Lactate-Sensors have a shelf life of at least 12 months at room temperature.
ConsumablesConsumables for the application Glucose | Lactate are listed in table 1:
Table 1: List of BioPAT® Trace consumables
Article Article number
Tubeset Dialysis BPT0003
Transport buffer 20x, for cell cultivations (Glucose | Lactate)
BPT0006
Transport buffer 5x, for microbial cultivations (Glucose | Lactate)
BPT0060
Membranes for Dialysis Probe; 5/pk
BPT0024
Calibration Standard 0.5 g/L Glucose, 0.25 g/L Lactate
BPT0011
Calibration Standard 2 g/L Glucose, 1 g/L Lactate
BPT0010
Calibration Standard 10 g/L Glucose, 5 g/L Lactate
BPT0007
Cleaning solution BPT0044
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