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BioPAT® Xgas Metabolic Calculations - 11 Pages

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BioPAT®  Xgas Metabolic Calculations

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sartoriusstedim biotech BioPAT® Xgas Metabolic CalculationsOnline Off-Gas Analysis Technical Note - General advice - Gassing strategy - Volumetric gas transfer - Gas measurement cases - Metabolic calculations - Data interpretation - Error determination The continued growth of the biotech and biopharmaceutical industry has been driven by the increasing use of therapeutics of large molecules produced by modified organisms. In biological manufacturing, validation and quality assurance documents submitted to regulatory agencies need to be compliant to a number of stringent criteria. Further, the costs related to any batch failure can become overwhelming. Therefore, accurate data on quality and performance aspects from process development and production is paramount. This has led to Lean Six Sigma and automation initiatives to improve process compliance | transparency and rationalize filing with the aid of electronic batch records and electronic signatures. Software technologies like BioPAT® SIMCA-online can use multi variant data analysis to produce these electronic batch trajectories. However, analytics must still be collected and fed into the software model to provide the assurance everything is on track. A non-invasive method for collecting critical online information comes from examining the changing gas composition as it passes through an aerobic cell process. The fermentation feed and oxygen is consumed at a measurable rate together with carbon dioxide, biomass and products being produced. These rates can be determined by using a Biostat® in combination with a BioPAT® Xgas. The gassing strategy control on the fermentor allows for accurate determination of gas composition input. The off-gas analyzer when placed outside the sterile barrier of the bioreactor exhaust vent determines the out gas composition. Herein, a detailed description is given on how this information can be used for metabolic calculations and practical advice for Biostat® u

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General advice for monitoring Off-gas The Biostat® hold up time and mixing efficiency determines the lag time and a change is detected from the inlet to the outlet. During this time the gases mixes with the culture and transfer back and forth into the various liquid, cell and gas phases. However, if the inlet oxygen or carbon dioxide gas flow rate is fluctuating in a narrow band of time (2–5 minutes) the hold-up time | mixing efficiency will result in a averaging of detected BioPAT® Xgas signal. This results in a higher degree of uncertainty and error. Therefore, it is advised to maintain a...

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Bubble Size Optimization Oxygen Demand Figure 1: three different modes of gas strategy possible with a Biostat® advance control. Biostat® gassing strategy The Biostat® advanced pO2 control allows parallel modification of all bioreactor parameters such as stirrer speed, flow rate for air and oxygen (and other parameters if configured). This simultaneous activation or change allows mimicking of all the common gassing strategies (figure 1) and allows the user to be resource efficient and optimize the gassing process control. Constant gas flow works by decreasing the flow of air and...

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Gas measurement cases Gassing with air Typical microbial applications will simply use process air through a ring sparger to aerate an aerobic cultivation in order to provide sufficient dissolved oxygen. The Biostat® vessel agitation rate and s ­ ystem pressure (only on steel vessel with pressure rating) can be used to increase the volumetric oxygen transfer rate when dissolved oxygen is limited or falls below the pO2 set point. Fortunately, these factors do not influence (or are accounted for by either the MFCs or BioPAT® Xgas) the measurement or control of the Biostat® gas flow rate and...

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O2-Enrichement The O2-Enrichment either uses a 3/2-way solenoid valve to select either air or O2 flow or individual MFCs to control the ratio of the two gases to the sparger. O2 is pulsed via a solenoid valve or MFC, enriching the oxygen percentage of the air to maintain the pO2 set point. FG,α N2,α = (FAir,α + 79.07 + FO2,α + 0a)/ FG,α O2,α = (FAir,α + 20.9 + FO2,α + 100b)/ FG,α CO2,α = (FAir,α + 0.03 + FO2,α + 0c)/ FG,α The MFCs can be integrated to measure and control the total gas flow rate via manual adjustment or automatically in conjunction with the controller. Therefore, the...

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Advanced additive flow Cell culture operations typically use 4 main gases; air, oxygen, carbon dioxide and nitrogen. Each gas servers a function and can be used at various stages throughout the process for controlling critical parameters. Advanced additive flow gassing strategy is furthermore systemdependent allowing gasses (Air, O2, N2 and CO2) to be directed to the sparger or routed to overlay. All gasses in use must be included in the inlet flow gas composition calculation. Please take note of the component percentages of a mixed composition gas cylinders as this can have a significant...

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Metabolic calculations Data interpretation In cases 1 to 3 the data from the inlet gas composition and the outlet is placed into the following equations to calculate; For a complete glucose conversion RQ will be 1 (six molecules of oxygen are used and six molecules of carbon dioxide produced). In other cases, the nutrient feed sources may have a different stoichiometric ratio of O2 consumption and CO2 production. Therefore, confirming the chemical decomposition of the nutrient source is needed to understand the theoretical output value of RQ. This calculation may also be dynamic as...

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Figure 2: Graphical plot of a 27 hour fed-batch fermentation of E.coli tracking the parameters in BioPAT® MFCS As the BioPAT® Xgas is dedicated to the 5L vessel exhaust gas outlet and the data from the Biostat® B’s MFC is logged into BioPAT® MFCS, this allows the dynamic calculation of OUR, CER and RQ shown in Figure 4. This calculation (combines; Case 1 and Case 2) factors in changes in bioreactor liquid volume, air and oxygen gas flow rate (inlet gas composition) as well as pressure and humidity compensation by the BioPAT® Xgas giving an accurate measure of cellular metabolic status as it...

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