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Steady State and Dynamic Control - 1 Pages

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Steady State and Dynamic Control

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Steady State and Dynamic Control Performance of the ambr® 15 Automated Micro Bioreactor System in a CHO Cell Batch Culture Sin Yee Yau PhD, Kenneth Lee PhD, Barney Zoro EngD Cell Culture Results Implementation of high throughput automated systems is recognised as a valuable approach in the field of bioprocess development (1).    Viabile Cell Count profiles (Vi-Cell) An efficient multi-parallel microscale bioreactor system can unlock development bottlenecks in a variety of bioprocess applications, such as cell line screening, media development, feed and process optimisation, QbD and DoE studies. Aims - Illustrate the capability of the ambr® 15 cell culture system with CHO cell cultures - Assess the degree of reproducibility for replicate ambr cultures - Investigate the resolution in CHO cell culture response for a variety of experimental conditions (signal to noise ratio) - Demonstrate process control performance - Steady State Control for standard culture operation - Dynamic Control for parameter shift applications Close grouping of replicates due to consistent conditions in ambr Lower cell growth at pH 6.9±0.05 Higher seeding density (sVCD) leads to lower VCD at day 10 DO tests (20,60%) very similar to standard conditions (not shown) (Slight scatter between replicates may be related to cell counting) Very close replicate grouping shows excellent culture repeatability Clear resolution between all three pH test conditions Higher sVCD leads to early glucose depletion (day 6) DO tests (20,60%) very similar to standard conditions (not shown) - 24 bioreactors run in parallel, as a single experiment - Standard Conditions: pH 7.1 ±0.1, DO 40%, 3x105 cell | mL - 6 (or 3) replicate cultures for standard (or test) conditions - CHO-K1S cells, Sigma ExCell ACF Medium, max volume 15 mL - Sigma Antifoam C, 20 uL added on days 0, 4, 7 - 37°C, 850 RPM (impeller tip speed 0.5 m | s) - Clear resolution between 3 and 8x105 cells | mL conditions - Slight scatter is observed in seeded cell density, as a result of the inherent variability in cell seeding. This is due to cumulative small errors in source cell count, sampling & pipetting, and final cell count - Very high viability with low scatter between replicates - Viability begins to decline after glucose is depleted, leading to lower viability for high seeding density, and pH 7.1 conditions - DO tests (20,60%) very similar to standard conditions (not shown) Cell Culture Conclusions - The ambr® 15 system performs very well with CHO cell cultures - Very good resolution in CHO culture response between test conditions (high signal to noise ratio) - High reproducibility between ambr® 15 CHO culture replicates demonstrates good process control performance within the ambr - Automated liquid handling substantially reduces laboratory work Results of various conditions reflect expected outcomes: - sVCD substantially influences the culture profile - A variety of pH conditions are possible; pH is a key parameter - DO was not a critical process parameter within this study The combination of tight process control performance, low replicate variation and high resolution between test results, illustrates the suitability of the ambr® 15 micro bioreactor system for parallel bioreactor tests in a wide range of cell culture applications. DO Control Test Setup Aim: Test a variety of DO values at steady state pH Control Test Setup Aim: Test a variety of pH values and ranges at steady state Dynamic Control Test Setup Aim: Test dynamic control, for parameter shifts (x3 bioreactors) pH Control Conclusions - Good steady state pH control, as 'wide range' or 'specific setpoint' - Individual vessel control of pH: CO2 gas and liquid base additions - Automatic liquid base addition as needed, e.g. through the night - Base settings are flexible e.g. gain, target pH, base concentration DO Control Conclusions - Good steady state control of dissolved oxygen at setpoint - Small deviations occur, due to gas exchange on cap removal - Rapid controller response compensates effectively - Individual vessel DO control: N2 | air ballast flow, plus O2 addition Dynamic Control Conclusions - Shifts occur smoothly and under control without overshoot - Control dynamics rapid compared to CHO cell culture - Enables common industry process 'shift' applications pH control test results: 7.1 ± 0.1 (x6 bioreactors) DO control test results (x12 bioreactors) (1) pH controlled down to upper limit by CO2 gas input (1b) an example CO2 gas flow trace (2) pH control at lower limit by liquid base additions (2b) an example pH trace, saw-tooth line due to base additions - Good DO control at setpoint - Good resolution between setpoints - Minor fluctuations occur due to bioreactor cap removal pH control test results: 7.0 ± 0.2 (x3 bioreactors) - (1a) pH controlled down to upper limit by CO2 gas - (1b) an example CO2 gas flow trace - pH generally fluctuates within the specified range - Only one base addition was required Base addition Temperature shift test - Temp. shift 37    33°C - Temp. shift time ~ 0.5 h - Time is independent of culture properties pH shift test (x3 bioreactors) (1) Excellent steady state DO control without cap removal (2) O2 addition rate increases with culture growth Total gas flow was approx. 0.02 vvm in this study - pH shift 7.2 -► 7.0 - pH shift time ~ 2 h - Time depends on culture properties e.g. metabolism DO shift test (x3 bioreactors) pH control test results: 6.9 ± 0.05 (x3 bioreactors) - pH controlled to setpoint by CO2 gas addition throughout - CO2 traces omitted for clarity - Small pH fluctuations due to minor pH offset adjustments (1) Small spikes occur due to cap removal Gas is exchanged between the headspace and sterile air in the laminar flow hood (2) The ambr 15 control system responds, adjusting O2 gas flow rate to compensate - DO shift 60 -► 40 % - DO shift time ~ 1 h - Time depends on O2 uptake rate TAP Biosystems is now part of the Sartorius Stedim Biotech Group Sartorius Stedim Biotech UK    Phone +44.1763.227200 York Way, Royston,    Fax +44.1763.227201

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