Dissolved Oxygen Control PID Tuning for Cell Culture via Chemically Produced Demand Simulation using the

BioFlo 320 Bioprocess Control Station

®
John Longsworth, Nina Schillings, and Ma Sha
Applications lab, Eppendorf Bioprocess Center, Enfield, CT, USA .
Contact: bioprocess-experts@eppendorf.com

a
Abstract
Eppendorf bioprocess controllers, such as BioFlo 320 Bioreactor Control System, use proportional– integral–derivative (PID) control mechanism for variety of processes. This includes DO control. The PID control loop mechanism for DO control is essential for optimal bioreactor cell culture. Although the D value is not user adjustable
in Eppendorf controllers, the P & I values can be changed. The default P and I values provided with the controller are a good starting point but may not be optimal for specific cell culture processes or vessels.
This protocol introduces a method developed at Eppendorf bioprocess applications lab to optimize the PI values on Eppendorf bioprocess controllers including BioFlo 320. The method is based on pre-programmed delivery of chemical oxygen scavengers to accurately simulate the oxygen demand throughout a bioreactor cell culture
process. This chemical simulation allows for quick and accurate tuning of PI values for cell culture DO control. The method can be adopted to fermentation PI tuning as well. However, a more aggressive gassing demand model will need to be established by the user to match the actual gassing demand from fermentation process.

Basic Simulation Procedure Using Time Profile Advanced Simulation Procedure Using BioCommand®
A B C D
100 100 7 100
A A
7 100 7 7
90 90 90 90 100 14 0.7
6 6 6 6
80 80 80 80 90
12 0.6
70 5 70 5 70 5 70 5 80
60 60 60 60 70 10 0.5

mL/min
mL/min
4

mL/min
4 4 4
mL/min

% DO
% DO

% DO
% DO

50 50 50 50 60
8 0.4

mL/min
% DO
3 3 3 3

SLPM
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50
30 2 30 2 30 2 30 2 6 0.3
40
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10
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10 10 10
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0 2 0.1
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DO Pump Speed
EFT (minutes) EFT (minutes)
Fig. 1: BioFlo 320 experimental Fig. 2: Percent dissolved oxygen vs. sodium sulfite pump speed, time profile simulation. Fig. 4: BioCommand® block
setup A: P= 5; I= 0.3 B: P= 3.5; I= 0.195 C: P= 2; I=0.09; D: P= 0.7; I= 0.06 diagram program for PFR. B
B
100 14 0.7
90
0.7 12
Table 1: BioFlo 320 settings
90 0.6
A B C D 80
1 1 1 1 80
0.6 70 10
Loop Mode Setpoint 0.9 0.9 0.9 0.9
70
60
0.5

Agitation On, 200 rpm 0.8 0.8 0.8 0.8 8 0.4

% DO
0.5

SLPM
60 50
0.7 0.7 0.7 0.7
Clockwise

mL/min
0.4 40 6 0.3
0.6 0.6 0.6 0.6 50

SLPM
% DO
SLPM

SLPM

SLPM
SLPM

Temperature On 37 °C 0.5 0.5 0.5 0.5 40 0.3

30 4 0.2
0.4 0.4 0.4 0.4 20
pH On 7.0, Deadband 0.1 0.3 0.3 0.3 0.3
30
0.2 10
2 0.1

Sparge Gas 3-Gas Mix 0.041 SLPM 0.2 0.2 0.2 0.2 20
0.1
0 0 0
0.1 0.1
minimum, 1 0.1 0.1 10

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0 20 40 60

DO On 50 % EFT (minutes) EFT (minutes) EFT (minutes) EFT (minutes)

DO [%]
EFT (hours)
Air [SLPM] O2 [SLPM]
DO Pump Speed O2 Air CO2
Volume On 3 L, Deadband 0.1 O2 Air CO2

Pump 4 On, Harvest 100 rpm Fig. 3: Gas flows over time, time profile simulation. Fig. 7: Gas demand for 10 L Fig. 5: Percent dissolved oxygen vs. sodium Fig. 6: Gas flows over time, BioCommand
(Scale) A: P= 5; I= 0.3 B: P= 3.5; I= 0.195 C: P= 2; I=0.09; D: P= 0.7; I= 0.06 CHO culture sulfite pump speed, BioCommand simulation. simulation.
A: P= 2; I= 0.09 B: P= 0.7; I= 0.06 A: P= 2; I= 0.09 B: P= 0.7; I= 0.06
Dsicussion Discussion
The step functions used in the time profile approximated the oxygen demand created by an actual CHO culture. This method allowed us to simulate CHO culture The use of a BioCommand block diagram program allows for a more precise simulation of cell culture oxygen demand. A comparison of Figures 5 and 6 to real CHO
oxygen demand sufficiently well to test different PI values efficiently. This basic PI tuning method does not require additional software or programming experience. culture gassing data (Figure 7) demonstrates the feasibility of this method. By using an exponential equation to control the pump flow rate, operators can more
In the CHO cell batch culture example above, values of P=2 and I=0.09 were found to be the most effective. These values reduced the fluctuations of the dissolved accurately mimic oxygen demand during the initial and log phases of cell growth. This example simulation was designed for CHO cell culture. But the methodolo-
oxygen around the setpoint and minimized fluctuations in the gas flows. gies described in this protocol can be easily adapted to any cell type or vessel size. Both simulation methods can be effective for tuning PI settings and optimizing
controller response to cell culture or fermentation oxygen demand. A more aggressive model may need to be established by the user to match the increased gassing
demand from a fermentation process, however.
Literature
[1] How to Measure and Calculated OTR Using a New BrunswickTM Fermentor. 2013, Applications Training Document, Eppendorf. Inc.
[2] “A Guide to Calibration on the BioFlo® 120 and BioFlo® 320: Dissolved Oxygen Sensors.” Short Protocol #40, Eppendorf. Inc. Your local distributor: www.eppendorf.com/contact · Eppendorf AG · Barkhausenweg 1 · 22339 Hamburg · Germany · www.eppendorf.com

[3] Eppendorf BioFlo® 320 Operating Manual, Eppendorf. Inc. Eppendorf® and the Eppendorf Brand Design are registered trademarks of Eppendorf AG, Germany. BioFlo® and BioCommand® are registered trademarks of Eppendorf Inc., USA. New Brunswick™ is a trademark of Eppendorf AG. www.eppendorf.com
[4] Dissolved Oxygen Control PID Tuning for Cell Culture via Chemically Produced Demand Simulation using the BioFlo® 320 Bioprocess Control Station. Short Protocol #32, Eppendorf. Inc. All rights reserved, including graphics and images. Copyright © 2020 by Eppendorf AG.