Evaluation of a Single-Use Bioreactor for the Fed-Batch Production of a Monoclonal Antibody - Despite different material, agitation, and aeration, the performance of the disposable bioreactor is simil

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Evaluation of a Single-Use Bioreactor for the Fed-Batch Production of a Monoclonal Antibody
Despite different material, agitation, and aeration, the performance of the disposable bioreactor is similar to that of stainless steel bioreactors.


BioPharm International Supplements


Material and Methods

Cell Culture and Bioreactor Operation


Figure 1. Description of the process and experimental scheme. The cell amplification was performed in T-flasks and shake-flasks, followed by a passage in a 2-L bag rocker and a 50-L bag rocker. The inoculum was splitted to inoculate the N-2 bioreactors (SUB and 5-L glass vessel bioreactor). The N-1 SUB seed train bioreactor was used to inoculate two 5-L glass vessel bioreactors and itself as production bioreactor with the remaining inoculum. The 5-L glass vessel seed train bioreactor was used to inoculate one other 5-L bioreactor and itself as production bioreactor.
The cell line used in this study was a Chinese hamster ovary (CHO) cell line developed for the production of a MAb. The cell culture process was performed using chemically defined cell culture media and feeds. A typical seed train was used for the production runs (Figure 1). After vial thawing, cells were grown in T-flasks and shake flasks for six days at 37C, 5% CO2, and then transferred into a 2-L Cultibag RM Optical (Sartorius Stedim Biotech GbmH), followed by a 50-L wave bag. The two last steps of the seed train were performed in bioreactors (N-2 and N-1 steps). To properly assess the performance of the SUB as a seed train bioreactor and a production bioreactor, the cell suspension from the 50-L wave bag was split in a 50-L SUB (Hyclone, Thermo Fisher Scientific Inc.) and a 5-L benchtop-scale glass bioreactor (BIOSTAT B-DCU Quad, Sartorius-Stedim Biotech GbmH) for the N-2 step. The N-1 step consisted of the passage of the N-2 bioreactor, removing part of the suspension, and adding fresh media. Then, the seed train in the 5-L glass bioreactor was used to inoculate two 5-L glass bioreactors for the production phase. The seed train in the SUB was used to inoculate, in parallel, one SUB and two 5-L glass bioreactors for the production phase. The experimental plan is described in Figure 1 and was performed twice.

The process developed in the 5-L glass bioreactors was adapted for the SUB as follows: pH, temperature, DO setpoints, and feeding strategy were unchanged compared to the 5-L bioreactor. Values for gas flow rates and agitation setpoint were scaled up by keeping the headspace renewal rate and the power per volume constant, respectively. The pH regulation was performed using CO2 in overlay and sodium hydroxide for the 5-L glass bioreactors.

Partial pressure of carbon dioxide (pCO2), pH, and oxygen (pO2) were controlled offline on ABL5 (Radiometer Medical). Viable cell density (VCD) and viability were measured using a Vi-Cell automatic cell counter (Beckman Coulter). Metabolites and electrolytes were controlled offline using a Nova Bioprofile 100+ (Nova Biomedical). Protein production was determined using a Protein A–based assay on the Gyrolab platform (Gyros).

Using a Disposable DO Probe

The 50-L SUB was operated using a TruLogic RDPD (R&D and process development bioprocess) controller (Finesse Solutions). The disposable TruFluor DO probe (Finesse Solutions) was compared to the InPro6800 polarographic probe (Mettler Toledo) at a setpoint of 40%. The disposable DO probe was inserted in a sleeve manufactured with the SUB 50-L bag. The sleeve contained the disposable sensor, and the probe contained a non-invasive reader connected to the transmitter. The standard DO probe was inserted with a Kleenpack connector (Pall Corporation) after sterilization. After insertion, the standard DO probe was calibrated as usual. The DO was controlled using the InPro6800 probe, but signals from both probes were recorded on the controller.


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