Scale-Up and Comparison Studies Evaluating Disposable Bioreactors and Probes
Acceleron Pharma has based its manufacturing platform on disposable technology for cell culture, harvest, and purification. Cell culture was originally performed in GE WAVE bioreactors; however, as the demand for material increased, the need for a more robust, more tightly controlled system became essential. Acceleron chose to maintain disposability and purchased the HyClone stirred-tank single-use bioreactor (SUB) for process development with the goal of implementing the SUB into a new manufacturing facility based on disposable technology. Four 2-L Applikon and two 5-L Sartorius Stedim glass vessel stirred-tank reactors were used for small scale development, process optimization, and comparison between the traditional bioreactor and the disposable technology. Temperature, pH, and dissolved oxygen (DO) were controlled, and nutrients, metabolites, and gases were monitored off-line using a Nova BioProfile Flex. The 50-L and 250-L SUBs were used for scale-up and pilot runs with the optimized process (a 14-day fed-batch culture). Data on cell density, fractional viability, and protein concentration were collected and compared across the scales and types of reactors. The oxygen mass transfer coefficient (kLa) was also compared from vessel to vessel to aid in scaling up from the glass vessels to the SUBs. More recently, a 1,000-L SUB, integrated with a Finesse controller, was used for engineering runs. In addition to disposable vessel technology, studies were performed that compared fiber optic DO probes and sensors (which have the option of being disposable), to autoclavable polarographic probes. The preliminary studies indicate that the fiber optic technologies are interchangeable with the conventional polarographic probes. Based on this work, disposable technology was determined to be a desirable option for biopharmaceutical manufacturing at Acceleron Pharma.
In addition, to simplify the manufacturing process and create a completely disposable environment for cell culture, disposable dissolved oxygen (DO) probes and the fiber optic technology associated with them were evaluated. The fiber optic probes were compared directly with the traditional polarographic DO probes.
Materials and Methods
Cell Culture and Bioreactor Operation
The cells used for protein production were Chinese hamster ovary (CHO) cells, and all data presented are from one protein, product 1. All cell culture was performed in custom media and feed developed by Irvine Scientific. Atypical seed train was used for manufacturing runs. The original seed train consisted of WAVE reactors up to the 100-L and 500-L working volume scale for manufacturing. The current seed train in the new facility uses WAVE bioreactors for scale-up and SUBs for production vessels at the 50-L, 250-L, and 1,000-L scale. Development runs were performed in four 2-L Applikon glass vessel reactors with ADI 1030 controllers and two 5-L Sartorius Stedim bioreactors with A+ and B+ controllers. Scaled-up runs were performed in 50-, 250-, and 1,000-L SUBs. The 50-L SUB was integrated with an Applikon I controller, and the 250- and 1,000-L SUBs were integrated with a Finesse TruLogic RDPD controller. The parameters that were logged and optimized included feeding strategy (i.e., schedule, volumes), pH, temperature, DO, and agitation set points, and flow rates. Partial pressure of carbon dioxide (pCO2) levels also were monitored and controlled. Viable cell density and viability were measured using a Cedex automatic cell counter (Innovatis) or a BioProfile Flex (Nova Biomedical). Protein production was determined using a Protein A assay on an Agilent 1100 series high performance liquid chromatography (HPLC) instrument. Some nutrients, metabolites, and electrolytes (L-glutamine, glucose, glutamate, lactate, ammonium, and sodium, calcium, and potassium ions) were monitored offline using the BioProfile Flex.
Multiple scale-up parameters were analyzed to transfer the optimized process from the glass vessels to the SUBs. Initial values for gas flow rates were calculated using vessel volumes per minute. For agitation speed, tip speed, power per volume, and torque, calculations were performed and evaluated. Power per volume was chosen to scale up the agitation speed. After the flow rates and agitation speed had been approximated, oxygen mass transfer coefficient (kLa) studies were performed in the 50-LSUB to determine the efficiency of the calculated values compared with small-scale studies.
Fiber Optic Autoclavable and Disposable Probes
Different configurations of fiber optic technology were evaluated. Autoclavable oxygen probes for in-line measurement from PreSens and Finesse (TruDO Optical) were studied in the 5-L and 2-L glass vessels, respectively. Measurements were taken every minute and compared directly to the Mettler-Toledo (InPro6800) polarographic probes integrated with the controllers. Preliminary studies also were performed in shaker flasks using the PreSens non-invasive oxygen sensors based on the same fiber optic technology. The values from these disposable probes were compared to offline measurements of the BioProfile Flex. Lastly, the disposable option available from Finesse (TruFluor DO) was evaluated in the 50-L SUB and compared directly with autoclavable polarographic DO probes. The TruFluor DO is available in a sleeve that is manufactured with HyClone's bioprocess container (bag) for the SUBs. The sleeve contains the disposable sensor, and inserted into the sleeve is a non-invasive optical reader connected to a transmitter.
Taking into account all parameters optimized in the 2- and 5-L vessels, the final process to transfer and scale-up to the SUBs was a 14 day fed-batch culture with a day 2 feed of 12% of the working volume, a temperature shift mid-culture, and a pH drift after day 9 when necessary. The SUBs have a dual sparger option (open pipe or frit). The open pipe sparger provides air to the culture while stripping excess CO2. If excess CO2 is stripped, the pH drift may not be necessary because the pCO2 levels will be decreased.
Comparing Traditional and Fiber Optic Probes
Finesse's TruDO Optical uses technology similar to that of the PreSens probe. However, the trends between the traditional probe and the fiber optic probe do not overlay exactly. As shown in Figure 7B, data from the TruDO Optical show a 5–8% upward shift in the pO2 level compared to the trend captured by the polarographic sensor. Based on discussions with Finesse, this shift is caused by the temperature at which the two different probes were calibrated. The TruDO Optical comes with a factory calibration at room temperature, whereas the polarographic probe is calibrated in-house at 37 °C. In addition, the TruDO Optical data appear to be more noisy. This may come from the number of readings (reading are taken every minute) compared with the number of readings taken from the traditional probe (every three minutes). Additional studies must be run to explain or correct for these differences.
Finally, the TruFluor DO was evaluated in the 50-L SUB. Figure 7C shows the pO2 levels over time in culture for both the disposable TruFluor DO and the traditional probe. The trends are identical between the two probes. Further studies are being performed to confirm direct comparability. These preliminary data show that disposable probe options available are interchangeable with the traditional technology.
An optimized and transferable process was developed in the 2- and 5-L glass vessels by monitoring, controlling, and adjusting process parameters such as pH, temperature, and partial pressure of carbon dioxide (pCO2). The final process, a 14 day fed-batch culture with a day 2 feed of 12% of the working volume, a temperature shift mid-culture, and a pH drift after day 9 when necessary, was transferred to 50-, 250- and 1,000-L stirred-tank single-use bioreactors (SUBs). The performance of the optimized process was comparable across all scales, determining that the HyClone SUB was a suitable disposable bioreactor for manufacturing at Acceleron.
In addition to implementing disposable bioreactors, fiber optic dissolved oxygen probes and sensors were evaluated. Autoclavable probes from both PreSens and Finesse were compared to the traditional polarographic dissolved oxygen probes, and the disposable sensor from Finesse was also evaluated. Although additional studies need to be performed, initial results showed that the fiber optic technology used in the disposable sensors was comparable to the technology used in the traditional sensors and was feasible for implementation in Acceleron's biopharmaceutical manufacturing process.
Alissa Fernald is a cell culture engineer, Anna Pisania is a senior cell culture engineer, Esam Abdelgadir is a manufacturing associate 2, Jesse Milling is a manufacturing associate 2, Tod Marvell is a cell culture manager, manufacturing, and Bob Steininger is the senior vice president of manufacturing, all at Acceleron Pharma, Cambridge, MA, 617.649.9326,
1. Trummer E, Fauland K, Seidinger S, Schriebl K, Lattenmayer C, Kunert R, et al. Process parameter shifting: Part I. effect of DOT, pH, and temperature on the performance of Epo-Fc expressing CHO cells cultivated in controlled batch bioreactors. Biotechnol Bioeng. 2006 Aug 20;94(6):1033–44.