Scalable perfusion cell culture using Xcellerex™ X-platform single-use bioreactors
This application note shows the Xcellerex™ X-platform single-use bioreactors to be suitably robust and scalable when performing a perfusion cell cultivation process.
Xcellerex™ X-platform single-use bioreactors have shown:
- Functional capability of high-performance cell growth and cell viability under difficult perfusion process conditions.
- Scalability from lab-to-manufacturing within the Xcellerex™ X-platform bioreactor range (50 to 200 L).
Introduction
Perfusion cultivation is undertaken by continuously feeding fresh medium to a bioreactor and continually removing the cell-free spent medium while retaining the cells in the reactor.
Xcellerex™ X-platform single-use bioreactors are designed for flexibility and scalability, with software features to ensure compatibility and functionality when using available perfusion technology. Perfusion ports on standard bag variants, standard bioreactor weight control functionality, and optional automated cell bleed functionality are keys to this enablement.
Materials and methods
Primary equipment for this study was the Xcellerex™ X-platform bioreactors (50 and 200 L) and the XCell ATF6 system (Repligen). Other equipment such as pumps and analytical instrumentation were common lab equipment and were not central to the output or conclusions of the study.
A CHO-K1 cell line (ExcellGene) was engineered to produce a mAb.
We used a cell culture medium comprised of: HyClone™ ActiPro™ cell culture media, HyClone™ Cell Boost™ 1 supplement (14% of 10% w/v), HyClone™ Cell Boost™ 3 supplement (16% of 5% w/v) modified for 10 g/L glucose in the final formulation.
The consumables were X-50 Perfusion II and X-200 Perfusion II cell culture bag assemblies. These single-use assemblies incorporate the novel 6B-R50 impellers with 115 µm and 203 µm porosity spargers, respectively. The XCell ATF6 system used a Cytiva 0.2 µm, 2.1 m2 hollow fiber (HF) filter (CFP-2-E-55SMO).
Perfusion cell culture process attributes
Cell culturing process layout
The perfusion process was run using the equipment layout represented in Figure 1. Key attributes of the design are a fixed feed rate and variable harvest rate pump configuration for control of the perfusion feed and permeate rates, respectively. The cell bleed rate was controlled using an integrated viable cell density (VCD) sensor (Hamilton) that triggered a bleed pump to remove cells from the bioreactor when the VCD exceeded a permittivity setpoint. The key bioreactor process set points are in Table 1. The ATF6 controller set points were 16 LPM for both the pressure cycle (P-cycle) and exhaust cycle (E-cycle).
| Parameter | Set point |
| Operating reactor volume | 200 L (kg) and 50 L (kg) |
| Dissolved oxygen (DO) | 40% |
| pH | 6.85 (X-50) 6.90 (X-200) |
| pCO2 | 70 mbar (Note: CO2 was monitored, not controlled) |
| Agitation | 90 W/m3 160 rpm, upflow (X-200); 216 rpm, upflow (X-50) |
| Temperature | 37°C |
Fig 1. Process layout for a perfusion run using an Xcellerex™ X-200 bioreactor.
pH control during the perfusion cell culture process
pH was controlled using a split range PID controller utilizing CO2 for acid control and 1M sodium carbonate for base control. A deadband of 0 and dead zones of 2% control variable (CV) on either side of the 50% midpoint were configured.
Gassing strategy during the perfusion cell culture process
The Xcellerex™ X-200 bioreactor dissolved oxygen (DO) control scheme (Fig 2) consisted of four segments and was mapped to Sparge 1. The first control segment ran between zero and 20% CV and ramped air from zero to 10 SLPM. The second control segment ran between 20% and 40% CV and ramped air from 10 to15 SLPM and oxygen from 0 to 5 SLPM for a total flowrate of 20 SLPM (0.1vessel volumes/min [VVM]) at the end of the segment. The third segment ran from 40% to 70% CV. It maintained 15 SLPM of air, and ramped oxygen from 5 to 15 SLPM for a total flowrate of 30 SLPM (0.15 VVM) at the end of the segment. The last segment ran from 70% to 100% CV. It maintained a constant total flowrate of 30 SLPM by ramping down air from 15 SLPM to zero and ramping up oxygen from 15 to 30 SLPM.
The Xcellerex™ X-50 bioreactor DO control scheme (Fig 3) was scaled from the established X-200 DO controller configuration to maintain the same oxygen transfer rate (OTR) and total scaled flow rate (in VVM) within each control segment. The OTR and calculated air and oxygen flow rates were estimated based on models of the characterized kLa (volumetric mass transfer coefficient) data for the two bioreactors used (data is available in the Bioreactor Scaler Tool software). The OTR at 0.3 VVM at 100% CV for the Xcellerex™ X-50 bioreactor was lower when compared to the Xcellerex™ X-200 bioreactor due to a higher kLa for the latter under the same agitation and flow rate conditions (data not shown).
Fig 2. Xcellerex™ X-200 bioreactor dissolved oxygen control scheme.
Fig 3. Xcellerex™ X-50 bioreactor dissolved oxygen control scheme.
Exhaust management during the perfusion cell culture process
Both bioreactors were outfitted with primary and secondary hydrophobic gas filters, heated to 55oC with onboard filter heaters. The secondary filter was isolated with a pneumatic pinch valve designed to open if the headspace pressure were to exceed 0.03 bar (0.44 psi, 3 kPa).
Hollow fiber (HF) filter life
Filter life was monitored by calculating an IgG sieving coefficient, which is derived from the ratio of measured IgG concentrations in the permeate line and in the bioreactor. An IgG sieving coefficient ≤ 40% indicates unacceptable fouling and either terminates the run or necessitates a filter switch, depending on timing.
Perfusion cell culture strategy
The media feed rate was adjusted daily to target a cell-specific perfusion rate (CSPR) of 20 pL/cell/day based on the measured VCD when sampled daily. The harvest was variable and automatically controlled by the bioreactor to maintain a specified load cell weight (target 200 kg or 50 kg). Perfusion was initiated when the culture reached a density of ~ 5 to 8 ×106 cells/mL (day 3 to 4 of a typical run). Bleed was started when the culture reached a density of 80 to 85 × 106 cells/mL. The bleed was self-regulated by the bioreactor using an in-line VCD sensor (Hamilton); a pump was used to remove cells and media from the reactor when the permittivity measurement exceeded a set trigger value and turn off when the permittivity dropped below a setpoint.
Antifoam strategy during the culturing process
A 3% stock of antifoam comprised of 30% Antifoam C Emulsion (Sigma) in DPBS was prepared and autoclaved. A single bolus of 0.1mL antifoam per liter of culture medium was added prior to inoculation. Subsequent bolus additions during the bioreactor run were made using an integrated peristaltic pump and the onboard liquid transfer control phases in the software. Antifoam addition at the beginning of a run occurred at a rate of one 8.5mL dose added daily. The addition frequency was manually increased, based on visual observation of the foam levels, to one dose approximately every hour in the Xcellerex™ X-200 bioreactor and every 2 h in the Xcellerex™ X-50 bioreactor at the end of the run.
Results and discussion
Cell culture growth and viability of perfusion runs
The cell growth and cell viability results for the five runs undertaken (three for the X-200 and two for the X-50) are displayed in Figure 4. Notably, cell viability remained above 95% for the duration of all the bioreactor runs.
The runs demonstrate a consistent VCD increase, each reaching ~ 85 ×106 cells/mL in the range of Day 8 to10 and maintaining a steady VCD through termination of the run. The dip in VCD for the 50 L run 2 is attributable to unintentional overbleeding caused by debris settling on the VCD sensor; once the permittivity SP was adjusted versus an offline VCD reading, the VCD was restored above 80 ×106 cells/mL.
Fig 4. VCD and viability for Xcellerex™ X-200 bioreactor and Xcellerex™ X-50 bioreactor perfusion cell culture runs.
Metabolites: Glucose and lactate concentrations during the bioreactor perfusion process
Glucose and lactate concentrations are used to determine if a perfusion run is operating in a steady metabolic state. The glucose concentrations displayed in Figure 5 show profiles with consistent trends within bioreactor size, and an offset between bioreactor sizes, which may be attributed to differences in the individual unit feed/harvest pumps and/or variability in pump calibrations. Initial glucose measurements were ~ 10 g/L and remained ≥ 1 g/L at steady-state, indicating the target perfusion rate was sufficient. The lactate profiles shown in Figure 6 were also similar for the Xcellerex™ X-200 bioreactor and Xcellerex™ X-50 bioreactor runs, with peak lactate concentrations remaining ≤ 3 g/L and remaining low for the duration of the run.
Fig 5. Glucose concentrations for each of five process runs.
Fig 6. Lactate concentrations for each of five process runs.
IgG concentration and IgG sieving coefficient measurement within the single-use bioreactors
IgG concentration is a measure of the accumulation of IgG produced by the cell culture, and the sieving coefficient measures the efficiency of retention of IgG by the perfusion device. Both measures (Fig 7 and 8, respectively) are consistent within bioreactor size, and there is an offset between bioreactor sizes. As both the 50 and 200 L runs use the same size of filter, we attribute this to filter fouling, with greater accumulation of fouling seen with the larger bioreactor system which operates at a higher absolute permeate flowrate across the filter membrane. As the fouling layer will have filtration capabilities, we can expect that the 200 L run would accumulate more IgG (i.e., the filter flux is four times higher in the Xcellerex™ X-200 bioreactor than in the Xcellerex™ X-50 bioreactor, for the same volumetric permeate rate).
Fig 7. IgG concentrations for each of five process runs (measured in reactor).
Fig 8. IgG sieving coefficients for each of five process runs (permeate concentration vs reactor concentration).
Dissolved oxygen control within the single-use bioreactors
The dissolved oxygen was well controlled for all the Xcellerex™ X-200 bioreactor runs (X-200 Run 2 shown in Fig 9) and Xcellerex™ X-50 bioreactor runs (X-50 Run 3 shown in Fig 10). The controller output ramped against viable cell density during the initial phase of the process and remained stable throughout the process once the target VCD was achieved and maintained. Throughout the entire process, the controller output was stable with very little oscillating behavior about the setpoint observed. Lastly, the controller reached a peak output of 40% CV, which corresponds with a predicted OTR of about 9 mmol/L/h. As the maximum possible OTRs of the bioreactors are approximately 55 mmol/L/h and 40 mmol/L/h, respectively, this suggests the systems have considerable mass transfer headroom and can support the oxygen uptake rate of more demanding processes.
Fig 9. IgG concentrations for each of five process runs (measured in reactor).
Fig 10. IgG sieving coefficients for each of five process runs (permeate concentration vs reactor concentration).
Perfusion attributes during the cell culture growth process
The perfusion media feed, permeate removal, and cell bleed were well controlled for all the bioreactor runs (Fig 11). The pump speed for the media feed was adjusted daily to maintain a CSPR (cell-specific perfusion rate) of approximately 20 pL/cell/d. The bleed pump was turned on and off at a rate equivalent to 70 mL/min or 18 mL/min for the 200 L and 50 L bioreactors, respectively, in response to a permittivity signal that correlated with the target VCD of approximately 80 million cells/mL (Fig 4) .The pump speed for the permeate removal pump was self-regulated by the vessel weight PID controller and maintained the reactor weight SP at 200 kg and 50 kg respectively.
The control strategy for maintaining a constant CSPR led to volumetric perfusion rate and permeate rate increases in conjunction with VCD increases until the VCD exceeded the target of 80 million cells/mL. This event occurred roughly on day 9 for both reactor sizes. When the target VCD was reached, the cell-bleed controller started, and the VCD was maintained at steady state through the end of the run. For the Xcellerex™ X-200 bioreactor run 2, the perfusion rate peaked at approximately 1.7 VVD (vessel volumes per day), the permeate rate peaked at approximately 1.4 VVD, and the cell bleed rate peaked at approximately 0.4 VVD. The reduction of the cell bleed rate from day 15 to day 16 is coincident with a pH excursion caused by a CO2 outage. The run was terminated at day 22 without incident. For the X-50 run 3, the perfusion rate peaked at approximately 2.0 VVD, the permeate rate peaked at approximately 1.5 VVD, and the cell bleed rate peaked at approximately 0.5 VVD. The reduction of the cell bleed rate on day 13 is coincident with a temperature excursion caused by a power outage that affected the TCU (temperature control unit). The run was terminated on day 21 when the perfusion filter fouled.
Fig 11. CSPR and bleed rate for four of five process runs. Note: X-200 run 1 data not shown, but was in line with runs 2 and 3.
Foam management during the perfusion bioreactor process
The foam quantity in the headspace ranged from trace amounts of foam with little surface coverage towards accumulation of foam not exceeding ~ 1 cm in height. No exhaust filter fouling was observed.
Post inoculation, single boluses of antifoam were dispensed daily before ramping to the following addition frequencies: every 1 h in the Xcellerex™ X-200 bioreactor and every 2 h in the Xcellerex™ X-50 bioreactor. Based on the peak settings, which were achieved roughly when max steady state gas flowrate was achieved on approximately day 9 in all process runs, the Xcellerex™ X-50 bioreactor required proportionally double the concentration of antifoam to manage foam levels. This is most likely attributed to the smaller porosity spargers used for DO and pH control (115 µm in the Xcellerex™ X-50 bioreactor and 203 µm in the Xcellerex™ X-200 bioreactor).
Conclusions
These experiments have shown that the Xcellerex™ X-platform single-use bioreactors are suitably robust in performing a perfusion cell culturing process with excellent results and reproducibility. The perfusion attributes confirmed the process had seeded well and any process foam was controlled adequately.
The results show that Xcellerex™ X-platform single-use bioreactors are:
- Functionally capable of high-performance cell growth and cell viability under difficult perfusion process conditions.
- Scalable from lab-to-manufacturing within the Xcellerex™ X-platform bioreactor range (50 to 200 L).
Functional capability of the single-use bioreactors
The oxygen mass transfer capabilities of the Xcellerex™ X-platform bioreactors can support a wide variety of process types, including seed cultures with minimal oxygen demand, to the most demanding steady-state perfusion processes. The reactors controlled well in this study from inoculation through harvest, representing a range of 1 to 80 MVC/mL. The 50 and 200 L bioreactors operated at 16% and 22% of their maximum available oxygen transfer rate at peak cell density, respectively, suggesting ample performance headroom for more demanding processes exists.
Bioreactor scalability of the perfusion cell culturing process
The process proved to be scalable between 50 and 200 L.
When scaled down from 200 L, the process required only a marginal increase in oxygen concentration for the Xcellerex™ X-50 bioreactor to provide equivalent oxygen transfer rates, when power density and total volumetric flow rate were conserved. When scaled based on OTR, power density, and total volumetric flow rate, the key performance metrics for the process (growth profiles, metabolite trends, etc.) were equivalent when the 200 L process was scaled down to 50 L. Coupled with the commercially available Bioreactor Scaler Tool software, process scaling within the Xcellerex™ X-platform family of bioreactors is straightforward.
Xcellerex™ X-platform single-use bioreactors are designed for flexibility and scalability, with software features to ensure compatibility and functionality when using available perfusion technology. The availability of perfusion ports on standard bag variants, standard bioreactor weight control functionality, and optional automated cell bleed functionality are keys to this enablement, and all serve to provide integrated, automated perfusion capabilities that reduce risk and save time during setup.
Appendix
Table 2. Bioreactor process deviations recorded during study
| Bioreactor/run | Event | Affect / Action |
| 200 L Run 1 | Laboratory upset not related to running of the bioreactor after 17 days. | None required |
| 200 L Run 2 | Short outage of CO2 | Did not affect process performance |
| 200 L Run 3 | Hollow fiber filter fouling on Day 15 | Run terminated |
| 50 L Run 1 | 8h O2 outage on Day 3 | Cell growth was suppressed, and the intervention step was to increase CSPR to 30 pL/cell*day until normal doubling times were achieved. Data not included for comparison due to significant deviations in cell growth and metabolite trends, though the target VCD of 80 million cells/mL was ultimately achieved and maintained. |
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