IVD Technology Magazine | IVDT Article Index
Originally published July 1996
Factors affecting antibody production efficiency in hollow-fiber bioreactors
Rudolf J. Czirbik, Steven M. Rosen, Diane M. Trunfio, Ellyn W. Fischberg-Bender, and Stuart M. Palmer
All cartridges produce mouse monoclonal antibodies, but some are more efficient than others.
The production efficiency of 10,000- and 30,000-MW cutoff hollow-fiber bioreactor cartridges using the Cellex, Inc., Acusyst Jr. and the Unisyn Technologies, Inc., CP2000 bioreactor systems was investigated. Both cartridge types produced significant quantities of mouse monoclonal antibody, but the production efficiency (milligrams of antibody produced per liter of medium consumed) was higher for the 30,000-MW cartridge. Also, the smaller pore size cartridges displayed a lengthened lag phase in antibody production and cell growth of 26 to 33 days versus 11 to 20 days for the 30,000-MW cartridge. High intracapillary medium feed rates (5.0 L/day for 30,000 MW and 8.4 L/day for 10,000 MW) may reduce overall antibody production efficiency, and lower feed rates (3.5 and 7.2 L/day, respectively) may produce more antibody per liter of medium consumed.
The periodic removal of excess cells from confluent bioreactor cartridges can augment antibody production and extend the length of the run. Batch harvesting of antibody may lead to more highly concentrated antibody product than continuous harvesting.
Bioreactor systems that use hollow-fiber cartridges as a matrix for the growth of hybridoma cells are an efficient way of producing multigram quantities of monoclonal antibody.13 Hollow-fiber cartridges can produce highly concentrated monoclonal antibody relatively free from contamination by cellular or serum proteins.4 These cartridges are available primarily in molecular weight pore sizes of 10,000 (e.g., renal dialysis cartridges) or in larger pore size configurations manufactured exclusively in bioreactor systems.
Using the Cellex, Inc. (Minneapolis), Acusyst Jr. and the Unisyn Technologies, Inc. (Tustin, CA), CP2000 bioreactor systems, we investigated the ability of several types of hollow-fiber cartridges to sustain the growth of mouse hybridoma cells and to produce antibody. We also studied the effects of several key operating parameters, including medium feed rate, antibody harvest method, cell removal, and glucose utilization rate (GUR) on antibody production.
Materials and Methods
Cell Lines. Mouse hybridomas VA1 18D7.3.2.4 (antivalproic acid) (IgG1), MOR 9B1 (antimorphine) (IgG1), METH7-4B6 (IgA) (antimethamphetamine), and PCP1.9D10 (IgG1) (antiphencyclidine) were grown in Iscove's Modified Dulbecco's Medium (IMDM) (Mediatech, Inc., Herndon, VA) supplemented with 2 mM L-Glutamine (Gibco, Grand Island, NY) and 10% fetal bovine serum (FBS) (Hyclone Labs, Logan, UT). All hybridomas were generated using the NSO fusion partner.
Baseline static culture productivity for each cell line was established by replicate seeding of 5 x 104 cells/ml in growth medium (described above) in standard T25 flasks (Corning, Corning, NY). The flasks were incubated at 37°C and 7% CO2 for 72 hours; then the medium was removed and centrifuged at 1000 rpm for 10 minutes. The resulting supernatant was assayed for total and specific immunoglobulin by ELISA (Table I), as described below in the section "Determination of Antibody Concentration." (Tables not yet available on-line.)
Bioreactor Systems and Hollow-Fiber Cartridges. The Acusyst Jr. was run with the Acucell 1100 (Cellex, Inc.) 10,000-MW cartridge with 19 sq ft of surface area and 120 ml of extracapillary space (ECS) (Table I). The cartridge uses a reverse-flow cycling process to actively transport medium back and forth between the intracapillary (ICS) and the ECS. The system was unique to the Acusyst Jr. and its cultureware and was not available for the other cartridges or systems tested.
The CP2000 was used to compare two other 10,000-MW cartridges: the Clirans T220 cuprammonium rayon kidney dialysis cartridge (19-sq-ft surface area, 100-ml ECS) (Terumo Corp., Somerset, NJ) and the BR2010 ampholytic copolymer membrane cartridge (35-sq-ft surface area, 210-ml ECS) (Unisyn Technologies, Inc., Tustin, CA). These were also compared to the BR3530 cellulose acetate 30,000-MW bioreactor cartridge from Unisyn Technologies, Inc. (Table I).
General Bioreactor Run Parameters. The mouse hybridoma cultures were grown in the IMDM formulation. Cells in log phase growth (>85% viable by trypan blue exclusion) were centrifuged at 400 x g for 5 minutes, then resuspended in fresh growth medium. Approximately 109 cells were then introduced into the ECS of each bioreactor cartridge by a large syringe.
The hollow-fiber bioreactor cartridges were tested in the two bioreactor systems with IMDM as described above. The FBS concentrations of the ICS medium for all the bioreactor experiments except those using MOR 9B1 and PCP1.9D10 were 3% for the first 20 L of media and 1% for the remainder of the experiment. The FBS concentration in the ICS for the MOR 9B1 and PCP1.9D10 runs was 3% throughout the course of the experiment. The serum concentration for the MOR 9B1 and PCP1.9D10 experiments was kept at 10% throughout. The FBS concentration of the ECS for the other cell lines was gradually reduced from 10 to 1% by introducing the ICS feed medium throughout the experiment.
The operating parameters of each bioreactor run were manipulated to maximize productivity. They varied according to the requirements of the cell line, bioreactor system, and bioreactor cartridge. The initial intracapillary recirculation rates for each of the CP2000 runs ranged between 350 and 550 ml/min and were gradually increased to the maximum setting of 999 ml/min. The recirculation rate of the Acusyst Jr. was between 200 and 400 ml/min. It could not be run with a recirculation rate >400 ml/min because of pump system limitations. Temperature was automatically maintained by the two systems at 37 ±0.5°C. The 100% air and carbon dioxide flow rates were held at approximately 100 and 510 cm3/min, respectively, throughout the course of each CP2000 run. The pH for the runs ranged between 6.9 and 7.3.
Determination of Glucose Utilization Rate. The GUR is an indicator of cellular metabolism and cell growth.5 The relative ability of the cartridges to sustain the growth of cells was assessed by measuring the amount of glucose from the ICS used by the cells. Glucose concentration in the circulating bioreactor ICS medium was determined using Roche Glucose Reagent and Roche Calibrator for Serum on the COBAS BIO instrument (all from Roche Diagnostic Systems, Inc., Somerville, NJ).
A bioreactor with a low GUR probably has too few metabolically active cells to demonstrate adequate antibody production. A high GUR may lead to rapid overgrowth and premature senescence. We have, therefore, established a target GUR for the different bioreactor configurations. At the target GUR, run parameters such as medium feed rates and recirculation rates are optimized to maximize antibody production. The target GUR is determined empirically by growing each cell line at different medium feed rates; it depends on the cell line and cartridge. A target GUR of 250 mg/hr for each bioreactor system was established.
The GUR (expressed as mg/hr) was determined by the formula:
GUR = {feed rate (ml/hr) x [glucose in (mg/L)glucose out (mg/L)]}/1000
where glucose in is the concentration of glucose in the feed medium entering the bioreactor system, and glucose out is the concentration of glucose in the recirculating medium.
Determination of Antibody Concentration. The concentrations of secreted antibody in the bioreactor systems were determined by ELISA. Ninety-six-well microtiter plates (Costar Corp., Cambridge, MA) previously coated with drugbovine serum albumin (BSA) conjugates were blocked to prevent nonspecific binding with 1% BSA in phosphate-buffered saline (PBS)/ azide. Serial dilutions of culture supernatant drawn from the bioreactor ECS were applied to the specific plate and a standard curve for assay quantitation was generated by incorporating known concentrations of purified antibody in parallel wells on the plate.
After incubation of the samples and standards, unbound antibody was removed by washing the plates in PBS/ Tween 20 (Sigma Biochemicals, St. Louis). The bound antibody was detected by the addition of alkaline phosphataselabeled antimouse antibody (Zymed, San Francisco). The concentration of antibody was determined spectrophotometrically at 405 nm with a CR340 plate reader (SLT Corp., Salzburg, Austria), and data analysis was performed using the ELISA version 3.0 computer software (Meddata, Inc., New York City).
Criteria for Cell Growth and Antibody Production. The rates of antibody production and cell growth in the Clirans T220, BR2010, and the Acucell 1100 hollow-fiber cartridges were compared to those of the Unisyn BR3530 cartridge. For consistency, arbitrary criteria were used for between-run comparisons. One criterion was the number of days required to reach a target GUR level of 250 mg/hr; another was the number of days required to produce 1 g of antibody. These criteria defined the lag phase in comparisons of the same cell line grown in different cartridges or different cell lines or conditions for a given cartridge.
The effect of pore size on antibody production was determined by measurement of the amount of antibody produced (in milligrams) per liter of ICS feed medium consumed over a period of time (production efficiency). Because the runs varied in duration, day 40 was chosen arbitrarily to be representative for each run. The last criterion was the total antibody produced (in grams) by day 40 of the run.
Using the VA1 18D7 cell line, we compared the rate of antibody production and cell growth in the CP2000 bioreactor system using the Clirans T220 (Terumo Corp., Somerset, NJ), the BR3530, and the BR2010 (Unisyn Technologies, Inc.) bioreactor cartridges. The MOR 9B1 cell line was used to compare the Acusyst Jr. bioreactor system and Acucell 1100 cartridge with the rates of antibody production and cell growth in the CP2000 bioreactor system using the BR3530 cartridge (Table I).
Effect of Medium Feed Rate on Antibody Production. The effect of medium feed rate on antibody production was examined in both bioreactor systems. In the CP2000 bioreactor system that effect was studied using the BR3530 cartridge inoculated with the METH7-4B6 cell line.
Cell Removal. The effect of cell removal on antibody production and on bioreactor run length was determined in the CP2000 with the BR3530 cartridge and the PCP1.9D10 cell line. Mammalian cell cultures grown in hollow-fiber cartridges have a finite life. It has been speculated that accumulating cells decrease the productivity of the system by inhibiting the transport of nutrients across the hollow-fiber membrane, causing cell death. Whether periodic removal of excess cells from the bioreactor cartridge ECS would extend run length and improve faltering run parameters such as GUR and antibody production was investigated.
Harvest Methods. Antibody was harvested from the ECS regularly beginning within two weeks after the cartridges were inoculated. Two harvest modes were used, both using the same equipment. In a continuous mode of harvest, a peristaltic pump (Cole-Parmer, Chicago) withdrew medium from the ECS at a slow, constant rate (50 to 200 ml/day). At the same time an equal volume of fresh medium was introduced into the ECS by the same pump by using a separate piece of tubing run through the pump. Harvest bottles were changed periodically, and their contents centrifuged at 1000 * g for 10 minutes to remove cells and debris. The antibody-containing supernatant was then stored at ¾20°C before testing. The second harvest mode was a batch method: A fixed volume of medium (50250 ml) was pumped at once into the ECS, displacing an equal volume of antibody-rich medium from the chamber. This was repeated two to three times per week. The harvests were then processed in the same way as they were with the continuous mode.
Results
Cell Growth and Antibody Production. For the MOR 9B1 cell line and the BR3530 cartridge, it was determined in two separate runs that an average of 19 days were required to produce the first gram of antibody. This same cell line in two experiments with the Acucell 1100 cartridge required 31 days to produce 1 g of antibody (Table I).
Similar experiments were performed with the VA1 18D7 cell line. The Clirans T220 cartridge needed 33 days to produce 1 g of antibody, an amount comparable to the other 10,000-MW cartridge (BR2010), which required 32 days to produce the same amount. But the same VA1 18D7 cell line grown in the BR3530 (30,000-MW) bioreactor cartridge required only 17 days to produce 1 g of antibody. Likewise, the 30,000-MW bioreactor cartridge required 17 days to reach the target GUR of 250 mg/hr, whereas the two 10,000-MW cartridges required 31 and 30 days, respectively, to reach this level.
Similar results--13 and 26 days, respectively--were obtained with the MOR 9B1 cell line in the BR3530 and Acucell 1100 cartridges. The average time a 10,000-MW-pore-size cartridge required to reach the target level (1 g of antibody production) was 32 days. This contrasted with an average of 16 days for the 30,000-MW cartridge. Similarly, the average time needed by a 10,000-MW-pore-size cartridge to reach the target GUR of 250 mg/hr was 28 days versus 18 for the BR3530 cartridge.
Antibody Production Efficiency. We compared the ability of each of the hollow-fiber cartridges to produce antibody as a function of the amount of intracapillary medium consumed by the system during the experiment. These values were then expressed as milligrams of antibody produced per liter of medium consumed over the course of the entire run.
Using the MOR 9B1 cell line, we determined the optimum production efficiency to be 50 mg/L for the BR3530 cartridge and 24 mg/L for the Acucell 1100 cartridge. The VA1 18D7 cell line grown in the 10,000-MW cartridges had a production efficiency of 35 mg/L in the Clirans T220 cartridge and 30 mg/L with the BR2010. However, when the same cell line was grown in the BR3530 (30,000-MW) cartridge, the efficiency increased to 44 mg/L of medium consumed.
Comparison of antibody production in the BR3530 hollow-fiber cartridge to that in 72-hour static tissue culture (Table I) reveals a correlation between the amount of antibody a cell line produces in 72-hour culture and the quantity produced in the 30,000-MW cartridge.
Feed Rate. The effect of feed rate on antibody production was measured in two experiments using the METH7-4B6-8C3.2.1 cell line grown in the BR3530 cartridge. In the first experiment, the intracapillary feed rate was slowly increased from 0.5 L/day to a maximum of only 3.5 L/day (Figure 1). In the second experiment, the intracapillary feed rate was increased more rapidly from 1 L/day to a maximum of 5.0 L/day. The run with the lower feed rate produced more antibody in less time than the run with the higher feed rate (Figure 2). At day 40, the production efficiency of the low-feed-rate experiment was 118 mg/L and the production efficiency of the high-feed-rate scheme was 58 mg/L (Table I).
The effect of feed rate on antibody production was measured in two other experiments using the MOR 9B1 F11 P1.1.1 cell line grown in the Acucell 1100 cartridge using the Acusyst Jr. bioreactor system. When the feed rate began at 1 L/day and was rapidly accelerated to 8.4 L/day, the efficiency of the run was increased by only 9 mg/L (Figures 3 and 4). With a lower feed rate strategy beginning at 0.6 L/day and accelerating slowly to 3.3 L/day, the production efficiency almost tripled to 24 mg/L (Table I). Total antibody production from each run was approximately the same.
Cell Removal. When the PCP1.-9D10 cell line was grown in the CP2000 bioreactor system using a BR3530 cartridge, the cells reached confluence after only 25 days. Then antibody production began to drop precipitously (Figure 5). Beginning on day 30, cells were removed from the cartridge twice weekly by attaching syringes filled with IMDM (without FBS) to the extracapillary ports and vigorously pushing and pulling the plunger of the syringes. Large numbers of cells, cell debris, and a significant amount of antibody were obtained with this procedure.
Immediately after each of the nine cell-removal procedures performed during this one bioreactor experiment, antibody production increased significantly. The GUR also had a corresponding elevation. (Figure 5).
Batch versus Continuous Harvests. How did the kind of harvest mode affect total antibody production? The batch mode of harvesting used either syringes or a faster pump rate to remove antibody. When the VA1 18D7 cell line grown in the CP2000 system was switched from continuous to batch mode harvesting, the concentration of antibody increased from an average of 413 to 3387 µg/ml (Figure 6).
Discussion
The 10,000-MW-pore-size hollow- fiber cartridges from Unisyn Technologies, Inc. (BR2010), Terumo Corp. (Clirans T220), and Cellex, Inc. (Acucell 1100), are each capable of producing significant amounts of monoclonal antibody if used in the bioreactor systems described above. However, when compared with the Unisyn Technologies, Inc., BR3530 cartridge, the BR2010 and Clirans T220 cartridges were shown to be less efficient in terms of amount of antibody produced per liter of medium consumed. Likewise, the BR3530 was also found to be more efficient than the Cellex, Inc., Acucell 1100.
All three of the lower-molecular- weight-pore-size cartridges experienced a significant lag phase (approximately 1 month) for antibody production and cell growth, as measured by GUR. The lag phase with the BR3530 cartridge was consistently about one-half that of the 10,000-MW cartridges. The cost of medium consumed during the lag phase and of the labor used to run the bioreactor for an additional two weeks may offset the savings derived from using the lower-cost Clirans T220 cartridge in a bioreactor system.
The shorter lag period for the 30,000-MW pore size cartridge may come from the ability of medium components to more easily traverse the hollow-fiber membrane. Increased mass transport associated with a larger pore size hollow-fiber membrane is well documented.1 The larger pore size fibers have higher ultrafiltration rates, which facilitate medium component exchange.6 The BR3530 cartridge provides a larger surface area for the growth of cells that may enhance cell growth and antibody production.
When low- and high-medium feed-rate strategies were compared, both the 10,000- and 30,000-MW cartridges showed that it was possible to overfeed cells in these bioreactor systems. High feed rates led to poorer cell growth and antibody production. By gradually increasing the feed rate throughout the run, more antibody was produced at a more efficient rate.
The high feed rates may dilute soluble factors that these cells require for growth. Some cell lines of the B cell lineage can stimulate autocrine growth.7,8 The factors secreted from cells may be small enough to pass through the hollow fibers into the ICS. Some manufacturers of hollow-fiber bioreactor equipment propose high feed rate strategies to maximize antibody production,4,6,9,10 but experience has shown that these strategies may not work well with all hybridoma cell lines.
The removal of cells from confluent hollow-fiber cartridges is useful to maintain the antibody production level of the bioreactor run. The cell-removal procedure rids the cartridge of large numbers of dead cells and cellular debris. This gives space for additional cell growth and clears the semipermeable fibers of cell components that may interfere with the efficient exchange of nutrients.
Harvesting the antibody in batch rather than in a continuous mode can increase the concentration of the antibody product if the batch harvests use small volumes of medium. This provides a reasonable alternative to continuous harvesting, especially for cell lines that do not secrete much antibody or if there is demand for a more highly concentrated product.
In the authors' experience, rapidly accelerating the medium feed rate in hollow-fiber bioreactor experiments may have an adverse effect on antibody production. It is recommended that once the GUR has begun to decrease, cells be periodically removed from the bioreactor cartridge to enhance antibody production. Inexpensive hollow-fiber cartridges, for kidney dialysis, can be a substitute for hollow-fiber cartridges used in antibody production with these instruments.
References
1. Evans TL, and Miller RA, "Large Scale Production of Murine Monoclonal Antibodies Using Hollow Fiber Bioreactors." Biotechniques, 6:762767, 1985.
2. Lowrey D, Murphy S, and Goffe RA, "A Comparison of Monoclonal Antibody Production in Different Hollow Fiber Bioreactors," J Biotech, 1994, in press.
3. Lowrey DM, Meslovich K, Murphy S, et al., "Small Scale in Vitro Antibody Production Using a Disposable Hollow Fiber Device," presented to the 12th Annual Meeting of the European Society for Animal Cell Technology (ESACT), Wurzberg, Germany, May 1993.
4. Muragachi A, Nishimoto H, Kawamura N, et al., "Cell Derived BCGF Functions as an Autocrine Growth Factor in Normal and Transformed Lymphocytes," J Immunol, 137:179, 1986.
5. Knazek R, Gullino PM, Kohler PO, et al., "Cell Culture on Artificial Capillaries: An Approach to Tissue Culture Growth in Vitro," Science, 178: 6567, 1972.
6. Gordon J, Ley SC, Melamed MD, et al., "Soluble Factor Requirements for the Autostimulatory Growth of B Lymphoblasts Immortalized by Epstein Barr Virus," J Exper Med, 159:154, 1984.
7. Hiefetz AH, Brantz JA, Wolfe RA, et al., "Monoclonal Antibody Production in Hollow Fiber Bioreactors Using Serum Free Medium," Biotechniques, 7:192199, 1989.
8. Lowrey D, Murphy S, and Goffe RA, "The Effect of Intracapillary Media Feed Protocols on Hollow Fiber Cell Culture," Biotech Lett, 15:10251030, 1993.
9. Caple MV, Fletcher TR, Owens WJ, , et al., "A Dual Formulation Serum Free Media System," Biopharm, 5:5260, 1992.
10. Hirschel M. and Keznoff S, "The Effect of Perfusion Rates on Hollow Fiber Bioreactors," presented to the Federation of Applied Scientists and Experimental Biologists, New Orleans, 1990.
Rudolph J. Czirbik, PhD, and Steven M. Rosen, PhD, are senior scientists; Diane M. Trunfio is an associate scientist; Ellyn W. Fischberg-Bender is research group manager; and Stuart M. Palmer, PhD, is director of research and development for the Therapeutic Drug Monitoring Business Unit of Roche Diagnostic Systems, Inc. (Somerville, NJ).
