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GENETIC ENGINEERING NEWS BIOPROCESS T U TORIAL

Volume 21, Number 7, April 1, 2001

Perfusion Culture in Disposable Bioreactors

Floating Filter Design to Simplify Operations
Ryo Ohashi, Vijay Singh, and Jean-Franois P. Hamel
t is well known that perfusion operations in cell culture are useful for increasing cell productivity. In this mode, feed solutions are fed to the bioreactor continuously, spent media and toxic byproducts are constantly removed, and nutrients are not wasted for growth. The key to successful perfusion is the retention of the majority of the cells in the bioreactor, allowing operation at relatively high flow rates without the danger of washout. But while perfusion is beneficial, it is not often used, due to the complexity of traditional perfusion bioreactors, in which cells are retained using one of three basic techniques: filtration, sedimentation, or centrifugation. Filtration methods require some means of keeping the filter from clogging over the required weeks of operation. Typically, high tangential velocity, generated by cross flow or spinning filters, is used to keep surfaces clean. These methods require pump-around loops and considerable mechanical complexity, which severely limits their applicability. Sedimentation devices need to be adjusted for specific settling characteristics and have found only limited commercial success. Centrifugal devices are difficult to keep sterile and not commonly used for cell culture. Most traditional systems pump the cells through the separation device and back to the bioreactor. In addition to difficulties in maintaining sterility, these devices subject the cells to damaging shear and oxygen depletion, often resulting in diminished product quality and quantity. The Wave Bioreactor introduced in 1998 by Wave Biotech (Bedminster, NJ) simplifies cell culture by providing a presterile, disposable bioreactor that can be scaled up to over 500 liters. In this

the Cellbag, and fed at the desired flow rate. At the same time, a second pump may be connected to the filtrate line on the Cellbag, to remove cellfree filtrate to a harvest container. No pump-around is required, nor are cells recirculated back to the bioreactor. As with all Wave Bioreactor systems, there is no need for cleaning or sterilization, with resulting savings in time and cost. Performance The Biotechnology Process Engineering Center at MIT (Cambridge, MA) has used the Wave Bioreactor perfusion system with a hybridoma cell line to produce a monoclonal antibody. Becton Dickinson (Sparks, MD) Cell Mab culture medium was used, with 10% (v/v) fetal bovine serum and 0.1% (w/v) pluronic F-68. Culture was done on a Wave Bioreactor System20 benchtop unit with integral temperature and CO2 control. Two two-liter Cellbags were run simultaneously from inoculum pooled from five T-175 flasks. One Cellbag was run as a batch culture, the other, equipped with a perfusion filter was operated in perfusion mode (Table 1). Results An analysis of the time-profile of cell density and viability using the Wave Bioreactor (Figure 3) demonstrates that, for the batch culture, total cell density peaked at 3x10 6 cells/mL and, after 9 days, viability declined rapidly. In the perfusion culture, the cells continued to grow to a maximum total cell density of 32x10 6 cells/mL. Viability decreased slowly towards the middle of the run, then stabilized at about 50% for 10 days. Results suggest that the perfusion bioreactor can support ten-

Figure 2 system, cell culture is performed in disposable Cellbags , which are inflated and rocked to provide oxygen transfer and mixing. These systems have been successful for suspension and microcarrier culture with a wide variety of cells, including insect, plant, animal, and virus cultures. The development challenge was to extend this system into a simple, disposable, presterile perfusion bioreactor suitable for biotechnology and medical applications. Floating Filter Wave Biotechs floating filter (Figure 1) (U.S. and European patents pending) takes advantage of the wave motion in the Wave Bioreactor to keep the filter surface from clogging. The underside of this filter consists of a flat cellretentive membrane; the upper surface is made of polyethylene and has a nozzle to draw off the cell-free filtrate. The filter is neutrally buoyant and floats on the liquid surface. The filtrate nozzle is connected by a flexible tube to the outside of the cultivation bag, enabling the filtrate to be pumped out. In normal operation, the Wave Bioreactor rocks back and forth at 1525 cycles/min. The resulting wave motion is necessary for oxygen transfer, and the rapid mixing whips the filter back and forth across the liquid surface. This tangential motion keeps the filter surface from clogging even after weeks of continuous operation, without need of external mechanical devices (Figure 2). This design makes possible a disposable perfusion Wave Bioreactor, which is simply a standard Wave Biotech Cellbag chamber with the special filter installed inside it. The entire assembly can be purchased presterilized by gamma radiation. The bioreactor is operated by placing the Cellbag that contains the perfusion filter on a Wave Bioreactor rocking platform, inflating the Cellbag, then filling it with medium. Next, the bag is inoculated with cells, which are permitted to grow to the desired cell density. The feed solution is then connected using a peristaltic pump to one of the inlet ports on

fold higher cell density, that the standard Wave Bioreactor (up to 30 x 106 cells/mL), and the perfusion bioreactor is able to maintain high viability for long periods. Figure 4 shows the time-profile of glucose and lactate concentrations of batch and perfusion cultures using the Wave Bioreactor. The dilution rate was adjusted daily to keep glucose and lactate concentrations constant. After 450 h, the glucose concentration in the reactor increased approximately 7-fold over 100 h. During this period, cells covered the top of the bag surface in significant amounts and cell density decreased after reaching its maximum level. Since cell viability started to decrease significantly at that time too, it is possible that the Cellbag reached its capacity to sustain viable cells, leading to cell death and cell adhesion to the bag. After 20 days, some cells leaked through the perfusion filter, perhaps due to the high rocking rates of the Wave Bioreactor and a weak membrane connection; however, even with this leakage, cell loss in the filtrate was 5x104 cells/mL, i.e., <1% of cells in the bioreactor, and the cell count in the bioreactor continued to increase, reaching over 30x106 cells/mL. The time-profile of glucose and lactate concentrations of batch and perfusion cultures shows that the total amount of medium used after 20 days culture was about 9 L, with the highest dilution rate corresponding to 450 mL/day or about one third of the maximum filtration capacity of 1.5 L/day. Using low flow rates and stepwise increases in the feed, the culture could be sustained for a long period, while maintaining a high glucose consumption rate.
See Bioprocess Tutorial on page 78

Table 1. Experimental Parameters

Sampling Agitation Aeration Batch Operation Perfusion Operation Cell density, pH, glucose and lactate concentrations were determined daily. Rocking rate was started at 8 rpm and then increased by 2 rpm per day up to a maximum of 35 rpm. 5% CO2 overlay. O2 was increased based on dilution rate to maximum of 50% O2. Initial volume 300 ml + 300 ml + 400 ml (final volume, 1 liter) Initial volume 500ml + 500ml (total, 1 liter) then start perfusion once cell density > 2 X 106 cells/ml. Initial dilution rate was 0.2/day. Dilution rate was adjusted to keep glucose/lactate concentrations constant.

Figure 3
Figure 1: Schematic of the floating perfusion filter

Figure 6
Figure 2: Filter movement in the Wave Bioreactor Figure 3: Time-profile of cell growth of batch and perfusion cultures Figure 4: Time-profile of glucose and lactate concentrations of batch and perfusion cultures Figure 5: Mab concentration and total amount of Mab Figure 6: Schematic of Perfusion Controller

Figure 4

Figure 5

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GENETIC ENGINEERING NEWS

Volume 21, Number 7, April 1, 2001

People. . . Bioindustry Appointments & Promotions

NewBiotics named Lorna Speid, Ph.D., vp of regulatory affairs and project management, and Thomas Lobl, Ph.D., vp of drug discovery...Xanthon made two appointments: Donald Haddock joined as quality engineer, and Dwayne J. Allen, Ph.D., joined as senior scientist. Alan Mendelson joined Valentis board of directors...Thermo Finnigan added Sally Swedberg, Ph.D., as senior director of bioanalytical products and solutions; Nelson Cooke, Ph.D., as senior director of marketing, and Paul Shieh, Ph.D., as director of LC systems. Gary A. Alianell, Ph.D., became COO of EraGen Biosciences... R. Borrmann, Leornard Pharm.D., was appointed president and CEO of Maret Pharmac e u t i c a l s . . . Biogen appointed Toshio Nakata, D.Sc., as president of Biogen Japan Ltd. and vp of Biogen...Transkaryotic Therapies appointed Anthony S. Murabito as vp of management information systems. Michael J. Brown, J.D., was named intellectual property counsel and technology transfer manager at the Institute for Genomic Research...Myriad Genetics elected Hugh A. DAndrade as chairman of its board of directors...Chiron named John A. Lambert president of Chiron Vaccines...Genzyme Corp. elected Connie Mack III to its board of directors. Gary Patou, M.D., is GeneSofts new president...Karl-Gunnar Hidinger, Ph.D., became vp of clinical development at Medicure...Amy Seltzer Hedison joined Exact Sciences Corp. as director of investor relations...Endorex Corp. named John R. McCracken vp of business development...Maxia Pharmaceuticals named Zofia E. Dziewanowska, M.D., Ph.D., as senior vp of clinical and regulatory affairs. Ann Merrifield was named executive vp of Genzyme Biosurgery...Alan M. Ezrin, Ph.D., was appointed CSO of Nortran Pharmaceuticals...Spherix elected Richard Levin to the newly established posts of CFO and vp for administration...Robert Garnick, Ph.D., was promoted to the position of senior vp of regulatory, quality, and compliance at Genentech. Antisoma appointed Adam Craig to the position of medical advisor of oncology...Charles D. Shively, Ph.D., was named director of product development at Zila Consumer Pharmaceuticals. NeoTherapeutics hired Frank Chiu, Ph.D., as director, biostatistics and data management...Donald D. Tartre joined Stressgen as vp and CFO. AtheroGenics made the following promotions: Dav id B. Edwards, senior director of toxicology and early development; Bruce Johnson, Ph.D., senior director of developmental chemistry; and Felicia Ford-Rice, director of quality assurance and compliance. ImmunoGen appointed Gregg D. Beloff vp and CFO...Peter A. Burke, Ph.D., joined Steris Corp. as vp and CTO...Paradigm Genetics appointed Mark Skaletsky to its board of directors. Lars Barfod was named executive vp of worldwide sales and services and CBO at MDL Information Systems...Structural Genomics hired Sheryl Silverstein, Ph.D., J.D., as senior director of intellectual property...William Matthews was appointed president and CEO at Deltagen...Alan Carpenter, Ph.D., joined Epix Medical as executive vp of R&D...Nina Ossanna, Ph.D., is the new senior director of business development at ImaRx Therapeutics...Larry Greller, Ph.D., has been appointed director of mathematical biology and systems biology at Molecular Mining Corp...Cytoclonal Pharmaceuticals named Robert J. Rousseau, Ph.D., vp of business development and licensing. Tatyana Touzova joined Xanthon as quality-control supervisor...Deltagen announced the appointment of William A. Scott, Ph.D., as a new board member and Erick J. Zimmer as director of commercial development...William R. Grant was named chairman of the board at Vasogen. Scott Chappel, Ph.D., was promoted to the position of executive vp of research and technology at Dyax Corp....Advanced Pharma appointed R. Gordon Douglas, M.D., and Wayne T. Hockmeyer, Ph.D., to its board of directors...Quintiles Transnational named Pamela J. Kirby, Ph.D., as its new CEO. Roy Kinoshita will fill the position of product manager for fluorescence microscopy at Omega Optical...CellExSys formed a board of directors whose initial members include: Edward O. Lanphier, Thomas G. Wiggans, David M. Schubert, and H. Stewart Parker, who will serve as chairman. Diversys Ltd. named Robert T. Connelly as CEO...David Kern was appointed director of program management at Protogene Laboratories...Vincent Gotz joined Epicyte Pharmaceutical as vp of business development...North Carolina SRT appointed John Laurence Thomas, Jr. as chief of operations. Robert J. Lefkowitz, M.D., was elected to the board of directors at Lexicon Genetics...Bernard Gibbs, Ph.D., M.Sc., joined MDS Pharma Services as research fellow in its bioanalytical applied research and development group...Immunex Corp. elected Robert J. Herbold to serve on its board. Inspire Pharmaceuticals made two announcements: Mary Bennett was appointed vp of operations and communications, and Mark A. Montgomery, J.D., was named senior director of intellectual property/legal. Oscar J.M. Goddijn, Ph.D., was named managing director of Syngenta Mogen BV...La Jolla Pharmaceutical Co. promoted Paul Jenn, Ph.D., to the position of vp of operations and Theodora Reilly to the position of vp of human resources.

Bioprocess Tutorial
Figure 5 shows the Mab concentration and total Mab production for batch and perfusion cultures. In the batch culture, Mab concentration started to increase after four days and reached a maximum level at end of culture. In the perfusion culture, Mab concentration was function of the dilution rate and reached 1.2 times higher concentration than in batch culture. After both feed and filtrate were stopped, Mab concentration continued to increase and reached 120 mg/L, twice the maximum level obtained in batch culture. In perfusion culture, with a one-liter working volume bioreactor, cumulative production of Mab was 450 mg, or about ninefold higher than in an equivalent batch culture. Table 2 shows cost and performance data for batch

Continued from page 40

and perfusion cultures. Cost analysis is based on catalog prices for cell culture medium and bioreactors and does not include labor costs. The perfusion bioreactor needed 10 liters of medium, and this number was used as a basis to normalize the Mab production data. While batch culture is slightly more efficient in media utilization, the perfusion system has half the production cost, with productivity per day about fourfold higher than with batch operation. This study shows the capacity of the Wave Bioreactor equipped with a perfusion filter to perform without clogging for over 25 days. The Cellbag 2L bioreactor (oneliter working volume) was capable of producing 18 mg Mab/day. It is feasible to fabricate Cellbags of larger volume on the same princi-

ple and work is under way with units of 10 liter and 100 liter working volume. Developments and Applications One of the major problems in perfusion control is the accurate metering of feed and harvest. Peristaltic pumps are not accurate enough to maintain a constant flow rate over days of operation. In addition, the bag cultivation chamber used in the Wave Bioreactor makes level control impossible. To address these operational problems, a new weight-based perfusion controller has been developed by Wave Biotech (Figure 6). Feed is contained in a sterile bag and suspended on a load cell. The feedbag is connected to the bioreactor by tubing routed through the feed peristaltic pump. Harvest is drawn through the perfusion filter by the harvest pump and collected in a harvest bag suspended on the same load cell. The feed pump runs until a preset volume, as determined by the load cell (e.g., 50 mL), is fed into the bioreactor. The harvest pump is that actuated until the load cell registers a net-zero gain or loss, ensuring that the bioreactor remains at

constant volume. The cycle then repeats with the feed pump. The alternating action can be made faster or slower to give the desired overall feed rate. The dispensed cumulative-feed and harvest volumes can easily be calculated by the controller, since only one pump operates at any given time. This simple perfusion controller allows precise feeding and volume control, regardless of pump accuracy, filtration rate, or tubing wear. By continuously monitoring weight loss and gain, the controller can be warned of tubing failure or filter clogging, and take appropriate action. Medical Applications In addition to use in biotech research and manufacturing, the Wave Bioreactor disposable perfusion culture system has novel applications in medicine. The lowcost, presterile design is ideal for the cultivation of patient-specific cells; the ability to feed nutrients and remove metabolic products without cell loss allows high cell densities to be achieved; and the disposable single-use bioreactor protects the patient from potential cross-contamination.

Table 2. Mab Production

Culture Time Reactor Cost Mab Production/reactor (mg) Production cost $/mg of Mab mg of Mab/10L medium mg of Mab/day

Wave Bioreactor/1 Liter Culture Volume

Batch 12 days $150 54.4 5.51 544 4.5 Perfusion 25 days $250 450 2.50 450 18.2

Furthermore, little infrastructure is needed to provide multiple bioreactors for individual patients. The floating perfusion filter design eliminates the traditional complex and leakage-prone cross-flow cell-retention filters required by traditional perfusion bioreactors, enabling multiple bioreactors to be operated by hospital personnel without the need for specialized training. All contact components are manufactured from USP Class VI qualified plastics and constructed under GMP conditions to ensure progeny-free operation. Applications include stem cell culture, ex vivo gene therapy, and replacement for bone marrow transplantation. Andrew Raubitschek, M.D., and Michael Jensen, M.D., at the City of Hope Medical Center in Duarte, CA, use the Wave Bioreactor perfusion system for ex vivo human therapy to expand patient specific T-cells for the treatment of pediatric cancer patients. GEN
Ryo Ohashi and Jean-Franois P. Hamel are based at MIT in Cambridge, MA. Vijay Singh is president of Wave Biotech in Bedminster NJ (www.wave biotech.com). Correspondence should be addressed to jhamel@mit.edu

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