Perfusion Cell Culture in Disposable Bioreactors

Ryo Ohashi, Vijay Singh, and Jean-Francois P. Hamel

Abstract
A novel disposable bioreactor design is described that incorporates a floating filter for the removal of cellfree perfusate. This design eliminates the need for traditional pumparound flow loops and external cell separators. Experiments with a hybridoma cell line producing a monoclonal antibody show that such a 7 device can support very high cell densities of over 3 x 10 cells/ml and produce up to 73 mg/day of product. This is 6 fold higher productivity than batch culture. The bioreactor was operated continuously for 25 days without any evidence of clogging. Applications include bioprocessing and human cell therapy.

Introduction
It is well known that perfusion operations in cell culture are very useful in increasing cell productivity. In this mode, feed solutions are fed to the bioreactor continuously, and spent media is constantly removed. Thus nutrients are not wasted for growth, and toxic byproducts can be removed. The key parameter for successful perfusion is the retention of the majority of the cells in the bioreactor. This allows operation at relatively high flow rates without the danger of washout. While perfusion is very beneficial, it is not often used due to the complexity of traditional perfusion bioreactors. In these bioreactors, cells are retained using one of three basic techniques: 1) filtration, 2) sedimentation, or 3) centrifugation. Filtration methods require some means of keeping the filter from clogging over the required weeks of operation. Typically, high tangential velocity generated by crossflow or spinning filters is used to keep the surfaces clean. These methods require pump-around loops and their mechanical complexity 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 perfusion culture 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 potential oxygen depletion, often resulting in diminished product quality and quantity. The Wave Bioreactor, introduced by Wave Biotech in 1998, has simplified cell culture tremendously by providing a presterile, disposable bioreactor that can be scaled up to over 500 liters. In this system, cell culture is performed in disposable bags (Cellbags) that are inflated and rocked to provide oxygen transfer and mixing. These systems have been proven for suspension and microcarrier culture with a wide variety of cells including insect, plant, animal, and virus culture. The development challenge was to extend this system into a simple disposable presterile perfusion bioreactor suitable for biotechnology and medical applications.

A New Concept - a floating filter

A unique floating filter was developed by Wave Biotech (US and European patents pending) that takes advantage of the wave motion in the Wave Bioreactor to keep the filter surface from clogging. The filter is shown in Figure 1. The underside of the filter consists of a flat cell-retentive membrane. The upper surface is made of polyethylene and has a nozzle to draw off the cell-free filtrate. The filter materials are chosen so that 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.

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Figure 1. Schematic of the Floating Perfusion Filter

Filtrate drawoff nozzle Polyethylene top

Cell retentive membrane In normal operation, the Wave Bioreactor rocks back and forth at 15 to 25 cycles/minute. The resulting wave motion necessary for oxygen transfer and mixing also rapidly 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 the need for any external mechanical devices.
Figure 2 Filter Movement in the Wave Bioreactor

Inflated Cellbag cultivation chamber

Floating perfusion filter moves with wave motion

Flexible filtrate draw-off tube

Rocking Motion

Cell culture media

Wave Bioreactor

Rocking Motion

This simple, yet ingenious design, makes it possible to produce a disposable perfusion that is nothing more than a standard bioreactor Wave Bioreactor Cellbag chamber with the special filter installed inside it. The entire assembly can be purchased presterilized by gamma radiation. To operate the bioreactor simple place the Cellbag containing the perfusion filter on a Wave Bioreactor rocking platform; inflate and fill with media. Next, inoculate with cells and allow them to grow to the desired cell density. Then connect the feed solution using a peristaltic pump to one of the inlet ports on the Cellbag and start the feed at the desired flowrate. At the same time connect a second pump to the filtrate line on the Cellbag to pump out the cell-free filtrate to a harvest container. No pump-around is required, nor are cells recirculated back to

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the bioreactor. As with all Wave Bioreactor systems, there is no need for cleaning or sterilization with resulting savings in time and cost.

Performance
Professor Jean-Francois Hamel and Ryo Ohashi at the Biochemical Process Engineering Center at MIT have tested the Wave Bioreactor perfusion system using a hybridoma cell line producing a monoclonal antibody. The culture media used was Becton Dickenson Cell Mab media with 10% fetal bovine serum and 1% pluronic F-68. Culture was done on a Wave Bioreactor SYSTEM20 benchtop unit with integral temperature and CO2 control. Two Cellbag2L bioreactors were run simultaneously from inoculum pooled from five T-75 flasks. One Cellbag was run as a batch culture; the other, equipped with a perfusion filter was operated in perfusion mode. Operating parameters are shown in Table 1:
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 5 rpm per day up to a maximum of 35 rpm. 5% CO2 overlay. O2 increased as per dilution rate to maximum of 50% O2. Initial volume 300ml + 300ml +400ml (final volume 1 liter) Initial volume 500ml+500ml (total 1 liter) then start perfusion once cell density > 2x106cells/ml. Initial dilution rate 0.1/day. Dilution rate was adjusted to keep glucose/lactate concentrations constant.

Results
Figure 3 shows the time-profile of cell density and viability of batch and perfusion cultures using the Wave Bioreactor. For batch culture, total cell density peaked at 3 x 106 cells/ml and, after 9 days, viability declined rapidly. This is typical for this cell line and media. In contrast, with perfusion culture, the cells continued to grow, and the maximum total cell density achieved was 32 x 106 cells/ml. Viability decreased slowly towards the middle of the run, and then stabilized around 50% for 10 days. These results suggest that: 1) the perfusion bioreactor can support ten fold higher cell density than batch operation; 2) the perfusion Wave Bioreactor has the capacity to support up to 30 x 106 cells/ml, and 3) the perfusion bioreactor is able to maintain high viability for long periods.
Figure 3. Time-profile of cell growth of batch and perfusion cultures.

350 300 Cell density (105cells/ml) 250 200 150 100 50 0 0 100 200

Perfusion viable Perfusion total Batch total Batch viable perfusion viability Batch viability

100 90 80 60 50 40 30 20 10 0 Viability (%) 70

300

400

500

600

700

Culture time (h)

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In this experiment 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 that period, it was observed that cells covered the upper bag surface in significant amounts, and that 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, the cells lost in the filtrate were only 5 x 104 cells/ml, i.e. less than 1% of cells in the bioreactor, and the cell count in the bioreactor continued to increase, reaching over 30 x 106 cells/ml. The total amount of medium used after 20 days culture was about 9 L. The highest dilution rate was 0.045h1 , corresponding to 450 ml/day or about one third of the maximum filtration capacity (1.5 L/day). Using low flow rates and step-wise increases in the feed, the culture could be sustained for a long period, while maintaining a high glucose consumption rate. Figure 4 shows the monoclonal antibody (Mab) concentration and total Mab production for batch and perfusion cultures. In the batch culture, Mab concentration started to increase after 4 days and reached a maximum level at the end of culture. On the other hand, in the perfusion culture, Mab concentration was function of the dilution rate and after both feed and filtrate were stopped, Mab concentration continued to increase and reached 480 mg/L, over three times the maximum level obtained in batch culture. The total Mab produced in perfusion culture was 1800 mg. This was almost 13 times more than the total Mab produced in batch mode.
Figure 4. Mab concentration and total amount of Mab.
600 500 Mab conc. (mg/L) 400 300 200 100 0 0 100 200 300 400 500 600 700 Culture time (h)
Perfusion conc. Batch conc. Perfusion total Batch total

Stop

2000 1800 1600 1200 1000 800 600 400 200 0 Total Mab (mg) 1400

Table 2 shows the cost and performance data for batch and perfusion cultures. The perfusion system has a third the production cost and the productivity / day is about 6 fold higher than batch operation.
Table 2. Mab production Wave Bioreactor - 1 liter culture volume Batch 12 days $150 140 1.63 12 Perfusion 25 days $170 1800 0.51 73

Culture time Reactor Cost Mab Production/reactor (mg) Production cost $/mg of Mab mg of Mab/reactor/day

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This study shows the capacity of the Wave Bioreactor equipped with a perfusion filter to perform without clogging for over 25 days. The Cellbag2L (1 liter working volume) bioreactor was capable of producing 73 mg Mab/day. It is entirely feasible to fabricate Cellbags of larger volume on the same principle and work is underway with units of 10 liter and 100 liter working volume.

Perfusion Control
One of the major problems in perfusion control is the accurate metering of feed and harvest. Peristaltic pumps do not have the accuracy to maintain a constant flowrate over many days of operation. A new weight-based perfusion controller has been developed by Wave Biotech. The basic design is shown in Figure 5. The feed is contained in a sterile bag and suspended on a load cell. The feed bag is connected to the Wave 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 that is also suspended on the same load cell. The system functions as follows: 1) the feed pump runs until a preset volume (say 50ml) as determined by the load cell is fed into the bioreactor; 2) the harvest pump is then actuated until the load cell registers a net zero gain or loss. This ensures that the bioreactor always remains at a constant volume. The cycle is then repeated starting with the feed pump. The alternating action can be made faster or slower to give the desired overall perfusion rate. It is possible to easily calculate the cumulative feed and harvest volumes dispensed since only one of the pumps operate 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 warn of tubing failure or filter clogging, and allow the user to take timely action. Figure 5. Schematic of Perfusion Controller Load Cell

Controller Feed bag Harvest bag

Harvest pump

Feed pump Wave Bioreactor

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Medical Applications
In addition to use in biotechnology research and manufacturing, the Wave Bioreactor disposable perfusion culture system has novel applications in medicine. The simple, low cost presterile design is ideal for the cultivation of patient-specific cells. The ability to feed nutrients, and remove metabolic products without the loss of cells allows high cell densities to be achieved. The disposable single-use bioreactor protects the patient from potential cross-contamination and very little infrastructure is necessary to provide multiple bioreactors for individual patients. The unique floating perfusion filter design eliminates the traditional complex and leakage-prone crossflow cell retention filters required by traditional perfusion bioreactors. This enables 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 pyrogen-free operation. Applications include stem cell culture, ex-vivo gene therapy and replacement for bone marrow transplantation. Drs Raubetshek and Jensen, at the City of Hope Medical Center in California are pioneers in using the Wave Bioreactor perfusion system for ex-vivo human therapy. They are using it to expand patient-specific T-cells for the treatment of pedriatic cancer patients. Early results look very promising and an IND is expected to be filed with the FDA in 2001. Ryo Ohashi and Jean-Francois P. Hamel are based at the Biotechnology Process Engineering Center at MIT in Cambridge, MA, USA. Vijay Singh is with Wave Biotech in Bedminster, NJ, USA. Correspondence may be addressed to jhamel@mit.com. Technical information can be obtained from www.wavebiotech.com.
Wave Bioreactor and Cellbag are registered trademarks of Panacea Solutions, Inc. US and European pending or granted on various technologies described in this paper.

Wave Biotech LLC Disposable Bioreactors for Cell Culture 999 Frontier Road Bridgewater, NJ 08807 USA Tel: (908) 707 9120 Fax: (908) 707 9161 Email: info@wavebiotech.com Web: www.wavebiotech.com

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