Use of Tangential Flow Filtration in Perfusion Propagation of Hybridoma Cells for Production of Monoclonal Antibodies

Daniel Velez* New Brunswick Scientific Company, Incorporated, Edison, New Jersey 088 17

Llonas Miller and James D. Macmillant Department of Biochemistry and Microbiology, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08903

Accepted for publication May 24, 1988

INTRODUCTION a stirred fermentor and were able to obtain antibody con-

Development of applications for monoclonal antibodies (MAb) in many areas of biology, medicine, agriculture, and industry has stimulated increasing interest in improv- ing methods for producing them in large quantities.’ Al- though amounts of MAb sufficient for many research and commercial uses are readily produced by growth of hy- bridoma cells in vivo (ascites fluid), this is a rather im- practical way to produce antibodies on a large scale as thousands of animals may be required. With expanding ap- plications in diagnosis, therapy, and immunopurification creating demand for larger amounts of MAb, there is need for efficient in vitro methods for MAb production.

A basic consideration in MAb production is that microgram-per-milliliter concentrations are typically found in conventional batch cultures compared to milligram-per- milliliter levels in ascites fluid. A variety of different cell propagation modes including batch, fed batch, and perfu- sion have been investigated for production of MAb in air- lift, stirred, and other types of bioreactors. ‘-I9 Because hybridoma strains vary considerably in growth characteris-

centrations of almost 1 m g / m ~ . ’ ~ The perfusion propagation system consisted of a 1-L fer-

mentor equipped with a cylindrically shaped filter screen mounted around the stirring shaft, which allowed for the removal of cell-free supernatant (containing MAb) at the same rate as fresh medium was added to the fermentor. With a feeding rate of 1100 mL/day, cell densities of up to 5 x lo7 mL-l and MAb productivity near 1.7 g/L of cul- ture/day were obtained. However, on scale-up of this sys- tem, the maximal size of a fermentor that can be used is limited by the ratio between the volume of the fermentor and the surface area of the filter screen.

In the present work we investigated the use of a tangen- tial flow filtration system for retaining cells during perfu- sion feeding. Since this filter is a separate unit operated externally to the fermentor itself, scale-up is not subjected to the limitations of the screen filter previously investi- gated. This type of filtration system has been previously used to draw off spent medium and recirculate mammalian cells in a continuous fermentation for virus p rod~c t ion .~~

tics and other factors that affect antibody production, it is difficult to compare yields for evaluation of the different EXPERIMENTAL - - systems. However, it seems clear from other investigations and from our own studieszozz1 that with appropriately se- lected and well-characterized hybridoma cell lines, propa- gation systems which improve antibody production can begin to approach levels found in ascites fluids.

We examined the cultural and environmental factors2’ which affected growth and antibody production in a mouse-mouse hybridoma line (VIII H-8) capable of pro- ducing MAb to a determinant on the surface of Brady- rhizobiurn japonicum cells.23 Based on these studies, we developed a perfusion system” for feeding hybrid cells in

The tangential flow filtration unit (Megaflow TM 100) employed in these investigations was provided by New Brunswick Scientific (Edison, NJ) (Fig. 1). The apparatus contained a 10-in.2 nylon membrane (0.2 p m pore size) sandwiched between a channel plate and a filtrate plate. Culture broth pumped through the channel plate passes over the surface of the membrane causing a pressure in- crease which forces cell-free liquid through the membrane. The concentrated cells are returned to the fermentor. The flow of liquid over the surface helps to clear the membrane and retards clogging of the filter pores.

Other components of the fermentation system (Fig. 2) included a 1.5-L Celligen fermentor (New Brunswick Sci- entific) with controls for temperature, agitation, pH, and

* Present address: Imclone Systems, Inc., 180 Varick Street, New York, NY 10014.

To whom all correspondence should be addressed.

Biotechnology and Bioengineering, Vol. 33, Pp. 938-940 (1989) 0 1989 John Wiley & Sons, Inc. CCC 0006-3592189/070938-03$04.00

Table I. tem using tangential flow filtration.

Hybridoma cell count and MAb production in perfusion sys- CONCENTRATED CULTURE CULTURE

I N OUT I t

1 ' 1

'1' CELL-FREE

FILTRATE OUT

Figure 1. Schematic drawing of tangential flow filtration module.

. c ---1 ~

i Figure 2. Schematic drawing of perfusion system using tangential flow filtration: (1) fermentor stirring base, (2) 1.5-L vessel, (3) pH electrode, (4) gas sparger, (5 ) dissolved oxygen electrode, (6) peristaltic pump recy- cling culture, (7) tangential flow filter unit, (8) fresh medium pump, (9) filtrate removal pump, (10) fresh medium reservoir, (1 1) spent medium reservoir.

dissolved oxygen; a pump (Watson Marlow, 601 S/R, New Brunswick Scientific) which recirculated the culture through the filtration unit; and two smaller pumps (Watson Marlow, 501S/R) set at the same flow rate used to add fresh medium to the fermentor and to remove cell-free cul- ture broth from the filtrate line to a collection reservoir.

Cell growth and immunoglobulin (IgG2a) production were evaluated in this system using the same cell line, cul- ture medium, and dissolved oxygen level (10%) employed in our previous studies with the rotating filter In the experiments described here, the fermentor was charged with 1100 mL culture medium inoculated at a cell density of 1.2 X lo6 mL-'. The culture was maintained at pH 7.2 and 37°C and agitated at 100 rpm. The culture liq- uid was recirculated through the tangential flow filtra- tion unit at 500 mL/min, a rate previously shown not to damage cells. After circulation for 24 h, cell viability was determined using the trypan blue exclusion method as outlined by Paterson.26 MAb in cell-free supernatant was measured using the radial immunodiffusion method previ- ously de~cribed.~'

RESULTS AND DISCUSSION

The results from this experiment are presented in Table I. Feeding with medium containing 4.5 mg glu-

Viable cells Dead cells Antibody Medium added Day ( x lo6 mL-') ( X lo6 mL-') (@g/mL) (mL)

~

2.4 3.7 8.2

14.0 22.0 28.0 25.0 22.0 21 .o

0.3 0.4 0.9 2.1 5.0 5.3 4.9 5.4 4.2

200 245 356 524

1147 I184 914

1051 914

350 350 350 350

1200 1200 1200 1200 I200

cose/mL was begun at a rate of 350 mL/day immediately after inoculation of the fermentor. On day 5 the rate was raised to 1200 mL/day and the glucose concentration was increased to 6.5 mg/mL to maintain the glucose level within the fermentor above 1000 pg/mL. By day 5, when the viable cell density had increased to above 2 X lo7 mI-' (5 X lo6 dead cells/mL) the MAb concen- tration in the filtrate reached about 1 mg/mL, where it re- mained until the experiment was terminated on day 9 due to a pump malfunction. Based on a culture volume of 1100 mL and a feeding rate of 1200 mL/day, this concen- tration of antibody indicated that the productivity was 1.2 g/L day.

Because of differences in culture volume and in feeding rates, these results are not directly comparable with those found in our earlier work using the fermentor equipped with the screen filter.24 The productivity there was 1.7 g/L day based on a culture volume of 565 mL and a perfusion rate of 1100 mL/day. However, the concentra- tion of antibody in the effluent of both systems was very comparable (i.e., -1 mg/mL), and since perfusion rates and average total cell numbers were about the same, specific antibody production rates were similar. Based on an average viable cel l concentrat ion of 2 .36 x lo6 cells/mL (days 5-9), antibody was produced at the rate of 46 pg/106 cells day compared to 38 pg/106 cells day in the earlier work.

The results presented here demonstrated the feasibility of using a tangential flow filtration unit for perfusion of hybridoma cells in a small fermentor. In another inves- tigation with a shear sensitive hybridoma cell line, we used a similar system for antibody production over a 22-day period.28

Use of tangential flow filtration for retaining cells within the fermentor has some advantages over the filter screen. Scale-up to larger fermentors should be possible, and since the filter unit is located outside the fermentor, it could eas- ily be taken off-line and replaced with another unit in the event of clogging.

We thank David Freeman for the collaborative arrangements that made this investigation possible. We are also appreciative of Nicholas Martin, who helped in initiating the research. New Jersey Agricultural Experiment Station Publication No. D-01406- 1-88 was supported by state funds and by funds from New Brunswick Scientific Company Incorporated.

COMMUNICATIONS TO THE EDITOR 939

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