[32] Production of monoclonal antibodies by agarose-entrapped hybridoma cells

[32] Production of monoclonal antibodies by agarose-entrapped hybridoma cells

352 PRODUCTION OF HYBRIDOMAS [32] Acknowledgments This work was supported by the National Health and Medical Research Council, Canberra, Australia...

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Acknowledgments This work was supported by the National Health and Medical Research Council, Canberra, Australia; by Grant Number AI-03958 from the National Institute of Allergy and Infectious Diseases, U,S. Public Health Service; and by the generosity of a number of private donors to The Walter and Eliza Hall Institute of Medical Research.

[32] P r o d u c t i o n of M o n o c l o n a l Antibodies b y A g a r o s e - E n t r a p p e d H y b r i d o m a Cells By K. NILSSON, W. SCHEMER, H. W. D. KATINGER, and K. MOSBACH

Mouse ascites culture is currently the most commonly used system for generation of monoclonal antibodies because it gives a much better yield than in vitro cultivation of hybridoma cells. However, this in vivo production is limited when scaling up, by the need for large numbers of animals. The contamination of ascites preparations by proteins of the host animal is another disadvantage, and in addition, human hybrids cannot be grown in ascites at all. On the other hand, the in vitro cultivation of hybridoma cell lines gives low antibody concentrations, only 1-10% of the levels normally reached in ascites fluid, and only a few cell lines have been grown in large scale. The reason for these disappointing results may be the extreme fragility of hybrid cells and/or the inadequate supply of nutrients and oxygen. It is likely, though, that large-scale operations using both suspension cultures as well as hollow-fiber microcarriers or ceramic systems will increase in importance. We have tried an alternate scheme to overcome some of the problems mentioned above by entrapment of cells in a matrix such as agarose. The matrix protects the cells from mechanical stress, and makes the preparations suitable for continuous operation in that the produced antibodies are excreted into the medium, thereby eliminating the need for separation of product from the cells. Preparation of Extracted Paraffin Oil Paraffin oiP (I00 ml) is extracted four times with PBS 2 (100 ml) in a separating funnel (500 ml). After the final extraction the mixture is alt Merck No. 7162 or BDH No. 29436. 2 K. Nilsson, W. Scheirer, O. W. Merten, L. 0stberg, E. Liehl, H. W. D. Katinger, and K. Mosbach, Nature (London) 302, 629 (1983).

METHODS IN ENZYMOLOGY, VOL. 121

Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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lowed to stand for 30 min to allow complete phase separation. The collected paraffin oil is either sterilized by heating for 2 hr at 160°, or autoclaved in closed bottles at 120° for 30 min. Each lot of paraffin oil should be tested for toxicity. This may be done, for instance, by comparing the growth curves of the cell line of interest in the presence or absence of paraffin oil (5%, v/v). If the extraction procedure does not remove all traces of toxic compounds paraffin oil from different manufacturers should be tested. Agarose Preparation

Two types of agarose are suitable for the entrapment of animal cells. Sigma type VII and FMC Sea Plaque both have a dynamic gelling temperature of 28 °, and Sigma type I or FMC SeaKem LE have dynamic gelling temperatures of 36° . As the dynamic gelling temperatures are determined when a solution is cooled at fixed rate, and are about 10° lower than the static gelling temperatures, one has to thermostat the different agarose solutions to about 37 and 45 °, respectively. The lower gelling temperature is obtained through a chemical modification of the agarose, which makes the polymer less porous and decreases its mechanical strength. Procedure. The required amount of agarose is mixed with PBS and autoclaved (it is not necessary to dissolve the polymer before steriliza-

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FIG. 1. Influence of agarose concentration on cell growth. Hybridoma cells (mousemouse against ovalbumin) were entrapped in agarose (Sigma type VII) of different concentrations at a cell concentration of 1.0 million cells/ml beads. Beads with entrapped cells (10 ml) were cultivated in a 50-ml spinner bottle with 50 ml medium, and 50% of the medium was replaced on days 3, 5, and 7. The number of free cells in the medium were counted in a Burker chamber each day. (O) 1%; (11) 2%; (,k) 3% (w/v) agarose.

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tion). After sterilization it can be stored at room temperature. Before use it is remelted by heating to 70 ° and thermostatted to either 37 or 45 °, depending on the type being used. Notes: The concentration of agarose is dependent on the type and the desired mechanical strength of the beads. For low temperature gelling agarose a final strength of at least 1% (w/v) should be used. The polymeric network will have an influence on cell growth, and therefore at higher agarose concentrations growth will be more restricted (Fig. 1). High temperature gelling agarose at 0.5% (w/v) will provide enough mechanical stability for small-scale culture. The most convenient way to immobilize cells is to prepare the agarose solution at twice the final concentration and mix it with an equal volume of suspended cells. Due to the viscosity of agarose solutions at high concentrations this is not practical for concentrations higher than 5% (low gelling temperature) or 4% (high gelling temperature). However, as the agarose solution is made up in PBS, it is possible to centrifuge down the required amount of cells and redisperse them directly into the agarose solution. Entrapment in Agarose Beads 2,3 General Procedure. The preparation of agarose beads from high or low gelling temperature agarose differs only in the mixing step: for low gelling temperature agarose, the paraffin oil, agarose solution, and cell suspension are all thermostatted to 37°, whereas for high gelling temperature agarose, the paraffin oil and cell suspension are thermostatted to 37° and the agarose solution is thermostatted to 45 °. After mixing the cell suspension and agarose, the mixture is dispersed in the paraffin oil and, when the desired bead size is reached, the dispersion is cooled to at least 10° below the gelling temperature. Medium 4 is added, and after sedimentation of the beads the oil phase and most of the washing medium are aspirated. The beads are further washed with medium until they are essentially free from oil. Small-Scale Procedure. The cell suspension (5 ml) is mixed with agarose (5 ml, 2% w/v) and poured into a beaker (100 ml) containing paraffin oil (20 ml). The mixture is dispersed with a magnetic stirrer, and when the desired bead size is obtained (100-300/zm), the beaker is cooled in an ice bath. After solidification, medium is added (30 ml) and the beads are allowed to sediment into the medium. They are further washed with several portions of medium until essentially free from oil. 3 All experiments described in this chapter were carried out with low temperature gelling agarose (Sigma type VII). 4 The culture medium for entrapped cells is the same as in the normal cultivation.

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Notes. (I) The whole procedure is carried out in a sterile hood. (2) Sedimentation of the beads can be speeded up by gentle centrifugation. (3) In the washing steps media can be replaced by PBS. Medium-Size Procedure. The cell suspension (50 ml) is mixed with agarose (50 ml, 2% w/v) and poured into a round-bottomed glass centrifuge tube (250 ml) containing paraffin oil (100 ml). The liquids are emulsified at room temperature, with a Vibromixer E1 fitted with a vibrator plate (P1, 54 mm diameter), to the desired bead size. The mixing vessel is cooled in an ice bath for 5 min and 50 ml growth medium is added. After 10 min at room temperature the tube is centrifuged at 1200 g for 10 min. The oil phase is removed by suction and approximately 100 ml growth medium is added. After mixing with the vibrator plate, the suspension is centrifuged again and the remaining oil removed. Note: If volumes larger than 100 ml of beads are needed the process may be repeated several times. The method is convenient for batches of beads up to 1000 ml, but if larger volumes are needed the method must be modified, either by using larger mixing vessels, or by making the dispersion directly in a reactor equipped with a stirring device, and thereby carrying out the whole procedure in the cultivating vessel. Cell Concentration With the entrapment method one has the possibility to obtain a wide variety of cell concentrations. There are actually two types of cell concentration that have to be considered: (1) the total cell concentration, i.e., the total number of cells divided by the total volume used, and (2) the concentration of cells per volume of beads. As the usual amount of beads is 10% (v/v), the cell concentration inside the beads will be 10 times higher than the total cell concentration. Entrapped cells will experience those small molecules provided by the medium at a concentration equal to the total cell concentration; however, molecules produced by the cells themselves will accumulate inside the beads and the cells will experience them at substantially higher concentrations than those seen in the medium. This may be advantageous if the cells produce a stimulator; but, if an inhibitory substance is produced by the cells, entrapment will have a negative influence on performance. This effect can be counteracted by increased mixing in the cultivating vessel and/or a more frequent exchange of media, thereby making the concentration gradient for the substance steeper. The method described here makes it possible to entrap cells at a low concentration and after a growth phase reach a high cell density, this will reduce the amount of cells needed for the inoculum. On the other hand it is also possible to entrap cells at an extremely high density and obtain a

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FIG. 2. Correlation between concentration of free cells (mouse-mouse hybridoma) and relative fluorescence after staining with mithramycin.

high catalytic activity per reactor volume. Cells have been entrapped in agarose at concentrations between 0.1 and 20 million cells/ml beads. As the entrapped cells have the same nutritional demands as free cells, one has to consider the extreme cell densities which can be reached when supplying medium and oxygen. Monitoring of Cell Growth Direct microscopic observation for monitoring of cell growth is impossible due to the high concentration of cells inside the beads. Attempts to free the cells from the beads result in partial destruction. Indirect measurement of cell concentrations must therefore be applied. A suitable method is to determine the amount of DNA and correlate it to cell concentration. This is conveniently done with the antibiotic mithramycin, which binds to double-stranded DNA and fluoresces in direct proportion to the DNA present. 5 Procedure. A 4-ml sample of suspended beads is centrifuged, and after decanting of the supernatant, 4 ml mithramycin solution is added (2.5 mg mithramycin in 250 ml PBS containing 15 m M MgC12), The sample is cooled in an ice bath and sonicated (70 W, microtip) for 30 sec to ensure destruction of the beads. The fluorescence is then measured directly (excitation wavelength 450 nm, emission wavelength 550 nm). 6 5 B. T. Hill and S. Whatley, F E B S Lett. 56, 20 (1975). 6 G. Himmler, G. Palfi, H. W. D. Katinger, and W. Scheirer, Dev. Biol. Stand. 60, 291 (1985).

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C a l i b r a t i o n is p e r f o r m e d b y e s t i m a t i o n b y t h e s a m e m e t h o d o f a susp e n s i o n o f f r e e c e l l s o f k n o w n c o n c e n t r a t i o n . I n this w a y a c a l i b r a t i o n c u r v e is o b t a i n e d ( s e e F i g . 2). R e s i d u e s o f p h e n o l r e d in t h e t i s s u e c u l t u r e media and polymeric material do not interfere. The useful range of the e s t i m a t i o n is b e t w e e n 0.05 to 2 m i l l i o n c e l l s / m l . T h e l i n e a r r e g r e s s i o n c o e f f i c i e n t h a s b e e n f o u n d to b e 0.98. Reactor Design F o r s m a l l - s c a l e p r e p a r a t i o n s (up to 50 ml o f b e a d s in 500 ml o f m e d i u m ) , it is u s u a l l y n o t n e c e s s a r y to p r o v i d e a n e x t e r n a l s o u r c e o f o x y g e n . T h e m o s t c o n v e n i e n t w a y to c u l t i v a t e t h e s e e n t r a p p e d cells is b y u s i n g s p i n n e r flasks o r r o l l e r b o t t l e s (see t a b l e ) , in w h i c h t h e p H is c o n t r o l l e d PRODUCTION OF MONOCLONAL ANTIBODIES BY AGAROSE-ENTRAPPED CELLS

Day

IgG" (reciprocal dilution)

IgM b (/zg/ml)

1 2 3 4 5 6 7

256 512 256 512 512 n.d. C 256

7.4 6.0 6.3 6.0 6.7 6.7 6.7

Monoclonal antibody (IgG) against herpes simplex type 2 glycoprotein was produced by a mouse-mouse hybridoma (LSP 21), which was entrapped in 2.5% (w/v) agarose at a concentration of 6.2 million cells/ml beads. Beads with entrapped cells (20 ml) were cultivated in 100 ml medium in a 250-ml spinner bottle, and 50% of the medium was replaced daily. Figures show IgG concentration in the harvested medium. b Monoclonal antibody (IgM) against human urokinase was produced by a mouse-mouse hybridoma (2T2 E8) which was entrapped in 1.0% (w/v) agarose at a cell concentration of 5.6 million cells/ml beads. Beads with entrapped cells (120 ml) were cultivated in a roller bottle with 400 ml medium. The medium was replaced daily. Figures show IgM concentration in the harvested medium. c n.d., Not determined.

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with a mixture of 5% COz and 95% air. Medium can easily be perfused by equipping the outlet tube with a sintered-glass filter. For a larger scale the most convenient reactor is the draft tube fermenter (Fig. 3), in which the beads are circulated by an axial marine-type impeller. Oxygenation is performed by direct titration with pure oxygen by gentle sparging. In addition, surface aeration can supply some oxygen and remove metabolic

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FIG. 3. Draft tube fermenter fitted with rotating basket for continuous peffusion with medium. Control circuits for oxygen and pH are shown.

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CO2 from the suspension to keep the pH in an appropriate range. Perfusion is performed by fitting a basket of stainless-steel mesh to the impeller shaft, thus making it possible to remove the medium from the inside of this basket without removing the beads. Growth Control If the cells are entrapped in low concentrations of agarose, the cell growth will be almost unaffected, and therefore, after a few days a large number of free cells will appear in the medium. To avoid problems associated with free cells (need for centrifugation, clogging of filters, etc.), it is thus necessary to control the cell growth inside the beads. This can be done in several ways: (1) Use of higher concentrations of agarose, as seen in Fig. 1. The cell growth can be slowed considerably by using a final agarose concentration of 3% (w/v). (2) Reduction of the temperature. As seen in Fig. 4, cell growth is reduced if the culturing temperature is

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FIG. 4. Production of monoclonal antibody (IgG) against influenza A, H3N2, by a human-mouse hybridoma. Cells were entrapped in 1.0% (w/v) agarose at a concentration of 3.3 million cells/ml beads. One liter of beads was cultivated in 10 liters of medium in a draft tube fermenter (shown in Fig. 3). Perfusion rate was 3.6 liters/day. At day 15 the cultivation temperature was lowered from 38 to 35°. (O) Cell concentration per total volume (determined by mithramycin staining); (©) tzg human IgG/ml.

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decreased from 38 to 35 °. In this case the reduction in temperature also results in increased product formation. The optimal temperature has to be determined for each cell line. The goal is to find a temperature at which the reduction in cell number (caused, for instance, by proteolytic enzymes) is balanced by net growth. This will provide a production of antibodies at a constant high level. (3) Reduction of the fetal calf serum concentration. Cell growth is also retarded if the concentration is decreased from the normally used 5 or 10% to 0.5 or 1%. This obviously may make product purification easier. Conclusions We have presented here methods for the entrapment of animal cells in beaded polymers which easily can be carried out in a normal laboratory without specialized equipment. These agarose-entrapped cells have been used for production of monoclonal antibodies (see table and Fig. 4). The method has been used both on a small scale (50 ml reactor volume) and on a large scale (10 liters reactor volume). The use of this method is of course not limited to hybridoma cells. Other cell lines which have been entrapped are Chinese hamster ovary cells (genetically modified to produce human interferon) and lymphoblastoid cells (producing interleukin 2). Experimental details, as well as entrapment matrices for anchorage-dependent cells, will be described in a forthcoming volume of this series. 7 7 K. Nilsson, W. Scheirer, H. W. D. Katinger, and K. Mosbach, this series, in press.

[33] A u t o m a t e d P r o d u c t i o n o f M o n o c l o n a l A n t i b o d i e s in a C y t o s t a t

By S. FAZEKASDE ST.GROTH A conventional tissue culture flask of 75-cm 2 growth area will support, at most, 4 × 10 7 hybridoma cells and may thus maximally yield around 1 mg of monoclonal antibody. Average, practical yields are about an order of magnitude lower. By using larger bottles, hollow-fiber systems, or suspension cultures with or without microcarrier beads, the number of cells per culture unit can be increased up to 10-fold, thus considerably reducing the labor per unit. But each of these systems is self-terminating METHODS IN ENZYMOLOGY, VOL. 121

Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.