Journal of Biotechnology, 2 (1985) 241-255 241 Elsevier

JBT 00156

Minireview

Large-scale cultivation of hybridoma cells

David H. Randerson Department of Chemical Engineering and Biotechnology Unit, University of Queensland, St Lucia, QId. 4067,

Australia

(Received 9 October 1984; accepted 21 February 1985)

Summary

The demand for large quantities of monoclonal antibodies has increased dramati- cally over the last few years to supply diagnostic kits for application areas with large markets, and for use in developing applications such as anti-tumour therapies and affinity chromatography. Scale-up from tissue and murine culture has not been a straightforward matter because of the special requirements of mammalian cells in culture. The sensitivity of hybridomas to their environment means that efficient mixing must be obtained at low shear rates, and that oxygen transfer must be adequate for high density cultures without aggressive sparging. The high fraction of serum required to satisfy undefined nutrient requirements needs to be reduced considerably in order to achieve economy of scale. This review discusses the technical barriers to scale-up and reports on successful attempts to produce large volumes of monoclonal antibodies.

hybridoma, monoclonal antibody, medium, oxygen demand, scale-up, growth rate, microencapsulation, membrane, chemostat

Introduction

The major commercial end-use of monoclonal antibodies is currently in the production of veterinary and human diagnostic kits, which are used to detect specific substances and cell types in biological fluids. The quantities of antibodies required are modest, and a single murine culture is usually sufficient to supply 10000 to 100000 assays. Nonetheless, production of monoclonal antibodies in a single, large-volume batch may be advantageous to ensure product consistency, as well as supplying the millions of assays performed in such areas as blood grouping,

0168-1656/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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pregnancy tests and sexually transmissible diseases, and other high volume applica- tions.

Monoclonals are being used increasingly for other novel applications such as: (a) precise targeting of pathogenic cells, in particular tumours, for imaging and

destruction (Hellstrbm et al., 1980; Ritz and Schlossman, 1982; Scheinberg et al., 1982; Levy and Miller, 1983),

(b) affinity purification systems for products of high commercial value, such as blood derivatives and pharmaceuticals (Secher and Burke, 1980; Tarnowski and Liptak, 1983),

(c) extracorporeal shunts of immobilized antibodies for therapeutic immunoadsorp- tion of circulating toxins and pathogens (Pak et al., 1984).

All of these applications potentially require tens of kilograms of antibody annually. The viability of monoclonal based cancer therapies, immunopurification systems and extracorporeal immunoadsorption will depend on the ability to large- scale cultivate hybridoma cells and at the same time reduce the cost of production.

Although in vitro cultivation of mammalian cells has been practised for almost a century, it is only in the last decade that there has been development of large-scale systems. These range in volume from 1 1 up to 1000 1 and are used for the production of vaccines (polio, mumps, measles, rubella, rabies and foot-and-mouth disease), plasminogen, interferon and hormones, such as human growth hormone, insulin and erythropoietin (Acton and Lynn, 1977).

Mammalian cells that multiply in suspension, i.e. that are not anchorage depen- dent, can usually be cultivated by techniques similar to those used in microbial biotechnology. However, there are several distinctions between mammalian cells and microbes which render conventional technologies inadequate. These include the sensitivity of mammalian cells to their milieu, and special nutrient requirements.

Hybridomas are routinely cultivated in 50-75 ml tissue culture flasks and in volumes up to 1 1 in spinner flasks and roller bottles. Such techniques yield antibody titres of 10-20/~g ml-i. To obtain a higher concentration of antibody hybridomas may be grown as ascites, that is, as a tumour within the peritoneal cavity of a histocompatible mouse or rat. This technique provides up to 15 ml of 5-20 mg ml- t of ascitic fluid before the host succumbs to the tumour (Kennett et al., 1980; Hurrell, 1982).

Restraints to large-volume cultivation of hybridomas include: (a) poorly defined nutrient needs, necessitating the use of a costly serum compo-

nent, (b) toxic metabolite accumulation, (c) high oxygen demand which must be supplied without aggressive sparging, (d) shear and environmental sensitivity of cells, and (e) lack of special equipment and process control.

There are also inherent problems in long-term culture of hybridomas, such as chromosomal stability, overgrowth by non-producing cells, and sterility and myco- plasma contamination which will not be addressed in this revue.

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Kinetics of cell growth

The growth of hybridomas can be expected to demonstrate similar growth characteristics to microbial cells, and modelling of growth kinetics will, in all likelihood, require an analogous approach.

Growth profiles for mammalian cells have been published by Acton and Lynn (1977), for a murine lymphoblastoid line, and by Fazekas de St. Groth (1983), for a murine hybridoma. Characteristic lag, exponential and stationary phases can be delineated. We have observed similar behaviour with hybridomas in which specific growth rates ranged from 0.03 to 0.04 h-1, corresponding to cell doubling times of 18-24 h.

Unrestrained cell growth (exponential phase) is sustained only while all nutrients are present in excess and no other factors are present which may inhibit growth (Bailey and Ollis, 1977; Fiechter, 1981). Hybridomas in small scale tissue culture enter the stationary phase after 60-80 h with maximum cell densities of 0.8-1.5 × 106 cells per ml-1. It has not been definitively established whether decline in growth of hybridomas in batch culture is the result of exhaustion of one or more substrate(s) necessary for cell metabolism; whether metabolic wastes, including H ÷ elevation; or whether oxygen depletion is responsible.

Cell culture media

It is not the intention of this section to present an extensive discussion of nutrient requirements, as this may be found in any good monograph on tissue culture (e.g. Jakoby and Pastan, 1979), and books specifically relating to hybridoma techniques (Kennett et al., 1980; Hurrell, 1982).

Media are based on balanced salt solutions (BSS) which, amongst other things, provide inorganic ions while maintaining physiological pH (ca. 7.2) and osmotic pressure. Glucose is supplied as a source of energy. Most media used in tissue culture of hybridomas are based on a BSS to which specific nutrients, vitamins, hormones, trace elements and growth factors have been added (for example, RPMI 1640 originally formulated for lymphocyte culture, and Dulbecco's modification of Eagle's medium, DMEM, as formulated for fibroblasts), and 5-20% animal serum or sera (horse, calf, or human) to supply 'undefined' components. Foetal or newborn serum is generally preferred due to the absence of any existing immunoglobulins which could interfere with the purification of the monoclonal antibodies, and the presence of mitogens or growth factors which stimulate cell division.

At least one report of suspension cultivation of hybridoma cells using standard formulations, such as RPMI 1640, and foetal calf serum (FCS) suggests that the level of serum can be reduced to 1-3% once an exponential growth has been obtained (Thomson and Birch, 1983)..Similar observations have been made for other animal ceils in continuous cultivation (Acton and Lynn, 1977; Tolbert and Feder, 1983).

Cultivation of cells in a totally defined, serum-free medium has several ad- vantages. Since antibody production by hybridomas is usually in the range of 10-20

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#g ml-t of culture fluid, the use of serum introduces a vast excess of heterologous protein that must be removed during antibody purification. Hence, additional production costs in a commercial operation. The cost of medium resulting from the addition of FCS is considerable, approximately $20-30 per 1 for FCS containing medium compared with $2-5 per 1 for serum-free. The supply of large volumes of FCS is likely to represent a major problem in most countries.

There has been considerable progress in attempts to grow hybridomas in serum- free media. The first reported serum-free medium used successfully with animal cells was Iscove's medium, which contains a specially prepared lipid stock (Iscove and Melchers, 1978; Andersson and Melchers, 1978). This formulation has been used for hybridoma cultivation by Fazekas de St. Groth and Scheidegger (1980). Cleveland et al. (1983) used a base of Iscove's medium for hybridoma growth to which were added trace elements, progesterone, thioglycerol, and in some studies transferrin, insulin and liposomes. However, the difficulties in preparing complete Iscove's medium and the higher cost of commercial preparations make it a less than desirable alternative to serum-containing media.

Chang et al. (1980) reported that some hybridoma lines grew, albeit with a marked reduction in growth rates, in a very simple medium consisting of enriched RPMI 1640 supplemented with only insulin, transferrin and non-essential amino acids. The addition of insulin, transferrin, 2-amino ethanol and selenium into a basal medium of DMEM was found by Murakami et al. (1982) to support particular hybridomas with growth rates comparable to those observed in serum-containing medium. Kov~ and Fran~k (1984) supplemented RPMI 1640 with insulin, transfer- rin, 2-amino ethanol, linoleic acid, albumin, ascorbic acid, hydrocortisone and 12 trace elements including selenium and magnesium. In all the above-cited studies various cell lines showed different responses to deprivation of serum.

Hana Biologics Inc. of California are marketing a serum-free medium which contains insulin, transferrin, fatty acid-linked BSA and 2-amino ethanol specifically for the cultivation of hybridoma cells (Steimer, 1982).

No information is available concerning use of the above serum-free formulations for large-scale cultivation of hybridomas. However, Iscove's medium has been used successfully in 500 ml continuous suspension culture by Fazekas de St. Groth (1983) and will be discussed subsequently.

Hybridomas have also been grown on dialyzable serum components (Klinman and McKearn, 1981) and within dialysis tubes (Adamson et al., 1983) with exclusion of substances greater than 10000 dalton, and in chambers separated from the nutrient by ultrafiltration membranes with a molecular weight cut-off of 50000 dalton (Wiemann et al., 1983). These findings demonstrate that higher molecular weight substances in serum, viz. proteins, may not be necessary for cell propagation (although it is conceivable that transport properties of proteins may be retained in the case of the ultrafiltration membrane perfusion system), and hence suggest the possibility for cultivating cells using ultrafiltered adult serum to supplement the basal medium.

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Oxygen transfer

Several investigations have determined oxygen demand for the maintenance of a steady-state culture of mammalian cells (see Glacken et al., 1983). Measurements range from 0.053 to 0.59 mmol O 2 1-1 h-1. Boraston et al. (1984) have determined oxygen demand for a hybridoma line in continuous culture as 0.22 mmol O 2 1- i h- at a cell density of 1.05 × 106, cells per ml. Since, in any culture broth, the maximum inventory of dissolved oxygen is relatively low, approximating 0.2 mmol O2 1-~ at 37°C, then it is obvious that oxygen must be added continuously to the liquid to maintain a viable cell population.

Glacken et al. (1983) calculated that oxygen deprivation would occur in a culture vessel with 107 HeLa cells m1-1 in which oxygen transfer is only from the header space air, if the culture volume exceeded 1 1. If oxygen replaced air in the header this volume could be increased to 3.5 1. Thus additional oxygen must be introduced directly to the medium for larger culture volumes. The authors go on to point out that conventional air sparging may be unsuitable for mammalian cells due to cell lysis and foaming of the medium. The latter restriction adds further impetus to the development of serum-free media to reduce the protein content. Other methods of oxygenation as discussed by Glacken et al. include: (a) recirculation of the medium through the culture vessel with oxygenation in an external vessel, (b) the use of silicone tubing which is permeable to oxygen, as gas delivery coils submerged in the medium, and (c) incorporation of a silicone-membrane reactor lining with air passed between the vessel walls and the membrane. All are apparently difficult to imple- ment and to scale-up.

Boraston et al. (1984) report the only detailed study of oxygen requirement for hybridoma cells in mass culture. They found that dissolved oxygen tension (DOT) had very little influence on cell growth rate when it was maintained above 870 of saturation. The oxygen utilization rate, which averaged 0.209 p, mol per 106 cells per h at maximum growth rate of 0.045 h-~, was essentially constant over a DOT of 8-6070, although it appeared to have increased slightly at 10070 DOT. They also determined that the oxygen required for cell maintenance was 0.056 #mol O 2 per 106 cells per h, which was about 3070 of the total oxygen used by cells growing at maximum rate. They suggested that the high fraction consumed for maintenance, compared to microbial cells, simply reflects the slower growth of mammalian cells.

Metabolic waste products

Mammalian cells in tissue are nurtured in a homeostatic environment from which waste metabolites are continually cleared by the kidneys and liver, and acid-base balance maintained by lungs and kidneys. Both functions are mediated via the circulation. In an in vitro static culture of cells, metabolic byproducts will accu- mulate, inhibiting cell function and ultimately resulting in death. (Cell death in tissue culture may be the result of nutrient depletion, as well as toxic inhibition, or both.) The toxic wastes, and the levels at which they become detrimental to cell survival, have not been specifically identified for cell culture systems; however, H +

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in the form of nonvolatile acids, e.g. lactic acid, and ammonia and urea, amongst others, may be implicated. It is difficult to obtain information concerning detrimen- tal ammonia, lactic acid and pH levels as most data have been acquired in static culture where all factors are changing to produce synergistically a hostile environ- ment. Glacken et al. (1983) report that prolonged exposure to ammonia levels higher than 4 mmol 1-i can inhibit growth, and found that HeLa cells were capable of generating in excess of 30 mmol 1-1 over 6 doublings. We generally find lactate levels of 12-15 mmol 1- i and pH of 6.7-7.0 at the termination of a static hybridoma culture in which cell densities reach ca. 1 × 106 cells per 1. Barton (1971) has suggested pH in animal cell cultures should be maintained between 7.0 and 7.5. Any level above 7.5 is definitely harmful to hybridomas.

Manipulations of medium in mammalian cell culture have been used to control lactic acid and ammonia production to some extent. These include the replacement of glucose by galactose as proposed by Eagle (Eagle et al., 1958; Glacken et al., 1983) to minimize lactic acid generation, and the maintenance of low glucose and glutamine levels by their continuous infusion to reduce ammonia production (Glacken et al., 1983). Such techniques may be of value in hybridoma cultivation.

Chemostat culture with continual removal of cell mass and nutrient, results in a steady-state of metabolite levels and may therefore be used to limit metabolite accumulation. The actual metabolite level depends on cell density, dilution rate and metabolite generation rate. The usual mode of operation is to have dilution rate equal to cell growth rate to maintain a stationary cell mass. However, any attempt to operate at high cell densities may be restricted by an elevation of metabolite concentration. Thus a chemostat with 3 x 106 hybrodoma cells 1-1 would have a lactate level of 35-50 mmol 1-1, which would most certainly be toxic. To raise cell mass beyond this requires nutrient throughput or dilution rate, to be greater than cell growth rate. This, of course, is the principle behind perfusion culture and hollow fibre cultivation systems.

Tolbert and Feder (1983) present data for the maintenance of rat carcinosarcoma in three culture modes: static culture, continuous perfusion in which cells are retained while nutrient perfuses the culture vessel, and in chemostat culture. In 4 1 static culture, cell growth entered the stationary phase after 50 h at which point lactic acid level was 39 mmol 1-1 and glucose had declined from 25 mmol 1- i to less than 5.5 mmol 1-1. In perfusion culture in which density reached 3.1 × 107 cells ml-1 after 67 h, lactate levels remained stable at 11-22 mmol 1-1 and glucose at 11-17 mmol 1-1 with a total nutrient throughput of 31.3 1. Although medium supply was progressively increased relative to biomass, the mean residence time was 8.6 h. Operation of a 12 1 chemostat with 1 × 107 cells ml- i utilizing a constant medium perfusion rate required a residence time of 24 h. Lactic acid and glucose !evels were stable at approximately 20 and 11-22 mmol 1-1, respectively.

The only report for hybridoma cells in chemostat culture was presented by Fazekas de St. Groth (1983) which cited residence times of 25.0, 25.6 and 27.1 h to maintain cell concentrations at 1 x 105, 3 × 105 and 1 x 106 cells m1-1, respectively. The higher residence times, and hence reduced cell growth rates, at the higher cell masses infer mild toxicity, or nutrient deprivation.

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Hardware for suspension culture

Mammalian cell membranes are thin (75-100/k), elastic bilipid structures con- taining protein intrusions, and as such they are readily ruptured by shear forces. Microbes, on the other hand, are generally smaller and have cell walls. They are therefore more durable. In addition, microbes have greater metabolic and prolifera- tion rates. Thus equipment designed for microbial cultivation is not always suitable for the growth of mammalian cells. There are very few dedicated systems available commercially, and more often than not special equipment has to be designed or existing equipment modified. Equipment produced by Bilthoven (The Netherlands) and New Brunswick Scientific (U.S.A.) has been developed specifically for mam- malian cells and may be suitable for hybridoma cultivation. LSL Biolaffitte (France) and Marubishi (Japan) market equipment which they consider suitable for hy- bridomas. Air-lift bioreactors have been modified by Celltech Ltd. (Great Britain) for in-house cultivation of hybridomas.

Mammalian cells are extremely sensitive to temperature variation, being unable to tolerate temperatures too much above 40°C, thus the avoidance of hot spots in the culture vessel is critical. Likewise, stagnant zones depleted of nutrient or with excessive metabolite build-up can be catastrophic. Hence, good agitation is a prime requirement of any system.

There have been no studies performed on the shear sensitivity of hybridoma cells. Fazekas de St. Groth (1983) cites a maximum impeller speed of 60 rpm above which cell multiplication was observed to be impaired. Although stirrer details are not explicit this would suggest that a maximum tip speed of approximately 20 cm s-~ is tolerable. The only other information available for mammalian cells was published by Augenstein et al. (1971) for HeLa and mouse L929 cells. Values for shear stresses of about 5 × 103 dyne cm -2 in a capillary tube caused significant cell disruption. This, however, is several orders of magnitude higher than one would expect from the findings of Fazekas de St. Groth, which may suggest an unusually high sensitivity of the fused cells.

Thomson and Birch (1983) have compared values for maximum shear stresses induced in vessels with stirred agitators and air-lift mixing. They employed the correlation developed by Bell and Dunnill (1982) for shear rate, R, in a vessel:

R = ( 2 7 r l z N y / V ) 1/2

where

y = 721 N2D

and/~ is the cell suspension viscosity, N is the rotational speed of an agitator with blade diameter D, and V is vessel volume. They determined a shear stress of 0.68 dyne cm -2 for a 15 1 vessel containing two Rushton turbine impellers. The calculation assumed an impeller speed of 100 rpm which was considered necessary to supply oxygen dissolution at the rate of 0.6 mmol 1-1 h- 1 at 0.05 11- i min- ~ oxygen

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perfusion. They go on to mention that, although this average stress is considerably below the critical value given by Augenstein et al., the impeller tip speed, which they calculated as 42 cm s-~ is above that suggested by Fazekas de St. Groth. Further- more, the calculated Reynold's number of 8200 implied turbulent conditions in the region of the impeller.

Although Thomson and Birch discuss the use of marine impellers, for which oxygen transfer rates are significantly poorer, and the Vibromixer, which has been implicated as responsible for low cell growth rates, they probably dismissed the use of mechanical agitators too summarily in favour of the air-lift system. It should not be a difficult matter to reduce the tip velocity of an impeller 3-fold and hence Reynold's number 9-fold to satisfy the observed maximum speed reported by Fazekas de St. Groth and still maintain high oxygen transfer.

New Brunswick Scientific Company fermenters have been used with mammalian cell cultivation, in which bottom mounted, off-centred, magnetically-driven, marine impellers have been installed (Lynn and Acton, 1975; Zwerner et al., 1981). These may be suitable for hybridoma cultivation.

Another possibility for providing adequate, gently agitation is the Vibromixer (Moore et al., 1968; Fontanges et al., 1971; Tolbert and Feder, 1983). This agitation system consists of one or more discs containing conical holes attached to a vertical shaft. The shaft and discs vibrate vertically with an amplitude of 1-3 mm at 60 cycles s- i . The shape of the conical holes causes vertical agitation of the suspension rather than the horizontal agitation as produced by turbine blades.

Tolbert and Feder (1983) of Monsanto (U.S.A.) developed a cultivation system utilizing large, flexible sail-like sheets which rotate slowly (down to 10 rpm) to provide agitation. Four sheets were incorporated into a 40 1 reaction vessel, and rotated by a magnetic coupling. A further novel aspect of their mammalian cell cultivation system was to utilize a second vessel in which medium was filtered from the cells via a stationary cylindrical porcelain 1.0 #m filter. Around this filter rotated a magnetically-driven, nine-vane sail agitator to prevent cellular clogging of the filter. These were rotated at 50-75 rpm. Cells and medium were pumped back and forth between the culture vessel and the smaller filter vessel. The latter was designed for easy replacement, because of the limited life of the filters, without requiring termination of the run. These authors have suggested cultivating hybridomas in such a system (Feder and Tolbert, 1983).

Other strategies for protecting cells from shear stresses are to encapsulate them in an artificial cell wall (Lim and Sun, 1980; Nilsson et al., 1983), and to culture them in the housing of a shell-and-tube style membrane system with the nutrient perfused through the capillary lumens (Knazek et al., 1972). These two techniques will be discussed in the next section.

Specific techniques for the cultivation of hybridomas

Hollow-fibre immobilization The rationale behind the hollow-fibre systems is to simulate cell tissue, normally

with densities of the order of 101° cells ml- i, and which are networked by capillaries.

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Blood perfusing the capillaries provides a highly efficient exchange of nutrients, gases and metabolites to the dense tissue masses.

Knazek et al. (1972) pioneered the application of shell-and-tube-like hollow-fibre polymeric membrane systems in which cells were grown in the shell while nutrient was recirculated from an external reservoir through the lumen of the fibres. The membranes used have molecular weight cut-offs of 20 000-100 000 dalton such that low molecular weight nutrients and dissolved gases can cross the membrane into the cell culture chamber and waste metabolites are convected back into the medium. High molecular weight products are retained in the cell chamber and may be flushed out on a regular basis in a relatively pure form. Serum levels of media can generally be reduced to 1-2% and adult serum may replace foetal because product contamina- tion is generally not a problem. Although cell division must eventually cease, the cells apparently continue to secrete macromolecules for weeks and even months. One would expect a non-uniform cellular environment with gradients in concentration of nutrients and metabolites as well as oxygen deprivation, and that this would be detrimental to product formation and maintenance of a viable long-term culture. This does not appear to be the case; however, it is probably true that only the cells adjacent to the fibres are productive, constituting only 0.1-1% of the total cell mass.

An adaptation of the original K.nazek system has been reported for monoclonal production by Calabresi and colleagues (Caiabresi et al., 1981; Wiemann et al., 1983). In one instance, capillaries with a 50000 dalton exclusion limit and a 2.5 ml chamber volume were inoculated with 5.5 × 107 hybridoma ceils. Fluid from the extracapillary space was removed every 2-3 days. Approximately 30 mg of antibody could be obtained weekly at levels of 6-8 mg ml-1.

Commercial hollow-fibre cartridges specifically designed for mammalian cell culture, are available from the Amicon Corporation (U.S.A.). Cartridges from other manufacturers (e.g. Romicon, Celanese, Asahi, Membrana, amongst others) could equally as well be used although they are produced for other purposes such as microfiltration or haemofiltration. Amicon quote a production cost of US$ 475 per gram of antibody compared with $ 850 for mouse ascites and $ 2800 for conven- tional tissue culture (Vitafiber II, 1983 product brochure).

Microencapsulation Entrapment of cells into small porous capsules enables high cell densities (107

cells m1-1) to be obtained in standard cultivation equipment. Both cells and the product (which is retained in the microcapsules) are protected by the encapsulation from the effects of shear. An easily purified protein is obtained within the micro- capsules as extraneous protein in the nutrient is restricted from entering the capsules, and low molecular weight nutrients and wastes diffuse in and out of the capsules.

Damon Biotech (Needham, MA, U.S.A.) have patented the ENCAPCEL Process for cultivation of monoclonal antibodies (Lim and Sun, 1980; Lim, 1983a, b). To produce the microcapsules hybridomas are suspended in sodium alginate in foetal calf serum and normal saline. Droplets extruded from a syringe gel immediately upon immersion into a solution of divalent cations (CaCI2). The mixture is then

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treated with a 0.16% solution of poly(L-lysine). The polymer adsorbs to the surface of the alginate gum where a cross-linking reaction occurs forming a semipermeable coat. The permeability of the coat, which is controlled by the molecular weight of the polymer and the duration of reaction, is chosen to have a limit of about 100000 dalton. The interior of the resulting capsules are then reliquified by immersion in a sodium citrate solution replacing the divalent ions in the gel.

The encapsulated cells are immersed in a culture medium containing 1-3% foetal calf serum in a batch type process for 7-14 days. 30-35 l-vessels produce ca. 10 g of antibody per run (Lim, 1983b). Capsules are harvested by centrifugation and disrupted using a polyanionic polymer, such as heparin, to recover the antibody. Damon are expanding to produce 10-20 kg per year of antibody using 100-1 vessels.

An apparent disadvantage of the system is the cost of encapsulation and product recovery. Damon, nonetheless, contend that their monoclonal antibody production costs for custom cultivation are competitive with other large-scale producers.

Damon have sizeable contracts with Hoffman-La Roche (Nutley, N J, U.S.A.) to produce an anti-interferon monoclonal antibody for interferon purification, and with the National Cancer Institute (Bethesda, MD, U.S.A.) to produce antibody to B-cell lymphoma antigen as part of a U.S. national cancer therapy trial.

From a technical point of view the cultivation process would appear to be oxygen limited, due to retarded diffusion into the centre of the capsule. As pointed out by Glacken et al. (1983) for a 500/~m capsule approximately 40% of cells in the capsule are oxygen limited. This calculation was based on a model for solute diffusion into a sphere presented by Bailey and Ollis (1977). A 170 ~m maximum diameter is required for adequate oxygenation, which theoretically should be possible, however this could add significantly to the cost of the process.

Air-lift fermenters Celltech Ltd. (Great Britain) use conventional air-lift technology (LH Fermenters,

Great Britain) with minor proprietary modification to grow hybridomas. The air-lift system consists of an inner baffle enclosed by a tall, cylindrical vessel (aspect ratio 12:1) which contains the cell and medium mixture. A nitrogen/oxygen/carbon dioxide mixture percolates from the base of the baffle. Ceils and nutrients are gently carried up and spill over the baffle recirculating down the annular space. Most hybridoma processes are run in batches, and cells are allowed to die off once the peak production of monoclonal antibodies has passed. Current production is in 30 and 100 1 vessels, and continuous cultivation is being developed.

The decision to use air-lift fermenters is based on shear and oxygen demand considerations as previously discussed (Thomson and Birch, 1983; Boraston et al., 1984; Birch et al., 1984). Suitable low levels of shear stress, while maintaining sufficiently high circulation rates, are attainable with more than adequate oxygen transfer rates.

Considerable information concerning the operation of these fermenters has been published compared to the previously mentioned techniques. In an example given for monoclonal IgM production, cells were grown in filter sterilized DMEM contain- ing 4.5 g glucose 1-1 supplemented with 3% (v/v) heat-inactivated FCS (Celltech

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have also grown cell lines with totally defined medium). The inoculum culture was grown in suspension in 200-600 ml volumes in 2-1 roller bottles. Cells in the exponential phase of growth were inoculated at 1.42 × 105 cells m1-1 (into a 30 1 bioreactor) and after a minimal lag phase grew exponentially to reach a maximum total population after 90 h of 2.68 × 106 cells ml-1 with a specific growth rate of 0.035 h -~. Cell death ensued rapidly culminating in zero viability after 137 h. Temperature was maintained at 37°C. pH was held constant at 7.1 by the inclusion of CO 2 in the gas stream or the addition of 1 M NaOH solution. Automatic addition of oxygen was used to maintain a dissolved oxygen tension of 10% of air saturation. Cell viability (by trypan blue exclusion) remained in excess of 90% up to the point of maximum population, and antibody titre increased linearly during cell growth and continued to increase during the stationary phase of culture.

Applications for bulk quantities of monoclonal antibodies have already been exploited by Celltech with anti-interferon antibodies for the purification and assay of human a-interferon and monoclonals for use in formulating ABO blood typing reagents. The price for contract manufacture is typically between $1000 and $ 4000 per g, for quantities over 10 g. Obviously, a limitation to the technology may be that small quantities (of the order 100 mg to 10 g) cannot be grown economically.

BioResponse Mass Culture Technique The Mass Culture Technique (MCT) uses fresh bovine lymph as the prime source

of nutrient. This is obtained by tapping into a live cow's lymph system via a catheter to the thoracic duct (Klausner, 1983). The lymph is filtered to remove cellular matter and then oxygenated and supplemented with additional nutrients. The cellular fraction is returned to the animal along with a saline make-up. The filtered lymph goes to the culture room where it passes through 5 to 10 chambers containing growing hybridoma cells.

The culture modules are, in all probability, hollow-fibre systems containing a molecular scale membrane which permits the passage of nutrients to the cell chamber while excluding proteins. A more porous, micron scale membrane is used to harvest antibodies on a daily basis (Klausner, 1983; Anonymous, 1983).

BioResponse is increasing its capacity with about 20 cattle and a potential output of 180-250 g monoclonal per day. The company estimates that the direct cost of producing antibodies is between $100 and $ 200 per g (Klausner, 1983). The process is extremely economical because of the low cost of the nutrient. Furthermore, since production is carried out in small continuously operating units, rather than one large batch system, production costs are likely to be similar for small amounts (ca. 10 g) as for large (1 kg). This may prove advantageous for example in the case of anti-tumour antibodies where they may be required on an individual basis. Furthermore, if a mishap occurs in one unit, the others are not affected and the problem can be easily corrected. The large-scale systems of Celltech and Damon do not have this flexibil- ity.

Chemostat culture By combining an effluent arrangement to a suspension culture vessel with a

supply of medium, and in turn matching these to the growth rate of ceils, a high

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density culture can be maintained in exponential growth in cytostat. Fazekas de St. Groth (1983) has demonstrated that hybridomas can be maintained in 500-ml jacketed, glass vessels with agitation provided by two teflon bars attached to a glass rod rotated magnetically. The system had no pH control, but relied on a high nutrient turnover to maintain lactic acid, and other metabolite levels, within a satisfactory range. Oxygen tension was maintained simply by dissolution from the header space of the reactor; however its perfusion rate was examined and a rate greater than 8 ml min -1 (of 7% CO 2, 10% 0 2 and 83% N2) was found to be necessary.

Cells were inoculated into the reactor at 1 × 105 cells ml -~. A comparison of media was made between RPMI 1640 and Iscove's (both with 10% newborn calf serum), Iscove's serum-free medium, and three simplified variants of Iscove's. There was no discernible difference in the growth rates of the cells in the two standard media (RPMI and Iscove's with serum); however the serum-free media did not support cells to equivalent densities. The three poorer media, Iscove's variants with omission of non-essential amino acids and vitamins, lacking lipids, and with only BSA and lipids as serum substitutes, were further distinguishable by reduced cell growth rates. In general, slower growth rates were observed in suspension cultures relative to static cultures, with doubling times averaging 15% less. Peak densities on the other hand were consistently about twice as high as in stationary cultures.

In three separate experiments cell densities were held at 1 × 105, 3 X 105 and 1 × 106 cells ml-1 with media residence times of 25.0, 25.6 and 27.1 h, respectively (dilution rates, 0.96, 0.94 and 0.89 d -1) and cell doubling times of 17.3, 17.7 and 18.7 h, respectively. All cultures remained viable over 3 weeks with the proportion of dead cells in the chemostat held at less than 3%. The three cultures yielded 5.9, 18.4 and 56.4 mg antibody per day. The yield in terms of media usage was considerably higher at the highest cell density (although not different in terms of yields per cell).

Antibody yields and cell doubling times varied considerably for a range of hybridoma types investigated.

By using a defined medium, Fazekas de St. Groth was able to couple the chemostat effluent to a protein-A column, preceded by a cell trap, and recover antibody on-line.

Summary

A review of the literature has shown that the technology, in terms of equipment capable of maintaining low shear and high oxygen transfer rates, is available for the large-scale cultivation of hybridomas for the production of monoclonal antibodies. Large-scale batch processes already exist capable of producing hundreds of grams of antibody in a run, and small-scale systems (hollow fibre and cytostat) for long-term continuous cultivation. The transition to a large-scale continuous system is not difficult to envisage.

Several studies have shown that in continuous culture the serum component can be drastically reduced to 1-3% of total medium, and other studies have demon-

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strated the ability to maintain hybridomas on totally defined media or filtered media. Thus economy of scale is apparent with a reduction in foetal serum coinponent, or through the ability to utilize filtered adult serum or in eliminating serum altogether. An additional advantage of the defined or filtered media is the vastly simplified antibody purification.

Nutrient studies have demonstrated that high molecular weight components are not necessary for cell growth, but that media should at least be supplemented with insulin, transferrin and 2-amino ethanol when serum is deleted.

Two studies (Fazekas de St. Groth, 1983; Boraston et al., 1984) have demon- strated that antibody synthesis is not totally growth dependent, i.e. the yield of antibodies does not seem related to the rate of replication. The same conclusion can be inferred from the studies with hollow fibre systems which have been maintained from 30 to 140 days with no physical possibility for increase in cell mass. This means that antibody can be obtained from restricted culture systems, and with lower nutrient usage than is already apparent. This follows from the fact that oxygen requirement, and hence metabolic rate, is of the order of 30% of oxygen requirement during maximal growth.

It is also obvious that hybridoma lines are very individual in terms of nutrient requirements, shear sensitivity, antibody production, and growth rates and hence it may be necessary to define each parameter for each line to optimize continuous culture in each case.

Although this report has focused on cultivation of hybridoma cells, large-scale mammalian cell culture will be required for other protein products such as hormones, enzymes and clotting factors, even though advances in recombinant DNA tech- niques with microorganisms has been impressive. Many biologically active proteins require glycosylation to be active, and posttranslational folding and the formation of appropriate cysteine bonds cannot always be achieved with recombinant organisms. Mammalian cell systems, and particularly with developments in cell fusion tech- niques, offer viable alternatives for preparing valuable molecules.

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