Journal of Biotechnology, 22 (1992) 21-30 0 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1656/92/$05.00
21
BIOTEC 00655
Animal cell culture processes batch or continuous? J.B. Griffiths Dioision
of Biologics,
PHLS
CAMR,
Porton
Down,
Salkbury,
Wiltshire,
U.K
(Received 2 October 1990; revision accepted 4 February 1991)
Culture process; Immobilized
culture; Porous microcarrier;
Perfusion
Introduction Batch culture is the traditional and, for most purposes, the preferred production technology for animal cells. The reasons for this are: (a) it developed from vaccine manufacture where lytic systems made batch culture the only option, human diploid cells were the only acceptable cell line for biologicals and they had a very limited life-span, and the technology available in the 1960’s was suited only to batch culture; (b) when scale-up was needed, particularly from multiple roller bottles, the simplest way forward was to adopt the bacterial-type stirred tank reactor. Suspension cells (which were not widely applicable for human products until the 1980’s for regulatory reasons) adapted well as exemplified by the multi-thousand litre production of FMDV vaccine in BHK cells (Radlett, 1987). The main adaptation from bacterial systems was to provide adequate oxygenation at low stirring speeds. The success of suspension cell culture was the basis for the development of microcarrier culture for anchorage dependent cells used for human vaccine production (van Wezel, 1984). However, this was again best suited to batch operation because of cell retention problems, changes to the microcarrier surface and breakdown of microcarriers. With the successful range of batch systems in operation (10,000 1 for tPA and IFN, 2000 1 for mAb’s, 4000 1 for microcarriers) (Nelson, 1988; Griffiths, 1990a) why does a continuous process need to be considered? In this paper the question of whether continuous processes offer a real production advantage, or whether it is Correspondence
SP4 OJG. U.K.
to: J. Griffiths, Division of Biologics, PHLS CAMR, Porton Down, Salisbury, Wiltshire
22
J
b time
Fig. 1. Choices of process for animal cells showing theoretical growth kinetics, cell yields, and idealised productivity values based on a monoclonal antibody model. See Table 1.
just an intellectual challenge to further physiology, is discussed.
our understanding
of cell growth and
Process options (a) Batch culture is a traditional and simple process with a great deal of user experience (from microbial processes) and with a proven record of reliability as a manufacturing process. It has a low productivity (see Fig. 1, Table 1) because conditions are continually changing and get progressively toxic, due to waste products. (b) Fed batch means that extra fresh medium is added which increases the volume, may increase the cell density, and increases productivity by extending the culture period (Fig. 1). Cc) Semi-continuous processes whereby a proportion of the culture is harvested at regular intervals, and replaced by an equal quantity of fresh medium, increases productivity by allowing multiple harvests (Fig. 1). It may be more cost effective in TABLE
1
Culture type (Millions)
Cell no. mg per week *
1. Batch 2. Semi-continuous batch 3. Fed batch 4. Continuous perfusion 5. Continuous-flow
3 6 30+ 2
* Values allow for turn-round
time of non-continuous cultures.
3
Product yield mg per month * 100 200 200 3000 300
Per litre Days 200 600 500 12000 1200
Length 7 21 14 > 30
> 100
23
that it reduces the down-time of a bioreactor (between batch cleaning and sterilisation), but it still suffers from many of the disadvantages of batch culture (changing conditions and gradual build-up of toxicity). (d) Continuous processes: These have evolved because medium perfusion maintains a semi-homogeneous environment which allows significantly higher cell densities to be achieved (Fig. 1, Table 1). In order to perfuse cells efficiently, immobilisation systems have had to be developed and these have conferred extra stability on cell populations thus leading to much longer culture periods (months). The high cell density (lo-20-fold) means that smaller bioreactors can be used, or at larger volumes a greatly enhanced production capability is achieved. Process advantages include less cell debris, reduced product hold up, and savings in the cost of media as simpler formulations can be used. The bioreactor is run for several weeks or months thus saving time both in equipment turn-around and the growing-up period. (e) Continuous-flow culture (chemostat). Cell yields are by definition below maximum, thus a chemostat is an invaluable tool for cell physiology studies, but not a serious contender as a production process.
Cost effectiveness of continuous processes The productivity data given in Table 1 (although idealistic for comparative purposes) do suggest that it would be considerably cheaper to use a continuous perfusion process. However, an analysis of costs by CellTech, U.K. (Birch and Rhodes, 1989) showed very little difference, whilst the data of Scheirer (1989) indicates a four-fold saving using a continuous process ($47 as opposed to $217 per g mAb). Obviously the final figure depends upon the factors costed in the equation (Table 2). If the factors listed in the example in Table 3 are used, a reason for discrepancies may be seen. The biggest factor is the capital costs of both the facility and the plant. Large volumetric systems need a large purpose built pilot plant whereas small high-density bioreactors will fit into smaller laboratory-type facilities which are cheaper to build and need less maintenance and manpower to operate. Continuous processes obviously make better use of the facilities with less turn-around and growing-up time, but, whatever the cell density approximately the TABLE 2 Process comparison factors 1. Investment/depreciation
- Facility - Bioreactor
2. Efficiency of utilization 3. Raw product costs 4. Manpower costs (numbers and quality) 5. Effect on DSP and QC costs 6. Reliability/reproducibility
24 TABLE 3 Batch vs. perfusion (costs fK)
Capital Equipment Plant
Batch 1000 I
Continuous Fibres/opticell 21
Fluid. bed 100 I
800 2000
100 500 600
100 1000 1100
2800 Per annum Capital (5 yr) Staff Media etc.
710 Yield (g) Cost per g Ratio
120 80 = 200
x2 140 100 =
220 150 =
240
370
400 500 1.4
800 300 0.85
Xl
560 150 zz
2000 355 1.0
3000 125 0.35
same amount of medium per cell is required. Thus raw material costs have been considered equal although there is strong evidence that simpler (cheaper) media can be used at high cell (> 5 X 10’ ml-‘) densities (Spier, 1988). The data in Table 3 indicate that batch culture can be cheaper (because of scale-up economies) unless multiple small-scale continuous processes are run in parallel. However, the type of savings indicated by Scheirer are possibly only achievable with a high volume and high cell density system, such as the fluidised beds of Verax etc. (Runstadler and Cernek, 1988). These methods are only on the threshold of being developed sufficiently to be considered by manufacturers.
Acceptability
of continuous processes
To be accepted as a production process a manufacturer must be satisfied that the performance will be reliable and reproducible (Table 4). The problem with many continuous processes is that they are too complex and usually cannot be scaled-up except by multiple units. They also need a lot of development and validation time. An additional factor is the problem of licencing a continuous process. For licencing purposes, a batch process is straightforward in that there are designated batches of raw materials at the start, and a finite batch of product at the end. Continuous processes, because of their longevity may have more than one batch of raw material, and with daily harvests of product, decisions have to be made as to how much constitutes a sub-batch or batch (NB it is very costly to QC a product batch). In addition, it is difficult to monitor the genetic stability and
25 TABLE Batch
4 (fed)
vs. continuous
Equipment
Process
Economics Licencing
(perfusion)
processes
Parameter
Batch
Operation Failure Sterility Handling Downtime
Simple + + + -
Residence time Development Unit productivity Unit cost Batch definition Cell stability Cell generation
f+), Advantage; t-j, disadvantage; system: f?), problem to be resolved.
(fed)
Perfusion Complex f f + + -
+ -
no. (+ ), likely
+ + -
+ + + to be a disadvantage
? ? but
depends
on
the
individual
number of cell passages in a dense immobilised culture-a mechanism of in-process sampling cells is invaluable to check for variations. Basically, for industry to accept a continuous process firstly, it has to have a cost advantage, which usually means a high density system, and secondly the process should be simple so that mechanical failure or contamination is minimised and the operation is reliable and consistent-a problem with complex perfusion systems. For the users of large volume batch systems, the development of culture systems based on their current bioreactors will give the confidence to change to continuous processes that much sooner. Meanwhile the small-scale continuous systems allow companies to get into production quickly and relatively cheaply, with the purchase of a ‘turn-key’ system in a high quality laboratory, and they are highly suitable where only gram quantities of product are needed (e.g. diagnostics). These factors are summarised in Table 4.
Continuous
culture systems
There are a large number of ,culture systems which have evolved out of the need to have different reactors to grow both suspension and anchorage-dependent cells, and the need to scale-up which has required design solutions to overcome limitations in oxygen, surface area, ability to withstand ‘shear’ damage, nutrients, and the build up of toxic waste products (Griffiths, 1988). This has led to the evolution of many bioreactors using perfusion (Griffiths, 1988, 1990a). Perfusion, particularly of suspension cells, can only be used efficiently if the cell is prevented from being washed out with the spent medium. Even in this specialised field of cell culture there is a wide range of systems, as exemplified in Table 5, which shows the chronological development using three different strategies of retaining the cells in
26 TABLE
5
Continuous bioreactors for cell retention showing developments in three conceptual strategies Separation
Immurement
Entrapment
Cytogenerator (1957) Glass helix (1962) Dialysis membrane (1965) Spin filters (1969)
Plates-stacked (1968) Heat exchanger (1984) Glass bead (1970) Hollow fibres (1972)
Loop reactors (1975) (e.g. hollow fibre)
Teflon membrane (1979) (e.g. IL-101 Microcapsules (1980) (e.g. Damon encapsulation)
Gravitational settling devices (1983) Membrane reactors (1987) (Membroferm, Dynacell)
Centrifugation (1989) (e.g. Centritech)
Glass tubing (1979) (Corbeil, Gyrogen) Ceramic cartridge (1983) (OpticelI) Aggregation (1985) (Fibres, Chemical) Sponge matrices (1987) (e.g. Polyurethane) Porous microcarriers (1988) Werax-collagen, Siranceramic, Cultispher-gelatin)
Details of above systems in reviews by Griffiths (1988, 1990b).
the culture. These strategies are immobilisation by immurement in a medium permeable chamber) or entrapment (the physical trapping of cells within a matrix), and separation either within (spin filter) or in a loop with a filtration or centrifugal device return the cells to the culture.
(retaining the cells or electrochemical the culture vessel to concentrate and
Separation
For commercial purposes the spin filter offers the best solution as it is a simple modification to existing stirred culture systems. However, it is mainly confined to microcarrier culture where the larger mesh size of the filter (70-120 microns) reduces the chance of filter blockage (Griffiths et al., 1987) compared to the 5-10 micron mesh needed for suspension cells. It has widespread use in developmental programmes (up to 200 1) but has not been seriously scaled-up or used in large-scale production systems. Gravitational settling (Butler et al., 1983; Griffiths, 1988) of cells before removing the medium is not efficient enough to permit the fast perfusion rates needed to maintain high cell densities for long periods. Loop reactors are a possibility, especially for scales under 100 1, but the rapid development in centrifuges (Gronvik et al., 1989; Tokashiki et al., 1990) holds promise that this technique will be possible for suspension cells.
21
Immurement
Hollow fibre systems (Hopkinson, 1985; Knight, 1989) have been the most successful production technologies of all the immobilised systems for mAb production. Cells are maintained at over lo* ml-’ in the bioreactor, a unit is capable of manufacturing over 20 g per month, and running continuously for many months. Solutions to the problem of concentration gradients leading to cell necrosis have been sought with some success, however, the main limitation is scale-up beyond the 1 1 unit (Griffiths, 1988). This can only be achieved using multiple units in parallel. The yields have proved very adequate for producing diagnostic quantities of mAb’s and animal trial quantities of recombinant proteins but that, at the moment, is the limit. Other systems listed in Table 5 have not proved\so successful: encapsulation (Duff, 1985) has not lived up to its promise on scale-up (above 70 1); and the membrane bioreactors (Scheirer, 1988; Brown, 1987) are difficult to use beyond laboratory scale-at which they can be very useful devices. Entrapment
The early devices listed in Table 5 were aimed at producing a large surface area bioreactor for attached cells. The need to have a system which is both scaleable, and supports high cell densities, led to the development of entrapment systems for suspension cells [Opticell, (Berg and Bodeker, 1988), and various matrices]. The perception was that this strategy, as opposed to immurement, had the potential to operate at large volumes. The extremely promising results with porous microcarriers look as if this confidence will be rewarded. The porous microcarrier is a sphere of 100-600 microns diameter, with a ‘honeycomb’ of interlinking channels, which can be stirred (using conventional microcarrier systems), or fluidised (air- or medium-lift reactors) giving higher carrier and cell density. Larger spheres (5 mm> can be successfully used in fixed beds (Looby and Griffiths, 1988a; Rather et al., 1990). They have many advantages (Table 6) which make them the possible dominant, and universal, culture system of the 90’s. Porous microcarriers are fabricated from collagen (Verax Corp., Fig. 2a, Runstadler and Cernek, 19881, gelatin (Cultispher, Fig. 2b, Nilsson, 19881, ceramic TABLE 6 The advantages of porous microcarriers as a continuous high productivity culture system Unit cell density 20-SO-fold higher than solid microcarriers Support both attached and suspension cells Suitable for stirred, fluidised or fiied bed reactors Short diffusion paths into a sphere Good scale-up potential by comparison with analogous systems (e.g. microcarrier at 4000 I) Cells protected from shear Capable of long term continuous culture 3-D configuration can be easily derivatised
Fig. 2. Porous
microcarriers: Siran (photo
(a) Verax microsphere; tb) Cultispher G, (photo by Perstop by Schott Glaswerke); fd) Informatrix (photo by Biomat Corp.).
Biolytica);
tc)
(Schott Glaswerke Siran, Fig. 2c> or glycosaminoglycan (Biomat Corp., Fig. 2d; Cahn, 1990). They support high densities of both suspension and anchorage dependent cells (Table 71, are capable of running continuously for at least 40 d, and so far have been scaled up to 20 1. Considering the similarity with conventional TABLE Cell yields
7 in porous
microcarriers
Microcarrier
Cell line
Fixed
Verax
CHO Hybridoma CHO CHO Hybridoma
NA NA NA 58 40
Cultispher Siran Yieldsx
G
10’ ml-’
bed volume;
NA,
carrier
bed
not optimised
Fluidised
Stirred
30-120 198 37 78 20
NA NA 20 NA NA
for culture
mode.
29
microcarrier systems, which are operating at 4000 1, and fixed bed at 200 I, the expectation for scaling-up is good (Griffiths, 1990b). Currently the Ver,ax system is operating as commercial production technology and this may be the strategy which gives manufacturers the confidence to move from batch to high density continuous processes. This will be especially so if the stirred, rather than the fluidised, approach is pursued.
Conclusion
Continuous processes for animal cells have a widespread and successful use for small quantity products (mAb’s, trial recombinant proteins). In particular, hollow fibre systems (e.g. Endotronics, Cell Pharm, Minn Tech, Kinetek etc.) and the ceramic Opticell cartridges are especially dominant in this area. For large scale production (kg’s) of biologicals, the large volume, low cell density systems still dominate. The reasons for this are that they are simple, reliable, well characterised, and many manufacturers are ‘locked in’ for reasons of resource investment or licencing. Currently the emphasis is shifting in Animal Cell Biotechnology from diagnostics to therapeutics/prophylactics, mainly as a result of recombinant products coming to near market readiness. The much higher quantities of product needed means that at some time manufacturers will have to enormously increase the productivity of their systems. Currently this is being done as a result of physiological studies, medium development and environmental control. However, as soon as a continuous and high density method becomes accepted enough in terms of process simplicity, reliability and reproducibility, the pressure will be on manufacturers to implement it. The most likely route to take will be extensions of existing large-scale plant rather than complete switches to new engineering technology. From this point of view the current system most likely to succeed is the porous microcarrier culture (Cahn, 1990; Looby and Griffiths, 1990).
References Berg,
G.J.
and
Bodeker,
B.G.D.
(1988)
Employing
a ceramic
matrix
for
the
immobilisation
mammalian cells in culture. In: Spier, R.E. and Griffiths, J.B. (Eds.1, Animal Cell Academic Press, London, pp. 321-325. Birch, J. and Rhodes, M. (1989) personal communication. Brown, B.L. (1987) Reducing costs upfront. In: Seaver, S. (Ed.), Commercial Production Antibodies. Marcel Dekker Inc., New York, pp. 35-48.
Biotechnology
J.B. (1988) Overview of cell culture systems (Eds.), Animal Cell Biotechnology 3, Academic
and their scale-up. In: Spier, Press, London, pp. 179-220.
R.E.
3,
of Monoclonal
Butler, M., Imamura, T., Thomas, J. and Thilly, W.G. (1983) High yields from microcarrier medium diffusion. J. Cell Sci. 61, 351-363. Cahn, F. (1990) Biomaterials aspects of porous microcarriers for animal cell culture. Trends 8, 131-136. Duff, R.G. (1985) Microencapsulation technology: a novel method of monoclonal antibody Trends Biotechnol. 3, 167-170. Griffiths, J.B.
of
cultures Biotechnol. production. and
Griffiths,
by
30 Griffiths, J.B. (1990~1) Animal Cells-the breakthrough to a dominant technology. Cytotechnology 3, 109-l 16. Ciriffiths, J.B. (1990bl Advances in animal cell immobilization technology. In: Spier, R.E. and Griffiths, J.B. (Eds.), Animal Cell Biotechnology 4, Academic Press, London, pp. 149-166. Griffiths, J.B.. Cameron, D.R. and Looby, D. (1987) A comparison of unit process system for anchorage dependent cells. Dev. Biol. Stand. 66, 331-338. Gronvik. K.O., Frieburg, H. and Malmstrom, U. (1989) Centritech Cell-a new separation device for mammalian cells. In: Spier, R.E., Griffiths, J.B., Stephenne, J. and Crooy, J.J. (Eds.), Advances in Animal Cell Biology and Technology for Bioprocesses, Butterworths, Guildford, pp. 336-344. Hopkinson, J. (1985) Hollow fiber cell culture systems for economical cell-product manufacturing. Bio/Technology 3, 225-230. Knight, P. (1989) Hollow fiber bioreactors for mammalian cell culture. Bio/Technology 7, 459-461. Looby, D. and Griffiths. J.B. (1988a) Fixed bed porous glass sphere (porosphere) bioreactors for animal cells. Cytotechnology 1, 339-346. Looby, D. and Griffiths, J.B. (1988b) In: Spier, R.E., Griffiths, J.B., Stephenne, J. and Crooy, J.J. (Eds.), Advances in Animal Cell Biology and Technology for Bioprocesses, Butterworths, Guildford, pp. 336-344. Looby, D. and Griffiths, J.B. (1990) Immobilization of animal cells in porous carrier culture. Trends Biotechnol. 8, 204-209. Nelson, K.L. (1988) Industrial-scale mammalian cell culture. Biopharm. Manuf., February. Nilsson, K. (1988) Macroporous gelatin microcarriers for animal cell culture. BioMedica (Biolytica) 1. 13-1.5. Rather, A., Looby, D. and Griffiths, J.B. (1990) Studies on monoclonal antibody production by a hybridoma cell line (CIE3) immobilised in a fixed bed, porosphere culture system. J. Biotechnol. 15, 129-146. Radlett, P.J. (1987) The use of baby hamster kidney (BHKI suspension cells for the production of foot and mouth disease vaccines. Adv. Biochem. Eng. Biotechnol. 34, 129-146. Runstadler, P.W. and Cernek, S.R. (1988) Large-scale fluidized bed, immobilized cultivation of animal cells at high densities. In: Spier, R.E. and Griffiths, J.B. (Eds.), Animal Cell Biotechnology 3. Academic Press, London, pp. 305-320. Scheirer, W. (1988) High density growth of animal cells within cell retention fermenters equipped with membranes. In: Spier, R.E. and Griffiths, J.B. (Eds.), Animal Cell Biotechnology 3, Academic Press, London, pp. 263-281. Scheirer. W. (1989) Workshop discussion at the ETCS Meeting, Graz, Austria. Spier, R.E. (1988) Animal cells in culture: moving into the exponential phase. Trends Biotechnol. 6, 2-6. Tokashiki, M., Arai. T., Hamamoto, K. and Ishimaru, K. (1990) High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge. Cytotechnology 3, 239-244. van Wezel. A.L. (1984) Microcarrier technology-present status and prospects. Dev. Biol. Stand. 55. 3-9.
21
BIOTEC 00655
Animal cell culture processes batch or continuous? J.B. Griffiths Dioision
of Biologics,
PHLS
CAMR,
Porton
Down,
Salkbury,
Wiltshire,
U.K
(Received 2 October 1990; revision accepted 4 February 1991)
Culture process; Immobilized
culture; Porous microcarrier;
Perfusion
Introduction Batch culture is the traditional and, for most purposes, the preferred production technology for animal cells. The reasons for this are: (a) it developed from vaccine manufacture where lytic systems made batch culture the only option, human diploid cells were the only acceptable cell line for biologicals and they had a very limited life-span, and the technology available in the 1960’s was suited only to batch culture; (b) when scale-up was needed, particularly from multiple roller bottles, the simplest way forward was to adopt the bacterial-type stirred tank reactor. Suspension cells (which were not widely applicable for human products until the 1980’s for regulatory reasons) adapted well as exemplified by the multi-thousand litre production of FMDV vaccine in BHK cells (Radlett, 1987). The main adaptation from bacterial systems was to provide adequate oxygenation at low stirring speeds. The success of suspension cell culture was the basis for the development of microcarrier culture for anchorage dependent cells used for human vaccine production (van Wezel, 1984). However, this was again best suited to batch operation because of cell retention problems, changes to the microcarrier surface and breakdown of microcarriers. With the successful range of batch systems in operation (10,000 1 for tPA and IFN, 2000 1 for mAb’s, 4000 1 for microcarriers) (Nelson, 1988; Griffiths, 1990a) why does a continuous process need to be considered? In this paper the question of whether continuous processes offer a real production advantage, or whether it is Correspondence
SP4 OJG. U.K.
to: J. Griffiths, Division of Biologics, PHLS CAMR, Porton Down, Salisbury, Wiltshire
22
J
b time
Fig. 1. Choices of process for animal cells showing theoretical growth kinetics, cell yields, and idealised productivity values based on a monoclonal antibody model. See Table 1.
just an intellectual challenge to further physiology, is discussed.
our understanding
of cell growth and
Process options (a) Batch culture is a traditional and simple process with a great deal of user experience (from microbial processes) and with a proven record of reliability as a manufacturing process. It has a low productivity (see Fig. 1, Table 1) because conditions are continually changing and get progressively toxic, due to waste products. (b) Fed batch means that extra fresh medium is added which increases the volume, may increase the cell density, and increases productivity by extending the culture period (Fig. 1). Cc) Semi-continuous processes whereby a proportion of the culture is harvested at regular intervals, and replaced by an equal quantity of fresh medium, increases productivity by allowing multiple harvests (Fig. 1). It may be more cost effective in TABLE
1
Culture type (Millions)
Cell no. mg per week *
1. Batch 2. Semi-continuous batch 3. Fed batch 4. Continuous perfusion 5. Continuous-flow
3 6 30+ 2
* Values allow for turn-round
time of non-continuous cultures.
3
Product yield mg per month * 100 200 200 3000 300
Per litre Days 200 600 500 12000 1200
Length 7 21 14 > 30
> 100
23
that it reduces the down-time of a bioreactor (between batch cleaning and sterilisation), but it still suffers from many of the disadvantages of batch culture (changing conditions and gradual build-up of toxicity). (d) Continuous processes: These have evolved because medium perfusion maintains a semi-homogeneous environment which allows significantly higher cell densities to be achieved (Fig. 1, Table 1). In order to perfuse cells efficiently, immobilisation systems have had to be developed and these have conferred extra stability on cell populations thus leading to much longer culture periods (months). The high cell density (lo-20-fold) means that smaller bioreactors can be used, or at larger volumes a greatly enhanced production capability is achieved. Process advantages include less cell debris, reduced product hold up, and savings in the cost of media as simpler formulations can be used. The bioreactor is run for several weeks or months thus saving time both in equipment turn-around and the growing-up period. (e) Continuous-flow culture (chemostat). Cell yields are by definition below maximum, thus a chemostat is an invaluable tool for cell physiology studies, but not a serious contender as a production process.
Cost effectiveness of continuous processes The productivity data given in Table 1 (although idealistic for comparative purposes) do suggest that it would be considerably cheaper to use a continuous perfusion process. However, an analysis of costs by CellTech, U.K. (Birch and Rhodes, 1989) showed very little difference, whilst the data of Scheirer (1989) indicates a four-fold saving using a continuous process ($47 as opposed to $217 per g mAb). Obviously the final figure depends upon the factors costed in the equation (Table 2). If the factors listed in the example in Table 3 are used, a reason for discrepancies may be seen. The biggest factor is the capital costs of both the facility and the plant. Large volumetric systems need a large purpose built pilot plant whereas small high-density bioreactors will fit into smaller laboratory-type facilities which are cheaper to build and need less maintenance and manpower to operate. Continuous processes obviously make better use of the facilities with less turn-around and growing-up time, but, whatever the cell density approximately the TABLE 2 Process comparison factors 1. Investment/depreciation
- Facility - Bioreactor
2. Efficiency of utilization 3. Raw product costs 4. Manpower costs (numbers and quality) 5. Effect on DSP and QC costs 6. Reliability/reproducibility
24 TABLE 3 Batch vs. perfusion (costs fK)
Capital Equipment Plant
Batch 1000 I
Continuous Fibres/opticell 21
Fluid. bed 100 I
800 2000
100 500 600
100 1000 1100
2800 Per annum Capital (5 yr) Staff Media etc.
710 Yield (g) Cost per g Ratio
120 80 = 200
x2 140 100 =
220 150 =
240
370
400 500 1.4
800 300 0.85
Xl
560 150 zz
2000 355 1.0
3000 125 0.35
same amount of medium per cell is required. Thus raw material costs have been considered equal although there is strong evidence that simpler (cheaper) media can be used at high cell (> 5 X 10’ ml-‘) densities (Spier, 1988). The data in Table 3 indicate that batch culture can be cheaper (because of scale-up economies) unless multiple small-scale continuous processes are run in parallel. However, the type of savings indicated by Scheirer are possibly only achievable with a high volume and high cell density system, such as the fluidised beds of Verax etc. (Runstadler and Cernek, 1988). These methods are only on the threshold of being developed sufficiently to be considered by manufacturers.
Acceptability
of continuous processes
To be accepted as a production process a manufacturer must be satisfied that the performance will be reliable and reproducible (Table 4). The problem with many continuous processes is that they are too complex and usually cannot be scaled-up except by multiple units. They also need a lot of development and validation time. An additional factor is the problem of licencing a continuous process. For licencing purposes, a batch process is straightforward in that there are designated batches of raw materials at the start, and a finite batch of product at the end. Continuous processes, because of their longevity may have more than one batch of raw material, and with daily harvests of product, decisions have to be made as to how much constitutes a sub-batch or batch (NB it is very costly to QC a product batch). In addition, it is difficult to monitor the genetic stability and
25 TABLE Batch
4 (fed)
vs. continuous
Equipment
Process
Economics Licencing
(perfusion)
processes
Parameter
Batch
Operation Failure Sterility Handling Downtime
Simple + + + -
Residence time Development Unit productivity Unit cost Batch definition Cell stability Cell generation
f+), Advantage; t-j, disadvantage; system: f?), problem to be resolved.
(fed)
Perfusion Complex f f + + -
+ -
no. (+ ), likely
+ + -
+ + + to be a disadvantage
? ? but
depends
on
the
individual
number of cell passages in a dense immobilised culture-a mechanism of in-process sampling cells is invaluable to check for variations. Basically, for industry to accept a continuous process firstly, it has to have a cost advantage, which usually means a high density system, and secondly the process should be simple so that mechanical failure or contamination is minimised and the operation is reliable and consistent-a problem with complex perfusion systems. For the users of large volume batch systems, the development of culture systems based on their current bioreactors will give the confidence to change to continuous processes that much sooner. Meanwhile the small-scale continuous systems allow companies to get into production quickly and relatively cheaply, with the purchase of a ‘turn-key’ system in a high quality laboratory, and they are highly suitable where only gram quantities of product are needed (e.g. diagnostics). These factors are summarised in Table 4.
Continuous
culture systems
There are a large number of ,culture systems which have evolved out of the need to have different reactors to grow both suspension and anchorage-dependent cells, and the need to scale-up which has required design solutions to overcome limitations in oxygen, surface area, ability to withstand ‘shear’ damage, nutrients, and the build up of toxic waste products (Griffiths, 1988). This has led to the evolution of many bioreactors using perfusion (Griffiths, 1988, 1990a). Perfusion, particularly of suspension cells, can only be used efficiently if the cell is prevented from being washed out with the spent medium. Even in this specialised field of cell culture there is a wide range of systems, as exemplified in Table 5, which shows the chronological development using three different strategies of retaining the cells in
26 TABLE
5
Continuous bioreactors for cell retention showing developments in three conceptual strategies Separation
Immurement
Entrapment
Cytogenerator (1957) Glass helix (1962) Dialysis membrane (1965) Spin filters (1969)
Plates-stacked (1968) Heat exchanger (1984) Glass bead (1970) Hollow fibres (1972)
Loop reactors (1975) (e.g. hollow fibre)
Teflon membrane (1979) (e.g. IL-101 Microcapsules (1980) (e.g. Damon encapsulation)
Gravitational settling devices (1983) Membrane reactors (1987) (Membroferm, Dynacell)
Centrifugation (1989) (e.g. Centritech)
Glass tubing (1979) (Corbeil, Gyrogen) Ceramic cartridge (1983) (OpticelI) Aggregation (1985) (Fibres, Chemical) Sponge matrices (1987) (e.g. Polyurethane) Porous microcarriers (1988) Werax-collagen, Siranceramic, Cultispher-gelatin)
Details of above systems in reviews by Griffiths (1988, 1990b).
the culture. These strategies are immobilisation by immurement in a medium permeable chamber) or entrapment (the physical trapping of cells within a matrix), and separation either within (spin filter) or in a loop with a filtration or centrifugal device return the cells to the culture.
(retaining the cells or electrochemical the culture vessel to concentrate and
Separation
For commercial purposes the spin filter offers the best solution as it is a simple modification to existing stirred culture systems. However, it is mainly confined to microcarrier culture where the larger mesh size of the filter (70-120 microns) reduces the chance of filter blockage (Griffiths et al., 1987) compared to the 5-10 micron mesh needed for suspension cells. It has widespread use in developmental programmes (up to 200 1) but has not been seriously scaled-up or used in large-scale production systems. Gravitational settling (Butler et al., 1983; Griffiths, 1988) of cells before removing the medium is not efficient enough to permit the fast perfusion rates needed to maintain high cell densities for long periods. Loop reactors are a possibility, especially for scales under 100 1, but the rapid development in centrifuges (Gronvik et al., 1989; Tokashiki et al., 1990) holds promise that this technique will be possible for suspension cells.
21
Immurement
Hollow fibre systems (Hopkinson, 1985; Knight, 1989) have been the most successful production technologies of all the immobilised systems for mAb production. Cells are maintained at over lo* ml-’ in the bioreactor, a unit is capable of manufacturing over 20 g per month, and running continuously for many months. Solutions to the problem of concentration gradients leading to cell necrosis have been sought with some success, however, the main limitation is scale-up beyond the 1 1 unit (Griffiths, 1988). This can only be achieved using multiple units in parallel. The yields have proved very adequate for producing diagnostic quantities of mAb’s and animal trial quantities of recombinant proteins but that, at the moment, is the limit. Other systems listed in Table 5 have not proved\so successful: encapsulation (Duff, 1985) has not lived up to its promise on scale-up (above 70 1); and the membrane bioreactors (Scheirer, 1988; Brown, 1987) are difficult to use beyond laboratory scale-at which they can be very useful devices. Entrapment
The early devices listed in Table 5 were aimed at producing a large surface area bioreactor for attached cells. The need to have a system which is both scaleable, and supports high cell densities, led to the development of entrapment systems for suspension cells [Opticell, (Berg and Bodeker, 1988), and various matrices]. The perception was that this strategy, as opposed to immurement, had the potential to operate at large volumes. The extremely promising results with porous microcarriers look as if this confidence will be rewarded. The porous microcarrier is a sphere of 100-600 microns diameter, with a ‘honeycomb’ of interlinking channels, which can be stirred (using conventional microcarrier systems), or fluidised (air- or medium-lift reactors) giving higher carrier and cell density. Larger spheres (5 mm> can be successfully used in fixed beds (Looby and Griffiths, 1988a; Rather et al., 1990). They have many advantages (Table 6) which make them the possible dominant, and universal, culture system of the 90’s. Porous microcarriers are fabricated from collagen (Verax Corp., Fig. 2a, Runstadler and Cernek, 19881, gelatin (Cultispher, Fig. 2b, Nilsson, 19881, ceramic TABLE 6 The advantages of porous microcarriers as a continuous high productivity culture system Unit cell density 20-SO-fold higher than solid microcarriers Support both attached and suspension cells Suitable for stirred, fluidised or fiied bed reactors Short diffusion paths into a sphere Good scale-up potential by comparison with analogous systems (e.g. microcarrier at 4000 I) Cells protected from shear Capable of long term continuous culture 3-D configuration can be easily derivatised
Fig. 2. Porous
microcarriers: Siran (photo
(a) Verax microsphere; tb) Cultispher G, (photo by Perstop by Schott Glaswerke); fd) Informatrix (photo by Biomat Corp.).
Biolytica);
tc)
(Schott Glaswerke Siran, Fig. 2c> or glycosaminoglycan (Biomat Corp., Fig. 2d; Cahn, 1990). They support high densities of both suspension and anchorage dependent cells (Table 71, are capable of running continuously for at least 40 d, and so far have been scaled up to 20 1. Considering the similarity with conventional TABLE Cell yields
7 in porous
microcarriers
Microcarrier
Cell line
Fixed
Verax
CHO Hybridoma CHO CHO Hybridoma
NA NA NA 58 40
Cultispher Siran Yieldsx
G
10’ ml-’
bed volume;
NA,
carrier
bed
not optimised
Fluidised
Stirred
30-120 198 37 78 20
NA NA 20 NA NA
for culture
mode.
29
microcarrier systems, which are operating at 4000 1, and fixed bed at 200 I, the expectation for scaling-up is good (Griffiths, 1990b). Currently the Ver,ax system is operating as commercial production technology and this may be the strategy which gives manufacturers the confidence to move from batch to high density continuous processes. This will be especially so if the stirred, rather than the fluidised, approach is pursued.
Conclusion
Continuous processes for animal cells have a widespread and successful use for small quantity products (mAb’s, trial recombinant proteins). In particular, hollow fibre systems (e.g. Endotronics, Cell Pharm, Minn Tech, Kinetek etc.) and the ceramic Opticell cartridges are especially dominant in this area. For large scale production (kg’s) of biologicals, the large volume, low cell density systems still dominate. The reasons for this are that they are simple, reliable, well characterised, and many manufacturers are ‘locked in’ for reasons of resource investment or licencing. Currently the emphasis is shifting in Animal Cell Biotechnology from diagnostics to therapeutics/prophylactics, mainly as a result of recombinant products coming to near market readiness. The much higher quantities of product needed means that at some time manufacturers will have to enormously increase the productivity of their systems. Currently this is being done as a result of physiological studies, medium development and environmental control. However, as soon as a continuous and high density method becomes accepted enough in terms of process simplicity, reliability and reproducibility, the pressure will be on manufacturers to implement it. The most likely route to take will be extensions of existing large-scale plant rather than complete switches to new engineering technology. From this point of view the current system most likely to succeed is the porous microcarrier culture (Cahn, 1990; Looby and Griffiths, 1990).
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G.J.
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Bodeker,
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