Systems for Suspension Cells or attached cells on microcarriers

10.3.2.1 Spinner flasks

These are cylindrical, agitated vessels, varying in capacity from around 100 ml to 36 litres. They represent an intermediate level of scale-up between flasks or dishes and fermenters, but are far cheaper to purchase and use than a small fermenter of equivalent size. However, far less instrumentation and control is available than on a fermenter, and thus they are not generally of great use as scaled-down models for fermenter process development. Where they may be useful is in the early stages of cell inoculum growth for large-scale systems, for the small-scale production of material, either during the 'proof of principle' or early clinical trial stage of development of a biological medicine, or for the production of diagnostics.

Many different formats are available (two examples are shown in Figure 10.4), with different methods of agitation, different stirrer configurations, different aspect-ratio vessels, different cap types, and different degrees and ease of access to the gas and liquid phases. In general, the options available in this last category increase with the size of the vessel.

10.3.2.2 Shake flasks

Erlenmeyer-style flasks have been used for the culture of mammalian cells since the 1950s. These flasks are secured to a shaker apparatus, which mixes the contents of the flask and keeps the cells in suspension. This culture method is particularly useful for small to moderate

The Gas Exchange System Insects
Figure 10.4 Two different types of spinner flasks. Reproduced from Doyle & Griffiths (2000) by permission of John Wiley & Sons Ltd.

volumes of cells having high oxygen requirements, such as insect cells (see Chapter 7). Both reusable and disposable flasks are generally available in sizes from 50 ml to 6 l. Baffles can be added to aid mixing, and vented caps can be used to increase gas exchange. Beyond about 6 litres (i.e. 2 litres of liquid volume) scale-up can only be achieved by the use of multiple flasks.

10.3.2.3 Culture bags

Bags suitable for use as vessels for cell culture have been available for at least 18 years (Schoof et al. 1988). Initial applications generally used static, gas-permeable bags in CO2 incubators, and culture volumes were consequently limited to a few litres at most by the need for oxygen to diffuse through the bag walls. A far more suitable system for use in scale-up was first commercialized by Wave Biotech in 1999, and basically consists of a sterile, single-use, non-gas-permeable pillow-shaped bag that is mounted on a rocking apparatus (see Figure 10.5). The bag is inflated with a CO2/air mixture suitable for the medium to be used, then the medium is introduced into the bag (and warmed, if necessary) after which cells are added. The bag retains a substantial head-space of CO2/air, typically equal in volume to that of the medium. Mixing is achieved and gas exchange facilitated by the reciprocating rocking motion of the motorized platform on which the bag is mounted. Shear levels are claimed

Gas Sparger

to be very low, as the cells mostly move with the bulk of the liquid, but the rocking must be regulated in order to avoid foaming. The bags come fitted with a fill tube, harvest tube, inlet filter, exhaust filter, sampling port, constant pressure relief valve, and ports for in situ pH and dissolved oxygen probes. The system has been used with animal cells both in suspension (Singh 1999; Fries et al. 2005) and on microcarriers (Namdev & Lio 2000), as well as plant and prokaryotic cells.

With retention of bag geometry and adjustment of the tilting parameters, linear scale-up is claimed from 100 ml up to 500 litres (www.wavebiotech.com; Pierce & Shabram 2004). Although the makers claim that there is no intrinsic limit to scale-up, other factors such as mechanical and safety problems associated with a large reciprocating mass could become a problem, as could temperature control which is still achieved by heat transfer through the bag wall.

The main advantages of the system, other than its simple principle and low-shear environment, are largely due to the disposable nature of the bag. Thus there is no cleaning or sterilizing (and validation of the cleaning and sterilizing procedures) to be performed by the user, nor is there any need to replace seals or undertake many of the other time-consuming maintenance procedures that are necessary for fermenters.

The manufacturer claims that the system is used by more than ten companies in facilities licensed for the production of human therapeutics. However, in at least some cases the system is used to grow cell inocula for seeding larger, more conventional culture systems rather than for the final full-scale manufacture of product. Nevertheless, up to a certain scale the system has attractive features, and many more systems might be in use but for two factors. The first has been a commercial decision by the manufacturer not to loan out equipment for trial, so some potential users not prepared to invest a substantial sum of money by buying a system 'sight unseen' have been unable to test its efficacy with their cell lines. The second has been problems with the in situ sensors (particularly for pH) and the resultant inability to monitor the culture environment accurately in real time and control it automatically. This has allowed the sensor, controller and bioreactor manufacturer Applikon, collaborating with the bag manufacturer Stedim, to bring to market the Appliflex system, which appears similar to the Wave system but has in situ pH, DO and temperature sensors interfaced with a controller that allows feedback control of the culture environment. At the time of writing, Appliflex systems are only available for volumes up to 50 litres. A similar system has also been introduced recently by Sartorius, the product of a collaboration with Wave Switzerland.

10.3.2.4 Fermenters

For the industrial-scale production of animal cells and their secreted products, by far the commonest method of culture is submerged culture in stirred-tank (Figure 10.6) or, less commonly, airlift (Figure 10.7) fermenters. This technology has been in use in the brewing and other industries for a great many years, and the principles and engineering involved are well understood (Bailey & Ollis 1986; van't Riet & Tramper 1991; Doran 1995; Asenjo & Merchuk 1994; Stanbury et al. 1999; Nielsen et al. 2002). Thus early attempts to use fermenters for large-scale mammalian cell culture employed the technology and designs that had been developed for microbial fermentation. However it soon became apparent that, although many of the issues and concerns remained the same [e.g. pH control, mixing (including gas/liquid mixing), oxygen mass transfer] the special characteristics of mammalian cells required that corresponding adaptations be made to fermenter hardware and control strategies. Some examples for a stirred-tank system are given in Table 10.2.

Although the basic issues were the same for airlift fermenters (Figure 10.7), fewer design changes were required, with the issue of cell damage by bubbles being the main problem.

Figure 10.6 Simplified diagram of a stirred tank fermenter. (A) Impeller drive; (B) marine impeller; (C) cell suspension; (D) water jacket; (E) pH probe; (F) DO probe; (G) removable headplate; (H) condenser; (I) gas filter; (J) headspace.

Vent

Vent

Research Culture Diagram

Figure 10.6 Simplified diagram of a stirred tank fermenter. (A) Impeller drive; (B) marine impeller; (C) cell suspension; (D) water jacket; (E) pH probe; (F) DO probe; (G) removable headplate; (H) condenser; (I) gas filter; (J) headspace.

Airlift Fermenter Plant Suspensions
Figure 10.7 Simplified diagram of an airlift fermenter. (A) Cell suspension; (B) headspace; (C) pH probe; (D) DO probe; (E) headplate: (F) condenser; (G) gas filter.

Table 10.2 Some changes in stirred-tank fermenter design/control required to accommodate animal cells rather than microbes.

Microbial characteristic

Animal Cell characteristic Resultant change

Low shear sensitivity

Low sensitivity to damage by bubble disengagement

High medium viscosity

High oxygen demands

High shear sensitivity

High sensitivity to damage by bubble disengagement

Low medium viscosity

Lower oxygen demands

Change impeller design (e.g. from Rushton turbine to marine impeller) and reduce rotation rate (tip speed); remove baffles Increase diameter:height ratio to maximize surface aeration (although effect decreases with culture volume); minimize gas bubble size used in sparging; add surface-active agent (but control foaming)

Efficient mixing can be achieved with low-shear impellers rotating at low speeds; curved fermenter bottom improves mixing at low impeller speeds; magnetic drive couplings can be used, overcoming seal problems Permits lower impeller speeds to be used; reduces the amount of (potentially damaging) sparging required

The stage at which one would change from using, say, a spinner flask to using a fermenter will depend on a number of factors, including:

• Handling: Although spinner flasks are available in sizes up to 36 l, manual handling becomes very difficult beyond about 20 l.

• Control: pH and DO control of spinner flasks may become difficult, as they cannot usually accommodate in situ probes, and are not supplied with feedback controllers.

• Availability of services: A fermenter may require some or all of the following services (although a small unit will probably only need a sub-set of these): 3-phase electricity; steam (possibly clean steam); cooling water; hot water; sterile water; drainage; CO2; O2; N2; compressed air.

If not already available, the expense of installing and running these services may discourage switching if product demand can still be met by using spinner flasks.

Thus the change from spinner flask to fermenter will generally occur when the required culture volumes are in the 10-100 l range, and a useful guide to purchasing fermenters at this scale has been published by Cino and Frey (1996, 1997). At the lower end of this range, glass vessels, and sterilization of vessels in an autoclave, are still an option, but manual handling, and the fragility and poor thermal conductivity of glass, rapidly become an issue. Consequently, all larger fermenters are made of stainless steel, and are sterilized in situ.

Scaling up of all fermenters, especially stirred tanks, cannot be proportional. For example, doubling a vessel's dimensions while retaining the same three-dimensional shape will increase its volume eightfold, but the air/liquid interface area of the headspace will only increase fourfold, decreasing the role that headspace gas exchange can play in oxygenation of the culture. Similarly, as a stirred tank increases in diameter it will usually be necessary to increase the diameter of the impeller in order to ensure good mixing. Yet if the impeller rotation rate is kept the same, the shear at the tips will increase, but of course the shear sensitivity of the cells will not change. Many other interacting factors will also vary to different extents. Thus at large scale a good understanding of the cells' physical and metabolic requirements (e.g. shear sensitivity, oxygen requirements) as well as the characteristics of the medium (e.g. density, viscosity, foaming properties) becomes essential in order to define the specification of the fermenter. This data should be supplied to the engineer designing the fermenter in order to help ensure that, when delivered and commissioned, the equipment performs satisfactorily. Currently, the largest stirred tank fermenters in use for animal cell culture have volumes of 20 000 l, whilst the largest airlifts are 5000 l.

The drive for optimum use of available capacity, as well as the desire to minimize both the volume of (expensive) medium to be purchased and the volume of spent, product-containing medium to be processed, has led to the intensification of fermenter processes. Originally, batch processes were employed, where a fixed volume of medium was added to the fermenter with the cells, and incubation was continued without addition of further medium until the endpoint of the culture was reached. However this was inefficient, as nutrient depletion tended to limit productivity. In principle this problem could be readily resolved by the addition of fresh medium and/or other nutrient solutions, and has led to the fed-batch process, which is now widely implemented in large-scale cell culture. This approach can ultimately become inefficient, however, due to the build up of inhibitory waste products. Overcoming the problem by removing a portion of the culture and replacing it with fresh medium led to the repeat fed-batch process. The logical extension of this is continuously to remove spent medium and replace it with fresh, to give a perfusion process. Perfusion processes in fermenters can be run either by removing cells from the system along with the medium in so-called chemostat mode

(where the rate of removal of medium, and thus cells, has to be carefully balanced against the multiplication rate of the cells in order not to deplete the cell population), or the cell suspension can be passed through a cell-retention device, with the medium harvested while the cells are returned to the fermenter. Where perfusion is used for the production of a secreted product (and most currently available cell-derived medicines are secreted products), the second of these approaches is favoured as it uncouples production rate from cell multiplication rate. Cell retention devices to permit fermenters to be used in this mode can take a number of forms, such as spin-filters within the fermenter, or tangential-flow filters, acoustic filters, gravitational settlers or continuous-flow centrifuges in an external recirculation loop, and each of these is dealt with in detail in Chapter 16.

Further discussion of the ramifications of culture mode on fermenter design, along with numerous other factors influencing fermenter design and construction, can be found in Chapter 14. For an assessment of some of the relative merits of the different culture modes in a real (i.e. uncertain) world, see Lim et al. (2006).

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  • Martha
    Why gas permeable culture bag needing remoing gas bubbles?
    4 months ago

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