Preparation and Storage of Culture Media and Supplement Solutions

Culture media and supplement solutions are normally prepared on site from dry powder materials, concentrated solutions and high-quality water. Water for injection (WFI), as defined in the US or EU Pharmacopeia, is normally used, although a quality such as USP purified water is also acceptable at this stage of the process (see Chapter 2).

Culture media and supplement solutions in small amounts and up to about 100 litres are typically prepared in mobile open containers, then sterilized and stored in bottles or disposable bags before use. The most common sterilization method is filtration (see Section 14.2.2.1); for some specific supplement solutions, heat sterilization is preferred or required (see Section 14.2.2.4). The batch size is typically chosen to be sufficient for several cell culture runs.

For volumes exceeding 100 litres, media and supplement solutions are usually prepared in a closed stainless steel tank (e.g. 316L grade, Dillon et al. 1992), either mobile or fixed, equipped with an agitator, internal baffles and a vent filter; powder components can be added through a manway or via an external recirculation loop. The solution is then usually sterilized by filtration and transferred either directly to the bioreactor or to a container for storage; traditionally, mobile or fixed stainless steel holding tanks have been used for this purpose; recently, however, sterile disposable plastic

Medicines from Animal Cell Culture Edited by G. Stacey and J. Davis © 2007 John Wiley & Sons, Ltd

Media Preparation And Storage

Figure 14.1 Example of sterile and viral filter barriers around a bioreactor. The characteristics of each filter type are discussed in the text (see Section 14.2.4 for gas filters). Dotted lines indicate an alternative flowpath. The size of the filter cartridges is not drawn to scale and their location is only approximate. For clarity, only the main culture medium, one supplement and one pH-control solution are represented. Not shown on the figure are the non-sterilizing vent filter on the media preparation tank, to prevent the release of powder in the room, and the tank(s) and filter(s) used for the preparation and sterilization of the two solutions (typically performed in advance in separate equipment, as discussed in Section 14.2.1).

Figure 14.1 Example of sterile and viral filter barriers around a bioreactor. The characteristics of each filter type are discussed in the text (see Section 14.2.4 for gas filters). Dotted lines indicate an alternative flowpath. The size of the filter cartridges is not drawn to scale and their location is only approximate. For clarity, only the main culture medium, one supplement and one pH-control solution are represented. Not shown on the figure are the non-sterilizing vent filter on the media preparation tank, to prevent the release of powder in the room, and the tank(s) and filter(s) used for the preparation and sterilization of the two solutions (typically performed in advance in separate equipment, as discussed in Section 14.2.1).

bags, for instance in ethylene-vinyl acetate or very low-density polyethylene, placed in mobile containers for mechanical support, have become increasingly popular. With mobile containers (tank or bag), the transfer of medium to the bioreactor is performed via flexible tubing, which is connected aseptically using special tube welders; with fixed holding tanks, medium is transferred via hard pipes that have been sterilized in place (see Section 14.4). A schematic flowsheet for the preparation, filtration and storage of media is illustrated in Figure 14.1, together with the main filters.

The choice of storage container - if any - should be based upon careful economic and technical analyses. The advantages of disposable bags over tanks are as follows. First, investment and qualification costs can be reduced. Second, since bags are disposable and can be purchased ready to use, i.e. already y-sterilized with a mounted sterilizing filter, the capacity of the cleaning and sterilization systems (see Sections 14.3 and 14.4) and of associated utilities (water, plant and clean steam) can be reduced, leading to further savings on both capital and operating costs. Furthermore, costly cleaning and sterilization validation studies can be avoided; this may be particularly advantageous in the case of a multi-product facility, where special care has to be taken with cleaning during product changeover. Additionally, since bag containers can be easily stacked, space and floor area can also be minimized compared with tanks. Finally, disposable bags also increase the flexibility of the plant (at least compared with fixed tanks) since the volumes of prepared media can be adapted easily if the process has to be modified or if a new process has to be introduced. Drawbacks are the absence of efficient mixing and poor heat transfer; bags also lead to additional handling, consumable and disposal costs. Sinclair and Monge (2002) performed a detailed economic comparison of disposable bags with fixed tanks for handling media and buffers in a 2000-l monoclonal antibody process; they claimed that disposable bags could reduce capital investment by about 20 % and cost of goods by about 9 %.

In addition to the aforementioned criteria, a key factor in determining the type of container for storage is, of course, the medium/solution batch size. Whereas, for a few hundred litres, disposable bags, mobile and fixed tanks can all be used, the last of these becomes the best or only practical solution above the 1000-l scale (although disposable bags are now available in sizes up to 3000 litres).

For perfusion processes, there is some flexibility with the medium batch size. Provided that the medium is stable, various combinations of prepared amount and frequency can be selected, which will then determine the most suitable container for storing the culture medium. One option is to prepare relatively small amounts (up to a few hundred litres) at a relatively high frequency (e.g. more than once a week); alternatively, larger amounts (e.g. above 1000 litres) can be produced in one batch, sufficient for more than one week of culture, and stored either in a large stainless steel holding tank or in several mobile tanks or bags that are then kept in a cold room. With a larger medium batch size, operational and handling costs are lower; however, investment costs are higher since a larger preparation tank is required, as well as either a storage tank with proper cooling capacity and serviced by cleaning and sterilization systems (see Sections 14.3 and 14.4), or a cold room for mobile containers. In either case, one holding tank or bag is normally dedicated to each bioreactor. However, only one preparation tank is required, which can serve several holding tanks sequentially since the preparation and filtration of the medium can be completed within a few hours.

For fed-batch processes, it is common, at least at large scale, to prepare the starting medium 'just in time', i.e. a few hours before it is needed, and then transfer it directly into the bioreactor through a sterilizing filter. By avoiding a storage tank, investment cost is reduced; however, operational costs tend to be higher since each cell culture batch requires the preparation of one batch of medium. Here too, one preparation tank, thanks to its relatively short cycle time, can serve several bioreactors. Feed and supplement solutions are either prepared 'just in time' and directly transferred to the bioreactor, or first stored, depending on the volume and feed rate (bolus vs continuous feeding over several days).

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  • marcus hearn
    How to filter litres of media for cel cuture?
    3 years ago
  • zahra girmay
    How to storage nemathode culture in media?
    2 years ago

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