Inactivation of biological wastes

At laboratory scale, manual autoclaving of biological solid and liquid waste is the simplest and most common method; alternatively, chemical sterilization using, for instance, iodine- or chlorine-containing agents can be performed. This second method is recommended for small-scale cultivation vessels, since heat treatment before washing may cause accumulation of cell debris as solid residues on surfaces. In a facility where a central liquid waste decontamination system is built for large-scale production (see below), a practical solution is to connect all the effluent from the laboratory sinks and automatic washers to this system, provided that dismantling prior to inactivation is allowed from a biosafety point of view; then only disposable solid waste will require autoclaving.

At large-scale, solid waste is commonly autoclaved whereas liquid waste is processed using a centralized and automatic liquid-waste decontamination system (LWDS). The LWDS should be located on the basement level so that the liquid waste from all process steps/CIP units can be drained by gravity to the system. The most common LWDS inactivation method is a heat treatment, either in batch or continuous mode (Carlson 2001; Miller & Bergmann 1993).

In a batch LWDS, the waste is collected in a so-called 'kill tank' and heated with plant steam, either via the tank jacket or via direct sparging, the latter being of course more efficient but leading to an increase in waste volume. It has been shown recently that exposing mammalian cells to 80 °C for 1 minute was sufficient to inactivate them all (Gregoriades et al. 2003). The waste is then cooled down to about 60 °C, either in the 'kill tank' or via an external heat exchanger. If neutralization is not performed in the LWDS, the waste must be directed to a neutralization system prior to discharge. A system with two 'kill tanks' in parallel, operating in a staggered way, is recommended to ensure uninterrupted service in a large-scale facility. To further enhance inacti-vation capacity, a sump tank for collecting liquid waste upstream of the two 'kill tanks' could be installed. With such a configuration, the waste, which is usually generated in a discontinuous and irregular way from process operations, accumulates in the sump tank. As soon as enough waste to fill one 'kill tank' is reached, it is rapidly transferred to one of the tanks and heating can follow

Mixing Tank

Figure 14.12 Example of a dual-tank batch LWDS without sump tank. The two tanks operate in parallel, in a staggered way. 'Pressurized, atmospheric and segregated systems' indicate various sources of biological liquid wastes. In this example, steam is injected both into the tank jacket and directly via a sparger; 'X' indicates the outlet for inactivated waste and 'Y' cooling water (reproduced with permission from Carlson 2001).

Figure 14.12 Example of a dual-tank batch LWDS without sump tank. The two tanks operate in parallel, in a staggered way. 'Pressurized, atmospheric and segregated systems' indicate various sources of biological liquid wastes. In this example, steam is injected both into the tank jacket and directly via a sparger; 'X' indicates the outlet for inactivated waste and 'Y' cooling water (reproduced with permission from Carlson 2001).

immediately. The idle time of the 'kill tanks' can thus be minimized. Figure 14.12 shows an example of a dual-tank batch LWDS without sump tank.

In a continuous LWDS, biological waste accumulates in a large collection tank connected to a continuous sterilizer; an additional sump tank upstream of the collection tank is sometimes installed, although in principle it is not required. Once a given level is reached in the tank, the liquid waste is pumped through a heat exchanger or mixed with steam, circulated through an insulated hold tube and then sent back to the tank during the heat-up phase. When the temperature set-point is reached over the whole hold tube, the inactivation step begins and the liquid is circulated in a flow-through mode. The aforementioned heat exchanger can be used at the same time for cooling down the inactivated wastes, thereby leading to significant energy savings. Figure 14.13 shows an example of a continuous LWDS.

The pros and cons of batch and continuous LWDS are discussed by Carlson (2001) and Miller and Bergmann (1993) with several configuration examples. In brief, batch systems tend to be more capital intensive (due to the requirement for larger equipment) and to consume more facility space and energy than do continuous systems; on the other hand, they are simpler to operate, require less maintenance and offer more flexibility if operating conditions, e.g. amounts of liquid waste, need to be changed. For these reasons, batch systems are the most common choice for pilot plants and multi-product facilities.

Alternatively, LWDS based upon chemical decontamination could also be used. The main problems, particularly at large-scale, are potential compatibility issues between the chemical agent and some materials, as well as the difficulty in ensuring adequate contact with all contaminated surfaces (Carlson 2001). For these reasons, this method is rarely used in the pharmaceutical and biotechnology industries.

Xlr Connector Dimensions

Figure 14.13 Example of a single-tank continuous LWDS. 'Pressurized, atmospheric and segregated systems' indicate various sources of biological liquid waste. Y indicates the stream of liquid waste, X the outlet of inactivated waste and Z cooling water. The liquid waste is recirculated until the deactivation temperature is reached throughout the whole hold tube. The waste is then eliminated, cooled down and sent to a neutralization tank. If needed, the tank itself and associated lines can also be decontaminated by heat (reproduced with permission from Carlson 2001).

Figure 14.13 Example of a single-tank continuous LWDS. 'Pressurized, atmospheric and segregated systems' indicate various sources of biological liquid waste. Y indicates the stream of liquid waste, X the outlet of inactivated waste and Z cooling water. The liquid waste is recirculated until the deactivation temperature is reached throughout the whole hold tube. The waste is then eliminated, cooled down and sent to a neutralization tank. If needed, the tank itself and associated lines can also be decontaminated by heat (reproduced with permission from Carlson 2001).

Sterile-filtration of exhaust gases from bioreactors is considered to be an adequate containment barrier. If required, systematic integrity testing might be performed after each SIP cycle, whereas for the purpose of aseptic operations only, it is normal practice to rely upon a validated number of sterilization cycles from the vendor. In a more hazardous operation, the exhaust gas can be incinerated as an added precaution; exposure to 370 °C for a few seconds kills all known microorganisms and viruses (Dream 1993).

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