Principles of biosafety regulations

There are numerous regulations and guidelines on biosafety and containment requirements for the cultivation of mammalian cells, with some differences between the US, the EU and other countries. Their analysis is further complicated by the fact that they are regularly updated, reflecting the continuously expanding knowledge and experience with recombinant DNA technologies.

In the US, some seminal guidelines were published by the National Institutes of Health (NIH, 1976) to provide a framework for conducting genetic engineering research in a manner that protects employees from infection and prevents adverse impact on the environment. These have since been amended (relaxed), in particular regarding large-scale applications. The current key documents on biosafety in the US that are applicable, among others, to mammalian cell culture for research and production are Guidelines for Research Involving Recombinant DNA Molecules (NIH, 2002) and Biosafety in Microbiological and Biomedical Laboratories (US Department of Health and Human Services, 1999). In brief, biological agents are classified into four risk groups according to their relative pathogenicity for healthy adult humans. A comprehensive risk assessment should be performed, taking into account the risk group of the agent and the way it is manipulated (including the amount and dose) in order to determine the appropriate safety practices and containment conditions. The guidelines differentiate small-scale from large-scale activities (defined as research or production involving more than 10 litres of culture), the latter requiring additional precautions. At small scale, four biosafety levels (BL1-BL4) are defined, with increasing requirements (BL4 being the most stringent level). At large scale, there are four biosafety levels: Good Large-Scale Practice (GLSP), BL1 large scale (BL1-LS), BL2 large scale (BL2-LS) and BL3 large scale (BL3-LS). The GLSP level, i.e. the least stringent level, is applicable to large-scale cultivation of non-pathogenic organisms that have built-in limitations to their survival in the environment and for which there is an extended history of safe large-scale use. The BL1-LS level applies, at large scale, to viable organisms that would require a BL1 level in the laboratory and do not qualify for the GLSP level; the BL2-LS and BL3-LS levels correspond, at large-scale, to the BL2, and BL3 levels in the laboratory. No provisions are made for large-scale activities using viable recombinant organisms that would require BL4 containment. These would need to be established by NIH on an individual basis.

In the European Union, the current key biosafety documents applicable to mammalian cell cultures are the European Council Directive 98/81/EC on the contained use of genetically modified microorganisms (which amends the Directive 90/219/EEC) and the European Council Directive 2000/54/EC on the protection of workers from risks related to exposure to biological agents at work (which replaces the Directive 90/679/EEC); additionally, the European Norm EN 1620:1996 provides biosafety guidelines on large-scale biotechnology activities. Like in the US, genetically modified microorganisms, including mammalian cells, are classified into four risk groups according to their level of pathogenicity to humans. A risk assessment, taking into account the classification of the organism together with the operating conditions, leads to the determination of the required containment level. There are four levels (1-4, 4 being the most stringent), at both small- and large-scale, which essentially mirror, with small differences, the US biosafety levels. A good comparative analysis of the US and EC biosafety regulations can be found in van Houten and Fleming (1993), although the latest amendments are not included. In brief, these regulations require biocontainment measures of two types:

(i) Operating procedures and practices These include good microbiology practices as well as specific handling techniques, for which the personnel must be regularly trained.

(ii) Physical containment barriers These can be further divided into two classes: (a) primary barriers, which aim to prevent or minimize direct exposure of personnel to biological agents and are provided by safety equipment (e.g. biological safety cabinets), personal protection devices and closed containers; (b) secondary barriers, which aim to minimize or prevent the escape of organisms into the environment, i.e. outside the laboratory or the facility. These barriers are integral parts of the design of the facility and include special air handling systems, contained work areas, and systems for waste inactivation.

A complete discussion on the requirements of each US and EU class and the implication for the design of laboratories, facilities and production equipment can be found elsewhere (Meslar & Geoghegan 1991; Miller & Bergmann 1993, Pearson 2003, Sinclair & Ashley 1995). Only the key elements applicable to mammalian cell culture are discussed briefly below.

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