Laboratory Biosafety Guidelines
2nd Edition 1996

CHAPTER 6

LARGE SCALE PRODUCTION OFMICROORGANISMS

6.1 INTRODUCTION

Since Canada is rapidly expanding its industrial base in the area of biotechnology, it is imperative that industrial fermentation and large scale manipulation of microorganisms be addressed so that all manipulations be conducted under conditions of containment that will ensure minimal risk to workers and to the environment. Large scale work is not necessarily more hazardous than laboratory scale work. In fact, it is often less of a concern since most of the procedures in large scale fermentation are automated, and are often conducted in closed systems, thereby reducing the probability of exposure of the operator. This has been adequately demonstrated with many highly infectious agents used for vaccine production. However, all organizations working with large scale fermentation must ensure that the appropriate equipment for such procedures and contingency plans are in place to minimize risks should there be a malfunction in the process.

This chapter is to be used as a guideline for work involving large scale processes. Previous chapters contain information that may be relevant. Large scale cultivation of microorganisms may involve hazards in addition to those that are discussed in this chapter. These must be identified and considered.

To address the issue of guidance for the large scale production of microorganisms, a working group consisting of the following persons prepared the following chapter:

6.2 SCOPE

The working group's objective was to provide a technical document for those who design, build, operate or work in research and industrial facilities where large scale manipulation of microorganisms is performed. The focus of this document is therefore on the use of microorganisms under conditions other than those described in previous chapters. The committee considers large scale to include fermenters and equipment that cannot be easily moved and sterilized in an autoclave, therefore, requiring in situ sterilization. This would normally apply to volumes greater than 10 litres.

A number of countries have addressed large scale work with recombinant organisms. The objective of this working group was to present guidelines to parallel those previously adopted by other countries, but which include large scale fermentation of all microorganisms.

The Committee is of the opinion that because a microorganism has been manipulated genetically, it does not necessarily pose an increased risk. For example, it is possible that a genetically altered microorganism (deletion mutants) may have a reduced potential to cause infections in humans or animals as a result of the deletion of a specific virulence gene. Similarly, insertion of foreign genes into an organism at a specific site may either reduce or increase its virulence. Each genetic manipulation must be evaluated on a case by case basis. If possible, an organism should be modified in such a way that its pathogenicity, dissemination and survival in the environment are reduced. Once the level of risk is established, each organism is required to be handled according to the criteria for that risk group.

Although this chapter does not address issues relating to the intentional environmental release of microorganisms, containment practices recommended should be sufficient to reduce the release of microorganisms which may be harmful to the environment.

6.3 SPECIAL CONSIDERATIONS

6.3.1 Biological Safety Officer:

When a production facility is engaged in activities with organisms in Risk Groups 2-4, it must appoint a Biosafety Officer (BSO), experienced and knowledgeable in handling pathogenic microorganisms and eukaryotic cells, with expertise in large scale containment.

6.3.2 Transgenic Plants and Animals

There is considerable potential for commercial production of bioproducts in transgenic plants or animals. The potential release of transgenics into the environment and transmission of novel genes to other plants and animals need to be considered when designing both the production system and facilities to contain the transgenics. In each case, the risk level needs to be determined in consultation with the appropriate Government agency.

This section will focus on transgenic plants and animals that are used for production of bioproducts and the containment required for these activities.

In the case of transgenic animals, the first consideration is that they be handled according to the Guidelines set forth by the Canadian Council for Animal Care. An important consideration is the ability of the animal to transmit genes by interbreeding with the same species or any related species. Under these conditions, it is important that the transgenics are well-contained to prevent the spread of genetic modifications. It is recommended that, if at all possible, transgenics be created using methodology which restricts the potential for transmission of the genes from one host to another.

Transgenic plants may transmit novel characteristics to other plants, thereby modifying the gene pool of existing species. Since this transmission is mediated by pollen, transgenic plants should be made sterile or contained in a growth chamber or greenhouse designed to prevent pollen release either by air or insects. If plants are allowed to mature, care must be taken to contain the seeds in the green house or growth chamber.

If live microorganisms are used as vehicles for transfection, the containment level for the plants or animals inoculated with these viable recombinant microorganisms must be at least as high as that required for work with that specific microorganism. Transgenics (eg. produced by micro-injection, by use of replication defective vectors, or other sequences that are not horizontally transmitted) can be normally handled at Containment Level 1. The following recommendations should be considered prior to the initiation of transgenic studies:

1. Complete copies of the genome or replication competent genome should not be used.

2. The constructs should not contain genes capable of causing neoplastic transformation of animals.

3. The probability of recombination with extraneous microorganisms should be minimal or non-existent.

6.3.3 Cell Lines

The biological hazards associated with the use of mammalian or other cells in culture fall into 3 categories:

1. Primary cultures of mammalian or other cells may harbour infectious agents or integrated DNA originally present in the animal or person from which the cultures were derived. Whenever possible, the donor should be tested for any suspect pathogens prior to the preparation of the culture, and the culture should be considered to be contaminated until proven to be free of suspect agents. Such primary cultures should be handled in a manner appropriate to the risk class of the suspected contaminant, and precautions should be taken against parenteral or other means of exposure of laboratory personnel.

2. Cell lines known to contain infectious agents or integrated DNA should be handled according to the risk class of the agent.

3. Cell lines that are deemed to be free of infectious agents would, rarely, pose a biological hazard. If there is unintentional parenteral inoculation, normal immune response should provide protection, prevent progressive growth and cause rejection of accidentally transplanted cells.

6.4 CONTAINMENT LEVELS AND PRACTICES

6.4.1 Containment Level Large Scale 1 (LS1)

Microorganisms that are demonstrated to be non-pathogenic, containing no adventitious agents and having a long history of safe industrial use are not considered in this containment level.

As the volume of cultivation and the quantity of biomass increases from laboratory to development or production scales, increased care must be taken to achieve control over the dispersal of Risk Group 1 agents, which is consistent with current large scale microbiological practice.

For genetically engineered organisms certain criteria are necessary to ensure continued safety. The host organism should be a non-pathogen with no adventitious agents, a history of safe use, and have limited capacity to survive in the environment. Vectors with known inserts should be well characterised and free from sequences that result in adverse effects to humans, animals, plants or the environment. The genomic insert should be limited in size to the smallest sequence required and should not increase the stability of the gene product in the environment.

Resistance markers should be transferred with caution to organisms, to prevent acquisition of resistance that might compromise therapeutic use of antimicrobials. The resulting recombinant organism should be non-pathogenic or alternatively possess limited survival characteristics and be without adverse environmental consequences.

Containment Level LS1 is suitable for large scale work with Risk Group 1 agents. In addition to the requirements of Containment Level 1, the following are required:

Physical Requirements

• The work area and equipment should be well planned and appropriate for the intended use.
• Process equipment should be designed to minimize aerosol generation.
• Process equipment must contain organisms within a closed system.
• Process equipment and unit operations must be designed to minimize environmental effects.

Operational Requirements

• All personnel should be adequately trained and knowledgable in workplace safety and protocols.
• A plan for the clean-up and, if required, disinfection of large scale spills must be developed and ready for use when required.

6.4.2 Containment Level Large Scale 2 (LS2)

Large scale cultivation or processing of Risk Group 2 agents requires greater containment than laboratory scale operations.

Containment Level LS2 is suitable for large scale work with Risk Group 2 agents. In addition to both the requirements for Containment Level LS1 and for Containment Level 2, the following are required:

Physical Requirements

• Process equipment must contain the organisms within a closed system, and must be provided with HEPA or equivalent filters, which have been tested for integrity to prevent releases of aerosols of the organism being processed. Alternatively equivalent procedures (i.e. incineration or off gassing through chemical disinfectants) may be employed to prevent release of microorganisms.

• Process equipment must be capable of being decontaminated with a validated inactivation procedure.

• Unit operations and transfers between operations must be designed to prevent the release of aerosols.

• Seals and mechanical devices associated with the process equipment shall be designed to prevent leakage or shall be enclosed in HEPA or equivalent filtered housings.

Operational Requirements

• Decontamination of process equipment and process effluent must be performed by a validated inactivation procedure.

• Decontamination must precede breach of containment.

• Sampling is to be performed in a controlled manner which prevents the release of infectious materials.

• Process equipment should be tested regularly for integrity of containment capability (i.e. after each use or operation) and records maintained of such testing.

• Unauthorized personnel are not permitted to enter process areas.

• Staff must be fully acquainted with emergency procedures to deal with spills or accidental release of viable organisms. Procedures should be posted and training documented.

• Equipment for emergency and decontamination response must be readily available in the process area and be maintained for immediate and effective use.

6.4.3 Containment Level Large Scale 3 (LS3)

Large scale production and processing of Risk Group 3 agents may result in serious hazard to people, animals and the environment. Containment Level LS3 is characterized by design features which involve primary and secondary levels of physical containment.

Containment Level LS3 is suitable for large scale work with Risk Group 3 agents. In addition to both the requirements for Containment Level LS2 and for Containment Level 3, the following are required:

Physical Requirements

• Air leaving the containment area is to be HEPA filtered.

• Provision must be made to contain the full volume of a complete release of all process fluids in the process area.

• All potentially contaminated liquids must be transferred in closed piping.

• Effluent must be taken to a decontamination system which is regularly validated to ensure efficacy of the process.

• Showers and change rooms must be provided.

Operational Requirements

• Entry to process area should be restricted to essential personnel while production is in progress.

• Upon entry, all personnel should exchange their outer clothing for dedicated laboratory clothing and shoes. Laboratory clothing shall be decontaminated before laundering or disposal.

• A personal shower should be used on egress.

• It is recommended that provision should be made for electronic transfer of all data from the containment area (e.g. computer networking).

6.4.4 Containment Level Large Scale 4 (LS4)

Specific requirements have not been outlined for large scale research or production of viable organisms classified in Risk Group 4. These requirements will be established on an individual basis, through consultation with the Office of Laboratory Security, Laboratory Centre for Disease Control, Health Canada.

6.5 TRANSPORTATION OF LARGE SCALE PRODUCTS

6.5.1 Transportation

Requirements for the shipment of infectious agents are outlined in Section 3.3. Specifically, all microorganisms classified in Risk Groups 2, 3 or 4 must be packaged, labelled and transported in accordance with the Transportation of Dangerous Goods Act and Regulations.

Further information may be obtained from the Transport Dangerous Goods Directorate of Transport Canada, 344 Slater Street, 14th Floor, Ottawa, Ontario K1A 0N3, tel.: (613) 958-0517.

6.5.2 Transportation On-site Between Containment Areas

Transportation of microorganisms of Risk Group 2 or higher within a facility must be accomplished within a closed system. Alternatively, process piping or a secure unbreakable, sealed container must be used. Containers or transfer lines must be capable of being decontaminated.

The facility's emergency response plan must include provision to respond to a spill, leak or accident during transportation between containment areas. All emergency response materials and equipment must be ready for use whenever needed.