The Laboratory Biosafety Guidelines: 3rd Edition 2004

Chapter 6
Large Scale Production of Microorganisms

6.1 Introduction

Canada has been steadily increasing its industrial base in the area of biotechnology. It is important that industrial fermentation and large scale manipulation of microorganisms be addressed in these Guidelines to minimize the risk to workers and the surrounding environment. Large scale work is not necessarily more hazardous than research/laboratory scale work, as many procedures and processes are conducted in closed systems, which thereby reduces the probability of exposure of the operator and the environment to the infectious material^(1,2). This has been adequately demonstrated with many highly infectious agents used for vaccine production⁽³⁾.

Fermentation processes have aerosol generating capabilities, and thus aerosols are likely to be the highest risk of exposure to pathogenic organisms and their products⁽⁴⁾. The scale of operations also poses a hazard for the potential release of large volumes of pathogenic organisms into the facility and/or the environment⁽¹⁾. Therefore, it is essential for facilities working with large scale fermentation to ensure that appropriate containment equipment is in place (both physical and operational) and that a detailed contingency plan is available for activation, so that potential exposures will be minimized if a malfunction in the fermentation process occurs.

6.2 Scope

Large scale processes involve fermenters and equipment that cannot be easily moved and sterilized in an autoclave and therefore require in situ sterilization and decontamination. However, as is discussed in detail in Chapter 2, Section 2.3 Risk Assessment, the volumes that define "large scale" and therefore require special consideration are based upon a detailed local risk assessment of the work being done and the organisms being manipulated. The volume of 10 L as the cut-off between laboratory scale and large scale is to be used as a guide only.

This chapter details the technical requirements for large scale processes involving organisms that require containment levels 1 to 3. Specific requirements have not been outlined for large scale research or production of viable organisms requiring containment level 4. These requirements should be established on a case-by-case basis, and assistance can be provided by the Office of Laboratory Security, Health Canada.

6.3 Operational Practices and Physical Requirements

The containment requirements listed here are the minimal requirements for large scale process areas and are to be used in addition to the corresponding laboratory scale containment facility operational procedures (see Chapter 3) and physical requirements (see Chapter 4).

6.3.1 Containment Level 1 Large Scale

1. Visual inspections of the integrity of the containment systems are important to detect small leaks.

2. Spills and accidents that result in exposures to organisms to be immediately reported to the facility director and facility Biological Safety Officer; medical attention and surveillance to be provided as appropriate; written records to be maintained.

3. Emergency plans and procedures to be readily available and to include appropriate equipment and training for emergency response to spills or accidental release of organisms (i.e., personal protective equipment, disinfectants); training to be documented.

4. Cultures of viable organisms to be contained within a closed system or other primary containment equipment (e.g., BSC) that is designed to reduce the potential for release of aerosols.

5. Culture fluids, except as allowed below, are not to be removed from a closed system or other primary containment equipment without prior inactivation of the organisms by a validated procedure. A validated inactivation procedure is one that has been demonstrated to be effective against the organism in use. Culture fluids that contain viable organisms intended as the final product may be removed from the primary containment equipment by way of closed systems for sample analysis, further processing or final fill.

6. Sample collection, the addition of materials and the transfer of culture fluids from one closed system to another to be performed in a manner that prevents the release of aerosols or contamination of exposed surfaces.

7. Process equipment, closed systems or other primary containment equipment to be provided with treatments (i.e., HEPA or equivalent filters, incineration, or gaseous decontamination through chemical disinfectants) to prevent the release of the viable organisms.

8. A closed system or other primary containment equipment that has contained viable organisms is not be opened for maintenance or other purposes without prior inactivation of the organisms by a validated procedure; a validated inactivation procedure is one that has been demonstrated to be effective against the organism in use.

9. Facilities to be designed to prevent the release of viable organisms to sanitary sewer (e.g., capping or raising of floor drains).

6.3.2 Containment Level 2 Large Scale

In addition to the requirements for containment level 1 large scale, the following describes additional requirements at containment level 2 large scale.

1. Cultures of viable organisms to be contained within a closed system or other primary containment equipment designed to prevent the release of aerosols.

2. Process equipment seals and other directly associated mechanical devices should prevent leakage or be fully enclosed in ventilated housings that are exhausted through HEPA or equivalent filters, or through other equivalent treatment technologies.

3. Process equipment to contain sensing devices (or equivalent), where possible, to monitor the integrity of containment during operations and alarm conditions leading to containment failure.

4. Process equipment to be tested for integrity of containment capability before initial use and after modifications or changes to the system that could affect the containment characteristics of the equipment; testing procedures and acceptance criteria to be appropriate for the process equipment and closed system design; records to be maintained of such testing.

5. Hazard warning signs (e.g., biohazard sign, containment level, contact information, entry requirements) must be posted at the entry to the process area. Consideration should be given to the addition of this signage to relevant process and primary containment equipment used to contain viable organisms.

6. Personal protective equipment requirements to be posted at entry.

7. Entry to process area to be restricted to authorized personnel while production is in progress.

6.3.3 Containment Level 3 Large Scale

In addition to the requirements for containment level 1 and level 2 large scale, the following describes additional requirements at containment level 3 large scale.

1. Personal protective equipment to include full change out of street clothes into dedicated process area garments (dedicated pants, shirts, shoes, socks, head covers, gloves) or complete coverage of street clothing with process area garments (dedicated jumpsuits, shoe covers, head covers, gloves); reusable garments to be removed upon exit, decontaminated and laundered after each use; single use garments to be removed upon exit, decontaminated and discarded after each use; respiratory protection may be appropriate depending on the organism being processed.

2. Hazard warning signs and identification to be used in all records relevant to the history of the equipment (e.g., testing, operation, maintenance).

3. Provision to be made in the process area to contain the full volume of a release of process fluids (e.g., process equipment in diked area).

References

1. Grinsted, J. Risk assessment and contained use of genetically modified organisms. In: Tzotzos, G.T. Genetically modified organisms: a guide to biosafety. Wallingford, UK: Centre for Agriculture and Biosciences (CAB) International, 1995;17-35.

2. Cipriano, M.L. Biosafety considerations for large-scale production of microorganisms. In: Fleming, D.O., and Hunt, D.L. Biological safety principles and practices. Washington, D.C.: ASM Press, 2000; 541-55.

3. Collins, C.H. Safety in microbiology: an overview. In: Collins, C.H., and Beale, A.J. Safety in industrial microbiology and biotechnology. Oxford, UK: Butterworth-Heinemann Ltd, 1992;1-5.

4. Brunius, N.G.F. In: Collins, C.H., and Beale, A.J. Safety in industrial microbiology and biotechnology.Oxford,UK: Butterworth-Heinemann Ltd., 1992;239-42.

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