Cumitech 36: Biosafety Considerations for Large-Scale Production of Microorganisms

36 Biosafety Considerations for Large-Scale Production of Microorganisms MARY L. CIPRIANO Cumitech 1B Cumitech 2B ...

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36 Biosafety Considerations for Large-Scale Production of Microorganisms MARY

L. CIPRIANO

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Blood Cultures III Laboratory Diagnosis of Urinary Tract Infections Quality Control and Quality Assurance Practices in Clinical

Microbiology

Laboratory Diagnosis of Gonorrhea Practical Anaerobic Bacteriology New Developments in Antimicrobial Agent Susceptibility Testing: a Practical Guide Laboratory Diagnosis of Lower Respiratory Tract Infections Laboratory Diagnosis of Bacterial Diarrhea

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Laboratory Laboratory Laboratory Laboratory

Diagnosis Diagnosis Diagnosis Diagnosis

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Laboratory Therapeutic Laboratory

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Cum/tech Coordinating

should be cited as follows, ed , B W McCurdy ASM

of of of of

Ocular Infections Central Nervous System Infections Viral Infections the Mycobacterioses

of Female Genital Tract Infections Viruses Diagnosis of ChlamydiatrachomatisInfections Drug Monitoring: Antimicrobial Agents Diagnosis of Viral Respiratory Disease Diagnosis

0fHepadtjs

Immunoserology of Staphylococcal Disease Infections of the Skin and Subcutaneous Tissues Rapid Detection of Viruses by Immunofluorescence Current Concepts and Approaches to Antimicrobial

Agent Susceptibility

Testing

Laboratory Diagnosis of Viral Infections Producing Enteritis Laboratory Diagnosis of Zoonotic Infections: Bacterial Infections Obtained from Companion and Laboratory Animals Laboratory Diagnosis of Zoonotic Infections: Chlamydial, Fungal, Viral, and Parasitic Infections Obtained from Companion and Laboratory Animals Laboratory Safety in Clinical Microbiology Selection and Use of Laboratory Procedures for Diagnosis of Parasitic Infections of the Gastrointestinal Tract Verification and Validation of Procedures in the Clinical Microbiology Laboratory Laboratory Diagnosis of Zoonotic Infections: Viral, Rickettsial, and Parasitic Infections Obtained from Food Animals and Wildlife Laboratory Safety, Management, and Diagnosis of Biological Agents Associated with Bioterrorism Laboratory Diagnosis of Mycoplasmal Infections Postmortem Microbiology Biosafety Considerations for Large-Scale Production

e g Press,

Caplan, M WashIngton,

Editorial Board for ASM Cumitechs: Alice S Weissfeld, Burken, Roberta Carey, Linda Cook, Lynne Garcia, Richard Sewell, Daniel Shapiro, James W Snyder, Allan Truant

J , and D C

F P

of Microorganisms

Koontz

2001

Cha/r, Maria D Appleman, M Jamlson, Karen Krlsher,

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35,

Postmortem

mIcrobIology

Vlckle Baselskl. B Kay Buchanan, Mitchell Susan L Mottlce, Michael Saubolle, David

I L

Effective as of January 2000, the purpose of the Cum/tech series IS to provide consensus recommendations regarding the judicious use of cllnlcal microbiology and Immunology laboratories and their role in patient care Each Cum/tech IS written by a team of clinlclans, laboratorlans, and other Interested stakeholders to provide a broad overview of various aspects of lnfectlous disease testing These aspects Include a discussion of relevant cllnlcal conslderatlons, collection, transport, processing, and Interpretive guldellnes, the cllnlcal utility of culture-based and non-culture-based methods and emerging technologies, and Issues surrounding coding, medical necessity, frequency limits, and reimbursement The recommendations In Cumltechs do not represent the official views or policies of any third-party payer Copyright 0 2002 ASM Press American Society for MIcrobIology 1752 N Street NW Washington, DC 20036-2904 All Rights

Reserved

Biosafety Considerations for Large-Scale Production of Microorganisms Mary L. Cipriano Abbott Laboratories, Department 3A4, APlA, 100 Abbott Park Rd., Abbott Park, IL 60064-6102 Introduction ......................................................................................... Risk Assessment ..................................................................................

1 2

Agent Considerations ................................................................................................. Process Considerations .............................................................................................. ..................................................................................... Enwronmental Considerations

2 2 3

General

Biosafety

Primary

Containment

Recommendations for Large-Scale Work ...................... ............................................................................

........................................................................................................ Special Practices Equipment Selection and Usage .................................................................................. Fermentors and Culture Vessels .................................................................................. ........................................................................................... Recovery and Purification ................................................... Mechanisms for Cleaning and Disinfecting Equipment

Secondary

........................................................................

Containment

3 3 3 3 3 4 5

5

Constructron and Finishes ........................................................................................... ..................................................................... Heating, Ventilation, and Air Conditioning Utilrtres and Maintenance Issues ................................................................................. ........................................................................... Facility Layout and Support Systems ....................................................................................................... Waste Treatment

6 6 7 7 8

...........................................................................................

8 8 15

Conclusion Appendix: References

............................................ Large-Scale Biosafety Guidelines .........................................................................................

However, none of them provides extensive detail for large-scale operations; they suggest consulting with a biological-safety professional. Although many biosafety professionals understand the agent, are capable of performing a risk assessment, and may also be knowledgeable about the equipment and processes used, they may have limited experience with the largescale processes. The purpose of this Cumitech (which is reprinted with minor modifications from chapter 35 of D. 0. Fleming and D. L. Hunt [ed.], Baological safety: Principles and Practices, 3rd ed., ASM Press, Washington, D.C., 2000) is to provide the Large-Scale Biosafety Guidelines that were developed by the ASM Subcommittee on Laboratory Safety. The subcommittee was chaired by Diane Fleming and Mary Cipriano; contributing and ad hoc members included Robert Hawley, Jonathan Richmond, Joseph Coggins, Benjamin Fontes, Christina Thompson, Stefan Wagener, Richard Rebar, and Paul Meechan. A draft of these guide-

INTRODUCTION

T

he notion of scale-up or large-scale processing of organisms is currently associated with recombinant DNA technology, but in fact, it has been common practice for many years. Microorganisms have been scaled up for the manufacture of foods and beverages for centuries. In the 20th century, largescale production of antibiotics and vaccines became commonplace. Although some laboratory infections have been associated with large-scale processing of infectious agents, their occurrence is very rare. This low incidence of infection may be partially due to the reduction in virulence of the cultured organism; however, it is likely that this lower level can, in part, be attributed to the use of proper handling techniques and primary or secondary containment measures, i.e., the appropriate equipment and facilities. There are a number of guidance documents addressing biosafety requirements (1, 2, 9, 14, 17, 18). 1

2

Cipriano

lines was presented for comment at the Centers for Disease Control and Prevention (CDC) Symposium on Biosafety in 1998, Rational Basis for Biocontainment. The guidelines were previously published as appendix A of chapter 35 of Biological Safety: Principles and Practices, 3rd ed. The initial section of this Cumitech provides a discussion of considerations for risk assessment and primary and secondary containment. There are few absolutes that can be applied across the board, because the decisions to be made are dependent on the risk assessment of the organism and the processes used. Fortunately, there are common biosafety principles, which are discussed below. In discussing large-scale processes, the term “large scale” must be defined. According to the National Institutes of Health (NIH) recombinant DNA guidelines (14) and the Canadian laboratory biosafety guidelines (9), more than 10 liters constitutes large scale. In Japan, more than 20 liters is designated as large scale. In the United Kingdom, the Advisory Committee on Dangerous Pathogens (2) states that it is not the volume but the intent of the work th at determines the scale. The CDC-NIH publication Biosafety in Microbiological and Biomedical Laboratories (BMBL) defines “production quantities” as a volume or concentration of infectious organisms “considerably in excess of those used for identification and typing” (18). There is no specific volume or concentration that can be universally cited. They recommend that the laboratory director must base the assessment on the organism, process, equipment, and facilities used. Certainly, in an ideal world that is the best solution. Unfortunately, not all laboratory directors have the depth of knowledge to make the assessments required and/or access to a biosafety professional for input. It is hoped that the ideas and suggestions discussed here will be of assistance to the individuals making those decisions.

CUMITECH

is generally not acceptable to the press and the public following an accidental release. This can force the institution to consider additional containment features for the facility to minimize the potential for such an incident. Three categories need to be taken into consideration: agent, process, and external environment. These considerations may not come into play for every assessment but should be reviewed to determine their relevance to the specific situation. In doing the risk assessment, a number of questions need to be answered about the organism(s) that will be used in the facility. These include, but are not limited to, the following: l

What is the highest biosafety level needed for containment of the agent(s) that wi 11 be used in the facility?

l

What is the mode of transmissi infectious dose?

l

How communicable

l

Is the agent an opportunistic pathogen that could infect immunocompromised individuals?

l

Does the agent produce any toxic, biologically tive, or allergen .ic compounds?

l

Are vaccines, prophylaxis, or therapeutic measures available to prevent or mediate an infection?

l

Is the agent endemic in the area?

l

How well does the agent survive outside the culture system?

l

Can the organism transfer genetic traits to other organisms in the environment?

l

How is the agent disseminated e.g., insects?

Process Agent

Considerations

An agent-based risk assessment is the starting point for any large-scale work (7). The scale of the work can influence the risk assessm .ent. For example, a nonpathogenic organi sm that produces an extracellular toxin may not pose a problem at 40 ml but can create significant concerns when one is dealing with 10,000 liters. Often, it is not even a real health risk that must be considered but the potential negative publicity from an event. The general public has a heightened awareness of infectious organisms, thanks to the media’s concept of “flesh-eating bacteria,” mad cow disease, the “hot zone,” and renewed threats of germ warfare. A rational scientific analysis of the situation

36

In? What

is the

is the agent?

through

ac-

vectors,

Considerations

Once that information is gathered, some specific information must be put together about the process (see reference 13). l

Will the facility be dedicated to one agent or will a number of agents be used?

l

What volume of active agents will be present in the facility, e.g., greater than 10 liters?

l

Will the process be continuous

l

Will the equipment

l

What type of equipment

l

What out?

be stationary

or batch? or movable?

will be used?

types of manipulations

need to be carried

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Biosafety

l

and Drug Administration (FDA) drug or device regulations, NIH recombinant DNA guidelines, or other governmental regulations? What type of cleaning, disinfection, tion equipment, etc., is needed? Environmental

decontamina-

Considerations

The last category can roughly be termed environmental considerations. This includes questions about the local environment external to the facility. What are the climatic conditions in the area, e.g., temperature and humidity? What is the geography of the site? What are the native flora and fauna? How close are the air supply intake and exhaust to other facilities? How near is the facility to private property? What is that property, e.g., industrial, school, or housing?

GENERAL BIOSAFETY RECOMMENDATIONS WORK

FOR LARGE-SCALE

Although it is not the intent to review here the exhaustive procedural requirements for work at each designated biosafety level, it is important to review the basic concepts to understand the criteria for the selection of the equipment and facility options. A copy of the large-scale biosafety guidelines is found in the appendix. l

l

l

Good large-scale practices (GLSI?) are designated for well-characterized organisms that are not pathogenic and do not produce compounds that are toxic, allergenic, or biologically active (12). Such agents will have been used safely over time or have been designated as safe, i.e., listed in appendix C of the NIH recombinant DNA guidelines (14). There are no specific biosafety containment requirements for GLSP facilities. Procedures should be done in a way that does not adversely affect the health and safety of the employee, i.e., splashing, spraying, and generation of aerosols are minimized. Biosafety level 1 -large scale (BSL-1LS) is for nonpathogenic organisms but can include organisms that can cause sensitization or are opportunistic pathogens. The goal at this level is to minimize release of viable organisms. Biosafety level 2-large scale (BSL-2LS) is used with common pathogens, i.e., risk group 2 agents. The operations should be designed to prevent re-

for Large-Scale

Production

of Microorganisms

3

lease and employee exposure to splashing and spraying. Biosafety level 3 -large scale (BSL-3LS) is for growing risk group 3 agents that may be aerosol transmissible, able to spread by insect vectors, cause serious diseases in humans or animals, etc. Equipment and facilities used for this level must be designed to prevent employee exposure and aerosol release of the agent within the facility and release of the agent outside the facility.

The requirements for large-scale production of risk group 4 agents are not addressed in this Cumitech because of their highly specialized requirements and limited use.

Special

Practices

Primary containment is provided by equipment and appropriate biosafety practices, administrative practices, and personal protective equipment. In general, all of the standard and special practices and safety equipment identified in the BMBL (18), along with the recommendations of the NIH Recombinant DNA Guidelines (14), are applicable to large-scale processes. However, additional requirements should be considered depending on the agent used and the processes involved. These issues are addressed in the large-scale biosafety guidelines in the appendix. They include respirators capable of protecting from the organism in use mandatory physicals, health screening, and/or immunizations, if appropriate written procedures for the process, e.g., standard operating procedures emergency response plans additional garbing as necessary to maintain product integrity, e.g., shoe covers, hair nets, and bunny Equipment

Selection

and Usage

The equipment used provides most of the containment required. When the containment is not adequate for the risks associated with the agents used, additional barriers may need to be utilized. Fermentors

and Culture

Vessels

To maintain the integrity of the culture, the culture vessel must provide an appropriate level of containment. The vessel must be constructed (3) to withstand rigorous cleaning and decontamination procedures. It must be insulated, have heating and cooling capabilities to maintain the proper growth temperature, and be capable of protecting the contents from contami-

4

Clpriano

nation. Although glass and plastic systems are used for smaller volumes, most large-scale units are constructed of metal, generally food quality stainless steel. Stainless steel minimizes corrosion, obviates the adverse effects of metallic ions on cultures, and is accepted by the FDA as suitable for direct contact with food and drugs, for which most of these processes are used. To facilitate cleaning and decontamination, the interior of the tank should be smooth, without dead legs, ledges, or inaccessible areas. The vessel may need to meet the applicable boiler or pressure vessel requirements, since it may be operated at a slight positive pressure or be pressurized during a sterilization cycle. Treatment or filtration of the exhaust from the culture system is not generally warranted for GLSP systems. Beyond that level, the exhaust gases must be filtered or treated. The filters used need to be capable of removing the organism, allergen, toxin, or biologically active compounds present. It may be desirable to pretreat the exhaust air before filtration by passing it through a condenser, separator, or preheating system, particularly if HEPA-rated filters must be used. The general practice is to use a single filter to reduce the potential for the escape of viable organisms (the requirement for BSL-1LS) and to use two filters in sequence to prevent the escape of viable organisms (the requirement for BSL-2LS and BSL-3LS). Most bioreactors use a mixing air lift or tower system to properly mix and aerate the culture. An air perfusion system or a magnetic coupling for the agitator facilitates the containment of the unit; however, their application may be limited by the size and/or viscosity of the material. Most fermentors utilize an agitator system that is connected to the tank via a rotating seal. For higher levels of containment, i.e., above BSL-2LS, a double mechanical seal should be used. There is some question regarding the increased reliability of a double versus a single seal (10,l l), but use of a double seal is specifically mentioned in the NIH recombinant DNA guidelines when prevention of release is necessary. When processes involve toxic or biologically active materials, or require additional containment measures because of the agents involved, liquids or steam can be used as the lubricant between the seals. The lubricant flow can be sent to a biowaste kill system. The location of the drive is another issue, i.e., bottom mounted versus top mounted. Although the bottom-mounted systems facilitate maintenance of the units, top-driven units provide better containment of the processor and should be the choice for BSL-2LS and BSL-3LS operations. It is not generally feasible to operate a fermentor or bioprocessor under negative pressure due to the obvious problems of foaming and product contamination. For processes where escape from the system must be

CUMITECH

36

prevented, the unit should be equipped with devices that monitor the pressure in the chamber and sound an alarm if the set level is exceeded. Pressure vessels must be equipped with a pressure relief system (PRS), which consists of a rupture disk and/or pressure relief valve. In dealing with risk group 2 or 3 agents, it is desirable to have the PRS located so that it releases away from the work area. Depending on the agent in use, some type of shrouding should be considered or the PRS should be vented to a kill tank or some other contained system. A better option might be to use a pressure sensor that will shut off the air supply when the unit exceeds the normal operating range. Sampling devices should maintain the integrity of the culture as well as meet the containment requirements. For GLSP and BSL-lLS, one can use a needle and septum or a steamable sampling valve, since the aim is minimizing release. The use of the needle needs to be evaluated for each agent in use to ensure that it does not create employee exposure problems. For work with pathogens, where preventing release is the goal, a sampling device that provides containment should be used. In some cases, secondary containment of the sampling device may be needed. All connections to the vessels are to be secured to prevent leakage or release. Depending on the agent and/or vessel size, hard piping may be indicated. All connections must be designed to facilitate cleaning and decontamination, e.g., flush mounted or steamable. If the fermentor does not have a sufficient degree of containment built into the unit for certain higher-risk materials, the entire unit may need to be placed in a containment device. Recovery

and Purification

In the downstream processing of the culture material, containment of the material is usually required to protect the product. If the organisms are inactivated in the culture system and there are no toxic, allergenic, or biologically active products (as is the case with GLSP processes), additional containment measures should not be needed. If the cultures are not killed before processing, BSL-1LS requires that the equipment being used for processing viable organisms be designed “to reduce the potential for the escape of viable organisms. ” At the higher containment levels, the equipment needs to be designed to “prevent release.” The risk assessment of the organism, which includes an analysis of any harmful characteristics of the organism, is the most important consideration in the choice of the containment features of the equipment. Downstream processing can be divided into three basic categories, although some of the equipment can be used for more than one purpose: cell separation,

CUMITECH

36

cell disruption, and purification. ment includes the following:

Biosafety

Some of the equip-

Production

of Microorganisms

5

accessibility to the equipment for operation, routine maintenance and servicing, materials loading and removal, and cleaning. This may necessitate the use of access panels, portholes, and gloves. Obviously, all of these issues need to be addressed in the design of the device so that it can be used in the manner intended. The topic of BSCs and the choice of the appropriate unit is discussed in references 4 and 19. For drug and device applications, all air coming into contact with open product must be class 100, which usually requires the unit to have a HEPA filter directly over the work surface. The use of horizontal laminar-flow clean-air stations should be limited to medium preparation. For applications where a BSC cannot be used but class 100 conditions are required, a vertical laminar-flow unit may be used, provided that additional shielding, curtains, low-level returns, etc., are used to reduce employee exposure.

cell separation equipment (filtration, membrane filtration, centrifuges, and presses) cell disruption equipment (homogenizers and sonicaters) purification equipment (chromatography columns and dialysis equipment) Rather than review each type of equipment separately, I will discuss general containment design approaches to achieve the different levels of containment. These approaches can be applied to a wide variety of equipment according to the required level. At BSL-lLS, the containment objective is to reduce the potential for release of viable organisms to minimize release. In more practical terms, that means that the equipment used should be designed to prevent spraying, splashing, or significant release of material. If not designed into the equipment itself, this level of containment may be achieved by shielding or putting barriers around the equipment or points where the release can occur. If the equipment generates aerosols, the use of shielding with an exhaust vent placed near the point of aerosol generation could be adequate to minimize release to the work area. At the higher biosafety levels, the requirement is to prevent release of aerosols. However, the rationale for doing that is different for respiratory transmissible agents (typically risk group 3) versus agents transmitted through contaminated fomites, e.g., surfaces or equipment (typically risk group 2). In the latter case, if the employees can be vaccinated and the agent is not transmissible by the aerosol route, the use of venting, shielding, or barriers may be adequate to prevent employee exposure. If prevention of aerosol escape is required, either because of the agent or the nature of the product, more rigorous containment measures must be used. One method is to place the entire piece of equipment in a containment device or room. For example, flowthrough centrifuges are known to generate aerosols, and where containment is needed, the unit can be placed in a separate room or containment device. Plastic isolators may be considered, but they are designed to be positive-pressure systems. Depen .ding on the agent or product being processed, the isolators might be used if fitted with a sensing device to detect leakage. Biological safety cabinets (BSCs) can be used for smaller equipment that does not generate much turbulence. In some instances, BSC manufacturers, or other specialty equipment fabricators, can make specialized containment devices for specific equipment. A containment device can consist of plastic shrouding or can be a cabinet with HEPA supply and exhaust to dissipate the heat load. The device needs to provide

for Large-Scale

Mechanisms Equipment

for Cleaning

and Disinfecting

All equipment is required to be cleaned and “sterilized” to prevent product contamination between runs, where the “sterilization” cycle has been validated to kill the organism being manipulated. A more appropriate term is “decontaminate,” since one does not always need to eliminate all viable organisms (15). Some facilities may use a clean-in-place (CIP) system that utilizes detergent and an extreme pH. In some cases, that treatment may be adequate to kill the organism in use, but that has to be validated. If the agent is not killed by the CIP process, all efluents must be piped to a tank for treatment and disposal. For some equipment, steaming or treatment with chlorine, other disinfectants, or acidic or caustic solution may be used for decontamination. For processes requiring containment above the GLSP level, the equipment must be decontaminated before being opened or cleaned.

SECONDARY

CONTAINMENT

The facility design and construction provide the secondary containment that protects people outside the immediate work area, both in other parts of the facility and in the community at large. The process, agent, and environmental-issue determinations, as outlined previously, will dictate many of the design parameters. It is important to keep the basic facility design criteria in mind throughout the process: l l

GLSP, no special facility requirements BSL-lLS, facility designed to contain large spills or releases of organisms

6

l

l

Cipriano

CUMITECH

BSL-2LS, facility designed to contain all spills or releases of organisms BSL-3LS, facility designed to contain all spills or releases of organisms, including aerosols

One of the most significant issues is whether the facility will be used for the manufacture of an FDAapproved drug or device. This issue is addressed in reference 13. Because there are a number of ways to achieve containment, various approaches to design issues are provided. These containment concepts are generally not applicable to GLSP facilities, but certain features may be usable, e.g., waste treatment. Construction

and Finishes

All surfaces within the facility should be designed to withstand regular cleaning and decontamination. Decontamination usually is by application of a disinfectant solution, but BSL-3LS facilities generally must withstand fumigation. The floors should provide a durable, slip-resistant, sanitary surface. If large equipment will be moved about, concrete floors with polyacrylate topping or an architectural epoxy can be used. Sheet vinyl flooring with welded seams or some other monolithic system should be considered where environmental release of pathogenic agents is a major concern or to meet good manufacturing practice (GMI?) requirements. The facility must be capable of containing releases from fermentors or bioreactors in case of a large spill. If the equipment is not movable, it can be surrounded by a diked area. If the propagation vessel is movable, containment can be achieved by placing the vessel in a diked area with a ramp or in an area with sunken or sloped floors. The dike or depressed area must be of sufficient volume to accommodate the contents of the tank and sufficient disinfectant to decontaminate the material if there is no liquid biowaste treatment tank. Similarly, drains in the containment area should be capped or raised unless connected to a biowaste treatment system. Walls and ceilings should be smooth, nonporous, and capable of withstanding cleaning and disinfection. Walls may need additional shielding to prevent damage if large equipment is moved around the area. Ceilings can be epoxy-painted hard plaster or inverted T-bar construction. Rigid, walkable ceiling panels provide enhanced containment for BSL-3 facilities. Penetrations into the floor, walls, and ceilings should be minimized to facilitate cleaning and prevent leakage through the floor. All penetrations into a BSL-3 facility must be sealed to prevent escape of aerosols and to allow fumigation of the facility. Some utilities, e.g., conduits, should be sealed internally. Work surfaces must be impervious to water and resistant to chemicals, particularly those used for de-

36

contamination. Work surfaces should be finished with smooth edges to minimize injuries to the employees. Furniture used in the facility must be sturdy and capable of being cleaned and decontaminated and should be positioned to facilitate cleaning of the area. Doors should be flush design of smooth, nonporous material that can withstand repeated cleaning and treatment with disinfectants. Doors should be self-closing and swing into the more hazardous room. Windows should be sealed to the frame. Sloping sills help promote cleanability. Heating,

Ventilation,

and Air Conditioning

Directional airflow created by negative pressure differentials is used to create an air barrier between production and adjoining areas. Although that is sufficient for BSL-lLS, work with pathogens requires additional containment, which can be achieved in a number of ways. Two basic designs are most commonly used (16). In the envelope system, the internal production areas are maintained at positive pressure and are completely surrounded by a negative-pressure corridor to prevent the migration of the agent or product from the facility. This design may be preferable for operations that are more vulnerable to contamination, for preventing product cross-contamination, and/or to meet stringent FDA regulations. In most cases, those same goals can be accomplished by using negative pressure gradients in the production area and “buffering” air locks, i.e., rooms that contain both air supply and sufficient exhaust to flush contaminants from the room. This maximizes the containment for the production area yet minimizes the ingress of environmental contaminants. For facilities with multiple rooms, the room pressure should be most negative in the area of highest hazard, which is usually the fermentor or bioreactor. Depending on the techniques or processes involved, culture starter areas may require a similar level of containment. The number of air changes per hour (ACH) significantly affects the quality of the air. In facilities that must meet class 100,000 conditions, 20 ACH is not uncommon. For facilities above BSL-lLS, 10 to 15 ACH should be targeted. The ventilation in the rooms should be designed to maximize the air exchange in the room, generally with the airflow directed from the entryway toward the interior wall of the room. The heating, ventilation, and air conditioning (HVAC) system should be sized to dissipate the heat load generated by the equipment and provide a comfortable atmosphere for employees wearing personal protective equipment. BSL-1LS facilities do not require any specialized supply or exhaust features. Most of the following features are critical for BSL-3LS facilities and may be considered for BSL-2LS.

CUMITECH

36

Biosafety

A dedicated air supply is desirable to facilitate tem control and balancing.

sys-

If the supply is shared with other areas, HEPA filters at the room supply vent or airtight dampers should be used to prevent contamination of the supply system. Supply air should be HEPA filtered if recirculated the facility.

in

If HEPA filters are used, suitable prefilters should be used to extend the life of the HEPA. Ports should be provided in the filter housing allow for periodic testing of the filters.

to

HEPA filtration of exhaust air should be considered depending on the agent and environmental concerns. Provision for testing and decontaminating HEPA filters or use of bag-in/bag-out assemblies should be made. Exhaust air from BSCs and other containment devices should be HEPA filtered prior to discharge, preferably to outside the facility. Exhausting these devices through the room exhaust system can create problems in air balance (8). Placing them on a separate exhaust system allows them to be used to maintain negative airflow in the facility should the room exhaust system fail. It is preferable to HEPA filter the air before it leaves the room to prevent contamination of the exhaust ductwork. When that is not feasible, consideration should be given to the use of redundant fans and/or an emergency power supply for the exhaust system to maintain the ductwork at negative pressure or, alternatively, to seal or weld the ducts up to the point of the HEPA filter to prevent contaminants from escaping the ductwork. The exhaust and supply systems should be interlocked to prevent the facility from being pressurized. Utilities

and Maintenance

Issues

Sufficient lighting should be provided for all activities, with efforts made to minimize reflections and glare. Lights should be covered with a cleanable surface and sealed for BSL-3LS facilities. Liquid and gas utility services, if not dedicated to the facility, should be protected with backflow preventers or other devices to prevent contamination, e.g., a bump tank for steamable distilled water systems, a liquid disinfectant trap, and a HEPA or equivalent filter at point of use for vacuum systems.

for Large-Scale

Facility

Layout

Production

of Microorganisms

and Support

Systems

Designating the flow of materials from “clean” to “dirty” can be ambivalent when referring to largescale facilities, since there are different meanings from a biosafety standpoint versus GMP requirements. One needs to specify whether the context is biosafety, where “dirty” signifies the highest concentration of organisms, or the GMP sense, where “dirty” signifies the crudest form of the product, i.e., raw materials. As long as the material and personnel flow is unidirectional, most of the biosafety and GMP criteria can be met. Facilities that are used to manufacture multiple agents at the same time need additional features to prevent cross-contamination. One way to accomplish this is to use an entry-exit corridor system, with each of the various rooms or suites having entrance and exit airlocks. Considerations need to be made for change rooms, storage areas for raw materials, equipment supplies, janitor’s closet for housekeeping supplies, equipment cleanup and decontamination, toilets and showers, freezer and refrigerator space, gas supplies and servicing, etc. In general, large-scale facilities utilize special garbing, so the entry airlock is typically designed as a change room. Office areas should be located outside of the large-scale facility or, at a minimum, in the “cleanest” area of the facility. It is understood that paperwork areas and computer terminals are necessary in the large-scale area, but office areas should be separated from production areas by full-height walls and doors. Large-scale facilities should be separated from high-traffic areas to help restrict access and to promote cleanliness. A controlled access system should be considered for all facilities above the GLSP level to protect the product and at higher levels to protect personnel from inadvertent exposures. These devices can range from an electronic card entry system to a combination lock or a key system. Each facility needs to contain all of the required safety equipment. Handwashing facilities, eyewash stations, and emergency showers must be provided. Sinks should generally be automatic or capable of being operated by foot, knee, or elbow. Larger facilities may also have a handwash sink in the change room. The sinks can be discharged to the sanitary sewer provided that they are not used for discharging viable materials. Provisions should be made to equip the area with telephones, computer terminals, fax machines, etc., to facilitate information and data transfer outside the facility and to minimize the need for personnel and paperwork to leave the area. If required, data packets can be autoclaved for removal from areas where pathogens are used. All critical equipment and systems supporting the facility should be placed on a

8

preventive maintenance program. Control panels and items that require regular maintenance should be positioned to allow repair and adjustments to be performed outside of the facility, where possible. An insect and rodent control program should be developed for the facility. All critical systems and equipment should have alarms. These alarms should be apparent from the outside of the facility so that people will not enter the facility unprepared if the containment has been breached. After completion, the facility must be commissioned or validated. These terms signify the same thing; h.owever, commissioni ng is used by the m litary and validation is used by industry. The points that need to be covered are HVAC, waste treatment systems, filter integrity, alarm function, and failure mode testing. Waste

CUMITECH

Cipriano

Treatment

Unless specified by local ordinance, there is no need to decontaminate discharges of viable organisms from GLSP facilities, although state and local regulations may require that stock cultures be decontaminated. There may be local regulations governing specific waste parameters, e.g., biological oxygen demand level of solids, that may require further processing before disposal. In the United States, all discharges of viable organisms and waste from BSL-1LS to BSL-3LS facilities require decontamination before disposal. For large-scale work with pathogens, an autoclave or other method for decontamination should be available to process waste within the facility. The material generally poses a higher risk because of the concentration and volumes involved. A double-door autoclave with access from inside and outside the facility is preferred. A decontamination tank or system may be needed to inactivate any viable organisms from the process, if that cannot be carried out in the fermentor or bioprocessor. The decontamination method typically involves the application of ’ heat or chemica Is to the material. Generally, some type of stirring or spraying system needs to be used in the tank to provide better heat or chemical distribution and reduce treatment time. Waste tanks may be used for equipment cleaning solutions and rinses, material from spills, etc. All of these factors should be considered in sizing these tanks.

Large-scale processes have the potential for increased risk of exposure because of the volume and concentration of the agents used. However, appropriate practices, equipment, and facility design can reduce

36

the risks significantly. Finally, it should be noted that there is no single, correct way to achieve an acceptable level of containment. Depending on the agent and the process, a number of techniques can be used. It is hoped that the concepts presented here will help in those decisions.

LARGE-SCALE

BIOSAFETY

GUIDELINES

Biosafety guidelines for work with small volumes of infectious agents, i.e., those amounts typically used for diagnosis, characterization, or basic research, have been established by the CDC and the NIH (18), the World Health Organization (20), the United Kingdom (l), and Canada (9). Guidelines for working with recombinant DNA molecules in large volumes exist in the NIH guidelines for research involving recombinant DNA molecules (14). Several other publications have dealt with large-scale requirements and guidelines in the United States (5,6). The United Kingdom has put together additional guidance for large-scale work (2). The following document is an attempt to compile a comprehensive biosafety guideline for dealing with various types of large-scale work with microorganisms. The pertinent features of the existing documents have been put together with specific recommendations for large-scale work and are included in this version. It is understood that the organism, quantity, and process have a significant impact on the choice of an appropriate biosafety level for the work to be conducted. There is no specific volume that constitutes “large scale” for microbial agents. Certain CDC-NIH guidance documents have referred to large scale as volumes typically in excess of those used for identification, typing, assay performance, or testing. The risk analysis (7) must include an assessment of the infectivity of the agent, the routes of transmission, the severity of infection, the availability of prophylaxis, the level of containment afforded by the process and equipment used, etc., not just the volume of material being handled. Similarly, there is little scientific evidence to support the premise that only volumes greater than 10 liters merit large-scale requirements. Certainly, that is not true for BSL-2 and BSL-3 organisms. The CDC-NIH guidelines (18) recommend raising the biosafety level for culturing and purifying many BSL-2 organisms; however, that was only done in an effort to provide considerations for the biosafety officer and scientists in the establishment of the appropriate level of protection. The NIH recombinant DNA guidelines provide guidance for the large-scale use of recombinant organisms to protect the environment but do not adequately address the level of containment necessary to protect the personnel working with infectious agents. These guidelines serve as an effort to collect best practices for maximizing safety for large-scale work and can be used by an institutional biosafety committee and/or a biological safety officer (BSO) to develop biosafety procedures for the work to be done. The guidelines cover four different levels for large-scale work: GLSP, BSL-lLS, BSL-2LS, and BSL-3LS. The con-

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Biosafety

36

tainment conditions for biosafety level 4 -large scale are not defined here but should be determined on a case-by-case basis. Only the biological hazard of the organism is addressed here. Other hazards, such as the toxicity or biological activity of the products produced, should be considered separately. These guidelines do not specifically address animal or plant pathogens; however, the containment principles and practices may be useful for some of those agents. All institutions that engage in large-scale research or production with microorganisms should appoint a BSO to oversee the procedures, facilities, and equipment used. A BSO is critical at BSL-2LS and above, where knowledge and experience with handling pathogenic organisms, biosafety practices, and containment design criteria are required.

for

LARGE-SCALE

PRACTICES

The GLSP level is recommended for certain risk group 1 organisms that are not known to cause disease in healthy adults, are nontoxigenic, are well characterized, and/or have an extended history of safe large-scale work. These organisms should not be able to transfer antibiotic resistance to other organisms. Examples of such organisms are Saccharomyces cerevisiae and Escherichia coli K-12. These organisms should have limited survival and/or no adverse consequences if released into the environment. Standard

Microbiological

Practices

1. Individuals wash their hands after handling viable material. Antiseptic hand cleanser may be used as an interim measure if a sink is not readily accessible. 2. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not allowed in the work area. 3. Mouth pipetting is prohibited. 4. Work surfaces are capable of being cleaned and disinfected. 5. An insect and rodent control program is in effect. Special

Practices

1. Institutions that engage in large-scale work should have a health and safety program for their employees. 2. Written instructions and training are provided for personnel who work in GLSP conditions. 3. Processing, sampling, transfer, handling, etc., of viable organisms are done in a manner that does not adversely affect the health and safety of the employees. 4. Discharges of viable organisms are disposed of in accordance with applicable local, state, and federal requirements. 5, The facility should have an emergency response plan that includes the handling of spills. Safety Equipment 1. Protective clothing, e.g., uniforms and laboratory coats, is provided to minimize the soiling of personal clothing. 2. Safety glasses are worn in the facility.

Production

of Microorganisms

9

Facilities Each facility contains a sink for handwashing or has an antiseptic waterless hand cleanser available. If present, the sink should be located near the exit doorway. An eyewash station and emergency shower are provided in the work area or easily accessible to it. BIOSAFETY

LEVEL

1 -LARGE

SCALE

BSL-1LS is recommended for the large-scale growth of risk group 1 organisms that are not known to consistently cause disease in healthy adult humans and pose minimal hazard to personnel and the environment but otherwise do not qualify for GLSP level. Standard

GOOD

Large-Scale

Microbiological

Practices

1. Access to the work area may be restricted at the discretion of the project manager when work is ongoing. A warning sign should be placed on the door that lists the agent(s) being used, the names and telephone numbers of persons knowledgeable about and responsible for the facility, and entry requirements, if any. 2. Persons wash or clean their hands after they handle viable organisms, after removing gloves, and on leaving the work area. Antiseptic hand cleanser may be used as an interim measure if a sink is not readily accessible. 3. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work area. 4. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only. 5. Mouth pipetting is prohibited. Only mechanical pipetting devices are used. 6. Work surfaces are decontaminated on a routine basis and after any spill of viable organisms. 7. Procedures are performed carefully in a manner that minimizes aerosol generation. 8. Policies for the safe handling of sharps are instituted. The use of sharps should be minimized. 9. All discharges of the viable organisms are disposed of in accordance with applicable local, state, and federal regulations. 10. An insect and rodent control program is in effect. Special

Practices

Institutions that engage in large-scale work have a health and safety program for their employees. Written procedures and training in basic microbiological practices are provided and documented. Medical evaluation, surveillance, and treatment are provided when indicated, e.g., to determine functional status or competency of employees’ immune systems when working with opportunistic pathogens. Spills and accidents that result in overt exposure to viable organisms are reported to the facility supervisor or manager. Medical evaluation, surveillance, and treatment are provided as appropriate, and written records are maintained. 5. Emergency plans include methods and procedures for handling spills and employee exposures.

10

Cipriano

6. Cultures of viable organisms are handled in a closed system or other primary containment equipment that is designed to reduce the potential for the escape of viable organisms. Sample collection and material addition to a closed system and transfer of culture materials from one closed system to another are conducted in a manner that minimizes employee exposure, the release of viable material, and the generation of aerosols. Cultures of viable organisms should be inactivated by a validated process before removal from the closed system or primary containment system, except as allowed in item 7 in this list or when the viable organism or viral vector is the desired product. In the latter case, the viable organisms should be removed from the closed system or other primary containment system in a manner that minimizes employee exposure, the release of viable material, and the generation of aerosols.

9 . Exhaust gasesremoved from a closed system or other primary containment system minimize the releaseof viable organisms to the environment bY the use of appropriate filters or procedures. 10. A closed system or other primary containment equipment that has contained viable organisms is not to be opened for maintenance or other purposes until it has been decontaminated. Safety

Equipment

1. Protective clothing, e.g., uniforms, laboratory coats, etc., is provided to prevent the contamination or soiling of personal clothing. 2. Safety glasses must be worn. 3. Gloves are worn if the skin on the hands is broken, irritated, or otherwise not intact. Facilities 1. Each facility contains a sink for handwashing or has an antiseptic waterless hand cleanser available. If present, the sink should be located near the exit doorway. An eyewash station and emergency shower should be provided in the work area or easily accessible to it. The work area has a door that can be closed when large-scale work is ongoing. The work area is designed to be easily cleaned. Floors are able to be cleaned and disinfected in case of spills of viable organisms. Rugs are not allowed. Work surfaces are impervious to water and resistant to acids, alkali, organic solvents, disinfectants, and moderate heat. Furniture in the work area is sturdy and placed so that all areas are accessible for cleaning. If the work area has windows that open, they are fitted with fly screens. Facilities are designed to contain large spills of viable materials within the facility until appropriately decontaminated. This can be accomplished by utilizing a dike or sloping or lowering the floor where the process vessels are located. The design should minimize the release of viable organisms directly to the sewer.

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BIOSAFETY LEVEL 2-LARGE

36

SCALE

BSL-2LS is recommended for the propagation and cultivation of risk group 2 infectious organisms and other organisms, such as attenuated strains from a higher risk group, that would be handled at BSL-2 in laboratory scale. The following guidelines have been developed for facilities that handle large volumes of these materials. Standard

Microbiological

Practices

1. Access to the work area is restricted to personnel who meet the entry requirements. 2. Persons wash their hands after they handle viable organisms, after removing gloves, and before leaving the work area. 3. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the facility. 4. Food is stored outside the facility in cabinets or refrigerators designated and used for this purpose only. 5. Mouth pipetting is prohibited. Only mechanical pipetting devices are used. 6. Work surfaces are decontaminated on a routine basis and after any spill of viable organisms. 7. Procedures are performed carefully in a manner that prevents aerosol generation. 8. All contaminated wastes are decontaminated by an approved method before disposal in accordance with local, state, and federal regulations. Wastes that need to be transported to a different area or facility are closed and placed in a durable, leakproof container for transfer. Material to be transferred off site for decontamination is packaged and labeled in accordance with the applicable regulations. 9. All discharges of viable organisms are inactivated by a validated process, i.e., one that has been demonstrated to be effective with the organism in question or with an indicator organism that is known to be more resistant to the physical or chemical methods used, e.g., Bacillus stearothermophilus for steam heat. 10. An insect and rodent control program is in effect. Special

Practices

1. Institutions that engage in large-scale work have a health and safety program for their employees. 2. Doors to the work area are kept closed when work is ongorng. 3. Access to the work area is restricted to personnel whose presence is required and who meet entry requirements, i.e., immunization, if any. Individuals who cannot take or do not respond to the vaccine, who cannot take the recommended prophylaxis in the event of an exposure incident, who are at increased risk of infection, or for whom infection may prove unusually hazardous are not allowed in the work area until their situation has been reviewed by appropriate medical personnel. The individuals are informed of the potential risks and sign an acknowledgment, consent form, or similar document, which indicates that they understand and accept the potential risk.

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36

Biosafety

4. Written procedures and policies for handling infectious organisms are provided. 5. Personnel are able to demonstrate proficiency in standard microbiological practices and procedures and the handling of human pathogens at biosafety level 2. This can consist of previous experience and/or training. Training in the hazards associated with the organisms involved and the practices and operations specific to the large-scale work area is provided and documented. 6. Appropriate immunizations, medical evaluation surveillance, and treatment are provided when indicated, i.e., immunization, survey of immune status, etc. the universal 7. A hazard warning sign, incorporating biohazard symbol, identifying the infectious agents, listing the names and telephone numbers of the persons knowledgeable about and responsible for the work area, along with any special requirements for entering the work area, is posted at the entry to the work area. 8. When appropriate, baseline serum samples or other surveillance samples are collected and stored for all personnel working in or supporting the work area. 9. A biosafety manual is available that details required safety practices and procedures, spill cleanup, handling of accidents, and other appropriate safety information. 10. The use of sharps is avoided. If required, additional safety devices or personal protective equipment is used to prevent accidental exposure. Plastic laboratoryware is substituted for glassware whenever possible. If glassware is used, it is coated or shielded to minimize the potential for breakage. 11. Viable organisms are placed in a container that prevents leakage during collection, handling, processing, and transport. 12. Viable organisms are handled in a closed system or other primary containment equipment that prevents their release into the environment. 13. Sample collection, material addition to a closed system, and transfers of culture materials from one closed system to another are conducted in a manner that prevents employee exposure and the release of viable material from the closed system. 14. Culture fluids are not removed from a closed system (except as allowed in item 13 in this list) unless the viable organisms have been inactivated by a validated procedure. In cases where the viable organism or viral vector is the desired product, the materials should be removed and processed in equipment that prevents employee exposure and release of viable material. 1.5. Exhaust gases removed from a closed system or other primary containment systems are filtered or otherwise treated to prevent the release of viable organisms to the environment. 16. A closed system that has contained viable organisms will not be opened for maintenance or other purposes unless it has been decontaminated. 17. Rotating seals and other mechanical devices directly associated with a closed system used for the propagation of viable organisms are designed to prevent leakage or are fully enclosed in ventilated housings that are

for Large-Scale

18.

19.

20.

21.

22.

23.

Production

of Microorganisms

11

exhausted through filters or otherwise treated to prevent the release of viable organisms to the environment. Closed systems, used for the propagation of viable organisms, and other primary containment equipment are tested for the integrity of the containment features before use and following any changes or modifications to the system that could affect the containment characteristics of the equipment. These systems are equipped with a sensing device that monitors the integrity of the containment while in use. Containment equipment for which the integrity cannot be verified or monitored during use is enclosed in ventilated housings that are exhausted through filters or otherwise treated to prevent the release of viable organisms. Closed systems that are used for propagation of viable organisms or other primary containment equipment is permanently identified. This identifier is used on all records regarding validation, testing, operation, and maintenance. Contaminated equipment and work surfaces are decontaminated with a suitable disinfectant on a routine basis, after spill cleanup, etc. Contaminated equipment is decontaminated before servicing or transport. Absorbent toweling and coverings used on work surfaces to collect droplets and minimize aerosols are discarded and decontaminated after use. Individuals seek medical attention immediately after an exposure incident. Spills and accidents that result in overt exposure to infectious materials are immediately reported to the facility supervisor or manager and the BSO. Appropriate medical treatment, medical evaluation, and surveillance are provided, and written records are maintained. Emergency procedures include provisions for decontamination and cleanup of all spills or releases of viable material, including proper use of personal protective equipment. Animals not involved in the work being performed are not permitted in the work area.

Safety

Equipment

Protective clothing, e.g., laboratory coats, protective coveralls, etc., is worn to prevent contamination of personal clothing. If the organism can be transmitted through the skin, the protective clothing is waterproof with solid-front, wraparound, or back- or side-tie coats. Protective clothing is removed when leaving the work area. Protective eyewear is worn at all times in the work area. Face protection, i.e., face shield or goggles and face mask or respirator, is worn for any procedures that may involve splashing or spraying. Respirators are worn if the agents involved are transmissible via the respiratory route. Impervious gloves are worn at all times in the work area when work is ongoing. Double gloving is considered if personnel are working for extended periods or with processes that may require direct contact with the infectious material. Gloves are discarded upon leaving the work area.

12

4. The selection of a respirator or face mask is based on the transmissibility of the agent. If the agent is transmitted through the respiratory route, a respirator with a filtration efficiency capable of protecting the individual from the organism is used, e.g., HEPA for viruses or N9.5 for Mycobacterium tuberculosis. If the agent is transmitted through mucous membrane contact, a face mask that prevents droplet penetration, e.g., plastic molded, is preferred. Personnel are trained in the use of respirators and face masks for procedures that may involve aerosol generation and for emergency situations that involve the release of viable organisms in the work area. 5. BSCs or other ventilated containment devices can be used to contain aerosol-generating processes or to prevent contamination of viable organisms when removed from a closed system. 6. Only centrifuge units with sealed rotor heads or safety cups that can be opened in a BSC are used, or the centrifuge is placed in a containment device. Facilities I

l

2. 3.

4.

5.

6. 7.

8.

9.

10.

11.

CUMITECH

Cipriano

Each facility contains a sink for handwashing, an eyewash station, and an emergency shower. The sink is foot, elbow, or knee operated, automatic, or otherwise not hand operated and is located near the door of each room in the work area. The work area has a door that is closed when largescale work is ongoing. The work area is designed to be easily cleaned and disinfected. Furniture and stationary equipment are sealed to the floor or raised to allow for cleaning and disinfection of the facility. Floors, walls, and ceilings are made of materials that allow for cleaning and disinfection of all surfaces. Light fixtures are covered with a cleanable surface. Work surfaces are impervious to water and resistant to acids, alkali, organic solvents, disinfectants, and moderate heat. Windows to the facility are kept closed and sealed while work is ongoing. General laboratory-type work areas are designed to have a minimum of 6 ACH. For large-scale facilities, the number of ACH will depend on the size of the area, the chemicals and agents handled, the procedures and equipment utilized, and the microbial or particulate requirements for the area. The ventilation in the work area is designed to maximize the air exchange in the area, i.e., the supply and exhaust are placed at opposite ends of the room, ceiling supply with low-level exhaust, etc. The work areas in the facility where the infectious organisms are handled are at negative pressure to the surrounding areas. Provisions are made to contain large spills of viable organisms within the facility until appropriately decontaminated. This can be accomplished by placing the equipment in a diked area or sloping or lowering the floors in those areas to allow sufficient capacity to contain the viable material and disinfectant. Drainage from the facility is designed to prevent the

36

release of large volumes of viable material directly to the sewer, e.g., floor drains are capped, raised, or fitted with liquid-tight gaskets to prevent release of untreated organisms to the sewer. BIOSAFETY

LEVEL

3 -LARGE

SCALE

BSL-3LS is recommended for the propagation and cultivation of infectious organisms classified as risk group 3, which are handled at BSL-3 at laboratory scale. The following guidelines have been developed for facilities that handle large volumes of these materials. Standard

Microbiological

Practices

1. Access to the facility is restricted to personnel who meet the entry requirements. Individuals who have not been trained in the operating and emergency procedures of the facility are accompanied by trained personnel at all times while in the facility. 2. Persons wash their hands after they handle viable materials, after removing gloves, and before leaving the work area. 3. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work area. 4, Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only. 5. Mouth pipetting is prohibited. Only mechanical pipetting devices are used. 6. Work surfaces are decontaminated on a routine basis and after any spill of viable material. 7. Procedures are performed carefully in a manner that prevents aerosol generation. 8. All contaminated wastes are decontaminated by an approved method before disposal in accordance with local, state, and federal regulations. Wastes that need to be transported to a different area or facility are closed and placed in a durable, leakproof container for transfer. Material to be transferred off site for decontamination is packaged and labeled in accordance with the applicable regulations. 9. All discharges of the viable materials are inactivated by a validated process, i.e., one that has been demonstrated to be effective with the organism in question or with an indicator organism that is known to be more resistant to the physical or chemical methods used, e.g., B. stearothermophilus for steam heat. 10. An insect and rodent control program is in effect. Special

Practices

1. Institutions that engage in large-scale work must have a health and safety program for their employees. 2. Doors to the facility are kept closed except for entry and egress. 3. Access to the facility is restricted to personnel whose presence is required and who meet entry requirements, i.e., immunization, if any, and comply with all entry and exit procedures. Individuals who cannot take or do not respond to the vaccine, who cannot take the recommended prophylaxis in the event of an exposure inci-

CUMITECH

4. 5.

6.

7.

8.

9.

10.

11.

12.

13.

14

15

36

Biosafety

dent, who are at increased risk of infection, or for whom infection may prove unusually hazardous are not allowed in the work area until their situation has been reviewed by appropriate medical personnel. The individuals are informed of the potential risks and sign an acknowledgment, consent form, or similar document, which indicates that they understand and accept the potential risk. Written procedures and policies for handling infectious materials are provided. All personnel working at a BSL-3LS facility must demonstrate proficiency in standard microbiological practices and techniques and in handling human pathogens at BSL-3. This can consist of previous experience and/or a training program. Training in the hazards associated with the materials involved and the practices and operations specific to the facility is provided and documented. Appropriate immunizations, medical evaluation surveillance, and treatment are provided where indicated, e.g., immunization and survey of immune status. A hazard warning sign, incorporating the universal biohazard symbol, identifying the infectious agents, listing the names and telephone numbers of the persons knowledgeable about and responsible for the facility, along with any special requirements for entering the work area, is posted at the entry to the facility. Baseline serum samples or other appropriate specimens are collected and stored for all personnel working in or supporting the facility. Additional specimens may be collected periodically depending on the agents handled. A biosafety manual is available that details required safety practices and procedures, spill cleanup, handling of accidents, and other appropriate safety information. The use of sharps is avoided. If required, additional safety devices or personal protective equipment is used to prevent accidental exposure. Plastic laboratoryware is substituted for glassware whenever possible. If glassware is used, it is coated or shielded to minimize the potential for breakage. Viable organisms are placed in a container that prevents leakage during collection, handling, processing, and transport. Viable organisms are handled in a closed system or other primary containment equipment that prevents their release into the environment. Sample collection, material addition to a closed system, and transfer of culture materials from one closed system to another are conducted in a manner that prevents employee exposure and the release of viable material from the closed system. Culture fluids are not removed from a closed system (except as allowed in item 13 in this list) unless the viable organisms have been inactivated by a validated procedure. In cases where the viable organism or viral vector is the desired product, the materials should be removed and processed in equipment that prevents employee exposure and release of viable material. Exhaust gases removed from a closed system or other primary containment systems are filtered or otherwise

for

16.

17.

18.

19.

20.

21.

22.

23.

Large-Scale

Production

of Microorganisms

13

treated to prevent the release of viable organisms to the environment. A closed system that has contained viable organisms will not be opened for maintenance or other purposes unless it has been decontaminated. Rotating seals and other mechanical devices directly associated with a closed system used for the propagation of viable organisms are designed to prevent leakage or are fully enclosed in ventilated housings that are exhausted through filters or otherwise treated to prevent the release of viable organisms. Closed systems used for the propagation of viable organisms and other primary containment equipment are tested for the integrity of the containment features before use and following any changes or modifications to the system that could affect the containment characteristics of the equipment. These systems are equipped with a sensing device that monitors the integrity of the containment while in use. Containment equipment for which the integrity cannot be verified or monitored during use is enclosed in ventilated housings that are exhausted through filters or otherwise treated to prevent the release of viable organisms. Closed systems that are used for propagation of viable organisms or other primary containment equipment is permanently identified. This identifier is used on all records regarding validation, testing, operation, and maintenance. Contaminated equipment and work surfaces are decontaminated with a suitable disinfectant on a routine basis, after spill cleanup, etc. Contaminated equipment is decontaminated before servicing or transport. Absorbent toweling and coverings used on work surfaces to collect droplets and minimize aerosols should be discarded and decontaminated after use. Individuals seek medical attention immediately after an exposure incident. Spills and accidents that result in overt exposure to infectious materials are immediately reported to the facility supervisor or manager and the BSO. Appropriate medical treatment, medical evaluation, and surveillance are provided, and written records are maintained. Emergency procedures include provisions for decontamination and cleanup of all spills or releases of viable material, including proper use of personal protective equipment. Animals not involved in the work being performed are not permitted in the work area.

Safety

Equipment

1. Persons entering the facility will change or completely cover their clothing with garments such as solid-front or wraparound gowns or coveralls. If the organism can be transmitted through the skin, the protective clothing must be waterproof. Head and shoe covers or dedicated shoes are provided. Protective clothing is to be removed when leaving the facility and decontaminated before disposal or laundering. 2 Protective eyewear is worn at all times in the work area. Face protection, i.e., face shield or goggles and face mask

14

CUMITECH

Clprlano

3.

4.

5.

6.

or respirator, is worn for any procedures that may involve splashing or spraying. Respirators are worn if the agents involved are aerosol transmissible. Impervious gloves are worn at all times in the work area when work is ongoing. Double gloving is considered if personnel are working for extended periods or with processes that may require direct contact with the infectious material. Gloves are discarded upon leaving the work area. The selection of a respirator or face mask is based on the transmissibility of the agent. If the agent is transmitted through the respiratory route, a respirator with a filtration efficiency capable of protecting the individual from the organism is used, e.g., HEPA for viruses or N9Ss for M. tuberculosis. If the agent is transmitted through mucous membrane contact, a face mask that prevents droplet penetration, e.g., plastic molded, is preferred. Personnel are trained in the use of respirators or face masks for procedures that may involve aerosol generation and for emergency situations that involve the release of viable organisms in the work area. Class II or III BSCs or other ventilated containment devices are used to contain processes of viable materials if removed from a closed system. Only centrifuge units with sealed rotor heads or safety cups that can be opened in a BSC are used, or the centrifuge is placed in a containment device. Continuous-flow centrifuges or other aerosol-generating equipment is contained in devices that are exhausted through filters or otherwise treated to prevent the release of viable organisms. Vacuum lines are protected with liquid disinfection traps and HEPA filters or equivalent, which are routinely maintained and replaced as needed.

Facilities

1. The facility is separated from areas that are open to

2.

3. 4.

5.

6.

7.

8.

unrestricted traffic flow within the building. The facility has a double-doored entry area, such as an airlock or pass-through. Each major work area contains a sink for handwashing that is not hand operated, e.g., automatic or foot, knee, or elbow operated. An eyewash station and emergency shower are available in the facility. The facility is designed to be easily cleaned and disinfected. Furniture and stationary equipment are sealed to the floor, raised, or placed on wheels to allow for cleaning and disinfecting of the facility. Work surfaces are impervious to water and resistant to acids, alkali, organic solvents, disinfectants, and moderate heat. Floors, walls, and ceilings are made of materials that allow for cleaning and disinfection of all surfaces. Light fixtures are sealed or recessed and covered with a cleanable surface. Penetrations into the containment facility are kept to a minimum and sealed to maintain the integrity of the facility. Windows to the facility are kept closed and sealed.

36

9. Liquid and gas services to the facility are protected from backflow unless they are dedicated to the facility. Fire protection sprinkler systems do not require backflow prevention devices. 10. The ventilation system for the facility is designed to control air movement. 11. The position of the supply and exhaust vents is designed to maximize the air exchange in the area, i.e., the supply and exhaust are placed at opposite ends of the room, ceiling supply with low-level exhaust, etc. 12. General laboratory-type work areas are designed to have a minimum of 6 ACH. For large-scale facilities, the number of ACH will depend on the size of the area, the chemicals and agents handled, the procedures and equipment utilized, and the microbial and particulate requirements for the area. 13. The facility is at negative air pressure to the surrounding areas or corridors. The system creates directional airflow that draws air from the “clean” areas of the facility into the “contaminated” areas. If there are multiple contaminated areas, the area of highest potential contamination is the most negative. 14. The exhaust air from the facility is not recirculated to any other area in the facility and is discharged to the outside through HEPA filters or other treatments that prevent the release of viable microorganisms. 15. The facility has a dedicated air supply system for the facility. If the supply system is not dedicated to the facility, it contains HEPA filters or appropriate dampers which can protect the system from potential backflow in the event of a system failure. 16. The supply and exhaust systems for the facility are interlocked to prevent the room from being pressurized in the event of power or equipment failure. The system is alarmed to indicate system failures or changes in desired airflow. 17. A visual monitoring device that indicates and confirms directional airflow is provided at the entry to the facility. 18. Visible or audible alarms are available to notify personnel of any HVAC system failure. 19. A method for decontaminating all wastes is available in the facility, i.e., autoclave, chemical disinfection, incineration, or other approved method. 20. Provisions are made to contain large spills of viable organisms within the facility until appropriately decontaminated. This can be accomplished by placing the equipment in a diked area or sloping or lowering the floors in those areas to allow for sufficient capacity to contain the viable organisms and disinfectant. 21. Drainage from the facility is designed to prevent the release of viable organisms directly to the sewer, e.g., floor drains are capped, raised, or fitted with liquidtight gaskets to prevent release of untreated organisms to the sewer. ACKNOWLEDGMENTS I thank Marian Downing, Diane Fleming, and Carol Onstad for their assistance in this work. The guidelines in the appendix were prepared by the ASM Subcommittee on

CUMITECH

For Large-Scale

36

Laboratory Safety (M. Cipriano and D. Fleming [chairs], R. Hawley, J. Richmond, J. Coggins, B. Fontes, C. Thompson, and S. Wagener). I extend special thanks to I?. Meecham and R. Rebar for their comments and suggestions. REFERENCES 1. Advisory Committee on Dangerous Pathogens. 1995. Categorization of Biological Agents According to Hazards and Categories of Containment, 4th ed. Her Majesty’s Stationery Office, London, United Kingdom. 2. Advisory Committee on Dangerous Pathogens. 1998. The Large Scale Contained Use of Biological Agents. Her Majesty’s Stationery Office, London, United Kingdom. 3. Bailey, J. E., and D. F. Ollis. 1977. Biochemical neering Fundamentals, p. 574-634. McGraw New York, N.Y.

EngiHill,

4. Centers for Disease Control and Prevention and National Institutes of Health. 1995. Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets. U.S. Government Printing Office, Washington, D.C. 5. Fleming, D. 0. 1995. Laboratory biosafety practices, p. 203-218. 1n D. 0. Fleming, J. H. Richardson, J. J. Tulis, and D. Vesley (ed.), Laboratory Safety: Principles and Practices, 2nd ed. American Society for Microbiology, Washington, D.C. 6. Fleming, D. 0. 1996. Large scale biosafety guidelines, p. 445-449. In B. A. Plog, J. Niland, and P. J. Quinlan (ed.), Fundamentals of Industrial Hygiene, 4th ed. National Safety Council, Itasca, N.Y. 7. Fleming, D. 0. 2000. Risk assessment of biological hazards, p. 57-64. In D. 0. Fleming and D. L. Hunt (ed.), Biological Safety: Principles and Practices, 3rd ed. ASM Press, Washington, D.C. 8. Ghidoni, D. A. 1999. HVAC issues in secondary containment, p. 63-72. In J. Y. Richmond (ed.), Anthology of Biosafety. American Biological Safety Association, Mundelein, Ill. 9. Health and Welfare Canada. 1996. Laboratory Biosafety Guidelines, 2nd ed. Laboratory Centre for Disease Control, Ottawa, Canada. 10. Leaver, G. 1994. Interpretation of regulatory requirements to large scale biosafetv-the role of the indus-

Production

of Microorganisms

15

trial biosafety project, p. 213-239. In I?. Hambleton, J. Melling, and T. T. Salisbury (ed.), Biosafety in Industrial Biotechnology. Blackie Academic & Professional, Glasgow, Scotland. 11. Liberman, D. F., R. Fink, and F. Schaefer. 1986. Biosafety and biotechnology, p. 402-409. In A. L. Demain and N. A. Solomon (ed.), Manual of Industrial Microbiology and Biotechnology. American Society for Microbiology, Washington, D.C. 12 McGarrity, G. J., and C. L. Hoerner. 1995. Biological safety in the biotechnology industry, p. 119-131. In D. 0. Fleming, J. H. Richardson, J. J. Tulis, and D. Vesley (ed.), Laboratory Safety: Principles and Practices, 2nd ed. American Society for Microbiology, Washington, D.C. 13 Meechan, I?. J., J. Gyuris, R. Greasham, and W. Herber. 2000. Biosafety in the pharmaceutical industry, p. 533-539. In D. 0. Fleming and D. L. Hunt (ed.), Biological Safety: Principles and Practices, 3rd ed. ASM Press, Washington, D.C. 14. National Institutes of Health. 1999. Guidelines for research involving recombinant DNA molecules. Fed. Regist. 64:25361 and revisions. Research Council. 1989. Biosafety in the 15. National Laboratory. National Academy Press, Washington, D.C. guidelines 16. Odum, J. 1995. Fundamental multiuse facilities. Pharm. Eng. 158 -20.

for biotech

for Economic Co-operation and Devel17. Organisation opment. 1992. Safety Considerations for Biotechnology. OECD Publications, Paris, France. 18. Richmond, J. Y., and R. W. McKinney (ed.). 1999. Biosafety in Microbiological and Biomedical Laboratories, 4th ed. U.S. Government Printing Office, Washington, D.C. 19. Stuart, D. G. 2000. Primary barriers: biological safety cabinets, fume hoods, and glove boxes, p. 313-330. In D. 0. Fleming and D. L. Hunt (ed.), Biological Safety: Principles and Practices, 3rd ed. ASM Press, Washington, D.C. 1993. Laboratory Bio20. World Health Organization. safety Manual, 2nd ed. World Health Organization, Geneva, Switzerland.

Cumitech 36: Biosafety Considerations for Large-Scale Production of Microorganisms

Electroporation Protocols for Microorganisms

Electroporation Protocols for Microorganisms

Electroporation Protocols for Microorganisms

Electroporation Protocols for Microorganisms

Considerations

Considerations

Considerations for Viral Disease Eradication

Molecular Epidemiology of Microorganisms

Considerations for Viral Disease Eradication

Uncultivated Microorganisms

Institutional Interplay: Biosafety and Trade

Uncultivated Microorganisms

36 Arguments for the Existence of God

36 propositions for a home

Challenges for Industrial Production

Bioethics and Biosafety in Biotechnology

Research for media production

Cumitech 35: Postmortem Microbiology

Cumitech 38: Human Cytomegalovirus

Arguing for Socialism: Theoretical Considerations: Revised Edition

Cumitech 12A: Laboratory Diagnosis of Bacterial Diarrhea

Cumitech 34: Laboratory Diagnosis of Mycoplasmal Infections

Tracking Genetically-Engineered Microorganisms

Biocommunication in Soil Microorganisms

Food spoilage microorganisms

Microorganisms and Bioterrorism

Uncultivated Microorganisms (Microbiology Monographs)

Modelling Microorganisms in Food

Considerations on Western Marxism

Considerations On Representative Government

Cumitech 36: Biosafety Considerations for Large-Scale Production of Microorganisms

Electroporation Protocols for Microorganisms

Electroporation Protocols for Microorganisms

Electroporation Protocols for Microorganisms

Electroporation Protocols for Microorganisms

Considerations

Considerations

Considerations for Viral Disease Eradication

Molecular Epidemiology of Microorganisms

Considerations for Viral Disease Eradication

Uncultivated Microorganisms

Institutional Interplay: Biosafety and Trade

Uncultivated Microorganisms

36 Arguments for the Existence of God

36 propositions for a home

Challenges for Industrial Production

Bioethics and Biosafety in Biotechnology

Research for media production

Cumitech 35: Postmortem Microbiology

Cumitech 38: Human Cytomegalovirus

Arguing for Socialism: Theoretical Considerations: Revised Edition

Cumitech 12A: Laboratory Diagnosis of Bacterial Diarrhea

Cumitech 34: Laboratory Diagnosis of Mycoplasmal Infections

Tracking Genetically-Engineered Microorganisms

Biocommunication in Soil Microorganisms

Food spoilage microorganisms

Microorganisms and Bioterrorism

Uncultivated Microorganisms (Microbiology Monographs)

Modelling Microorganisms in Food

Considerations on Western Marxism

Considerations On Representative Government

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