ISSUE AREAS:

Quick Reference Guide to Biological Technology Equipment

By David Isenberg, Senior Analyst

Table of Contents

• Introduction

• The Reference Guide

  • Guide limitations

  • Design of a Production Facility

  • Containment

  • Purification

  • Sterilization

  • Ventilation

  • Storage

  • Replication

  • Equipment

    • Bioreactor (fermenter)

    • Clean Rooms

  • General Guidelines for Identifying BW Equipment

    • Centrifugal Separators

    • Cross-Flow Filtration equipment

    • Equipment that is often contained in BL-3 or BL-4 containment housing

    • Aerosol Inhalation Chambers

Introduction
Currently, pursuant to United Nations Security Council Resolution 1441 of 8 November 2002 inspectors of the United Nations Monitoring, Verification and Inspection Commission (UNMOVIC) are in Iraq seeking evidence as to whether Iraq has developed chemical, biological, and nuclear weapons, ballistic missiles, and other delivery systems such as unmanned aerial vehicles and dispersal systems designed for use on aircraft, in violation of its obligations under UN resolutions to rid itself of such programs. This briefing offers an insight into the equipment that UNMOVIC inspectors will be looking for as they look for evidence of biological weapons.

Nuclear weapons programs, given their large and complex infrastructure are relatively easy to detect. In contrast, much of the equipment used in civilian, non-military chemical and biotech industries can also be used to produce chemical and biological weapons.

This is especially so with biological weapons (BW). It is generally thought that any well-equipped microbiological laboratory with normally skilled technical personnel can produce large quantities of pathogens, if the desired microorganism is available.

Still, there are some things that must be available to those seeking to develop BW. Bacteria and viruses must be cultured and then produced in quantity. They must be stabilized and stored. Facilities where agents are produced usually have certain types of ventilation systems in order to ensure the health of the personnel working there.

These things require certain types of material and equipment, which to an inspector who knows what to look for, can help determine whether a facility is or is not being used to produce BW. Nevertheless, the dual-use nature of production facilities makes the inspection exercise a significant challenge.

The Reference Guide

Guide Limitations
Equipment with the same name does not always look or appear the same, and equipment with the same function does not always have the same name.

This guide is focused only on research and production equipment. There are other indicators of potential BW activity, which are not covered here, such as inspection of end product, documentation of the paper trail for legitimate products, and munitions activity.

Design of a Production Facility
The design of a production facility provides important information regarding whether the facility is intended to produce pharmaceutical grade products or biological weapon grade materials. Relevant design elements include containment, purification equipment, sterilization equipment, ventilation and filtration systems and storage.

Containment
Containment measures are one of the prime methods for distinguishing Biological and Toxin Weapons (BTW) and countermeasure production. When producing BW there is an over-riding need to protect the environment from the agent because of its infectious nature. In contrast, in the production of vaccines, biological response modifiers, antibiotics, and antiviral agents, for defensive military activities, the requirement is to protect the processed biomaterial from being contaminating by materials in the environment.

Purification
Achieving high levels of purity enhances the effectiveness of biopharmaceutical products. This concern is reflected in the nature of the sealing joints, positive or negative pressure chambers, and containment of venting systems. Utilities involving clean steam, sterile air, and inert gas supply are most critical for containment in the processing of biologically based materials for human use, which must meet good manufacturing practices (GMP). Clean steam, generated from a purified water supply, must be supplied to all processing equipment having direct contact with the product to ensure sterility and prevent the influx of environmental contaminants.

By contrast, an unpurified biological agent that will be used in offensive BW is generally more stable than the purified agent that is needed to produce vaccines and biological response modifiers (BRMs); it does not require a high level of purity.

To attain a higher level of containment, many bioprocessing industries have employed greater degrees of automation. Potential contamination of purified product, human exposure to toxic products or constituents, and the risk of human error are minimized. Processing facilities make use of state-of-the-art computerized distributed control systems which allow automatic control, control from remote locations, and automatic data logging and trending.

Sterilization
Steam sterilization is a time proven and economical process of killing microorganisms through the application of moist heat (saturated steam) under pressure. Heat damages the cell's essential structures including the cytoplasmic membrane, rendering the cell no longer viable. The rate by which bacterial cells are thermally inactivated depends on the temperature and the time of heat exposure to which they are exposed.

Steam sterilization is accomplished before product processing by direct supply to the equipment. Steam is supplied to the equipment seals (e.g., sample ports, agitator shafts, raw material addition ports) during processing as a primary barrier. Equally important is the removal of collapsed steam or condensate formed on the equipment. This prevents the formation of pockets of standing water, which promote bacterial growth, and maintains the high temperature necessary for sterilization. The collected contaminated condensate can be channeled to an area for final sterilization or inactivation before it is released into the environment. Efficient steam supply and condensate removal requires pressure regulators, pressure relief devices, venting, and the capability for free draining of all lines.

Supplying sterile, inert gases to processing equipment is a method of containment. This can protect oxygen-sensitive biomaterials and prevent aerosol generation of toxic products. Inert gases, such as nitrogen, helium, and argon, are usually supplied directly to processing equipment through sterile, in-line filters, maintaining a pressurized system or providing an inert blanket over the product in processing vessels.

Ventilation
Another component in bioprocessing is the design of ventilation within the primary barriers (separating product from operators) and secondary barriers (separating the product from contamination). Primary barriers involve dedicated, in-line air/gas membrane filters. Secondary barriers require more complicated air handling systems to maintain clean areas (rated by the number of particles per volume of air) and pressure differentials. Equipment used in these designs includes high efficiency fans and high efficiency particulate air (HEPA) filters.

Storage
The primary means of stabilization for storage or packaging are:

initial concentration: (such as vacuum filtration, ultrafiltration, precipitation, and centrifugation); and

drying: (such as direct freeze-drying (lyophilization); direct spray-drying; formulation into a special stabilizing solid, liquid, or sometimes gaseous solution; and deep-freezing).

Freeze drying is the preferred method for long-term storage of bacterial cultures because freeze-dried cultures can easily be rehydrated and cultured via conventional means. Deep or ultra freezing of biological products is another long-term storage technique for species and materials not amenable to freeze-drying. The method involves storage of the contained products in liquid nitrogen refrigerators (-196° Celsius) or ultra-low temperature mechanical freezers (-70° Celsius). Mechanical freezing systems usually have precautionary back-up freezers and electrical generators. Infectious biological agents are generally stabilized and then spray dried. A toxin agent is most effective when prepared as a freeze-dried powder and encapsulated. Such encapsulation, however, is not necessary for weaponization.

Agents such as dimethyl sulfoxide (DMSO), glycerol, sucrose, lactose, glucose, mannitol, sorbitol, dextran, polyvinylpyrollidone, and polyglycol are required to ensure cell viability during storage.

Replication
The procedure used for the actual replication of an organism is a function of the organism itself. Techniques include cell culture, fermentation, viral replication, recombinant DNA, and powdering and milling.

Cell culture (such as chick embryos or tissue cultures) is necessary for the reproduction of pathogenic viruses and rickettsia since they will not reproduce outside a living cell. (Rickettsia is a type of microorganism that, like viruses, require other living cells for growth but, like bacteria, use oxygen, have metabolic enzymes and cell walls, and are susceptible to antibiotics)

Single cell growth chambers, including fermentation, are used for the production of bacteria and bacterial toxins, although some bacteria (such as plague bacteria) can also be cultivated in living animals.

Recombinant DNA techniques are a preferred method to produce rare animal toxins. Because of the complexity of this technique, the capability is not widespread.

Powdering and milling is generally used to produce BTW, as particles having diameters less than 10 microns can easily be absorbed by the human lung.

Equipment
Toxins and pathogens that affect animals, such as anthrax, brucella, plague and tularemia, are widespread. As a consequence, vaccines are widely produced and administered. Often, the same toxic agent is required for both BTW and countermeasure vaccine production. Indeed, initial processing of agents and processing of their associated vaccines may only differ by a few steps, and then often only in the degree of care taken and subtle differences in method (such as the degree of purification and the type of containment used).

The facilities required for the production of BTW agents are the same as those used in legitimate vaccine or pharmaceutical plants. Both include equipment and materials for microbial fermentation, cell culture or egg incubation, followed by harvesting, purification, and lyophilization. Both would require a source of pharmaceutical-grade distilled water free from bacterial contaminants, which would interfere with the growth of desired microbial agents. And both require autoclaves to sterilize the growth media and decontaminate the equipment after production. Standard biological laboratory equipment, such as fermenters, centrifugal separators, large-scale lyophilizers, or freeze dryers, Class II or III safety hoods, High-Efficiency Particulate Air (HEPA) filters and centrifuges could easily be converted to a weapons program. This dual-use equipment, similar to the equipment used in making beer, can be acquired commercially without raising suspicion.

Some typical equipment you would find in many research facilities would include: PCR thermocyclers, ultracentrifuges, clinical centrifuges, microfuges, Sorvalls, hybridization ovens, water baths (stationary and shaking), tissue culture facilities (hoods with CO2, incubators, liquid nitrogen freezing tanks), fume hoods, autoclaves, -20 and -80 freezers, cold rooms, incubators for growing bacterial or yeast cultures (usually 25 and 37 degrees C), scintillation and gamma counters, electrophoresis equipment (gel boxes, power sources etc.), and special cabinets for solvents, acids, bases etc.).

Bioreactor (Fermenter)
Bioreactors, the heart of the fermentation process, are the places where microorganisms are made. They can be vessels made of plastic, glass or steel in which cells are cultured or, alternatively, whole plants or animals can be genetically engineered to produce a particular protein. The reactor is designed to meet the specific needs of the cells, namely optimal mixing, temperature, and acidity, along with a supply of nutrients or precursors. The bioreactor will often be under computer control. They can be operated in three ways:

• Discontinuous, or batch reactors are simplest type. In this mode, the reactor is filled with medium and the fermentation is allowed to proceed. When the fermentation has finished the contents are emptied for downstream processing. The reactor is then cleaned, re-filled, re-inoculated and the fermentation process restarted.
  
• Anaerobic, or feed batch, is the most common type of reactor used in industry. In this reactor, fresh media is continuously or periodically added to the bioreactor. The fermenter is emptied or partially emptied when reactor is full or fermentation is finished. High productivities are achieved by controlling the flow rate of the feed entering the reactor.
  
• Continuous reactors feature the continuous addition of fresh media, and the bioreactor fluid is continuously removed. As a result, cells continuously receive fresh medium and products and waste products and cells are continuously removed for processing. The reactor can thus be operated without interruption. Continuous reactors can be many times more productive than batch reactors, as the growth rate of the bacteria in the reactor can be more easily controlled and optimized. Cells can also be immobilized in continuous reactors to prevent their removal and thus further increase the productivity of these reactors. Continuous reactors are as yet not widely used in industry except in wastewater treatment.

A standard, general purpose fermenter consists of a cylindrical metal vessel (usually stainless steel) with a 2:1 height to diameter ratio with either a cone shaped or sloping bottom to facilitate emptying. The fermenter also has a number of ports for adding nutrients, removing content samples, and inserting control probes. Larger fermenters have integrated steam systems for cleaning and sterilization. The tank may be fitted with openings for venting or collecting waste gases. Most are equipped for agitation by baffle plates fitted inside the fermentation tank and intermeshing motor-driven impellers. The general types of fermenters include:

Stirred tank and heavy-ton vessels, which have all the features described above. Stirred tank vessels use mechanical stirrers (using impellers) that mix the reactor to distribute heat and materials (such as oxygen and substrates). The heavy-ton vessels are much larger and are commonly used commercially for Single Cell Protein (SCP) production-a microbial-based product used for animal feeds. Both of these systems are well suited for most BTW agent production

Airlift vessels, which use bubbling air from the bottom of the vessel to stir the broth instead of an agitator. Airlift systems include an inner tube, which improves circulation and oxygen transfer and equalizes shear forces in the reactor. These systems would be well suited for fragile organisms but could not be used in anaerobic fermentation.

Chemostatic fermenters, that are designed to facilitate the continuous fermentation process.

The cell, immobilized cell and hollow-fiber fermenters, designed to provide a small growth surface for the cells by physically separating the cells from the growth media while allowing diffusion of nutrients and end products through membranes. Typical systems include fluidized bed (small particles that move in the fluid) and packed bed (larger particles that do not move) on which the cells are immobilized. These systems allow greater and more efficient yields and are more commonly used with animal cell systems, which have greater growth regulation requirements than bacterial cells.

Clean Rooms
Clean rooms are defined as a specially constructed enclosed area environmentally controlled with respect to airborne particulates, temperature, humidity, air pressure, air flow patterns, air motion, vibration, noise, viable (living) organisms, and lighting.

These facilities require special coordination of the integrated segments of HVAC, controls, room finishes, process equipment, room operations, utilities, and flow of equipment, personnel and product.

For product containment issues (where dangerous pathogens are involved), the suite must be at a lower, or negative, pressure than the surrounding areas. In this case the air lock must be maintained at least one pressure level difference higher than the adjacent areas and also higher than the contained suite. The airlock must be designed at the same class as the contained suite but kept at a pressure so that the airflow from the lock will always be out of the air lock into each space.

General Guidelines for Identifying Possible BW Equipment and Related Technology
There are subtle signs that would leave clues for inspectors. Equipment that would be of particular interest to inspectors include: Air compressors, air tanks, or lines for air-protective suits as a means of enhancing physical containment.

Some specialized equipment for the lyophilization, milling, or microencapsulation of BTW agents, though such machinery is ubiquitous in the pharmaceutical industry.

There is no equipment unique to BTW agent proliferation, although the Australia Group has defined parameters for equipment, which would be of particular utility for BW purposes:[i]

Centrifugal Separators
Centrifuges are used to separate heavier from lighter molecules and cellular components and structures. Essentially, it whirls test tubes around rapidly to force the heavier suspended molecules and macromolecules (in the solutions in the test tubes) to the bottom. A high-speed centrifuge can attain speeds up to 85,000 rpm and centrifugal fields up to 500,000 times gravity.

• Flow rate greater than 1000 liters per hour
• Components of polished stainless steel or titanium
• Double or multiple sealing joints within the steam containment area
• Capable of in situ sterilization in a closed state
• Capable of processing 5-liter batches.
• Critical materials = smooth surface; aerosol containment

Freeze Drying Equipment
Steam sterilizable freeze-drying equipment with a condenser capacity between 50kg and 1,000kg of ice in 24 hours.

• Critical materials = stainless steel, titanium, glass

Cross-Flow Filtration equipment

• Equal to or greater than 5 square meters
• Capable of in situ sterilization
• Capable of processing 20-liter batches.
• Critical materials = Smooth surface; Aerosol containment

Equipment that is often contained in BL-3 or BL-4 containment housing.
(Microorganisms are categorized by biosafety level risk group [1 2, 3, and 4]. Laboratories are designated according to the group and are designed accordingly in terms of construction, and containment facilities. The two most dangerous groups are 3 and 4. Group 3 refers to pathogens that usually cause serious human or animal disease but does not ordinarily spread from one infected individual to another. Effective treatment and preventive measures are available. Group 4 refers to those pathogens that usually cause serious human or animal disease and that can be readily transmitted from one individual to another, directly or indirectly).

Specifically:

• Independently ventilated protective full or half suits.
• Class III safety cabinets or isolators with similar performance standards.

Aerosol Inhalation Chambers

• Chambers designed for aerosol challenge testing with pathogenic microorganisms, viruses, or toxins and having a capacity of 1 cubic meter or greater.

Other Equipment
Equipment for the microencapsulation of live microorganisms and toxins in the range of 1 to 10 microns particle size, specifically:

• Interfacial polycondensors
• Phase separators
• Fermenters having a capacity of greater than 100 liters but of less than 300-liter capacity with special emphasis on aggregate orders or designs for use in combined systems. Multiple sealing joints capable of in situ sterilization in a closed state.
• Critical materials = stainless steel; titanium; glass
• Conventional or turbulent air-flow clean-air rooms and self-contained fan HEPA, filter units that may be used for P3 or P4 containment facilities.
• Critical materials = HEPA filters

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