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The stages involved in the production of biological agents include selection of the organisms, large-scale production of organisms from small starter cultures, and stabilization of the organisms.
The introduction of modern biotechnology during the past 25 years has markedly changed the qualitative and quantitative impact that biological warfare, or the threat of such warfare, can have on military forces and urban communities. This new technology provides the potential capability of (1) developing biological agents that have increased virulence and stability after deployment; (2) targeting the delivery of organisms to populations; (3) protecting personnel against biological agents; (4) producing, by genetic modification, pathogenic organisms from non-pathogenic strains to complicate detection of a biological agent; (5) modifying the immune response system of the target population to increase or decrease susceptibility to pathogens; and (6) producing sensors based on the detection of unique signature molecules on the surface of biological agents or on the interaction of the genetic materials in such organisms with gene probes. The specific technologies used in realizing these capabilities include (1) cell culture or fermentation; (2) organism selection; (3) encapsulation and coating with straight or crosslinked biopolymers; (4) genetic engineering; (5) active or passive immunization or treatment with biological response modifiers; (6) monoclonal anti-body production; (7) genome data bases, polymerase chain reaction equipment, DNA sequencers, and the rapid production of gene probes; and (8) the capability of linking gene probes and monoclonal antibodies on addressable sites in a reproducible manner.
New technologies related to biological warfare are emerging rapidly. The technology of monoclonal antibody production has existed only since 1975, while the technology of genetic engineering has existed since the 1980’s. Technology for sequencing the genomes of organisms has changed so dramatically that the rate of sequencing has increased by several orders of magnitude since 1994. All of these reflect the enormous change in information databases and in technology including biotechnology, computer equipment, processes, and networking of research teams.
The rapid rate of development reflects to some degree the national and international investment in this technology. The level of federal spending in the United States in the entire biotechnology area during 1994 approximated 4 billion dollars. The private sector invested approximately 7 billion dollars during the same year. This investment and the rate of information accrual indicates that biological technology that can be used for peaceful and military purposes is increasing in capability at a rate exceeding most other technologies. The pharmaceutical industry is relying on biotechnology for new therapeutic products to improve prophylaxis and therapy for many different diseases.
New technologies, such as genetic engineering, are more likely to affect fabrication, weaponization, or difficulty of detection than to produce a “supergerm” of significantly increased pathogenicity. Seed stocks for biological agents are readily available in the natural environment and from culture collections in the industrialized and in some devel-oping nations. The recent outbreaks of Ebola in Africa and Hanta virus infections in Asia and North and South America are evidence of this. In addition, these organisms may be obtained from national collections (e.g., American Type Culture Collection [ATCC] and European collections). Most industrialized nations manufacture equipment and materials necessary for the production, containment, purification, and quality control of these materials. The biological technology industry is information intensive rather than capital intensive. Data on technologies involved in biological production are widely avail-able in the published literature. These technologies are dual use, with applications in the pharmaceutical, food, cosmetic, and pesticide industries. While laboratory-scale capability for production of biological agents is sufficient for achieving most terrorist purposes, large-scale production for military purposes can be achieved easily in dual-use facilities. All of the equipment needed for large-scale production of offensive biological agents is dual use and available on the international market. Although a typical vaccine plant costs in excess of $50 million, a less elaborate fermentation plant that could produce biological agents could be built for less than $10 million. The term "containment" is used in describing safe methods for managing biohazardous agents in the environment where they are being handled or maintained. Primary containment, the protection of personnel and the immediate laboratory environment from exposure, is provided by good technique and the use of appropriate safety equipment. Secondary containment, the protection of the environment external to the laboratory from exposure to biohazardous agents, is provided by a combination of facility design and operational practices. The four biosafety levels for containment purposes and the types of agents placed in them are:
- Monoclonal Antibodies - In the early 1970’s, Kohler and Milstein developed a procedure to produce antibodies for a single antigenic epitope. An epitope is the region of a molecule that initiates the production of a single antibody species. The dimensions of an epitope approximate a surface area 50 ´ 50 Angstroms. These anti-bodies are called monoclonal antibodies. With quality control, these antibodies can be produced in gram quantities in a highly reproducible manner, and therefore, they are suited for industrial uses. The industries currently using monoclonal antibodies include medical diagnostics, food, environmental protection, and cosmetics.
- Combinatorial Chemistry - This is a technique for rapidly synthesizing large numbers of peptides, polynucleotides, or other low molecular weight materials. These materials are synthesized on a solid-state matrix and in an addressable form so that materials of known sequence can be accessed readily. The materials can function as receptors, pharmaceuticals, or sensor elements. The technique, developed by Merrifield in the 1970’s, has been essential for the growth of combinatorial chemistry.
- Gene Probes - These are polynucleotides that are 20–30 units bend, under stringent conditions, complementory nucleic acid fragments characteristic of biological agents. These units provide the basis of rapid detection and identification.BL-1 Biosafety Level 1 – suitable for work involving well-characterized agents of no known or of minimal potential hazard to laboratory personnel and the environment. The laboratory is not necessarily separated from the general traffic patterns in the building. Work is generally conducted on open bench tops using standard microbiological practices. Special containment equipment is not required or generally used. This is the type of laboratory found in municipal water-testing laboratories, in high schools, and in some community colleges. BL-2 Biosafety Level 2 – suitable for work involving agents of moderate potential hazard to personnel and the environment. Agents which may produce disease of varying degrees of severity from exposure by injection, ingestion, absorption, and inhalation, but which are contained by good laboratory techniques are included in this level. Biosafety Level 2 practices, containment equipment and facilities are recommended for activities using clinical materials and diagnostic quantities of infectious cultures associated with most biological warfare agents. BL-3 Biosafety Level 3 – applicable to clinical, diagnostic, teaching, and research or production facilities involving indigenous or exotic strains of indigenous agents which may cause serious or potentially lethal disease as a result of exposure by inhalation. All procedures involving the manipulation of infectious material are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. The laboratory has special engineering and design features. A ducted exhaust air ventilation system is provided. This system creates directional airflow that draws air from "clean" areas into the laboratory toward "contaminated" areas. The High Efficiency Particulate Air (HEPA)-filtered exhaust air from Class II or Class III biological safety cabinets is discharged directly to the outside or through the building exhaust system. The typical HEPA filter removes 99.97% of all particles that are 0.3 micron or larger in size, which means that all microbial agents will be trapped in the filter. Biosafety Level 3 practices, containment equipment and facilities are recommended for manipulations of cultures or work involving production volumes or concentrations of cultures associated with most biological warfare agents. BL-4 Biosafety Level 4 – required for work with dangerous and exotic agents which pose a high individual risk of life-threatening disease. The United States has two BL-4 laboratoriess, used by the US Army at Fort Detrick, MD, and the Centers for Disease Control and Prevention in Atlanta, GA. The facility is either in a separate building or in a controlled area within a building, which is completely isolated from all other areas of the building. Walls, floors, and ceilings of the facility are constructed to form a sealed internal shell which facilitates fumigation and is animal and insect proof. A dedicated non-recirculating ventilation system is provided. The supply and exhaust components of the system are balanced to assure directional airflow from the area of least hazard to the area(s) of greatest potential hazard. Within work areas of the facility, all activities are confined to Class III biological safety cabinets, or Class II biological safety cabinets used with one-piece positive pressure personnel suits ventilated by a life support system. The Biosafety Level 4 laboratory has special engineering and design features to prevent microorganisms from being disseminated into the environment. Personnel enter and leave the facility only through the clothing change and shower rooms, and shower each time they leave the facility. Personal clothing is removed in the outer clothing change room and kept there. A specially designed suit area may be provided in the facility to provide personnel protection equivalent to that provided by Class III cabinets. The exhaust air from the suit area is filtered by two sets of HEPA filters installed in series. Supplies and materials needed in the facility are brought in by way of the double-doored autoclave, fumigation chamber, or airlock, which is appropriately decontaminated between each use. Viruses assigned to Biosafety Level 4 include Congo-Crimean Hemorrhagic Fever, Ebola, Junin, Kumlinge, Kyasanur Forest Disease, Lassa, Machupo, Marburg, Omsk Hemorrhagic Fever, Russian Spring-Summer encephalitis, and Tick-borne encephalitis virus complex (Absettarov, Hanzalova, Hypr).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, and ventilation and filtration systems. The design of a biochemical processing plant is an important signal of covert biological agent production. Containment of the biological material during processing is of special interest. There is a clear distinction between processing materials for biological or toxin agent weaponization and processing protective agents to be used for countermeasures or personnel performance enhancement. For the production of biological agents for offensive military activities, the processing containment requirement is to protect the environment from the agent because of its infectious nature. For the production of biomaterials, such as vaccines, biological response modifiers, antibiotics, and anti viral agents, for defensive military activities, the containment require-ment is to protect the processed biomaterial from contaminating materials in the environment. Effectiveness of countermeasures is enhanced by achieving high levels of purity and cleanliness in the product before it is administered to friendly personnel. By contrast, an unpurified biological agent that will be used in BW is generally more stable than the purified agent that is needed to produce vaccines and biological response modifiers (BRMs). Consequently, a proliferant does not require a high level of purity if production is for BW use only. Generation of biological agents requires fermenters or single cell production capabilities including smooth, highly polished stainless steel surfaces, self-containment capability, and negative pressure conditions. The primary difference between the production requirements for biological weapons and non-military commercial purposes lies in containment and contamination. During biological agent production, efforts are generally made to avoid contaminating the environment with the organism. Less concern arises about the contamination of the product. Conversely, the pharmaceutical, brewing, and biotechnology industries are most concerned about protecting the purity and quality of the product. 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. 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.
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 (ABB, Modicon, Allen Bradley Corp.), which allow automatic control, control from remote locations, and automatic data logging and trending.
Another component in bioprocessing is the design of ventilation within the primary and secondary barriers of a process area. Ventilation at primary barriers (i.e., barriers separating product from equipment operators and the rest of the processing area) is accomplished with dedicated, in-line air/gas membrane filters. Ventilation across secondary barriers requires more complicated air handling system design to allow for the maintenance of clean areas (rated by the number of particles per volume of air) and maintenance of positive or negative pressure between the processing area and the outside environment or between different processing areas in the same facility. Equipment used in these designs includes high efficiency fans and high efficiency particulate air (HEPA) filters.
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 is necessary for the reproduction of pathogenic viruses and Rickettsiae since they will not reproduce outside a living cell (e.g., chick embryo or tissue cultures). Single cell growth chambers, including fermentation, are used for the production of bacteria and bacterial toxins, although some bacteria (e.g., 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 as widespread as the others. Powdering and milling is the technique generally used to produce BW and toxin weapons (TW) agent particles having diameters less than or equal to 10 mm, the size most effective for respiratory delivery.Toxins and pathogens that affect animals, such as anthrax, brucella, plague, and tularemia, are widespread. Vaccines are widely produced and administered. The issue of the need for the same toxic agent for either BW/TW production or countermeasure vaccine production emphasizes the dual-use nature of the technologies. Indeed, initial processing of agents and processing of their associated vaccines only differ by a few steps (e.g., the degree of purification and the type of containment used).