Turning research into products
By JOHN HAYSTEAD
Approximately 20 years ago, breakthroughs in gene splicing and cloning technology gave rise to the modern biotechnology industry which today produces over 40 biotechnology-based drugs and vaccines, hundreds of diagnostic products and numerous agricultural products.1 To manufacture these products, biotechnology companies require highly-controlled process environments and facilities that must not only protect the products from contamination, but also protect their personnel and the environment at large from the products.
Biotechnology is sometimes defined as any technology that relies on living organisms or biological systems, but this would include textiles and other products such as bread, yogurt, cheese, wine, beer and vinegar — all of which are produced with the help of cultured micro-organisms. Instead, the term “biotechnology” is more properly restricted to only those processes based on genetic engineering and associated techniques, resulting in new combinations of (recombinant) genetic material.
While biotechnology is one of the world`s fastest growing and investment-intensive industries, there are not nearly as many companies involved in actual manufacturing as in research. The industry is generally segmented into three arenas: pure research, limited development and full-scale manufacturing, and according to Hank Rahe, director of technology at Contain-Tech (Indianapolis, IN), “there is a tremendous technology gap between research and development and manufacturing requirements.” The companies that have successfully bridged this gap have done so with the aid of sophisticated and flexible contamination control technology at both the process and production environment level.
Biotechnology requirements
Class levels for biotech manufacturing facilities begin at Class 10,000 environments but quickly move up for many processes and products to Class 100. Since most biotech products are parenteral (injectable) products, they are regulated according to the FDA`s aseptic processing guidelines which mandate Class 100 environments for critical areas (anywhere product is, or could be, exposed to the environment) as well as overall support by a minimum Class 10,000 cleanroom. According to Rahe, many manufacturers have, in fact, opted for Class 1,000 cleanrooms.
One particular category of biotechnology product is particularly sensitive to contamination. According to Fred Marsik, of the FDA`s Anti-infectives Group (Rockville, MD), “although the allowable levels of contamination in the biotech industry for anti-sera and other types of less-sophisticated diagnostic devices are generally higher than those in the pharmaceutical industry, DNA-related products such as probes and test kits require comparable controls and cleanroom environments to prohibit the introduction of extraneous DNA or RNA.” These products usually require Class 100 environments throughout their manufacturing process, and in fact, DNA probe and diagnostic kits are so sensitive to contamination that, to prevent end-users from themselves introducing contamination, some manufacturers have designed them as completely self-contained units — in effect, cleanrooms in a box.
Containment is crucial
In addition to protecting product, biotech facilities must also provide containment and isolation. In fact, “very high levels of containment are necessary,” says Marsik, “since some of the organisms being used to extract beneficial antigens or DNA material are infectious disease agents themselves.” Often biotech employees work with some type of biological safety cabinet or isolation system, and when working with highly-infectious agents, are not only fully gowned and masked but equipped with respirators as well.
Each containment unit must be individually exhausted through a sophisticated filtration system with negative pressure the standard for many live-process environments. Positive pressure can only be used if 100 percent of the air is HEPA-filtered prior to being exhausted and in some cases, double-HEPA or HEPA/ULPA filtered prior to being released to the outside environment.
Drugs are drugs
Because several large pharmaceutical companies have their own biotechnology operations and many biotech companies also produce products for pharmaceutical companies, the biotech industry and its contamination control requirements are often lumped together with those of the pharmaceutical industry. In fact, however, biotechnology and pharmaceutical production facilities are in most cases very different animals, and according to Scott Mackler, marketing manager at Clestra Cleanroom Inc. (North Syracuse, NY), “the requirements of the two industries should be viewed as separate and unique entities.”
Containment is a key distinguisher between biotech and most pharmaceutical cleanroom requirements, points out Mackler. “If you don`t have a solid basis of design to account for this, you will be in trouble, which is why we see many biotech cleanrooms built that won`t validate and end up as trophies.” For example, while the requirements of classical chemical-based, pharmaceutical sterile fill rooms are more or less standard from product to product, biotech facilities require independent pressure/humidity/temperature controls as well as special attention to cross-contamination issues such as corridor cleanliness, pressure differentials and air flows, batching and lots — all of which add complexity and cost.
Containment and cross-contamination issues can easily double the cost of a biotech facility cleanroom, says Mackler. “Except for the very big firms, biotech companies generally require cradle-to-grave solutions including complete process design, development and scale-up, process support services, process equipment, validation and service.” FDA`s Marsik agrees, “although because biotechnology is generally a fairly sophisticated industry, smaller companies often don`t fully realize what is necessary to manufacture their products.”
Isolation technology
Because of the dual contamination control/containment requirements of biotechnology production, isolation technology holds particular promise for the industry. But at the same time, the challenges of implementing such systems are amplified.
According to Bob Torregrossa, manager of biotechnology at Lockwood Greene Engineers (Somerset, NJ) “Although isolation technology has been talked about quite a bit, so far, the number of real-world applications is somewhat limited.”
Although isolation systems are becoming the standard today for sterility testing and other specific processes, actual isolation-technology-based fill lines are only now being tested in some biotech facilities, and according to Torregrossa, “there aren`t a lot of validated systems out there.”
As observed by Torregrossa, isolation systems require a lot of extra time and money to design, procure equipment for and validate.
For example, many validation issues remain with regard to direct sterilizing isolation units through vapor-phase hydrogen peroxide and other methods, and manufacturers almost have to work hand-in-hand with process equipment manufacturers to design the systems.
In addition, isolation technology is just now reaching the level of maturity where systems can be seen and evaluated as standard products and the debate continues to rage over the level of additional cleanroom protection required when isolators are being used. Although some European countries have relaxed their requirements to Class 100,000 (M6.5), the FDA is still typically looking for Class 10,000 unless the manufacturer can unequivocally demonstrate that this is not required.
Flexibility is key
According to Shelley Henderson, director of business development for BioMetics (Waltham, MA), a biotech facility design and validation firm, “One of the common attributes of biotechnology products is that they tend to be low-volume and high-value.” As a result, manufacturing facilities must be capable of efficiently producing several different types of products, making flexibility a key parameter of cleanroom and process design.
According to Wadi Farach, project manager at Chiron Corp. (Emeryville, CA) “Designing flexibility into your manufacturing facility is crucial.” Chiron is the world`s second-largest biotechnology company, manufacturing diagnostic, vaccine and therapeutic products for treatment of diseases such as AIDS, Hepatitis C and cancers. (See related article, “Contamination control down the process line.”)
“Particularly in the diagnostics business where there is such severe competition, manufacturers really need to be able to get a product line to market within a year, and flexibility is crucial since you can`t predict the type of product or requirements that will be coming,” Farach says.
Fortunately, however, the process requirements for diagnostic products are not as intense as other biotechnology areas, such as therapeutic products, making it easier for manufacturers to use less fixed equipment.
The demand for flexibility is also a principle reason for the success of contract manufacturing in the biotech industry, Henderson says. “Although this has entailed a fairly major change for the FDA, they have made the transition rather well, and it is reflected in the facilities being used.”
Contract manufacturing
Among the biotechnology facilities designed and validated by BioMetics is the Lonza Biologics plant in Portsmouth, NH. Lonza is the world`s largest contract manufacturer of mammalian-cell-derived therapeutic proteins.
Lonza produces recombinant therapeutic proteins such as monoclonal antibodies and growth hormones by growing specially-engineered living animal cells in large growth tanks called fermenters. The products produced by the cells are then isolated from the culture media and purified. (See related article, “The biotechnology process.”) According to David Jackson, Lonza`s vice president of operations, “Given that these are living systems with cycle times in the weeks and months, clean environments are critical. Any contamination (bacterial or viral), or carryover from one cell line to the next, could be catastrophic to the product.”
As Jackson points out, cleanroom equipment plays a key role in the company`s ability to simultaneously maintain and control the integrity of numerous production environments.
Classification levels at Lonza run from Class 100,000 down to Class 100. “Per the FDA`s guidelines,” Jackson says, “we increase classification levels as we move from one process to the next, increasing our controls and improving cleanliness levels to ensure our ability to avoid cross-contamination.” Initial fermentation activities involve a number of protein types, including media and others unique to the cell, etc., but “by the time you isolate and purify at the last step, it is critical that you have maintained integrity at every point,” says Jackson.
Lonza officials currently rely more on the company`s cleanroom facilities and the design of process equipment than on isolation technology to control contamination and maintain containment. “By design, we limit our areas of exposure, minimizing the number of steps where we have to deal with the requirements of exposing cell material to the environment,” Jackson says.
Still, he does see an eventual place in the biotechnology industry for isolation technology. He points out, however, that “you also have to take into account the scale of each operation. If you`re dealing with a 2,000- to 10,000-liter fermenter system, for example, you need to determine at what point it is appropriate to introduce isolation technology. Today, you can`t economically build an isolation system around a huge fermenter, and even if you could, it might not be the best choice.”
Air management
In terms of basic biotechnology cleanroom design, air management systems are clearly one of the most challenging elements.
As BioMetics` Henderson describes, “Because in biotech, you are dealing with low-volume production, there is little difference between the design requirements of small and large facilities, however, when you have many different areas in one building with different pressure requirements, HVAC becomes much more challenging.”
Explains Clestra`s Mackler, “The need for flexibility and prevention of cross-contamination often results in a more costly HVAC system for these facilities than a typical Class 10,000 cGMP area.” For example, to accomplish both aseptic conditions and containment, most biotechnology facilities are designed for positive pressure with 100 percent-once-thru fresh air suites and negative pressure entry/exit vestibules. Individual room pressure is normally controlled by variable-frequency, direct-drive-controlled exhaust fans. Also 100 percent-once-thru cGMP containment facilities require 95 percent ASHRAE DOP-tested prefiltration. Overall, the total installed cost of a biotech containment cleanroom could be as much as $100 to $150 more per square foot than a standard Class 10,000 positive-pressure facility.
FDA regulation/validation
In general, biotechnology is regulated under the FDA`s Center for Biologics Evaluation and Research (CBER), which regulates biological and related products including blood, vaccines and biological therapeutics. However, Henderson points out that regulation is actually a complex topic, since biotechnology products such as hormones and recombinant insulin are also regulated under the FDA`s drugs.
The FDA reviews all applications for new biotechnology medical products, and manufacturers are required to submit detailed documentation on the facilities in which the products will be manufactured, Marsik explains. The documentation must include the sources of equipment as well as detailed descriptions of airflow management systems and contamination measurement procedures and techniques. Any changes to an existing product line, such as a new laminar flow hood, must also be approved in advance by the FDA.
New product lines require multiple qualification lots to be submitted to the FDA before full production can begin, and this can take up to six months.
Although biotechnology applications often challenge contamination control systems, the regulatory requirements primarily involve standard monitoring of process and facility cleanliness and operation. Particulate and microbial monitoring is a critical element of FDA licensing but standard microbial testing procedures for air and surfaces are used.
Molecular contamination
Other than monitoring for the presence of large particulates, biotechnology companies have had to test only for the presence of microorganisms. Today, however, there is growing concern for molecular level contamination (DNA material) in some biotech process environments. As observed by FDA`s Marsik, “Ten years ago, we didn`t even think about this, but today, in the preparation of certain vaccines, for example, we`re trying to get a handle on the possible presence of extraneous DNA that could itself potentially cause a disease.”
Not only is little known about the possible implications of this level of contamination, but the level of equipment required to detect these materials is also much more sophisticated, and in fact does not exist.
Chiron`s Farach says molecular contamination is currently not a problem in most processes, but acknowledges it does pose some challenges in their Polymerase Chain Reaction (PCR) DNA areas. “Here, any level of cross-contamination can be a problem, and since we haven`t seen any adequate monitoring devices available, we deal with it through segregation, air-handling and pressurization techniques.”
Biosafety requirements
The National Institutes of Health (NIH) has established biosafety requirement levels for containment. Biosafety Level 1 (BL-1) is for normal laboratory operations involving agents, not known to cause disease in healthy adults, while Level 2 is for agents associated with human disease. Hazards at Level 2 include auto inoculation, ingestion and exposure to mucous membranes. Level 3 is used to protect against indigenous or exotic agents with potential for aerosol transmission of diseases which may have serious or lethal consequences and BL-4 is reserved for highly-infectious agents for which there is no known cure such as the Ebola virus. BL-4 is never a factor in biotechnology facilities, however.
Usually biotechnology applications fall under NIH biosafety levels (BL) 1 and 2, with most production classified under BL-2.
As Chiron`s Farach describes, each type of product, whether therapeutic, diagnostic or vaccine will have a different level of containment specified for its handling. In general, diagnostic products require the least degree of containment, while viruses or other BL-3 classification organisms will require the use of a localized-containment biosafety cabinet with laminar flow HEPA modules maintaining Class 100 conditions.
Reference
1. Biotechnology Industry Organization (BIO) — “Profits, promises and positive results,” 1996, Time, Inc.
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A large-scale continuous centrifuge is used in primary recovery of biopharmaceutical product from a cGMP cell structure.
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A Lonza Biologics operator places in-process samples into an airlocked transfer chamber.
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Lonza Biologics`s distributed control system assists in assuring cGMP compliance when manufacturing biopharmaceuticals.
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A Class 100 aseptic fill line.
Contamination control down the process line
At Chiron Corp. (Emeryville, CA), therapeutic products typically go through three different types of processes — fermentation/recovery; purification; and fill/finish. (See related article, “The biotechnology process.”)
Typically fermentation and bi-product recovery areas are class 100,000 and are under negative pressure with exhaust HEPA filtration systems to deal with the containment concerns of the recombinant organisms. Personnel gowning requirements are typical of standard laboratory environments in this area.
Once in the purification areas, however, in addition to Class 100,000, Class 100 laminar flow areas are introduced. All chromatography and reduction, for example, is done under Class 100 hoods. As observed by Wadi Farach, Chiron project manager, “Although the air does not actually come in direct contact with the product, and it is not an aseptic process, we still filter at 0.2 microns at different locations such as where we are preparing buffers, etc.”
Once in the fill/finish area, conditions move to full-blown aseptic processing, following standard pharmaceutical guidelines such as a Class 10,000 parts-prep area. Critical parts cannot go directly from this area to the aseptic area, without passing through double pass-through autoclaves or sterilization tunnels. Personnel and materials must also pass through air locks, and personnel must also fully gown to enter the aseptic areas. Farach notes, “We try to maintain temperatures at 62 degrees Fahrenheit to keep personnel comfortable while working in the environment.”
Chiron doesn`t incorporate air showers in its pass-throughs, but as described by Farach, “Since we`ve found that particle counts are highest within the air lock areas, we increase air circulation within these areas to a much higher level.”
Depending on the product`s destination, the aseptic area is either Class 10,000 or 1,000. Products intended for the European market can be processed at Class 10,000 (M5.5) while the U.S. requires Class 1,000 conditions. In both cases, the fill lines are full-laminar flow Class 100 areas. Chiron does not currently utilize isolation systems in its manufacturing lines other than that which is provided as part of certain process equipment itself, such as chromatography columns.
Vinyl curtains or Plexiglas are sometimes used to physically separate areas. As noted by Farach, there are applications for both approaches, but “some people feel vinyl curtains are easier to operate and maneuver around than Plexiglas, and in California, we also have to take earthquakes into consideration, which means we have to ensure any Plexiglas structures will withstand seismic activity.”
In terms of containment, most of the production areas are at Biosafety level-2 (BL-2) levels with BL-3 areas typically only used for R&D purposes. Since most of Chiron`s product lines involve bacterial fermentation, dealing with E. coli or yeasts, these areas are maintained at negative pressure. This differs from mammalian cell fermentation which is typically more sensitive to contamination but less of an environmental hazard, and is typically done in positive pressure environments. As emphasized by Farach, however, “If you are dealing with both types of fermentation, you absolutely must have complete segregation and, in fact, entirely different systems.”
Even between yeast and bacterial processes, or within different types of bacterial hosts, Chiron provides complete segregation of areas. Farach notes that there has been a push recently to allow multi-host environments by means of Clean-In-Place (CIP) or Sterilization-In-Place (SIP) systems, but only if you can prove you have completely cleaned the equipment. “This approach still needs to be approved by the FDA,” however, Farach says.
Within the negative pressure room, a cascade system provides decreasingly negative pressure areas, with the highest level of negative pressure at the fermenter, slightly less negative at the recovery areas and less again at the in-process-testing area.
Purification areas are usually always positively pressured, separated by corridors, gowning areas and airlocks. Similar to the fermentation/recovery, there is also a cascade arrangement of increasingly positive pressure zones.
In the aseptic area, cascade zones are increasingly frequent with the fill and lyophilization areas being the most positive. A Class 100 laminar flow area is installed directly at the opening of the lyophilization unit. Farach says they typically like to maintain 0.05 pressure differentials between each adjacent area which is monitored by a computer system that will sound an alarm at any deviations of ۪.02 to prevent any possible reversal of air flow. — J.H.
The biotechnology process
The biotechnology manufacturing process starts with a fermenter or “reactor” where live organisms or cells are grown that will generate the desired product. The reactor provides the correct environment and nutrients to allow the organisms to thrive and “express” the byproduct being sought such as insulin or growth hormone. Commercial-scale fermenters produce large quantities of host cells, each cell producing the target protein from its recombinant DNA blueprint.
At the end of growth cycle, the organisms are killed by heat, steam or chemical means. This part of the process is particularly closely regulated to ensure that none of these bio-engineered cells survive and enter the natural environment. After the organisms are killed, the product is “harvested.” In this process, manufacturers protect both the purity and integrity of the “broth” by transferring it either in closed piping or through a critical laminar flow environment or barrier-isolator.
After any filtration necessary to remove organism debris from the product, the material is then collected in separation columns. The columns segment out the desired material, providing multiple separations or “cuts” of solution. Acceptable levels of purified solution are saved for further processing while the rest is either repurified or discarded. Purification involves several steps in a defined order that take advantage of the unique characteristics of the product to separate it from other substances.
The purified protein is mixed (formulated) in preparation for filling. In some cases, the material will be freeze-dried (lyophilized) to a powder before being bottled and labeled for use. — J.H.
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A Chiron Corp. fermenter.
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