Contamination means death for live tissue products
Tissue banks and researchers rely on frequent testing and strict cleanroom controls to keep human cell- and tissue-based products alive and free from contamination.
By Sarah Fister Gale
Every year, more than 1.5 million units of allograft tissues harvested from deceased donors are distributed annually for transplant in the U.S. While the risk of bacterial infection from an allograft tissue transplant is extremely low, in an industry where the lives of human patients are at stake, any contamination risk is a serious concern. Fortunately, the industry has established strict protocols for the screening and handling of tissue in the manufacturing environment to effectively manage that risk.
“The current risk of an allograft infection to the average patient appears to be much less than the risk of infections surrounding the operation itself,” notes Joel Osborne, vice president of quality assurance for the Musculoskeletal Transplant Foundation (MTF), an American Association of Tissue Banks (AATB) accredited tissue bank in Edison, NJ. Still, the allograft industry and the FDA are making constant strides to more strictly control contamination in the handling and processing of human tissue-based products.
Patients receiving allograft tissues put their lives in the hands of the companies supplying those products, relying on them to enforce strict tissue reviews, cleaning and disinfecting processes, and monitoring strategies to ensure donor tissue is healthy and free from infection, and that it does not encounter contaminants during processing.
Human tissues intended for transplantation have been regulated by the U.S. Food and Drug Administration (FDA) since 1993 under 21 CFR 1271. All human cells, tissues and cellular, and tissue-based products (HCT/Ps) used for transplant fall into this category, including bone, ligament, skin, and other soft tissues. These requirements were designed to prevent the introduction, transmission, and spread of communicable diseases via HCT/Ps by ensuring that the HCT/Ps do not contain communicable disease agents, are not contaminated, and do not become contaminated during manufacturing.
As part of the ongoing effort to enforce rigorous safety methods, in May 2005, FDA put three new, comprehensive regulations into effect that address manufacturing activities associated with HCT/Ps. The first requires companies that produce and distribute HCT/Ps to register with FDA. The second provides criteria that must be met for donors to be eligible to donate tissues (the “Donor Eligibility” rule). The third rule defines current Good Tissue Practices (cGTPs), which govern the methods used in, and the facilities and controls used for, the manufacture of HCT/Ps; recordkeeping; and the establishment of a quality program.
Many tissue banks also receive accreditation from the AATB, a voluntary accreditation organization that sets standards for tissue banking.
Every step of the way
“HCT/Ps are unlike pharmaceutical products because they are composed of living functional cells,” notes Dr. Scott Burger, principal of Advanced Cell and Gene Therapy, a consulting firm in Chapel Hill, NC. “They can respond to the microenvironment of the patient, taking advantage of their intrinsic biological functions and capabilities, but they are very complex and challenging materials to handle.”
The success of a tissue-based product relies on its ability to stay alive and functional, which dramatically limits sterilization options and requires strictly controlled transportation and handling conditions to maintain that viability. Those limits in larger part define the processing environment.
“A tissue manufacturing facility cannot be a sterile operation because you are working with biomass,” notes Mark Hallworth, pharmaceutical business manager for Particle Measuring Systems in Boulder, CO. “Human tissue is a living organism that can’t be sterilized, so you’ve got to be able to prove you are in control of your processes at all times.”
Figure 1. A CryoLife technician dissects a human heart, retrieving valve and vascular tissue for preservation and eventual implantation. Photo courtesy of CryoLife.
Proving that drives a lot of the environmental controls used by the HCT/P industry, and it begins with evaluating the donor tissue before it ever comes through the door.
The Donor Eligibility rule requires comprehensive screening of the donor’s medical and social histories, and testing of the donor for risk factors and/or clinical evidence of infection due to communicable disease agents and diseases, such as HIV, Hepatitis, or Strep A. The rule also includes requirements for recordkeeping, quarantine, storage, and labeling of the HCT/Ps.
If a donor meets the eligibility requirements, the tissue is removed in a hospital setting using aseptic surgical techniques. At the time of recovery, cultures of the tissue are taken to look for the presence of bacteria, mold, and fungi, with those test results later determining whether the tissue can and will be processed, says MTF’s Osborne. While every processing facility is different, at MTF the donor tissue is frozen at ???70??C until those test results come back 14 days later.
“In some cases the culture results will show some organisms that normal aseptic processing can’t remove,” he says. Those could include E. coli, Pseudomonas, or other organisms that are typically present in the gut. When that occurs, Osborne’s team may pre-treat the tissue with low-dose gamma radiation to eliminate these organisms prior to processing; or if the tissue can’t be irradiated, they may use chemical cleaning, validate the aseptic processing to prove the safety of the tissue, or discard it altogether.
“All of our processes are validated to ensure we are not using contaminated tissue and that tissue doesn’t encounter cross-contamination during handling,” he says.
Most facilities runs a lengthy list of additional tests on materials when they are delivered to the facility, notes Dave Fronk, vice president of regulatory affairs and quality assurance for CryoLife, a biological medical device company in Kennesaw, GA.
“The tissue arrives submerged in transport media, in a double-bag system, in a cooler full of wet ice,” he says. Before it is processed, his team tests the tissue and the transport solution for aerobic, anaerobic, and fungal contaminants; verifies that the tissue has been completely submerged in the media at all times; and that it was maintained in a 1-to-10??C temperature range.
At CryoLife, these test results take 14 days to be completed, but processing will take place during that time. “All of our processing is done at risk,” he says, noting that to protect personnel from any potential contaminants in the tissue, all dissection of material is done in an ISO 5 (Class 100) biosafety cabinet.
Once tissue passes initial inspections at MTF, it is brought into the ISO 4 (Class 10) cleanroom processing space and thawed in warm purified water. The tissue is then cut and shaped for specific surgical needs, then it goes through a delipidization machine that uses high degree agitation and water rinses to shake loose any lipids in the material, which can contain bacteria.
“At that point the tissue is ready to be treated,” Osborne says.
Processing under control
How tissue is treated depends on the material and its ultimate use. It may be as simple as shaping or cutting the material for final use, or it may require more complex, multi-day processing steps to expand, separate, modify, or activate donor cells.
Bruce Levine, director of the Clinical Cell and Vaccine Production Facility and research associate professor at the University of Pennsylvania School of Medicine in Philadelphia, processes T-cells, which requires cell separation steps as well as expanding, accelerating, and re-infusing cells over several days of processing steps before they are ready for use in an application.
His facility relies largely on single-use disposable bags, tubing, and needles to maintain contamination control and to isolate the tissue throughout the processing steps. The process is conducted in ISO 5 (Class 100) laminar flow hoods in an ISO 7 or 8 (Class 10,000 or 100,000) cleanroom.
“Most processing steps are conducted in closed systems to minimize cross-contamination,” Levine says, noting that his team also decontaminates surfaces between processing steps to further control the environment.
Levine’s facility includes a primary ISO 8 (Class 100,000) environment with gown-in and gown-out rooms, as well as eight open processing rooms, two of which are ISO 7 (Class 10,000), linked by a common hallway. Six of the processing rooms feature positive pressure in relation to the hallway, while two of the rooms, in which HIV cell handling is conducted, are negative pressure in relation to the hallway. These pressure designs were chosen to ensure the highest safety levels for materials and personnel.
Tests prove success
Throughout the processing of tissue, most manufacturing facilities rely on frequent testing, to identify whether contaminants are present or to validate that no contaminants have been introduced into the process as part of their quality assurance program.
Fronk notes that tissue media is tested multiple times throughout the processing steps at CryoLife and tissue is placed in an antimicrobial wash at the end of processing for up to 38 hours as a further safety step.
“We need assurances that tissue is reasonably free from contamination,” he says of the multistep process. “It can’t be sterilized but we can test as much as possible to achieve an acceptable level of assurance.”
Along with testing tissue, manufacturers also keep an eye on technicians who can be unwitting carriers of contamination. Levine notes that skin contaminants, along with rare cases of fungus or mold, are a constant con-cern for the lab. “Most of the contamination we see is from skin, which we believe comes from personnel,” he says. His biggest challenge in managing this source of contamination is in effective training of personnel to prevent it. “You can have appropriate gowning, but if people don’t follow protocol you have problems.”
His team struggles to find the balance between maintaining total contamination control and making sure personnel are comfortable and free to move around in the manufacturing environment. “You can cover every inch of their skin,” he says, “but you’d need a space suit to do it.”
The key is getting the most coverage to allow for sufficient comfort and movement with minimal skin exposure. Training on good gowning practices is also coupled with frequent gown changes and testing of gloves and gown surfaces for contamination.
Frequent reviews or testing of personnel is also employed to ensure they are performing proper aseptic gowning and good handling practices. MTF’s Osborne conducts quarterly surveys of every technician’s gowning performance. “They are required to be 100 percent free of viable organisms to pass the test,” he notes.
End of the line
Before being packaged for use, tissues are subjected to one or more sets of disinfecting processes that are designed to kill or remove any bacteria, fungi, or viruses that might be present. Different companies use different methods to accomplish this goal, including rinsing with antimicrobial chemicals, sometimes under pressure; antibiotic washes; or in the case of more sturdy materials, irradiation may be used. In some instances, multiple methods might be employed to achieve the desired level of cleanliness as various methods have different levels of effectiveness in killing or removing infectious organisms for different kinds of tissue.
At MTF, Osborne uses a chemical wash for bone tissue that includes peroxide and alcohol, but he notes that soft tissue, such as ligaments, can’t survive the harsh chemicals. In those cases he relies on a cocktail that combines three antibiotics and was designed by MTF. To ensure its effectiveness, the soft tissue is placed in a vessel, submerged in the cocktail, then rotated and agitated at near body temperature conditions.
“The rotation and agitation ensure that all surface area is exposed to the antibiotics, and the body temperature conditions maximize the effectiveness of the drugs–which are not as effective at room temperature,” explains Osborne.
The tissue then goes through a series of rinses with a combination of purified water and a phosphate buffered saline. The tissue and/or the rinse aid is tested one more time before the tissue is packaged in an ISO 4 (Class 10) cleanroom within an ISO 4 biosafety hood.
“After the rinse the tissue still needs to be measured, which requires some contact with the operator–which is why we use the safety hood,” Osborne says. “It adds another layer of protection to the process.”
Larger amounts of tissue are then preserved in a freezer at ???40??C, while smaller materials are freeze dried and stored until the 14-day test results come back.
The wet cleanroom
As any cleanroom operator knows, frequent rinsing and water usage in a critical environment presents additional challenges to maintaining a safe manufacturing environment and MTF is no exception, Osborne notes. “Managing our water system is critical to our contamination control process.”
The organization uses 200 to 300 gallons of water per donor, and his team must ensure the water that comes in contact with the tissue is pure and not carrying contaminants, and no harborages for bacteria growth are established in the manufacturing space as the result of moisture build-up.
To prevent contaminants from the water source, MTF produces its own USP validated purified water on site, putting the water through an osmosis system and a series of filters down to a 0.2-µm filter at the make-up tank, where it is then ozonated. Once in the system all of the make-up water is continuously sanitized with ozone, with an ozone flush through the system conducted every night. “Ozone is very effective against any organism in the water system,” he says.
The water system was also engineered with no dead legs to prevent contamination build-up; drains and sink traps are regularly sanitized with a sporicidal agent and monitored for any waterborne organisms that could potentially contaminate the tissue.
Figure 2. Fully processed CryoValve SG decellularized human pulmonary heart valve. Photo courtesy of CryoLife.
“Monitoring is important, especially when there is water in the environment,” he says. “We are constantly looking for organism growth in our environmental monitoring and our tissue tests.”
Osborne also pays close attention to any equipment using air compression or compressed nitrogen, as these are difficult to clean and monitor. “Moisture in the air lines in particular can be a problem,” he says.
Filters are employed within the equipment and at the point of use to control contamination, and Osborne’s team regularly samples the air around that equipment for viable particulates. “If something arises, that’s a big issue because the only way to clean those systems is to take them apart,” he points out. As a preventive maintenance strategy, his team dismantles and cleans the units every six months and again if the operation goes through an extended downtime.
Any shutdown as the result of construction or a modification to the cleanroom requires additional cleaning and monitoring before manufacturing can resume. In these cases Osborne’s team relies on chlorine dioxide, gassing the entire room prior to use.
“Chlorine dioxide permeates the room, getting at hard-to-clean areas,” he points out. “It reduces problems especially in getting rid of Bacillus subtilis, which is hard to kill.”
Osborne has found chlorine dioxide to be so effective that he is considering using it on a more regular basis as part of the cleaning and disinfecting routine of the cleanroom. “It’s more effective than normal disinfecting agents: It dissipates easily and isn’t harmful to the environment,” he explains. “It’s becoming more accepted for these kinds of uses.”
Isolation is key
While managing contaminants that come into the environment from donors, handlers, and materials is critical to cGTPs, tissue handlers are also always concerned about cross-contamination among unique tissue products. “The underlying regulation from the FDA centers on the prevention of contamination and cross-contamination,” notes Fronk.
Cleaning strategies, room design, and handling procedures are all engineered around avoiding cross-contamination. In most facilities that means only one donor’s tissue is allowed into the manufacturing space at any given time, and many facilities build their clean manufacturing environments so that two donors’ tissues will never cross paths.
“One of the best ways to achieve that is with a ‘clean in’???‘dirty out’ room design so that product A and product B never come in contact with each other,” says Hallworth of Particle Measuring Systems. In that design, all tissue, manufacturing materials, devices, and personnel come in through one corridor and entrance and out through another, creating a natural flow that does not turn back on itself.
Figure 3. One measure to prevent contamination of delicate tissues is to incorporate automated equipment in the cleanroom for advanced tissue processing. Photos courtesy of the Musculoskeletal Transplant Foundation (MTF).
“Without this kind of design, you have to closely limit the movement of product and staff in the facility,” Hallworth says.
Along with keeping the materials apart, filtered air handling systems, positive-pressure cleanrooms, cleaning and disinfecting processes, and closed manufacturing environments further contribute to controlling cross-contamination. Most processing rooms are maintained at positive pressure to the entryways to prevent the escape of contaminants, with built-in alarms and pressure monitors to indicate differential pressure changes.
However, Advanced Cell and Gene Therapy’s Burger points out that if the tissue being processed is dangerous to personnel due to potential infections or diseases, it may be handled in a negative-pressure room. “Sometimes it’s a matter of establishing a negative-pressure process cleanroom within a positive-pressure processing suite to prevent material from being released and to protect the product from contamination.”
To prevent leftover contaminants from being transferred to new material, the cleanrooms and cabinets are cleaned and sanitized after each tissue processing batch is complete, using disinfectants and sporicides, and those cleaning steps are documented as part of the quality assurance process. Air and surface samples are taken to monitor for the presence of microbial contaminants as well as for viable and non-viable particulates before and after disinfecting.
“It’s a very proactive monitoring process,” says CryoLife’s Fronk. “On a daily basis we look for trends that might indicate a need for action.”
Burger adds that the processing rooms are kept at a bare minimum with easy-to-clean walls and surfaces, a few built-in pieces of equipment, and biosafety cabinets that are vented to the outside.
“There is a heavy reliance on pre-sterilized, single-use materials, particularly disposable closed systems, and mobile equipment,” he says. “That makes it difficult for contamination to hide.”
Packaging of the finished product is also conducted in a separate processing area to further protect the tissue from contamination during this high risk step, says Kevin Bachtel, senior manager of the tissue processing lab at CryoLife. At CryoLife, packaging is done in an ISO 7 (Class 10,000) room within an ISO 5 (Class 100) cabinet.
“We do one final microbial test at that point, then the tissue is packaged and sealed,” he says. “We want to be sure that step in the process has the cleanest handling possible. It’s critical for product release.”
Although the industry seems to have a strong grasp on managing contamination, it’s a constantly evolving process, says Burger. “This is a young field, but it’s growing fast. The field as a whole needs more support infrastructure, and a great deal of work remains to be done. The International Society for Cellular Therapy is an especially valuable resource, bringing together industry, academic programs, regulators, and standard-setting organizations.”
Bachtel notes that the progress the industry has already made in defining and validating contamination control strategies for tissue manufacturing has come about because industry leaders are so proactive and willing to work together to define the most effective control protocols for everyone involved. “The industry and the American Association of Tissue Banks are very forthcoming with best practices and good at open dialog,” he says. “That’s important because this industry is susceptible to negative press, and we need to do everything we can to protect ourselves and to protect the patients who use these products.”
References and contacts
Advanced Cell and Gene Therapy
Chapel Hill, NC
American Association of Tissue Banks
International Society for Cellular Therapy
Vancouver, BC, Canada
Musculoskeletal Transplant Foundation (MTF)
Particle Measuring Systems
University of Pennsylvania School of Medicine
Clinical Cell and Vaccine Production Facility