Class 10 cleanroom is key to safe and sterile bone transplants
Donated tissue — bone, tendons, ligaments and cartilage — is processed in a Class 10 cleanroom for use in surgical procedures.
By Susan English-Seaton
Construction is nearly complete on a brand new, state-of-the-art cleanroom facility in Edison, NJ, for the Musculoskeletal Transplant Foundation (MTF), the largest tissue bank in the country. MTF processes donors in Class 10 cleanrooms to provide implantable human allograft tissue — bone, tendons, ligaments and cartilage — for patients undergoing orthopedic surgery and neurosurgery. Started back in 1986 by a group of orthopedic surgeons who did bone transplants for cancer patients, the new facility — an unusual, octagonal suite in an office park setting — contains 2,500 ft2 of cleanroom space. The full complement includes a Class 100,000 preparation room and three separate Class 10 rooms, two Class 10,000 gowning rooms a Class 1,000 corridor, a Class 100 sterile supply room and a Class 100 packaging room.
The concern for sterility begins in the operating room where the tissue is recovered. Each piece of tissue is swabbed for microbiological testing. All tissue is kept at -70 degrees C in a quarantine state until microbiological and blood testing is completed, which usually takes several weeks. It arrives in a refrigerated state, along with the documentation, including the donor record, family history, donor screening information, medical records, autopsy record and biological tests. These microbiological and seriological tests are reviewed internally at three different levels before the tissue can be released into the cleanroom for processing. The collection and review of data to ensure tissue safety could take anywhere from three weeks to six months.
All equipment in the facility is either autoclaved or sterilized — everything from trays and cutting blades to paper records and pens. Operating room technicians, experienced in sterile handling, and a trained phlebotomist work with the primary tissue in a Class 10 cleanroom. After a preliminary washing, personnel don scrubs, and emerge into a Class 100,000 room, where smocks, shoe covers, hairnets and/or beardnets are worn. From there, they proceed into a gowning room inside the Class 100,000 room, where a full scrub procedure takes place. At this point, they will have put on three layers of apparel: scrubs, reusable sterile polyester bunny suits, full hoods, boots, goggles, masks, aprons and double-gloves in preparation for the first step in processing the tissue.
Debridement and lyophilization
In the first step, a special process called debridement is used to remove non-viable tissue from the bone surface. The heart of the process is in a custom-built Class 10 debridement chamber — a steel glovebox with a glass front — where technicians use high pressure, sterile WFI water pumped through a sterile hose to remove the bone`s soft periosteal tissue. Enclosed in an extra protective box, the tissue is placed in clamps, and water flowing at a high velocity is applied. The glovebox is essential to controlling the water level, keeping water contained inside the chamber in order to maintain a Class 10 level of cleanliness throughout the process. Pneumatic equipment such as drills and saws are powered by sterile air. Rooms are cleaned after each donor processing, and a HEPA-filtered vacuum is used to take up any water.
Because of the level and type of activity — sawing, drilling and hosing with high pressure water — air temperature is kept at 63 degrees F ۭ degrees, humidity at 50 䔮 percent RH. Arthur Gertzman, MTF`s vice president of technology, explains: “The reason we`re making it that cold is that our operators are going to be wearing first the scrub suit, the jumpsuit, then a waterproof apron over that to keep themselves dry and away from the risk of contamination from the operator to the tissue. With all that on — the three layers of clothing and standing and moving around and vigorously cutting — the temperature is important.”
After debridement, the tissue is removed from the chamber, immersed in an antibiotic solution, put on a cart and brought through a Class 1,000 corridor into the Class 10 processing room. Here, processing technicians cut the tissue according to an individual plan for each donor. Processing includes various proprietary chemical treatments, including solvents and aqueous wash steps. Each piece of bone is soaked in an aqueous antibiotic solution to deactivate any bacterial remnants. They are mechanically and ultrasonically washed to remove any remaining non-bone components, using a combination of WFI water, antibiotic solution, organic solvents and aqueous solutions in various forms.
After cutting and cleaning, the bone is air dried in a Class 10 room and each piece is swabbed for microbiological sterility before being placed in its own pre-sterilized plastic tray package. They are then prepared for lyophilization, or vacuum freezing. Pre-freezing the tissue speeds up the process, so the packages are first placed in a deep freezer in the Class 100 packaging room at -70 degrees C for 24 to 48 hours. In the lyophilizer, vacuum is applied, then a small amount of heat dries any water.
After lyophilization, the tissue is put into another quarantine chamber — under ambient conditions — and held until the microbiological swab comes back from micro-testing at a laboratory. It must be labeled as sterile, in compliance with the United States Pharmacopeia`s USP 23 Monograph, Section 71. Even at the end of the cycle, after the bone has been processed, says Gertzman, it`s very important to protect against cross-contamination from donor to donor. Therefore, cleanroom protocol dictates that tissue from one, and only one, donor be allowed in the cleanroom at any given time. Generally, the process can be completed in a 10-hour shift. The room is then broken down and all equipment is removed, washed, and autoclaved.
The design/build challenge
Air Energy Systems (Willow Grove, PA) is the design/build firm in charge of the MTF project. Sales Manager Ralph Melfi says, “The challenge was the mix of design and space requirements, utilizing the best possible components to keep the system modular, keeping cost under control for the client, and making it an easy system to service.” Design and space problems included attempting to fit the whole floorplan into an octagonal space; low ceilings, which would not allow room for a conventional ducted air system; a roof that would not support the weight of a cleanroom ceiling, an air handling unit and a 100-ton chiller; and extra heavy demands on the HVAC and water systems as a result of the low temperatures and quantity of water used in the debridement chamber. A modular cleanroom system was selected because of flexibility and cost savings, and because of the cleanability factor in meeting the FDA`s GMP requirements. Although FDA GMPs are not currently mandatory for tissue banks, the facility was built in anticipation of possible future requirements, according to Gertzman.
From a design perspective, says Melfi, the challenge was to transform plans for a rectangular-shaped facility into what was ultimately an octagon design in a rented office building, which posed its own set of problems. Once the lease was signed, the floor plan had to be reworked to accommodate the necessary room sizes, the flow of people and product, windows, etc., while working within the octagonal space. A low clearance problem — only ten feet clearance from the floor to the bottom of the steel — with Class 10 rooms and 100 percent ULPA filter coverage ruled out a conventional ducted air system, so Air Energy dealt with the static pressure of the ULPA filters and the low-profile space by installing filter fan modules.
Joe D`Aquila, Air Energy Systems` process design coordinator, says: ” The bad news was, we had no ceiling clearance; the good news is, there was a full basement, which allowed us to put the water system and related liquid equipment downstairs.” The basement also allowed room for the drain piping and looping off of the water system. The other major issue was that the roof of the building could not support either the weight of the cleanroom ceiling or the air-handling system. The solution was a self-supporting wall and ceiling system with built-in plenum, which provided 18 ft. to 60 ft. clear spans, and eliminated the need to build a steel superstructure from which to suspend the ceiling.
“The next trick was the chiller,” says D`Aquila. “The only place the landlord would allow the 100-ton chiller package to go was on the roof, with appropriate cosmetic screening. A structural steel platform had to be built and tied into the building columns to support the chiller package.”
Another design issue was the extra capacity requirements for the HVAC system, particularly in the Class 10 debridement room. Class 10 process rooms were to be positive pressure, cascading from the cleanest space outward. Based on the double-gowning procedures and some of the process requirements, a temperature of 63 degrees F ۬ degrees within the facility and 50 percent relative humidity 䔮 percent was determined as the optimum for performance. This necessitated a huge air-handling system, one that generated 63,000 cfm of recirculated filtered air, says Melfi. Because of process requirements for chilled water at the lyophilizer, it was decided to put in a 100-ton central chiller system to service the cleanroom.
During the transition from operation mode to clean mode between donor patients, 100 percent outside air must be maintained to supply HEPA-filtered air coming into the facility, and at the same time afford protection against vapor buildup within the space. Therefore, process rooms had to be set up on a 100 percent exhaust cycle. Recovery for pressure and temperature, humidity and cleanliness takes place within a very short time frame. Hence, it is imperative that personnel, rooms, and equipment be properly prepared for the next donor.
Modular construction was selected so that the entire facility could be demounted and relocated if needed — important because they were moving into leased space and would need the option to relocate. Besides the financing advantages and the depreciation factor, modular construction was chosen because the entire facility, including the windows, needed to meet GMP requirements for cleanliness. A monolithic design would afford easy cleanability and eliminate crevices that could harbor bacteria. The wall partition system features galvanized steel panels with a smooth, epoxy polyester powder-filled finish, making it chemical and rust resistant. Radius coves within the corners provide no extrusions or lips to create crevices for potential microbial growth. Also, the finished steel panels provide an inert surface against the aggressive materials used in the required full washdown procedures — a pressure wash with purified water and ethanol. To complete the monolithic design, all the floors were trowel-epoxied under the wall, providing wipedown capability uninhibited by ledges. Windows around the perimeter of the room served a double purpose: cleanroom personnel could view the outside world, while visitors on tour could see into the cleanrooms without having to gown up and enter the space, tracking in outside contaminants.
The water system
The water purification system is key to MTF`s processes. Says Gertzman: “Not only do we have the Class 10 cleanroom, which is unusual for pharmaceutical facilities — most are Class 100 — we have a water system that is based on ozonation to sterilize the water. The water system is basically filtration, ultraviolet, RO, and ozone. We`re using microfiltration, ultrafiltration, UV, two-pass RO and ozone. Our endpoint is a 0.2 micron microfilter.” A loop system ensured that the system would have no “dead legs” and that velocity could be closely controlled — two key requirements. D`Aquila says, “We treated this as though it were a WFI system, with full-loop treatment, velocity of over 7 feet per second at all times, no dead legs, slope for draining, sanitary pipe PVDF piping, in order to meet all the GMP requirements. We pre-piped the water system and the loop runs in such a direction that we could tap into it for future expansion.
The debridement station presented its own challenges: the water had to be at a very high pressure (1200 psi) but at a very low flow — about 1.5 gallons a minute — in order to strip the tissue. “It`s not like a fire hose, where you`d have a tremendous quantity of water,” says Melfi. Known as “hydrocutting,” it is analogous to deck-washing or house-washing with a high pressure washer. Because the debridement station is self-contained, water is pumped through the walls of the glovebox into the nozzles of the gun, which is held through the gloves of the glovebox. D`Aquila worked closely with the manufacturer in developing the custom glovebox technology. He says, “The unit was really built to the same standards as an isolator, but without the laminar flow aspects.” Made of shatter-proof glass and 360-grade stainless steel, it features radius coves and can be totally washed down between processes.
MTF`s practices ensure safety in tissue recovery
MTF is a not-for-profit membership organization composed of 40 medical schools, and 25 tissue recovery agencies, such as organ procurement organizations (OPOs), eye banks, and tissue banks, who recover tissue on behalf of the Foundation. A tissue donor can donate either bone, tendons and ligaments (musculoskeletal tissue), or skin and cardiovascular tissues such as heart valves or veins. In MTF`s case, musculoskeletal tissues are processed, then delivered back to hospitals to be used in surgical procedures such as spinal fusion or to replace portions of limbs afflicted with cancer of the bone. In 1996, it is estimated that over 500,000 tissue transplants were performed in the U.S. alone compared to 16,000 organ transplants.
Unlike organs such as kidneys, hearts and livers, musculoskeletal allograft tissue is transplantable to other humans with minimal rejection, eventually becoming biointegrated into the body. According to Arthur Gertzman, MTF`s vice president of technology, this allows MTF and other tissue banks to make a large number of allografts available to thousands of recipients throughout the U.S. “You don`t have the problem that you have with livers, hearts and kidneys. Patients do not have to go on anti-rejection drug therapy. That`s the end point. The beginning is, how do we get the donors? How do we make sure we have safe tissue?”
Sterility and screening key
Sterility and safety of the tissue is of primary concern. Donors undergo an extensive screening process to ensure that no infectious diseases, such as AIDS or Hepatitis, are transmitted. Also ruled out as a potential donor is anybody who has been treated for cancer, used IV drugs, or had a tattoo that is less than a year old. (Tattoo parlors are not regulated, and needle reuse may spread Hepatitis.) Says Gertzman, “Our goal is to ensure that all of our processes, from donor screening to seriological testing through chemical and physical processing, eliminate the possibility of disease transmission. We don`t rely on just one part of the process to make sure that our tissue is the safest we can make it, we take every step possible throughout the chain of events to keep the tissue, clean, safe, and sterile.”
Some of the Foundation`s rules are mandated by the FDA, while others were established years ago by the American Association of Tissue Banks, a voluntary standard-setting association of organizations similar to MTF. In addition, MTF has devised many of its own protocols and rules, which are even more stringent than those mandated by FDA, says Gertzman. Martha Anderson, vice president of donor services, says, “We are driven by an orthopedic/academic perspective. Our protocols and criteria are devised by the orthopedic surgeons using the tissue. This allows us to provide the kinds of tissues they need for their patients. We are also driven by the need of donor families to know that their selfless gifts bring new health to tissue recipients, and we guard this gift very carefully.” — SES
The HVAC system, including DDC control system with PC monitoring. Extra capacity requirements in the Class 10 debridement room meant that a temperature of 63 degrees F ۬ degrees and 50 percent relative humidity 䔮 percent had to be maintained throughout the process.
The 2-door pass-thru autoclave affords service access from outside the cleanroom.
The monolithic design and open cavity return allow total room washdown. Window details allow for visibility in every room.
The Class 10 process room features roomside-accessible ULPA filters, variable speed control, all 316L stainless steel sinks and tables, GMP cove details, and point-of-use sterile air and water.