Because of the vulnerability and short shelf life of harvested human tissue, isolation is one of the key strategies for ensuring its sterility

By Sarah Fister Gale

Tissue engineering is still a relatively new field that has only developed over the last ten to fifteen years. Described as regenerative medicine or cell therapy, tissue engineering involves the combination of live and/or synthetic cells that are manipulated using biochemical techniques and additives. To produce more complex tissue-engineered products, harvested human cells are sometimes implanted onto scaffold material, which enhances the cells’ structural properties, delivers biochemical factors or cell nutrients, or exerts mechanical or biological influences to modify the behavior of the cells or tissue. The resulting biological products can be used in medical procedures to promote skin, bone or tissue growth, support organ function and create other medical miracles that were previously not possible.

The first and most important contamination concern in the tissue engineering industry is the integrity of the raw materials. Unlike other industries that use prepackaged sterile materials or ingredients that have been certified clean as the foundation for their manufacturing processes, tissue engineers base their end products largely on live tissue harvested from human organs, skin, bone cartilage and other living human materials.

Click here to enlarge image

Because it is viable, tissue has a short and vulnerable shelf life and must be processed as soon as it’s harvested. Maintaining sterility during processing is critical, yet challenging, as conventional sterility testing can take weeks to complete. As a result, most biotech firms are forced to collect, process, and store each individual piece of tissue and resulting end product under the assumption that it could potentially be contaminated. It must therefore be kept completely isolated from other processing materials to ensure strict contamination control and lot segregation.

Isolation is mandatory

Whether manufacturers are producing complex cell therapies as treatments for existing diseases, or procuring and processing tissue for use in clinical applications, isolating and testing any raw tissue used in the manufacturing process is a critical first step. While all human tissue is evaluated for bacteria and viruses prior to use, every tissue engineering facility has additional testing guidelines tofurther explore and verify the results of previous tests. These tests primarily look for antibody-derived human viruses, such as HIV, says Jim Embree, senior vice president of development and manufacturing for Nephros Therapeutics, a cell therapy company in Lincoln, Rhode Island.

Nephros produces Renal Tubule Assist Devices (RAD) to treat multi-organ failure associated with Acute Renal Failure. The product is based on the renal tubular precursor stem (progenitor) cell, which plays a central role in the maintenance of metabolic and endocrine equilibrium and the management of a patient’s immune surveillance and defense systems. To create the RAD, Nephros begins with viable human kidneys that cannot be used for transplants. Operators isolate and expand the kidney-derived stem cells from the kidney’s cortex for therapeutic use concurrent with dialysis.

Even though the kidneys used by Nephros are initially tested for viral entities when harvested, there is a window of time after exposure to a virus in which tests result in a false negative, Embree points out. Therefore, Nephros also conducts donor background assessments to identify potential risk factors that might indicate a greater likelihood for certain viruses. “For patient safety and to meet our own patent criteria, it’s important that we do exhaustive testing,” explains Embree.

The viral and bacterial tests are conducted by a third party testing company outside the Nephros facility immediately upon receipt of the kidneys. Using an external testing company further isolates the Nephros manufacturing areas from potentially contaminated tissue. Once the initial
testing has been completed, the kidneys are delivered to Nephros for processing. However, because it can take up to nine weeks for tests results, each kidney is considered a risk and must be isolated from other raw tissue and final
products to avoid potential cross-contamination. To
maintain this isolation, every kidney is processed in its own Class 100 biosafety cabinet, which resides in a Class 100,000 cleanroom with standard air locks, relative pressure to protect the environment, and interlocking doors.

Once inside the cabinets, the kidneys are primarily manipulated by human operators. They begin by isolating and removing the kidney’s cortex, which is rich in the proximal tubular cells, through a surgical-like procedure. The cortex is then macerated to release the cells, which are sieved into a disposable sterile T Flask. This process is repeated four times to collect the maximum number of cells, then the flasks are moved to a 37°F CO2 incubator for seven to ten days. During the incubation period, operators perform two changes of media in the cabinets to verify and maintain sterility of the cell cultures.

Once the cells have expanded, they are moved back to the biosafety cabinet where they are trypsinized to disrupt their adherence to the plates and transferred to double the number of plates for further expansion in the incubators. The cells undergo this transference four times before they are ready for harvesting.

The cells, and ultimately the final RAD device, remain isolated throughout the entire manufacturing process.

“The entire manufacturing process for the kidney is kept separate from any other kidney tissue, ensuring that no cross-
contamination will occur,” Embree says. And, no product is released for use until test results prove it is free from any harmful contaminants.

The finished product is stored in a Class 100,000 cleanroom in a 37°F incubator with a closed loop sterile tubing system that recirculates media. Twice a week, it is moved to a biosafety cabinet for media changes and the removed media is tested to ensure ongoing sterility.

In the final step of the process, the cells from 120 plates are aseptically inoculated into the intracapillary space of a hollow fiber membrane in a monolayer using a sterile disposable syringe. The product is then ready for use in the treatment of patients experiencing acute renal failure.

Ultimately Nephros would like to move to a completely automated, closed-system manufacturing process in an
effort to remove human error from the equation. “Fatigue is always a factor in contamination,” Embree says. “When you take the people out of the process you remove fatigue and achieve uniformity of results.” He hopes to accomplish this goal in the next year and a half. In the meantime, Nephros puts operators through extensive training and employs all disposable tools, from gloves and sleeves to petri dishes, culture flasks and screw top gamma radiated containers, to reduce cross-contamination risk.

Short shelf life makes sterility testing tricky

Fortunately for Nephros, under its current maintenance guidelines, its RAD device can be stored for five months before use. But for other tissue engineering facilities, the short shelf life of the end product prohibits lengthy
storage and creates final testing challenges, says Gary C. du Moulin, vice president of quality systems at Genzyme, a biotech firm in Cambridge, Mass., whose tissue engineering division developed Carticel (autologous cultured chondrocytes), a cell-derived product used to regrow a patient’s own cartilage cells to replace damaged cartilage in the knee; and Epicel (cultured epidermal autografts), an autologous cell therapy for severe burn victims.

Genzyme employs a patient’s own cells to develop products with unique properties designed just for their use. Carticel uses cells derived from “tic-tac-sized” cartilage biopsies taken directly from an injured patient’s knee. The biopsies are received in closed tubes filled with media and antibiotics. Each biopsy is carefully placed in a Class 100 (ISO 5) biosafety cabinet in a Class 10,000 (ISO 7) cleanroom (see Fig. 1), where all processing and manipulation by trained technicians occurs (see Fig. 2). The necessary cells, called chondrocytes, are isolated and placed in cell culture flasks. Once capped, they are moved from the cabinet into a dry incubator to avoid risk of bacterial growth.

Instead of using humidified CO2 incubators, which require a pan of water, Genzyme prefers a dry environment and uses media that can grow without the presence of moisture, says Rick DiOrio, director of engineering for Genzyme. “We are very strict about water in the cleanroom. Standing water is a great place for bugs to develop.” The only water that is allowed into the room is used for cleaning. “We really drill that home with all of our operators.”

Once the cells are grown, expanded and manipulated, the final Carticel product has a shelf life of only 72 hours and each vial of cells receives a date and time stamp to
ensure freshness. “Carticel must be put into the patient before the shelf life expires,” du Moulin says. Yet, the final sterility test, which is a required part of their standard operating procedure, takes 14 days. “It’s a unique aspect of this field. Since we don’t fit typical regulatory paradigms for biological products, we have to work closely with the FDA on a product-by-product basis.”

Like Nephros, Genzyme tests each patient’s cell culture after the first cell expansion and technicians conduct up to three additional sterility tests during the roughly three week manufacturing process, ensuring that no bacteria have contaminated the cell culture during production. “Basic sterility tests are somewhat problematic because of the short shelf life of the product,” du Moulin says. “That’s why the cleanroom environment is so critical. The adage ‘You have to build quality in, you can’t test it out’ has never been more important.”

As with most tissue engineering facilities, the biggest contamination risk for Genzyme is contaminated patient tissue. Because sterility tests take so long, and because Genzyme handles nearly three thousand samples per year and processes up to one hundred samples on any given day, it is vital that each sample of cartilage be kept completely separate from other tissue. Each sample is given two forms of identification, besides the patient’s name, that can be traced directly to the patient to ensure that every final product is derived entirely from cells harvested from that individual.

Besides contaminated tissue, another major contamination concern for Genzyme is the possibility of human error in the facility. “This process can’t be automated. At certain points you have to open the flasks and manipulate the cells, and people can sometimes contribute to a contamination event,” says John Hefferman, vice president of operations for Genzyme.

To reduce and control those errors, Genzyme operators go through an intensive three- to six-month program that includes classroom and performance-based training in aseptic technique and testing before an operator ever works with a patient’s tissue. The company also has a vigorous daily personnel monitoring system that tracks who performed which operations on which product lots, and includes regular monitoring of performance. When operators are linked to a contamination problem, they go through additional training in aseptic technique and they work one-
on-one with the supervisor to understand the risks of certain behaviors and to improve their performance techniques, says Michelle Eldridge, Genzyme’s training manager.

Regular cleaning and sanitizing of the cleanroom and biosafety cabinets further ensures sterility. Genzyme has a dedicated team of five trained cleaners who are
responsible for maintaining the cleanroom. The floors are sanitized and biohazard waste is removed on a daily basis; the walls are cleaned and sanitized weekly; and the ceilings are cleaned quarterly, all with a rotating supply of chemicals to prevent bacteria from building resistance to a cleaner. Twice a week the quality control group takes hundreds of samples from the floors, walls and equipment surfaces to verify sterility and the environmental monitoring system constantly samples air quality.

If a spike in bacteria growth does occur somewhere in the cleanroom, pinpointing the cause can be difficult. By the time a positive test result is observed, the implicated area may have gone through two or three disinfecting cycles, du Moulin says. “It’s a continuous process.”

What tissue engineers dream about

Ideally, du Moulin imagines finding a new way to conduct analytic testing that isn’t based on incubating and growing samples, such as a marker that immediately indicates the presence of bacteria. “If we had an assay that could determine the sterility of a vial of cells before it’s even packaged, that would be the Holy Grail,” he says. Genzyme is already working with testing vendors to find faster testing techniques. Because the test results from the final inspection are not received until well after the medical procedure is complete, Genzyme’s quality control organization has spent over four years developing an automated sterility testing technology that can detect a microbial contaminant in approximately 72 hours.

Du Moulin also hopes the industry will soon come together to create a set of cleanroom standards specific for tissue engineering that acknowledge and accommodate the manufacturing needs of these unique processes. He is optimistic about the imminent implementation of the FDA’s Good Tissue Practices (GTPs) to regulate the manufacturing of human tissue products. The final rule, entitled “Current Good Tissue Practice for Human Cell, Tissue, and Cellular and Tissue-Based Establishments,” becomes effective on May 25, 2005. It requires manufacturers who recover, process, store, label, package, and distribute tissue to use safeguards to prevent introduction, transmission, or spread of communicable diseases. Hospitals and surgery centers would fall under the rule only if they procure and “manufacture” tissue but not if they simply transplant tissue.

The rule also requires that tissue have a unique identifying code that can be tracked from the donor to the consignee and that significant adverse reactions and manufacturing deviations be reported to the FDA.

Figure 1. Carticel Class 10,000 Cleanroom. Source: Genzyme Click here to enlarge image

“Hundreds of new cell therapies are developed every year, and most will require a cleanroom setting,” du Moulin says. “A set of global standards would allow us to harmonize the cleanroom with our manufacturing requirements to create the safest environment we can.”

Figure 2. Carticel technician in a biosafety cabinet. Source: Genzyme Click here to enlarge image

Until then, Genzyme relies on its cycle of sterility tests, extensive training and monitoring, and a well-maintained cleanroom to avoid accidental contamination-and it works. In its six years of producing Carticel for more than 10,000 patients, Genzyme has had a contamination rate of less than 0.06 percent. If a contamination event occurs, Genzyme’s procedures require immediate contact with the surgeon, who will keep a close eye on the patient.

Figure 3. Encapsulated Cell Technology (ECT) products for the treatment of retinal diseases. Source: Neurotech USA Click here to enlarge image

Working with live tissue restricts sterilization options for cell therapy end products, notes Bill Tente, vice president of manufacturing and quality systems for Neurotech USA (Lincoln, RI), a biotechnology company that is currently developing Encapsulated Cell Technology (ECT) products for the treatment of retinal diseases (see Fig. 3). “Our products can’t be terminally sterilized, which is common for any cell-based product,” he says. “That’s why our company depends on robust aseptic procedures for raw materials, processes and people.” (See Fig. 4.)

Figure 4. Strict contamination control guidelines are followed during processing of ECT products. Source: Neurotech USA Click here to enlarge image

Neurotech has rigorous qualification specs for bioburden and endotoxin levels in any raw materials such as culture media, biological fluids, and other products used in the manufacturing process. Relying on highly reputable vendors is the key to building quality in from the beginning, says Tente. “It’s challenging to manufacture products in the cell therapy industry,” he says. “You don’t want to deal with someone who doesn’t have a track record in early development, and you
really don’t want to deal with the unknown.”

Tente prefers to avoid vendors who specialize only in research products because they may not have systems in place to provide the high sterility assurance necessary for commercial products. He chooses vendors, such as Invitrogen (Carlsbad, Calif.), an ISO-certified materials supplier, based on reputation and preliminary visits to assess the facility’s ability to meet his quality needs. Neurotech verifies all the testing provided by the vendor, paying particular attention to sterility and endotoxin results.

Invitrogen won Tente’s approval with its strict aseptic policies, operator training, environmental monitoring processes, and raw materials testing program. “When we procure raw materials we look for USP grade,” says Rick McAvoy, business area manager for Invitrogen. Before it purchases materials, Invitrogen performs a four-tiered quality audit and uses Good Manufacturing Practices (GMPs) to verify results. “Not everyone does this, but it adds assurances to the quality of our products,” he says. “That’s what the customers want.”

Creating that quality chain in the materials supply further reduces the potential for product contamination down the line, and if something does happen, the paperwork is available and the communication lines are open, making it easier to solve the problem, he says.

Microbiology experience preferred

Once materials are in-house, Neurotech imposes strict guidelines for contamination control during processing. “The cleanroom is our battleship,” Tente says. “We make sure it always runs smoothly and we protect it at all costs.”

Neurotech manufactures its cell therapy products in Class 100 laminar flow units in a Class 10,000 cleanroom. Its manufacturing processes are based on the successful production model employed in live viral vaccine manufacturing. Currently Neurotech’s process is semi-automated, using proprietary equipment to inject sterile cells into the interior of a small capsule, which is then sealed with a light-cured glue-all within the laminar flow unit. In a separate unit, the capsule is placed in a package filled with tissue culture media. A secondary sterile foil is then heat-sealed over the top of the primary package, which is then placed into a secondary outer package.

Once the product is finished, storing it in tissue culture media further helps to verify sterility and prevent contaminated products from being administered. If bacteria are present there is a very high probability that they will bloom in the storage media, Tente says. “We train our quality assurance staff as well as end users at clinical sites to look for indicators of microbial contamination, such as the presence of turbidity or a change of color in the media.” The color change occurs when a pH indicator in the fluid detects by-products of microbial growth. For comparative purposes, a color sample chart is provided with the product to facilitate inspection.

Neurotech has also built in a long enough shelf life to allow for results from the 14-day final sterility test to be returned before the product is used. The company assures that if a problem does arise, the capsule, which is inserted directly into a patient’s eye, can be easily removed. “We have a lot of confidence in our aseptic processes,” he says, “but if there were ever an unanticipated infection or adverse event as a result of our product, it could be retrieved in a simple procedure.”

Eventually, Tente would like to automate the entire manufacturing process because, he says, “The easiest way to reduce risk is to eliminate human contact.” When that’s not possible, training and experience are the best ways to eliminate error, he says, adding, “We’ve been very lucky with employees. The company inherited a group of seasoned veterans when it
acquired the intellectual property from the original company involved with ECT.” And of the few employees Neurotech has since hired, most have Bachelor of Science degrees with tissue culture background and a good understanding of
microbiology. “I feel it’s important, not just to have experience in processing tissue, but also with contamination
control,” Tente says. “That’s a product of the coursework and training for people in the microbiology field.” III