Disinfection and Sterilization of Living and Scaffold Tissue Engineered Medical Products
Kelvin G.M. Brockbank
Alyce Linthurst Jones
The use of human allogeneic tissues, such as arteries, bone, heart valves, skin, tendon, veins, and organs in medicine has become standard practice. Allogenic refers to biological material from the same species with a different genetic composition, with an allograft being biological material intended for transplantation into another individual of the same species. In contrast, autologous is related to self (or belonging to the same organism), where autografts are biological material intended for implantation into the individual from whom they were recovered. Finally, xenograft s are from nonhuman animal source and implanted into a human recipient. Although clinical demand for organs and tissues continues to grow, the supply of these valuable human resources is a limiting factor. The United Network for Organ Sharing reported over 33 000 organ transplants in 2016, which represents a 20% increase since 2011.1 The American Association of Tissue Banks (AATB) estimates that there are more than 39 000 deceased tissue donors annually in the United States and more than 3.2 million tissue grafts (allografts) are implanted annually2; however, there remains over 113 500 people in the United States alone waiting on the organ transplant list and the number of people awaiting tissue grafts is not maintained. As a result, the development of scaffolds and living engineered constructs has become an important discipline in biomedical science. Many tissue engineered products are under development, including wound covering and repair systems, orthopedic tissue repair systems, encapsulated tissues, cardiovascular products, and organs on a chip. Tissue engineering in this context refers to the development, design, and implantation of devices consisting of materials and living cells to replace defective or diseased organs and tissues. The long-term goal of tissue engineering is essentially to being able to replace or repair virtually every tissue and organ system. Four examples of marketed living engineered tissue products are Apligraf® (Organogenesis, Inc, Canton, MA) for wound repair, CarticelTM (Genzyme Tissue Repair, Inc, Cambridge, MA) for articular cartilage repair, DeNovo® NT Natural Tissue Graft by Zimmer Biomet (Warsaw, IN) (particulate, juvenile human cartilage for the repair of articular cartilage damage), and ViviGen® Bone Matrix (LifeNet Health, Virginia Beach, VA) for new bone regeneration (cryopreserved, viable cortical cancellous bone matrix, and demineralized bone). Two of these engineered tissues are co-formulated with a cellular component of either an allogeneic material with different genetic composition within the same species (Apligraf®) or autologous material (CarticelTM) along with one or more natural or synthetic biomaterials. Apligraf® is a bilayered human skin equivalent consisting of an epidermal layer made of human keratinocytes (the cells forming the keratinized epidermal layer of skin) and a dermal layer made of human fibroblasts (the most commonly found cell in connective tissues and are responsible for the synthesis of collagen, extracellular matrix, and can differentiate into a variety of cells types), in a bovine collagen matrix. CarticelTM is an implant consisting of concentrated autologous human chondrocytes (differentiated cells found in all types of cartilage) in culture medium that is injected beneath a periosteal flap into an articular cartilage defect.3 DeNovo® NT is a minimally manipulated tissue product, regulated as a 361 human cells, tissues, and cellular and tissue-based product (HCT/P) by the US Food and Drug Administration (FDA) and not a medical device or biologic. It purports its efficacy in repairing articulating joints based on a large density of chondrocytes due to the donors being juveniles.4 Additionally, there are many tissue engineered products that are regulated as 361 HCT/Ps under the US 21 CFR Part 1271 and do not require premarket approval prior to commercial availability. Some examples are live bone products (Osteocel®, Trinity Evolution®, and ViviGen®) used to stimulate new bone formation through osteoconduction (support new bone formation by providing an environment in which preexisting osteoid cells capable of synthesizing and secreting components essential to the formation lead to growth of
new host bone), osteoinduction (the ability of a substance to stimulate cells to differentiate along some osteoprogenitor pathway resulting in those capable of synthesizing and secreting components essential to the formation of bone), and osteogenic (the presence of cells capable of synthesizing and secreting components essential to the formation of bone) properties of the bone in orthopedic, spine applications, and trauma repair. Decellularized dermis is another tissue engineered product requiring no premarket approval in the United States (AlloDerm®, DermaMatrix®, DermACELL®, FlexHD®, and AlloMaxTM) for breast augmentation postmastectomy, burns, soft tissue augmentation, and wounds. The FDA held a panel meeting for the General and Plastic Surgery Devices on March 25 to 26, 2019, indicating that premarket approval may be required in the future for decellularized dermis indicated for breast reconstruction because its homologous use for this indication is in question.5
new host bone), osteoinduction (the ability of a substance to stimulate cells to differentiate along some osteoprogenitor pathway resulting in those capable of synthesizing and secreting components essential to the formation of bone), and osteogenic (the presence of cells capable of synthesizing and secreting components essential to the formation of bone) properties of the bone in orthopedic, spine applications, and trauma repair. Decellularized dermis is another tissue engineered product requiring no premarket approval in the United States (AlloDerm®, DermaMatrix®, DermACELL®, FlexHD®, and AlloMaxTM) for breast augmentation postmastectomy, burns, soft tissue augmentation, and wounds. The FDA held a panel meeting for the General and Plastic Surgery Devices on March 25 to 26, 2019, indicating that premarket approval may be required in the future for decellularized dermis indicated for breast reconstruction because its homologous use for this indication is in question.5
REGULATION OF HUMAN TISSUESHuman tissues are regulated in the United States by the FDA in the Center for Biologics Evaluation and Research. This jurisdiction and the regulations are outlined in 21 CFR Part 1271 and were promulgated May 25, 2005.6 These products may have different or similar regulatory pathways in other parts of the world. For instance, in Canada, some tissues are similarly regulated, where tissue bank are assigned a Human Cells, Tissues and Organs number to include in their labeling7 and in other stances, like cryopreserved heart valves, they are considered class IV medical devices. In the European Union, tissues only require registration, but some member states can require additional information above and beyond the European Union Tissue and Cells Directives.8 Germany and India are special cases where human tissues are regulated as drugs, which can be exceptionally complex in compliance given how different drugs are from human tissues in that there is no active pharmaceutical ingredient in tissues.
The US regulations are often referred to as Good Tissue Practices (GTPs). FDA has issued many guidance documents to provide its current thinking on the implementation of 21 CFR 1271 across such topics as donor screening and eligibility,9 minimal manipulation, homologous use, use of donor screening tests, current GTPs, small entity compliance guide, and validation.10 Allografts that are regulated as 361 HCT/Ps are deemed to be
Minimally manipulated, for homologous use.
The manufacture of the HCT/Ps does not involve the combination of cells or tissues with another article, except for water, crystalloids, or a sterilizing, preserving, or storage.
The HCT/P does not have a systemic effect and is not dependent on the metabolic activity of living cells for its primary function.
The HCT/P has a systemic effect or is dependent on the metabolic activity of living cells for its primary function and (1) is for autologous use, (2) is for allogeneic use in a first-degree or second-degree blood relative, or (3) is for reproductive use.
These products are regulated under section 361 of the US Public Health Services Act as 361 HCT/Ps. If the tissue does not meet all four of these definitions, then it is either regulated in the United States as a device or a biologic; however, the tissues are still subject to the rules and regulations outlined in 21 CFR 1271. To further elaborate on two key definitions, minimal manipulation and homologous use, the FDA has further defined these two attributes.11 Minimal manipulation is (1) for structural tissue, processing that does not alter the original relevant characteristics of the tissue relating to the tissue’s utility for reconstruction, repair, or replacement, and (2) for cells or nonstructural tissues, processing that does not alter the relevant biological characteristics of cells or tissues, as defined in 21 CFR 1271.3. Homologous use means the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor, 21 CFR 1271.3. Therefore, Center for Biologics Evaluation and Research regulates products whose primary mode of action is the result of its cellular component, such as cultured cartilage cells where the cells are expanded in culture, whereas a product whose primary mode of action is structural in nature, such as a decellularized heart valve, is regulated by the Centers for Devices and Radiological Health.
The safety standards for tissue engineered products are driven by their regulatory status; however, one unifying theme for human tissues, regardless if regulated as a 361 HCT/P, a medical device, or a biologic, is that they do not introduce, transmit, or communicable diseases. Therefore, products without viable cells are often terminally sterilized using a validated sterilization dose via electron beam or γ irradiation (see chapter 29).12 Grafts with viable cells cannot be irradiated without killing the constituent cells via formation of free radicals and/or direct damage to the DNA that is unable to be repaired (see chapter 5). For example, the typical cumulative dose of irradiation for a breast cancer patient is 50 Gy, and a typical absorbed dose for tissue being terminally sterilized is 15 kGy or 15 000 Gy; thus, mammalian cells that absorb this much irradiation are likely to be necrotic and proinflammatory once implanted as a result of the lethality of the terminal sterilization dose. Often allografts are irradiated on dry ice to minimize free radical damage from free water in the product, thereby overcoming many of the limitations reported in older literature (1970s-1990s) of the loss of functionality or early failure because the tissues were γ irradiated at 25 kGy (2.5 Mrad) at ambient temperatures. In addition to irradiation in the presence of dry ice, there are other strategies that may be employed.
The traditional terminal sterilization requirement of a sterility assurance level (SAL) of 10-6 is giving way to less-stringent SALs as a compromise to achieve a sterility claim for regenerative medicine products but at a dose or treatment process that does not negatively impact the product for its intended surgical purpose (see chapter 5). The difference between SALs is in the theoretical probability of a viable microorganism, such as in 1 in a 1000 for an SAL 10-3 versus 1 in a million for an SAL 10-6. Guidance documents such as International Organization for Standardization (ISO)/TS 19930 and, specific for radiation processes, AAMI TIR 76 provide some instructions for alternatives.13,14 The AAMI TIR 76 provides instruction supplementary to the radiation sterilization standard ISO 11137 for additional options for VDmax doses and charts to achieve alternative SALs (10-5, 10-4, 10-3, and 10-2) using lower doses of irradiation.12 The radiation validation method 1 (see chapter 29) can also be used to achieve alternative SALs to 10-6 like 10-3, but at this time, this approach requires minimally 139 samples (30 bioburden, 100 verification dose, 6 for method suitability testing to validate the test of sterility, and 3 for bioburden recovery efficiency test to determine the correction factor to be applied to bioburden count) to execute verses 29 for the VDmax protocols (10 bioburden, 10 test of sterility, and the balance of 9 for the aforementioned test method validations). To achieve the lowest possible dose at a desired SAL, method 2B can be used; however, it requires around 300 samples to execute the validation as described in AAMI TIR 37.15 To determine the optimal validation strategy and SAL, knowledge of the maximum absorbed dose the product can withstand through functional testing such as biomechanical integrity or other appropriate functional testing is important. The range between the minimum absorbed dose, maximum absorbed dose, dose uniformity ratio, and the correction factor to a reference location in the carrier must be considered because commercial irradiators generally require a 35% spread between the minimum and maximum specified delivered doses (minimum or maximum dose location in the product payload container multiplied by the reference to minimum ratio or reference to maximum ratio). Many considerations for terminal irradiation of allografts are further discussed in guidances such as AAMI TIR 37.15 The other option for nonviable and viable grafts is end point microbiological testing of the lot that conforms to the United States Pharmacopeia (USP) chapter 71 or 21 CFR Part 610.12 to demonstrate no bacterial growth after a 14-day incubation period in appropriate media.16,17 But it is important to note that USP <71> microbiological testing is intended to be an adjunct to a validated process demonstrated not to introduce bioburden and to reproducibly disinfect the graft. One significant difference between processed tissue-based products and pharmaceuticals is that the tissue entering the process is not sterile, whereas pharmaceutical components are typically sterile and need only be aseptically combined without cross-contamination. USP <71> testing generally involves testing 10% of the lot aerobically and 10% of the lot anaerobically. There are strategies that involve incubating the same sample at 25°C to 30°C for 3 to 5 days and then moving the sample to 35°C for the remaining 9 days on test (see chapter 64). Samples can be assessed through direct immersion into nutrient media or rinsates, filtered and the filter placed on agar or nutrient media for 14 days. The final result from USP <71> testing is often mistakenly referred to as demonstrating an SAL of 1 × 10-3, but this is incorrect. It is only appropriate to use SAL in the context of products that have been terminally sterilized by a validated method in the final packaging and not handled until needed for implantation. USP <71> can provide the probability of a nonsterile unit, which is generally 10-3 (1 in 1000), and not to be confused with a SAL 10-3 because it is typically arrived upon by conducting an aseptic fill validation and not having a positivity rate of more than 1 in 1000. This level of performance for aseptic tissues is highly unlikely—only 1 in 1000 donors found to be culture-positive during end point microbiological testing.
To provide additional guidance to the allograft tissue community, the AATB has established standards and guidance documents that provide minimum performance requirements for all aspects of tissue banking activities.18,19 These standards and guidance documents include requirements for donor suitability, handling of transplantable human tissue, and a guidance document focused on microbial surveillance testing of donor tissue and environmental monitoring of dedicated recovery suites as well as processing clean rooms.19 The intent is to ensure allograft tissue recipients receive disease and contaminant-free implants and optimal clinical performance of transplanted tissues.
Regardless of whether the transplantable material is a US 361 HCT/P allograft, 351 HCT/P allograft, or a tissue engineered medical device, recipient safety must be ensured by starting with the administration of strict donor screening criteria along with stringent quality control measures that encompass the entire tissue preparation protocol and highly controlled environments, such as an ISO 14644-1 class 7 cleanroom or cleaner that are designed for unidirectional flow from clean to dirty to minimize cross-contamination.20 The gowning of processing technicians has advanced from gowns, gloves, and surgical masks to surgical togas and contained head gear with fans to reduce skin contaminants from entering into the sterile field.21 Various tools and aseptic training techniques can be used to reduce cross-contamination risks in these areas (see chapter 58). Overall, approaches involve employing the essential requirements in ISO 13485 for quality management systems and ISO 14871 for risk management to ensure that high-quality and microbiologically safe grafts are accomplished and brought to market with the risk to the patient as low as reasonably practicable and thus the risk-benefit ratio being in the patients’ favor.22,23
ENGINEERED TISSUE GRAFTSThere is no current need for modification from existing best clinical practices in the autologous situation, in which cells or tissues are removed from a patient and transplanted back into the same patient in a single surgical procedure. The FDA recently issued guidance on this subject.24 If the autologous cells or tissues are banked, transported, or processed with other donor cells or tissues, then there exist opportunities for the introduction of transmissible disease. When this additional manipulation occurs, good manufacturing practices and GTPs should be implemented and it becomes necessary to screen for infectious agents (21 CFR 1271 Subpart C: Donor Eligibility).6 For example, in the case of the CarticelTM process, in which biopsies of healthy cartilage are used as a source of chondrocytes, the biopsies are minced, washed, and cultured with cell culture medium containing antibiotics. As prescribed in USP <71>, method suitability testing, previously referred to a bacteriostasis/fungistasis (B/F) testing, must be performed prior to using a nutrient growth-based culture method to confirm the culture negative status of the lot for at least detectable bacteria/fungi (see chapter 64). To confirm that the test method will not result in a false-negative result at low numbers of bacterial colony forming units (CFUs), tissue products processed at the maximum residuals of antimicrobials (maximum concentration, maximum exposure time, and minimum rinses) are placed into nutrient media and inoculated with 10 to 100 CFUs of typical human flora and assessed for growth within five day of inoculation.16 The turbidity of the test grafts must closely approximate the turbidity of the positive control flasks that contain no tissue but were inoculated with the same number of CFUs. Other methods such as filtration of the final solution are also appropriate; however, the time the tissue is incubated prior to the fluid being tested should be validated with a dwell time study to ensure if there were microorganisms present, they would be removed from the tissue into the fluid during the extraction period. A microbiologist should be consulted to ensure proper conductance of these studies. Incorrect performance of method suitability testing could give in false-negative culture results and the potential for disease transmission in the recipient.
Tissue Grafts and Prevention of Infectious Agent Transmission
All tissues, whether they be allografts, autografts, and/or engineered grafts, should be delivered to patients with the highest possible assurance that they are free of pathogens. The most effective and common methods for the prevention of infectious agent transmission are thorough donor screening, donor serological testing for relevant viruses, donor tissue microbiological testing, and adherence to sterile techniques during procurement, transport, processing, and terminal sterilization if applicable. There are six pillars to ensure tissue safety (1) medical suitability determination through a detailed medical history, physical examination, and detailed social history; (2) serology testing for viral diseases such as human immunodeficiency virus (HIV), hepatitis B, hepatitis C, and the bacterial disease syphilis; (3) microbiological testing of recovered tissues prior to disinfection; (4) validated processing to clean and disinfect tissues; (5) validated end point microbiological testing or a validated terminal sterilization process; and (6) validated packaging to maintain the sterile barrier for the shelf life of the graft.
In United States, the first step in confirming eligibility of a potential donor is to obtain authorization for organ and/or tissue donation from the donor’s legal next of kin. This is the case even if a potential donor has registered as an organ and tissue donor with the Department of Motor Vehicles, Donate Life State Registry, various health applications (eg, on mobile phones), or legal agreements such as advanced directives. Alternatively, authorization may be obtained via telephone on a recorded line and documented as such in the donor record. Once authorization for donation is obtained, the donor must be screened to minimize the potential for disease transmission as a result of health status, travel, social influences such as drug and alcohol, or sexual orientation. The AATB, for example, has developed a standardized process for its member banks to use, the Uniform Donor Risk Assessment Interview (DRAI) that ensures compliance with FDA’s requirements.25 Some AATB member banks add additional questions to the DRAI to aid medical directors in making medical suitability determinations. These determinations are living documents and account for emerging infectious diseases such as Zika virus. The DRAI consists of flowcharts, guidance documents, and questionnaires to assess medical history, behavioral history, travel history, and social history. The DRAI also covers a range of age groups, newborns, donors younger than 12 years, and those 12 years or older. This age range accounts for maternal contribution to the disease/health status of the child and in the instance of children younger than 12 years there are differences in hemodilution (explained in the following text) calculations due to their size. The DRAI is completed by a knowledgeable historian such as a family member or partner and assessed in conjunction with the donor’s medical records. The next step in assessing medical suitability is a physical examination of the donor by the recovery teams prior to recovery as detailed in Appendix III of the AATB standards.18 Some of the elements of this are, but not limited to, conclusive identification of the donor and authorization for donation, documentation of trauma, nonmedical needle punctures, presence of jaundice, presence of infection in tissue recovery areas, skin lesions, enlarged lymph nodes, enlarged liver, genital and anal lesions, tattoos, and piercing. The next critical element in determining donor suitability is serological testing for viral load
using FDA-approved/cleared test kits for use on cadaveric samples by laboratories that are registered with the FDA as a tissue establishment to perform infectious disease testing and that hold a current Clinical Laboratory Improvement Amendments certification. Prior to testing, the donor record is reviewed for evidence of hemodilution, which if present would result in the donor’s blood being significantly diluted (>2 L) by blood donor products, colloids, or crystalloids within 48 hours of asystole. Getting an accurate assessment of viral load in the donor under such conditions is impossible with current technology, and thus, the donor tissue is discarded. It would be difficult to demonstrate freedom from relevant communicable virus with deference to the window period where virus is present in the blood below the detection limit of the nucleic acid test (NAT), which is maximally 60 days for hepatitis C. But once the specimen is determined to be suitable, the following tests shall be performed:
using FDA-approved/cleared test kits for use on cadaveric samples by laboratories that are registered with the FDA as a tissue establishment to perform infectious disease testing and that hold a current Clinical Laboratory Improvement Amendments certification. Prior to testing, the donor record is reviewed for evidence of hemodilution, which if present would result in the donor’s blood being significantly diluted (>2 L) by blood donor products, colloids, or crystalloids within 48 hours of asystole. Getting an accurate assessment of viral load in the donor under such conditions is impossible with current technology, and thus, the donor tissue is discarded. It would be difficult to demonstrate freedom from relevant communicable virus with deference to the window period where virus is present in the blood below the detection limit of the nucleic acid test (NAT), which is maximally 60 days for hepatitis C. But once the specimen is determined to be suitable, the following tests shall be performed:
Antibodies to the HIV, type 1 and type 2 (anti-HIV-1 and anti-HIV-2)
NAT for HIV-1
Hepatitis B surface antigen
NAT for the hepatitis B virus
Total antibodies to hepatitis B core antigen (anti-HBc—total, meaning immunoglobulin G and M)
Antibodies to the hepatitis C virus (anti-HCV)
NAT for hepatitis C virus
Tests for syphilis (a nontreponemal or treponemalspecific assay may be performed)
All tissue from donors who test repeatedly reactive on a required screening test shall be quarantined and shall not be used for transplantation.
The next step in reducing the risk of disease transmission is determination of the microorganisms on the tissue postrecovery, prior to the use of an antimicrobial or other solution that could obscure a positive result, false negative. Theoretically, other than skin, the tissue should be sterile; however, surface contamination can and does occur during recovery in spite of detailed and thorough disinfection of the donor skin and aseptic practices employed during donor recovery (Appendix IV, AATB Standards18). These risks become greater in the recovery of animal tissues (eg, in an abattoir), as compared to a surgical suite. The AATB maintains a list of objectionable microorganisms “considered to be pathogenic, highly virulent microorganisms that shall result in tissue discard unless treated with a disinfection or sterilization process validated to eliminate the infectivity of such organisms.” Those organisms include Clostridium species, fungi (yeasts and molds), and Streptococcus pyogenes (group A streptococci). Some tissue types have additional specific high-risk microorganisms as well, such as with cardiovascular tissues.26 Additionally, each member bank can develop a list of additional organisms that their medical directors and laboratory professionals find to be objectionable in spite of the bank’s validated cleaning, disinfection, and possible terminal sterilization validations. The development of such a list should take the cleaning and disinfection reagents, concentration, contact time, and temperature into account as well as the susceptibility of the organisms to method of terminal sterilization and pathogenicity should there be survivors detected. The FDA and AATB require all processing and sterilization activities to be validated through the demonstration of documented, objective evidence. Terminal sterilization validations are verified minimally on an annual basis per applicable ISO standard to ensure the susceptibility of the remaining organisms to the mode of sterilization has not changed.
Many tissue processors have proprietary or in-licensed cleaning and disinfection processes to help ensure the safety of the allografts distributed for implantation. One of the most widely used is the Allowash® technology that was developed by Lloyd Wolfinbarger, PhD, at LifeNet Health Inc (Virginia Beach, VA) and was protected by multiple US patents. Given the interest of allograft safety in the United States, this process was further out-licensed to other tissue banks. The Allowash® process had demonstrated superior cleaning and bactericidal/virucidal capabilities, which was of the upmost concern in at the height of the HIV epidemic in the 1980s and 1990s. The process involves 3% hydrogen peroxide, nonionic detergents, antibiotics, isopropanol combined with centrifugation, ultrasonics, and elevated temperatures to provide the desired cleaning, disinfection, and adventitious agent removal. Another major cleaning and disinfection process developed to address the threat of disease transmission and improve throughput efficacy with the hope of donor pooling was the BioCleanse® process, developed by RTI Surgical Holdings Inc (Marquette, MI).27
Ultimately, in these cases, the initial goal of donor pooling was not realized due to FDA requirements (defined in 21 CFR 1271.220) that prohibit the pooling of donor tissues.6 The rationale for this was if a pathogen does get through screening, serology, processing, and/or terminal sterilization by limiting each processing event to one donor, the harm to recipients is finite and limited by the number of grafts one donor can produce that range anywhere from 1 to in excess of 500 if demineralized particle bone for dental implants are considered. These regulations represent the state of science at the time they were developed, the 1990s, and may not properly consider the many technological advances in disease detection, treatment, and efficacy of terminal sterilization processes; however, they have prevented the spread of disease as the result implantation of allograft tissues.
The next step in providing microbiologically safe tissues is terminal sterilization that can be achieved through several methods. The use of physical or chemical inactivating methods has been supported by laboratory data and clinical experience.28,29,30,31 Despite this, the primary means of terminal sterilization today uses low
dose, <20 kGy/2.0 Mrad, electron beam irradiation or γ irradiation at dry ice temperatures to reduce free radical formation and prevent damaging the tissues (see chapter 29). The international standards, ISO 11137 and associated standards in that series, define the minimum requirement for the definition, validation, and maintenance of radiation sterilization methods.12 There are at least two mechanisms by which ionizing radiation is effective (see chapter 5).32 First, it causes lethal strand breaks in the nucleic acids of the infecting microorganism rendering it incapable of proliferation or infectivity. Secondarily, irradiation induces several sublethal chemical alterations through free radicals and their propagation in an aqueous, room temperature environment that collectively become lethal. The required dose of γ radiation to achieve sterility varies with the number and types (radiosensitivity) of microorganisms (bioburden) present on the tissue and may also be impacted by the temperature at which sterilization is performed (see chapter 29). As discussed previously, an SAL of 10-6 is the typical validation target for such processes, but alternative SALs may be acceptable. Because absolute sterility is not achievable, it is determined through mathematical probability of a single organism surviving based on the collective D10 (dose of irradiation to cause a 90% decrease the number of viable microorganisms on a graft) value for a diverse population of organisms. The ISO 11137 standard refers to this concept as “population C.”
dose, <20 kGy/2.0 Mrad, electron beam irradiation or γ irradiation at dry ice temperatures to reduce free radical formation and prevent damaging the tissues (see chapter 29). The international standards, ISO 11137 and associated standards in that series, define the minimum requirement for the definition, validation, and maintenance of radiation sterilization methods.12 There are at least two mechanisms by which ionizing radiation is effective (see chapter 5).32 First, it causes lethal strand breaks in the nucleic acids of the infecting microorganism rendering it incapable of proliferation or infectivity. Secondarily, irradiation induces several sublethal chemical alterations through free radicals and their propagation in an aqueous, room temperature environment that collectively become lethal. The required dose of γ radiation to achieve sterility varies with the number and types (radiosensitivity) of microorganisms (bioburden) present on the tissue and may also be impacted by the temperature at which sterilization is performed (see chapter 29). As discussed previously, an SAL of 10-6 is the typical validation target for such processes, but alternative SALs may be acceptable. Because absolute sterility is not achievable, it is determined through mathematical probability of a single organism surviving based on the collective D10 (dose of irradiation to cause a 90% decrease the number of viable microorganisms on a graft) value for a diverse population of organisms. The ISO 11137 standard refers to this concept as “population C.”
It may be suggested that freedom from viral contamination cannot be assumed under validated SAL conditions with radiation because this has been particularly demonstrated with bacteria and to a much lesser extent with viruses. Some viruses may have higher D10 values than do bacteria,33 but also bacteria themselves can range in radiation sensitivity (see chapter 29). The reduction of viruses is also considered a mathematical probability; however, the kinetics of kill (as with other microorganisms) can vary relative to the antimicrobial agents and the substrate involved and thus do not lend themselves as readily demonstrable as the mathematical modeling that occurred with bacteria and fungi (as the basis in ISO 11137).12 As an example, there is an international standard defining requirements for viral inactivation/removal validation, ICH Q5A (R1).34 The first step is to identify the relevant viruses or model viruses to test and then identify those steps in the tissue processing regime to test keeping in mind solutions with the same mode of kill, for example, oxidation, may not have their levels of log kill combined because the virus may become recalcitrant to solutions with the same mechanisms of action. The ideal outcome is a 6 log10 reduction above, which is expected on the incoming tissues achieved through two to three different steps. Minimally, regulatory agencies prefer to see at least 4 logs of kill/removal for each relevant virus. For human tissues, the relevant viruses/model viruses represent a variety of DNA and RNA viruses, as listed in Table 50.1.
TABLE 50.1 Examples of relevant model viruses used in demonstrate viricidal efficacy in tissues | ||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ||||||||||||||||||||||||||||
Briefly, after the appropriate method, validations for toxicity and ensuring the method of inactivation can be stopped (neutralized) to get an accurate log kill (see chapter 61), the tissue is seeded with a known amount of virus, exposed to one step in the process, and the amount of virus remaining on the tissue and solution are determined through plaque formation assays with susceptible cell lines. Ideally, this is repeated over multiple time points to determine the consistency of the inactivation kinetics and whether or not it is first order. With respect to kill by γ irradiation, many researchers have tried to determine the amount of radiation lethal to HIV. Results have been equivocal and range from 10 to 40 kGy.32,35,36 Given the structure of HIV, it is not considered to be highly resistant to inactivation (see chapter 3). The γ radiation doses greater than 25 kGy have been recommended to inactivate most other viruses, including those of similar structure and nucleic acid type to HIV; therefore, procedures have been developed that incorporate multiple antiviral treatment methods in order to produce the optimum possibility of a disease-free graft. It should be recalled that bacterial and viral safety are part of a continuum starting with donor screening, medical, behavioral, and social history as well as bacteriological/fungal testing of the recovered tissues and viral testing of the donor plasma and serum. During chemical treatments, each viral inactivation step will not only depend on the chemical type and exposure process conditions (including concentration, time, and temperature) but may also involve physical removal through centrifugation, ultrasonic cavitation, or
other mechanical means. If there are multiple steps that inactivate by the same mechanism, such as oxidation, only the kill from one of the oxidation steps can be counted toward total kill per the ICH guidance. The reason for this, despite its limitations, is the suggestion that viruses can become recalcitrant to kill from the same mechanism of action in subsequent steps and thus cannot be reliably counted on to result in the presumed kill. This approach is considered very conservative.
other mechanical means. If there are multiple steps that inactivate by the same mechanism, such as oxidation, only the kill from one of the oxidation steps can be counted toward total kill per the ICH guidance. The reason for this, despite its limitations, is the suggestion that viruses can become recalcitrant to kill from the same mechanism of action in subsequent steps and thus cannot be reliably counted on to result in the presumed kill. This approach is considered very conservative.
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