Processing must not change the physical properties of the tissue so as to make it unacceptable for clinical use. Processing steps must be validated to demonstrate that the final product does not have any clinically significant residual toxicity.
Facilities for aseptic processing must comply with the Rules and Guidance for Pharmaceutical Manufacturers and Distributors 20075 and EC Guidelines to Good Manufacturing Practice.6 They must provide separate work areas with defined physical and microbiological parameters. Facilities must have:
Critical work areas are those where tissue is manipulated openly either following a disinfection or sterilisation step or in those cases where tissue has been procured aseptically and will not be further disinfected or sterilised. Critical work areas on which sterile containers, aseptically procured tissue or disinfected tissue are exposed to the environment, must have an air quality of Grade A and should have a Grade B background. (For further information see Rules and Guidance for Pharmaceutical Manufacturers and Distributors 20075 and the EC Guidelines to Good Manufacturing Practice.6) Any lowering to this standard in the background environment (as long as it is compliant with EU requirements) must be documented and it must be demonstrated that the chosen environment achieves the quality and safety required, at least taking into account the intended purpose, mode of application and immune status of the recipient.
Wherever possible, representative samples of tissue should be removed and tested for bacterial and fungal contamination using protocols authorised by the designated medical officer or designated microbiologist. Swabs or other validated non-destructive sampling methods should be used where it is impossible to remove tissue without damaging the graft. Microbiological inclusion/exclusion criteria should be developed by the designated medical officer or designated microbiologist in accordance with national policy.
Where tissues are processed in batches, procedures must ensure that no cross-contamination between batches can occur. Key process parameters and acceptance limits must be identified and validated. A full record of each process applied to each tissue or batch must be retained.
Work areas in which tissue materials and containers are prepared should have an environment with air quality of at least Grade C in the vicinity of exposed tissue.
Terminal antimicrobial processing must follow the filling of the final container. The procurement, processing and filling environment must be of sufficient quality to minimise the microbial contamination of the tissue to ensure that the subsequent antimicrobial processing is effective.
The tissue in its final container must be subjected to a validated procedure utilising an agent such as gamma irradiation. The processing method and dose of the sterilant should be validated as sufficient to bring about at least a six logarithms reduction in a recognised marker resistant organism (e.g. Clostridium sp. for irradiation).
Sterilisation is a statistical phenomenon, expressed as the probability of microorganisms surviving the procedure. The sterility assurance level (SAL) is the probability of a microorganism on one item within a batch or within a defined population. The accepted level for considering medical devices to be ‘sterile’ is a SAL of 10–6 (i.e. less than one item per million items will have a surviving microorganism on it). For medical devices, the microorganisms under consideration are contaminants (i.e. bacteria and fungi and their spores). Unless specifically stated, viruses are not routinely considered.
Because of the large numbers involved, demonstrating SAL of 10–6 must use procedures that extrapolate from smaller batches. For sterilisation procedures that show a log10/linear decrease in microbial viability, extrapolation can be achieved using the D-value (decimal reduction value) concept.
Figure 21.1 An example of increasing inactivation of bacteria related to increasing the dose of the sterilant
In the example shown in Figure 21.1, each log reduction requires an additional unit of the sterilant to be applied, hence D-value = 1.0. Therefore moving from an initial bioburden of 106 bacteria to a SAL of 10–6 would require 12 × D-value of the sterilant.
In practice, the processing that is applied to tissue grafts prior to application of the terminal sterilisation step often reduces the bioburden to close to zero. Therefore application of a sterilisation procedure sufficient to provide a 6-log reduction of bacteria is often satisfactory to achieve a SAL of 10–6.
Very often, validation studies will be carried out using the microorganism that is known to be most resistant to the sterilisation procedure (often bacterial spores). This is therefore a ‘worst-case’ validation. Achieving a SAL of 10–6 for this microorganism will guarantee a significant overkill for more sensitive microbes.
Whenever a novel terminal sterilisation step is introduced the following validations need to be addressed:
Gamma irradiation must be performed in a controlled manner to ensure that all tissue receives at least the minimum specified dose of radiation. This requires the use of standard packaging materials and irradiator load configuration and is usually validated using calibrated dosimeters placed throughout the load. The dose should never be less than 15 kGy, unless pre-irradiation processing has been validated to consistently yield a low microbial bioburden such that there is the required assurance, in accordance with medical device standards, that the dose will result in the tissue being sterile.
Tissue must be irradiated in its final packaging, which must bear a suitable indicator to demonstrate that it has been irradiated. This must be checked before release of the tissue.
If a dose in excess of 25 kGy is required, then consideration must be given to the possible detrimental effect on the biological and physical properties of the tissue.
Many viruses are resistant to irradiation and therefore any claim of viral inactivation must be supported by validation data obtained using appropriate marker viruses.
Pooling of tissues from different donors is not permitted.
Where specific attributes of a tissue are claimed, the process should be validated to show these attributes are preserved.
For the purposes of this guidance this term applies to tissues that are frozen and stored under conditions that are unlikely to be compatible with preservation of cells. Frozen tissue must be stored below –20°C and the length of storage permitted depends on the temperature the tissues are stored at (see Table 21.1).
For the purposes of this guidance this term applies to tissues that are treated with a cryoprotectant and/or cooled at a controlled rate in order to preserve cells. Cryopreserved tissue must be stored below –135°C. For storage at higher temperatures, validation must be performed to demonstrate that the required properties of the graft are maintained for the stated expiry.
Where tissues are freeze-dried, a sample of each type of tissue from each freeze-drying run must be analysed for residual water which must be less than 5% (weight/weight) of the dry weight of the graft or equivalent residual water activity of between 0.2 and 0.5 Aw.
Where tissues are preserved by high concentrations of glycerol the procedure should be validated to demonstrate achievement of the specified glycerol concentration within the tissue or an acceptable range within the tissue.
Rinse solutions, antibiotic mixtures, nutrient media and cryopreservation solutions must be stored at a specified temperature and with a storage period consistent with functional requirements. They must be discarded if not used within 24 hours of opening. Any solutions coming into direct contact with tissues during retrieval or processing must be sterile.