The Principles of ProcessValidation and EquipmentQualifi cation Martell Winters1 and Richard Lomas2
1Nelson Laboratories, Salt Lake City, UT, USA 2NHS Blood and Transplant, Liverpool, UK
7
Introduction
This chapter explains the principles of validation and qualifi cation as applied to cell and tissue banking, and, by the use of worked examples, demonstrates their practical application. To understand the importance of validation, the meaning of the term must fi rst be clearly understood. In its current Good Cell and Tissue Practice (cGTCP) guidelines, the United States Food and Drug Administration (FDA) defi nes process validation as:
Establishing by objective evidence that a process consistently produces a result or human cell/tissue product meeting its pre -determined specifi cations [1].
A similar defi nition is employed in European Directive 2006/86/EC:
Validation (or “qualifi cation ” in the case of equipment, environments or person-nel) means establishing documented evidence that provides a high degree of assurance that a specifi c process, piece of equipment or environment will con-sistently produce a product meeting its pre -determined specifi cations and quality attributes; a process is validated to evaluate the performance of a system with regard to its effectiveness based on intended use [2].
Key words in both these defi nitions are “consistently” and “predeter-mined”. It is well established that cell and tissue grafts that meet prede-termined criteria of safety and quality are profoundly life -enhancing, and in some cases life -saving. Conversely, grafts that do not achieve these cri-
The Principles of Process Validation and Equipment Qualifi cation 125
teria can have profoundly negative effects on the health of the recipient. Validation (for processes) and qualifi cation (for equipment) is the method of demonstrating, both to ourselves and to the regulatory authorities, that the protocols reliably and consistently produce grafts with these criteria.
While validation and qualifi cation have always been good scientifi c and clinical practice, they are also increasingly a statutory requirement. In Europe, the Cell and Tissue Directives require that all critical equipment and technical devices be identifi ed and qualifi ed, and that all critical processing procedures must be validated so as not to render tissues and cells clinically ineffective or harmful to the recipient. In the USA, the FDA cGTCP rule requires establishments to validate processes that cannot be verifi ed through subsequent inspection and testing, and that the validation activities and results be documented [3].
There are also operational advantages resulting from robust process vali-dation. It can reduce or obviate the requirement for in -process or fi nished tissue monitoring, which can require the destructive testing of a proportion of the grafts from each production run, for example for microbiology assess-ment. If a process is validated to consistently achieve the desired result, the assessment requirement may no longer exist and a greater proportion of the donated material will be available for clinical use.
Process validation has a crucial role in insuring that the grafts supplied by an establishment are safe and fi t for purpose, is a key tool to improve effi -ciency and reduce wastage, and as such must be a core function for any cell and tissue bank.
Validation and qualifi cation principles
Validation versus qualifi cation In performing process validation and equipment qualifi cation it is important to clearly distinguish between the concepts of validation and qualifi cation. The introduction provided a defi nition for process validation which can also be used for equipment qualifi cation. Unfortunately it is too common to use the term “validate” or “validation” any time that testing is being performed to evaluate a process, equipment, environments, personnel, or a product. Thus, the clarifi cation in the introduction that validation is performed on a process whereas qualifi cation refers to equipment, environments, and per-sonnel is appropriate.
The term “validation” can be used whether the evaluation is being per-formed on one step of a process (e.g., a particular soaking step) or an overall process. It should be noted, however, that after validation of process steps it is still expected that the overall process be validated.
In practice, equipment qualifi cation entails an evaluation of the variables which exist in the equipment to determine acceptable operational ranges
126 Tissue and Cell Processing
for those variables. Once the acceptable set points have been determined, it should be demonstrated that the set points remain valid regardless of when they are observed (e.g., beginning of morning shift, end of evening shift, after a weekend, etc.). This determination should involve evaluation of the set points at those various times to verify their appropriateness.
Research and development and process characterizationIt is also important to understand is the difference between the terms “research and development, ” “process characterization, ” and “validation”.Other terms are often used to describe these activities and the intent here is not to standardize the terms used, but to clarify the different activities that occur in each of these phases. As validation has been addressed previously, the other two are addressed below.
In the research and development phase of a product the primary purpose is to try many options to determine what provides the best result to the process or to the product. Specifi c protocols are usually not required to allow for fl exibility in the trial and error process, but good documentation is critical so that the data generated can be used in subsequent phases.
Process characterization takes the rough process determined in research and development and prepares it for validation. This is the time to evaluate the effects the process variables have on the process or product (e.g., time, temperature, concentration, pH, etc.). Usually specifi c protocols are gener-ated and the protocols may focus on the variables of individual steps rather than an entire process together.
Alternatively, if the potential variables or procedures in a process are already defi ned, it is possible that research and development, process charac-terization, and validation can be designed to occur concurrently. In this case a protocol must be in place before the evaluations are begun. In a situation where research and development and process characterization are already well understood, it becomes possible to write the protocol with suffi cient detail at an earlier point in the process and it is less likely that much trial and error will occur.
The point here is not to require that these three phases be followed strictly, but to clarify the idea that going directly from process or product concept to validation, without appropriate evaluations to understand the variables, may result in frustration or failure.
Benefi ts of process validationPerforming process validation allows for great fl exibility regarding how a process is evaluated on a routine basis. Historically it has not been common to validate tissue processing, and as a result the primary method of assurance that the process is functioning properly was to test a certain percentage of processed tissues for sterility (typically 10%). Although this practice was and in some cases still is common, it must be understood that without proper
The Principles of Process Validation and Equipment Qualifi cation 127
data it is diffi cult to provide a correlation between the 10% that was tested and the 90% that was not.
Process validation allows for a more scientifi c determination regarding which process aspects best represent the microbiological quality of the batch of fi nished tissue. Under this approach it may be possible not to test any tissue after processing. Rather, the process validation data may indicate that other aspects of the process are a better process indicator than testing 10% of the processed tissue.
For example, the process validation data may indicate that the fi nal rinse
solution, in which the entire batch of tissue is soaked before packaging,
provides better microbiological data on the entire batch compared to individual
tissue testing and therefore more accurately demonstrates the microbiological
quality of that batch. In this situation it may be appropriate to routinely test
the fi nal rinse solution for microorganisms rather than a certain percentage of
fi nished tissues.
For example, the process validation data may indicate that that there are three
key aspects of the process which best describe the microbiological status of the
tissue: testing of rinse solution #2, testing of tissue trimmed off during Step
5, and fi ve pieces of companion tissue after packaging. In this case, all three
aspects of the process are tested on a routine basis but no fi nished tissue that
could be clinically used is tested.
Validation data may indicate that more than one aspect of the process should be tested to obtain an accurate vision of the fi nished tissue microbiol-ogy. Ideally these aspects which are tested should be something other than tissue that could be clinically used. This may result in more microbiologi-cal testing than was performed previously, but it should also result in more tissue being distributed rather than being used in testing.
Process validation complexityIt is not necessary that every aspect or variable of a process be characterized or validated individually. A particular step of a process may be important but because of its lack of complexity or because there is no need, it may not be evaluated as an individual step and may simply be verifi ed as appropriate by being included in the overall process validation. This concept is also applied to equipment qualifi cation when one or more operational ranges are estab-lished without evaluation in an effort to simplify the qualifi cation process.
In both process validation and equipment qualifi cation, simultaneous assessment of multiple variables can cause studies to increase in complexity. Selection of which variables should be addressed in validation/qualifi cation
128 Tissue and Cell Processing
and which can be established without evaluation can be a useful tool, but must be done carefully. It is acceptable to refer to pre -existing data and basic scientifi c principles to narrow down the number and range of variables using a risk assessment approach.
Risk assessmentRisk assessment has become an increasingly useful tool in process validation and equipment qualifi cation. There are many approaches to risk assessment but basically the process is broken into its many components, the risk associ-ated with each component is considered, and a value is applied to each one. This allows for determination of which components of a process are most critical to evaluate and how to prioritize their evaluation.
In a perfect world it would be nice to validate all aspects of every process. However, reality dictates that one works with limited resources and decides how best to utilize them. A useful tool, both to prioritize validation studies and to decide which aspects of a process should be evaluated, is risk assess-ment. The risk assessment process is detailed elsewhere in this book, but can be described concisely as quantifying the risk of events happening, and the severity of the consequences of these events, to decide if particular events need to be considered and if so, the priority in which they should be consid-ered. This is perhaps best demonstrated with an example.
Table 7.1 shows an example of a risk assessment matrix. There are two components to the matrix: the risk of an event occurring and the sever-ity of the consequences if the event occurs. Numerical values are assigned to likelihood and consequence, which when multiplied together produce a risk fi gure for that event; the higher the fi gure, the higher the priority for addressing it. Note that the values assigned to action and priority are for this example only. Those values must be determined based on the situation and by those involved.
Likelihood 0 –3, no action required; 4 –6, low priority; 7 –12, high priority.
Table 7.1 Example of a risk assessment matrix
Consequence Risk likelihood
Negligible (0) Very unlikely (1)
Unlikely (2) Likely (3)
Mild (1) 0 1 2 3
Moderate (2) 0 2 4 6
Severe (3) 0 3 6 9
Catastrophic (4)
0 4 8 12
The Principles of Process Validation and Equipment Qualifi cation 129
Table 7.2 Examples of calculated risk for heart valve allografts
Risk Likelihood Consequence Risk figure
Priority
Biomechanical properties of the valve are adversely altered
Unlikely (2) Catastrophic (4)
8 High
Burst pressure of the vessel is adversely altered
Unlikely (2) Catastrophic (4)
8 High
Suture pullout strength of the tissue is adversely altered
Very unlikely (1)
Catastrophic (4)
4 Low
The viability of the tissue is reduced
Unlikely (2) Moderate (2) 4 Low
Residual toxic processing chemicals present
Very unlikely (1)
Moderate (2) 2 No action
Next, the process is considered and the risks defi ned. In this example the process is for banking heart valve allografts, using antibiotic decontamina-tion followed by cryopreservation for long -term storage. Having mapped out the process and considered how it might affect the properties of the graft, fi ve ways are identifi ed in which the processing may damage the tissue. It is then considered how likely it is that the damage might occur, and what the consequences might be of the damage occurring. Then the results of the Likelihood and Consequence columns are taken to the matrix to determine the priority. This is by its nature a subjective process, best accomplished by seeking as wide an opinion as possible from experts in the fi eld. Having com-pleted the analysis, the results can be summarized (Table 7.2).
The assessment indicates that in validating the overall process, the bio-mechanical properties and burst strength of the vessel should defi nitely be validated. The suture pullout strength and tissue viability should also be vali-dated, but as a lower priority, and it is not necessary to validate the residual toxic processing chemicals.
Sterility assurance levels and log reductions
Upon completion of process validation from a microbiological perspective, it is common to have obtained log reduction values for particular micro-organisms. It is often misunderstood that the number of log reductions obtained directly corresponds to the sterility assurance level (SAL). It is important to understand the distinction between log reductions and SAL.
130 Tissue and Cell Processing
Sterility assurance levelsUse of the term SAL is only appropriate when a sterilization cycle has been implemented, whether it be chemical (liquid or gaseous) or ionization (radi-ation) based, and when the microbial effect of the sterilant has been charac-terized to be predictable with respect to time or dose. The predictable nature of a sterilant allows for extrapolation to a theoretical value (i.e., the SAL) based on empirical data (data actually obtained from testing using a frac-tional cycle or dose).
Other documents (e.g., Association for the Advancement of Medical Instrumentation (AAMI) and International Organization for Standardization (ISO) 11137 [4] for radiation sterilization and AAMI/ISO 14160 [5] for liquid chemical sterilization) describe appropriate methods for determina-tion of sterilization cycles, so it is not covered in detail here. Suffi ce it to say that although microbiological log reduction data are critical to understanding the capability of a process, it does not automatically result in an equivalent SAL value.
Additive nature of log reductionsAlso often misunderstood is the idea that log reductions obtained from various process steps are automatically additive. This is often not the case, for the following reasons (at a minimum):
• The mode of microbial inactivation of the various process steps may be similar enough that additional inactivation is not obtained beyond a certain level.
• The effectiveness of the various process steps may vary based on the type of microorganisms tested. For example, process A may show greater effec-tiveness against microorganism Q, but process B may show greater effec-tiveness against microorganism R. In this situation addition of the highest log reduction value obtained from each process is not appropriate.
Generally, if log reductions are determined to be additive, it is appropri-ate to verify their additive nature in a study which includes all processes in question.
For example, this could be accomplished by fi rst determining the most
resistant microorganism to each process step. If this determination results in
more than one type of microorganism, all microorganism types should be
used in the evaluation. Tissue could be inoculated with a known number
of these most resistant microorganism(s) and subjecting the tissue to the
entire process followed by testing of the tissue to determine how many
viable microorganisms remain. This data can demonstrate the microbiological
reduction capability of the entire process for each microorganism in question.
The Principles of Process Validation and Equipment Qualifi cation 131
In this type of test it is critical to demonstrate that proper neutralization has occurred and to understand the effi ciency of the microorganism removal process (recovery effi ciency). The recovery effi ciency process is explained in AAMI/ISO 11737 - 1:2006 [6] .
Sterile l abel c laims In most parts of the world it is valid to apply a sterile label claim to a product under three primary circumstances. The fi rst two options are common and are described in various AAMI/ISO documents. The last is not as common, but is also described in AAMI/ISO documents.
1. Application of a validated terminal sterilization process to a product which is sealed in its fi nal packaging (e.g., AAMI/ISO 11137 for radiation sterilization).
2. Application of a validated aseptic process where all product components are sterilized in some manner prior to assembly or fi lling (i.e., AAMI/ISO 13408 - 1 and - 7) [7, 8] .
3. Application of a validated liquid chemical sterilization or disinfection process followed by a validated aseptic sealing process (i.e., AAMI/ISO 14160 and AAMI/ISO 13408 - 1 and - 7).
Usually a disinfection or decontamination process on its own does not qualify for a sterile label claim. In these cases the appropriate label claim, if any, is dictated by the country or countries in question.
It is also important to distinguish between use of the term “ sterile ” based on a validated sterilization process and its use on the basis of culture - negative sterility test results (also called destructive testing) of processed, packaged tissue. In the former approach the term “ sterile ” is properly used and is clear, but in the latter approach use of the term can be misunderstood. In the USA there has been a push to more clearly distinguish the proper use of the term and this will likely result in a clearer defi nition of the term as well as having specifi c requirements in place in order to use the term on a label.
Practical a spects of v alidation
There are many aspects to a process validation and it can be easy to over-look some aspects that must be considered. The proper way to ensure that the process validation covers all important aspects is to have a validation plan in place before initiating the validation. Its authors should include, at a minimum, personnel with responsibility for quality, procurement, produc-tion, distribution, and marketing as these groups can all provide good input regarding the fi nished output of the process.
132 Tissue and Cell Processing
Sample size and microorganism panelTwo important aspects of the validation plan are determination of sample sizes and the panel of microorganisms to be used for testing. There is no interna-tionally established sample size to be used for process validation, but testing in replicates of 3 –10 tissues or samples per test is common. Determination of sample size per test can be based, for example, on the quantity of batches to be evaluated, the criticality of the data being gathered, the availability of the tissue or sample in question, and/or how much previous or concurrent data on the process are already available. Although statistics can be used to determine an appropriate sample size based on the expected quantity of tissues per batch, it is more common not to use statistics but rather to make the determination based on other information, such as those items men-tioned previously.
The microorganisms used in a process validation can have a signifi cant impact on the results, thus the microorganisms must be selected appro-priately. Most process validation approaches do not have a pre -establishedmicroorganism panel; rather, it must be determined by the individual proc-essor. Most regulatory bodies prefer to have, at a minimum, all main types of microorganisms present in the panel: i.e., a Gram -positive coccus, a Gram-positive rod, a Gram -negative rod, a yeast, and a mold. Most of those selected are aerobic organisms, but it is also important to evaluate anaerobes, Clostridium and Propionibacterium being among the more common.
Some microorganisms have the ability to form spores and the spore form of a microorganism will be inactivated differently than its vegetative form (usually they are more resistant). Thus if a microorganism to be used can form spores, a conscious decision must be made whether to include the microorganism in its spore form or its vegetative form and the testing must be performed accordingly.
It is also common to consider selecting commonly occurring microorgan-isms from either the tissue and/or the environment. In most cases these can be considered while selecting which microorganisms are to represent the main types (e.g., Gram -negative rods, as mentioned earlier), in an effort to keep the number of microorganisms in the panel to a minimum.
This selection process may result in more microorganisms being selected than is desirable from a fi nancial or tissue availability standpoint. It is not required that all microorganisms selected in the panel be used for the entire validation. If early studies in the validation demonstrate clearly that one or two of the microorganisms in the panel are more resistant than the others, it is possible to rationalize discontinuing use of the less resistant microorgan-isms as the validation progresses.
The last consideration in selecting the panel is based on whether the process in question has an established or understood most resistant micro-organism. If the process being validated is known to be particularly resistant to a specifi c microorganism it may be benefi cial to also include that micro-
The Principles of Process Validation and Equipment Qualifi cation 133
organism in the panel. On the other hand, inclusion of this microorganism may be too much of a worst -case scenario and may not represent the types of microorganisms expected to occur in the process, so this approach should not be used without good rationale.
A determination of which microorganisms to use in a process validation might follow steps as in the following example.
1. The following microorganisms are commonly found on the tissue or in the
environment:
a. E. coli (Gram -negative rod) b. P. aeruginosa (Gram -negative rod) c. E. faecalis (Gram -negative rod) d. E. faecium (Gram -negative rod) e. S. aureus (Gram -positive coccus) f. M. luteus (Gram -positive coccus) g. C. albicans (yeast) h. B. cereus (Gram -positive rod, spore -former)i. B. thuringiensis (Gram -positive rod, spore -former)j. C. perfringens (Gram -positive rod, spore -former, and anaerobe)
2. Selecting from these microorganisms to obtain a panel including all general
microorganism types results in:
a. E. coli (Gram -negative rod) b. S. aureus (Gram -positive coccus) c. C. albicans (yeast) d. B. cereus (Gram -positive rod, spore -former)e. C. perfringens (Gram -positive rod, spore -former, and anaerobe)
3. In the literature it is well explained that spores are the most resistant
type of microorganisms to the process being evaluated. Spore -forming
microorganisms (i.e., Bacillus and Clostridium) are found on the tissue and
in the environment, so they should be included in the microorganism
panel for the validation. It is critical that the state of the spore -forming
microorganisms be defi ned for use in the test (e.g. spore -forming
microorganism in its spore state).
Acceptance criteriaThe validation plan must also include established acceptance criteria. In most cases these acceptance criteria are not pre -established values and must be determined based on the expected or desired results of the tissue bank. Acceptance criteria for a process may include various facets. For example, a process validation may be evaluating microbiological log reductions, lack of addition of new microorganisms (from the environment and personnel), and specifi c physical characteristics (e.g., consistency of collagen structure).
134 Tissue and Cell Processing
In this example, each of these critical facets must have specifi c acceptance criteria established in the validation plan. Note, however, that one should not enter a process validation being blinded as to what the general outcome should be for these facets. In research and development and in process char-acterization the general outcome of the process for each critical facet is being addressed. The data from these initial studies should help in understanding the ability to obtain the acceptance criteria.
This lack of established acceptance criteria is usually not the case if a sterile label claim is desired. Although it is possible to obtain a sterile claim on tissue which is not terminally sterilized, (e.g., by aseptic processing; see AAMI/ISO 13408-1), it is most common that a sterile claim is based on a terminal sterili-zation process (e.g., by irradiation; see AAMI/ISO 11137 -1). In both of these cases, requirements or guidance is established in international standards and specifi c national standards may also be in place.
Conditions of a validation studyWhen planning a validation study, it is important to ensure that the protocol accurately refl ects the process as it is actually performed. When working in a controlled laboratory setting, it is tempting to work to tight tolerances that may not be practical in a “real world ” situation. For example, when validat-ing a transportation container, working in an air -conditioned laboratory at ambient temperature will not necessarily refl ect the extremes of tempera-ture that the container may be exposed to while being transported.
The principle of modeling worst -case conditions should be applied as a matter of course to all aspects of validation design, including temperatures, times, reagent specifi cations, and so on. It is vital, especially where there is a division between the staff who perform validation work and the operational staff who routinely perform a process, that operational staff approve the vali-dation protocol as being an accurate refl ection of what is capable of being achieved operationally.
In the same way that Good Manufacturing Practice (GMP) principles should be applied to the production of all cell and tissue products, the prin-ciples of Good Laboratory Practice (GLP) should be applied to all validation studies. This may include the following:
• All equipment used for validation studies should be appropriately cali-brated and maintained.
• The specifi cations and batch number of any reagents and consumables used should be recorded.
• All information and data should be recorded contemporaneously and signed by the operator and manager responsible for the study.
• Any variation in the data generated should also be considered, and the number of replicates used in any follow -up studies adjusted accordingly.
The Principles of Process Validation and Equipment Qualifi cation 135
As a general rule, each study should be performed with at least three rep-licates, or repeated at least three times as appropriate. If high variation is encountered, this number may need to be increased to ensure that valid results are obtained.
Commercially available processes In situations where a new or custom process is being implemented, it is appropriate to validate that process from beginning to end. However, there are instances where personally performing full validation of a process may not be necessary.
In the tissue banking world there are some commercially available proc-esses that have been completely validated by the manufacturer. Usually the manufacturer provides validation details along with the purchased product. In this circumstance full validation at the tissue bank may not be necessary. It may be possible to only perform verifi cation that the process functions as indicated by the manufacturer. If the in -house verifi cation data agrees with the manufacturer ’s validation data, it is safely assumed that the process is functioning as intended and that it is providing the full process benefi t to the tissue.
It is usually not advisable to implement a validated, commercially avail-able process without any on -site verifi cation. Small differences in concentra-tion, handling, time, equipment, temperature, etc., may result in a difference in the benefi t to the tissue when implemented on -site. The on -site verifi ca-tion usually does not need to include all variables and for a microbiological process may not require evaluation of a microorganism panel. The manufac-turer can usually prescribe what the variables were in their validation and, in a microbiological reduction process, which microorganisms were used. In this situation it is usually acceptable to perform verifi cation at the “worst-case” scenario with respect to the prescribed variables (e.g., at or near the lower end for temperature and chemical concentration) and with a single microorganism (i.e., the most resistant microorganism as determined by the manufacturer). Whether the question is with respect to a piece of equipment or something other than a microbiological reduction process, the concepts discussed here still apply.
With a piece of equipment, the manufacturer may or may not have fully qualifi ed the item before shipping. This information should be obtained before purchasing the equipment. It appears to be most common for individ-ual pieces of equipment not to be fully qualifi ed before shipping. This leaves all qualifi cation work on the back of the purchaser. However, in these situ-ations it is common that the manufacturer can provide general information regarding typical parameters for different variables. This type of information can simplify qualifi cation work.
136 Tissue and Cell Processing
Historical and published dataThe use of historical data is appropriate for validation or qualifi cation. This is called retrospective validation or qualifi cation and is useful in a situation where something has been done for some time and it has now been decided that it should be validated or qualifi ed.
For a process, the historical data are gathered and reviewed in the context of what would be required to complete a validation. If all of the required data are present, a report is written to compile the data and demonstrate comple-tion of the validation. It is important to consider whether any aspect of a validation is missing in the retrospective data. It may be required to proceed as described previously for a process which was validated by the manufac-turer, where a small number of verifi cation studies are done to assure that all variables have been addressed.
It should also be noted that published data can be a valuable resource in reducing the amount of work to be done in validation or qualifi cation. As with manufacturer ’s data, it must be understood that published data can also vary from in -house data due to small differences in carrying out the process or handling the equipment.
ResponsibilityExact designation of who is responsible for which aspects of validation or qualifi cation is not established by regulatory bodies; rather, it must be deter-mined by the company performing the work. High -level oversight is often assigned to a particular person, but various duties should be assigned to others depending on the scope of the work. This information is usually pro-vided in the protocol and should be given in enough detail that there is no doubt about who is responsible for each activity in question. This approach can often result in a better validation or qualifi cation performed in a shorter period of time because there are more people involved in an organized manner.
For example, overall responsibility for a process validation may be assigned to the quality assurance (QA) manager, but coordination may be assigned to a member of QA. Primary protocol development may be assigned to the process engineer with all testing being coordinated by the produc-tion manager and performed by personnel in production. In this situation, if coordination and oversight are not perceived as critical it can result in prob-lems with the validation, so care must be taken to assure that coordination and oversight are correctly and frequently performed.
Change control In any situation where validation or qualifi cation has been performed it is critical that change control be strictly enforced. A process or component change that may seem minor to an engineer may be critical to a microbiol-ogist, and vice versa. The formal change control process should include a
The Principles of Process Validation and Equipment Qualifi cation 137
review by all who are potentially impacted by the change and must be for-mally documented. Determination of who will be involved in change control reviews must be made wisely since all potential issues must be addressed in a situation when not all potential issues may be known by those making the change.
NonconformanceThere will inevitably be occasions where a process does not conform to its specifi cation. This is termed a “nonconformance” or “quality incident. ” It is incumbent on the tissue bank to investigate these incidents, both to deter-mine their effect on the suitability of the tissue for release, and also to inves-tigate why the incident occurred, with a view to taking appropriate action to prevent recurrence.
Take an example where a bone graft is being washed to remove marrow
contents. The specifi cation for the process dictates that the wash be performed
at a temperature of 55 –60°C, but it is noticed during review of the batch
manufacturing record that the wash temperature reached 65 °C for a short
period. This is recorded as a nonconformance that must be addressed before
the bone graft may be issued. The initial investigation will address the effects
of the temperature excursion on the quality of the tissue, and the effi cacy of
the process. Is it likely to have damaged the osteoinductive, osteoconductive
or biomechanical properties of the bone? Will it have affected the effi ciency of
the wash? Some or all of this data may already be available from the process
characterization step.
The second investigation does not relate to batch release, but will investigate
the reasons why the temperature excursion occurred. What is the level of
confi dence that the temperature readout was true? Is the washing incubator
malfunctioning? Were the staff appropriately trained to operate it?
Serious and persistent nonconformances may benefi t from thorough root cause analysis to identify the true underlying causes. For example, the super-fi cial cause may be determined as staff error; however, deeper investigation may reveal the error was due to insuffi cient training and supervision, which would point towards wider defi ciencies in the quality system.
Revalidation and requalifi cation Usually full revalidation or requalifi cation should not be done if the process or equipment trending continually provides acceptable data. Trending of process or product output (e.g., fi nished product dimensional or functional-ity testing) as well as trending of process monitors (e.g., temperature, con-centration, contact time) should provide data that the process or equipment
138 Tissue and Cell Processing
continues to function properly. In these situations periodic, full revalidation or requalifi cation is not required.
It usually is appropriate to require a formal review of the available data on a periodic basis (e.g., yearly) to determine if any additional testing should be done. In some instances, such as when frequent product or process testing is not carried out, it is wise to automatically perform occasional verifi ca-tion testing to demonstrate that the process is still in compliance. However, this testing should be substantially less than that required for validation or qualifi cation.
In the situation of tissue sterilization, the frequency of tissue revalidation is often established by the method being used. For example, in radiation sterilization it is required to perform a dose audit every 3 months. This is a simplifi ed version of the initial radiation validation which is intended to verify that the current sterilization dose is still providing the proper sterility assurance.
As a general rule if suffi cient data are available and trended from process/product output testing and from process monitoring testing to demonstrate continued compliance with acceptance criteria, it will be less necessary to require periodic revalidation/requalifi cation or verifi cation testing.
When revalidation/requalifi cation testing is to be performed, it may be as simple as testing of products from a single batch which was processed using the same worst -case conditions as used in the original validation/qualifi cation. Depending on available data the testing may need to be more complex, such as inoculating product with the worst -case microorganism obtained in the validation and putting it through the process or testing of product from multiple batches.
Certainly when re -evaluating due to a change (as part of a change control process) the testing may more closely approximate a full revalidation or requalifi cation, depending on the number of variables which the change potentially impacts.
Examples of validation studies
The fi rst part of this chapter discussed why it is necessary to perform valida-tion and qualifi cation, and the general principles for good design and control of studies. Now these principles are applied by reference to actual studies.
Example 7.1: Tissue transportationThe fi rst practical example is of a process that will be common to most if not all tissue banks: the need to transport tissue from one place to another, e.g., from the site of procurement to the processing facility, or from the bank to the end user. The control of transportation conditions is critical to ensur-ing tissue quality. This particular example is of transport of skin allografts
The Principles of Process Validation and Equipment Qualifi cation 139
from the site of procurement to a tissue bank at refrigerated temperatures; however the principles are the same for all types of transport.
The fi rst stage is to defi ne the process in detail. This address the following questions:
• What is the format of the tissue, and what is the maximum volume that will be transported?
• How is the tissue contained; what is the nature/volume/temperature of any transport solution, and what type of packaging is used?
• What refrigerant is used? What is its specifi cation and volume? • What are the specifi cations of the transport container: dimensions, insula-
tion, etc.? • What are the worst -case transportation conditions, in terms of time of
transport and ambient temperature?
Once the process has been defi ned, the acceptance criteria must be defi ned. In this case they were:
• That the temperature of the tissue must remain within a range of 0 –10°Cfor the duration of the transit.
• That the integrity of the tissue packaging must be maintained during transit.
• That the integrity of the transport container must be maintained during transit.
• That the pH of the transportation fl uid must be within the range 7.0 –7.5 at the end of the transport.
For some tissues, it may be advisable to go further to validate the quality of the tissue following transit, e.g., to assess its viability, or histological structure.
It was determined that the maximum amount of skin that would be transported would be 6000 cm2 immersed in a minimum volume of 300 mLtransport fl uid. The specifi cations of the packaging, transport container, and refrigerant were also documented. The worst -case transportation conditions were defi ned as an ambient temperature of 40 °C (e.g., a hot summer day in a vehicle) for a maximum of 12 hours with the minimum volume of refriger-ant and transport solution, and the maximum volume of tissue.
A protocol was written and a model prepared using skin obtained from donors unsuitable for clinical donation, using the defi ned transport solution, refrigerant, packaging, and container. A calibrated data -logging thermom-eter was used to record the temperature on the external surface of the tissue packaging, and the container was placed into a shaking incubator set at an ambient temperature of 40 °C. A shaking incubator was used to model agita-tion of the container during vehicular transit – the model should as closely as practically possible approximate the real -life conditions.
140 Tissue and Cell Processing
The study was repeated three times, with acceptable results being obtained each time. As all of the results were well within the predefi ned acceptance criteria, the process was accepted on the basis of these three replicates. Note also, however, that it may be necessary to compromise between an “ideal”validation and operational practicalities – it may not be possible or ethical to obtain and sacrifi ce large amounts of tissue for validation studies. In these cases, an acceptable compromise should be reached using risk assessment principles, e.g., use of animal tissue as a substitute.
The application of suffi ciently robust validation, by challenging a trans-port process with extremes of time and temperature, obviates the need for routine temperature monitoring of the process. It enables us to reliably conclude that if the physical conditions identifi ed by the validation study are complied with (the correct container, containing at least the minimum amount of refrigerant in transit for less the maximum modeled time), then the process itself has been correctly performed. Therefore, to demonstrate compliance with the validated process all operatives need to do is confi rm that they have complied with relevant datasheets and standard operating protocols.
Example 7.2: Transport of deceased donorsThis study was performed by a tissue bank who wished to transport deceased donors from hospital mortuaries to a centralized facility where tissue pro-curement could be carried out under controlled conditions [9]. The tissue bank aimed to demonstrate that the donor bodies could be transported in vehicles that were not equipped with active cooling, without resulting in an unacceptable rise in core body temperature which could result in increased proliferation and migration of enteric bacteria.
The fi rst part of the study was to thoroughly defi ne the process and worst -case conditions. In this case, the worst -case scenario was determined to be a low body weight donor (which would warm quicker), a maximum transport time of 2 hours (based on the maximum transportation distance and pre-vailing rush hour levels of traffi c) and a maximum ambient temperature of 30°C (based on the extremes of temperature that could be expected in the particular geographic location during the height of summer).
The next step was to design an appropriate model system. For ethical and logistical reasons, it was not practical to use actual cadavers for the study, so an appropriate substitute was sought. A gel composed of water, glycerol, and agar that mimicked the density and thermal characteristics of human fl esh was identifi ed, and poured into a diver ’s wetsuit to approximate the shape of a human torso. Different -size wetsuits were used to model donors of low (40 kg) and moderate (70 kg) body weights (Figure 7.1). A calibrated temperature probe was inserted so that it was positioned in the center of the model torso, to record the core temperature. To control ambient tempera-ture, the models were placed in a large incubator.
The Principles of Process Validation and Equipment Qualifi cation 141
The acceptance criteria for the validation were predefi ned as that the core temperature of the model cadaver should not exceed 20 °C, a temperature below which enteric bacteria do not proliferate at a signifi cant rate. As the initial core temperature of the cadavers could vary considerably depending on the size of the cadaver, and the time it had been in storage in a hospital mortuary, the validation also addressed the impact of different initial core temperatures. The results of the validation are shown in Table 7.3.
All studies were repeated in triplicate, with minimal variation encoun-tered. The results of the validation show (unsurprisingly) that core tempera-ture rise is a function of the starting temperature of the cadaver and the ambient temperature of the transport. However, in all scenarios including the “worst case ” (the 40 kg cadaver with a starting core temperature of 15 °Ctransported at 30 °C) the fi nal temperature remained below the predefi ned
Figure 7.1 Gel-fi lled dummy, utilizing a wetsuit as a torso -shaped mold. A thermocouple probe to measure core temperature can be inserted.
Table 7.3 Core temperature increase (in °C) in model cadavers following 2 hours simulated transport at different ambient temperatures
Ambienttemperature
Initial core temperature ( oC)
40kg model 70kg model
5 10 15 5 10 15
20°C 4.2 3.3 2.0 2.6 1.3 0.5
30°C 7.2 6.4 3.7 5.5 5.2 2.4
142 Tissue and Cell Processing
maximum acceptable temperature. The validation was therefore successful and enabled routine monitoring of the cadaver transportation process by a simple confi rmation that the body weight of the cadaver was above the defi ned minimum, that the initial core temperature was at 15 °C or less, and that the transportation time was less than the defi ned maximum of 2 hours.
Readers may have noticed that this example is in effect 12 different vali-dation studies in one; it could reasonably have been assumed that if the worst-case scenario had passed, all the other would have too. The advantage of including different scenarios is that if the worst case had failed, other sce-narios might still have passed, permitting validation of the process but with more stringent criteria, e.g., a minimum acceptable body weight or starting core temperature. This is an example of how development and validation can be combined.
Example 7.3: Process validation and determination of microbial surveillanceThere are many approaches which can be taken in setting up and performing a process validation and in determining appropriate microbial surveillance for that process. This example is meant to provide some of the main concepts that are often implemented in a process validation and is not meant to be prescriptive. The information is provided as more of an outline with some detail where it may be helpful rather than including all of the necessary documentation and verbiage.
The process is chemical disinfection of cortical/cancellous bone blocks. A minimum of a 4 log reduction is desired for the process. Chemical ABC is to be used in the process, but the variables are not yet established.
1. Develop process validation master schedule. This should include all major milestones in the validation.
2. Perform literature search to determine what information is available regarding the chemical process to be used. This information can be used to potentially speed up the process characterization step.
3. Determine what personal experience employees or industry contacts may have regarding the chemical process to be used. This may include use of a consultant who is an expert in the type of chemical process.
4. Determine any potential effect (whether positive or negative) which the chemical process might have on the tissue. Some of this informa-tion might be gathered in the literature search or review of personal experiences.a. Perform a simple evaluation to verify that the information gathered is
accurate. This can consist of a single time point, single concentration, etc. (perhaps “worst case ”) to verify full functionality of the tissue
The Principles of Process Validation and Equipment Qualifi cation 143
prior to spending more resources on the validation. If the chemical process is well characterized this step may not be necessary.
5. Determine the appropriate microorganism panel for the testing. This approach is well described in the text of this chapter.
6. Perform process characterization. With a more novel process this step may be more involved. With a well -established process this step may be minimal or perhaps even skipped. a. Design protocol for process characterization. This is where the full
scope of process characterization for this tissue is well established as well as all steps necessary to fully understand the process. Exact details regarding test conditions may be left out as they might change as the process becomes more characterized.
i. Defi ne the initial fl ow of the tissue process based on assumptions gathered during previous steps. This initial process might also change as more information regarding the process is gathered.
b. Perform process design studies to fully understand the effects which different variables will have on the tissue. Risk management steps like those explained in the chapter can be used to help determine which of the many variables should be evaluated.
i. Different variables to address might be chemical concentration, life cycle of chemical once mixed and once it makes contact with the tissue, chemical temperature, chemical pH.
ii. Time kill studies might be performed. These studies assist in dem-onstrating that the chemical is capable of providing the desired log reduction and what the appropriate soak times may be for the tissue. Time kill studies may be performed on something other than tissue (e.g., a stainless steel coupon) to avoid use of too much tissue and to evaluate the chemical capability with fewer variables.
Because of their simplicity, time kill studies are an ideal way to deter-mine which of the microorganisms in the panel are more resistant to the chemical process. This information can be used for selecting appropriate microorganisms in future evaluations, e.g. the process validation.
c. Data determined in these steps are also used to determine what the “worst case ” or “worse case ” conditions are. These conditions are usually implemented in the process validation.
7. Perform process validation. Once the chemical process is well character-ized it is then ready for validation. a. Design protocol for process validation. This is where the full scope
of process validation for this tissue is well established as well as all steps necessary to validate the process. In this case exact details of the various steps are expected to be present and any alteration to those details (whether intentional or not) is usually treated as a deviation.
144 Tissue and Cell Processing
i. The protocol must be clear regarding which tissue types it encom-passes. It is not uncommon to validate the same process for mul-tiple tissue types. As the surface characteristics of various tissue types can cause a change in the chemical effectiveness, it is usually not appropriate to validate a tissue process for one type of tissue and then apply it to another.
ii. The process validation must address which type of testing is to be performed after the process is applied to the inoculated tissue (i.e. qualitative or quantitative recovery). A qualitative test is more sensitive and can resolve down to 1 cfu. This is good if the tissue process is robust and expected (based on the data obtained in process characterization) to reduce the bioburden to 0 cfu or close to it. A quantitative test is more appropriate if a high inocu-lum is being used on the tissue and the process is not expected to reduce the bioburden to 0 cfu (again based on the data obtained in the process characterization). One disadvantage of the quantita-tive test is that the recovery method must be validated since the microorganisms are being removed from the tissue for testing. If possible the qualitative test should be used because of the greater test sensitivity.
b. Validate the recovery method to be used. Whether qualitative or quantitative testing is to be performed in the validation, the test method selected must be validated. For qualitative testing this usually means performing a bacteriostasis/fungistasis test (B/F) and for quan-titative testing this usually means performing a recovery effi ciency test.
i. In both test method validations an acceptance criteria is estab-lished and must be met. For qualitative testing, the B/F test must pass. For quantitative testing there is no established percentage recovery which must be met. It is common to desire greater than 50% recovery, but any value is acceptable as long as it is repeata-ble and provides the level of sensitivity required in the validation.
c. Perform the process validation. i. Inoculate the tissue and process the tissue using worst case or
worse case conditions as established in the protocol. ii. Perform the recovery method on tissue.
Evaluate the test data. Do the test data meet the acceptance criteria? If not, additional review and testing may be required to determine the cause. This is where it may be noted that additional process characterization should have been performed. For this reason it is often good prac-tice to spend a little more time in process characterization prior to going into process validation.
iii. Write the process validation report.
The Principles of Process Validation and Equipment Qualifi cation 145
8. Establish the microbial surveillance details. Information gathered in the process characterization and process validation can assist in understand-ing which aspects or steps of the process are most critical, and which most accurately represent the microbiological status of the fi nished tissue. It is ideal if it can be determined that aspects or steps of the process can be routinely tested rather than having to routinely test fi nished tissue. a. Determine what will be tested for microbial surveillance. b. Determine how frequently microbial surveillance testing is to be
performed.c. Determine the microbial surveillance acceptance criteria. d. Perform necessary training for those involved.
9. Establish change control procedure for this process. In a validated process any change, regardless of how small, can have a signifi cant impact on the results of the process. For this reason it is critical that a strict change control process be implemented. It should include members of many teams to ensure that all aspects of a change are addressed before that change is implemented.
10. Establish the frequency of process verifi cation or revalidation. A review of the characterization and validation data should shed light regarding how frequently revalidation should occur. This determination of fre-quency should include which aspects or steps of the process should be revalidated and if the revalidation requires testing of if it can consist of a review of historical data.
KEY LEARNING POINTS
• Validation and qualifi cation are a critical step in ensuring that tissue grafts
are safe and effective.
• Evidence of adequate validation and qualifi cation will be required by
regulators; both are mandatory under current legislation in the USA and the
EU.
• Protocols for validation and qualifi cation may, and ideally should, be
formalized as an integral part of the quality system.
• It is essential that acceptance criteria are predetermined before validation
and qualifi cation commence.
• Sterility assurance levels (SAL) and log reductions are two different
concepts.
• Selection of the proper microorganism panel for a tissue disinfection/
sterilization process validation is critical.
• Risk assessment is an indispensable tool in determining the scope and
criticality of validation studies.
146 Tissue and Cell Processing
References
1. US FDA Federal Register. Current Good Tissue Practice for Human Cell, Tissue and
Cellular and Tissue Based Product Establishments, 21 CFR 1271.3(kk), April 2009
