In November 2011, Nelson Laboratories conducted a webinar entitled, Tissue Process Validation Concepts. (You can see a recording of the webinar here: http://www.nelsonlabs.com/KnowledgeCenter/Education/Webinars.aspx) Following the webinar, Nelson Labs provided responses to a myriad of questions from participants. Today we would like to provide those questions and responses on our blog.
The webinar was conducted to provide those who perform or review process validations with the concepts and knowledge needed to properly set up the validation and interpret the data. The concepts are included in the upcoming Microbiological Surveillance document from AATB.
Q: Do you have suggestions for process validation for tissue that is aseptically processed and not terminally sterilized?
A: Those types of validations usually involve a chemical soak. This was part of what we covered in the presentation.
Q: Can you go through an example of a neutralization process?
A: Neutralization has multiple steps involved; the first step includes processing one tissue sample through your disinfection/decontamination procedure and then extracting the tissue sample in a neutralizer. The extracts from the neutralizer are then plated through membrane filtration on agar plates. A positive control, which is just the organism that is to be tested, is used to spike the control and the test replicate.
Q: How do you validate an aseptic process?
A: Validation of an aseptic process, per the traditional tissue bank definition, is performed as Alpa described in the webinar. However the FDA and other industries (i.e. medical device and pharmaceutical) use the term Aseptic Processing very differently. They view an aseptic process as a means of maintaining a product sterile during handling and assembly, resulting in a sterile product.
Q: Is this presentation specific to aseptic processing or would it also apply to terminally sterilized tissue?
A: This presentation was specifically for aseptic processing, we do have testing for soft tissue that can’t be terminally sterilized through radiation tested for sterility testing. This is usually accomplished through a chemical soak or multiple chemical soaks for an extended period of time (i.e. 10-15 hrs. at 35°C).
Q: What do you normally use for neutralization if inhibition is encountered?
A: Most of our neutralization techniques are from USP <1227>, Table 1. That section of USP also provides information on other techniques such as membrane filtration or larger volumes, which we also use.
Q: How do you approach process validation for aseptic processing when no claims are made for elimination of microorganisms? Sterilization methods are not indicated as cell viability is a key requirement of the tissue.
A: When no claims are needed for elimination of microorganisms in a tissue process it is usually just demonstrated that the company is capable of performing the process without contaminating the product. This usually entails testing of actual product or a surrogate product, or a solution (depending on the situation) during and at the end of a typical or worse case process.
Q: What are you seeing as a trend from the FDA for acceptable viral clearance? For example, what log reduction of a panel of viruses does the FDA count as acceptable?
A: The FDA does not require testing for viruses. You do screen for them upfront, but then bacteria are tested. There is no requirement that the tissue process be capable of reducing viruses.
Q: The tissue we process is NOT sterile (cornea). Would bacteriostatis/fungistasis studies be more appropriate for this tissue for process validation?
A: These are two different things. B/F is testing sample for inhibition after sterility testing, process validation includes testing your internal process for how to clean the cornea before you use it, if you only do rinses or use antibiotics that is what we would do as well, to ensure that your process is effective.
Q: Are there methods to sterilize tissue? Or can it only be aseptically processed?
A: Many types of tissue are routinely sterilized, especially bone. Most types of sterilization are detrimental to tissue, but radiation can often be used. Historically radiation sterilization has received a bad reputation for damaging tissue, but that irradiation was usually performed under uncontrolled conditions. When irradiation occurs under controlled conditions (e.g. low temperature, anoxic conditions) it can often provide sterile tissue without noticeable damage to tissue.
Q: Can real-time PRC techniques be considered for quantification of fastidious microorganism with difficult plaque counts or growth-no growth approach?
A: There is no reason that PCR techniques and other rapid microbiological methods could not be used for tissue as long as they have been properly validated.
Q: Should you test for aerobic & anaerobic organisms during bioburden testing?
A: Yes, generally both aerobic and anaerobic testing should always be performed during bioburden testing on tissue.
Q: How do you account for tissue variation?
A: We usually recommend performing important tests (e.g. B/F or other neutralization studies for testing which will be done routinely) on at least three donors to demonstrate consistency.
Q: What would you do if your challenge organism doesn't grow in the validation study that you are doing?
A: If you have inhibition in the test study then it would not allow the organism to grow. Also if the tissue has some inhibitory properties it will not allow the organism to grow.
Q: Why Clostridium sporogenes is a particular cause of concern to the tissue industry, in contrast with all other clostridium species?
A: C. sporogenes is easy to work with in a laboratory setting whereas other species of Clostridium can be very difficult. Thus, it is more of a functional and practical issue than a scientific issue.
Q: If bacteria are destroyed in the manufacturing-process, are there any provisions to monitor Residual LPS or teichoic acids in the tissue as probable source of shock reaction?
A: LPS, or bacterial endotoxin, from Gram negative rods are easily detected using an LAL test. Teichoic acids from Gram positive microorganisms are not commonly screened for and we are not aware of standardized screening test.