Recently we learned of a case where a single use device was tested in an academic institution in Europe. It was concluded that the two different, sterile, non-cytotoxic, virgin, single use devices contained protein burden [proburden] levels of 4.72 and 11 pg/mm2. It was also observed that substantial industrial contaminants like fibers, hair, grease and other chemicals were present in single use devices from a variety of manufacturers. This finding raised a public safety concern and MHRA (Medicines and Healthcare products Regulatory Agency, UK) required a response from the sponsor pertaining to the levels of protein observed. The following comments are provided by Gibraltar Laboratories Inc. [GBL] in an attempt to help sponsors to anticipate this issue and to provide a reasoned approach to understand the meaning of the results.
A possible inference from the MHRA communication is that the device in question is unacceptable because trace levels of molecular markers were observed. Total protein/amino acids detected were 4.72 and 11 pg/mm2 (equivalently 0.472 and 1.1 x 10-3 µg/cm2) for two devices respectively. Currently there is no residual protein value published by regulatory authorities as to the cleanliness benchmark for single-use device. Therefore we choose a comparative approach to interpret these data.
The established regulatory guidance for residual protein, as an indicator of organisms or tissues, in the evaluation of the efficacy of reprocessing reusable devices is published [AAMI TIR No.30: 2011 – A compendium of processes, materials, test methods, and acceptance criteria for cleaning reusable medical devices.] The benchmark value is less than 6.4 µg/cm2 for protein after a device has been cleaned. This acceptance criterion is >1000-fold higher than what was detected from the single-use devices by MHRA. Then the essential question is whether this 1000-fold less protein residue is properly interpreted as a criterion for cleanliness of single-use devices.
The number one safety concern is infection caused by contamination of medical devices. This is particularly more critical in reusable devices then single-use ones because certain reusable devices such as endoscopes inevitably become contaminated with great load of bioburden (bacteria, fungi and viruses) during usage and all procedures are performed on patients not guaranteed free of infectious agents. For single-use devices, bioburden can be introduced during the entire manufacturing process from initial raw materials to final packaging. However, the types of environmental bioburden associated with single-use devices does not pose a risk as high as the infectious agents, like HIV, HBV, TB, etc, associated with reusable devices where the acceptable limit of cleanliness is less than 6.4 µg/cm2 protein.
Regardless, reusable or single-use devices all have to be sterilized before usage. Given the high sterilization assurance level which can be achieved in modern sterilization technology, infectious agents [except for prions] can be inactivated completely. Since protein is ubiquitous in all living organisms and tissues it not unexpected that it remains on the devices after infectious agents are completely inactivated. The present study showed devices passed sterility test. What perspective should be drawn from the trace levels of protein reported to be present by the academic institution? For bacteria, the protein content is estimated to be in the range of 60 to 330 fg per cell (M. Zubkov et al. Determination of Total Protein Content of Bacterial Cells by SYPRO Staining and Flow Cytometry, Appl Environ Microbiol. 1999 July; 65(7): 3251–3257). Based on this data, TIR30 protein acceptance criterion 6.4 µg/cm2 can be converted to protein content from 19.4 – 106.7 x 106 bacterial cells/cm2 or 0.2-1.1 x 106 bacterial cells/mm2, which is likely to be a visible level. If this is safe for reusable devices then a 1000-fold less level found on single use devices should be even safer.
Additionally, in the case of reusable medical devices it is known that residual levels of protein accumulate after repeated use. The reprocessing procedure usually consists of chemical and thermo treatments; protein residue becomes less and less soluble and begins to build up inside devices. This can lead to malfunction during the operation and a life-threatening event. However, for single-use devices, residual accumulation is out of the question. If 6.4 µg/cm2 protein level is acceptable for repetitive use and under the risk of malfunction caused by accumulation, then a 1000-fold less level portends to be unnecessarily burdensome without additional safety.
Having said all of the above, we are not arguing that quality control on single-use devices should be neglected. In the opposite we would like to suggest more research, better industrial guidance and a more practical approach for quality control.
The protein detection method used in the evaluation of cleanliness of reusable devices and acceptable to the FDA is an ‘in situ’ assay using a modified OPA/NAC reagent spray. Compared to other common protein assays such as BCA or Bradford, which is colorimetrical testing protein in liquid form, it is more sensitive because the analyte doesn’t need to be further diluted with either reagent or extraction buffer. As a practical matter it will be difficult for manufacturers to implement routine monitoring with this technology. We suggest using total organic carbon (TOC) analysis instead as provided at Gibraltar Laboratories.
A TOC analyzer has extremely low limit of detection (ppb). It is fully automated and has been utilized in quality control in the pharmaceutical industry for decades for water quality, cleanliness monitoring, etc. TOC analysis is available through many contract laboratories like GBL. Many pharmaceutical products are administrated intravenously to patients daily. The risk is considerably higher, if they are contaminated, than single-use devices. TOC criteria from the pharmaceutical industry should be stringent enough for manufactures to adopt into its routine quality program for single-use devices. A TOC assay is superior to a protein assay because it can detect all critical components of life, i.e. protein, carbohydrate, lipid and nucleic acid. Additional benefits provided by TOC also include detecting residues from certain mechanical lubricants and cleaning detergents, etc. TOC can help to track the trend of these residues before they reach a harmful level. An extra assurance level can be provided by TOC analysis due to its ability to detect things which protein assays cannot.
With respect to possible biological reactivity we recommend the ISO 10993 Biocompatibility procedure or USP <87> Biological Reactivity tests, in vitro. Both “cytotoxicity tests” are very sensitive, rapid and inexpensive official methods to assess the safety of medical devices. In addition, other in vitro tests are available at our laboratory for specialized purposes such as for ophthalmic exposure [e.g., MATTEK].
In summary, the residual protein level found on single-use devices discussed here do not appear to pose any risk to a patient based on comparative analysis with the acceptance criterion published for reusable devices. TOC and cytotoxicity analyses may be a practical alternative for manufacturers to implement in their routine quality control program.