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Specifications for Endotoxin levels for Single Use systems

As endotoxin contamination on a drug product can be difficult to destroy or remove, measures built into the manufacturing process can help prevent endotoxins entering the production chain.  Therefore, these single-use systems, used during production, are required to contain low levels of endotoxins. 

In a previous blog we discussed the endotoxin levels for medical devices. Endotoxin limits for medical devices are often (mis-)used as limits for Single Use Systems for biopharmaceutical manufacturing processes. The underlying thought for a limit of 20 EU/unit is that exposure to an entire device (like an implant) that contains less than 20 EU is still safe for the patient. Single use systems for manufacturing are, per definition, not directly exposed to the patient. 

To demonstrate the shortcoming of an EU/unit limit, let’s take the example of disposable bioreactors of different volumes. A bioreactor might have a volume ranging from 1 L up to 1000L. A limit of 20 EU/unit is much more challenging to meet for the system that is 1000 times larger. But for an example, assume that both systems are contaminated around the 20 EU/unit limit, and we assume that when filled, 100% of the endotoxin contamination will be extracted into the contents. When used as intended and both bioreactors are filled up to their nominal volume, the liquid inside the small reactor will contain 0.02 EU/mL while the contents of the larger container will contain 2 x 10-5 EU/mL. That means that the smaller container will pose a significantly larger risk to introduce endotoxins in the end product. 

Additionally, it may not be feasible for the lab to obtain a result below 20 EU/unit when evaluating these larger systems. Referring back to the hypothetical 1000L bioreactor, regardless of the extraction method utilized, we will use a very conservative figure of 5 L needed for the extraction. When that extraction volume is considered with the endotoxin test sensitivity of 0.005 EU/mL the lowest result that could be obtained would be 25 EU/unit. As a result below 20 EU/unit could not be obtained, a different standard of evaluation is needed. There are no regulations or guidelines in place for single use systems. However, it is clear that setting limits according to the limits applied to devices does not take into account the size or volume of the unit. The Pharmacopeial endotoxin limit for Water for Injection (WFI), 0.25 EU/mL, is often a better standard to utilize for single use systems as pharmaceutical water used during production will also have the endotoxin limit of WFI. 

In practice, we will fill the unit with an amount of endotoxin free water, usually lower than the nominal volume. During extraction we ensure to include parts of the system at higher risk of endotoxin contamination (cutting edges, tubing connectors, etc.) and express the endotoxin contamination not per device but per mL.  

A limit of 0.25 EU/mL will be appropriate for most systems; however, this is not a blanket limit to be applied to all systems. The Hazard and Risk analyses of the manufacturing process of a pharmaceutical should be considered and may require more strict limits. 

Peter Cornelis, Ir.

Senior Expert Microbiology

Peter Cornelis graduated from the Catholic University of Leuven (Belgium) in 2000 as a Master in Applied Biological Sciences (Major Biotechnology). In 2003 he started working for Toxikon Europe (now Nelson Labs) as a study director Microbiology and in-vitro Toxicology. From 2007 until 2016, he was department supervisor for Microbiology and in-vitro Toxicology. Since 2016, he is responsible for research, validation, and development of new microbiological and in-vitro toxicological methods. Peter is a member of the ISO committee TC 194 WG5, Cytotoxicity and WG 8, Irritation and sensitization. As an expert, he was involved in the adaptation of ISO/TC 194 ISO/DTS 11796:2022(E) Biological evaluation of medical devices — Guidance for interlaboratory studies to demonstrate the applicability of validated in vitro methods to assess the skin sensitization of medical devices.

Nathan Pett

Study Director BET

Nathan Pett graduated from Utah State University in 2018 with a bachelors in Biochemistry. In 2019 he began work for Nelson Labs as a Lab Analyst and in 2021 transitioned to the role of Study Director in the BET department.

Emily Spackman, B.S., RM(NRCM)

BET Consulting Study Director

Emily Spackman has worked for Nelson Laboratories in the Bacterial Endotoxins Lab for over 16 years. She graduated from the University of Utah and holds a bachelors degree in Biology. She is a member of The National Registry of Certified Microbiologists and has experience with both medical devices and pharmaceutical products.