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Bacterial Endotoxin Limits And Calculations For Drugs And Biologics

What are drugs and biologics?

Drugs in this context are considered compounds that contain both the active substance(s) within a drug compound, known as the active pharmaceutical ingredient (API) and the non-active drug product delivery system for the API. The FDA considers biologics gene therapies, vaccines, and recombinant therapeutic proteins. Biologics are also products that contain blood or blood components, tissues, cells, glycosylation, nucleic acids, proteins, or other tissue components. Biologics can come from biotechnology methods or isolated from natural sources (humans, animals, microorganisms).  Biologics are difficult to characterize as they are complex mixtures, unlike most chemically synthesized drugs with known structures. Most biologics require aseptic practices through all manufacturing steps and cannot be sterilized using heat. Thus, ensuring packaged biologics and drugs meet regulatory endotoxin limits is of the utmost importance for patient safety. Bacterial endotoxin limit calculations for USP endotoxin limits on drugs and biologics are covered in this article along with endotoxin unit information.

How are calculations performed for endotoxin limits for drugs and biological products?

Endotoxins are pyrogens that can cause fever and illness in the human body. An endotoxin limit is the amount of endotoxin activity that can be safely contained in a product. These limits are based on experimental evidence and depend upon how a product is administered (i.e., aerosol, eye drops, intramuscular injection, etc.), product dosage per kilogram of body weight, and the duration of use of the product. Bacterial endotoxin tests (BETs) measure a product’s endotoxin concentration using horseshoe crab limulus amebocyte lysate (LAL). In rare instances where LAL testing is not possible, alternatives to LAL testing may be needed to accurately assess endotoxins within a product. 

Endotoxin limits are calculated using the equation below. In this equation, the way a drug or biologic is administered primarily defines the threshold pyrogenic dose “K.” The maximum recommended dose “M” relates to the potency of the product’s active ingredient. If a product is used for a long time (such as if a product is infused or injected at frequent intervals over an extended time), M is based on the maximum total dose administered in 1 hour. If you have a combination product with a drug or biologic, please see our other article on endotoxin limits for combination products.

Bacterial Endotoxin Limit Calculation:

Endotoxin Limit= K/M

Where: 

K = threshold pyrogenic dose of 5 EU/kg for most routes of administration

or 0.2 EU/kg for intrathecally administered drugs (see Table 1 below)

M = maximum recommended dose of product per kilogram of the patient’s body weight.

When calculating the USP endotoxin limit, bodyweight is defined by the intended patient population. The average adult in the United States is assumed to weigh 70 kg. The average weights for children can be found on the Centers for Disease Control and Prevention Clinical Growth Charts. Where the product package insert describes multiple patient populations, indications, and routes of administration, endotoxin limits should be calculated for all indications and modes of administration.

Endotoxin limits established at the early stages of product development may change based on dosing or changes to the formulation before commercialization. If a dose has not been established, the endotoxin limit should be calculated based on the highest dose anticipated for the patient population and route of administration. A summary of endotoxin limits by route of administration is shown in Table 2 of USP 1085, reproduced as Table 1 below.

Table of defined values for K In terms of route of administration
Table 1. Defined values for K In terms of route of administration

Relevance of Limits for Compounded Sterile Preparations

Bacterial endotoxin limits can be exceeded if care is not taken when compounding pharmacies prepare injections or infusions. Additional quality control steps must be taken to avoid the addition of endotoxins to preparations made by compound pharmacies. For example, the compound pharmacy should only use materials with pyrogens removed in-house or they have received as sterile and free of detectable endotoxins. Only diluents from commercial vendors which meet the compendial limits for endotoxin (most often 0.5 EU/mL) should be used. The compound pharmacy must make intrathecal (IT) preparations following strict IT endotoxin limits. Endotoxin limits are more stringent for IT formulations because the cerebrospinal fluid will be exposed to the IT preparation following injection.

Compounded sterile preparations. Disease control. Scientists examining medical product samples

Reducing Endotoxins in Active Substances and Excipients

Endotoxin contamination can be reduced or eliminated in active substances and excipients through assessing raw materials, controlling production methods, and monitoring storage conditions. Natural materials such as sugars, heparins, and enzymes are at the highest risk of containing significant levels of endotoxins or glucans that interfere with BET testing. Suppliers of raw materials should be audited regularly to ensure control of microbial and endotoxin contamination during the manufacturing of these materials. Additional information on endotoxin control can be found HERE. Further, many biologics and drugs can depyrogenate products using filtration

Summary

Overall, endotoxin limits are calculated for both drugs and biologics. An endotoxin limit is the amount of endotoxin activity that can be safely contained in a product. These limits depend upon various factors, including the way a product is administered, product dosage, and the duration of use of the product. Care should be taken to assess raw materials used for excipients and active substances to prevent endotoxin and glucan contamination. It is vital to reassess endotoxin limits for your drug or biologic if the route of administration, indication, dosage, or target patient population changes.

Ethide Labs is a contract testing organization that specializes in Bacterial Endotoxin Testing. Ethide Labs also offers Bioburden Testing, Environmental Monitoring, Sterilization Validations, Microbiology Testing, EO Residual Testing, Package Integrity Testing & Cytotoxicity Testing services for medical device companies and allied industries. Ethide is an ISO 13485 certified facility. 

References

United States Food And Drug Administration. What Are “Biologics” Questions And Answers. February 6, 2018.

United States Pharmacopeial Convention. <1085> Guidelines On The Endotoxins Test. Rockville, MD, USA. 2021. (USPC <1085>).

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