How Do You Set Endotoxin Specs on API?

By Dr. Lori Nixon

For parenteral drug products, setting a specification limit for endotoxin is a relatively straightforward exercise. If you know the maximum hourly dose and route of administration, the endotoxin limit can be readily derived from the equations set out in USP<85>.


But how do you determine an appropriate limit for your API? Don’t make the mistake of applying the same limit as product (in terms of EU/mg of active). And don’t make the mistake of blindly applying the same limit as a similar API, that may be dosed differently. Since the drug product is typically a combination of active ingredient along with excipients, solvents and container components—each of which may potentially contribute to the total endotoxin level—you should account for other potential contributions and set your drug substance limits accordingly. By setting the appropriate limit in API, you avoid the risk of an endotoxin failure that doesn’t show up until your precious API has been formulated and filled into vials—a business catastrophe!

Consider the following example:

Drug product at 50 mg/mL, intended for iv delivery in adults.

For this product, the maximum human dose/hour is 250 mg, delivered as a 5 mL volume.

From USP<85>, the endotoxin limit for iv delivery is 5 EU/kg. For a 70 kg adult, this corresponds to 350 EU maximum, or 70 EU/mL.

Tabulate the potential endotoxin contributors from your drug product formulation, container and manufacturing process. Look up the endotoxin limits (specifications) for each component you listed. In some cases it’s not possible to get a hard number, but you can at least estimate or list a “worst-case”. Some components will have negligible contribution based on their low ratio of surface contact to total formulated volume (such as the needle/tubing in the fill line); or low overall volume contribution (such as an acid/base used in a titration step). In this scenario, the sum of endotoxin contributions from all components (including API) cannot exceed 350 EU. “Solve” for the allowable endotoxin contributions from the API (X, in the table).

*Notice that in the table, drug substance is shown as 60 mg/mL (as powder) even though the formulation strength was listed as 50 mg/mL. Recognize that endotoxin measurements are usually based on powder weight rather than active ingredient weight. In this example let’s assume that the API has a moisture specification of NLT 95% and purity specification of NLT 88%; so 50 mg/mL of active (in the worst-case) actually corresponds to 60 mg/mL of API powder. So a 5 mL dose may contain as much as 300 mg of API powder.


You won’t be able to nail down everything exactly, and should recognize that relying on release specifications for some of the components above will clearly result in overestimations for endotoxin contribution. For example, the specification limit for the vials may not take into account additional reduction from depyrogenation; filters would be pre-rinsed and any subsequent endotoxin contributions would be largely diluted within the total product volume; and so forth. Nonetheless, this can be a convenient and systematic way to start your evaluation from a “worst-case” standpoint.

The allowed endotoxin contribution from the API can be calculated by subtracting the sum of the other component contributions (far right column of the table) from the total of 350 EU. In this case, X = 235.7 EU. Divide by the largest amount of drug substance powder that could go into the maximum dose (in this case 300 mg), and you have the drug substance endotoxin limit (0.79 EU/mg).

Now, do a reality check: If this drug substance limit looks like it may be problematic for any reason (analytical or process capabilities), look again at the table to see other possible ways to reduce the overall endotoxin (or revisit some of the worst-case assumptions that were clearly overly exaggerated). In the example, Salt 2 appears to be a large potential contributor, with a fairly loose specification for endotoxin. It may be possible to specify a different grade of this excipient (low-endotoxin or parenteral grade). Remember that compendial grades don’t necessarily mean low-endotoxin grades or even endotoxin-controlled grades.