Endotoxins or Endospores?

At one point I was a little mixed up by these two very different terms. They start with the same prefix; they’ve got to be closely related. Well, sort of. But they’re extremely dissimilar when you break each down. So what are the differences?

Endospores (Spores)

Endospores form typically when bacteria (usually gram positive bacteria – the most commonly found bacteria in cleanroom environments) go through a period of starvation or when experience conditions that are not ideal. It is a survival mechanism that allows the bacteria to survive through those periods and when conditions are better they can become a “normal” gram positive bacteria again and thrive. They can “live” in this state for very long periods of time also; there are various reports of how long but we can be sure they’re able to outlive a human’s lifespan for sure. Another important point to note is that spores are highly resistant to disinfection; they’re harder to kill than Bruce Willis in this state. Yet, these are the most prevalent type of bacteria found in cleanroom environments, is this a losing battle? Well, let’s not give up all hope just yet. There are ways to kill them but you have to bring out the big guns to do it.

Killing Endospores

There are a couple of ways of effectively killing endospores. First, using an autoclave with the proper time, pressure and temperature will do the trick; but the key there is proper time, pressure and temperature. Using an exposure time of at least 15 minutes and 15 PSI at 121 celsius will usually do the trick. Gamma irradiation has been known to work as well. However, what about the surfaces and air of our cleanroom environments? We certainly can’t autoclave an entire room.

There are chemicals that can be used to kill spores as well. We should be routinely incorporating their use into our cleaning regimens. How often should be dictated by our environmental monitoring. Every six months may be too long between sporicidal cleans and weekly is probably overkill since these can also be toxic and noxious for humans to be exposed to. Again, a good tracked and trended environmental monitoring program will give you an idea of how often you should be using sporicides. To give you an idea my pharmacy will use a sporicide once a month during our entire room clean; it’s preceded by the use of two other disinfectant (this is called a triple clean – 2 x clean with disinfectant followed by a sporicide). One thing to note is that given the ability of sporicides to degrade surfaces they need to be followed up with a rinse of some kind, usually with sterile water or IPA; of course this is after the proper contact time has been observed (read instructions for the particular sporicide you use).

Sporicides are typically composed of one of two chemicals or a combination of them: Hydrogen Peroxide and Acetic Acid. The combination of those two compounds is Peroxyacetic Acid. Acetic acid of course is also known as vinegar and will have a fairly strong noxious smell when used for a whole room clean. It should be noted that bleach too has sporicidal activity, however is not an EPA approved cleaning agent.

 Hi there!

Endotoxin

Ok, so an endospore is a gram positive bacteria in a starved state that becomes a survival machine (like a terminator? Mmm sort of I guess, sure). What’s an endotoxin? An endotoxin is actually a PART of a gram NEGATIVE bacteria, specifically it is a portion of their cell wall. I’m sure if you’ve taken a microbiology course you’ve at least heard the term “lipopolysaccharide” (made up of a lipid and polysaccharide – LPS). It is a constituent of the cell wall of gram negative bacteria and is released after the cell has been destroyed. Even with the cell destroyed endotoxin can still be harmful if injected into humans or other animals. Endotoxin is known to be pyrogenic (fever inducing) at least and in high enough concentrations injected into an person could lead to sepsis which can be fatal; especially for the immunocompromised.

A little about gram negative bacteria: they’re typically found in water sources. Which means even at very low levels they could be found in tap water. While using a 0.2 micron sterilizing filter will remove bacteria, remember this is a small piece of a gram negative bacteria (a portion of the outer membrane) and potentially will not be eliminated with filtration. One similarity endotoxin has with endospores: it is also difficult to deactivate and get rid of. Endotoxin is extremely heat and pH stable; meaning it can endure through conditions most other parts of the cell may not.

Getting rid of endotoxin

Endotoxin from a destroyed bacterial cell is elusive to say the least. The most ideal way to avoid endotoxin contamination is to prevent it from entering into your processes in the first place. Most likely glassware is being used at some point in your compounding process. Glassware is of course reusable and is most likely cleaned with detergent and water. Since water is involved therein lies endotoxins potential point of entry. As I’ve already said, endotoxin is stable at high temperatures but it can be deactivated with heat. USP Chapter <797> recommends using a dry heat oven at 250 degrees celsius for a minimum of 30 minutes to achieve sterility and depryogenation. Of course it should be noted that the time can vary based on the load of the cycle and the oven. A proper depryogenation oven should also be used, this is not meant to be done in a kitchen style oven. I’d also like to note that the exposure time is the key here; 30 minutes minimum at 250 degrees, however it will take time for the oven to heat up to that point so your cycle may be over 2 hours to achieve that 30 minute time.

To verify that your cycle actually did depyrogenate your glassware you should be using biological indicators. One of my first posts on this site I made a video about bio indicators: check it out. They’re simple little “things” that are inoculated with a difficult to kill bacteria, or in this case endotoxin, that are also inside the chamber of your depyrogenation oven during a cycle. There’s something very interesting about endotoxin; it reacts with a component of the blood of a horseshoe crab: Limulus Amebocyte Lysate (LAL – amebocyte is the blood component from the crab). When  LAL comes in contact with endotoxin it forms a gel clot and will actually adhere to a vial in such a way that the gel will stick to the glass even if it’s inverted. This particular biological indicator for endotoxin is known as an endotoxin challenge vial. They’re available for in-house analysis and are fairly easy to perform or alternatively you can send off to a third party to have analyzed and reported to you.

Using endotoxin challenge vials is a good way to validate your oven and the typical cycle times you use for depryogenation. In order for the oven to pass the validation it must be able to at least have a 3-log reduction in endotoxin of the vial. Proper validation of a dry heat oven (by GMP standards) is quite a bit of work and goes beyond the scope of this blog post. Just to point you in the direction if your interested in going down that rabbit hole check out the Parenteral Drug Associations technical report No. 3 (here is the table of contents; members of PDA can access).

Calculating Endotoxin Limits

Minimally, all of the sterile compounds produced by compounding pharmacies should be tested for sterility and endotoxin. Although it seems like many topics in the analytical laboratory realm elude us. Endotoxin is one of those tests that we know we might have to give some numbers to perform a math problem but we may not know what all of those numbers actually mean.

First thing you should know, the pyrogenic threshold (the point at which humans will have a response) for endotoxin is 5 endotoxin units (EU – about 0.5 ng of endotoxin) per kilogram of body weight. This is called the K value and is why you have to give the average patient weight with your compounds when requesting a USP <85> analysis. However, this level can be much lower for intrathecals; so the route of administration matters as well. Intrathecals have their own lower K value of 0.2 EU/kg, so be sure to make the correct calculation based injection site. You’ll also need to know the Maximum dose for the particular drug you’re compounding; this is expressed as M.

Again, what you need is:

  1. The weight of patient
  2. Max dose of the particular drug (M)
  3. Route of Administration (Parenteral K = 5 EU/kg, Intrathecal K = 0.2 EU/kg)

The equation to calculate the Endotoxin Limit (EL) is:

EL = K/M

K=Pyrogenic Threshold for humans (see above for limits given route of admin)

M=Max Dose

The first thing to do here is calculate the maximum endotoxin limit per hour (assuming 70 kg patient and a non-intrathecal route of administration) for a drug where our dose will be 5 mL once a day:

5 EU/Kg  x 70 KG = 350 EU (per hour)  then divide by 5 mL (max dose) = 70 EU/mL

How endotoxin limit results will be reported to a compounding pharmacy from a third party lab will be expressed in EU/mL. Based on the above calculation will determine whether the results “pass” or not. Last thing to mention is that this is a standard analysis that should be performed on EVERY batch (like sterility) for your sterile preparations; the corresponding USP chapter that talks about Endotoxin is USP <85>.

Summary

So there’s a big difference between endotoxin and endospores, starting first with they’re from two different kinds of bacteria. The differences don’t stop there but hopefully the confusion about which is which has been clarified. It’s extremely important to be aware of both as they can have detrimental, if not deadly effects to a person who’s been injected with a compound that’s been contaminated with either. Have further questions? Reach out to me! seth@pharmacyinspection.com

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References

http://www.eagleanalytical.com/uploads/groups/117/documentgallery/pep_allowendotoxlimits_04-06.pdf

http://www.acciusa.com/pdfs/whitepapers/EndotoxinLimits_SeanJH-PR17-012.pdf

http://www.acciusa.com/pdfs/newsletter/LAL_Vol.13No.2.pdf

https://www.fda.gov/ICECI/Inspections/InspectionGuides/InspectionTechnicalGuides/ucm072918.htm

https://compoundingtoday.com/Newsletter/Science_and_Tech_1307.cfm

 

About the author:

Seth DePasquale is a pharmacist and co-owner of BET Pharm, LLC in Lexington, KY; a compounding pharmacy specializing in long-acting injectable hormone formulations for equine reproduction. Seth is a 2002 graduate of Albany College of Pharmacy in Albany, NY and is a Registered Pharmacist in New York, Kentucky, Michigan, Oklahoma, Texas, West Virginia, Virginia, Alabama, Tennessee, Mississippi, Arkansas, Nebraska, Louisiana and Oregon.


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