Learning Objectives

1. Discuss the importance of low levels of endotoxins in parenteral preparations
2. Discuss the limits for endotoxins for various routes of administration
3. Discuss ways to control the levels of endotoxins in final preparations

In this weeks' 483 we're going to be talking about Bacterial Endotoxins and the testing related to endotoxins. Let's first take a look at the observation then we'll discuss what endotoxins are and why it's important to keep them out of your preparations.

First thing that should be talked about specific to this 483 is that we're dealing with preparations that are intrathecals or will be injected into the spinal fluid. One thing to know about intrathecal injections in particular is that they have a lower limit for endotoxins than other injections.

When performing final testing for preparations one of the tests is a bacterial endotoxin test. This test is performed according to USP Chapter <85> Bacterial Endotoxins Test. For parenterals that are not intended for intrathecal administration the limit is 5 EU/kg (Endotoxin Units). For intrathecal injections the limit is 0.2 EU/kg. The per kilogram is the weight of the patient.

The fact that there is a much lower limit of endotoxin specifically for intrathecal injections should give an indication as to the importance of having very low levels of endotoxin in the final preparation.

This particular pharmacy is probably compounding the intrathecal in either very low batch sizes (less than 5) or one at a time as they're ordered by a physician. By not batch compounding the intrathecals there's not enough of the final preparation to send off for testing. My guess is that these are being made one at a time per order and are delivered to be administered within 24 hours.

What are Bacterial Endotoxins and where do they come from?

Most common, endotoxins are by-products of the gram negative bacterial cell lysis. They are lipopolysaccharides that make up part of the cell wall. Gram negative bacteria are commonly found in water sources. So if at any point you are using water either in the preparation itself or using materials that have been washed (i.e. glassware), there is a very high risk of having bacterial endotoxins in the preparation if there aren't any controls in place to limit the level of bacterial endotoxins.

If glassware is being washed and re-used for processing the preparation, the glassware should go through a depyrogenation cycle meaning they would be held at a temperature of 250 degrees celsius for a minimum of 30 minutes. The time may vary depending on the glassware and oven that's being used for the depyrogenation process. Depyrogenation cycles of glassware should be validated to show that the process of depyrogenation is producing a 3-log reduction in bacterial endotoxins.

Even if the process for limiting endotoxins in glassware in place you still need to consider any of the raw materials you're using (i.e. the active pharmaceutical ingredient or API). Water is used quite frequently in the manufacturing of pharmaceutical ingredients and the certificates of analysis for your API should include the limit for endotoxin and the results of testing for endotoxins. It's pointed out in this 483, that the certificates of analysis (COAs) do not include results of endotoxin limits for the APIs.

Why are Endotoxin limits so critical?

When a sufficient amount of endotoxins are injected this could result in a fever or can be as severe as death; particularly if injected into the spinal fluid where there is no immune system. Since this pharmacy doesn't have COAs that show the endotoxin results and are not testing for endotoxin in their final preparation, they really have no way of showing that their intrathecal injections are meeting the required limits for endotoxins.

There are several ways that this pharmacy could increase the level of assurance that their process is limiting the endotoxin in the final preparation. According to USP Chapter <1228> there are 3 ways of controlling endotoxin in parenteral products:

• Indirect Control, "which is comprised of a series of preventive measures that control bioburden, the potential endotoxin contribution by formulation components (e.g., raw materials, APIs, excipients), water, primary packaging components, equipment, and the manufacturing environment, including personnel."
• Process Control, "in which endotoxin is monitored at Critical Control points (CCP) during processing to ensure that there is no increase in endotoxin. These process control elements are subject to validation or qualification."
• Direct Control, "or the direct destruction or removal of endotoxins from product streams, equipment, and primary packaging materials. As with controls on processing, direct measures of endotoxin destruction or removal must be validated."

First, you'd need to evaluate the process for compounding the preparation. At any point is water being used in the process (i.e. glassware and other materials for compounding)? If so, are you using the proper process to control endotoxins? Are there ways to possibly eliminate the need for glassware and materials that may have endotoxins; could single use depyrogenated and sterilized materials and equipment be used?

Lastly, is there a way to compound the intrathecals in batches so that a percentage of the batch is sent off for testing to increase the level of assurance that endotoxins are at low enough limits for the injectable being compounded.

References

1. FDA Inspection Technical Guide: Bacterial Endotoxins/Pyrogens
2. USP Chapter <85> Bacterial Endotoxins Test
3. Associates of Cape Cod BET White Paper: ENDOTOXIN LIMITS For Parenteral Drug Products
4. USP Chapter <1228> Depyrogenation
5. USP Chapter <1228.1> Dry Heat Depyrogenation
6. USP Chapter <1228.3> Depyrogenation by Filtration
7. USP Chapter <1228.4> Depyrogenation by Rinsing