Depyrogenation Lethality Passed VS Endotoxin Challenge Test Failed

Dear All,
I faced a problem recently while performing Heat Penetration & Endotoxin Challenge Test on Dry Heat Depyrogenation Oven. Need help for finding the root cause.
I was performing heat penetration by using half load loading patterns. (Heat Distribution Test is already passed and met all the criteria.) My QC department is using gel clot method to perform the challenge test. Below are my results:
i) Run 01 (vial size 3ml) : Endotoxin Challenge Test passed
ii) Run 02 (vial size 5ml): all three samples are failed
iii) Run 03 (vial size 5ml): Endotoxin Challenge Test passed but sample 3’s result is not so satisfying.
iv) Run 04 (vial size 10ml): 2 out 3 samples are failed
But all of the runs above reach 230 degree Celsius for 75 mins during depyrogenation phase and the lethality values are above 30 according to the Kaye validator result. Sensors calibration verification also done and passed.
So, now my problem is, I don’t know the root cause of the failure in Endotoxin Challenge Test while the oven and Kaye validator shows good results. Can anyone help me with this? What is key to pass the Endotoxin Challenge Test and what criteria should be considered during the preparation and testing of endotoxin?

Dear Jasonljt,
A lot of reasons can be cause of your problem.
First, maybe 230oC is not appropriate for your load and oven and try to raise temperature at 250oC for 75 minutes. In this case you will be able to compare log reduction with the previous tests and it should be greater.
Second, what is your acceptance criteria for log red? Per USP it is more than 3 log reduction. Your cycle 230oC/75 minutes must achieve at least 3log reductions. We have cycle 250oC/60 minutes and for this cycle we have more than 6logs.
Try to investigate next items: is equipment calibrated, is appropriate parameters input in Kaye validator (z value), are thermocouples positioned in the ampoules (bottom of the ampoule) and for me the most important , what Endotoxin and which target concentration was used? Did they contaminated after tests. Can operater make a mistake when performing lab tests?
Usually target concentration is 10.000EU. From two positive and from exposed ampoules you calculate log red and it should be more than 3 log according to USP.

[quote=Jasonljt]Dear All,
I faced a problem recently while performing Heat Penetration & Endotoxin Challenge Test on Dry Heat Depyrogenation Oven. Need help for finding the root cause.
I was performing heat penetration by using half load loading patterns. (Heat Distribution Test is already passed and met all the criteria.) My QC department is using gel clot method to perform the challenge test. Below are my results:
i) Run 01 (vial size 3ml) : Endotoxin Challenge Test passed
ii) Run 02 (vial size 5ml): all three samples are failed
iii) Run 03 (vial size 5ml): Endotoxin Challenge Test passed but sample 3’s result is not so satisfying.
iv) Run 04 (vial size 10ml): 2 out 3 samples are failed
But all of the runs above reach 230 degree Celsius for 75 mins during depyrogenation phase and the lethality values are above 30 according to the Kaye validator result. Sensors calibration verification also done and passed.
So, now my problem is, I don’t know the root cause of the failure in Endotoxin Challenge Test while the oven and Kaye validator shows good results. Can anyone help me with this? What is key to pass the Endotoxin Challenge Test and what criteria should be considered during the preparation and testing of endotoxin?[/quote]

Please be very careful that you are looking at this properly. The lethality calculation is based on making sure we are getting adequate kill while the Depyrogenation is based on breaking down the cellular structure of the endotoxins to ensure they cannot harm. If we consider that the act of the kill can release the endotoxins, it can be understood that Lethality does not play as important of a role in regards to Depyrogenation as it does in steam sterilization. You may be calculating the lethality at the start of your exposure, if you have the Kaye set up to do so, but if not, you are still getting lethality at much lower temperatures than it takes to get the Endotoxin reduction. Consider taking a look at the time and temperature needed for your endo challenge vials. I see that you are using different ones through the study. There could be issues with that as different sources may have different strengths. For a study like this you should ensure that you are using test indicators of the same type, size and lot. I would also take a look at the temperature profile of the unit. I do not know how well you have it mapped, or how your study is set up, but if the cycling of the heat allows some areas to get cooler than the control probe, that would impact your indicators. Consider calculating the temperature and time needed based on the kill of the indicators and you may find greater success.

[quote=Jasonljt]Dear All,
I faced a problem recently while performing Heat Penetration & Endotoxin Challenge Test on Dry Heat Depyrogenation Oven. Need help for finding the root cause.
I was performing heat penetration by using half load loading patterns. (Heat Distribution Test is already passed and met all the criteria.) My QC department is using gel clot method to perform the challenge test. Below are my results:
i) Run 01 (vial size 3ml) : Endotoxin Challenge Test passed
ii) Run 02 (vial size 5ml): all three samples are failed
iii) Run 03 (vial size 5ml): Endotoxin Challenge Test passed but sample 3’s result is not so satisfying.
iv) Run 04 (vial size 10ml): 2 out 3 samples are failed
But all of the runs above reach 230 degree Celsius for 75 mins during depyrogenation phase and the lethality values are above 30 according to the Kaye validator result. Sensors calibration verification also done and passed.
So, now my problem is, I don’t know the root cause of the failure in Endotoxin Challenge Test while the oven and Kaye validator shows good results. Can anyone help me with this? What is key to pass the Endotoxin Challenge Test and what criteria should be considered during the preparation and testing of endotoxin?[/quote]

Dear Jasonljt,

I agree with Mr. Zeljko. Consider to set the temperature higher or equal 250 degree.
Is Z value in KAYE validator set on 46.4? Base temperaature 250 degree.
Make sure that SOP for preparation endotoxin and check the result is correct refer to latest USP.
Check the distribution temperature, coldest and hottest. From reference of validation of depyrogenation is set temperature +/- 15 degree (static oven) and +/- 30 degree (dynamic oven / tunnel).

[quote=Jasonljt]Dear All,
I faced a problem recently while performing Heat Penetration & Endotoxin Challenge Test on Dry Heat Depyrogenation Oven. Need help for finding the root cause.
I was performing heat penetration by using half load loading patterns. (Heat Distribution Test is already passed and met all the criteria.) My QC department is using gel clot method to perform the challenge test. Below are my results:
i) Run 01 (vial size 3ml) : Endotoxin Challenge Test passed
ii) Run 02 (vial size 5ml): all three samples are failed
iii) Run 03 (vial size 5ml): Endotoxin Challenge Test passed but sample 3’s result is not so satisfying.
iv) Run 04 (vial size 10ml): 2 out 3 samples are failed
But all of the runs above reach 230 degree Celsius for 75 mins during depyrogenation phase and the lethality values are above 30 according to the Kaye validator result. Sensors calibration verification also done and passed.
So, now my problem is, I don’t know the root cause of the failure in Endotoxin Challenge Test while the oven and Kaye validator shows good results. Can anyone help me with this? What is key to pass the Endotoxin Challenge Test and what criteria should be considered during the preparation and testing of endotoxin?[/quote]

Dear Jasonljt,
calculate the lethality at the start of depyrogenation cycle for this purpose u need to set the temperature in the set up and u will get the actual lethality , getting the lethality of 19.95 at 250 degrees is the criteria we used to follow but since some of the product needs a lower range so please ensure that the raise in temp. does not pose a threat to quality of product for more please reply.

regards
nitesh
validation

Sorry for late reply. I was discussing with my superior and investigating lately.

Dear Mr Zeljko, actually I’m performing version 2 PQ, just changing loading patterns for better heat distribution. 230ºC 75min should be fine because the endotoxin test was passed that time in ver 1 PQ. Plus, the filter of my oven cannot support 250 ºC, and the machine got setting for emergency cooling if the temp reaches 260 ºC. So, I’m unable to raise the temp.
The equipment all calibrated. Sensors are put inside the vials. According to Kaye validator setup, base temp is 250 ºC, D value= 1, Z value= 46.4.
The QC dept. are using target concentration is 10.000EU. Their testing method should be no problem because the entire positive control and negative control results seems normal while the sample results failed.

Dear Guru, thanks for the info about the lethality. And yes, I did have the Kaye to set up to do so.

Dear Mr Good, about the Kaye setting, I’ve mentioned above. The heat distribution results are meeting the criteria of temp which temp are within ±10 ºC with mean temp.

Dear Mr Nitesh, according to my site’s previous protocol, the lethality value limit must above 30. I have checked my processes, even the coldest spots are above 40 in every run.