I'm currently working on purification process. In final step of purification (after anion exchange step of purification (q-sepharose)) different fractions were collected. Endotoxin test by use of LAL and serial dilution was carried out. result shows that in final pooled fractions there was not any endotoxin.
After 3 days endotoxin test was repeated with the same procedure, the result showed that our sample have had endotoxin!!!.
For further sterility we passed sample through the microfiltter 0.22 micron for storage the sample for 3 days. we made sure that our bottles was sterile too. unfortunately the result was repeated. I mean after 3 days endotoxin was find out in our sample which was stored .could any one help me to find out the reason? what is the source or the reason for formation endotoxin.?
thanks in advance.
Hi Hadi, as you know endotoxin is everywhere, with or without the bugs that make them.
Storage of endotoxin-free samples in tubes containing endotoxin will contaminate your sample.
0.22 micron filtration might get rid of some of the bacteria, but endotoxin will pass through them.
Dear Stefansson
I said the sample passed through the filter to declare that it was sterile, the glass bottle was sterile too and endotoxin free. the sample was endotoxin free.
I don't know where did it come from. the API is hGH and base of the buffer is tris in pH 6.5
Hi Hadi, so the endotoxin free sample was put into a previously baked glass container?
Was the buffer you used from a vendor that tested it for endotoxin?
Hi
I have dedicated glass bottle for this purpose, I prepared it with this procedure:
at first it was washed with NaOH 1 M then washed with enough WFI (water for injection, distillated water) to remove NaOH, then it was autoclaved and put into furnace with 250 C for 2.5 hour to be prepared as container for storage.
All of buffer were tested about endotoxin content!!!
:(((((
Hi there,
I think the simplest explanation would be that your first detection assay hasn't actually worked and the following ones have properly worked. BTW are the results consistent in the second ad third tests?
Hi again,
Have you got the way to repeat the experiment and then the first detection assay on the IEX chromatography purified fraction and in parallel to run an positive control assay the purified sample you have in hands?
It looks like leakage from an unknown source. Working endotoxins free include to be sure that everything is free from them. caps of the bottles, tips used to get samples.... I recommend to increase the oven treatment for the bottle to 4 hours and let the caps in NAOH overnight before rinsin with water for injection . take the samples and do the filtration in a laminar flow not used for microbiology. Hope you manage.
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