Can anyone help me know what I should expect as an acceptable range of endotoxin in EU/ml from my purified recombinant protein sample, purified using E.coli expression vector.
Dear Kamalika Samanta
it depends from the downstream application that you would like to perform with this protein.
The limit for human dose is
5.0 EU/kg
https://pubmed.ncbi.nlm.nih.gov/17847072/
https://www.fda.gov/inspections-compliance-enforcement-and-criminal-investigations/inspection-technical-guides/bacterial-endotoxinspyrogens
therefore the EU/ml acceptable depends from both, injected protein dose and protein concentration
for example if you use a mouse model and you consider the mice weight 20g, to fulfill the 5EU/kg limit you have to inject less than 0,1EU.
if you would like to inject 200ug in 100ul , you protein do not have to contanin more than 1EU/ml or 2EU/mg
im my experience, generalle a purification step with endotoxin removal coloumns is necessary when you are producing protein from E.coli.
best regards
Manuele
Hello Manuele, thankyou so much for the explanation. I have long purification using expression vector and conventional anion exchange chromatography by packing my own columns, what i observed through LAL test that I see a range of OD405 of 0.3-0.7 EU/ml. After purification i use spin column for detergent removal but have not used endotoxin removing column. I use a total of 200ug rProtein in 600 ul.
Kamalika Samanta In industry, we express the Endotoxin concentration in EU/mg of protein. The regulatory requirement of Endotoxin levels depends upon the mode of administration. In our case, the FDA approved limit is 1 EU/mg of protein.
You can convert from EU/ml to EU/mg by dividing the Endotoxin conc. (in EU/ml) with the protein conc. in (mg/ml). So, in your case (considering protein conc. is 0.33 mg/ml and ET conc. is 0.5 Eu/ml) the final Endotoxin conc. is 0.66 EU/mg of protein which should be within the limit for most of the applications.
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