[Sabine Suppmann]

Dear Colleagues
welcome to the forum !
My first and very urgent question:
can you recommend reagents succesful in endotoxin removal?
Thank you very much in advance
Best
Sabine

[Anonymous]

HI Sabine,

We bought the following strain for a similar purpose (purification of a protein without endotoxin) But the project ended up being canceled, so I can’t exactly say if they work or not.

http://www.lucigen.com/ClearColi-BL21-DE3-Electrocompetent-Cells/#subcat-tabs2

But I would assume that you would still have to be careful about endotoxin contamination from other sources – flask, columns etc.

Dr. Mario Lebendiker was a nice description of available kits (see link below). Not sure if it is up to date.

http://wolfson.huji.ac.il/purification/extraction_and_clarification.html#endotoxin

Good luck.

Best, Andrea

[Kim Remans]

Hi Sabine,

We had to do some endotoxin-free purifications for an immunology group last year. We “decontaminated” the Akta with NaOH and Triton-X114 and used fresh columns. We measured the LPS content after each step (Ni-NTA, tag removal, second Ni-NTA and ion exchange) and after the HiTrapQ virtually no LPS remained (0,5 EU/ml = 0,05 ng LPS). So now we often just run a HiTrapQ to remove LPS.
Another thing we tried were the endotoxin removal beads from Pierce. They worked quite nicely as well, but it didn´t give a better result than the HiTrapQ in our hands.

Cheers, Kim

[Sabine Suppmann]

Dear Andrea and Kim
we had immediately tested the Clear E coli when it was launched (even before as ß tester) Apart from growth problems, the Endotoxin content was equally high with this strain !!
Thanks Kim for your suggestions, the strict NaOH has not solved the problem here. The last product I had seen was Hyglos.
So we would go for HiTrapQ
Best
Sabine

[Anders Olsson]

Dear Sabine;

This may be out of scope for you but here is an inexpensive method developed in my previous lab for endotoxin removal from mAb’s.

J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Sep 1;856(1-2):343-7. Epub 2007 Jun 29.
Endotoxin reduction in monoclonal antibody preparations using arginine.
Ritzén U1, Rotticci-Mulder J, Strömberg P, Schmidt SR.

Best,
Anders

[Patrick Duriez]

Hi Sabine,

There is a certain number of columns available to remove endotoxins but they are often expensive.

We decided to try out the use of Triton X114 and we have had good results. The Triton X114 has peculiar properties as it is liquid at 4C and solid at room temperature due to its unusual melting point temperature. In short the endotoxin is carried away during washes with Triton X114 at low temperature. We usually have our protein (GST-, His-, MBP- …) captured on loose beads (we don’t have a chromatography system in the cold) and work in the cold room. The beads are washed with buffer + Triton X114 in the cold (a minimum of 30 min on rollers) then washed extensively with buffer without detergent before proceeding to the elution of the protein.
There are other protocols with the purified protein mixed directly with the Triton X114 and endotoxin removed by successive centrifugations at higher temperature to have 2 phases (solid with Triton X114/endotoxin and liquid with the protein) but I have never tried this.

Immunologists requiring endotoxin removal would like to be sure that there is no traces of Triton X114, particularly if the protein is to be injected into animals, so the washes are critical.

Here are the papers describing the method:
Single step protocol to purify recombinant proteins with low endotoxin contents. Protein Expression and Purification 46 (2006) 483–488
Simultaneous metal chelate affinity purification and endotoxin clearance of recombinant antibody fragments. Journal of Immunological Methods 314 (2006) 67–73

There are also some links on Mario’s website:
http://wolfson.huji.ac.il/purification/Protocols/Endotoxin_Clearance.html

I hope this helps.
Best wishes,
Patrick

[Barbara Giabbai]

Dear Sabine,
In the pharma company where I worked we were routinely producing and purifying an injectable protein expressed in E.coli. So the risk of killing the animals after was very high!
To remove the LPS from glassware we were sterilizing at 180°C for 5 hrs (no steam, dry autoclave).
To eliminate the endotoxins from the product we introduced a chromatographic step of Anion Exchange, but the best resin was the EMD TMAE from Merck-Millipore, and then testing with the LAL assay from Lonza. This type of resin gave us the best results, and the price was good.

Good luck,
Barbara

[Patrick Duriez]

Hi Sabine,

Just an additional piece of information. If your protein binds to a HiTrapQ, you can probably proceed to TritonX114 washes as well. We are currently working on removing endotoxin from commercial ovalbumin this way and the initial tests are promising.

BW

Patrick

[Nick Berrow]

Hi All,
Relating to some projects that we have on the go where proteins will be made in E.coli and used in mammalian cell culture but also for inclusion in the QC guidelines (as Raquel pointed out to me we are asking people to perform this QC test without giving them any details!) :
When is it necessary to test for/assay LPS?
How to test?
What would be acceptable levels of LPS contamination for cell culture?
How to remove LPS if contamination levels do not meet your tissue culture quality criteria?
Thanks
Nick

N.B. David started a parallel thread on endotoxins on 13/06/2018 as well…

[Mario Lebendiker]

I agree with you Nick, we must add it in our guidelines. I talked about it in one of our phone meetings, but we did not add

[Jean-Philippe Gaudry]

Dear all,
As mentioned in the previous comments, HiTrap Q is an efficient way to remove endotoxins.
In my case I work with a strong anion exchanger membrane format (Sartobind Q, MA15, Sartorius stedim), which is quite easy to use. The sample is passed through the membrane with a syringe and the flowthrough (FT) is recovered. The negatively charged endotoxins will bind the membrane and the protein goes in the FT (in the best case…). The membrane can be reused several times after the 1M NaOH cleaning procedure described in the instructions.
You have to be careful with the isoelectric point of your protein and the buffer used. Negatively charged proteins will also bind to the membrane and you can lose protein. In this case, you can play with the salt concentration of your buffer (LPS binds strongly to the membrane) and still work with negatively charged proteins. Otherwise you have to test another method.
This Q membrane saved many protein batches (specially after a long expression and purification effort, you realize that your protein is contaminated)!
Best,
Jean-Philippe

[Nick Berrow]

Hi All,
Thanks for all of the tips and protocols for endotoxin removal but how is everyone testing for endotoxins?
I’ve recently come across the Genscript kit:

https://www.genscript.com/kit/L00350-ToxinSensor_Chromogenic_LAL_Endotoxin_Assay_Kit.html?position_no=3&sensors=search%20product%20box

that seems to be pretty simple and cost-effective.
Has anyone used this kit?
Thanks
Nick

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