- This topic has 5 replies, 3 voices, and was last updated 3 months, 3 weeks ago by Mario Lebendiker.
April 5, 2021 at 09:46 #3705
Hi to all
Here is a nice technical article how to Reduce Endotoxin / Lipopolysaccharides to FDA limit:
Triton X-114 and Amine-Based Wash Strategy Reduces Lipopolysaccharides to FDA Limit and Achieves Purer, More Potent Recombinant Immunotoxin – Rebekka George et al. – Bioconjugate Chem.2021 – https://doi.org/10.1021/acs.bioconjchem.1c00013
Accepted LPS limits for therapeutics as well as for substances used in immunological studies in animals must be very low. In this article, the authors shows that in order to obtain LPS contamination well below FDA limits, the use of the popularly known Triton X-114 wash or the polyamine-based wash strategies are not enough in many cases. Only the combination of both technologies can considerable reduce endotoxin levels. The poly(L-lysine)-based or polyamine-based spin columns can be replace by on-column Arginine wash for upscaling as well as automated procedures (but not for IEX)
April 5, 2021 at 15:12 #3706
Thank you very much for pointing out this article.
Can I ask you what you mean by replacing the spin columns with “on-column Arginine wash”? Could you give an example of the procedure?
April 5, 2021 at 15:36 #3707
for the Arg on-column washing they cite: Ritzén, U., Rotticci-Mulder, J., Strömberg, P., and Schmidt, S.
R. (2007) Endotoxin reduction in monoclonal antibody preparations
using arginine. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 856
Arg as other polyamines reduce endotoxin. Arg wash can be use for protein A or G chromatography, and some other affinities, and HIC
What is nice in the article is that they show 1) that there are some differences between different endotoxin spin columns 2) that spin columns alone or Triton X114 wash alone is not enough. Apparently, they are eliminating different endotoxins
April 5, 2021 at 17:17 #3708
Many thanks for your reply Mario. I apologize for not reading the paper in full before asking my question!
I usually use Triton X114 alone so this is indeed very interesting.
April 6, 2021 at 09:09 #3709
thank you for this article, really helpful. We never managed to reduce LPS enough in our NLS-CAS9 preps which for certain cell lines is too high.
Worth trying this
April 6, 2021 at 09:32 #3710
Endotoxins are a very heterogeneous populations, so the possibility that you need more than one method to completely eliminate them is not a surprise.
In the article they do not mention the use of Anion exchange, that is another alternative, cheap and very popular in the industry
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