I'm currently working on purification of protein process. In final step of the purification I found out endotoxin in the product, to eliminate it I use ultrafiltration by use of cut off 10 and 30 kDa but it wasn't applicable,
In the second attempt I applied protein solution on Q sepharose resin, the pH of solution was 6.5 and the PI of target protein in 5.22 then I set a gradient between 0 mM NaCL and 250 mM NaCL but in wasn't applicable too. I know that Lipid A has negative charge so target protein and endotoxin can absorb to the ligands simultaneously and both of them detach by increase ion strength so we failed in this attempt.
Is it proper to decrease the pH of protein solution to 4 and applied it on Q-sepharose resin, then collect the target protein in flow through ? I think that Lipid A in LPS has negative charge so it can capture to the ligand of Q sepharsoe.
Thanks in advance for your kind attention.
Dear Sir. Concerning your issue about the endotoxin removal from protein solutions. Endotoxins liberated by gram-negative bacteria are frequent contaminations of protein solutions derived from bioprocesses. Because of their high toxicity in vivo and in vitro, their removal is essential for a safe parenteral administration. A general method for the removal of endotoxins from protein solutions is not available. Methods used for decontamination of water, such as ultrafiltration, have little effect on endotoxin levels in protein solutions. Various techniques described in the patent literature are not broadly applicable, as they are tailored to meet specific product requirements. Besides ion-exchangers and two-phase extraction, affinity techniques are applied with varying success. Also, taylor-made endotoxin-selective adsorber matrices for the prevention of endotoxin contamination and endotoxin removal are discussed for this purpose in the attached paper. I think the following below links and the attached file may help you in your analysis:
http://archives.njit.edu/vol01/etd/2000s/2000/njit-etd2000-023/njit-etd2000-023.pdf
http://microsite.sartorius.com/en/biotechnology/laboratory/products_applications/membrane_adsorbers/literature/pdfs/Appl_Sartobind_Endotoxin_Removal_SL-4040-e.pdf
Thanks
Dear Elgailani
thanks for your kind attention it was applicable in our process.
Hello,
Endotoxin can be removed with three possible mechanisms:
1. Affinity based : Affi-Prep Polymyxin resin from Bio-rad
http://www.bio-rad.com/en-in/product/affi-prep-polymyxin-resin?ID=5c53a1f3-b0da-4799-8a54-7e94f090e0f9
2. Charge based : MacroPrep High Q ( When your Protein have basic pI and operating at neutral pH) , You can try Ceramic Hydroxy Apatite also.
Selecting right resin for endotoxin removal :
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6813.pdf
3. Size based : If your desired protein size less then 5kda then you can try TFF (10kda) in flowthrough (permeate mode), Endotoxin have higher size so endotoxin will retain in retenate and your product will come in flow-through.
Hope this is useful.
Please contact me for any more info or support.
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