I get endotoxins in my antibodies that are purifed on Protein A column in a chromatography system. The sample are tested before introduced into the system and are negative. I have tried to wash the system and column with NaOH (1M) but it does not help. What should I do?
Are you cleaning just your system or your column as well? It can be contaminated with endotoxins. If so you can wash it with 0.5 M sodium hydroxide with a contact time
of 15 minutes.
Are you performing a quick wash or a long wash on your system? You should run NaOH on your system for at least 2h.
If you cannot solve this problem, you can use an endotoxin removal column as a final step. Here is one example:
https://www.thermofisher.com/order/catalog/product/88270
Hi,
I agree with Andre that removing endotoxin from the column is going to take time and since column and matrix are washed with 1M NaOH the removal of NaOH from the column takes even more time. Moreover removal of Endotoxin by this method will require endotoxin free water (or WFI) which again is expensive, and at times can be rate limiting.
I will advice you to use endotoxin removing filters or columns as suggested by Adam.
Thank you for your interesting answers!
I clean the column with 0.5M or 1M NaOH with a contact time of 15 minutes before every run.
I perform system cleaning whith 1M NaOH, contact time 30 minutes. Is that too short then? And rinse with sterile water that I suppose is free from endotoxins, but I will definitely check that!
Yes, please check the water that you are using for cleaning the system should be free from endotoxin.
I know that you might be aware that not necessarily sterile water is endotoxin free, that's why I asked you to use either endotoxin free water or Water for injection.
Also please check that all the glass wares/ plastic wares that come directly in the contact of your product and buffer should be strictly depyrogenated.
Understand the source first. : Container , Raw materials , Water or process it self
For container : use glass container (DHS) - Depyrogented
For Water : you can use positively charge membranes or anion ex-changer sorbent to remove endotoxin from water source.
If it is in process carry over then : If your molecule is antibody , know the pI and use Anion exchange sorbent for endotoxin, HCP , aggregates and DNA removal. depending on pI of your antibody, select pH of step in flowthrough mode. (desired product in flowthrough and endotoxin bind to the column)
e.g. PolyQUAT , PolyPEI for more info , please go through with attachment.
Cleaning Protein A column with 1M NaOH will damage the column as protein A is not stable at 1M NaOH. Please dont use that much conc. you can use 0.1-0.5M NaOH for 15min hold time.
For system you can use , 0.5 - 1M NaOH solution and hold time as per system manufacturer recommendations.
http://www2.avantormaterials.com/l/44622/2016-05-17/7b97yp/44622/100576/Process_Chrom___Sales_Flyer_10.02.15.pdf
If the article/Material is heat resistant then according to Pharmaceutical guidelines it required to bake it at 250°C for min 30 min.
If the material is heat labile then ,chemical treatment with 1n NaOH for a validated period of contact time needs to be establiehed.
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