Hello! I have been trying to purify an antibody using protein A column chromatography but noticed that as soon as the antibody equate hits the neutalisation buffer (Tris, NaOH, ph 9) it forms a cloudy precipitate. Any advice?
Dear Anna
which antibody format?
Purified from? ibridoma? CHO?
which was you elution buffer? and how much neutralizzation buffer did you added? How long after elution? Can please provide me some more details?
best
Manuele
Dear Manuele,
I used Expi293 cells. It is a human IgG1 monoclonal antibody
You are eluting with low pH glycine, correct? I don’t see why you need to neutralize with a pH 9 buffer. I typically elute with 0.1M glycine, pH 3.0 and then neutralize with addition of 1/20th volume of 1M Tris, pH 7.5. Should work fine. Alternatively, you can apply the eluate immediately to a PD-10 column to buffer exchange into your buffer of choice.
Dear Thomas,
yes that’s correct.
thank you very much for your reply!
Just to make sure I understand correctly, how will the change of pH in the neutralisation buffer change eliminate the precipitation?
Dear Anna Meister
i purified a lot of different mabs using protA from Expi293 surnatants using Triua 1M pH9 for neutralizzationever observed precipitation after elution
Generally what i do is:
1) Dliute directly in a falcoon containig the Tris 1M pH9 (1/20 of the ELution volume is ok) and after this change the buffer in PBS using desalting coloumn
2) ProtA elution could be performed more genlty than protG using citrate pH3,0 instead glycine pH2.7
in the attached link you can find the exact buffer composition than i'm using;
https://proteocool.blogspot.com/2022/07/tips-of-week18-mab-select-cheaper.html
just a comment: in my experience ExpiCHO work much better in term of yields than Expi293 for the production of antibodies. Those cells are more expensive in terms of cost for liter and you need a second shaker working at 32°C for incubate the cells during transfection but in my experience the productivity is quite higher (3-4 fold)
Here you can find an example
https://proteocool.blogspot.com/2021/06/tips-of-week-8-expi-cho-powerful-cell.html
good luck
Manuele
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