At the first, take care and hope you are doing well in this pandemic situation.
Recently, I face the problems during protein A purification for mAb, Fc fusion and others.
The scheme of experiment is described below.
Media harvest with centrifugation -> Protein A purification with step elution condition.
The elute sample reveals yellow colored and makes the precipitation during neutralization(at pH 5.0~6.0).
I check that this neutralized sample forming pellet after centrifugation and supernatant contains target protein(protein concentration is decreased slightly).
I think the problem derived from below things and expect 1 is reasonable thing.
1. Target makes heteroaggregation with cell related components such as HCP, HCD and others.
2. Media components
3. Unexpectable issue
How do you think about this issue and how can I solve it ?
In my guess, depthfilter after harvest , optimization for elition condition and harvest condition is best way for solving them.
If you have others suggestions or know the state of arts could be applied, please let me know.
Thank you.
Dear Daniel Son
which cell line did you use for the mab production?
I have similar issue using the ExpiCHO after protein A and or proteinG purification some mab sample are yellow coloured and this colour cannot be removed with dialisys or ultrafiltration.
However the colour seem to stack of the 0,22uM filter and/or we can separate it by centrifugation at high speed after mab incubaction O/N at 4°C.
We suspect that it is some media component but sincerelly we are not sure.
best regards
Manuele
Dear Daniel
i forgot to say that However i forgot to say that, Thermo for protA purification relaase a note that suggest to slightly modify the elution buffer for prot A https://www.thermofisher.com/it/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/protein-biology-application-notes/optimizing-protein-a-purification-monoclonal-antibodies.html i tested it, and it seems to work. instaed PBS I used Tris 25mM, Nacl25mM ph 7.1 as washing buffer Citrate 100mM, Nacl 60mM pH=3 as elution buffer. in this way the mab eluted from the coloumn, while the yellow colour not. The yellow colour was detached from the coloum performing a short washing with NaOH 0,1MI think the yellow is someting present on the feed. However i forgot to say that, Thermo for protA purification relaase a note that suggest to slightly modify the elution buffer for prot A https://www.thermofisher.com/it/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/protein-biology-application-notes/optimizing-protein-a-purification-monoclonal-antibodies.html i tested it, and it seems to work. insted PBS is used Tris 25mM, Nacl25mM ph 7.1 as washing buffer Citrate 100mM, Nacl 60mM pH=3 as elution buffer. in this way the mab eluted from the coloumn, while the yellow colour not. The yellow colour was detached from the coloum performing a short washing with NaOH 0,1M
I am trying to remove the yellow colour from the pellet after ultracentrifuge of expicho supernatant. can I filter it too?
do you know what is making this colour?
thanks
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