Recently I had my CHO cells cultured in a basal medium (Hycell CHO medium). During the fed batch process, a small amount of high concentration of feed containing Tyr and Cys, which has a high pH (>11), was added into flasks or bioreators. When the two media got touched together, the cultures became black instantaneous around the adding point, then when the cultures got homogeneous after shaking or stirring,the black would disappear. Now the question is that what reactions have happened? Does it impact on cell growth and how to prevent that? Has anyone experienced this? Thanks!
I don't know the media you are using but could the colour change been due to pH changes? You could test that easily yourself, this would also explain while the colour disappears after stirring.
Could this be cysteine SH binding to Fe? The color is visible because of the transient high concentration that quickly dissipates.
Dear Michael B LoMonaco, maybe you are right, I have tested the sample, first the sample pH was adjusted to about 11 with NaOH, the colour did not change,then Cys was added alone, soon the medium bacame black, after a long time the black became shallow, then the sample pH was adjusted to neutral, the black dissipates soon. If Cys SH binding to Fe, does it impact on the synthesis of antibody? and how to prevent that binding? Could we add some reagent like 2-Mercaptoethanol to protect SH which do not impact on the synthesis of antibody and cell growth? I also want some references about the reaction mechanism. Thanks
I think your HyCell medium contains a lot of extra additives besides metal ions (assuming it is a chemically defined medium). In this case rather the pH is the problem causing some metal ions to precipitate transiently. I am not an expert but i think it is not problem if the precipitate dissolved. Try to bring the feed pH to around physiological as possible might be worth. However, reducing agents are able to destroy IgG quaternery structure: i would not suggest to use them.
I don't completely understand, but I would try to adjust the solution that is pH 11 with some type of buffer before combining it with your media in case precipitation does destroy something. Also, as mentioned, make sure the complete medium is still at physiological pH.
I think the question is if there is a reaction happening due to the high pH and the colours disappears because of the dilution effect (which would mean you have something in your media you do not want) or do you have a reaction which is reversible and the moment you have a homogenous pH again the problem has solved itself. If possible the easiest solution would be to have a lower pH in your feed medium to avoid whatever reaction is going on.
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