I have cultivated some hybridoma clones for about two months, since I thaw them. I have tested for antibody production by Flow Cytometry and ELISA gathering positive results. Then, I expanded them in order to obtain enough cells (about 25 milion cells) and put them in a biorreactor for bigger production (350 mL of medium). After 1 week, I tested for antibody production (by flow cytometry) and the result was negative for all clones. Can it be possible that all cells lost their potencial for antibody production? It looks like very weird for me. I have checked and the cells are still alive inside the biorreactor.
There is anything that I can do to avoid this issue?
I think it is a common problem when trying to upscale production. I would try incremental approach. Cells require contact.
Hi Priscilla,
It is well known that after long culture period, hybridoma cells loose their production capacity for the mAb. They are not totally stable. The first option is to subclone your hybriboma and look for subclones that will maintain the expression of the mAb for a longer time than you need for your scale up.
The other option is to prepare a recombinant cell line out of your hybridoma. This last option has the advantage that you will never loose your mAb because since you will have sequenced the antibody coding sequences thanks to molecular biology approaches such as 5'RACE, you will know the sequence. This last option has also the advantage that you can manipulate your recombinant mAb by changing its affinity or abolishing the binding to FcgR that can be a problem if you just need a tool or a blocking antibody.
hi
i agree with Fontayne , you can do :
1- you should prepare enogh back up from mother clone in nitrogen tank.
2- before scale up ,you must be at least 4 step sub cloning to achieve pure single clone & stable clone
3- test clones by PCR for mycoplasma infection .
4- you can used ascites technique for Lab scale of Mab production.
good luck
I agree with Alexandre answer, we got the same results with long term cultivation of hybridoma cells, thus we start again regularly from frozen cell stock.
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