We have established several cell lines for expressing mAb with GS/CHO systems. The expression level decreased obviously in > 50L bio-reactors. Some papers say that it's because of the effect of methylation in the CMV promoter region. Does anybody have related experiences?
Yes, Methylation does affect CMV promoters, and we have observed silencing upon passaging of such promoters. However all is not lost, since 5-Azacytidine can reverse these effects, especially effectively in cell lines. here is one such example of the drug being used http://www.ncbi.nlm.nih.gov/pubmed/20933502
What exactly is your question - could you please formulate it a bit clearer?
To answer what I understand so far:
1.) "We have established several cell lines for expressing mAb with GS/CHO systems. The expression level decreased obviously in > 50L bio-reactors."
- I would expect that the availability of nutrients, oxygen, etc. decreases with bioreactor size. Are you sure it is the expression of the (trans)gene? Have you checked for reduced cell viability?
2.) "some papers say..."
- I could only find papers involving CHO and CMV methylation, but also involving bioreactors and upscaling. Please gives some references.
3.) "...that it's because of the effect of methylation in the CMV promoter region."
- Yes, the CMV promoter can be silenced by methylation. Yet, why should it be silenced only in large reactors, and not also in small reactors or normal cell culture flasks? (The only explanation I can think of is as a reaction reduced nutrient avalability, so basically the same as above)
In short:
Regardless of whether the CMV promoter is methylated or not, I think the cause of yout problem is likey to be the upscaling/ bioreactor size. Have you tested the upscaling on wild type CHO cells?
Good luck
Nick
we met with this problem in scalling up the production of therapeutic antiboy candidate. So we have test the culturing condition and cell viability. Now methylation of CMV promoter is proposed to be one possible cause for the result.
Also, one friend used virus derived promoter in controlling the IgG expression in CHO cells and never met with the phenomenon even using >50L reactor. I think it is interesting to find out the relationship between methylation and the promoter sequence.
Indeed, this is very interesting - but (i) there are probably already others who are investigating this, and (ii) your goal is to upscale, and not to find out why upscaling does not work sometimes. So your time may be better invested in recloning the mAb with the virus-derived promoter from your friend :-)
Good luck!
thanks for the advices.
my interest is to upscal the production of mAb with CHO stable cell lines established within a short time. virus-derived promoter can help me to do it. even though, I wish to see the progress on the topicof promoter methylation.
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