I have a protein of interest that is expressing in Pichia. It is expressing well in the shaker flask, but when I apply the same expression methodology but now in a bioreactor I get minimal expression.
Any logical reason for this? Is the density inhibiting the expression somehow. I would think that that more cell mass = more protein expression but could there be some kind of quorum inhibition by pichia?
There is room for more biomass production in the bioreactor, but I keep the feeding rate such that it does not go to the maximal limit to allow for protein synthesis.
The reactor has many parameters which are absent in the shaker flask. For instance, the impeller rotational speed could affect your organism negatively. The environment in a reactor is well controlled than in flask
http://www.mdpi.com/1420-3049/23/1/125/pdf. Article Agitation effects in yeast propagation
These articles might help you.
Yeah I figured you'd be talking about shearing forces but this doesn't apply.
The article shows streptomyces which are filamentous bacteria that can shear easily.
You can see in the paper cell mass decreasing as RPM go up. That's due to shearing. You would have to go pretty high to shear yeast.
I have no evidence of shearing as cell mass increases at highest RPM and then stays constant at lower RPM to sustain a constant density. SDS-PAGE gels don't support shearing either.
Hi,
as mentioned above there is NO real linear upscale from shake flask to fermentor, for a variety of reasons .... In short up-scaling has many considerations and limitations, intrinsic and extrinsic, from the used yeast strain, the vector used, the expressed proteins, oxygen transfer, aeration, agitation, pH, clonal stability, temperature, and more. and it usually takes time to calibrate these.
I don't know what is your final aim, for now I will just simplify for you your decision:
1) Do you just need to upscale from 1-2 liters to 10 liters to express and get several fold more from your protein of interest? If your answer is yes, then I suggest you to make several 5 liters flasks and continue as you have done so far.
2) If this is a long-term project where you need a lot of protein, either for biotech, pharma or research, then you should start process development which may take a few months to complete.
To give you a flavor see the following recent reviews that directly discuss this topic.
Article Cultivation strategies to enhance productivity of Pichia pas...
Article Engineering strategies for enhanced production of protein an...
Best of luck.
Ghil
I completely agree with what Parise and Ghil mentioned above. The flask reaction system does not exemplify what happens in a bioreactor system.
As a suggestion, there are stirred tank reactors (magnetically) that best exemplify a bioreactor ... you can find them as stirrer culture flask and there are different small volumes. If you can get one of those and try it there rather than continue in large volume bioreactors, it would be preferable.
In my experience, I have worked on the scaling of enzymatic systems from 1 mL to 5 L bioreactors. I do not know how many times you have tried to perform the experiment in the bioreactor system, but you have to take into account that it is very possible that the first few times the experiment goes wrong. Sometimes it is required to perform the experiment more times.
Cheers!!!
I appreciate the responses, but obviously I considered that there are different parameters between the shaker flask and the fermenter. I understand the general differences and the global variables to consider, but I am looking for specifics.
I am really looking to see if anyone has practical experience with this to give a few insights.
And yes I have performed the fermentation multiple times. It's not a one time thing and rush to researchgate.
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