[Karine Bernardeau]

Hi everybody,
I have a question about screening buffer for antibodies formulation. I find 96 plate with various different buffers to test stability of antibody but no information about time for incubation of the antibody in each buffer before test stability/aggregation by nanoDSF? If someone have already use this sort of plate and after how many time do you estimate that antibody have reach a stable state in the new buffer?
Thanks a lot for help.
Best regards.
Karine Bernardeau

[Mario Lebendiker]

Hi Karine,
I have very low experience with nano-DSF, nevertheless, my opinion is: you are checking there thermal stability from RT till 99 C, so you do not need theoretically incubation time, just mix the Ab with the different buffers and check the Tm of each mixture. This information will give you the best buffer to stabilize your protein.
But, since aggregation and stability is time dependent, incubation before checking Tm could be highly relevant. Perhaps there is some information in the web, if not, could be a very good idea to incubate each mixture for several hours or a few days at 37C or higher temperature (mimicking a few months at -80C), and only then to check Tm
Here are some other ideas from OptiSol (http://www.dilyx.com/protein_solubility_screen_home2 )
Elevated Temperature Incubate 24 hours at 37°C
Long-Term Storage Store 2 weeks at room temperature
Ice/Liquid Transition Freeze and thaw 20 times
Shear Force Force material 20 x through narrow syringe need
Intense Light Expose samples to direct sun-light or UV light for 1 h
Chemical Compatibility Add 10 mM of caustic reagent (i.e. heavy metal)
Surface Exposure Add 5 uL of 10 um diameter glass beads
Air Oxidation Bubble 10 ml of air through sample
Chemical Oxidation Add Hydrogen Peroxide

[Susanne Witt]

Dear Karine,

I worked with therapeutical antibodies for quite some time in pharmaceutical industry. Except when working with some extreme mutations that e.g. inhibit dimerisation, antibodies tend to be extremely stable. Of course the stability is dependent on the temperature. When considering stability of the antibodies in various buffers we looked at the stability in a time course of at least weeks storing them at 4 C up to room temperature. Those to be further developed into a product needed to be stable for month. To speed up the process you can keep the antibody at elevated temperature to reduce the half life. Still, the half life should normally be in the range of days. Obviously, the situation is different when you try to mimic their PKPD in vitro and look for their stability in lysate or even blood. But even in such systems you need a half life of hours or better days to have a suitable antibody for further development. I hope this is the information you’re after. Kind regards, Susanne

• This reply was modified 8 months ago by Susanne Witt. Reason: deleting

[Karine Bernardeau]

Hi, thanks a lot for your answer Mario and Susanne.
I agree, I think we will test first the effect of differents buffers without incubation time but in a second step we have to check long term stability of the antibody at various temperature and time course. Thanks again.
Best regards.
Karine

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