Possibly different glycosylation patterns.
What kind of linkers are you using and which protocol do you follow?
How do you determine the success of your reaction? Do you know the composition of the liquid the ABs are dissolved in?
Can you check if the reduction and coupling work with an unrelated protein, e.g. BSA?
There are many parameters to control:
Possibly you have to titrate TCEP and incubation parameters (time, temperature, pH). Then, Maleimides react with -SH groups best at pH 6.8 (useful range 6.5 to 7.0).
In worst case, you have too much payload on your AB which could render it non-fonctional. Or the TCEP has broken your AB into pieces.
The thioether formed with the maleimide is somewhat susceptible towards hydrolysis (the reaction is reversible under certain conditions, esp. in biosystems). To cope with this, there are self-stabilizing linkers as an alternative (see, e.g., https://www.iris-biotech.de/de/blog/maleimide-crosslinkers/). If this should be the problem, you probably need to construct and assemble your own linker/payload molecule. A different strategy to attach the payload, e.g. by aqueous copper-free Click Chemistry, also might be an option. The company I have mentioned above has lots of building blocks for this purpose and can procure the MMAE and TCEP.
What is your preparation scale, and how do you test the functionality of your construct ?
Edit: Maybe some hints in this article: Article Influence of disulfide bond isoforms on drug conjugation sit...
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