I use the refolding buffer with the following specifications for the refolding of brolucizumab protein.
l-Argenine, Sorbitol, EDTA, Tris, and fresh Cystein and Cistine.
I have already used these compounds to refold this protein and I was getting a proper protein refold. Currently, although I use the same compounds and with the same concentrations, the answer I get at the end is not the same. In fact, the protein disappears after being solubilization and added to the refolding buffer.
The issue really confuses me and I don't have an answer for it. Can anyone have an answer for me?
Luckily having a previous positive result is VERY encouraging! You must assume that something has changed, is the protein exactly what was used previously? (even a few mutated amino acids can entirely change folding and thus refolding) and are the regents entirely the same? Then you need to find where protein is going, it cant really disappear, so is it precipitated, absorbed onto container surface? With several careful control experiments you should be able to figure out what has changed and where the protein is. Good luck!
something must have been modified in upstream processing (assuming recombinantly produced mab is the case), leading to scrambled refolding products. Are you able to confirm the protein is holding fully the same PTMs and PMF profile...As Edward Michelini indicated, if all the reagents and even containers, concentrations, and experimental conditions are the same, there might be any change occurred at your protein level.....Since w/o refolding antigen binding capacity, efficacy, and titer cannot be tracked, Mass spec analysis can tell much at this point and may give some clues about your unsuccessful assay reasons.
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