I inherited a poorly working method for a 93kDa protein that needs significant revision. Column is a waters XBridge BEH C4 300A and mobile phase is 0.1% triethylamine in water (A) and ACN (B); diluent is 1% SDS (also confusing me). During development when mobile phase was modified with TFA or formic acid it lead to significant and persistent carryover. When TEA was tried (~pH 12) the carryover got better to be workable but when i titrated the TEA with acetic acid for TEAA to get the pH to a more reasonable operating range the separation fell apart; at 0.1% TEA i am burning through columns rapidly as i am at the operating limit. Why could this be happening; it seems more dependent on the pH than the ion-pairing reagent? Also the protein is in an oil-in-water emulsion and a liquid/liquid extraction is used to isolate the protein and remove the vehicle, is there any other methods to be used to possibly dilute and shoot? any guidance on approach from the community is greatly appreciated!
I would eliminate TEA altogether. I would also redevelop and validate a 'new' method that meets the requirements of ICH Q2(r1).
My 1st idea would be to use IPA instead of ACN since the protein is very fat soluble. In addition BEH is a 'very' polar column so you may want to evaluate silica or amino columns.
A 'very' alkaline mobile phase will cleave the ODS linkages and thus kill the column.
Thank you Bruce for your suggestion.
Have you experience using the Proteomix RP column from Sepax Technologies; the phase is phenyl and substituted phenyl with 1000A pore size. Thoughts on that column for my particular problem?
Never used Proteomix column though as its name suggests it may be designed for 'regular' proteins like globins. I would look around because your protein is 'very' strange. Is it found in a cellular membrane? What is its regular environment?
very strange doesn't begin to explain this protein and the protein has no native environment; it is an engineered hodgepodge of several antigens expressed as a single protein.
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