Low protein recovery

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chemjmh1's picture
Low protein recovery

I am getting very low recoveries of protein from an HPLC assay.  The method is for a protein drug product with the analytical level around 37-40 ug/mL.  Protein recoveries are fine at 40ug/mL, but lower than that-problem.  The request has been made to get the LOQ (S/N of 10) at 0.1%.  My best S/N at this level has been 9, while recoveries have ranged from 15-30%, with poor reproducibility.
Tried Tween 20 (0.01%) and silicanized HPLC vials with slight success.  Any thoughts on protein blocking agent or how to obtain better recoveries?  Assumption is that I'm losing protein to glassware, etc. at these very low levels. 

Dr. Analytical
Dr. Analytical's picture
What kind of filters are you

What kind of filters are you using?  Some are known to bind proteins.

And have you verified that you are not losing anything on the column?