Method for detecting polysorbates

A method for identification of a protein sample that contains from about 10 ppm about 5000 ppm of intact polysorbate, wherein the method comprises:

• (a) measuring polysorbates in a sample comprising a protein, comprising
• (i) precipitating the protein by exposing said sample to an organic polar solvent,
• (ii) separating the protein from the precipitated sample by centrifuging the precipitated sample to pellet the protein and to obtain a liquid supernatant comprising polysorbates,
• (iii) separating subspecies of the polysorbates by subjecting the liquid supernatant to chromatography, wherein the chromatography comprises applying the liquid supernatant to a stationary phase column comprising an immobilised cyano group, and eluting the polysorbates using a mobile phase composition gradient, and
• (iv) detecting the separated subspecies of the polysorbates using a chromophore-lacking detector and quantitating intact polysorbates;
• (b) identifying the sample from (a) which contains from about 10 ppm to about 5000 ppm of the intact polysorbates, and
• (c) isolating and recovering said sample identified in step (b).