Proteolysis has been identified as the main cause of target loss during the purification of a 75 kDa glycosylated enzyme from Chinese Hamster Ovary (CHO) culture harvest. Acidic pH and metal chelating agents were found effective for the suppression of proteolytic degradation of this glycoprotein. We have therefore developed a three-column procedure for its purification. This enzyme was captured from culture supernatant with a hydrophobic cation exchange column under mild acidic condition, followed by two additional polish steps consisting of high capacity anion exchange and hydroxyapatite chromatography. This new purification scheme, with the combination of in situ inhibition of proteolysis and chromatographic condition optimization, has delivered a ten-fold increase of target protein yield.

Protecting target glycoenzyme from proteolysis during purification

249th National Meeting (2015)

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