Comparison of strong anion-exchangers for the purification of a PEGylated protein.
We have studied the effect of protein PEGylation on ion-exchange adsorption using bovine serum albumin as a model system. The free sulfhydryl group of BSA, located on cysteine 34, was PEGylated using the maleimido-PEG chemistry. Several different BSA preparations were screened for extent of reaction using a 30 kDa PEG reagent. The highest yielding BSA preparation was PEGylated using linear 12 kDa and 30 kDa PEG reagents at the 1 liter scale. The PEGylated reaction mixture was purified by anion-exchange gradient elution chromatography to remove native protein and aggregates. Purity following anion-exchange chromatography was >90% as determined by analytical size exclusion chromatography. The elution salt concentration decreased with increasing PEG chain length. Breakthrough studies on six commercially available anion-exchange stationary phases with purified PEG-BSA conjugates confirm a very large decrease in dynamic binding capacity compared to the native protein. The decrease in dynamic binding capacity is likely due to modulation of electrostatic interactions caused by the neutral PEG chain and increased mass transfer resistance associated with the large size of the molecule. Of the stationary phases evaluated, the open porous structure of the agarose based ion-exchangers resulted in the highest dynamic binding capacities for the PEG-BSA conjugates. Frontal analysis experiments demonstrate use of this technique for purification of PEGylated proteins. A stationary phase that tended to exclude the large PEG-BSA conjugate was very efficient in removing native protein from a crude reaction mixture by frontal analysis.
Pabst
Timothy M
TM
Pfizer Inc., Global Biologics, 700 Chesterfield Village Parkway, Chesterfield, MO 63017, USA.
Buckley
John J
JJ
Ramasubramanyan
Natraj
N
Hunter
Alan K
AK
D000837:Anion Exchange Resins; D002850:Chromatography, Gel / Q000295:instrumentation; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation; D002852:Chromatography, Ion Exchange / Q000295:instrumentation; D004591:Electrophoresis, Polyacrylamide Gel; D011092:Polyethylene Glycols / Q000737:chemistry*; D011506:Proteins / Q000302:isolation & purification*
D000837:Anion Exchange Resins; D011506:Proteins; D011092:Polyethylene Glycols