Separation of protein charge variants with induced pH gradients using anion exchange chromatographic columns.
This work concerns the chromatographic separation of protein charge variants using pH gradients generated by step changes in buffer composition with weak base anion exchange columns. A local equilibrium model is first developed to describe pH transitions occurring in the column using buffers comprising neutral, zwitterionic or positively charged species. Model predictions, based solely on the resins' titration curves and obtained with the method of characteristics, show, in excellent agreement with experiments, that induced pH gradients of varying durations and shapes can be obtained with a broad range of buffer systems including Tris, Bis-Tris propane, histidine, and their mixtures and ethanolamine. The separation of protein charge variants is then demonstrated for bovine apo-transferrin and for a monoclonal antibody. The resolution of the charge variants present in these proteins, demonstrated via isoelectric focusing analyses, is obtained for conditions amenable to scale-up for preparative purposes; that is larger particle sizes (90 mum), higher flow rates (100-600 cm/h), and higher protein loads (2-5 mg/mL). Because the approach requires only step changes in buffer composition and commonly available, unretained buffers species, practical implementation is straightforward. The focusing effect of the induced pH gradient results in relatively sharp peaks and substantial resolution even for these conditions.
Pabst
Timothy M
TM
Dept of Chemical Engineering, University of Virginia, Charlottesville, VA 22904-4741, USA.
Carta
Giorgio
G
Ramasubramanyan
Natrajan
N
Hunter
Alan K
AK
Mensah
Paul
P
Gustafson
Mark E
ME
D000818:Animals; D000837:Anion Exchange Resins / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification*; D001059:Apoproteins / Q000737:chemistry / Q000302:isolation & purification*; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D011199:Potentiometry; D017186:Titrimetry; D014168:Transferrin / Q000737:chemistry / Q000302:isolation & purification*
D000837:Anion Exchange Resins; D000911:Antibodies, Monoclonal; D001059:Apoproteins; D014168:Transferrin; C028284:apotransferrin