A modified IMAC method for the capture of target protein from mammalian cell culture harvest containing metal chelating species.
Although immobilized metal affinity chromatography (IMAC) offers high capacity and protein selectivity it is not typically used commercially for the capture of native proteins from mammalian cell culture harvest. This is due mainly to the potential for low target recovery due to the presence of strong metal ion chelating species in the harvest that compete for the metal immobilized on the resin. To address this issue a buffer exchange step, such as tangential flow filtration (TFF), is added after harvest clarification and prior to IMAC to remove the interfering harvest components. The addition of a TFF step adds process time and cost and reduces target protein recovery. The elimination of the TFF might make IMAC competitive with other orthogonal methods of protein capture. In this study, we developed a modified IMAC method to allow the direct loading of clarified mammalian harvest without prior buffer exchange (direct IMAC). Although the target enzyme recovery was lower than that from standard IMAC the elimination of the buffer exchange step resulted in a 19% increase in overall enzyme recovery. The target enzyme capacity in direct IMAC was higher, in our experience, than the capacity of hydrophobic interaction (HIC) and ion-exchange (IEX) for protein capture. An economic evaluation of using direct IMAC as a capture step in manufacturing is also discussed.
Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia, USA.
Aming
A
Zhang
Cheng
C
Zhang
Veena
V
Warikoo
Jonathan
J
Forstrom
Frank
F
Riske
D000818:Animals; D001709:Biotechnology / Q000379:methods*; D016466:CHO Cells; D002614:Chelating Agents / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D008670:Metals / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism
D002614:Chelating Agents; D008670:Metals; D011994:Recombinant Proteins