Evaluation and comparison of alternatives to Protein A chromatography Mimetic and hydrophobic charge induction chromatographic stationary phases.
In this paper Protein A mimetic and hydrophobic charge induction chromatographic (HCIC) stationary phases are characterized in terms of their protein adsorption characteristics and their selectivity is compared with Protein A chromatography using a set of Chinese hamster ovary-derived monoclonal antibodies and Fc-fusion proteins. Linear retention experiments were employed to compare the selectivities of these resins for both non-IgG model proteins as well as antibodies and the fusion proteins. While none of the non-IgG model proteins were observed to bind to the Protein A resin, most of them did in fact bind to the alternative resins. In addition, while the elution pH was similar for the model proteins and antibodies on the HCIC resin, the mimetic resins did exhibit higher binding for the antibodies under these linear pH gradient conditions. A mixed mode preparative isotherm model previously developed for HCIC was shown to accurately describe the adsorption behavior of the mimetic materials as well. Host cell protein clearance profiles were also investigated under preparative conditions using complex biological feeds and the results indicated that while some selectivity was observed for both the HCIC and the mimetic materials, the purification factors were in general significantly less than those obtained with Protein A. It is important to note, however, that the selectivity of the mimetic and HCIC materials was also observed to be antibody specific indicating that further optimization may well result in increased selectivities for these materials.
Ghose
Sanchayita
S
Purification Process Development, Amgen, WA, USA.
Hubbard
Brian
B
Cramer
Steven M
SM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000302:isolation & purification; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D002919:Chymotrypsinogen / Q000032:analysis; D006224:Cricetinae; D003412:Cricetulus; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000032:analysis; D015394:Molecular Structure; D011993:Recombinant Fusion Proteins / Q000032:analysis; D015203:Reproducibility of Results; D013205:Staphylococcal Protein A / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D002919:Chymotrypsinogen