title: Evaluation of CNBr, FMP and hydrazide resins for immunoaffinity purification of factor IX.
abstract: The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation Factor IX to high levels of purity for therapeutic treatment of hemophilia B. The resin currently used in this process is Sepharose CL2B, a cross-linked 2% agarose, which is activated with cyanogen bromide to immobilize an anti-Factor IX monoclonal antibody. This study evaluated two alternative resins and coupling chemistries, a synthetic polymer bead activated by 2-fluoro-1-methyl-pyridinium toluene 4-sulfonate (FMP) and a cross-linked 2% agarose bead with free hydrazide groups for site-specific coupling. The cyanogen bromide and FMP chemistries immobilize the monoclonal antibody in a random orientation. In hydrazide coupling, the monoclonal antibody is immobilized by the non-antigen-binding part of the molecule which, theoretically, should increase the amount of immobilized monoclonal antibody able to bind antigen. To examine this, the capacity of the resins to bind Factor IX and the purity and recovery of Factor IX eluted from the resins were measured. The FMP-activated resin exhibited the lowest capacity, binding only 2% of the Factor IX feed. Sepharose CL2B bound 87% of the loaded protein, while the hydrazide resin bound 43%. These results suggest that (a) hydrazide activation may be insufficient to orient monoclonal antibody and (b) other factors such as steric hindrances and diffusional resistances during immobilization may be important. Neither of the other resins tested demonstrated improved performance compared with cyanogen bromide-activated Sepharose CL2B for the immunoaffinity purification of Factor IX.
authors.affiliation: University of Maryland.
authors.forename: F
authors.initials: F
authors.lastname: Highsmith
authors.forename: T
authors.initials: T
authors.lastname: Regan
authors.forename: D
authors.initials: D
authors.lastname: Clark
authors.forename: W
authors.initials: W
authors.lastname: Drohan
authors.forename: J
authors.initials: J
authors.lastname: Tharakan
mesh_terms: D000911:Antibodies, Monoclonal; D001709:Biotechnology; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D003488:Cyanogen Bromide; D005069:Evaluation Studies as Topic; D005164:Factor IX / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D012117:Resins, Synthetic
chemical_list: D000911:Antibodies, Monoclonal; D012117:Resins, Synthetic; D005164:Factor IX; D003488:Cyanogen Bromide