Characterization of Noncovalent Complexes of Recombinant Human Monoclonal Antibody and Antigen Using Cation Exchange, Size Exclusion Chromatography, and BIAcore

The binding of fully human monoclonal antibodies (MAbs) D2E7 and 2SD4 to their antigen, human tumor necrosis factor-alpha (TNFalpha), was investigated by BIAcore, cation exchange (CIEX), and size exclusion liquid chromatography (SEC) using ultraviolet and laser light scattering detectors. D2E7 has a higher affinity for TNFalpha than 2SD4 and the two antibodies (Abs) differ by 12 amino acids in the antigen (Ag) binding regions. A BIAcore biosensor instrument was used to determine the association, k(on) and dissociation, k(off), rate constants for the binding of TNFalpha to D2E7 and 2SD4. The HPLC methods were used to resolve and to study D2E7, 2SD4, and TNFalpha molecules and the noncovalent complexes of D2E7 and 2SD4 with TNFalpha. The CIEX method demonstrated that all D2E7 charged-variants bound TNFalpha equally well. There was no preferential binding for any one of D2E7 charged-variants to TNFalpha. D2E7 and 2SD4 Abs were resolved by the CIEX method. When a mixture of D2E7 and 2SD4 was mixed with excess TNFalpha, D2E7. TNFalpha complexes were formed before any 2SD4. TNFalpha complexes. Thus, the CIEX method was able to rank the affinities of the MAbs. D2E7 and TNFalpha formed complexes of 600-5000 kDa. The molecular weights of various D2E7. TNFalpha complexes were determined by a SEC method with light scattering (LS) and refractive index (RI) detectors. Upon overnight incubation, a 598-kDa complex emerged as the most stable and the only D2E7. TNFalpha complex. The molar ratio of D2E7 to TNFalpha in this complex was approximately 1:1. Based on molecular weights and the molar ratio, an immune complex, consisting of alternating three D2E7 and three TNFalpha molecules, is proposed as the most stable complex.