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

doi:10.1006/abio.2001.5380

Analytical Biochemistry

Volume 299, Issue 2, 15 December 2001, Pages 119-129

https://doi.org/10.1006/abio.2001.5380 Get rights and content

Abstract

The binding of fully human monoclonal antibodies (MAbs) D2E7 and 2SD4 to their antigen, human tumor necrosis factor-α (TNFα), 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 TNFα 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 TNFα to D2E7 and 2SD4. The HPLC methods were used to resolve and to study D2E7, 2SD4, and TNFα molecules and the noncovalent complexes of D2E7 and 2SD4 with TNFα. The CIEX method demonstrated that all D2E7 charged-variants bound TNFα equally well. There was no preferential binding for any one of D2E7 charged-variants to TNFα. D2E7 and 2SD4 Abs were resolved by the CIEX method. When a mixture of D2E7 and 2SD4 was mixed with excess TNFα, D2E7 · TNFα complexes were formed before any 2SD4 · TNFα complexes. Thus, the CIEX method was able to rank the affinities of the MAbs. D2E7 and TNFα formed complexes of 600–5000 kDa. The molecular weights of various D2E7 · TNFα 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 · TNFα complex. The molar ratio of D2E7 to TNFα 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 TNFα molecules, is proposed as the most stable complex.

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

Abstract

Anal. Biochem.

J. Biol. Chem.

J. Chromatogr.

Trends Anal. Chem.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

Anal. Biochem.

J. Biol. Chem.

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