Analytical ultracentrifugation with fluorescence detection system reveals differences in complex formation between recombinant human TNF and different biological TNF antagonists in various environments

Abstract

A number of studies have attempted to elucidate the binding mechanism between tumor necrosis factor (TNF) and clinically relevant antagonists. None of these studies, however, have been conducted as close as possible to physiologic conditions, and so the relationship between the size distribution of TNF-antagonist complexes and the antagonists' biological activity or adverse effects remains elusive. Here, we characterized the binding stoichiometry and sizes of soluble TNF-antagonist complexes for adalimumab, infliximab, and etanercept that were formed in human serum and in phosphate-buffered saline (PBS). Fluorescence-detected sedimentation velocity analytical ultracentrifugation analyses revealed that adalimumab and infliximab formed a range of complexes with TNF, with the major complexes consisting of 3 molcules of the respective antagonist and one or 2 molcules of TNF. Considerably greater amounts of high-molecular-weight complexes were detected for infliximab in human serum. The emergence of peaks with higher sedimentation coefficients than the adalimumab monomer as a function of added human serum albumin (HSA) concentration in PBS suggested weak reversible interactions between HSA and immunoglobulins. Etanerept exclusively formed 1:1 complexes with TNF in PBS, and a small amount of complexes with higher stoichiometry was detected in human serum. Consistent with these biophysical characterizations, a reporter assay showed that adalimumab and infliximab, but not etanercept, exerted FcγRIIa- and FcγRIIIa-mediated cell signaling in the presence of TNF and that infliximab exhibited higher potency than adalimumab. This study shows that assessing distribution profiles in serum will contribute to a more comprehensive understanding of the in vivo behavior of therapeutic proteins.

Considerations for the Design of Antibody-Based Therapeutics

“…Intriguingly, differences in antibodyantigen affinity, stoichiometry, and complex size have been noted based on serum matrix effects. 327,328 Thus, measures of binding affinity in dilute buffers may not accurately represent the binding that occurs in vivo. Furthermore, mixing of formulated antibodies with serum can result in aggregation of antibodies with serum proteins in an antibody-and excipient-dependent manner.…”

Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodies

“…The applications of AUC-FDS are summarized in Table 3. In addition, AUC-FDS offers unique means for studying macromolecules in complex biological environments, such as blood serum (Demeule et al 2009;Kingsbury et al 2012;Hill and Laue 2015;Krayukhina et al 2017) or cell lysates (Kroe and Laue 2009). A number of differences in the experimental setup and technical aspects exist between AUC-FDS and conventional detection using absorbance or interference detection.…”

“…9 Comparison of the results of conventional c(s) and c(s) with inhomogeneous option analyses applied for human serum albumin formulation containing 5% sorbitol. The result of conventional c(s) analysis (blue) and that for c(s) with inhomogeneous option analysis (red) are indicated demonstrated at nanomolar and micromolar concentrations (Krayukhina et al 2017). The loading signal at 500-pM adalimumab is approximately 174.5 counts, whereas the rmsd are 31.1 counts.…”

“…A recently reported approach using photoswitchable fluorescent proteins demonstrated characterization of heterogeneous multi-protein complexes at previously unthinkably low concentration ranges Chaturvedi et al 2017b). In addition, to address growing concerns from regulatory agencies regarding potential immunogenicity of therapeutic proteins, AUC-FDS can be conveniently employed to evaluate antibody-antigen interactions in close to in vivo conditions (Krayukhina et al 2017).…”

“…However, an important analytical milestone was the recent measurement of size distributions of ICs for the three TNFantagonists at a clinically relevant concentration of a therapeutic antibody (i.e. close to that in blood serum), as revealed by AUC with a fluorescence detection system (AUC-FDS) (Krayukhina et al 2017). The elucidated size distributions may help identify a role of ICs in immunogenicity of therapeutic antibody in patients.…”