Surrogate potency assays: Comparison of binding profiles complements dose response curves for unambiguous assessment of relative potencies

Surface plasmon resonance (SPR) systems are widely used for detailed characterization of antibody activities including antigen and Fc-receptor binding. During the later stages of development, where the focus is to ensure that established critical quality attributes (CQAs) are maintained during cell culture, purification and formulation processes, analysis is simplified, and relative potencies are often determined. Here, simulation of binding data revealed that relative potency values, determined via parallel line analysis (PLA) and half maximal effective concentration (EC50) analysis accurately reflect changes in active concentration only if binding kinetics remain unchanged. Changes in the association rate constant shifted dose response curves, and therefore relative potencies, in the same way as changes in analyte concentration do. However, for interactions characterized by stable binding, changes in the dissociation rate constant did not result in any shift, suggesting that this type of change may go unnoticed in the dose response curve. Thus, EC50 and PLA analyses of dose response curves obtained with an anti-TNF-α antibody were complemented with the Biacore functionality for sensorgram comparison analysis, whereby changes in antigen and Fc-receptor binding profiles could be detected. Next, analysis of temperature stressed TNF-α antibody revealed that calibration free concentration analysis (CFCA) data correlated perfectly with relative potency values. Together, these results demonstrate that combinations of SPR based dose response curves, sensorgram comparison and CFCA can be used to strengthen the confidence in relative potency assessments, and suggest that SPR can potentially be used as a surrogate potency assay in the quality control of biotherapeutic medicines. Keywords: Surface plasmon resonance, EC50, Sensorgram comparison, Calibration free concentration analysis, Surrogate potency assay, TNF-

Does protein binding modulate the effect of angiotensin II receptor antagonists?

Introduction Angiotensin II AT 1-receptor antagonists are highly bound to plasma proteins (≥ 99%). With some antagonists, such as DuP-532, the protein binding was such that no efficacy of the drug could be demonstrated clinically. Whether protein binding interferes with the efficacy of other antagonists is not known. We have therefore investigated in vitro how plasma proteins may affect the antagonistic effect of different AT1-receptor antagonists. Methods A radio-receptor binding assay was used to analyse the interaction between proteins and the ability of various angiotensin II (Ang II) antagonists to block AT1-receptors. In addition, the Biacore technology, a new technique which enables the real-time monitoring of binding events between two molecules, was used to evaluate the dissociation rate constants of five AT1-receptor antagonists from human serum albumin. Results The in vitro AT 1-antagonistic effects of different Ang II receptor antagonists were differentially affected by the presence of human plasma, with rightward shifts of the IC50 ranging from one to several orders of magnitude. The importance of the shift correlates with the dissociation rate constants of these drugs from albumin. Our experiments also show that the way that AT1-receptor antagonists bind to proteins differs from one compound to another. These results suggest that the interaction with plasma proteins appears to modulate the efficacy of some Ang II antagonists. Conclusion Although the high binding level of Ang II receptor antagonist to plasma proteins appears to be a feature common to this class of compounds, the kinetics and characteristics of this binding is of great importance. With some antagonists, protein binding interferes markedly with their efficacy to block AT1-receptors