Determining the Binding Affinity of Therapeutic Monoclonal Antibodies towards Their Native Unpurified Antigens in Human Serum

Abstract

Monoclonal antibodies (mAbs) are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen’s endogenous concentration, by combining the kinetic exclusion assay and Biacore’s calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9), progranulin (PGRN), and fatty acid binding protein (FABP4). We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.

The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

“…Analytes were prepared by buffer-exchanging them into the running buffer using spin columns and their "active" or "effective" concentrations determined under conditions of mass transport limitation using a CFCA method on a Biacore T200, as described elsewhere. 38 CFCA experiments confirmed that all analytes used herein were >70% active relative to their nominal or total protein concentration as determined by light absorbance at 280 nm with use of an appropriate extinction coefficient. All kinetic and affinity calculations used active analyte concentrations, rather than nominal ones, as input values.…”

Antibodies Targeting Closely Adjacent or Minimally Overlapping Epitopes Can Displace One Another

“…Serum experiments were performed as described previously [15, 16] using a Dylight-labeled anti-PGRN mAb 27D5 (M14) as secondary detection, chosen because its epitope does not overlap with that of mAb 14C7 (M4) or mAb 28H6 (M27).…”

A Hybrid Approach Enabling Large-Scale Glycomic Analysis of Post-Golgi Vesicles Reveals a Transport Route for Polysaccharides

“…Cell wall glycan-directed Abs are an elegant alternative for the identification of plant cell carbohydrates (Moller et al, 2008;Pattathil et al, 2010;Pedersen et al, 2012). Monoclonal Abs (mAbs) can bind to their epitopes with high selectivity and affinity (K d ;10 -6 M; Müller-Loennies et al, 2000;Bee et al, 2013) and are therefore highly sensitive, specific, and efficient probes. The latter aspect is of paramount importance when the quantity of the sample is severely limited, such as in the caseof endomembrane vesicles.…”