Purpose: Complex sample matrixes present a significant challenge for most biomolecular binding assays, and, thus, require several purification steps for obtaining reliable assay results. This leads to lengthy assay development times, a need for time-consuming purification steps, and ultimately increased operational costs. Flow induced dispersion analysis (FIDA) is a novel capillary-based technology for characterizing ligand-protein interactions and quantifying biomolecules in complex solutions (e.g., plasma). FIDA is based on Taylor dispersion in a pressure driven flow of a ligand (termed indicator, here Protein-A) interacting selectively with the analyte of interest (here human IgG). FIDA and Taylor dispersion analysis is performed in narrow capillaries and is used for measuring diffusion coefficients and hydrodynamic radii of molecules. In FIDA, the apparent change in hydrodynamic radius of the indicator molecule upon binding to the analyte is exploited for determination of analyte concentration. Here, we present a FIDA method for quantification of human IgG directly in complex F12 fermentation media used in antibody production with CHO cells, without any need for purification.
Alexa488-labelled protein-A (Product no. 11047; Molecular Probes, Eugene, OR, US) served as the indicator (100 nM) for quantification of IgG from human serum (analyte; Product no. I 4506; Sigma) spiked (0-1000 nM) into 80% (v/v) F12/DMEM fermentation medium. The advanced F12/DMEM fermentation medium (Product no. 12634, Thermo Fischer Scientific) is commonly used in the production of therapeutic proteins using Chinese Hamster Ovary (CHO) cells as the host cells.
FIDA analyses were performed using a FIDAlyzer instrument with LED fluorescence excitation at 480 nm and emission collection at λ > 510 nm (FIDA-Tech ApS, Copenhagen, Denmark) using a coated fused silica capillary (i.d.: 75 µm, LT: 100 cm, Leff: 88 cm) at 25 oC.
Figure 1 shows how the peak width (variance) of the protein-A-Alexa488 indicator increases with increasing IgG concentration. Figure 1 shows that human IgG can be detected in the F12 fermentation medium without sample preparation even though the medium is complex containing amino acids, vitamins, salts, proteins and sugars. The auto-fluorescence of the medium is apparent as base line shift but does not affect the peak shape. The FIDA approach is characterized by a short analysis time (3 min) and selective fluorescence detection allowing the protein-A-Alexa488 indicator to be determined at a relatively low concentration (100 nM). The indicator (MW 425000 Da) is small relative to the IgG antibody, but upon binding, the diffusivity will decrease corresponding to a larger apparent hydrodynamic radius. The apparent hydrodynamic radius of protein-A-Alexa488 is plotted as a function of IgG concentration in Figure 2. The change in apparent size forms the basis for a measure of the interaction and the IgG (analyte) concentration. The binding of protein-A to IgG was expected (protein A binds to the Fc portion of many subclasses of immunoglobulins) and is apparent from the size increase. The unbound Protein-A had a hydrodynamic radius of 5.5 nm, but upon binding to one or two IgG molecules it increased gradually to approximately 11 nm. The data points were fitted to a 1:1 binding isotherm (red line in Figure 2), and a dissociation constant (Kd) for the protein-A-IgG complex in 80% F12 fermentation media of 60 nM was determined. The results document the ability of FIDA to characterize interactions in native medium. Furthermore, the binding curve serves essentially as a calibration curve allowing the quantification (working range 200 – 1000 nM) of the IgG in F12 fermentation media upon determination of the protein-A-Alexa488 apparent hydrodynamic radius.