Purpose: Over time Fc-fusion proteins have gained great interest due to their beneficial biological and pharmacological properties, markedly increasing drug half-life which prolongs therapeutic activity along with slow renal clearance. Many bioanalytical assays designed to quantify these therapeutics require reagents. Due to the accelerated timelines in early drug development, the availability of a generic method that avoids sourcing and screening reagents will significantly expedite program support driving scientific decision making and improving POS. Fortebio has established a Bio-Layer Interferometry (BLI) technology for the quantitation of human Fc-fusion protein in the cell culture supernatant. We further explored this technology for the measurement of human Fc-fusion proteins in plasma from preclinical species. Herein, we present a generic, direct, and high throughput quantitative method that can be used irrespective of the size and selectivity of the Human Fc-fusion proteins in mouse plasma.
Methods: The assay was performed using Anti-Human IgG Fc and Streptavidin Biosensors. Standards were analyzed in 96 well black half area plates and antigen was spiked in 10% BALB/C mouse K2EDTA plasma corresponding to an MRD of 10. The assay plate temperature was set at 30 °C and the agitation at 1000 rpm for 600s. Experimental data were analyzed using SoftMax Pro software version 7.0.3.
Results: The dynamic range of 0.08 – 50 µg/mL was established. Accuracy of the assay was evaluated by recoveries of control samples with known concentration at 0.25 and 17.07 µg/mL. Dilution linearity and recovery were within +20%.
Conclusion: We have successfully developed an assay that can rapidly and accurately quantitate the drug in mouse plasma samples, with acceptable recovery values and CV’s. The sensitivity of the assay can be further improved using a multistep assay. To ensure the acceptability of the method, parameters like accuracy, precision, selectivity, sensitivity, and reproducibility are evaluated.