(T1530-02-10) Development of a Generic, Automated, Microfluidic Human IgG-FcRn Affinity Assay In-Solution for Use in Antibody Engineering

Purpose: The relatively long half-life of IgG-based antibody therapeutics (~21 days) is primarily due to their protection from degradation by interaction with the neonatal Fc receptor (FcRn) at acidic pH (6.0).¹ Robust IgG Fc-FcRn binding and affinity assays are increasingly important to support antibody therapeutic engineering targeted to the Fc region of IgGs to extend serum half-life.² Measuring the affinity of this interaction can be challenging due to the acidic pH required and the relatively low K_D values. While ligand-binding and SPR affinity assays are available to measure FcRn-IgG affinity, an assay that measures unlabeled reactants in-solution that is also fast, accurate, and automated would provide workflow and convenience advantages. In this study, a proof-of-principle assay was developed to measure in-solution affinity of IgG-FcRn interactions using an automated, microfluidic ligand-binding assay platform.

Methods: The IgG-FcRn affinity assay was developed on an automated, CD-based Gyrolab® microfluidic platform. The affinity assay model system consisted of three different IgG-based therapeutics, Keytruda (pembrolizumab), Remsima (inflixmab), and Rituxan (rituximab), along with a recombinant His-tagged FcRn heterodimer: FcGRT (Fc fragment of IgG receptor transporter neonatal) and B2M (Beta 2 microglobulin). A dilution series of IgG therapeutic Remsima and FcRn was prepared in Rexxip H, pH 6 (3.3E-5 M to 3.2E-8 M) and combined with a fixed concentration FcRn-His (180 and 90 nM). The prepared samples were incubated for one hour to reach equilibrium. The IgG-FcRn binding assay was automatically carried out by the Gyrolab platform within the microfluidic Gyrolab Bioaffy™ 1000 CD for the introduction of capture (biotinylated antibody therapeutic), therapeutic:FcRn-His sample, and detection reagent (Alexa-Fluor 647^® labelled mouse anti-His IgG) as well as buffers and wash solutions. The assay is built onto the flow-through affinity column with streptavidin-coated beads step-wise (Figure 1). Finally, the integrated laser-induced fluorescent (LIF) signal automatically detects column fluorescence for each data point. Free (unbound) FcRn-His was measured for each dilution to calculate K_D values for the FcRn-His:IgG therapeutic interaction using the Gyrolab Software Affinity Module. The titration assay was completed for Remsima using both a 1-hour and 4-hour pre-incubation for confirmation of the 1-hour incubation time. The K_D values for Remsima (inflixmab), Rituxan (rituximab) and Keytruda (pembrolizumab) were determined using this assay to compare results to published values.

Results: For the 1- and 4-hour pre-incubations of reactants prior to measuring free FcRn, the K_D values were comparable, K_D 595 nM (1 hour) and K_D 740 nM (4 hours) see Figure 2 and the 1-hour pre-incubation was used to determine affinity Calculated K_D values were Remsima, 629 nM; Rituximab, 468 nM; and Keytruda, 209 (Figure 3). The values for Remsima and Rituximab were in line with literature values of 708 and 508 nM, respectively (Keytruda FcRn affinity value not available). Manual pipetting was needed to set up the dilution series prior to the offline incubation followed by the automated Gyrolab affinity assay. The run time for the complete assay including the 1-hour offline incubation was about 2 hours.

Conclusion: Gyrolab automated microfluidic platform was a suitable assay approach to determine in-solution FcRn-IgG affinity of the reactants in their native state by measuring free FcRn from a dilution series of FcRn (fixed concentration) with the IgG therapeutic. The approach was suitable to determine these relatively low affinity interactions and for use in antibody engineering screening or development. The flow-through format of the Gyrolab assay allows a snapshot of the sample state. The short binding time to the flow-through column in the microfluidic CD (~6 seconds) prevents equilibrium shifts during the assay, providing an accurate determination of unbound analytes in a short 2-hour total assay time. The K_D values of FcRn binding determined for three IgG-based therapeutics, Keytruda (pembrolizumab), Remsima (inflixmab), and Rituxan (rituximab) were consistent with published values. This proof of principle affinity assay merits further optimization. The generic format can be used on any IgG based therapeutic, and multiple affinity assays can be run in parallel for higher throughput.

References: 1. Suzuki T, Noritaka H, Minoru T et al, MAbs. 2021 Jan-Dec;13(1)
2. Chadha GS and Morris M. Current Pharmacology Reports 2 (2016): 45 - 56.
3. Suzuki T, Ishii-Watabe A, Tada M, et al. J Immunol. 2010 Feb 15;184(4):1968-76.

Microfluidic automated assay showing flow-through 15 nL affinity column within microstructure segments on the Bioaffy CD. Immunoassays are built on the streptavidin bead within the column with fluid movements regulated by centrifugal forces (spin).

Titration curve of Remsima dilution series with fixed concentration of FcRn using a 1 hour and 4 hour pre-incubation.

Titration curves of a dilution series of three approved IgG therapeutics, Keytruda (pembrolizumab), Remsima (inflixmab), and Rituxan (rituximab) with a fixed FcRn concentration to determine affinity by measuring free FcRn.