(M1030-01-01) Measuring Free IgE in Low-Affinity Interaction with Omalizumab Using an Automated Microfluidic Immunoassay to Support Pharmacokinetics and Pharmacodynamics Studies

Purpose: Omalizumab (Xolair®) is a humanized IgG1 monoclonal antibody that specifically binds to free human immunoglobulin E (IgE) in the blood and interstitial fluid and to the membrane-bound form of IgE of B lymphocytes. Assays are needed to measure free IgE to support the study of omalizumab pharmacokinetics (PK) and pharmacodynamics (PD). The omalizumab–IgE interaction has a low affinity. The equilibrium between free and bound IgE is therefore easily perturbed by the long incubation times and sample dilution required by plate-based immunoassays, resulting in overestimation. IgE levels can also change dramatically during treatment with omalizumab (1). A microfluidic immunoassay was therefore developed to measure IgE levels in 100% matrix with short contact times (~6 seconds) to improve accuracy and with a broad analytical range to meet the needs of PK studies.

Methods: The nanoliter-scale immunoassay was run on Gyrolab® system using Gyrolab Bioaffy™ CDs with biotinylated omalizumab immobilized on streptavidin beads as capture reagent and anti-human IgE labelled with Alexa Fluor® 647 as detection reagent. The omalizumab – IgE interaction was investigated by solution-based affinity determinations performed by pre-incubating a fixed concentration of unlabeled omalizumab with variable concentrations of IgE and using the Gyrolab immunoassay to measure free IgE.

Results: The free IgE assay using the high capacity (HC) Gyrolab Bioaffy CDs such as Gyrolab Bioaffy 1000 HC showed good parallelism (Table 1). Inter-assay performance testing using, for example, Gyrolab Bioaffy 1000 HC showed an analytical range (lower limit of quantitation, LLOQ — upper limit of quantitation, ULOQ) of 0.2 –430 IU/mL with a CV of < 12%. Relative error was < 13% and total error was in the range -1 to 9.5 for five QC samples (n=18). The affinity determination based on fixed IgE levels of 125, 400 and 5000 pM gave K_D values of 20, 18, and 32 nM respectively (Figure 1). Based on this data, omalizumab concentrations were selected for a drug interference experiment in human serum samples using the free IgE assay (Table 2). The drug interference experiment showed that the level of interference was approximately 50% at the approximate K_D value, indicating that the assay measures free IgE.

Conclusion: The affinity assay confirmed the low affinity of the IgE-omalizumab interaction and the need for minimal sample dilution and short incubation times to minimize equilibrium shifts and ensure accurate determination of free IgE levels. This could be achieved using the microfluidic automated immunoassay system, which enables the assay to be run on neat samples and with very short column contact times. The IgE immunoassay could be quickly performed and gave good parallelism, precision, and accuracy. It would be possible to run the assay using different Gyrolab Bioaffy CD types to maximize the analytical range without diluting the sample and meet the needs of PK/PD studies on omalizumab.

References: 1. Çildağ S, Şentürk T. The effect of omalizumab treatment on IgE and other immunoglobulin levels in patients with chronic spontaneous urticaria and its association with treatment response. Postepy Dermatol Alergol. 2018 Oct;35(5):516-519. doi: 10.5114/ada.2017.71422. Epub 2018 Jul 19. PMID: 30429712; PMCID: PMC6232555.


Figure 1. Affinity curve for IgE-omalizumab interaction based on fixed concentrations of IgE of 125, 400, and 5000 pM.


Table 1. Free IgE assay parallelism in Gyrolab Bioaffy 1000HC


Table 2. Assessment of omalizumab drug interference of free IgE assay in serum samples.