Solving selectivity issues in LBAs: case study using Gyrolab to quantify CB307, a bispecific Humabody in human serum

<b>Aim:</b> Endogenous interferents can cause nonselectivity in ligand binding pharmacokinetic assays, leading to inaccurate quantification of drug concentrations. We describe the development of a Gyrolab immunoassay to quantify a new modality, CB307 and discuss strategies implemented to overcome matrix effects and achieve selectivity at the desired sensitivity. <b>Results:</b> Matrix effects were mitigated using strategies including increasing minimum required dilution (MRD) and lower limit of quantification, optimization of antibody orientation, assay buffer and solid phase. <b>Conclusion:</b> The strategies described resulted in a selective method for CB307 in disease state matrix that met bioanalytical method validation (BMV) guidance and is currently used to support clinical pharmacokinetic sample analysis in the first-in-human POTENTIA clinical study (NCT04839991) as a secondary clinical end point. Selectivity is the ability of the ligand-binding assay (LBA) to quantify accurate levels of drug in the presence of nonspecific matrix components and is a requirement within the scope of the ICH M10 bioanalytical method validation guidelines adopted by regulatory authorities. This paper will present the development of a Gyrolab-based LBA for the determination of a new biological modality, CB307, in human serum from patients with prostate-specific membrane antigen (PSMA) positive tumors, as a case study of approaches for the successful resolution of selectivity issues in a LBA pharmacokinetic method development. CB307 is a novel half-life extended bispecific Humabody^® therapeutic molecule that binds to CD137, PSMA and human serum albumin. CB307 is designed to deliver CD137 agonism in a tumor-specific context by targeting PSMA expressing cells in the tumor microenvironment. A three-step sequential Gyrolab method was developed for determination of CB307 in individuals with PSMA positive tumors using two anti-idiotype antibodies targeting the αCD137 V_H and αPSMA V_H domains on CB307. The original assay format was developed on the Bioaffy 1000 CD using a tenfold minimum required dilution (MRD) in Rexxip HX, however nonselectivity was observed. Nonselectivity of a method can be caused by various endogenous matrix components such as circulating drug ligand, closely related endogenous compounds, human antispecies antibodies and more, which can bind to or block epitopes on the capture and detection antibodies or drug. Method development variables for the Gyrolab format can include standard parameters for an immunoassay, such as buffer choice, MRD and reagent concentrations. In addition, there can also be instrument-specific parameters, such as a variety of different CD types (varying capture volume and affinity) plus spin speed, number of wash cycles and incubation times. Strategies were implemented to overcome the matrix effects including increasing the MRD, raising the assay LLOQ, testing an alternative antibody orientation, optimizing assay buffer and selecting the most suitable solid phase for the assay. Use of the Gyrolab Bioaffy high-capacity CDs can be particularly helpful for lower affinity capture and detection reagents and can result in improved binding within the affinity column. Alongside selectivity in healthy individuals, assessing selectivity in disease state matrix is recommended as early as possible in method development to ensure the assay is fit for measuring drug in the target population. Use of an 80-fold MRD in Rexxip HX with a 100 ng/ml LLOQ on the Bioaffy 1000 HC CD eliminated matrix effects and led to a robust method for determination of CB307. The method was validated to local BMV guidelines and supports clinical study POTENTIA (NCT04839991). The strategies described within this research article can be used for all ligand-binding assays to overcome undesirable matrix effects.