MSACL 2022 Abstract

Introduction:
Immunoassays based on label-free technologies (LFIA), such as surface plasmon resonance (SPR) or biolayer interferometry (BLI) offer an innovative approach to clinical diagnostics. BLI detects a wavelength shift in incident white light resulting from a change in thickness caused by biomolecular interactions at the measurement surface on a sensing probe. This allows for real-time immunometric measurement of the initial binding rate of antigen-antibody complex formation without the need for signal generation from a secondary reporter/label (enzyme, chromophore, fluorophore).

The use of monoclonal antibody (mAb) drugs for conditions ranging from oncology to autoimmune disorders has increased substantially in the past 3 decades to become the predominant class of new therapeutic agents. For anti–tumor necrosis factor-a (anti-TNF-a) mAb drugs, initial response rates vary from 50-90% and responses are lost over time in a subset of patients due to the formation of antidrug antibodies (ADAs), which impair efficacy and increase the clearance of mAb drugs.

Objectives:
Using BLI technology, open-access LFIAs were developed and validated for the quantification of the mAb drugs adalimumab (ADL) and infliximab (IFX) and for the detection of the antidrug antibodies (ADAs) to the mAb drugs (ADL-ADAs and IFX-ADAs).

Methods:
The LFIAs directly measured antibody–antigen interactions using streptavidin coated sensing probes loaded with biotinylated TNF-a to quantify ADL and IFX or with biotinylated ADL and IFX to detect ADL-ADAs and IFX-ADAs. Each LFIA consisted of 3 steps: (1) equilibrating a sensing probe in PBS at pH 7.4 with 0.05% Tween 20, 0.5% BSA, and 0.05% NaN3 (PBST-B); (2) loading the ligand (the first binding partner) onto the sensing probe; and (3) measuring the interaction between the ligand and the analyte (the second binding partner) on the sensing probe. The time course of signals acquired on a sensing probe is called a sensorgram. ADL and IFX biosimilars and monoclonal anti-ADL and anti-IFX IgG antibodies were used as calibrators in their respective assays. The LFIAs for active mAb drugs and for ADAs were evaluated for imprecision, recovery, limit of quantification (LOQ), interferences, and analytical measurement range (AMR). The LFIAs for active mAb drugs (ADL and IFX) and ADAs (ADL-ADAs and IFX-ADAs) were compared with cell-based reporter gene assays.

Results:
The analytical measurement range (AMR) for both ADL and IFX was 2-100 µg/mL, allowing for quantification of the target therapeutic trough concentrations for ADL (7.5 µg/mL) and IFX (5 µg/mL). The AMR for ADL-ADAs and IFX-ADAs was 5-100 µg/mL and 10-100 µg/mL, respectively. Intra- and inter-assay precision ranged from 4-12%. There were no interferences from protein samples (TNFα, ADL-ADA-mIgG, IFX-ADA-mIgG, DARA, His-tagged CD38, mouse antihuman IgG), diseased clinical samples, biotin, or hemolyzed, lipemic or icteric samples. In the comparison of LFIAs and reporter gene assays, the correlation coefficient was 0.972 for the quantification of ADL and 0.940 for the quantification of IFX. The concordance rate was 90% for the detection of ADL-ADAs and 76% for the detection of IFX-ADAs.

Conclusions:
All current methods for the TDM of mAb drugs are laboratory-developed tests. Most methods are complex in their development and application in routine practice. The LFIAs based on BLI technology offer unique advantages including an open-access, automated experimental process, and rapid measurements, by allowing for quantification on the initial binding rate. No pretreatment of samples is required prior to analysis and a 96-well plate can be analyzed in 2 hours.