Development and validation of a recombinant human TNF-α based ELISA to detect and quantify adalimumab

Abstract

Adalimumab, a humanized IgG1 monoclonal antibody is currently used to treat inflammatory diseases. However, a sensitive, in-house ELISA for evaluating inter- and intra-individual pharmacokinetic variability of adalimumab remains limited. In this study, an ELISA was developed to measure adalimumab levels, using recombinant human TNF-α (rhTNF-α) as capture antibody. Initially, surface plasma resonance showed acceptable binding kinetics (K_D) of 2.38x10^-07 nM for adalimumab. Next, a standard curve of adalimumab (1.54 ng/ml to 300 ng/ml), with five quality control points (5.2, 16, 27, 150, and 200 ng/ml) was evaluated for inter and intra-assay accuracy and precision, using serum matrix, by four independent validations. The linear range of the validated assay was 5.2 ng/ml to 200 ng/ml, upper limit of quantification (ULOQ) and lower limit of quantification (LLOQ) were 200 ng/ml and 5.2 ng/ml, respectively. The assay specificity was validated by testing cross-reactivity of rituximab with rhTNF-α, which was found to be non-reactive. Further, the hook effect was over-ruled by diluting the highest concentration of adalimumab tested to assay linear range, and dilution integrity was observed for entire concentrations within linear range (%RE ≤ 20 %), as recommended by European Medicines Agency. Collectively, this rhTNF-α binding-based ELISA method is highly sensitive, reproducible, and useful for monitoring adalimumab.

Keywords: Adalimumab; Arthritis; Bioanalysis; ELISA; Inflammation; Pharmacokinetics; TNF-Alpha.

Fig. 1

Construction and expression of TNF-α in E. coli. The rhTNF-α used as coating reagent in ELISA was commercially cloned using pET28 a (+) vector from Gene Universal Inc. USA. (A) pET28a (+) vector map with human TNF-α gene insert of 486 bp between Nde-I and Hind-III restriction sites. (B) 1 % agarose gel electrophoresis of pET28a (+)-TNF-α DNA construct after restriction digestion with Nde-I and Hind-III enzymes (lane 1- DNA molecular weight marker, lane 2- digested pET28a (+)/TNF-α plasmid, lane 3- undigested pET28a (+)/TNF-α plasmid). (C) pET28a (+) with TNF-α construct transformed into E. coli colonies and selected on kanamycin antibiotic.

Fig. 2

Purification of TNF-α protein by affinity chromatography (A) SDS-PAGE (12 %) of purified rhTNF-α by affinity chromatography. Lane 1- protein molecular weight marker and lane 2- purified rhTNF-α. (B) Western blot analysis of purified rhTNF-α. Lane 1- Confirmation of rhTNF-α protein expression by immunoblotting, using an adalimumab antibody, and lane 2- pre-stained protein molecular weight marker.

Fig. 3

Affinity of adalimumab for TNF-α (A) Binding of adalimumab on immobilized TNF-α protein; segment-1 (5000 ng/ml), segment-2 (10000 ng/ml), segment-3 (20000 ng/ml) and segment-4 (40000 ng/ml). (B) Linear fit of nM concentration of adalimumab drug vs RU shift.

Fig. 4

Standard calibration curve of adalimumab for ELISA. The calibration curve generated using fourteen calibrator standards was fitted using the 4 PL model. Calibration curve was prepared using calibrators ranging from 1.54 ng/ml to 300 ng/ml, by diluting the analyte adalimumab in a diluent buffer spiked with human serum matrix.

Fig. 5

Precision and accuracy of rhTNF-α-binding ELISA (A) Intra-assay accuracy and precision. (B) Inter-assay accuracy and precision analysis. Intra and inter assay %CV, %RE and %total error variance of quality controls (ULOQ, HQC, LQC and LLOQ) within and between the analyst run was analyzed using 4 PL. The QC samples were tested in twelve replicates on a single plate. Five different concentrations of QC samples such as ULOQ, HQC, MQC, LQC, and LLOQ within calibration range were calculated.

Fig. 6

Specificity of rhTNF-α-binding ELISA. Adalimumab showed the specific binding to rhTNF-α, and the cross reactivity of rituximab with rhTNF-α was not observed. A non-specific drug, rituximab was tested against rhTNF-α-coated ELISA plate to ensure the specific binding of adalimumab to rhTNF-α.

Fig. 7

Dilution linearity of rhTNF-α-binding ELISA.