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. 2024;16(14):735-745.
doi: 10.1080/17576180.2024.2360363. Epub 2024 Jun 17.

Unique challenges required reassessment and alterations to critical reagents to rescue a neutralizing antibody assay

Blake A Rowe  1 , Katie Medina-Carle  1 , Keguan Chen  1 , Kimberly J Reese  1 , Kenneth M McCarthy  1 , Amy A Concannon  1 , George R Gunn  1 , Andrew P Gehman  2 , Yong Jiang  3 , Erik Meyer  1
Affiliations

Unique challenges required reassessment and alterations to critical reagents to rescue a neutralizing antibody assay

Blake A Rowe et al. Bioanalysis. 2024.

Abstract

Aim: To redevelop a neutralizing antibody (NAb) assay to be much more drug tolerant, have a large dynamic range and have high inhibition when using high levels of positive control (PC).Materials & methods: Early assay data suggested that typical biotin labeling of the capture reagent (Drug 1, produced in a human cell line) was blocking it from binding with the PC or the detection target, and that the detection target was out competing the PC. Methodical biotin labeling experiments were performed at several challenge ratios and an Fc linker was added to the detection target.Results & conclusion: A larger dynamic range, high inhibition and higher drug tolerance were achieved by adding an acid dissociation step to the assay, performing atypical biotin labeling of Drug 1 and switching to a detection target that contained an Fc linker to increase steric hinderance and decrease its binding affinity to Drug 1.

Keywords: acid dissociation; anti-drug antibody; biolayer interferometry; biotinylation; competition assay; critical reagents; immunogenicity; ligand binding; monoclonal antibody therapeutics; neutralizing antibody.

Plain language summary

Many of the drugs available today are produced by a living organism and these are called biologics. Biologics are larger than chemical drugs and the human body can detect them as foreign and create antibodies against them. This is called immunogenicity. When the antibodies created against the biologic blocks the drug's ability to work correctly, they are called neutralizing antibodies (NAbs). Testing for NAbs is one of the requirements of regulatory agencies for biologics. Here we describe challenges encountered developing an assay to test for NAbs against a biologic.

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Conflict of interest statement

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.

Figures

Figure 1.
Figure 1.
LC/MS analysis of LC-Bt-Drug-1 comparing 5:1 and 50:1 Bt-Drug challange ratios. (A1) Reduced LC-MS of the light chain of the final reagent preparation of the large-scale Bt-Drug 1 reagent at a challenge ratio of 5:1, showing an average of 1.11 biotins on the light chain. (A2) Reduced LC-MS of the heavy chain of the same 5:1 Bt-Drug 1 reagent showing no biotin labels on the heavy chain. (B1) Reduced LC-MS of the light chain of a Bt-Drug 1 reagent at a challenge ratio 50:1 showing an average of 2.94 biotins on the light chain. (B2) Reduced LC-MS of the heavy chain of the same 50:1 Bt-Drug 1 reagent showing an average of 3.54 biotins on the heavy chain.
Figure 2.
Figure 2.
LC-MS comparison of the drug biotinylated at two different challenge ratios to unlabeled drug.
Figure 3.
Figure 3.
Drug tolerance tested at 500, 600, 750 & 1000 ng/ml positive control and up to 15 μg/ml drug.
Figure 4.
Figure 4.
Sensitivity curves. (A) Sensitivity curve showing ECL vs positive control concentration, (B) A logarithmic curve of the sensitivity tested up to 10 μg/ml shows that the validated assay has over 90% inhibition at the HQC (5000 ng/ml). ECL: Electrochemiluminescence.
Figure 5.
Figure 5.
Target tolerance data shows that the assay has target tolerance at the LQC (750 ng/ml) up to 1000 pg/ml.
Figure 6.
Figure 6.
Final configuration of the neutralizing antibodies assay. NAb: Neutralizing antibody; PC: Positive control.
See this image and copyright information in PMC

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