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

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.

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.

TWEETABLE ABSTRACT

Development of a ligand binding neutralizing antibody (NAb) assay offered some unique challenges. The biotin conjugated capture drug and the ruthenylated detection target both required atypical labeling to achieve desired results.

Article highlights

• Unusual light chain biotin labeling of Drug 1 was interfering with its ability to bind to the positive control (PC), thereby lowering the PCs ability to inhibit the electrochemiluminescence (ECL) signal by blocking detection target binding.

• Labeling with high challenge ratios (i.e., 50:1 & 100:1) over labeled the light chain of the antibody (Ab), inhibiting Drug 1 from conjugating with the PC and the target, resulting is very low ECL signal and very little inhibition by the HQC.

• Given enough time, some of the detection target was able to colocalize with bound PC, so competition was not completely exclusive. By adding an Fc linker to the detection target, we were able to increase its size and steric hinderance (reducing its binding affinity), which reduced colocalization with bound PC.

• Having the ability to assess critical reagents with LC-MS and biolayer interferometry we were able to elucidate what was causing most of the problems we encountered and make appropriate adjustments to produce a better neutralizing antibody assay.

Keywords: :

• acid dissociation
• anti-drug antibody
• biolayer interferometry
• biotinylation
• competition assay
• critical reagents
• immunogenicity
• ligand binding
• monoclonal antibody therapeutics
• neutralizing antibody

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17576180.2024.2360363

Author contributions

All authors were involved in the overall design and execution of the study and authoring of the manuscript. In addition, B Rowe and K Carle conducted the experiments described, A Concannon was involved in reagent preparation, B Rowe, K Carle, Y Jiang, K Reese and E Meyer were involved in the analysis of data. E Meyer was responsible for data review, and A Gehman was responsible for statistical analyses. K Chen and K McCarthy were involved in assay design and validation.

Financial disclosure

All authors are employees of GSK PLC. Some authors are owners of GSK PLC shares. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

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.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval for all experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.