One of our sponsors requested immunoassay development and validation to measure a humanized therapeutic monoclonal antibody (mAb) fragment (Fab) that inhibits a human growth factor involved in the pathogenesis of a common degenerative disease of aging.
Antibody fragments offer numerous advantages over full-length antibodies in terms of production cost, increased penetration into the target tissue, and reduced potential for inappropriate activation of immune effector mechanisms. However, because antibody fragments lack many common domains typically targeted (e.g., Fc regions) by immunoassay reagents for binding and detection, they may also present unique challenges for bioassay design, potentially impacting sensitivity since fewer antigens/antibodies can bind to each fragment.
We could try to optimize the concentration of coating and detection reagents to improve sensitivity, but this strategy carries the risk of increasing background noise due to specific and nonspecific interactions within the various assay components (e.g., the therapeutic molecule, the blocking agents, and/or the coating antigen). The potential for off-target specific interactions is especially concerning when the matrix belongs to a closely related species, in this case a non-human primate.
In this case, our team leveraged their decades of collective experience in immunoassay development and troubleshooting to explore multiple assay configurations.
Ultimately, a robust and reproducible indirect ELISA using the antigen target of the monoclonal antibody fragment for capture and a secondary antibody against the mAb’s Fab fragment for detection was determined as the best approach to assay this therapeutic. Since only a portion of the mAb was detected, increased background noise proved challenging.
Thus, a checkerboard approach was used to determine the optimal concentrations for coating and detection antibodies. Additionally, our veteran team fine-tuned multiple aspects of the initial immunoassay, including a novel approach with preincubation of the detection antibody with a small percentage of a homologous matrix. This approach tremendously aided the immunoassay development, GLP validation, and subsequent sample analysis by enhancing signal-to-noise and boosting sensitivity.
An immunoassay that met the sponsor-mandated sensitivity and specificity requirements was successfully developed and validated as per the FDA bioanalytical guidelines, and bioanalysis of the mAb was completed in plasma and ocular fluid matrices for GLP PK study in NHPs.
Over the last decade, mAbs, already well known for their use in immunodetection-based assays, have also found their place as a therapeutic agent for treating various diseases. To either provide a differentiated therapeutic advantage or overcome the side effects, modified mAbs are increasingly being developed and put forward for FDA authorization. Therefore, novel approaches to assay these therapeutics that build on traditional immunoassay methods are of high value to facilitate regulatory approval.
