One of our sponsors requested GLP mAb bioanalysis to measure a humanized therapeutic monoclonal antibody fragment (Fab) that inhibits a human growth factor involved in the pathogenesis of a common degenerative disease of aging. Several considerations come into play to deisgn and execute a high performing PK ELISA assay for this crucial mAb bioanalytical testing.
Monoclonal 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 monoclonal 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. Still, this strategy risks 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.
The development and validation of immunoassays for mAb bioanalysis require careful consideration of various factors, including the unique properties of monoclonal antibody fragments, optimization of assay components for sensitivity and specificity, and mitigation of risks associated with off-target interactions. Through meticulous mAb bioanalytical testing, optimal assay performance can be achieved to meet the sponsor’s requirements for measuring the therapeutic efficacy of the monoclonal antibody fragment.
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 for mAb bioanalysis. Our rigorous mAb bioanalytical testing protocols ensured accurate assessment of the therapeutic’s efficacy and safety profiles. Through meticulous optimization and validation, we secured reliable data crucial for advancing the development of this important therapeutic.
An immunoassay that met the sponsor-mandated sensitivity and specificity requirements was successfully developed and validated as per the FDA bioanalytical guidelines, and mAb bioanalysis 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 monoclonal antibody fragments 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. Such advancements in mAb bioanalysis not only streamline the approval process but also ensure the safety and efficacy of novel treatments, thus contributing significantly to advancements in healthcare.
mAb bioanalytical testing is crucial to accurately assessing therapeutic efficacy and safety profiles. By employing state-of-the-art techniques and methodologies, researchers can gain deeper insights into the pharmacokinetics and pharmacodynamics of monoclonal antibody fragments, paving the way for more informed clinical decision-making and regulatory submissions. Furthermore, the development of robust mAb bioanalytical testing enables researchers to explore novel therapeutic modalities and optimize treatment strategies, ultimately benefiting patients and advancing the field of medicine.