How APBio Builds Multispecific Analytical Target Profiles
A conversation with Jhong-Jhe You, Ph.D., AP Biosciences
The biomanufacturers’ standard analytical toolkit is filled with gear for traditional monospecific antibodies, not multispecifics. As this segment matures, multispecific developers are looking hard at ways those legacy assays remain relevant, and also where they need to change.
In December, AP Biosciences (APBio) Vice President of Antibody Discovery Jhong-Jhe You, Ph.D., told us about the complexity in making multispecific antibodies. The conversation sparked more questions about analytical target profiles (ATPs) and potency and release testing. Here’s that conversation in case you missed it.
He told us then that multispecific antibodies are still antibodies, and measuring critical quality attributes in multispecifics has many similarities to conventional antibodies. He emphasized the importance of using established technology as much as possible. Inventing new processes, after all, gets expensive.
We followed up with APBio’s You to dig deeper into what parameters his team considers when building an ATP, how it decides when new assays are warranted, and how they approach the conundrum of potency testing when animal models offer the only way to measure a multispecific’s synergistic effects. Here’s what he told us.
What does the ATP look like for APBio's multispecific candidates or multispecifics in general? How is it different from a conventional mAb ATP?
Though multispecific antibodies are fashioned to acknowledge several epitopes or targets, they are designed and identified as one molecular part. Thus, numerous aspects of a typical mAb ATP, like identity, purity, potency, and safety, still apply to APBio’s multispecific candidates.
Due to the structural complexity and functional diversity of multispecific antibodies, certain attributes need to be elevated in consideration. Specifically, attributes related to chain pairing, folding fidelity, and aggregation must be more rigorously monitored during development and manufacturing. The consequences of mispairing or misfolding are reduced production yield as well as diminished potency and increased safety risks in clinical use.
In addition, potency assays for multispecific antibodies must not only confirm activity at individual binding sites but also demonstrate synergistic or additive biological effects that reflect their intended mechanism of action. These can include physiochemical binding assays or robust cell-based functional assays designed to record dual or cooperative effects. As such, the ATP for multispecifics must be both comprehensive and tailored to capture the unique functionalities and critical quality attributes inherent to their design.
How does APBio determine that developing entirely new assays is unavoidable or more practical than modifying existing ones?
At APBio, our assay development strategy is based on combining scientific rigor with efficiency of development. Therefore, we seek to adapt or optimize existing validated assay platforms, either internal or those widely adopted in the industry, to meet the analytical needs of our candidates. This practical approach greatly reduces the time necessary for both validation and alignment with regulations, thus enabling faster movement through the development pipeline. But when it comes to complicated formats, like multispecific antibodies with different binding epitopes, targets, or mechanisms of action, current tests might not be enough to completely assess key active traits.
For instance, our multispecific antibody AP203, which was made to block PD-1/PD-L1 and activate CD137 signaling simultaneously, needed a custom-built reporter assay. This assay was developed to evaluate the synergistic engagement of both pathways in a controlled quantitative manner, something that could not be assessed using conventional single-target reporter systems.
The AP203 reporter assay incorporated two distinct signaling pathways in the reporter cell. One was to measure the blockade of the PD-1/PD-L1 axis and the other to detect CD137-mediated T cell co-stimulation. The design allowed us to assess the synergistic enhancement that defines AP203’s therapeutic rationale. Developing this assay de novo was necessary to ensure accurate potency profiling.
Previously, you mentioned that the synergistic effects of multispecifics can be demonstrated only in animal models. How does that limitation affect assay design, especially when it comes to potency and release testing?
For some multispecific antibodies, therapeutic action is only evident in in vivo models because of spatial, temporal, or mechanistic separation at the level of their individual targets. This poses a challenge to potency assay design, particularly when combined mechanisms of action cannot be recapitulated completely in one cell-based assay system.
At APBio, AP505 serves as a clear example. It is a multispecific antibody targeting both PD-L1, an immune checkpoint regulator, and VEGF, a key factor in angiogenesis. These two pathways operate in distinct biological contexts: PD-L1 modulation mainly occurs in the immune compartment, while VEGF blockade acts on the tumor vasculature. Moreover, the timing and microenvironmental conditions required for each mechanism to become functionally relevant differ significantly during tumor progression. That’s why it’s difficult to design a single cell-based test that realistically records both functions of AP505. Instead, we take a mixed approach. We first verify molecular integrity and dual-target binding via SPR or ELISA, followed by separate cell-based assays to confirm PD-1/PD-L1 blockade and VEGF neutralization.
This assay design allows us to robustly support release testing and comparability studies, even if in vitro models don’t totally show the effective match of our therapies. While it does not replace the need for in vivo validation to demonstrate combined efficacy, it enables consistent batch control and ensures alignment with ATP.
How do you ensure that modified assays retain sufficient sensitivity, specificity, and reliability for their intended purpose, especially when dealing with potential impurities like host cell proteins or demonstrating distinct mechanisms of action?
We ensure that modified assays maintain sufficient sensitivity, specificity, and reliability through rigorous assay qualification and validation in accordance with regulatory guidelines. This process includes testing across multiple production batches to confirm consistency and robustness.
For example, we performed an integrated cell-based assay that simultaneously measures two signaling pathways for AP203: PD-1/PD-L1 blockade and CD137 activation, thereby enabling a comprehensive functional assessment of the bispecific antibody in a single assay format. After implementation, we monitor assay performance by trend analysis, system suitability checks, and periodic revalidation to ensure long-term robustness under conditions that evolve with process scale, formulation, or manufacturing parameters.
About The Expert:
Jhong-Jhe You, Ph.D., is the vice president of antibody discovery at AP Biosciences. He joined the company in 2013 as its first employee and pioneered its OmniMab human antibody library. He received his Ph.D. in molecular medicine from the National Taiwan University and continued his research in antibody engineering at the Genomic Research Center, Academia Sinica.