New Analytical Tool Helps Probe Higher-Order Structure of Bispecific Antibody Therapeutics
The development of a new tool that combines native ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) could lead to a better understanding of bsAbs and aid in the development of effective treatments for diseases that remain challenging for conventional mAb therapeutics to access.
A team of researchers from the University of Michigan in Ann Arbor, Michigan, developed a new analytical tool to probe the higher-order structure (HOS) of bispecific antibodies (bsAbs), which are emerging as important therapeutics capable of targeting two different antigens simultaneously. The tool, which combines native ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU), has demonstrated the ability to provide detailed and quantitative data sets on the HOS of bsAbs.
Virus protection. Antibodies and viral infection. Immune defense of the body. Attack on antigens 3D illustration | Image Credit: © Siarhei - stock.adobe.com
In practice, bsAbs are designed to bind to two different antigens, making them valuable tools in various therapeutic and diagnostic applications. The HOS of bsAbs refers to the overall three-dimensional (3D) arrangement of the two antigen-binding domains and the linker region that connects them. HOS is critical for the stability, specificity, and functionality of bsAbs, and it can significantly affect their pharmacokinetics and pharmacodynamics.
IM-MS is a technique that combines the separation capability of ion mobility with the mass measurement accuracy of mass spectrometry. This allows for the determination of the mass-to-charge ratio (m/z) and the collision cross-section (CCS) of ions in the gas phase, which provides information about their size, shape, and charge distribution. CIU is a technique that utilizes the energy from collisions with an inert gas to unfold or denature proteins and other biomolecules in the gas phase. CIU can be coupled with IM-MS to provide additional structural information about proteins and protein complexes, such as their stability, folding pathways, and subunit interactions.
The researchers utilized a knob-into-hole (KiH) bsAb model system and its corresponding parent monoclonal antibodies (mAbs) to gather data on the global KiH bsAb stability and the stability of its constituent domains. The CIU data indicated that the global KiH bsAb stability occupied an intermediate space between the stabilities of its parent mAbs. The hole-containing half of the KiH bsAb construct was identified as the least stable, driving much of the overall stability of the KiH bsAb.
The team also associated the first and second CIU transitions observed for the intact KiH bsAb to the unfolding Fab and Fc domains, respectively. The results suggest that changes in the first CIU transition were primarily connected to the Fab region of the hole-containing halfmer, while the second CIU transition was likely strongly linked to the Fc region of the knob-containing halfmer.
Overall, the IM-MS and CIU tool proved to be effective in probing the HOS of bsAbs and could be used to evaluate the domain-level stabilities and HOS of both KiH bsAbs and mAb constructs.
IM-MS and CIU are analytical tools used to study the HOS of bsAbs and mAbs. In this study, IM-MS and CIU were used to evaluate the domain-level stabilities and HOS of a KiH bsAb model system and its corresponding parent mAbs. Quantitative analysis of CIU data revealed that global KiH bsAb stability occupies an intermediate space between the stabilities recorded for its parent mAbs. The analysis of both intact bsAb and enzymatic fragments allowed the researchers to associate the first and second CIU transitions observed for the intact KiH bsAb to the unfolding Fab and Fc domains, respectively. This provides a road map for evaluating the domain-level stabilities and HOS of both KiH bsAbs and mAb constructs using CIU.
The study's main author, Brandon T. Ruotolo, noted that current analytical tools for tracking the bsAb construction process have demonstrated a limited ability to robustly probe the HOS of bsAbs (1). The development of this new tool could lead to a better understanding of bsAbs and aid in the development of effective treatments for diseases that remain challenging for conventional mAb therapeutics to access.
Reference
(1) Villafuerte-Vega, R. C.; Li, H. W.; Slaney, T. R.; Chennamsetty, N.; Chen, G.; Tao, L.; Ruotolo, B. T. Ion Mobility-Mass Spectrometry and Collision-Induced Unfolding of Designed Bispecific Antibody Therapeutics. Anal. Chem. 2023, ASAP. DOI: 10.1021/acs.analchem.3c00344
AI and GenAI Applications to Help Optimize Purification and Yield of Antibodies From Plasma
October 31st 2024Deriving antibodies from plasma products involves several steps, typically starting from the collection of plasma and ending with the purification of the desired antibodies. These are: plasma collection; plasma pooling; fractionation; antibody purification; concentration and formulation; quality control; and packaging and storage. This process results in a purified antibody product that can be used for therapeutic purposes, diagnostic tests, or research. Each step is critical to ensure the safety, efficacy, and quality of the final product. Applications of AI/GenAI in many of these steps can significantly help in the optimization of purification and yield of the desired antibodies. Some specific use-cases are: selecting and optimizing plasma units for optimized plasma pooling; GenAI solution for enterprise search on internal knowledge portal; analysing and optimizing production batch profitability, inventory, yields; monitoring production batch key performance indicators for outlier identification; monitoring production equipment to predict maintenance events; and reducing quality control laboratory testing turnaround time.
2024 EAS Awardees Showcase Innovative Research in Analytical Science
November 20th 2024Scientists from the Massachusetts Institute of Technology, the University of Washington, and other leading institutions took the stage at the Eastern Analytical Symposium to accept awards and share insights into their research.
