Stability Testing Reveals Photoselective Clipping of Heavy Chain C-Terminal Amino Acids That Leads to Fragmentation and Aggregation of an Antibody Fab Fragment.

We built a custom device to subject an antibody fragment A33 Fab to controlled stress conditions that combined pH, temperature, agitation, and LED-based light exposure in polypropylene microplates; to simulate the real-world challenges it may encounter during storage and transportation and to evaluate the key degradation routes in Fab formulations. We also explored the addition of Tween 80 as a surfactant and the impact of plate surface siliconisation. Monomer loss and fragmentation was monitored by size-exclusion chromatography, aggregate formation determined by changes in hydrodynamic radius in DLS, and chemical modifications identified through intact mass analysis by LC-MS, and N-terminal sequencing. The findings indicated that the light exposure conditions often interacted with other factors. In particular, light exposure in the UV to blue range led to chemical degradations that led to greater susceptibility to aggregation, particularly at elevated temperatures. Interestingly, while Tween 80 provided stabilization, particularly within siliconized plates, the presence of Tween 80 also promoted losses due to light exposure, consistent with previous findings that Tween 80 could act as a photosensitizer. Exposure of A33 Fab to light led to sequential losses of amino acids selectively from only the heavy chain C-terminus, indicating a photosensitive hotspot in that region of the Fab structure. These also suggested that photoinduced clipping of the heavy chain C-terminus increased the susceptibility of A33 Fab to fragmentation into heavy and light chains and aggregation, consistent with previous work in which flexibility-suppressing mutations of the hinge region decreased the aggregation kinetics.