Evaluating the Impact of Antibody Fragments on Aggregation using FabALACTICA®
Scientists at Biogen have developed an approach to better understand monoclonal antibody (mAb) aggregation by characterizing fragments that may form during the production process. Fab/c fragments – common impurities generated through hinge digestion and loss of a Fab subunit – can offer valuable insights into aggregation mechanisms. To support this work, FabALACTICA, which specifically digests human IgG1 above the hinge, was used to generate structurally relevant Fab/c fragments. Its ability to produce consistent and defined mAb fragments makes FabALACTICA a powerful tool for studying mAb aggregation through this approach.
Aggregation is a significant product-related impurity that can impact the stability and safety of therapeutics, potentially reducing efficacy and triggering adverse immune responses. It is driven by factors such as post-translational modifications, disulfide bond changes, and hydrophobic interactions, and can be exacerbated by external conditions like temperature and pH. While size-exclusion chromatography (SEC) with UV detection remains the gold-standard analytical method for identifying and quantifying aggregates, native mass spectrometry (nMS) combined with SEC (SEC-nMS) offers a powerful approach for accurately characterizing aggregate heterogeneity with minimal sample preparation.
Figure 1. FabALACTICA (IgdE) is a cysteine protease that digests human IgG1 at a specific site above the hinge, generating intact and homogenous Fab and Fc fragments.
In this study, scientists examined the stability and aggregation behavior of two monoclonal antibodies (mAb1 and mAb2), focusing on their fragments – particularly the Fab/c fragment, which could be consistently generated using a limited FabALACTICA digestion approach. The results indicated that the presence of Fab/c led to the formation of heterogeneous dimers, including mAb-Fab/c, Fab/c dimers, and mAb dimers, though the formation kinetics varied between the two mAbs. The study also highlighted the utility of the SEC-UV-nMS method for assessing mAb stability, as it enables the identification and quantification of low-level aggregates and offers detailed insight into the structural heterogeneity of aggregates.
The glycosylated mAb1 and aglycosylated mAb2 were incubated under accelerated stability conditions at 25°C. After three months, differences in aggregation patterns emerged between the two mAb samples. mAb1 showed the presence of heterogeneous aggregates, including Fab/c-associated dimers, while mAb2 exhibited fewer aggregated species. The study demonstrated that Fab/c fragments – particularly from mAb1 – contribute significantly to aggregate formation under stress. Additionally, N-glycosylation in mAb1 was found to promote Fab/c-associated dimer formation, suggesting a role in enhancing aggregation potential.
This study investigates the impact of Fab/c fragments, generated through limited FabALACTICA digestion, on the thermal stability and aggregation of mAbs. The results show that the presence of Fab/c fragments contributes to the formation of heterogeneous dimers, including mAb-Fab/c and Fab/c dimers, alongside mAb dimers. The aggregation rate correlates with the abundance of Fab/c fragments, which varies between different mAbs. The study also highlights that Fab/c fragments are more susceptible to further degradation. The developed methodology provides a promising platform for studying fragment-influenced mAb aggregation. Combining limited FabALACTICA digestion with a SEC-UV-native MS platform proved valuable in analyzing mAb aggregation and could be further optimized and applied to expand our understanding of mAb stability.
Reference
Xu et al., 2024. Evaluation of the impact of antibody fragments on aggregation of intact molecules
via size exclusion chromatography coupled with native mass spectrometry mAbs.
FabALACTICA® – Above hinge digestion of human IgG1