In Silico Analysis of Therapeutic Antibody Aggregation and the Influence of Glycosylation

The aggregation of therapeutic antibodies is a major issue for the pharmaceutical industry leading to loss of drug quality, increased dosage, and unwanted immune responses such as the production of anti-drug antibodies (ADA). As aggregation can occur at various stages of development and storage, much work has been performed to reduce or eliminate it. In this report we analyzed four antibodies available in the PDB (1IGT, 1IGY, 1HZH, and 5DK3) using the online software UCSF Chimera to study the structural features of the proteins and the associated N-linked glycans in the CH2 domains of the Fc region. To study antibody aggregation in silico we used the online software TANGO and AGGRESCAN to identify aggregation prone regions (APR) in the antibodies and the influence of the Fc glycans on hydrophobic and aromatic residues present in the APRs. In the 3D structures of 1IGT and 1IGY the glycan chains are in close enough proximity to influence and protect these hydrophobic regions. However, in the 3D structures of 1HZH and 5DK3 the glycans do not appear to influence the likely APRs of the antibodies. Therefore, in these structures we modified the Fc glycan regions by adjusting the glycosylated asparagine side chains and glycosidic bonds. We successfully adjusted the glycan chains of 1HZH and 5DK3 and reduced the distance between them and the APRs to show potential influence on aggregation. However, similar to 5DK3, the influence of glycosylation on the APRs of the antibody was limited due to the size of the glycans present in the 3D structure. This report is based on in silico studies to show how antibody glycans can influence aggregation.