Consequences of glycan truncation on Fc structural integrity

Abstract

Effective characterization of protein-based therapeutic candidates such as monoclonal antibodies (mAbs) is important to facilitate their successful progression from early discovery and development stages to marketing approval. One challenge relevant to biopharmaceutical development is, understanding how the stability of a protein is affected by the presence of an attached oligosaccharide, termed a glycan. To explore the utility of molecular dynamics simulations as a complementary technique to currently available experimental methods, the Fc fragment was employed as a model system to improve our understanding of protein stabilization by glycan attachment. Long molecular dynamics simulations were performed on three Fc glycoform variants modeled using the crystal structure of a human IgG1 mAb. Two of these three glycoform variants have their glycan carbohydrates partially or completely removed. Structural differences among the glycoform variants during simulations suggest that glycan truncation and/or removal can cause quaternary structural deformation of the Fc as a result of the loss or disruption of a significant number of inter-glycan contacts that are not formed in the human IgG1 crystal structure, but do form during simulations described here. Glycan truncation/removal can also increase the tertiary structural deformation of CH2 domains, demonstrating the importance of specific carbohydrates toward stabilizing individual CH2 domains. At elevated temperatures, glycan truncation can also differentially affect structural deformation in locations (Helix-1 and Helix-2) that are far from the oligosaccharide attachment point. Deformation of these helices, which form part of the FcRn, could affect binding if these regions are unable to refold after temperature normalization. During elevated temperature simulations of the deglycosylated variant, CH2 domains collapsed onto CH3 domains. Observations from these glycan truncation/removal simulations have improved our understanding on how glycan composition can affect mAb stability.

Keywords: Fc; glycans; glycosylation; molecular dynamics; monoclonal antibodies; oligosaccharides; stability.

Figure 1. Fc structure (A) indicating the locations of structural domains, two attached oligosaccharides colored cyan, and the position of Gly237 residues used to calculate the C_H2-C_H2 distance. For chain H, the C_H2 domain is red and the C_H3 domain is orange. For chain K, the C_H2 domain is green and the C_H3 domain is blue. The hinge, which was present in all glycoform variant simulations, has been left out of this representation for the sake of clarity. C_H2 domain schematic (B) with β-strands labeled and residue indices for strands in Kabat numbering scheme. Two phenylalanine residues 241 and 243, important for C_H2 domain stability, are located on β-strand A. The oligosaccharide attachment residue, Asn297, is located in loop C’E.

Figure 2. Structure, composition, and branches are labeled for oligosaccharides of G2FS2. The glycan has three terminal carbohydrates: FUC-2, and two N-acetyl neuraminic acids or sialylic acids, labeled S. Desialylating G2FS2 produces G2F. Alternate glycan compositions and structures are shown using a simplified notation. Note that MN2, not shown, can be formed by removing MAN5 and MAN8 from M3N2.

Figure 3. The average C_H2-C_H2 distance during simulations at (A) 300 K and (B) 375 K is plotted. Distances are averaged over a 5 ns window and plotted in the window center. Even though glycoform variant starting structures are the same, initial C_H2-C_H2 distances for the three glycoform variants are slightly different because the first data points reported are at 2.5 ns due to averaging. Figure insets (α) and (β) represent DEGLYCO structures along the trajectory. At 300 K, simulations began with DEGLYCO in an open conformation. As DEGLYCO simulations progressed, the C_H2-C_H2 distance decreased and the FcR binding site closed. At 375 K, the C_H2-C_H2 distance increased as C_H2 domains collapsed onto C_H3 domains. The FcR binding site was also closed in the collapsed structure.

Figure 4. Residue-wise RMSDs for all residues of C_H2 domains calculated in simulations at (A) 300 K and (B) 375 K. Residue-wise RMSD values for each glycoform variant were averaged between the two independent simulation runs at each temperature and then averaged over a 5 ns window. Local structure including β-strands and loops are illustrated with black bars.

Figure 5. RMSF values are plotted for individual carbohydrates of G2F and M3N2. RMSF values were averaged between the two independent simulation runs at each temperature. RMSF values for data points simulated at 300 K are connected by lines. RMSF values for simulations preformed at 375 K are not connected by lines due to the absence of terminal GAL and NAG carbohydrates.

Figure 6. Glycan-glycan conformation for G2F in the (A) crystal structure of PDB entry 1HZH with GAL10 of chain K and FUC2 of chain H modeled in using MOE. (B) The preferred glycan-glycan conformation in 300 K simulations (see Materials and Methods). Carbohydrates (black hexagons) are represented using Twister and Paper Chain representations in VMD.^, Gray outlines show the surface of carbohydrates. Yellow dotted lines indicate inter-glycan contacts (identified at ≤ 4.5 Å for non-hydrogen atoms) between chains H and K in the crystal structure. Red dotted lines indicate inter-glycan contacts in the preferred glycan-glycan conformation that were formed during simulations at 300 K and are not formed in the crystal structure for 1HZH.