Physical Stability Comparisons of IgG1-Fc Variants: Effects of N-Glycosylation Site Occupancy and Asp/Gln Residues at Site Asn 297

Abstract

The structural integrity and conformational stability of various IgG1-Fc proteins produced from the yeast Pichia pastoris with different glycosylation site occupancy (di-, mono-, and non- glycosylated) was determined. In addition, the physical stability profiles of three different forms of non-glycosylated Fc molecules (varying amino acid residues at site 297 in the CH2 domain due to point mutations and enzymatic digestion of the Fc glycoforms) were also examined. The physical stability of these IgG1-Fc glycoproteins was examined as a function of pH and temperature by high throughput biophysical analysis using multiple techniques combined with data visualization tools (three index empirical phase diagrams and radar charts). Across the pH range of 4.0 to 6.0, the di- and mono- glycosylated forms of the IgG1-Fc showed the highest and lowest levels of physical stability respectively, with the non-glycosylated forms showing intermediate stability depending on solution pH. In the aglycosylated Fc proteins, the introduction of Asp (D) residues at site 297 (QQ vs. DN vs. DD forms) resulted in more subtle changes in structural integrity and physical stability depending on solution pH. The utility of evaluating the conformational stability profile differences between the various IgG1-Fc glycoproteins is discussed in the context of analytical comparability studies.

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

“…30 It is possible that the increased flexibility of the peptide backbone disrupts the packing interactions between the peptide backbone and the glycan chains, leading to unfolding of this hydrophobic segment. This unfolded apolar segment, in turn, may act as a hotspot for subsequent structural alterations that could lead to irreversible aggregation in IgG1 mAbs, [55][56][57] for example, as observed by SEC analysis in this study. Limiting the flexibility of this particular segment may therefore serve as a good indicator for designing mAbs (and mAbrelated products such as IgG-Fc, fusion proteins, or antibody drug conjugates) with higher thermostability and resistance to aggregation.…”

Production, Characterization, and Biological Evaluation of Well-Defined IgG1 Fc Glycoforms as a Model System for Biosimilarity Analysis

“…Spinner flask expression was utilized for the production of N297Q-Fc in a glycosylation-deficient strain of SMD1168 P. pastoris (Clone produced in previous study 1 ) as follows. A starter culture of 2 mL was inoculated in YPD that contained 100 µg/mL Zeocin.…”

“…Protein therapeutics are inherently structurally complex biological drugs whose active components are not a single, well-defined molecule, but a mixture of similar molecules which can differ by type and extent of post-translational modification, 1–5 chemical modifications, 6–11 and three dimensional conformations. 12,13 In addition, batch to batch variation of active components and production impurities can further complicate defining of the analytical characteristics of a protein drug.…”

“…The Fc region of an IgG1 is also important in antibody clearance because binding of the Fc region to the neonatal Fc receptor (FcRn) increases in vivo half-life 28–31 . In addition, N-linked glycosylation at asparagine 297 (N297) in the Fc region is known to modulate the biological activity 19,32–39 and physical properties of IgG1 Fc, 1,32,40–48 and this can be used to establish similarities and differences between the members of this model system. A series of sequentially truncated glycoforms of IgG1 Fc (Figure 1), which differ only in the size of the N-linked glycan at N297 or a single conservative amino acid mutation (N297Q), were chosen as members for the model system.…”

“…A series of sequentially truncated glycoforms of IgG1 Fc (Figure 1), which differ only in the size of the N-linked glycan at N297 or a single conservative amino acid mutation (N297Q), were chosen as members for the model system. Previous studies have indicated that these glycoforms would display a range of biological activities and physical properties 1,32,45 that would be advantageous for our biosimilarity studies.…”