Effects of glycosylation on the stability of protein pharmaceuticals

Abstract

In recent decades, protein-based therapeutics have substantially expanded the field of molecular pharmacology due to their outstanding potential for the treatment of disease. Unfortunately, protein pharmaceuticals display a series of intrinsic physical and chemical instability problems during their production, purification, storage, and delivery that can adversely impact their final therapeutic efficacies. This has prompted an intense search for generalized strategies to engineer the long-term stability of proteins during their pharmaceutical employment. Due to the well known effect that glycans have in increasing the overall stability of glycoproteins, rational manipulation of the glycosylation parameters through glycoengineering could become a promising approach to improve both the in vitro and in vivo stability of protein pharmaceuticals. The intent of this review is therefore to further the field of protein glycoengineering by increasing the general understanding of the mechanisms by which glycosylation improves the molecular stability of protein pharmaceuticals. This is achieved by presenting a survey of the different instabilities displayed by protein pharmaceuticals, by addressing which of these instabilities can be improved by glycosylation, and by discussing the possible mechanisms by which glycans induce these stabilization effects.

Figure 1

Molecular models for the α-chymotrypsin (α-CT) glycoconjugates engineered through chemical glycosylation. α-CT at center and α-CT glycoconjugates with glycosylation degree increasing clockwise from top (Lac_n-α-CT with varied n: 1, 3, 5, 7, and 14). Protein represented in CPK style with standard atom coloring, glycans represented in stick style with green coloring. Reproduced with permission of Springer, from Solá et al.

Figure 2

Changes in energetic parameters and in solvent accessible surface area (SASA) for the overall glycoprotein and for the protein portion of the glycoprotein as a function of glycosylation degree. Results were derived from calculations performed on the molecular models constructed for the various α-CT glycoconjugates engineered through chemical glycosylation (see Fig. 1). Glycosylation degree is equal to the number of glycan molecules chemically attached to the protein surface. Adapted with permission of Wiley-Blackwell, from Solá and Griebenow.

Figure 3

Changes in thermodynamic unfolding parameters as a function of glycosylation degree and glycan size (lactose (○) and dextran (Δ)) for the various α-CT glycoconjugates engineered through chemical glycosylation. Reproduced with permission of Springer, from Solá et al.

Figure 4

Changes in protein structural mobility (〈ΔG^mic〉⁻¹) as a function of glycosylation degree and glycan size (lactose (○) and dextran (Δ)) for the various α-CT glycoconjugates engineered through chemical glycosylation. Reproduced with permission of Springer, from Solá et al.