Abolition of aggregation of CH₂ domain of human IgG1 when combining glycosylation and protein stabilization

The CH₂ domain is a critical element of the human Immunoglobulin G (IgG) constant region. Although the CH₂ domain is the least stable domain in IgG, it is also a promising scaffold candidate for developing novel therapeutic approaches. Recently, we succeeded in preparing glycosylated and non-glycosylated CH₂ domain in the host organism Pichia pastoris. Herein, we verified that glycosylation of the CH₂ domain decreased both, its tendency to aggregate and its immunogenicity in mice, suggesting that aggregation and immunogenicity are related. In addition, we have produced in P. pastoris a stabilized version of the CH₂ domain with and without glycan, and their propensity to aggregate evaluated. We found that stabilization alone significantly decreased the aggregation of the CH₂ domain. Moreover, the combination of glycosylation and stabilization completely suppressed its aggregation behavior. Since protein aggregation is related to immunogenicity, the combination of glycosylation and stabilization to eliminate the aggregation behavior of a protein could be a fruitful strategy to generate promising immunoglobulin scaffolds.