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.