Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab

Abstract

The aggregation of biotherapeutics is a major hindrance to the development of successful drug candidates; however, the propensity to aggregate is often identified too late in the development phase to permit modification to the protein's sequence. Incorporating rational design for the stability of proteins in early discovery has numerous benefits. We engineered out aggregation-prone regions on the Fab domain of a therapeutic monoclonal antibody, bevacizumab, to rationally design a biobetter drug candidate. With the purpose of stabilizing bevacizumab with respect to aggregation, 2 strategies were undertaken: single point mutations of aggregation-prone residues and engineering a glycosylation site near aggregation-prone residues to mask these residues with a carbohydrate moiety. Both of these approaches lead to comparable decreases in aggregation, with an up to 4-fold reduction in monomer loss. These single mutations and the new glycosylation pattern of the Fab domain do not modify binding to the target. Biobetters with increased stability against aggregation can therefore be generated in a rational manner, by either removing or masking the aggregation-prone region or crowding out protein-protein interactions.

The Emerging Importance of IgG Fab Glycosylation in Immunity

“…It was proposed that glycosylation can induce a conformational change in these proteins and trigger fibril formation (67), thereby contributing to amyloidosis; however, this hypothesis needs further research to substantiate. In contrast, a recent study described a decreasing effect of Fab glycans on Ab aggregation (68).…”

De Novo Sequencing and Resurrection of a Human Astrovirus-Neutralizing Antibody

“…Interestingly, engineering of a Fab glycosylation site into the therapeutic antibody bevacizumab has been used to mask hydrophobic amino acids, thereby increasing the antibody stability and decreasing its aggregation. 17 …”

Biopharmaceuticals from microorganisms: from production to purification

“…Among the several techniques used for gene manipulation, we highlight site-directed mutagenesis (SDM). This technique allows rational protein engineering based on its three-dimensional structure 23, 24. Using SDM, one can replace, delete, or insert one or more amino acids in the sequence of a protein.…”