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.

Keywords:

• aggregation
• bevacizumab
• biobetter
• glycosylation
• monoclonal antibody
• protein engineering
• spatial aggregation propensity
• stability

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors would like to acknowledge the financial support provided by the Singapore-MIT Alliance.

The Biophysical Instrumentation Facility for the Study of Complex Macromolecular Systems (NSF-0070319) is also gratefully acknowledged.

Supplemental Material

Supplemental data for this article can be accessed on the publisher's website.