Structure-Based Prediction of Asparagine and Aspartate Degradation Sites in Antibody Variable Regions

Jasmin F Sydow,Joerg T Regula,Apollon Papadimitriou,Bjoern Mautz,Patrick Cramer,Maximiliane Hilger,Stefan Klostermann,Hans R Voelger,Jan Kuentzer,Hubert Kettenberger,Florian Lipsmeier,Juergen Schoch,Vincent Larraillet,Michael Mølhøj

doi:10.1371/journal.pone.0100736

Abstract

Monoclonal antibodies (mAbs) and proteins containing antibody domains are the most prevalent class of biotherapeutics in diverse indication areas. Today, established techniques such as immunization or phage display allow for an efficient generation of new mAbs. Besides functional properties, the stability of future therapeutic mAbs is a key selection criterion which is essential for the development of a drug candidate into a marketed product. Therapeutic proteins may degrade via asparagine (Asn) deamidation and aspartate (Asp) isomerization, but the factors responsible for such degradation remain poorly understood. We studied the structural properties of a large, uniform dataset of Asn and Asp residues in the variable domains of antibodies. Their structural parameters were correlated with the degradation propensities measured by mass spectrometry. We show that degradation hotspots can be characterized by their conformational flexibility, the size of the C-terminally flanking amino acid residue, and secondary structural parameters. From these results we derive an accurate in silico prediction method for the degradation propensity of both Asn and Asp residues in the complementarity-determining regions (CDRs) of mAbs.

Highlights

Monoclonal antibodies and new antibody domain-based molecules constitute the majority of protein therapeutics under clinical investigation [1,2] for severe malignancies such as cancer, viral and inflammatory diseases. mAbs are potent in a diverse range of therapeutic indications, and are readily generated against promising new targets
Spontaneous degradation of Asn and Asp residues in therapeutic proteins can occur during production, storage, and in vivo
In case of involvement in target binding, the formation of the degradation products succinimide, isoAsp, and Asp embedded in the complementarity-determining regions (CDRs) can lead to loss of function or potency

Summary

Introduction

Monoclonal antibodies (mAbs) and new antibody domain-based molecules constitute the majority of protein therapeutics under clinical investigation [1,2] for severe malignancies such as cancer, viral and inflammatory diseases. mAbs are potent in a diverse range of therapeutic indications, and are readily generated against promising new targets. The process of selecting the clinical candidate mAb typically starts with large-scale screening for functional properties. To ensure optimal technical development and in vivo stability, potentially instable mAbs have to be identified and excluded during the lead selection process. Amongst the most frequently occurring degradation reactions in proteins are the chemical degradation of Asn [4] and Asp residues [5,6]. While these reactions may be kept under control by appropriate storage and formulation conditions [7,8,9,10]

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