Enhancing the stability of adalimumab by engineering additional glycosylation motifs

• Published in: International journal of biological macromolecules Vol. 158; pp. 189 - 196
• Main Authors: Reslan, Mouhamad, Sifniotis, Vicki, Cruz, Esteban, Sumer-Bayraktar, Zeynep, Cordwell, Stuart, Kayser, Veysel
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2020
• Subjects: Aggregation; antigens; binding capacity; Biobetters; engineering; glycosylation; half life; immunogenicity; melting point; Monoclonal antibodies; mutation; neoplasms; receptors; therapeutics; Biobetters; SAP; APR; SE-HPLC; AdmAb; Tagg; BCM; mAb; Aggregation; Monoclonal antibodies; Tm; KD; SLS; WT
• ISSN: 0141-8130; 1879-0003; 1879-0003
• DOI: 10.1016/j.ijbiomac.2020.04.147

Monoclonal antibodies (mAbs) are of high value in the diagnostic and treatment of many debilitating diseases such as cancers, auto-immune disorders and infections. Unfortunately, protein aggregation is one of the ongoing challenges, limiting the development and application of mAbs as therapeutic products by decreasing half-life, increasing immunogenicity and reducing activity. We engineered an aggregation-prone region of adalimumab, the top selling mAb product worldwide - with additional glycosylation sites to enhance its resistance to aggregation by steric hindrance as a next generation biologic. We found that the addition of N-glycans in the Fab domain significantly enhanced its conformational stability, with some variants increasing the melting temperature of the Fab domain by >6 °C. The mutations tested had minimal impact on antigen binding affinity, or affinity to Fcγ receptors responsible for effector function. Our findings highlight the significant utility of this rational engineering approach for enhancing the conformational stability of therapeutic mAbs and other next-generation antibody formats.